Journal of the Association of Genetic Technologists

The Journal

of the Association of Genetic Technologists

Volume 43 • Number 1 • First Quarter 2017

Brain Tickler

Column Editor: Helen Lawce

Brain Tickler

Submitted by:

Bone marrow was received in the laboratory from a 54-year-old man with refractory AML, presenting with leukocytosis and 90% circulating blasts. Ten of 25 metaphases exhibited this karyotype.

Craig Davis, Helen Lawce, and Rikki Harris, Oregon Health & Science University KDL Cytogenetics Laboratory, Portland, Oregon

The answer to this Brain Tickler appears on page 16.

The Journal of the Association of Genetic Technologists First Quarter 2017 Volume 43, Number 1

Table of Contents

The official journal of the AGT

Brain Tickler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside Front Cover Column Editors and Review Board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 A Note from the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Editorial Information Editor Mark Terry, BSc Associate Editors Turid Knutsen, MT(ASCP), CLSp(CG) Helen Lawce, BSc, CLSp(CG) Heather E. Williams, MS, CG(ASCP)CM Su Yang, BSc, CLSp(CG) Book Review Editor Helen Lawce, BSc, CLSp(CG) Copyright © 2017 by the AGT. All rights reserved. Contents are not to be reproduced or reprinted without permission of the AGT Editor.

The Journal of the Association of Genetic Technologists is published four times a year and is available to individuals and libraries at a subscription rate of $115 per year. The subscription rate for members of the AGT is included in the annual membership dues.

Back issues can be purchased for members at $25 per issue as long as supplies are available.

Material intended for publication or correspondence concerning editorial matters should be sent to the editor.

JAGT Editor Mark Terry 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) 248-628-3025 (phone/fax) Email: markterry@charter.net

Case Study Report of a Triploid Fetus Identified in a Pregnancy with Oligohydramnios Ghanbarian Alavijeh Maryam, Saber Siamak, Fazeli Bavand-pour Fatemeh Sadat, Mirzaloo Masoud, Mirzaie Fatemeh, Zare-abdollahi Davood, Ebrahimi Ahmad . . . . . . . 6 Case Report MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/Deletions Helen Lawce, Elina Szabo, Yumi Torimaru, Craig Davis, Karin Osterberg, Susan Olson and Steve Moore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Brain Tickler Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Molecular Diagnostics The Development of NGS Technologies, Looking Back at 2014 Michelle Mah. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Case Report Novel Cytogenetic Findings in a Case of Mixed Phenotype Acute Leukemia within the Context of a Complex Karyotype David Shabsovich, Gary Schiller, Yalda Naeini, Robert Collins and Carlos A. Tirado. . . . 20 Review Lost in Interpretation: Evidence of Sequence Variant Database Errors Adam Coovadia.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Continuing Education Opportunities Test Yourself #1, 2017. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 AGT Journal Clubs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Association Bu siness Association of Genetic Technologists BOD Contacts. . . . . . . . . . . . . . . . . . . . . . . . . . 40 Letter from the President . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Placement service items of less than 150 words and advertisements, requests for back issues, reprint orders, and questions about subscriptions and advertising costs should be sent to the AGT Executive Office at AGT-info@kellencompany.com. Acceptance of advertisements is dependent on approval of the editor-in-chief.

New Membership Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Product Order Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

ISSN 1523-7834

The Journal of the Association of Genetic Technologists is indexed in the life sciences database BIOSIS and in the National Library of Medicine’s PubMed. The Journal of the Association of Genetic Technologists 43 (1) 2017

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The Journal of the Association of Genetic Technologists Staff

Column Editors Abstract Reviews/Genetics in the News Jaime Garcia-Heras, MD, PhD Director of Cytogenetics The Center for Medical Genetics 7400 Fannin, Suite 700 Houston, TX 77054 713-432-1991 713-432-1661 FAX jgarcia@geneticstesting.com Brain Tickler/Book Review Editor Helen Lawce, BSc, CLSp(CG) Clinical Cytogenetics Laboratory Oregon Health Sciences University 3181 SW Sam Jackson Parkway MP-350 Portland, OR 97201 503-494-2790 503-494-6104 FAX lawceh@ohsu.edu

Genetics, Government & Regulation Helen Bixenman, MBA, CLSup, CLSp(CG) San Diego Blood Bank 3636 Gateway Center Avenue, Suite 100 San Diego, CA 92102 619-400-8254 hbixenman@sandiegobloodbank.org Jennifer Crawford-Alvares Cytogenetic Technologist II Section of Hematology/Oncology The University of Chicago Medicine 5841 S. Maryland Ave., Rm. I-304 Chicago, IL jen.crawford34@gmail.com Office: 773-702-9153

Meeting Notices Jun Gu, MD, PhD, CG(ASCP)CM University of Texas MD Anderson Cancer Center School of Health Professions Cytogenetic Technology Program 1515 Holcombe Blvd., Unit 2 Houston, TX 77030 713-563-3094 jungu@mdanderson.org Molecular Diagnostics Michelle Mah, MLT, MB(ASCP)CM Advanced Diagnostics Lab Princess Margaret Cancer Centre University Health Network 610 University Ave., Rm 7-707 Toronto, Ontario Canada M5G 2M9 416-946-4501 ext.5036 michelle.j.mah@gmail.com

Letters to the Editor Mark Terry, JAGT Editor 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) 248-628-3025 (phone/FAX) markterry@charter.net

Special Interests Turid Knutsen, MT(ASCP), CLSp(CG) 17836 Shotley Bridge Place Olney, MD 20832 301-570-4965 turid.knutsen@verizon.net Test Yourself Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

Profiles & Perspectives Hon Fong Louie Mark, PhD, FACMG President KRAM Corporation 2 Pine Top Road Barrington, RI 02806 401-246-0487 HonFong_Mark@Brown.edu

Review Board Linda Ashworth, BSc, CLSp(CG) (Cytogenetics, Molecular genetics) Helen Bixenman, BSc, CLSp(CG), CLSup (Prenatal diagnosis) Judith Brown, MS, CLSp(CG), CLSp(MB) (Cytogenetics) Kim Bussey, PhD (Cancer genetics, Molecular genetics, Microdissection/PCR/DNA) Mona CantĂş, BSc, CLSp(CG) (Cytogenetics) Anthony Ciminski, CG(ASCP)CM Molecular Genetics, Molecular Cytogenetics Adam Coovadia, CLSpP(CG, MG) (Traditional, Molecular, Regulatory) Philip D. Cotter, PhD, FACMG (Prenatal diagnosis, Chromosome rearrangements, Molecular genetics) Jennifer Costanzo, MS, CLSp(CG) (Cytogenetics, Molecular genetics) Janet Cowan, PhD (Cytogenetics, Cancer genetics, FISH, Solid tumors) Lezlie Densmore, BSc, CLSp(CG) (Cytogenetics, Molecular genetics) Janet Finan, BSc, CLSp(CG) (Hemic neoplasms, Somatic cell hybridization) Lakshan Fonseka, MS (Cytogenetics, Molecular genetics)

Sue Fox, BSc, CLSp(CG) (Bone marrow cytogenetics, Prenatal diagnosis, Supervisory/Management) Jaime Garcia-Heras, MD, PhD (Clinical cytogenetics) Robert Gasparini, MS, CLSp(CG) (Prenatal diagnosis, Cytogenetics) Barbara K. Goodman, PhD, MSc, CLSp(CG) (Molecular cytogenetics)

Hon Fong Louie Mark, PhD, FACMG (Molecular genetics, Somatic cell genetics, Cancer cytogenetics, Breast cancer, Trisomies, Laboratory practices, Regulatory practices, FISH) Jennifer L. McGonigle, BA, CLSp(CG) (Cytogenetics) Karen Dyer Montgomery, PhD, FACMG (Cancer cytogenetics, Cytogenetics, Molecular cytogenetics)

Debra Saxe, PhD (Prenatal diagnosis, Cytogenetics) Jack L. Spurbeck, BSc, CLSp(CG) (Cancer cytogenetics, Molecular genetics) Peggy Stupca, MSc, CLSp(CG) (Cytogenetics, Prenatal diagnosis, Breakage syndromes, FISH, Regulations/ QA)

Nancy Taylor, BSc, CLSp(CG), MT(ASCP) (Cytogenetics, Cancer cytogenetics) Stephen R. Moore, PhD, ABMG Michelle M. Hess, MS, CLSp(CG) (Clinical cytogenetics, radiation biology, (Cytogenetics, Cancer cytogenetics) Thomas Wan, PhD toxicology; clinical molecular genetics) (Cytogenetics, Molecular genetics, Lynn Hoyt, BSc, CLSp(CG), CLSup Cancer genetics) Rodman Morgan, MS, CLSp(CG) (Classical cytogenetics) (Cancer cytogenetics) James Waurin, MSc, CLSp(CG) Peter C. Hu, PhD, MS, MLS(ASCP), CG, MB (Prenatal diagnosis, Counseling) Susan B. Olson, PhD (Cytogenetics, Molecular cytogenetics, (Cancer cytogenetics, Molecular Sara Wechter, BSc Education) genetics, Prenatal diagnosis, OB/GYN, (Cytogenetics, Cancer) Counseling, Cytogenetics) Denise M. Juroske, MSFS, MB(ASCP)CM (Cytogenetics, Molecular, Education) Heather E. Williams, MS, CG(ASCP)CM Jonathan P. Park, PhD (Cytogenetics, Molecular Genetics) (Cytogenetics, Molecular genetics, Julia Kawecki, BSc, CLSp(CG) Cell biology) (Cytogenetics, Molecular genetics) Su Yang, BSc, CLSP(CG) (Education, Traditional Cytogenetics) David Peakman, AIMLT, CLSp(CG) Turid Knutsen, MT(ASCP), CLSp(CG) (Prenatal diagnosis) (Cancer cytogenetics, CGH, SKY) Jason A. Yuhas, BS, CG(ASCP)CM (Cytogenetics, Molecular cytogenetics) Carol Reifsteck, BA Brandon Kubala, BSc, CLSp(CG) (Breakage syndromes, Fanconi’s (Traditional Cytogenetics) James Zabawski, MS, CLSp(CG) anemia, Prenatal diagnosis) (Education, Traditional Cytogenetics) Anita Kulharya, PhD Gavin P. Robertson, PhD (Molecular genetics, Clinical (Cytogenetics, Molecular genetics, cytogenetics) Somatic cell genetics, Tumor suppressor Helen Lawce, BSc, CLSp(CG) genes, Cancer genes) (Prenatal diagnosis, Solid tumors, FISH, Laurel Sakaluk-Moody, MSc, MLT(CG) Chromosome structure, Evolution) (Cytogenetics, Developmental biology, Prenatal cytogenetics)

The Journal of the Association of Genetic Technologists 43 (1) 2017

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A Note from the Editor

Welcome Back My Friends…

Welcome to the first issue of The Journal of the Association of Genetic Technologists for 2017. It’s filled with quite a few technical articles, which cover quite a range of topics. I hope you enjoy.

The Usual Suspects And as we start our new year, I want to mention the other people who help me get the journal to you every quarter. I absolutely could not do it without them.

A Quick Look Adam Coovadia, with Evolve Gene in Saint Petersburg, Florida, brings us an in-depth look at problems found in numerous online sequence variant databases, an extraordinarily important topic as we increasingly move toward personalized medicine and next-generation sequencing.

Turid Knutsen, Helen Lawce, Heather Willliams and Su Yang are my Associate Editors, who help me proof galleys, recruit writers, and sometimes review articles. Helen also provides the Brain Ticklers and is the Book Review Editor. From the bottom of my heart, thank you.

Jaime Garcia-Heras writes an occasional column, Abstract Reviews/Genetics in the News. Helen Bixenman and Jennifer Crawford-Alvares have us covered with Genetics, Government & Regulation, Jun Gu handles Meeting Notices, Michelle Mah writes a column on Molecular Diagnostics, Hon Fong Louie Mark writes Profiles & Perspectives, and Sally Kochmar handles all our education-related activities, including the Test Yourself.

Thank you to everyone!

Helen Lawce, one of my long-time Associate Editors, along with her colleagues Elina Szabo, Yumi Torimaru, Craig Davis, Karin Osterberg, Susan Olson and Steve Moore, at the Oregon Health and Science University in Portland, Oregon, presents a case study on MECOM (EVI1) rearrangements in patients with myelodysplastic syndrome.

David Shabsovich and Yalda Naeini, with UCLA, Carlos Tirado with Laboratory of Cytogenetics, Allina Health in Minneapolis, Gary Schiller with UCLA, and Robert Collins from UT Southwestern in Dallas, bring us an article on mixed phenotype leukemia within the context of a complex karyotype.

Behind the scenes, Raven Hardin is our Publications Coordinator, who along with Diane Northup, Christie Ross and Monica Evans-Lombe keep the association’s executive office and publications on track. Thank you!

And this is a first for the journal in the years I’ve been editing it. Maryam Ghandbarian Alavijeh, Saber Siamak, Fazeli Bavand-pour Fatemeh Sada, Mirzaloo Masoud, Mirzaie Fatemeh, Zare-Abdollahi Davood and Ebrahimi Ahmd from Yet Hospital, part of Tehran University of Medical Sciences in Iran, report on a triploid fetus. I don’t believe we’ve ever had submissions from Iran before. Welcome.

Heads-Up! And as an initial reminder, the AGT’s 42nd Annual Meeting will be held in St. Louis, Missouri from June 15-17, 2017 at the St. Louis Union Station Hotel. It’s quite a spectacular-looking hotel and a look at the meeting schedule shows it’ll be yet another great meeting filled with interesting talks and useful workshops!

Start making your plans now!

And Michelle Mah, from McMaster University Medical Centre in Hamilton, Ontario (Canada) writes her regular column on next-generation sequencing.

Cheers, Mark Terry, Editor

AGT Website: www.AGT-info.org Member’s Only area – User Name: First initial, Last initial and AGT ID# (Ex. CR12345) Password: genetics

The Journal of the Association of Genetic Technologists 43 (1) 2017

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Case Study

Report of a Triploid Fetus Identified in a Pregnancy with Oligohydramnios Ghanbarian Alavijeh Maryam1, Saber Siamak1, Fazeli Bavand-pour Fatemeh Sadat1, Mirzaloo Masoud1, Mirzaie Fatemeh1, Zare-abdollahi Davood2*, Ebrahimi Ahmad3* *Corresponding authors 1. Medical Genetics Laboratory, Yas Women's Hospital, Tehran, Iran.

2. University of Social Welfare and Rehabilitation Sciences, Genetic Research Center, Tehran, Iran 3. Kowsar Human Genetics Research Center, Tehran, Iran

Abstract This report describes a pregnancy with a triploid fetus identified from a scan for anomalies at 18 weeks and confirmed by amniocentesis.

A 29-year-old, primigravida woman was referred to our clinic for genetic counseling at 18 weeks of gestation because of a mild oligohydramnios due to amniotic fluid index (AFI) less than the fifth percentile in her 18th week. The woman underwent amniocentesis, which revealed a karyotype of 69,XXX. There was no consanguinity in this family. In postmortem evaluation, we encountered a hydrocephalic fetus with an aberrant skull shape, including dysplastic calvaria with large posterior fontanel, marked intrauterine growth retardation (IUGR) in addition to syndactyly of third and fourth fingers, exophthalmia, cleft palate, low set malformed ears, micrognathia and club foot. Early prenatal diagnosis of this syndrome would provide women an opportunity to terminate a pregnancy earlier, which can prevent the risks associated with late induced abortion or obstetric complications. The results of this study also provides useful information for prenatal genetic counseling.

Keywords Triploidy, Amniocentesis, Olygohydramnios, IUGR, Syndactyly, Genetic counseling

Introduction

upon the parental origin of the extra chromosome set, and placental and/or ultrasound findings. In type I, the additional chromosome set is of paternal origin (diandric) and the placenta is enlarged and partially multicystic (molar), while the fetus is relatively well-grown with either proportionate head size or slight microcephaly (Engelbrechtsen et al., 2013). In type II, which is the most common, the extra set of chromosomes is of maternal origin (digynic) and is characterized by a small, normal-looking placenta and a severely growth-restricted fetus with pronounced wasting of the body and sparing of the head (Engelbrechtsen et al., 2013).

In the context of the first trimester combined screening, although the fetal NT in triploidy does not markedly deviate from that observed for euploid fetuses, the biochemical markers show large deviations. Diandric triploidy is characterized by a combination of increased fb-hCG MoM and moderately decreased PAPP-A MoM, similar to the biochemical profile of cases of trisomy 21. On the other hand, in cases of digynic triploidy, the biochemistry demonstrates markedly decreased fb-hCG and PAPP-A MoMs (Engelbrechtsen et al., 2013; Torring, 2016).

This report describes a pregnancy with a triploid fetus identified from an 18-week anomaly scan and underscores the potential of first trimester combined screening to detect this chromosomal aberration earlier in pregnancy.

Several studies have investigated the usefulness of first trimester combined screening, and confirm its acceptable common aneuploidies detection rate, including: trisomies 13, 18 and 21, and monosomy X (Engelbrechtsen et al., 2013; Baer et al., 2015;). It is well established that trisomy 21 pregnancies are associated with increased maternal age, increased fetal nuchal translucency (NT) thickness, increased maternal serum free b-human chorionic gonadotrophin (fb-hCG) and decreased maternal serum pregnancy-associated plasma protein A (PAPP-A) (Kazerouni et al., 2011). In the context of screening for trisomy 21 at 10-14 weeks, several studies reported that about 85-90% of affected pregnancies are identified for a screen positive rate of 5%. Trisomies 13 and 18 are characterized by increased fetal NT and decreased maternal serum fb-hCG and PAPP-A. Increased NT, normal maternal serum free b-hCG and low PAPP-A is the pattern ascribed to the Turner syndrome that is identified in over 90% of cases (Engelbrechtsen et al., 2013; Baer et al., 2015;).

Beside this acceptable high detection rate for common aneuploidies mentioned above, some studies highlighted the usefulness of this screening strategy to detect a much broader range of chromosomal aberrations, including triploidy and rare numerical and structural chromosomal abnormalities. Fetal triploidy is estimated to occur in 1% of all conceptions, but the affected fetus typically does not survive past the first trimester.

The majority of these pregnancies abort spontaneously in the first trimester. Occasionally, the pregnancy progresses to the second and third trimesters. The prevalence of triploidy at the 11th to 14th week scan is approximately 1:3300 (Jauniaux et al., 1997). The rate continues to decrease from 14th week of gestation onwards. The incidence of triploidy in liveborns is approximately 1:10,000 (Ferguson-Smith and Yates, 1984).

Triploidy can be classified into two phenotypes dependent

Case: A 29-year-old, primigravida Iranian woman was referred to our clinic (Yas Hospital, Tehran University of Medical Sciences) for genetic counseling at 18 weeks of gestation because of a mild oligohydramnios due to an amniotic fluid index (AFI) less than the fifth percentile in her 18-week anomaly scan. Her first trimester combined screening included: nuchal translucency (NT=1.4 mm) and reduced PAPP-A and fb-hCG (0.38 MoM and 0.25 MoM, respectively). Despite these reductions of the biochemical markers,

The Journal of the Association of Genetic Technologists 43 (1) 2017

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Case Study Report of a Triploid Fetus Identified in a Pregnancy with Oligohydramnios

her final screening results were negative for 13, 18 and 21 trisomies, and neural tube defects (NTDs). According to the approved national pregnancy screening program, the 18-week anomaly scan is all that is required. We recommended amniocentesis.

The woman underwent amniocentesis, which revealed a karyotype of 69,XXX (Figure 1). After genetic counseling, the parents decided to terminate the pregnancy and agreed to a postmortem examination. In the postmortem evaluation, we encountered a hydrocephalic fetus with an aberrant skull shape, including dysplastic calvaria with large posterior fontanel, marked intrauterine growth retardation (IUGR), in addition to syndactyly of the third and fourth fingers, exophthalmia, cleft palate, low set malformed ears, micrognathia and club foot (Figure 2).

Fig 1. Karyotype of 69,XXX

Among chromosomal disorders, triploidies are most commonly observed at conception. However, the majority of these triploid cases abort during the first trimester or even later as spontaneous abortions. This reinforces the rarity of triploidy as a chromosomal aberration in cases that undergo amniocentesis. With continued decreases during pregnancy progression, the incidence of triploidy in liveborns is approximately 1:10,000 (Ferguson-Smith and Yates, 1984). Regardless of the lethality of the triploidy as a general rule, as shown in our case, some triploid pregnancies can continue until the later stages of pregnancy and impose great concern to families.

As mentioned, a strategy of routine first trimester screening

has the potential to detect a much broader range of chromosomal aberrations, including triploidy, and rare numerical and structural chromosomal abnormalities. In this regard, despite the normal range of the NT in triploid pregnancies, to a large degree, the double marker biochemical pattern is characteristic. In diandric triploidy, a combination of increased fb-hCG MoM and moderately decreased PAPP-A MoM can be seen. On the other hand, in cases of digynic triploidy the biochemistry demonstrates markedly decreased Fb-hCG and PAPP-A MoMs (Engelbrechtsen et al, 2013; Torring, 2016). In our report, we emphasize the potential of this combination to predict and/or confirm a triploid pregnancy in earlier stages of pregnancy or in confronting a triploid karyotype result during amniocytes culture.

(a)

(c)

Discussion

(b)

(d) Fig 2. (a) club foot, (b) low set malformed ears, (c) cleft palate, (d) syndactyly fingers The Journal of the Association of Genetic Technologists 43 (1) 2017

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Case Study Report of a Triploid Fetus Identified in a Pregnancy with Oligohydramnios

To the best of our knowledge, this is the second related report from Iran. In comparing the aforementioned report and the present study, the mother in the first report underwent amniocentesis due to high risk second trimester maternal serum screening for trisomy 18 (low unconjugated estriol 3 (uE3), normal alpha fetoprotein (AFP) and human chorionic gonadotropin (hCG) levels). The reported karyotype was 69, XXX (Bagherizadeh et al., 2010).

According to the second trimester quadruple maternal serum screen, and based on the largest studies, cases with triploidy fall into two major groups. Those with elevated hCG (>2.5 MoM) and hence at high risk for Down syndrome, and those with elevated AFP (>2.5 MoM) and hence screen positive for open neural tube defect (ONTD), with or without low/normal uE3, and probably elevated inhibin-A. Pregnancies in the second group are identifiable as high risk for trisomy 18 by very low hCG and with or without low/normal AFP, uE3, and inhibin-A (Kazerouni et al., 2011).

Together, using combined, quadruple marker and sequential prenatal screening strategies, the detection rate of the triploid pregnancies was defined as 85%, 78.1% and 91%, respectively (Kazerouni et al., 2011; Engelbrechtsen et al., 2013; Baer et al., 2015). These findings confirm that triploidy is a disorder that can incidentally be detected as part of a screening program for trisomy 21 and/or NTDs and highlight the usefulness and high detection rate for triploidy using these screening strategies.

Many malformations have been reported in association with triploidy, including hydrocephalus, holoprosencephaly, dysplastic calvaria, gastroschisis, encephalocele, myelomeningocele, syndactyly, low set ears, low nasal bridge, exophthalmia, cleft lip/palate, talipes equinovarus, clubfeet, heart defects, and renal agenesis (Doshi et al., 1983; Daniel et al., 2001; McFadden and Robinson, 2006; Toufaily et al., 2016). As mentioned, a postmortem examination found a hydrocephalus fetus with an aberrant skull shape, including dysplastic calvaria with large posterior fontanel, low set ears, low nasal bridge, exophthalmia, cleft palate and limb abnormalities including 3-4 syndactyly of the hands, and club feet.

The most common of the associated malformations have been syndactyly, especially of the third and fourth fingers, which was present in about 65% of the fetuses and regarded as a distinctive feature of triploidy (Toufaily et al., 2016).

Although information on parental origins was not available in this study, more cases with the serum marker patterns of digynic than diandric triploidy were seen in amniotic samples (Engelbrechtsen et al., 2013). Based on this premise and normal looking placenta in our case, it seems that the extra chromosome set is of maternal origin, although this should be verified through short tandem repeats (STRs) study.

Overall, although there is a questionable advantage in screening for triploidy from the view of birth defect prevention, prenatal diagnosis of this syndrome would provide women an opportunity to terminate a pregnancy, which has a high risk of associated obstetric complications. The results of this study also provide useful information for prenatal genetic counseling.

and sequential screening. Obstet Gynecol. 2015;126(4): 753-9.

Bagherizadeh E, Oveisi M, Hadipour Z, Saremi A, Shafaghati Y, Behjati F. Triploidy in a fetus following amniocentesis referred for maternal serum screening test at second trimester. Indian J Hum Genet. 2010 May;16(2): 94-6.

Daniel A, Wu Z, Bennetts B, Slater H, Osborn R, Jackson J, Pupko V, Nelson J, Watson G, Cooke‐Yarborough C, Loo C. Karyotype, phenotype and parental origin in 19 cases of triploidy. Prenat Diagn.

2001 Dec;21(12): 1034-48.

Doshi N, Surti U, Szulman AE. Morphologic anomalies in triploid liveborn fetuses. Hum Pathol. 1983 Aug;14(8): 716-723.

Engelbrechtsen L, Brøndum‐Nielsen K, Ekelund C, Tabor A, Skibsted L. Detection of triploidy at 11-14 weeks' gestation: a cohort study of 190,000 pregnant women. Ultrasound Obstet Gynecol. 2013 Nov;42(5): 530-5.

Ferguson‐Smith MA, Yates JR. Maternal age specific rates for chromosome aberrations and factors influencing them: report of a collaborative European study on 52,965 amniocenteses. Prenat Diagn. 1984 Spring;4 Spec No: 5-44.

Jauniaux E, Brown R, Snijders RJ, Noble P, Nicolaides KH. Early prenatal diagnosis of triploidy. Am J Obstet Gynecol. 1997 Mar;176(3): 550-4.

Kazerouni NN, Currier RJ, Flessel M, Goldman S, Hennigan C, Hodgkinson C, Lorey F, Malm L, Tempelis C, Roberson M. Detection rate of quadruple-marker screening determined by clinical follow-up and registry data in the statewide California program, July 2007 to February 2009. Prenat Diagn. 2011 Sep;31(9): 901-6.

McFadden DE, Robinson WP. Phenotype of triploid embryos. J Med Genet.

2006 July;43(7): 609-612.

Torring N. First trimester combined screening - focus on early biochemistry.

Scand J Clin Lab Invest. 2016 Oct;76(6): 435-47.

Toufaily MH, Roberts DJ, Westgate MN, Holmes LB. Triploidy: variation of phenotype. Am J Clin Pathol. 2016 Jan;145(1): 86-95.

Correspondence to: Zare-abdollahi Davood and Ebrahimi Ahmad Yas Hospital, Tehran University of Medical Sciences, Tehran, Iran Email c/o: Maryam Ghanbaryan, ae35m@yahoo.com

References Baer RJ, Flessel MC, Jelliffe-Pawlowski LL, Goldman S, Hudgins L, Hull AD, Norton ME, Currier RJ. Detection rates for aneuploidy by first-trimester The Journal of the Association of Genetic Technologists 43 (1) 2017

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Case Report

MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/Deletions Helen Lawce, Elina Szabo, Yumi Torimaru, Craig Davis, Karin Osterberg, Susan Olson and Steve Moore, Knight Diagnostics Laboratories, Oregon Health and Science University, Portland, Oregon

Abstract Acute myelogeneous leukemia (AML) with inv(3)/t(3;3)(q13q25) is associated with aberrant expression of the stem-cell regulator MECOM (aka EVI1). Two bone marrow samples received in the OHSU Knight Diagnostic Laboratories (KDL) Cytogenetics Laboratory for chromosomes and FISH for a question of progression of myelodysplastic syndrome (MDS) to AML showed complex abnormalities including a deletion of chromosome 3q, one with del(3)(q13q25) and the other with del(3)(q22q25). In light of the prognostic importance of the activation of the MECOM oncogene and the concurrent inactivation of the GATA2 tumor suppressor that occurs with the classic inversion of chromosome 3q, fluorescence in situ hybridization (FISH) was performed using two different probe designs to better define the 3q deletions in the two cases. Using the Abbott Molecular Laboratories dual fusion MECOM/RPN1 probe, interphase and metaphase cells in both patients showed a variant single fusion (orange/green/fusion) signal pattern consistent with fusion and deletion.

Using the three-color (red/green/aqua) Cytocell EVI1 probe, interphase cells in both cases showed a split red/green signal with the aqua signal remaining with the green signal. The distance between the split signals was generally less than is usually seen in the commonly described inverted chromosome 3. These findings are therefore consistent with a complex inversion and concurrent deletion/deletions of chromosome 3q. Thus, the deletion 3q seen in G-banded chromosomes from bone marrow from these two patients is most consistent with the activation of MECOM and the inactivation of GATA2.

Keywords: 3q21q26 syndrome, AML, MDS, deletion 3q, enhancers, EVI1, FISH, GATA2, inversion 3q, MECOM, oncogene, promoters, RPN1, topologically associated domain (TAD), tumor suppressor.

Introduction

deletion 7q or monosomy 7 is present (Lugthart et al., 2010). There is no difference in survival between t(3;3) and inv(3). AML with 3q21q26 syndrome may occur simultaneously with monosomy 7 (66% of cases), deletion 7q (3%), and deletion 5q (5%) (Huret, 2005). Complex karyotypes are common (21% of cases). The inversion is more common than the translocation, and in rare cases, there can be an insertion of one 3q into the other, ins(3;3) (q26;q21q26) (Smol, in press; Huret, 2005). Also reported are variant translocations of MECOM with other chromosomes such as a t(3;21)(q26;q11), t(3;10)(q26;q21) (Jancuskova et al., 2014), and der(7)t(3;7)(q26;q21). There are at least 12 partners involved in MECOM rearrangements (Wieser, in press). Some of these rearrangements can be cryptic by chromosome analysis (Baldazzi et al., 2016). The partial gain of 3q26-29 in Fanconi anemia has been shown to be associated with overexpression of EVI1 (Meyer et al., 2007, 2011).

The breakpoints for 3q21 and 3q26 are quite variable (Bobadilla et al., 2008; Groschel et al., 2013) (Figure 1). In addition, concurrent microdeletions are not uncommon and can partially remove parts of the probe target yielding aberrant signal patterns (Bobadilla et al., 2008). These factors contribute to the difficulties of FISH probe design. A break-apart probe needs to be designed to span the wide range of breakpoints and microdeletions. The dual fusion design can fail if microdeletions occur and remove some or all of the fusion signals, making true fusions and chromosome overlap difficult to distinguish in interphase. Certain MECOM rearrangements may require more than one probe design to detect or define.

The two most common MECOM rearrangements, the translocation (3;3) and the inversion (3q21q26) are depicted at

The World Health Organization (WHO) classifies acute myelogeneous leukemia (AML) into several groups depending upon the recurrent cytogenetic findings, such as t(8;21), inv(16), t(15;17) and so on. One of the classifications is “AML with a translocation or inversion in chromosome 3.� Termed 3q21q26 syndrome, the common finding is rearrangements of MECOM at 3q26, especially the paracentric inversion of the long arm, inv(3)(q21q26), as well as the less common translocation between homologous long arms, t(3;3)(q21;q26). MDS (greater than 5% of blasts present in bone marrow/blood) is reclassified as AML when the percentage of blasts increases above 20%.

The MECOM gene complex (MDS1 And EVI1 Complex Locus) codes for a protein that regulates transcription and is involved in differentiation, proliferation, and apoptosis (Huret, 2005). It is expressed at high levels in normal CD34 positive cells (lineage-independent). There are two alternative forms of the protein. One is generated from EVI1 (ecotropic viral integration site 1), and the other from the MECOM complex with EVI1 and MDS1 (140 kb upstream of EVI1). It maintains hematopoietic cells and can inhibit myeloid differentiation (Goyama et al., 2008).

Overexpression of the MECOM gene is responsible for 6-11% of adult AML (Hinai et al., 2016), and it is also overexpressed in MDS and other tumor types (Wieser et al., 2003). The frequency of MECOM rearrangements is about 1-4% of AML and MDS patients (Huret, 2005; Lugthart 2010) but represents 32-52% of all 3q abnormalities in AML (Huret, 2005; Baldazzi et al., 2016). The prognosis for patients with 3q21q26 syndrome is very poor, with a CR of 31% (Smol, in press). Overall five-year survival rate is 5.7% with a median survival of 10.3 months. Survival is even shorter if

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Case Report MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/ Deletions

Figure 1. Normal position of the MECOM, EVI1, MDS1, and G2DHE genes in chromosome 3q21 and 3q26 with the breakpoint regions indicated for the inv(3)(q21q26) and the t(3;3)(q21;q26). ISCN (McGown-Jordan et al., 2016) chromosome 3 figure shown below.

A. Breakpoints in the inversion of the MECOM gene generally occur within the EVI1 region or slightly distal to it and in the MDS1 region and distal to it for the translocation 3;3. After Bobadilla et al., 2007.

B. Breaks in 3q21 are generally within the G2DHE enhancer or slightly distal to it. After Grรถschel et al., 2014.

the molecular level in Figures 2 and 3. There are several FISH probes available with various designs for the detection of EVI1 and MECOM rearrangements. Standard two-color break-apart probes, dual color, dual fusion probes, and three-color break-apart probes are all available. Each has advantages and disadvantages for these complex aberrations, as will be discussed below. For our two cases with deletions of 3q, the Abbott Molecular Laboratories (Chicago, IL USA) dual color dual fusion MECOM/RPN1 probe (Figure 4 shows the usual inversion and translocation patterns) and the Cytocell (Cambridge, UK) three-color break-apart (Figure 5 shows the usual inversion and translocation patterns)

were used to determine the presence of an inversion in the deleted chromosomes 3. The three-color probe break-apart probe design has a proximal green signal, a central aqua signal, and a distal red signal. If the 3q26 breakpoint occurs distally, the red signal moves away from the green/aqua, as in a t(3;3). If the breakpoint occurs proximally, the green signal moves away from the red/aqua, as in the inversion 3q (Figure 5).

Here we report two cases of high grade MDS with very unusual signal patterns using MECOM probes.

Materials and methods Probe designs used: For our two cases with deletions of 3q, the Abbott Molecular Laboratories dual color dual fusion MECOM/

Figure 2. Translocation breakpoints, t(3;3).

A. Schematic of the breakpoints of the t(3;3)(q13;q16) at the molecular level (above) and on the ISCN diagram (below).

B. The der(3;3) with activated MECOM on the longer derivative chromosome, seen on the right in the G-banded pair below. The GATA2 enhancer G2DHE is moved from the 3q21 of one homolog to the 3q26 of the other, up-regulating the MECOM. The Journal of the Association of Genetic Technologists 43 (1) 2017

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Case Report MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/ Deletions locus. If the 3q26 breakpoint occurs distally, the red signal moves away from the green/aqua, as in a t(3;3). If the breakpoint occurs proximally, the green signal moves away from the red/aqua, as in the inversion 3q (Figures 3 and 5).

Cultures: Bone marrow was received on both patients and was cultured for 24 and 48 hours using standard cytogenetic methods.

Cells were then harvested for cytogenetic preparations as per standard protocol. Please contact authors if additional information is required.

FISH: The KDL AML FISH panel consists of probes for inv(3)/t(3;3), -5/del(5q), -7/del(7q), and rearrangements of MLL (11q23), BCR/ABL, PML/RARA, RUNX1/RUNX1T1, and CBFB (16q22).

Patient information: Patient 1: Our laboratory received bone marrow from an 80-year-old female diagnosed with myelodysplastic syndrome with a question of progression to AML. She originally presented with macrocytic anemia and thrombocytosis with bone marrow exhibiting sideroblasts and an outside laboratory report showing chromosome deletions of 5q and 12p. From diagnosis, she has been treated with lenalidomide. Four months after her diagnosis she presented with progression to high grade MDS with a hypercellular marrow with trilineage dysplasia and increased aberrant myeloid blasts (15% of white blood cells). At this time her bone marrow

Figure 3. Schematic of the breakpoints of the inv(3)(q13q16) at the molecular level, above, and on the ISCN figure, below. The GATA2 enhancer G2DHE is moved during the rearrangement from 3q21 to 3q26, up-regulating MECOM.

RPN1 probe and the Cytocell three-color break-apart probe were used to determine the presence of an inversion in the deleted chromosomes 3. Figures 2 and 3 show the usual inversion and translocation patterns using the two probe designs. The Abbott dual fusion probe yields the typical 1 orange, 1 green, 2 yellow (fusion) signals seen in this type of probe, with the inversion displaying both fusion signals on the same 3q (Figure 2). The translocation again exhibits the dual fusion classical pattern with one fusion on each derivative, and both the orange and green signals on the longer derivative 3q (Figures 2 and 4). The three-color Cytocell probe break-apart design has a proximal green signal, a central aqua signal, and a distal red signal at each

Figure 5. Abnormal FISH patterns seen using the Cytocell three-color break-apart probes. The probe consists of three colors (center), a proximal aqua, central green, and distal red signal. Since the MECOM breakpoints are spread widely (see Figure 1), this scheme can detect the break-apart pattern in proximal or distal regions, and is often able to define which rearrangement is present in interphase cells (A, B).

A. The inversion 3q213q26 moves the proximal aqua signal away from the green and red due to the more proximal breakpoint.

B. The translocation moves the distal red signal away from the green and aqua due to the more distal breakpoint.

Figure 4. Abbott Molecular Laboratories dual fusion MECOM/ RPN1 abnormal patterns for the inversion (A), shown with the corresponding G-banded homologs in the center, and for the translocation (B). The longer derivative 3 in B is the homolog that has the up-regulated MECOM due to its proximity to the G2DHE GATA2 enhancer (near RPN1, see Figure 1) from the other homolog. C shows the abnormal dual fusion interphase pattern seen with either the inversion or the translocation.

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Case Report MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/ Deletions was submitted for cytogenetics and FISH at KDL. Patient 2: Our laboratory received bone marrow on a 61-yearold male with history of complex cytogenetics and high-risk myelodysplasia. He had pancytopenia with 14% myeloblasts and had been treated with Vidaza. DNA tests showed a TP53 frameshift mutation p.M169fs*12 conferring a higher risk prognosis. He was recommended for stem cell transplant therapy. Unfortunately, the patient died shortly after diagnosis.

probe results were normal. The 3q deletion was investigated using two commercial probes for EVI1 and MECOM/RPN1. See Figures 6 and 7. Results are discussed below.

MECOM/EVI1 FISH results: In light of the prognostic importance of the activation of the EVI1 (MECOM) oncogene and the concurrent inactivation of the GATA2 tumor suppressor, the Abbott Molecular Laboratories dual fusion probe was used to better define the 3q deletion abnormality. In both patients, approximately 25% of interphase cells and most metaphases showed a single fusion of MECOM (3q23) at the 3q21 region containing the GATA2 and RPN1 genes, confirming the interpretation of a deletion (Figure 5). However, in order to determine if the MECOM would be activated and the GATA2 inactivated by a simple deletion, FISH was performed using the three-color Cytocell EVI1 probe. In both cases, the interphases showed a split red/green signal with the aqua remaining with the green signal, consistent with an inversion/deletion rearrangement (Figure 6). Metaphase cells did not exhibit enough distance between the EVI1 FISH signals to be of diagnostic use. See Figure 7 for a schematic of how the abnormal chromosomes 3 in these two cases could have occurred.

Results Both patients showed complex abnormalities including a deletion of 3q. Note that both also had a deletion of the long arm of chromosome 5.

Patient 1: The karyotype was described from the G-banding as follows: 46,XX,del(5)(q13q33),del(12)(p12p13)[2]/46,idem,del(3) (q13q25),del(8)(p21p23)[18] AML FISH panel results: 74% of nuclei exhibited a missing signal for EGR1 (5q31). Other probe results were normal. The 3q deletion was investigated using two commercial probes for EVI1 and MECOM/RPN1. Results are discussed below.

Patient 2: The karyotype was described from the G-banding as follows: 47~50,XY,del(3)(q22q25),del(5)(q14q34),-18,-20,add(21) (p11.2),+22,+22,+1~4mar[cp20] AML FISH panel results: 64% of cells exhibited a missing signal for EGR1 (5q31); 70% of cells had three signals for BCR (22q11.2) consistent with the trisomy 22 seen in the karyotype. All other

Discussion The increased expression of MECOM in the 3q21q26 syndrome was at first thought to be due to its activation by the enhancer of RPN1 at 3q21, but recently the promoter of a nearby gene, GATA2,

Figure 6. Abnormal FISH patterns seen for Patients 1 (A) and 2 (B) using the Abbott Molecular Laboratories dual color dual fusion and the Cytocell break-apart probes.

Using the Abbott Molecular Laboratories dual fusion MECOM/RPN1 probe design, both patients 1 and 2 showed a single fusion pattern on the deleted chromosome 3q. If this was the only probe design available, it would not clarify whether the single fusion was a true MECOM rearrangement or artifact due to the deletion bringing the MECOM and RPN1 close together.

Using the Cytocell EVI1 three-color break-apart design: the distal red separated from the green/aqua in both patients due to one or two deletions that occurred during the inversion event. The interphase cells showed the abnormal pattern, while the metaphase cells had the signals so close to each other that the separation was not clear. The G-banded abnormal chromosomes 3 from each patient are shown at the right for each pair.

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Case Report MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/ Deletions expression: the HoxA cluster of 11 genes normally expresses only Hoxa1 through Hoxa6, but when an intervening CTCF-binding site is knocked out, the next gene in line, Hoxa7, turns on (Narendra et al., 2015). DNA sequences within a TAD interact with a high frequency, and share histone chromatin signatures, expression levels, DNA replication timing (Pope et al., 2014), lamina association, and chromocenter association. These findings suggest that TADs are structural and functional determinants of gene expression and regulation (Sexton et al., 2012). The inversion/ translocation MECOM gene rearrangements have been shown to cause oncogenesis, not by forming a fusion protein such as is the case for BCR/ABL rearrangements in the t(9;22) or RUNX1/ RUNX1T1 in the t(8;21), but rather by up-regulation of MECOM due to close juxtaposition with an enhancer from a different gene. The gene that loses its enhancer is GATA2 in 3q21, a known tumor suppressor, so the rearrangement is oncogenic by moving an enhancer from one TAD to another, up-regulating an oncogene and down-regulating a tumor suppressor. In other words, the 3q21q26 syndrome is caused by moving an ectopic enhancer into an erroneous TAD (Groschel et al., 2014) containing the MECOM gene (Figure 1).

MECOM rearrangements demonstrate how simple movement of a gene from its TAD (topologically associated domain) can change its expression without a true gene fusion by placing the genes in a new genetic compartment under the control of new promoters/enhancers. The apparent deletions of 3q in both patients were instead found to be complex inversion/deletions. These two cases and the literature indicate that it is important to use these probe sets to investigate any abnormality of 3q in AML/MDS, especially if it is concurrent with 5 or 7 monosomy/deletion. Since the rearrangement can also occur as an insertion 3;3, and would be cryptic, our laboratory routinely performs MECOM interphase FISH on AML and MDS cases. In addition, our laboratory policy would be to reflex to MECOM if the karyotype exhibits monosomy or deletion of 7/7q and/or 5/5q and/or the quality of the material is insufficient to see subtle rearrangements of 3q.

Figure 7. Possible schematic for the complex inversion/ deletion(s) to explain the results. The aqua, green, and red circles represent the Cytocell three-color probe signals as seen in the patients (right, shown with G-banded chromosomes 3 from the patient). While the separation of the red signals from the green/aqua usually indicates a translocation of the homologous chromosomes, in this case it indicates that the MECOM breakpoint was more distal than the usual inversion.

Both Patients 1 and 2 lost the q22 to q26.3 segment, and Patient 1 lost additional segment(s) proximally (q13.2 to q13.3).

called G2DHE, has been implicated (Dixon, 2012; Yamazaki, 2014).

GATA2 is a critical hematopoietic stemness factor (Grรถschel, 2014).

Chromosome translocations and inversions play an important role in cancer pathogenesis. Commonly, these rearrangements drive the abnormal cell growth through rearrangement of coding sequences, creating a hybrid protein. The mechanism of carcinogenesis has been more difficult to explain in malignancies with inversions/ translocations that do not create fusion products. However, recently the role of non-coding, regulatory sequences has become easier to detect, and is beginning to be recognized as another driver of cancer. Involvement of such non-coding regulatory sequences was known through the example of the expression of MYC in lymphoid cells due to close proximity with the immunoglobulin heavy chain regulatory regions after a translocation between chromosomes 8 and 14, driving aberrant MYC expression (Polack et al., 1993).

More recently, the role of topologically associated domains (TADs) in gene transcription has become known (Dixon et al., 2012; Perkel, 2015).

TADs are physical compartments within chromatin that allow organization of genomes into discrete neighborhoods with boundaries between that disallow interaction between the genes in that TAD and the regulatory elements of neighboring genes.

The boundaries, enriched for binding sites for cohesion and CTCF binding factor thought to act as insulators, can disrupt gene regulation if they are altered. For example, in mouse embryonic stem cells, knocking out a TAD boundary in a gene cluster with CRISPR/Cas-mediated genome editing can lead to atypical

Glossary AML: Acute myelogeneous leukemia is the most common adult acute leukemia, characterized by a rapid growth of abnormal white blood cells. AML has several subtypes; treatment and prognosis vary among subtypes. AML arising from MDS is often refractory to treatment due to complex genetic changes that are present.

CTCF binding factor: a protein that was discovered originally as a negative regulator of the chicken c-myc gene. It also attaches to chromatin to form loops and to anchor it to the nuclear lamina.

Ecotropic virus: a retrovirus that does not produce disease in its natural host but does replicate in tissue culture cells derived from the host species.

MDS: Myelodysplastic syndromes are a group of clonal myeloid neoplasms characterized by ineffective hematopoiesis that presents

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Case Report MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/ Deletions clinically as cytopenia(s), dysplasia in one or more hematopoietic cell lines in the bone marrow, and risk of transformation to acute myeloid leukemia (AML).

associated 31 gains in leukemic transformation consistently target EVL1, but do not affect low TERC expression in FA. Blood. 2011;117(22):60476050.

Narendra V, Rocha PP, An D, Raviram R, Skok JA, Mazzoni EO, Reinberg D. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science. 2015;347: 1017-21.

Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot T, van Berkum NL, Meisig J, Sedat J, Gribnau J, Barillot E, Bluthgen N, Dekker J, Heard E. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature. 2012;485: 381-385.

Perkel J. Mapping Chromosome Neighborhoods. BioTechniques. 2015;58: 280-284.

Polack A, Feederle R, Klobeck G, Hörtnagel K. Regulatory elements in the immunoglobulin kappa locus induce c-myc activation and the promoter shift in Burkitt’s lymphoma cells. EMBO J. 1993;12: 3913–3920.

Pope BD, Ryba T, Dileep V, Yue F, Wu W, Denas O, Vera DL, Wang Y, Hansen RS, Canfield TK, Thurman RE et al. Topologically associating domains are stable units of replication-timing regulation. Nature.

2014;515: 402-405.

Sexton T, Yaffe E, Kenigsberg E, Bantignies F, Leblanc B, Hoichman M, Parrinello H, Tanay A, Cavalli G. Three-dimensional folding and functional organization principles of the Drosophila genome. Cell.

2012;148: 458-472.

Sexton T, Cavalli G. The Role of Chromosome Domains in Shaping the Functional Genome. Cell. 2015;160: 1049-1059.

Smol T. inv(3)(q21q26) RPN1/MECOM; t(3;3)(q21;q26) PRN1/MECOM; ins(3;3)(q26;q221q26) RPN1/MECOM. Atlas Genet Cytogenet Oncol Haematol. In Press.

Wieser R, Schreiner U, Rieder H, Pirc-Danoewinata H, Grüner H, Loncarevic I.F, Fonatsch C. Interphase fluorescence in situ hybridization assay for the detection of rearrangements of the EVI-1 locus in chromosome band 3q26 in myeloid malignancies. Haematologica. 2003;88: 25–30.

WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (IARC WHO Classification of Tumours) 4th Edition, 2008 by The International Agency for Research on Cancer (Author), S. Swerdlow (Editor), E. Campo (Editor), N. Lee Harris (Editor), E.S. Jaffe (Editor), S.A. Pileri (Editor), H. Stein (Editor), J. Thiele (Editor), J.W. Vardiman (Editor) Publisher: World Health Organization.

Yamazaki H, Suzuki M, Otsuki A, Shimizu R, Bresnick E, Engel JD, Yamamoto M. A remote GATA2 hematopoietic enhancer drives leukemogenesis in inv(3)(q21;q26) by activing EVL1 expression. Cancer Cell. 2014;25: 415–427.

Topologically associated domain (TAD): a three-dimensional chromosome neighborhood, ranging in size from hundreds of kilobases to a megabase, that functions to organize genes together with those genes and regulatory elements with which it requires contact and insulating them from those it should not contact.

Chromosome rearrangements can influence the position of a gene and move it to an ectopic TAD, changing its expression. TADs disappear in metaphase. The nature of the TAD boundaries is still being investigated, but is thought to involve CTCF binding factors and cohesin.

References Baldazzi C, Luatti S, Zuffa E, Papayannidis C, Ottaviani E, Marzocchi G, Ameli G, Bardi MA, Bonaldi L, Paolini R, Gurrieri C, Rigolin GM, Cuneo A, Martinelli G, Cavo M, Testoni N. Complex chromosomal rearrangements leading to MECOM overexpression are recurrent in myeloid malignancies with various 3q abnormalities. Genes Chromosomes Cancer. 2016;55(4): 375–388.

Bobadilla D, Enriquez E, Alvarez G, Gaytan P, Smith D, Slovak M. An interphase fluorescence in situ hybridisation assay for the detection of 3q26.2/EVI1 rearrangements in myeloid malignancies. Br J Haematol.

2007;136: 806–813 Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012;485: 376-80.

Gröschel S, Sanders MA, Hoogenboezem R, de Wit E, Bouwman BA, Erpelinck C, van der Velden VH, et al. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia. Cell. 2014;157: 369–81.

Goyama S, Yamamoto G, Shimabe M, Sato T, Ichikawa M, Ogawa S, Chiba S, Kurokawa M. Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells. Cell Stem Cell. 2008;(3): 207–220.

Hinai AA, Valk PJ. Review: Aberrant EVI1 expression in acute myeloid leukemia. Br J Haematol. 2016;172(6): 870-878.

Huret JL. 3q21q26 rearrangements in treatment related leukemia. Atlas Genet Cytogenet Oncol Haematol. 2005;9(3): 234-235.

ISCN 2016: An International System for Human Cytogenomic Nomenclature (2016). Editors: McGowan-Jordan J, Simons A, Schmid, M. Cytogenetic and Genome Research 2016;149, No. 1-2.

Jancuskova T, Plachy R, Zemankova L, Hardekopf D, Stika J, Zejskova L, Praulich L, Kreuzzer KA, Rothe A, Othman MA, Kosyakova N, Pekova S. Molecular characterization of the rare translocation t(3;10)(q26;q21) in an acute myeloid leukemia patient. Molecular Cytogenetics. 2014;7: 47 Lugthart S, Gröschel S, Beverloo H, Kayser S, Valk P, van Zelderen-Bhola S, et al. Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and Various Other 3q Abnormalities in Acute Myeloid Leukemia. J Clin Oncol. 2010; 28(24): 3890-3898.

Meyer S, Fergusson WD, Whetton AD, Moreira-Leite F, Pepper SD, Miller C, Saunders EK, White DJ, Will AM, Eden T, Ikeda H, Ullmann R, Tuerkmen S, Gerlach A, Klopocki E, Tonnies H. Amplification and translocation of 3q26 with overexpression of EVI1 in Fanconi anemiaderived childhood acute myeloid leukemia with biallelic FANCD1/ BRCA2 disruption. Genes Chromosomes Cancer. 2007;46(4): 359-72.

Meyer S, Bristow C, Wappett M, Pepper S, Whetton AD, Hanenberg H, Neitzel H, Wlodarski MW, Ebell W, Tonnies H. Fanconi anemia (FA)-

Correspondence to: Helen Lawce Oregon Health and Sciences University, Portland, Oregon lawceh@ohsu.edu

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Brain Tickler

Brain Tickler Summary (see inside front cover)

46,XY,t(4;12)(q12;p13)[10]/46,XY[10] Because of the abnormal chromosome 12 short arm, FISH with an ETV6 break-apart probe was performed. 52/100 interphase cells scored exhibited an ETV6 rearrangement signal pattern, consistent with the breakpoints on 12p. Metaphase FISH demonstrated the proximal green signal to be retained on the derivative 12 with the distal red signal present on the derivative 4. Based on the 4q breakpoint, ETV6/CHIC2 fusion is predicted, consistent with AML. (Note: CHIC2 FISH was not performed). Interphase FISH with the ETV6 break-apart probe will be useful in following the patient for residual disease.

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Molecular Diagnostics

Column Editor: Michelle Mah, MLT, MB(ASCP)CM

The Development of NGS Technologies, Looking Back at 2014 Happy first quarter of 2017 everyone! I would like to start off with some follow-up on a brief review of technologies I wrote about in 2014. It has been a full two years of advancements and clinical integration of next-generation sequencing (NGS) across genetic laboratories. For those joining us for the first time, welcome and to everyone else, welcome back. This column is a quasi-sequel to the technical progressions of genetic sequencers I wrote about two years ago.

The grandest Illumina platforms are the HiSeq X Systems, which were first reported to break the $1,000 human genome barrier in 2014. The cost strictly covered consumables, sequencing power plus labour at the time; it did not include the data processing, project management, quality control and other extended analyses. According to the National Human Genome Research Institute's update last summer, the cost to generate one high-quality whole human genome sequence was reported to be less than $1,500 in 2015. Interestingly, in the first quarter of 2016, a US-based genetics startup, Veritas Genetics (Danvers, MA, USA), advertised consumer-based whole genome sequencing on the HiSeq X Ten system for $999. The requirements include a doctor’s consent and very little else. This kind of consumerism opens great potential for data generation from the public domain, but it may create channels of confusion and misinterpretation without fully supported medical guidance.

Another biotech giant with a prominent presence in clinical genetics is Thermo Fisher Scientific and its Ion Torrent series of benchtop sequencers. The series starts with the smallest Ion PGM System, which provides output of up to two GB, and its diagnostic counterpart, the Ion PGM Dx instrument designed for regulated clinical environments. The larger systems include the Ion Proton, with throughput of 10 GB, and its newest members, the Ion Proton S5 and S5 XL with throughput of up to 15 GB. Many Ion Torrent applications mirror the Illumina assays but one main difference is the sequencing speed. The sequencing time on Illumina instruments can generally take over 24 hours, whereas the sequencing on the Ion sequencers takes two to four hours. Sequencing run times are just one factor when deciding on the best choice of platforms; other factors include total cost, application types and data analysis workflows. For those interested in more details between the two different platforms, please refer to an application review by Goodwin S et al. (2016).

Finally, the world leader in nucleic assay and sample extraction methodologies, Qiagen (Hilden, Germany), launched a complete NGS system from sample extraction to bioinformatics analysis near the end of 2015. The platform, called the Qiagen GeneReader NGS System, consists of several components designed to complete a workflow from automated NGS sample setup to data analysis. GeneReader has been made available to markets outside North America, and recently Qiagen announced broader commercialization should take place at the start of 2017.

NGS chemistries in the clinical lab The most relevant NGS platforms implemented in diagnostic laboratories are benchtop sequencers, most accompanied by same-vendor NGS targeted gene panels to optimize sequencing performance and genetic results. The two largest providers of NGS products to date are Illumina, Inc. (San Diego, CA, USA) and Thermo Fisher Scientific (Waltham, MA, USA).

Both technologies are based on massively parallel sequencing where millions of amplified single stranded nucleic acid templates are anchored on a solid matrix and followed by sequencing by synthesis technology (SBS). SBS technology is based on the use of DNA polymerase to extend template strands one nucleotide at a time. The incorporation activity is monitored and then processed for base calling. Illumina uses optics to capture the addition of four differently labeled fluorescent nucleotides to newly synthesized template strands, and images of fluorescent intensities are used for base calling. Thermo Fisher uses semiconductor sequencing technology which measures the change in pH upon the incorporation activity: base calling is dependent on the release of hydrogen ions when nucleotides are successfully incorporated into the growing complementary template strand. There are millions of these reactions occurring simultaneously in a very small surface area, the very definition of massively parallel or high-throughput sequencing. There is a good review by Fuller CW et al (2009) for those interested in learning more about SBS approaches.

Illumina is still considered the leader in providing sequencing information, and carries several benchtop sequencers that mainly differ in sequencing capacity. The smallest and most recent MiniSeq System released at the beginning of 2016 has complete onboard informatics analysis. The versatile MiSeq and FDA-cleared MiSeqDx system are both slightly larger in size, but has a sequencing output of 15 gigabytes (GB), which is approximately twice the output of the MiniSeq. Both systems are sufficient for small to medium sized labs to perform many targeted gene panels that can interrogate hundreds of diseases associated genomic regions. Next in line is the NextSeq System, which has a maximum output of 120 GB and is sufficient for sequencing one complete human genome. Finally, the larger sequencers which require significantly more informatics support are the HiSeq instruments. The maximum output of the largest model is 1,800 GB of data; these sequences are less common in routine diagnostics and most frequently used in genomics centers to perform large-scale research projects like whole -omics sequencing and population studies.

Developing NGS platforms In that same column, I also wrote about the 2013 announcement by global healthcare company Roche (Basel, Switzerland) to develop diagnostic products for the clinical market based on single-molecule real time (SMRT) Sequencing technology devised by Pacific Biosciences (Menlo Park, CA, USA). Unlike massively parallel sequencing technologies, SMRT Sequencing does not require amplification and hence reduces PCR-associated biases with increased sensitivity.

The partnership between Roche and Pacific Biosciences was

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Molecular Diagnostics

Column Editor: Michelle Mah, MLT, MB(ASCP)CM

The Development of NGS Technologies, Looking Back at 2014

terminated last year and the news was in one of the top ten most-read articles of 2016 published by online news organization GenomeWeb (https://www.genomeweb.com/). Despite the announcement, Pacific Biosciences introduced the Sequel System last year with capabilities suited for research endeavors such as whole genome de novo assemblies. The company has stated that it will forge ahead with the commitment to advance clinical research. Roche, on the other hand, has acquired another kind of single molecule sequencing technology based on nanopore sequencing methods. Essentially, sequencing takes place through synthetic or biological nano-sized channels. This is considered an example of molecular diagnostics coupled with nanotechnology, which is a topic I introduced in my last column of 2016.

Finally, Bio-Rad Laboratories (Hercules, CA, USA) announced the development of a fully-integrated microdropletbased desktop sequencer called the GnuBio. The technology is based on proprietary chemistry where individual sequencing reactions take place in single aqueous droplets. It is designed as a single workflow application touted as the first fully integrated NGS platform where laboratorians simply load genomic DNA into the system and obtain real time analysis from one machine.

However, aside from the website gnubio.com current updates are scarce. Mentions of these and more upcoming technologies are highlighted in the review by Goodwin et al. (2016).

Jovanovich SB, Nelson JR, Schloss JA, Schwartz DC, Vezenov DV. The challenges of sequencing by synthesis. Nat Biotechnol. 2009;27(11): 101323.

Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet. 2016;17(6): 333-351.

Institute for Quality Management in Health Resources. Available on the World Wide Web: https://iqmh.org/Resources NextSeq Series of Sequencing Systems. System Specification Sheet: Sequencing, Illumina. 2015. Available on the World Wide Web: http:// www.illumina.com/content/dam/illumina-marketing/documents/products/ datasheets/datasheet-nextseq-500.pdf Precision Medicine initiative. Draft Guidance: Use of standards in FDA regulatory oversight of next generation sequencing (NGS)-based in vitro diagnostics (IVDs) used for diagnosing germline diseases.

U.S. Department of Health and Human Services, Food and Drug Administration. 2016. Available on the World Wide Web: http://www. fda.gov/ScienceResearch/SpecialTopics/PrecisionMedicine/default.htm The Cost of Sequencing a Human Genome. National Human Genome Research Institute. 2016. Available on the World Wide Web: https://www. genome.gov/sequencingcosts/.

Looking forward I described NGS-based clinical assays as a disruptive shift in 2014 because of unfamiliarity and lack of standardizations.

Fortunately, from my observations of my surrounding work environment over the last two years, the growing implementation of NGS has accumulated different expertise and built collaborations between different working groups.

I have also noticed astonishing developments in sequencing chemistries and availability of clinical applications. Workflows are becoming more efficient, sequencing runtimes have decreased while generating high quality genetic data and more accurate results. This maturity of NGS technologies has assisted many labs around us to move pass the initial lack of familiarity.

As labs successfully incorporate NGS assays, not only have concerns like quality controls and informatics challenges been continually addressed, the focus has evolved to more downstream issues like improving the design of assay content and pathways to better data processing. Efforts to standardize the implementation and quality management of NGS technologies are gaining traction; examples include the FDA draft of regulatory oversight for NGS-based tests issued last July, and the release of more comprehensive accreditation checklists by CAP and IQMH.

Resources Aziz N, Zhao Q, Bry L, Driscoll DK, Funke B, Gibson JS, Grody WW, Hegde MR, Hoeltge GA, Leonard DG, Mercker JD, Nagarajan R, Palicki LA, Robetorye RS, Schrijver L, Weck KE, Voelkerding KV. College of American Pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med. 2015;139(4); 481-493.

Fuller CW, Middendorf LR, Benner SA, Church GM, Harris S, Huang X, The Journal of the Association of Genetic Technologists 43 (1) 2017

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Case Report

Novel Cytogenetic Findings in a Case of Mixed Phenotype Acute Leukemia within the Context of a Complex Karyotype David Shabsovich2, Gary Schiller3, Yalda Naeini2, Robert Collins4 and Carlos A. Tirado1 1. Laboratory of Cytogenetics, Allina Health, Minneapolis, MN 2. Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, CA 3. Department of Medicine, UCLA, Los Angeles, CA 4. Department of Internal Medicine, UT Southwestern, Dallas, TX

Abstract Background Mixed phenotype acute leukemia (MPAL) is a rare hematological malignancy characterized by combinatorial aberrations involving cells of the myeloid, T-, and/or B- lineages, most often diagnosed by means of immunophenotyping in order to assess lineage-specific markers, which can still yield inconclusive diagnoses. MPAL with a complex karyotype (three or more chromosomal abnormalities) is a cytogenetic subtype of MPAL associated with a poor prognosis, but limited data is available about the cytogenetic abnormalities present in this context.

Findings Herein, we present the case of a 67-year-old female whose bone marrow biopsy revealed an extensive blast population showing dual morphologic differentiation, including lymphoblasts and larger myeloblasts with monocytic differentiation. Multiparametric immunophenotyping by flow cytometry revealed a blast population that was positive for CD45, CD19, CD22, CD34, CD38, and HLA-DR. The blast populations were also immunereactive for both myeloperoxidase and TdT; thus, a diagnosis of mixed phenotype acute leukemia was rendered. Conventional cytogenetic analysis revealed a hyperdiploid composite karyotype with numerical abnormalities involving chromosomes 2, 6, 8, 10, 11, 14, 19, 20, 21, and 22, as well as structural abnormalities involving 1p, 1q, 9p, 16p, 17p, 19q, 20q, and a marker chromosome. Concurrent interphase and metaphase FISH studies were able to detect a deletion of CDKN2A/p16 at 9p21 and corroborated the presence of extra copies of chromosomes 8, 11, 20, and 22.

Conclusions This case provides further insight into the plethora of cytogenetic abnormalities not involving BCR-ABL1 and/or MLL present in MPAL with a complex karyotype and adds to the pool of cytogenetic information about this rare subset of hematological malignancies.

Keywords: mixed phenotype acute leukemia, cytogenetics, FISH

Introduction

was performed consequently in August 2014, which demonstrated a mixed phenotype acute leukemia, B/myeloid, with extensive blast population showing dual morphologic differentiation including lymphoblasts and larger myeloblasts with monocytic differentiation.

Multiparametric immunophenotyping by flow cytometry revealed a blast population that was positive for CD45, CD19, CD22, CD34, CD38, and HLA-DR. The blast population was also immunoreactive for both myeloperoxidase and TdT; hence, the designation of mixed lineage acute leukemia was rendered. FISH was positive for 9q34 deletion and gain of 11q23 (MLL). Cytogenetic analysis showed a hypodiploid, complex karyotype. No BCR-ABL1 translocation was observed.

She was started on chemotherapy in August 2014. Bone marrow biopsy collected in March 2015 showed no evidence of blasts on flow cytometry and a normal female karyotype, indicating a good response to her treatment. She was then referred to UCLA Medical Center for consideration of allogeneic stem cell transplant in April 2015 where she was diagnosed with relapsed mixed-phenotype (B/myeloid) leukemia by means of bone marrow biopsy with approximately 80-90% involvement of the marrow cellularity. She was then treated with two cycles of salvage chemotherapy from June 2015 to August 2015 at which time peripheral blood flow cytometry and immunophenotyping showed no evidence of disease. She has again returned to UCLA for consideration of the allogeneic stem cell transplant.

Mixed phenotype acute leukemia (MPAL) is a rare hematological malignancy characterized by combinatorial aberrations involving cells of the myeloid, T-, and/or B- lineages, most often diagnosed by means of immunophenotyping in order to assess lineage-specific markers. The most recurrent cytogenetic correlates of MPAL are t(9;22)(q34;q11.2) resulting in the BCR-ABL1 fusion gene or rearrangements involving MLL (11q23), the former of which is associated with a poor prognosis (Matutues et al., 2011). MPAL with a complex karyotype (three or more chromosomal abnormalities) is likewise associated with a poor prognosis, but limited data is available about the cytogenetic abnormalities present in this context.

Herein, we present the case of a 67-year-old female diagnosed with MPAL (myeloid/B lineage) bearing a complex karyotype with various numerical and structural abnormalities. Conventional cytogenetics and fluorescence in situ hybridization analyses were used in conjunction to elucidate gene-specific abnormalities, and their independent and synergistic prognostic impacts are discussed.

Clinical Presentation A 67-year-old female with a past medical history only significant for hypothyroidism, presented to the emergency department in July 2014, where she reported a three-month history of weakness, fatigue, multiple bruising, and shortness of breath. Initial lab work obtained at that time revealed leukocytosis with WBC at 41.0 × 103 μL, hgb 7.4 g dL−1, and Plt 29.0 × 103 μL. Bone marrow biopsy

Materials and Methods

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Case Report Novel Cytogenetic Findings in a Case of Mixed Phenotype Acute Leukemia within the Context of a Complex Karyotype Conventional cytogenetics Conventional cytogenetic analysis was performed using standard cytogenetic techniques and karyotypes were conveyed using ISCN 2013 guidelines for nomenclature (Shaffer et al., 2013).

Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) analysis was performed using the following probes on interphase nuclei and/or metaphases: Vysis LSI MLL Dual Color, Break Apart Rearrangement Probe, Vysis BCR/ABL1/ASS1 Tri-Color DF FISH Probe Kit, Vysis LSI MYC Dual Color Break Apart Rearrangement Probe, and Vysis CDKN2A/CEP9 FISH Probe Kit, all purchased from Abbott Molecular.

corroborating extra copies of chromosome 8 (Figure 4). Metaphase FISH revealed monoallelic loss of p16/CDKN2A (9p21) signal as a result of a structural abnormality involving 9p (Figure 5).

Results

Figure 2. Interphase and metaphase FISH studies using the Vysis LSI MLL Dual Color, Break Apart Rearrangement Probe shows additional intact MLL (11q23) signals in 220/300 nuclei.

Conventional cytogenetics Conventional cytogenetic analysis revealed a complex karyotype with multiple numerical and structural abnormalities. Numerical abnormalities included gains of chromosome X, 2, 6, 8, 10, 11, 19, 20, 21, and 22 and a loss of chromosome 14. Structural abnormalities involved additional material of unknown origin on chromosomes 1p, 9p, 16p, 17p, 19q, 20q, an isodicentric chromosome 1q, and a marker chromosome (Figure 1). This constellation of findings was conveyed as follows: 61-63,XX,+X,+X,+add(1)(p32),+idic(1)(q21),+2,+6,+8,add(9) (p21),+10,+11,-14,add(16)(p11.2),add(17)(p11.2),+19,+add(19) (q13.3),+20,+20,+20,+del(20)(q11.2q13.1),+21,+21,+21,+21,+22, +22,+22,+mar[cp11]/46,XX[9]

Figure 3. Interphase and metaphase FISH studies using Vysis BCR/ABL1/ASS1 Tri-Color DF FISH Probe Kit shows additional BCR (22q11.2) signals in 232/300 nuclei, corroborating the presence of additional copies of chromosome 22.

Figure 4. Interphase FISH reveals extra copy of intact MYC (8q24) signal in 17/300 nuclei, corroborating extra copies of chromosome 8.

Figure 1. Representative karyotype depicting numerous numerical and structural abnormalities. Only 9 metaphases were analyzed due to low mitotic index.

Fluorescence in situ hybridization Interphase and metaphase FISH studies using the Vysis LSI MLL Dual Color, Break Apart Rearrangement Probe showed additional intact MLL (11q23) signals in 220/300 nuclei, corroborating the presence of additional copies of chromosome 11 (Figure 2).

Interphase and metaphase FISH studies using Vysis BCR/ABL1/ ASS1 Tri-Color DF FISH Probe Kit showed additional BCR (22q11.2) signals in 232/300 nuclei, corroborating the presence of additional copies of chromosome 22 (Figure 3). Interphase FISH revealed extra copy of intact MYC (8q24) signal in 17/300 nuclei,

Figure 5. Metaphase FISH reveals monoallelic loss of p16/ CDKN2A (9p21) signal as a result of a structural abnormality involving 9p.

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Case Report Novel Cytogenetic Findings in a Case of Mixed Phenotype Acute Leukemia within the Context of a Complex Karyotype

Discussion

analysis of similar cases is warranted in order to elucidate additional information about MPAL with a complex karyotype, not bearing abnormalities involving the Philadelphia chromosome or MLL.

This case highlights the complex plethora of numerical and structural abnormalities that can be present in mixed phenotype acute leukemia (MPAL), and raises questions about the prognostic impact their synergistic effect can have. Many of these findings have been observed in individual cases of MPAL, but not in conjunction with one another. Additionally, molecularly confirmed (by FISH) loss of CDKN2A/p16 has never been reported in MPAL, and is likely associated with a poor prognosis as it is a key tumor suppressor gene involved in numerous pathways and is associated with a poor prognosis in both AML and B-ALL. Among other abnormalities observed in this case, structural abnormalities involving 1p32 (e.g., deletions) have been observed in about 1% of MPAL cases, and can result in the SIL-TAL1 fusion gene.

To our knowledge, an idic(1)(q21) has not been reported in MPAL, and it is unclear what role gain of 1p/loss of 1q may play (Al-Seraihy et al., 2009; Yan et al., 2012). In this case, additional material of unknown origin on the short arm of chromosome 1 may have resulted in deregulation of the SIL (1p32) or TAL1 (1p32) genes, although abnormalities of these genes have been most widely implicated in T-cell-related leukemias, providing evidence against its involvement in a myeloid/B-cell leukemia (Aplan et al., 1991; Mansur et al., 2009).

In the context of such SIL or TAL1 disruptions, there is generally a negative prognostic impact (Mansur et al., 2009). Given the lack of information regarding isodicentric chromosome 1q in MPAL, we are not able to determine the possible prognostic significance of this abnormality in this context.

Abnormalities involving the short arm of chromosome 17 have been reported in the context of i(17)(q10), a very rare abnormality in MPAL. In the present case, there appears to be additional material of unknown origin at 17p11.2, leading to partial deletion of 17p distal to this breakpoint. 17p abnormalities can lead to loss of TP53, which is associated with a poor prognosis in AML and B-ALL, and would likely be associated with a poor prognosis in MPAL (Hoehn et al., 2012).

In this case in particular, the synergistic effects of the loss of TP53 and p16/CDK2NA would likely lead to an even greater worsening of the prognosis.

Hyperdiploidy is seen in nearly a quarter of cases of MPAL, and is typically associated with a good prognosis and considerable response to therapy. This case bears numerous numerical gains, leading to a modal number of about 62, which would be considered hyperdiploid.

However, the presence of numerous structural abnormalities in the context of a hyperdiploid karyotype may offset the positive prognostic impact (Matutes et al., 2011; Manola, 2013). Despite this information, given the complex and mixed phenotype of this leukemia, the exact prognostic impact of hyperdiploid remains equivocal.

All in all, this case provides further insight into the plethora of cytogenetic abnormalities not involving BCR-ABL1 and/or MLL present in MPAL with a complex karyotype, and adds to the pool of cytogenetic information about this rare subset of hematological malignancies. Although particular abnormalities may have certain prognoses in contexts independent of other ones, this case highlights the synergistic prognostic impact that cytogenetic abnormalities can have, such as the loss of multiple tumor suppressor genes nullifying the positive prognostic impact of hyperdiploidy. Further investigation and

References Al-Seraihy AS, Owaidah TM, Ayas M, El-Solh H, Al-Mahr M, Al-Ahmari A, Belgaumi AF. Clinical characteristics and outcome of children with biphenotypic acute leukaemia. Haematologica. 2009;94: 1682–1690.

Aplan PD, Lombardi DP, Kirsch IR. Structural characterization of SIL, a gene frequently disrupted in T-cell acute lymphoblastic leukemia. Mol Cell Biol. 1991 Nov;11(11) :5462-9.

Hoehn D, Medeiros LJ, Chen SS, Tian T, Jorgensen JL, Ahmed Y, Lin P. CD117 expression is a sensitive but nonspecific predictor of FLT3 mutation in T acute lymphoblastic leukaemia and T/myeloid acute leukaemia. Am J Clin Path. 2012;137: 213–219.

Manola KN. Cytogenetic abnormalities in acute leukaemia of ambiguous lineage: an overview. Br J Haematol. 2013 Oct;163(1): 24-39.

Mansur MB, Emerenciano M, Brewer L, Sant'Ana M, Mendonça N, Thuler LC, Koifman S, Pombo-de-Oliveira MS. SIL-TAL1 fusion gene negative impact in T-cell acute lymphoblastic leukemia outcome. Leuk Lymphoma. 2009 Aug;50(8): 1318-25. doi: 10.1080/10428190903040014.

Matutes E, Pickl WF, Van't Veer M, Morilla R, Swansbury J, Strobl H, Attarbaschi A, Hopfinger G, Ashley S, Bene MC, Porwit A, Orfao A, Lemez P, Schabath R, Ludwig WD. Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood. 2011 Mar 17;117(11): 3163-71.

Shaffer L, McGowan-Jordan J, Schmid M (Eds): ISCN (2013): An International System for Human Cytogenetic Nomenclature. Basel: S. Karger; 2013.

Yan L, Ping N, Zhu M, Sun A, Xue Y, Ruan C, Drexler HG, Macleod RA, Wu D, Chen S. Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 adult patients with mixed-phenotype acute leukaemia defined by WHO-2008 classification. Haematologica. 2012;97: 1708–1712.

Competing interests The authors declare that they do not have competing interests.

Corresponding author: Carlos A. Tirado, Ph.D., FACMGG Carlos.Tirado@allina.com

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Review

Lost in Interpretation: Evidence of Sequence Variant Database Errors Adam Coovadia, Laboratory Operations Director, Genomic Scientist, EvolveGene, Saint Petersburg, Florida

Abstract Variant databases serve as a resource for clinical molecular genetics laboratories. There is evidence of widespread interpretive and syntactic errors within the entries of both small and large-scale variant databases used for germline clinical molecular genetic interpretation reports. The over-dependence on variant databases for variant annotation, classification and reporting may be a potential source of error to clinical molecular genetics laboratories. Recent evidence suggests 12-50% of clinical test reports are in significant conflict with clinical reports from other laboratories. A non-systematic literature review of evidence of discrepancies within frequently used genetic variant databases used for generating clinical genetic tests is provided. The implications of and recommendations for addressing variant annotation, classification and interpretive errors are discussed.

Introduction

Phenomenon (the observation of rapidly alternating extreme research claims and extremely opposite refutation) has extended from molecular genetics research to clinical molecular genetics with an estimated 12-50% of clinical test reports being in significant conflict with clinical reports from other laboratories (Pepin et al., 2015; Rehm et al., 2015; Harrison et al., 2016).

Herein is a literature review of evidence of discrepancies within frequently used genetic variant databases used for generating clinical genetic test reports using the PubMed database and keywords and phrases including variant interpretation, variant classification, database curation, annotation errors, and specific names of variant databases.

There are hundreds of genetic variant databases (HGVS, 2016) that collectively detail over 87 million human variants (NCBI, 2015). Some are freely available to the public, while others are commercial and/or private. Some are peer reviewed while others contain unpublished material. The Human Genome Variation Society (HGVS, 2016) maintains a comprehensive list of approximately 1,646 variant databases that are categorized as Locus Specific Mutation Databases, Disease Centered Central Mutation Databases, Central Mutation & SNP Databases, National & Ethnic Mutation Databases, Mitochondrial Mutation Databases, Chromosomal Variation Databases, Clinical & Patient Aspects Databases and Other Mutation Databases. In addition, there are countless lab-based and population frequency databases which may or may not be integrated into or shared with other databases.

Central Mutation & SNP Databases Human Genome Mutation Database (HGMD®) With over 183,000 variants reported, HGMD® (HGMD.cf.ac. uk) is a semi-commercial comprehensive collection of germline mutations in nuclear genes that underlie, or are associated with, human inherited disease (HGMD Product, 2016). A limited, two-year-old version of the database is freely available to the public while the current version is available as a subscription.

The website describes the database as the gold standard (HGMD Product, 2016) and is recommended by the American College of Medical Genetics as a resource use for genomic tools such as reference transcripts (Richards et al., 2015). The database is routinely accessed and utilized by human molecular geneticists, molecular biologists, clinicians, genetic counselors and bioinformaticians (Stenson et al., 2013). In the clinical laboratory setting, HGMD is widely utilized by many involved in diagnostic next generation sequencing and human genome sequencing and is integrated into a number of bioinformatic algorithms including MutPred, PROVEAN, CAROL, CRAVAT and FATHMM (Stenson et al., 2013).

A 2016 study of 23 FBN1 variants associated with Marfan Syndrome (MFS) showed HGMD® incorrectly classified all of the variants as disease-causing mutations. Manual review of the evidence of the 23 variants based on defined current medical diagnostic criteria known as Ghen II nosology (a standard which includes clinical manifestation and family history) indicated that three variants were “maybe MFS,” 14 variants were inconclusive,

Table 1: Types of Genetic Databases per HGVS (HGVS, 2016) Database Type

# of Databases

Locus Specific Mutation Databases

1,653

Disease Centered Central Mutation Databases

10

Central Mutation & SNP Databases

21

National & Ethnic Mutation Databases

11

Mitochondrial Mutation Databases

3

Other Mutation Databases

4

Clinical & Patient Aspects Databases Total

10 1,712

Variant databases serve as a resource for clinical molecular genetics laboratories. Given that it has been estimated that most published research findings are false or exaggerated (Ioannidis, 2005; Moonesinghe, 2007; Ioannidis, 2014) the over-dependence on variant databases for variant annotation, classification and reporting may be a potential source of error to clinical molecular genetics laboratories. Recent evidence suggests that The Proteus

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Review Lost in Interpretation: Evidence of Sequence Variant Database Errors

and six were “not MFS” (Groth et al., 2016). Furthermore, the database classified a MFS variant as pathogenic by referencing a publication with an incidental findings of MFS (Groth et al., 2016). Interestingly, one researcher suggested that of the 23 variants investigated, none may be a monogenic cause of MFS (Yang et al., 2014). The same study also showed that HGMD®’s assertion of the 23 variants’ clinical significance was inconsistent with three other databases included in the study (UMD-FBN1, ClinVar and Uniprot).

According to a 2013 report by Stenson et al., of 539 HGMD® mutation records that were reexamined based on a frequency of <1% in a population database (1000 Genomes Project), 33 mutations were removed, 109 were re-categorized and 220 had additional comments or references added for further justification (Stenson et al., 2013). Indeed, curators at HGMD ® routinely “retire” a variant listing, when appropriate. However, despite statements made by the database’s representatives (Stenson et al., 2013), a listing of removed variants is not publically available.

NIH funded public variant databases (dbSNP, dbVar and OMIM) and enables direct submission by individuals and organizations. It is funded by the National Center for Biotechnology Information (NCBI), National Library of Medicine (NLM) and the National Institutes of Health (NIH). With over 137,916 unique variant records representing 26,790 genes (ClinVar Submission, 2016) it is second only to HGMD® in size. The ClinVar website has more than 5,000 hits per day (Rehm et al., 2015). ClinVar collects but does not require detailed evidence to support any interpretation of clinical significance (Rehm et al., 2015). Moreover, despite its official description, ClinVar does not provide phenotypic data associated with any variant. ClinVar contains over 200,000 records but ~44% lack the criteria by which submitters arrived at the clinical significance of the variant and ~10% lack interpretations (Ray, 2016). Additional concerns include the fact that the database contains unpublished (non-peer reviewed) mutation data (Stenson et al., 2013) and that the submitter can choose to be anonymous.

A 2014 ClinVar study of the 118,169 unique variants showed 12,895 (11%) of the variants had accompanying clinical interpretation submitted by more than one laboratory. Of those, 2,229 (17%) were interpreted differently by the submitters with one or two degree differences between any of the three major levels of variant classification, that being, pathogenic, variant of unknown significance and benign (Rehm et al., 2015).

More recently, a 2016 study of 6,169 variants involving four clinical labs (Ambry Genetics, GeneDx, Laboratory for Molecular Medicine (LMM), and University of Chicago) showed that 12% (724 variants) had one or two-step classification differences (Harrison et al., 2016).

Table 2: Summary of HGMD Mutation Evaluation by Stenson et al. (2013) Action

# of Variants

% of Variants

Removed

33

6

Re-categorized

109

20

Further Justification Added

220

41

Verified

177

33

Total

539

100

As expressed in a journal article featuring the HGMD database, classification of the variants is complicated by the genetic phenomena of penetrance. HGMD® cautions that many “disease-causing mutation,” (DM) will display reduced or variable penetrance for a variety of different reasons and that a considerable number of DM appearing in apparently healthy individuals may be relevant as they may represent low-penetrance, mild or late onset or more complex disease susceptibility alleles (Stenson et al., 2013).

In addition to curation errors regarding interpretation, syntactic errors have been noted. A 2011 study of 58,182 HGMD® variants revealed inconsistencies with standard genomic coordinates, nomenclature and gene structure within HGMD® Professional v2010. Discrepant (“unresolved”) mutation annotations were found to be present at a frequency of 18.8%.

A study by Bowdin et al. summarized three database studies from 2011-2013. Collectively, these studies indicated that HGMD® overestimated variant pathogenicity with false positive error frequencies of between 4-23% (Bowdin et al., 2016).

Online Mendelian Inheritance in Man (OMIM) With approximately 25,000 variants (Rehm et al., 2015), OMIM (http://www.omim.org/) is a “comprehensive, authoritative compendium of human genes and genetic phenotype.” OMIM is a freely available public database authored and maintained by McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine and updated daily (OMIM Homepage, 2016).

OMIM records only a limited number of variants per gene (Stenson et al., 2013) and is, despite its claims, not comprehensive.

Like the other large variant databases, there is evidence of discrepancies within OMIM. A 2015 study of the OMIM database identified 220 “pathogenic” variants that were later reinterpreted as benign, likely benign or variant of unknown significance (VUS) (Rehm et al., 2015). While there is a mechanism to track new gene and disease information, there is no public list of variants that have been reinterpreted by OMIM. A 2011 study of 10,054 OMIM variants for reference accuracy (inconsistencies with standard genomic coordinates, nomenclature and gene structure) resulted in 23.2% unresolved mutation annotations (Tong et al., 2011). Of the 2,332 variants with curation errors, five distinct categories with corresponding frequencies were revealed: 1) amino acids assignment problems

ClinVar According to its website, ClinVar (http://www.ncbi.nlm.nih. gov/clinvar/) is “a public archive of reports of relationships among medically important variants and phenotypes.” It builds upon other

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Review Lost in Interpretation: Evidence of Sequence Variant Database Errors where the amino acids were not present at the described location in any of the known gene product isoforms (44.2%); 2) alias gene symbols (31.5%); 3) codon numbers that were greater than protein length (3.2%); 4) omitted genomic coordinates (<1%); and, 5) incorrect HGVS nomenclature (<1%) (Tong et al., 2011).

The significance of the syntactic errors is particularly troubling given the widespread use of automated bioinformatic algorithms that may not recognize or account for the curation errors. These types of errors could lead to inappropriate annotation and characterization, thereby leading to incorrect interpretation and inadequate or incorrect patient diagnosis.

of variant interpretation (Bell et al., 2011). Consequently, the authors suggested that without reference database improvements, clinical utility of comprehensive carrier testing would be limited.

In addition, they proposed the establishment of an authoritative disease mutation database (Bellet al., 2011).

UniProtKB/Swiss-Prot The UniProtKB/Swiss-Prot database is a public, manually curated database maintained by the European Bioinformatics Institute (EMBL-EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). Its primary source of funding is an NIH grant (About Uniprot, 2015). It is self-described as “a high quality annotated and non-redundant protein sequence database, which brings together experimental results, computed features and scientific conclusions.” (http://web. expasy.org/docs/relnotes/relstat.html, 2016). The database houses over 20,198 human variants (Documents, 2016).

A 2013 study of FBN1 variants within the UniProtKB/SwissProt database found that among 23 variants reviewed, five UniProt records were incorrectly labeled as pathogenic; causing Marfan syndrome (Groth et al., 2015). The same study also showed that UniProtKB/Swiss-Prot’s assertion of the clinical significance was inconsistent with three other databases included in the study (UMD-FBN1, ClinVar and HGMD®).

Table 3: Summary of Tong et al. (2011) Study of Syntactic Errors within OMIM Error Type

% of Variants

Amino Acids Not Present at Described Location in Any Isoforms of Gene

44

Alias Gene Symbols

32

Codon Numbering Greater Than Protein Length

3

Genomic Coordinates

<1

Nomenclature

<1

Total (10,054 Variants)

23.2

Locus Specific Databases (LSDBs) LSDBs are independently maintained, gene-specific variant databases. There are a relatively large number of locus specific databases with 1,646 LSDBs being listed by HGVS (HGVS Databases, 2016). LSDBs may contain unpublished (non-peer reviewed) mutation data that is submitted directly to the database curators (Stenson et al., 2013). Consequently, the published data within the database may be inaccurate.

Single Nucleotide Polymorphism Database (dbSNP) With over 87 million variants (“dbSNP Summary,” 2016), dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) is a central repository for both single base nucleotide substitutions and short deletion/insertion polymorphisms. The database contains over 23,973 pathogenic or probable pathogenic germline variants (Stenson et al., 2013). The database is a collaborative project involving The National Human Genome Research Institute and The National Center for Biotechnology Information (dbSNP Overview, 2016).

A 2011 study of 2,646 dbSNP variants for reference accuracy (inconsistencies with standard genomic coordinates, nomenclature and gene structure) resulted in an unresolved mutation annotation of 12.7% (Tong et al., 2011), i.e., variants that could not be annotated by the employed bioinformatic workflow.

Universal Mutation Database Fibrillin 1 Gene (UMDFBN1) With approximately 1847 variant records (FBN1 Homepage, 2016), UMD-FBN1 (http://www.umd.be/FBN1/) is a freely accessible, public database that presents molecular and clinical data on causative mutations of Marfan syndrome and type 1 fibrillinopathies. The database does not accommodate complex mutation events such as double mutants (Collod et al., 2003). Like many LSDBs, the database does accept unpublished (non-peer reviewed) data.

A 2015 study of variants within the UMD-FBN1 database showed that all 23 variants investigated were incorrectly registered as mutations even though the variants were associated with a variety of (up to 7 different) phenotypes (Groth et al., 2015). The study also showed that UMD-FBN1’s assertion of the clinical significance was inconsistent with three other databases included in the study (UniProtKB/Swiss-Prot, ClinVar and HGMD ®) (Groth et al., 2015).

HGMD®, OMIM, dbSNP Collectively A 2011 study of carrier testing for severe childhood recessive diseases unexpectedly found that literature-annotated disease mutations within HGMD ®, dbSNP, OMIM and the literature “were insufficient arbiters of whether variants are disease mutations” (Bellet al., 2011). In fact, 74% of the “disease mutation” calls were found to be substitutes with an incidence of greater than 5% (Bell et al., 2011). In addition, almost 50% of those variants were homozygous; a finding that is inconsistent with severe carrier status of a recessive disease, per ACMG guidelines

AlzGene Database (AlzGene)

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Review Lost in Interpretation: Evidence of Sequence Variant Database Errors The AlzGene Database (www.alzforum.org/alzgene) covers a set of over 1,200 case-control studies on nearly 2,300 different genetic markers for Alzheimer’s disease (Pfeiffer et al., 2011).

The database is operated by a subsidiary of Fidelity Investments and “strives to produce unbiased content to rigorous editorial standards” (“Mission ALZFORUM,” n.d.).

Results of a 2011 meta-analysis indicated that initial studies within the AlzGene Database were more biased than subsequent investigations (Pfeiffer et al., 2011). The researchers suggested the results were indicative of the Proteus Phenomena (Pfeiffer et al., 2011).

on frequency-based filters as they do not exclude all common benign variations and they do not maintain all pathogenic variations (Tong et al., 2013).

Discussion An estimated 12-50% of clinical test reports conflict significantly with clinical reports from other clinical laboratories (Pepin et al., 2015; Rehm et al., 2015; Harrison et al., 2016). Indeed, there are reports of people who have been falsely diagnosed by clinical genetic CAP/CLIA certified labs for a specific genetic tests (Predham et al., 2016). The implications of an incorrect variant interpretation that leads to a misdiagnosis may be profound and involve life-changing decisions including costly follow-up testing, health management, and decisions regarding procreation including termination. The implications of incorrect variant classification extend to misdiagnosis of family members.

By way of data sharing and integration via database reporting and curation, diffusion of the variant’s interpretation can introduce bias and potential clinical interpretation errors worldwide.

Since 2000, the ACMG has issued four comprehensive guidelines regarding variant interpretation. Interestingly, there is no professional organization guidance regarding rectifying the interpretation of reclassified variants. However, there is at least one molecular genetic software reporting system (GeneInsight) that alerts the client and/or ordering physician (among others that are designated) of post-report variant reclassification along with classification details, as appropriate. Although the legal ramifications may be considerable, an automated solution, such as this, should be recommended if not mandated by the ACMG. A major component of clinical reports is derived from the knowledge base of previously identified pathogenic variants (Tong et al., 2013). While ACMG has provided recommendations of standards for interpretation of sequence variation since 2000, it was not until 2007 that the ACMG urged caution when using variant database information in isolation. The paradigm of uncritical acceptance of the database assertions that was established over a decade and a half ago may still persist today.

Regardless, while other sources of evidence are necessary for the classification for any variant, bias introduced from database investigation may pose a large risk of clinical variant interpretation error. As an example, using the justice system as an analogy, if a variant is listed in a database as definitely pathogenic, is the variant pathogenic until proven benign or vice versa? As more laboratories move to the dependence on automatable annotation and characterization, establishing clinical testing standards for variant database curation are necessary in order to reduce the risk of clinical interpretation error. ClinVar is a database that is tasked with this goal. However, as previously described, errors within the database abound. Part of the problem may reside in the automatic integration of poorly curated data from OMIM and dbSNP databases.

Most disease-related variant databases are limited by their ability to account for multigenic results and this can be a severe

Lab-Based Databases Commercial and academic genetic laboratories maintain proprietary databases of variant interpretations with data that may or may not be shared with other laboratories and/or the public. Indeed, variant databases for known mutations, benign polymorphisms and variants of undetermined significance are mandated by the College of American Pathologists (CAP), a regulatory body that oversees the federal regulatory standards know as Clinical Laboratory Improvement Amendments (CLIA).

There are approximately 680 CAP-approved clinical genetic laboratories that, collectively, offer 78,997 tests for 4,548 disorders, encompassing a total of 5,386 genes (GeneTest Content, 2016).

Thus, it is reasonable to estimate that there are at least 680 lab-based variant databases. Because there is no data sharing mandate, there is a concern that a “silo effect” may be tainting these databases (Rehm et al., 2015).

While it is not possible to assess the level of inter- and intradiscrepancy of variant interpretations for such closed databases, for labs that do share data, a 2015 study showed that 415 variant interpretations from currently operating clinical labs and expert consortia had differing assertions (Rehm et al., 2015). These discrepant assertions were clinically significant to a level that is anticipated to have a differential effect on medical decision making (Rehm et al., 2015).

Population Frequency Databases Population databases are helping to identify, clarify and resolve discrepancies within database-literature. Per ACMG variant interpretation guidelines, with notable caveats of the disease and gene being investigated, generally, germline variants that are less than 5% in population frequency databases in the Exome Aggregation Consortium (ExAC), Exome Sequencing Project (ESP) or 1000 Genomes (Richards et al., 2015). However, like other types of variant databases, there are limitations to population databases. For example, the Exome Sequencing Project database (ESP), phenotypic information is not publically available and verifying in which each cohort each variant was detected is not possible (Gorth et al., 2015). A detailed discussion of possible errors and/or discrepancies within population databases are beyond the scope of this review. However, as one researcher has pointed out, the variant filtering process cannot be simply based

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Review Lost in Interpretation: Evidence of Sequence Variant Database Errors problem for evidence synthesis. As discussed by one researcher, when disease follows more complex patterns and differs for initial studies and subsequent ones, it is particularly difficult to correct for them in the literature (Pfeiffer et al., 2011). Most certainly, penetrance is a phenomenon that is proving to be a complex problem as well, possibly, a convenient “scapegoat” for many controversial variant classifications.

Greater regulation of the databases may be required, either by a watchdog group or governmental agency or a combination thereof. The great majority of genetic tests are laboratory developed tests. In July 2014, the FDA notified congress of its intent to oversee clinical laboratory developed tests (U.S. Food and Drug Administration, 2015). There was and continues to be significant controversy around this proposal with notable opponents such CAP (CAP, 2015) and the Association of Molecular Pathology (AMP, 2015). However, an area of regulatory oversight that may be of generally mutual acceptance within the industry may be variant databases. A representative of the FDA recently suggested that the organization may exempt NGS tests from premarket review of clinical validity if developers cite data from FDA-recognized public databases (Ray, 2016). Indeed, standardization by such an authority would further reduce existing subjective interpretation and bias that variant databases are likely contributing. In addition to standardization and regulatory oversight, a mandate to deposit clinical variant interpretation along with demographic, ethnic and phenotypic information is recommended. Zygosity data and multi-genic data could also be shared. The ability to identify and report the discrepancies is also necessary. However, there are risks when the variants are reclassified, legal implications notwithstanding.

In July 2016, the FDA published draft guidance on how they might establish the clinical validity of genetic variants.

The conceptual framework addressed how public databases recognized by the agency could be designed and used. According to the publication, FDA recognition for a database would have to submit documentation on aspects, such as standard operating procedures, data privacy and security, variant curation, interpretation, reinterpretation, personnel qualifications, and conflicts of interest (Ray, 2016). Interestingly, with the possible exception of Johns Hopkins Medicine’s CFTR2 database, which was used to develop Illumina’s FDA premarket clearance of a 139 variant CFTR diagnostic test, most other databases, including ClinVar, could not be FDA-approved based on these guidance standards (Ray, 2016).

Clinical-grade databases exist for other types of genetic data.

The Database of Genomic Variation and Phenotype in Humans Using Ensemble Resources (DECIPHER, Wellcome Trust Sanger Institute) is such an example and is routinely used for the interpretation of copy number variation, translocations, and inversions (Yohe et al., 2015).

A systematic and meta-analysis of the discrepancies within variant databases is warranted. Such a study would help quantify the level of erroneous variant reporting. In addition, a baseline of the extent of the problem will allow researchers to track the

nature of the respective database’s evolution, possibly providing greater resolution and further categorization of the sources of errors along with the ability to identify and track specific metrics.

The importance of manual review per current ACMG guidelines as the gold standard may need to be highlighted.

Indeed, the industry is beginning to recognize this as exemplified by advent of a newly designated scientific roles such as “Variant Scientists” that are dedicated to annotation and curation of genetic variants. Possible recommendations of continuous review of variants for all clinical genetic tests should be considered by laboratories and regulatory agencies.

Conclusion The finding of this review support the growing body of evidence that the level of error within variant databases may be above clinical grade acceptance and unreliable. Moreover, this literature reaffirms and expands on the findings that pathogenic variants and entries in both small and large scale databases appear to be incomplete and generally overestimate variant pathogenicity. In addition, as evidenced by aforementioned widespread interpretive and syntactic errors, self-regulation of the content of variant databases appears to have failed.

Conflicts of Interest Adam Coovadia subscribes to HGMD® Professional and has published variants that appear in the HGMD ® and ClinVar databases.

References “About UniProt.” (August, 28, 2015). Retrieved April 19, 2016, from http:// www.uniprot.org/help/about ACMG recommendations for standards for interpretation of sequence variations. Genet Med. 2000 Sep;2(5): 302-303. doi:10.1097/00125817200009000-00009.

ALZFORUM. (n.d.). Retrieved April 03, 2016, from http://www.alzforum.org/ about-us/mission AMP Press Release. (December 16, 2015). Retrieved July 23, 2016, from http:// www.amp.org/emailads/AMPPressRelease121615.html Bell CJ, Dinwiddie DL, Miller NA, Hateley SL, Ganusova EE, Mudge J, Langley RJ, Zhang L, Lee CC, Schilkey FD, Sheth V, Woodward JE, Peckham HE, Schroth GP, Kim RW, Kingsmore SF. Carrier testing for severe childhood recessive diseases by next-generation sequencing. Sci Transl Med. 2011 Jan;3(65). doi:10.1126/scitranslmed.3001756 Bowdin S, Hayeems R, Monfared N, Cohn R, Meyn M. The SickKids Genome Clinic: Developing and evaluating a pediatric model for individualized genomic medicine. Clin Genet. 2015 Mar;89(1): 10-19. doi:10.1111/cge.12579 “ClinVar Submission.” (n.d.). Retrieved April 04, 2016, from http://www.ncbi. nlm.nih.gov/clinvar/su College of America n Pat hologists, CAP Submits Comments on LDT Oversight (Febr uar y 3, 2015). Retrieved July 23, 2016, from http://www.cap.org/web/submenu/news/press-releases/pressrelease?contentID=11072207&_afrLoop=259925631732866#!@@?_afrLoo p=259925631732866&contentID=11072207&_adf.ctrl-state=hii03vqp9_30 Collod-Béroud G, Bourdelles SL, Ades L, Ala-Kokko L, Booms P, Boxer M,

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Review Lost in Interpretation: Evidence of Sequence Variant Database Errors Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015 Mar;7(5): 405-423. doi:10.1038/gim.2015.30 Stenson PD, Mort M, Ball EV, Shaw K, Phillips AD, Cooper DN. The Human Gene Mutation Database: Building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet. 2013 Sep;133(1): 1-9. doi:10.1007/s00439013-1358-4 Tong MY, Cassa CA, Kohane IS. Automated validation of genetic variants from large databases: Ensuring that variant references refer to the same genomic locations. Bioinformatics. 2011 Jan;27(6): 891-893. doi:10.1093/ bioinformatics/btr029.

U.S. Food and Drug Administration. (November 17, 2015). Retrieved Ju l y 2 3, 2 016 , f r o m h t t p :// w w w. f d a . g o v/ M e d i c a l D e v i c e s / ProductsandMedicalProcedures/InVitroDiagnostics/ucm407296.htm Yang R, Jabbari J, Cheng X, Jabbari R, Nielsen JB, Risgaard B, Chen X, Sajadieh A, Haunso S, Svendsen JH, Olesen MS, Tfelt-Hansen J. New population-based exome data question the pathogenicity of some genetic variants previously associated with Marfan syndrome. BMC Genet.

2014;15(1): 74. doi:10.1186/1471-2156-15-74 Yohe SL, Carter AB, Pfeifer JD, Crawford JM, Cushman-Vokoun A, Caughron S, Leonard D. G. Standards for clinical grade genomic databases. Arch Pathol Lab Med. 2015 Nov;139(11): 1400-1412. doi:10.5858/arpa.2014-0568-cp

Child A, Comeglio P, De Paepe A, Hyland JC, Holman K, Kaltila L, Loeys B, Matyas G, Nuytinck L, Peltonen L, Rantamaki T, Robinson P, Steinmann B, Junien C, Beroud C, Boileau C. Update of the UMDFBN1 mutation database and creation of an FBN1 polymorphism database. Hum Mutat. 2003 Sep;22(3): 199-208. doi:10.1002/humu.1024 “dbSNP Overview” (n.d.). Retrieved April 19, 2016, from http://www.ncbi. nlm.nih.gov/SNP/get_html.cgi?whichHtml=overview dbSNP Summary (April 14, 2016). Retrieved April 19, 2016, from http://www. ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi Documents. (n.d.). Retrieved April 04, 2016, from http://web.expasy.org/docs/ relnotes/relstat.html “FBN1 Homepage.” (August, 28, 2014). Retrieved April 19, 2016, from http:// www.umd.be/FBN1/ GeneTests Content. (2016). Retrieved April 19, 2016, from https://www. genetests.org/img/content/chart2.png Groth KA, Gaustadnes M, Thorsen K, Østergaard JR, Jensen UB, Gravholt CH, Andersen NH. Difficulties in diagnosing Marfan syndrome using current FBN1 databases. Genet Med. 2015 Mar;18(1): 98-102. doi:10.1038/ gim.2015.32 Harrison S, Dolinsky J, Vincent L, Knight Johnson A, Rehm H, Bale S, Azzariti D, Das S, Chao E. (2016, March). Cinical laboratories implement the ACMG/AMP guidelines to resolve differences in variant interpretation submitted to ClinVar. Poster presented at the annual meeting of the American College of Medical Genetics., Tampa, Fl. HGMD Product. (2016). Retrieved April 19, 2016, from http://www.biobaseinternational.com/product/hgmd “HGVS Databases.” (April 6, 2016), Retrieved April, 19, 2016, from http:// www.hgvs.org/locus-specific-mutation-databases Ioannidis JP. How to make more published research true. PLoS Med. 2014 Oct;11(10). doi:10.1371/journal.pmed.1001747 Ioannidis JP. Why most published research findings are false. PLoS Med. 2005 Aug;2(8). doi:10.1371/journal.pmed.0020124 “Mission ALZFORUM.” (n.d.). Retrieved April 03, 2016, from http://www. alzforum.org/about-us/mission Moonesinghe R, Khoury MJ, Janssens AC. Most published research findings are false—but a little replication goes a long way. PLoS Med. 2007 Feb;4(2). doi:10.1371/journal.pmed.0040028.

“OMIM Homepage.” (April 18, 2016). Retrieved April 19, 2016, from http:// www.ncbi.nlm.nih.gov/omi). Retrieved April 19, 2016, from http://www. ncbi.nlm.nih.gov/omi Pepin MG, Murray ML, Bailey S, Leistritz-Kessler D, Schwarze U, Byers PH. The challenge of comprehensive and consistent sequence variant interpretation between clinical laboratories. Genet Med. 2015 Apr;18(10): 20-24. Doi:10.1038/gim.2015.31 Pfeiffer T, Bertram L, Ioannidis JP. Quantifying selective reporting and the proteus phenomenon for multiple datasets with similar bias. PLoS ONE. 2011 Mar;6(3). doi:10.1371/journal.pone.0018362 Predham S, Hamilton S, Elliott AM, Gibson WT. Case Report: direct access genetic testing and a false-positive result for long QT syndrome. J Genet Counsel. 2015 Aug;25(1): 25-31. doi:10.1007/s10897-015-9882-0 Ray T. FDA plans to use public genetic variant databases for NGS test regulation but not many may qualify, GenomeWeb, July 12, 2016.

Retrieved July 13, 2016, from https://www.genomeweb.com/sequencing/ fda-plans-use-public-genetic-variant-databases-ngs-test-regulation-notmany-may-qualify Rehm HL, Berg JS, Brooks LD, Bustamante CD, Evans JP, Landrum MJ, Ledbetter DH, Maglott DR, Martin CL, Nussbaum RL, Plon SE, Ramos EM, Sherrry ST, Watson MS. ClinGen — The Clinical Genome Resource. N Engl J Med. 2015 Jun;372(23): 2235-2242. doi:10.1056/ nejmsr1406261 Relating variation to medicine. (n.d.). Retrieved April 04, 2016, from http:// www.ncbi.nlm.nih.gov/clinvar/submitters/ Richards CS, Bale S, Bellissimo DB, Das S, Grody WW, Hegde MR, Lyon E, Ward BE. ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med. 2008 Apr;10(4): 294-300. doi:10.1097/gim.0b013e31816b5.

For correspondence: Adam Coovadia, Laboratory Operations Director, Genomic Scientist EvolveGene, Saint Petersburg, Florida adam.coovadia@evolvegene.com

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Continuing Education Opportunities

Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

Test Yourself #1, 2017 Readers of The Journal of the Association of Genetic Technologists are invited to participate in this â&#x20AC;&#x153;open book testâ&#x20AC;? as an opportunity to earn Contact Hours. AGT offers 3 Contact Hours for this Test Yourself based on articles in Volume 42, Number 4, Fourth Quarter 2016 of the Journal.

Test Yourself is free to AGT members and $30 for non-members. To take this exam, send a copy of your completed Answer Sheet along with the completed Contact Hours Reporting Form to the AGT Education Committee Representative in your region. The list of representatives can be found on the AGT website. Non-members should submit a check payable to AGT for $30 with their answer sheet. Entry material must be postmarked on or before June 6, 2017.

Passing score is 85% or 17 out of 20 questions answered correctly. Compiled by Doina Ciobanu and Sally Kochmar.

The following questions are from Owen M et al. A new Rhesus Macaque Karyotype based on Human-rhesus Syntheny. J Assoc Genet Technol. 2016;42(4): 178-179.

The following questions are from Gardner J et al. Acute Myeloid leukemia with inv(16)(p13.1q22). J Assoc Technol.

2016;42(4): 180.

1. Rhesus Macaques have ____ chromosomes.

6. AML with inv(16)(p13q22) accounts for______ of all AML cases.

a. 46 b. 40 c. 44 d. 42

a. 35-55% b. 55-65% c. 50-80% d. 5-8%

2. According to this article:

7. All of the following are true, except:

I. The rhesus genome is studied on its own by primatologists.

II. Rhesus models are used in biomedical research.

III. Demand for rhesus cytogenetics is growing.

IV. Rhesus models require the same routine cell line authentication and analysis as human cell lines. a. b. c. d.

a. The patient presented with fatigue, flu-like symptoms and gum bleeding. b. The hypercellular marrow had 58% blasts. c. A blood smear demonstrated 58% blasts. d. AML is associated with a good prognosis in the absence of KIT mutation.

I, II and III I, III and IV I and II only all of the above

The following questions are from Liu K et al. Ring chromosome 7: a rare structural abnormality in acute myeloid leukemia (AML). J Assoc Genet Technol. 2016;42(4): 182-186.

3. Rhesus Macaque research is done with the intention of extrapolating the findings to human disease.

8. The ring chromosome of this patient was characterized by FISH as:

a. True b. False

a. r(7)(p32q32) b. r(7)(p11q11) c. r(7)(p12q23) d. r(7)(p13q32)

4. All of the following are correct, except: a. Rhesus macaque is the most common non-human primate model used today. b. Rhesus models have been used to work on an HIV vaccine. c. Rhesus cells for chromosome analysis were treated with colcemid overnight. d. Rhesus metaphases were analyzed using CytoVision software.

9. FISH results on this patient indicated monosomy 7 in ___ % of the cells. a. 6 b. 16.6 c. 7.2 d. 5.3

5. What is the most obvious difference between rhesus and human chromosomes? a. b. c. d.

10. Complete or partial del(7) is associated with a poor prognosis, especially when there is deletion of 7q.

Chromosomes 1 and 6 Chromosomes 5 and 8 The fusion of chromosomes 14 and 15 The fusion of chromosomes 2a and 2b giving rise to human chromosome 2

a. True b. False

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Continuing Education Opportunities

Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

11. Choose the incorrect statement: a. b. c. d.

19. The amount of nuclear DNA in a human cell is about:

De novo monosomy 7 occurs in about 10% of adult AML. Monosomy 7 rarely occurs as a sole abnormality.

Monosomy 7 is an initiating event in AML. Chromosome 7 deletions may be important in leukemogenesis.

a. 0.006 picograms b. 60 picograms c. 6 picograms d. 10 picograms The following questions are from Dr. Hon Fong L. Mark.

Claudia Wiersch, B.S. J Assoc Genet Technol. 2016;42(4): 188-189.

12. There are ___ cases of r(7) in the Mitelman Database, ___ % of which have r(7) as a sole abnormality. a. b. c. d.

35 and 26% 33 and 26% 35 and 20% 35 and 66%

20. All of the following are true, except: a. Claudia Wiersh was a happy stay-at-home mom. b. She thinks she inherited her curiosity from her mother. c. She once thought that a family is enough for her. d. She found out she wanted to be more engaged in the world.

13. The most common ring chromosome 7 abnormality in the Mitelman Database is r(7)(p22q22).

21. Claudia Wiersch worked for a while at the Mystic Aquarium in Connecticut for Dr. Trac Romano.

a. True b. False

a. True b. False

14. All of the following are correct, except: a. r(7) is associated with a good prognosis in AML. b. The marker chromosome of this patient was present in all 20 metaphase examined. c. Acquired ring chromosomes are rare in hematological malignancies. d. After undergoing treatment, the patient showed a normal karyotype.

22. She was encouraged to explore the sciences by her professors at: a. Mystic Aquarium in Connecticut b. Pharmaceutical company in Rhode Island c. Brown University d. Mohegan Community College

15. In the Mitelman Database, the most common abnormality to occur in conjunction with r(7) was: a. b. c. d.

Trisomy 21 Monosomy 17 Monosomy 7 Loss of 5q

16. The case presented in this article has a 7q31 deleted region.

Answer Sheet

a. True b. False

1.____

6.____ 7.____ 8.____ 9.____ 10.____

2.____ 3.____ 4.____ 5.____

The following questions are from Mah M and Haasen A. A brief reflection. J Assoc Genet Technol. 2016;42(4): 187.

17. According to this article, the manual part of technologist workflow in a whole genome sequencing protocol has remained largely unchanged. a. True b. False

Please Print Clearly 11.____ 12.____ 13.____ 14.____ 15.____

16.____ 17.____ 18.____ 19.____ 20.____

Answers to Test Yourself #4, 2016 Passing Score: (passing score is 21/24 or 87.5%)

18. Choose the incorrect statement: 1.d 2.a 3.b 4.d 5.c 6.d

a. Computer programs for sequencing have largely improved. b. Development and streamlining of postflow sequencing has improved productivity. c. Variant call files are also called VFCs. d. Large targeted gene panels are now increasingly integrated into routine.

7.c 8.a 9.c 10.c 11.a 12.d

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13.b 14.d 15.c 16.b 17.a 18.d

19.c 20.c

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Continuing Education Opportunities

The AGT Education Committee’s Journal Club Journal Clubs are a great way to earn Contact Hours without leaving your home or lab! Journal Clubs can be completed as a group or individually. Each Journal Club includes a reading list, several discussion questions and a post-test. The discussion questions provide a starting point for group discussion and give individuals taking a Journal Club questions to consider while reading the articles. The post-test is taken after reading the articles and is returned to the regional representatives of the Education Committee to be graded.

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READING LIST 54 – General Content Area: Chromosome Breakage Syndromes–2006 1. Chromosome Breakage Syndromes and Cancer 2. DEB Test for Fanconi Anemia Detection in Patients with Atypical Phenotype 3. Nijmegen Breakage Syndrome: Clinical Manifestation of Defective Response to DNA Doublestrand Breaks

READING LIST 55 – General Content Area: Array Based Prenatal Genetics–2006 1. Array-based Comparative Genomic Hybridization Facilitates Identification of Breakpoints of a Novel der(1)t(1;18) (p36.3;q23)dn in a Child Presenting with Mental Retardation 2. Detection of Cryptic Chromosome Aberrations in a Patient with a Balanced t(1;9)(p34.2;p24) by Array-based Comparative Genomic Hybridization 3. Jumping Translocations in Multiple Myeloma

READING LIST 56 – General Content Area: Leukemia–2007 1. Fluorescence in situ Hybridization Analysis of Minimal Residual Disease and the Relevance of the der(9) Deletion in Imatinib-treated Patients with Chronic Myeloid Leukemia 2. Characterization of the t(17;19) Translocation by Gene-specific Fluorescent in situ Hybridizationbased Cytogenetics and Detection of the E2A-HLF Fusion Transcript and Protein in Patient’s Cells 3. Combination of Broad Molecular Screening and Cytogenetic Analysis for Genetic Risk Assignment and Diagnosis in Patients with Acute Leukemia

READING LIST 57 – General Content Area: Premature Chromosome Condensation–2007 1. Premature Chromosome Condensation in Humans Associated with Microcephaly and Mental Retardation: A Novel Autosomal Recessive Condition 2. Chromosome Condensation: DNA Compaction in Real Time 3. Phosphatase Inhibitors and Premature Chromosome Condensation in Human

Peripheral Lymphocytes at Different CellCycle Phases

READING LIST 58 – General Content Area: Solid Tumor and FISH–2007 1. Methylthioadenosine Phosphorylase Gene Deletions are Frequently Detected

by Fluorescence in situ Hybridization in Conventional Chondrosarcoma 2. Solid Pseudopapillary Neoplasms of the Pancreas are Associated with FLI-1 Expression, but Not with EWS/FLI-1 Translocation 3. High Incidence of Chromosome 1 Abnormalities in a Series of 27 Renal Oncocytomas: Cytogenetic and Fluorescent In Situ Hybridization Studies

READING LIST 59 – General Content Area: Treatment of Prader-Willi Syndrome with Growth Hormone–2008 1. Two Years of Growth Hormone Therapy in Young Children with Prader-Willi Syndrome: Physical and Neurodevelopmental Benefits - American Journal of Medical Genetics Part A, Volume 143A, Issue 5, pages 443-448, 1 March 2007 2. Growth Hormone Therapy and Scoliosis in Patients with Prader-Willi Syndrome 3. Cause of Sudden, Unexpected Death of Prader-Willi Syndrome Patients with or without Growth Hormone Treatment

READING LIST 60 – General Content Area: Generics of Autism–2008 1. 15q11-13 GABAa Receptor Genes are Normally Biallelically Expressed in Brain yet are Subject to Epigenetic Dysregulation in Autism-Spectrum Disorders 2. Characterization of an AutismAssociated Segmental Maternal Heterodisomy of the Chromosome 15q1113 Region 3. 15q Duplication Associated with Autism in a Multiplex Family with a Familial Cryptic Translocation t(14;15)(q11.2;q13.3) Detected Using Array-CGH

READING LIST 61 – General Content Area: Genetics of Nicotine Addiction–2008 1. Fine Mapping of a Linkage Region on Chromosome 17p13 Reveals that

GABARAP and DLG4 are Associated with Vulnerability to Nicotine Dependence in European-Americans 2. Genomewide Linkage Scan for Nicotine Dependence: Identification of a Chromosome 5 Risk Locus 3. Genetic Linkage to Chromosome 22q12 for a Heavy-Smoking Quantitative Trait in Two Independent Samples

READING LIST 62 – General Content Area: Somatic Mutation Detection–2007 1. Inferring Somatic Mutation Rates Using the Stop-Enhanced Green Fluorescent Protein Mouse 2. Paternal Age at Birth is an Important Determinant of Offspring Telomere Length 3. Genome-Wide SNP Assay Reveals Structural Genomic Variation, Extended Homozygosity and Cellline Induced Alterations in Normal Individuals

READING LIST 63 – General Content Area: Polyglutamine Neurodegenerative Disorders–2007 1. CAG- Encoded Polyglutamine Length Polymorphism in the Human Genome 2. Polyglutamine Neurodegenerative Diseases and Regulation of Transcription: Assembling the Puzzle 3. Pathogenesis and Molecular Targeted Therapy of Spinal and Bulbar Muscular Atrophy

READING LIST 64 – General Content Area: Clinical Applications of Noninvasive Diagnostic Testing–2008 1. Digital PCR for the Molecular Detection of Fetal Chromosomal Aneuploidy 2. Noninvasive Testing for Colorectal Cancer: A Review 3. Novel Blood Biomarkers of Human Urinary Bladder Cancer

READING LIST 65 – General Content Area: Diabetes–2010 1. The Development of c-MET Mutation Detection Assay 2. Molecular Mechanisms of Insulin Resistance in Chronic Hepatitis C 3. A Genetic Diagnosis of HNF1A Diabetes Alters Treatment and Improves

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Continuing Education Opportunities Glycaemic Control in the Majority of InsulinTreated Patients

READING LIST 66 – General Content Area: Diabetes–2010 1. Distribution of Human Papillomavirus Genotypes in Invasive Squamous Carcinoma of the Vulva 2. Distribution of HPV Genotypes in 282 Women with Cervical Lesions: Evidence for Three Categories of Intraepithelial Lesions Based on Morphology and HPV Type 3. Evaluation of Linear Array Human Papillomavirus Genotyping Using Automatic Optical Imaging Software

READING LIST 67 – General Content Area: Pancreatic Cancer and its Biomarkers–2010 1. Molecular Profiling of Pancreatic Adenocarcinoma and Chronic Pancreatitis Identifies Multiple Genes Differentially Regulated in Pancreatic Cancer 2. Effect of Recombinant Adenovirus Vector Mediated Human Interleukin-24 Gene Transfection on Pancreatic Carcinoma Growth 3. Highly Expressed Genes in Pancreatic Ductal Adenocarcinomas: A Comprehensive Characterization and Comparison of the Transcription Profiles Obtained from Three Major Technologies

READING LIST 68 – General Content Area: Influenza A(H1N1) Virus–2010 1. Detection of Influenza A(H1N1)v Virus by Real-Time RT-PCR 2. Economic Consequences to Society of Pandemic H1N1 Influenza 2009 – Preliminary Results for Sweden 3. Response after One Dose of a Monovalent Influenza A (H1N1) 2009 Vaccine — Preliminary Report

READING LIST 69 – General Content Area: The Development of c-MET Mutation Detection Assay–2010 1. Somatic Mutations in the Tyrosine Kinase Domain of the MET Proto-Oncogene in Papillary Renal Carcinomas 2. Expression and Mutational Analysis of MET in Human Solid Cancers 3. Role of cMET Expression in Non-Small-Cell Lung Cancer Patients Treated with EGFR Tyrosine Kinase Inhibitors

READING LIST 70 – General Content Area: Molecular Cardiology–2010 1. Identification of a Pleiotropic Locus on Chromosome 7q for a Composite Left Ventricular Wall Thickness Factor and Body Mass Index: The HyperGEN Study 2. Novel Quantitative Trait Locus is Mapped to Chromosome 12p11 for Left Ventricular

Mass in Dominican Families: The Family Study of Stroke Risk and Carotid Atherosclerosis 3. Genome-Wide Association Study Identifies Single-Nucleotide Polymorphism in KCNB1 Associated with Left Ventricular Mass in Humans: The HyperGEN Study

READING LIST 71 – General Content Area: Detection of Clarithromycin Resistance in H. Pylori–2010 1. Rapid Detection of Clarithromycin Resistance in Helicobacter Pylori Using a PCR-based Denaturing HPLC Assay 2. Rapid Screening of Clarithromycin Resistance in Helicobacter Pylori by Pyrosequencing 3. Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

READING LIST 72 – General Content Area: Werner Syndrome Gene–2010 1. Telomeric protein TRF2 protects Holliday junctions with telomeric arms from displacement by the Werner syndrome helicase 2. WRN controls formation of extrachromosomal telomeric circles and is required for TRF2DeltaBmediated telomere shortening 3. Sequence-specific processing of telomeric 3' overhangs by the Werner syndrome protein exonuclease activity

READING LIST 73 – General Content Area: Diagnosis of Melanoma Using Fluorescence in Situ Hybridization–2011 1. Using Fluorescence in situ Hybridization (FISH) as an Ancillary Diagnostic Tool in the Diagnosis of Melanocytic Neoplasms 2. Transcriptomic versus Chromosomal Prognostic Markers and Clinical Outcome in Uveal Melanoma 3. Detection of Copy Number Alterations in Metastatic Melanoma by a DNA Fluorescence In situ Hybridization Probe Panel and Array Comparative Genomic Hybridization: A Southwest Oncology Group Study (S9431)

READING LIST 74 – General Content Area: Role of Short Interfering RNA in Gene Silencing–2011 1. Highly Specific Gene Silencing by Artificial miRNAs in Rice.

2. Gene silencing by RNAi in mouse Sertoli cells.

3. Retrovirus-delivered siRNA.

READING LIST 75 – General Content Area: Multiple Myeloma: Molecular Markers and Tests–2010 1. Multiple Myeloma: Lusting for NF-B 2. Functional Interaction of Plasmacytoid Dendritic Cells with Multiple Myeloma Cells: A Therapeutic Target 3. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients

READING LIST 76 – General Content Area: Colorectal Cancer and Loss of Imprinting of IGF2–2010 1. Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer 2. Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene.

3. Epigenetics at the Epicenter of Modern Medicine

READING LIST 77 – General Content Area: Health Effects Associated with Disruption of Circadian Rhythms–2011 1. Circadian Polymorphisms associated with Affective Disorders 2. A new approach to understanding the impact of Circadian Disruption on Human Health 3. Circadian Rhythm and its Role in Malignancy

READING LIST 78 – General Content Area: Role of Hedgehog Signaling Pathway in Diffuse Large BCell Lymphoma–2010 1. Sonic hedgehog signaling proteins and ATP-bindig cassette G2 are aberrantly expressed in diffuse large B-cell lymphoma 2. Sonic Hedgehog Signaling Pathway is Activated in ALK-Positive Anaplastic Large Cell Lymphoma 3. Sonic Hedgehog is Produced by Follicular Dendritic Cells and Protects Germinal Center B Cells from Apoptosis

READING LIST 79 – General Content Area: Whole Genome Amplification & 1986 Chernobyl, Ukraine Nuclear Power Plant Accident–2010 1. BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer.

2. Whole Genome Amplification Technologies - Eliminating the Concern Over Running Out of DNA Samples Mid Experiment.

3. A break-apart fluorescence in situ hybridization assay for detecting RET translocation in papillary thyroid carcinoma.

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Continuing Education Opportunities READING LIST 80 – General Content Area: Expression of miRNA in Diffuse Large B-Cell Lymphoma–2010 1. Differentiation stage specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas.

2. Coordinated Expression of MicroRNA-155 and Predicted Target Genes in Diffuse Large B-cell Lymphoma.

3. Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma.

READING LIST 81 – General Content Area: The Genetics of Bipolar Disorder–2010 1. Gene-wide analyses of genomewide association data sets: evidence for multiple common risk alleles for schizophrenia and bipolar disorder and for overlap in genetic risk 2. Subcortical Gray Matter Volume Abnormalities in Healthy Bipolar Offspring: Potential Neuroanatomical Risk Marker for Bipolar Disorder?

3. Genetic and Environmental Influences on Pro-Inflammatory Monocytes in Bipolar Disorder

READING LIST 82 – General Content Area: Role and Detection of Human Endogenous Retroviruses in Rheumatoid Arthritis–2011 1. Increase in Human Endogenous Retrovirus HERV-K(HML-2) Viral Load in Active Rheumatoid Arthritis.

2. A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis 3. Lack of Detection of Human Retrovirus-5 Proviral DNA in Synovial Tissue and Blood Specimens From Individuals With Rheumatoid Arthritis or Osteoarthritis.

READING LIST 83 – General Content Area: Roles of Oncogenes in Breast Cancer–2010 1. The Nuclear Receptor Coactivator Amplified in Breast Cancer-1 Is Required for Neu (ErbB2/HER2) Activation, Signaling, and Mammary Tumorigenesis in Mice.

2. Dysregulated miR-183 inhibits migration in breast cancer cells.

3. Current and emerging biomarkers in breast cancer: prognosis and prediction

READING LIST 84 – General Content Area: Elevated Levels of Human Endogenous Retrovirus-W in Patients With First Episode of Schizophrenia–2010 1. Elevated Levels of Human Endogenous Retrovirus-W Transcripts in Blood Cells From Patients With First Episode Schizophrenia.

2. Endogenous Retrovirus Type W GAG and Envelope Protein Antigenemia in Serum

of Schizophrenic Patients.

3. Reduced Expression of Human Endogenous Retrovirus (HERV)– W GAG Protein in the Cingulate Gyrus and Hippocampus in Schizophrenia, Bipolar Disorder, and Depression.

Strategies for Rapid Molecular Resource Development from an Invasive Aphid Species.

3. Evaluation of next generation sequencing platforms for population targeted sequencing studies.

READING LIST 85 – General Content Area: Esophageal Cancer–2010

READING LIST 91 – General Content Area: Hutchinson-Gilford Progeria Syndrome–2011

1. The Changing Face of Esophageal Cancer 2. Epidermal Growth FactorInduced Esophageal Cancer Cell Proliferation Requires Transactivation of-Adrenoceptors 3. Esophageal cancer risk by type of alcohol drinking and smoking: a casecontrol study in Spain

READING LIST 86 – General Content Area: p53 Family and Its Role In Cancer–2010 1. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence.

2. Shaping genetic alterations in human cancer: the p53 mutation paradigm.

3. p53 polymorphisms: cancer implications.

READING LIST 87 – General Content Area: Proteins Involved with Chronic Myleloid Leukemia and Other Myleoprolifertive Disorders–2011 1. Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders.

2. Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects.

3. Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML).

READING LIST 88 – General Content Area: DNA Topology–2010 1. The why and how of DNA unlinking.

2. Bacterial DNA topology and infectious disease.

3. DNA topoisomerase II and its growing repertoire of biological functions.

READING LIST 89 – General Content Area: LPL Waldenstrom Macroglobulinemia–2010 1. Spontaneous splenic rupture in Waldenstrom's macroglobulinemia.

2. How I Treat Waldenstrom's Macroglobulinemia.

3. International prognostic scoring system for Waldenström Macroglobulinemia.

READING LIST 90 – General Content Area: Next Generation Sequencing Platforms–2010 1. Rapid whole-genome mutational profiling using next-generation sequencing technologies.

2. Combining Next-Generation Sequencing

1. Epidermal expression of the truncated prelamin a causing Hutchinson– Gilford progeria syndrome: effects on keratinocytes, hair and skin 2. Defective Lamin A-Rb Signaling in Hutchinson-Gilford Progeria Syndrome and Reversal by Farnesyltransferase Inhibition 3. Increased expression of the Hutchinson– Gilford progeria syndrome truncated lamin a transcript during cell aging.

READING LIST 92 – General Content Area: Severe Combined Immunodeficiency Screening and Patient Studies–2011 1. Long-term Outcome after Hematopoietic Stem Cell Transplantation of a Singlecenter Cohort of 90 Patients with Severe Combined Immunodeficiency.

2. Why Newborn Screening for Severe Combined Immunodeficiency Is Essential: A Case Report.

3. Development of a Routine Newborn Screening Protocol for Severe Combined Immunodeficiency.

READING LIST 93 – General Content Area: Biological and Physical Hazards Encountered in the Laboratory–2011 1. Lab Safety Matters.

2. Virus Transfer from Personal Protective Equipment to Healthcare Employees’ Skin and Clothing. Emerging Infectious Diseases.

3. Prevalence of Hepatitis C Virus Infection Among Health-Care Workers: A 10-Year Survey.

READING LIST 94 – General Content Area: Rapid whole-genome mutational profiling using nextgeneration sequencing technologies–2011 1. Comparison of next generation sequencing technologies for transcriptome characterization.

2. ShortRead: a bioconductor package for input, quality assessment and exploration of highthroughput sequence data.

3. Next-Generation Sequencing: From Basic Research to Diagnostics.

READING LIST 95 – General Content Area: Cell Death–2011 1. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3.

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Continuing Education Opportunities 2. Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxiainduced cell death.

3. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains.

3. Evidence for Three Loci Modifying Ageat-Onset of Alzheimer’s Disease in EarlyOnset PSEN2 Families.

READING LIST 96 – General Content Area: Genetic Associations of Cerebral Palsy– 2011

1. A multiplex one-step real-time RT-PCR assay for influenza surveillance.

2. Taking New Tack, PrimeraDx Offers MDx Tech as Open Platform for Test Developers.

3. Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children.

1. Mannose-binding lectin haplotypes may be associated with cerebral palsy only after perinatal viral exposure.

2. Methylenetetrahydrofolate Reductase Gene Polymorphisms and Cerebral Palsy in Chinese Infants.

3. Apolipoprotein E genotype and cerebral palsy.

READING LIST 97 – General Content Area: Treatments for HIV/AIDs–2011 1. Early Antiretroviral Therapy Reduces AIDS Progression/Death in Individuals with Acute Opportunistic Infections: A Multicenter Randomized Strategy Trial.

2. Asia can afford universal access for aids prevention and treatment.

3. Trends in reported aids defining illnesses (adis) among participants in a universal antiretroviral therapy program: an observational study.

READING LIST 98 – General Content Area: Myosin Light Chain Kinase (MYLK) Gene Mutation Affect in Smooth Muscle Cells– 2012 1. Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice.

2. Mutation in myosin light chain kinase cause familial aortic dissections.

3. Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

READING LIST 99 – General Content Area: Chromosome 6 and Its Associated Diseases–2011 1. Novel Cleft Susceptibility Genes in Chromosome 6q.

2. A susceptibility locus on chromosome 6q greatly increases risk lung cancer risk among light and never smokers.

3. The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer.

READING LIST 100 – General Content Area: Early onset of autosomal dominant Alzheimer disease–2011 1. Genetics of Alzheimer Disease.

2. New mutation in the PSEN1 (E120G) gene associated with early onset Alzheimer’s disease.

READING LIST 101 – General Content Area: Multiplex PCR and Emerging Technologies for the Detection of Respiratory Pathogens–2011

neuronal trigger for inflammation and Alzheimer’s pathology.

3. The inflammasome: a caspase-1activation platform that regulates immune responses and disease pathogenesis.

READING LIST 106 – General Content Area: DNA Barcoding–2011 1. Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles.

2. Mutational Patterns and DNA Barcode for Diagnosing Chikungunya Virus.

3. The Barcode of Life Data Portal: Bridging the Biodiversity Informatics Divide for DNA Barcoding.

READING LIST 102 – General Content Area: Single Nucleotide Polymorphism (SNP) Array Analysis–2011

READING LIST 107 – General Content Area: HERV-K and Its Correlation With Melanoma Cells–2011

1. A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data.

2. SAQC: SNP array quality control.

3. Calibrating the performance of SNP arrays for whole-genome association studies.

1. Expression of human endogenous retrovirus K in melanomas and melanoma cell lines Cancer.

2. Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells cancer.

3. An endogenous retrovirus derived from human melanoma cells.

READING LIST 103 – General Content Area: Research of BRAF Gene Related to Cancer–2011

READING LIST 108 – General Content Area: Refractory Myeloma–2011

1. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. 2. Distinct patterns of DNA copy number alterations associate with BRAF mutations in melanomas and melanoma derived cell lines.

3. Pharmacodynamic Characterization of the Efficacy Signals Due to Selective BRAF Inhibition with PLX4032 in Malignant Melanoma.

READING LIST 104 – General Content Area: Microarray Single Nucleotide Polymorphism (SNP) Troubleshooting–2011 1. Model-based clustering of array CGH data.

2. Application of a target array comparative genomic hybridization to prenatal diagnosis.

3. A model-based circular binary segmentation algorithm for the analysis of array CGH data.

READING LIST 105 – General Content Area: Inflammasome Activation by Proteins–2011 1. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1 2 in type 2 diabetes.

2. ER stress in Alzheimer’s disease: A novel

1. Pomalidomide plus low-dose dexamethasone in myeloma refractory to both bortezomib and lenalidomide: comparison of 2 dosing strategies in dual-refractory disease.

2. Relapse/Refractory Myeloma Patient: Potential Treatment Guidelines.

3. Emerging role of carfilzomib in treatment of relapsed and refractory lymphoid neoplasms and multiple myeloma.

READING LIST 109 – General Content Area: Short Tandem Repeat (STR) Technology in Forensic Community–2011 1. An integrated microdevice for highperformance short tandem repeat genotyping.

2. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

3. Generating STR profile from "Touch DNA".

READING LIST 110 – General Content Area: Methods of Screening and Evaluation of Hepatitis C Virus–2011 1. Hepatitis c virus: prevention, screening, and interpretation of assays.

2. Serial follow-up of repeat voluntary blood donors reactive for anti-hcv elisa.

3. Comparison of fibrotest-actitest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis b and c.

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Continuing Education Opportunities READING LIST 111 – General Content Area: Pharmacogenomics–2011 1. Pharmacogenomic testing: Relevance in medical practice: Why drugs work in some patients but not in others.

2. Clinical assessment incorporating a personal genome.

3. Genomics and drug response.

READING LIST 112 – General Content Area: Adrenoleukodystrophy–2011 1. Novel exon nucleotide deletion causes adrenoleukodystrophy in a Brazilian family.

2. X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism.

3. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with Xlinked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes.

READING LIST 113 – General Content Area: Quality Assurance and Quality Control of Microarray Comparative Genomic Hybridization–2011 1. Customized oligonucleotide array-based comparative genomic hybridization as a clinical assay for genomic profiling of chronic lymphocytic leukemia.

2. Comparison of familial and sporadic chronic lymphocytic leukaemia using

high resolution array comparative genomic hybridization.

3. Microarray-based comparative genomic hybridization.

READING LIST 114 – General Content Area: mFISH–2012 1. Human interphase chromosomes: a review of available molecular cytogenetic technologies.

2. Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization.

3. CD5-negative Blastoid Variant Mantle Cell Lymphoma with Complex CCND1/ IGH and MYC Aberrations.

READING LIST 116 – General Content Area: Autism - 2015 1. Intellectual disability and autism spectrum disorders: Causal genes and molecular mechanisms.

2. Aberrant tryptophan metabolism: the unifying biochemical basis for autism spectrum disorders?

3. Decreased tryptophan metabolism in patients with autism spectrum disorders

READING LIST 115 – General Content Area: Cystic Fibrosis - 2014 1. Rapid Detection of the ACMG/ACOGRecommended 23 CFTR DiseaseCausing Mutations Using Ion Torrent Semiconductor Sequencing 2. Long-Term Evaluation of Genetic Counseling Following False-Positive Newborn Screen for Cystic Fibrosis 3. Rapid Transport of Muco-Inert Nanoparticles in Cystic Fibrosis Sputum Treated with N-acetyl cysteine

Copyright law prohibits AGT from supplying readers with the actual journal articles (electronically or otherwise). Availability of articles online does not imply the service is free.

Some journals require a subscription or impose a fee. The web addresses are included for the convenience of those wishing to obtain the articles in this way.

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Continuing Education Opportunities

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Association Business

AGT, The Organization for Cytogenetic & Molecular Professionals AGT, originally founded in 1975 as the Association of Cytogenetic Technologists, serves to: • promote the scientific and professional development of all areas of genetics; • foster the exchange of information between those interested in genetics; • encourage cooperation between those persons actively or formerly engaged in genetics; and • stimulate interest in genetics as a career.

AGT has approximately 1,000 members. Membership is open to all who are employed or interested in genetics. All regular members are entitled to hold office, vote in elections, attend all AGT meetings, and receive The Journal of the Association of Genetic Technologists and access the AGT International Online Membership Directory.

Board of Directors Officers President Patricia K. Dowling, PhD Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 PDowling@pathlinelabs.com President-Elect Jason A. Yuhas, BS, CG(ASCP)CM Mayo Clinic Division of Laboratory Genetics Cytogenetics Lab 200 First St. SW Rochester, MN 55905 507-538-7634 yuhas.jason@mayo.edu Secretary-Treasurer Denise Juroske Short, MSFS, MB(ASCP)CM 219 Timberland Trail Lane.

Lake City, TN 37769 dmj4565@gmail.com Public Relations Director Ephrem Chin MBA, MB(ASCP)CM, QLC Oxford Gene Technology 520 White Plains Road, Suite 500 Tarrytown, NY 10591 404-579-9995 nzelfman@gmail.com Education Director Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

Annual Meeting Director Jennifer N. Sanmann, PhD, FACMG UNMC Human Genetics Laboratory 985440 NE Med. Center Omaha, NE 68198-5440 402-559-3145 jsanmann@unmc.edu Annual Meeting Co-Director Christina Mendiola, BS, CG(ASCP)CM University of Texas Health Science Center – San Antonio 7703 Floyd Curl Dr. San Antonio, TX 78229 210-567-4050 mendiolac@uthscsa.edu

Council of Representatives Representative to CCCLW Term: 7/14 – 6/20 Hilary E. Blair, BS, MS, CG(ASCP)CM Mayo Clinic 200 First St. SW Rochester, MN 55905 507-255-4385 blair.hilary@mayo.edu Representatives to BOC Term: 10/12 – 9/18 Helen Bixenman, MBA/HCM, CHC, CG(ASCP)CM, DLMCM, QLC San Diego Blood Bank 3636 Gateway Ctr. Ave., Ste. 100 San Diego, CA 92102 619-400-8254 hbixenman@sandiegobloodbank.org Term: 10/11 – 9/16 Amy R. Groszbach, MEd, MLT(ASCP)CM, MBCM Mayo Clinic Molecular Genetics Laboratory – Hilton 920 200 First St. SW Rochester, MN 55905 507-284-1229 groszbach.amy@mayo.edu

Representative to NAACLS Term: 9/12 – 9/16 Peter C. Hu, PhD, MS, MLS(ASCP)CM, CGCM, MBCM University of Texas M.D. Anderson Cancer Center School of Health Sciences 1515 Holcomb Blvd., Box 2 Houston, TX 77030 713-563-3095 pchu@mdanderson.org Representative to Foundation for Genetic Technology Term: 7/10 – 6/16 Patricia LeMay, MT(ASCP), CG(ASCP)CM Monmouth Medical Center Department of Pathology 300 Second Ave. Long Branch, NJ 07740 732-923-7369 plemay1945@aol.com Representative to CAP/ACMG Term: 1/16 – 12/21 Jun Gu, MD, PhD, CG(ASCP)CM University of Texas MD Anderson Cancer Center School of Health Professions Cytogenetic Technology Program 1515 Holcombe Boulevard, Unit 2 Houston, TX 77030 (713) 563-3094 jungu@mdanderson.org

Publications AGT Journal Editor Mark D. Terry 1264 Keble Lane Oxford, MI 48371 248-628-3025 markterry@charter.net

Visit AGT’s Website at www.AGT-info.org The Journal of the Association of Genetic Technologists 43 41 (4) (1) 2015 2017

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Other Contacts Liaison to ASCLS Governmental Affairs Committee Kathryn Sudduth, BA, CG(ASCP)CM, DLMCM 2713 Brookmere Road Charlottesville, VA 22901 434-973-0690 kas3m2@embarqmail.com FGT Board of Trustees President Robin A. Vandergon, CG(ASCP)CM, DLMCM 8767 E. Los Altos Ave. Clovis, CA 93619 559-392-0512 rrink@quixnet.net

Executive Office AGT 4400 College Blvd, Suite 220 Overland Park, KS 66211 913-222-8665 913-222-8606 FAX agt-info@kellencompany.com www.AGT-info.org

Staff Contacts: Monica Evans-Lombe, Executive Director 913-222-8636 mevanslombe@kellencompany.com Christie Ross, Education Program Coordinator 913-222-8626 cbross@kellencompany.com Diane Northup, Administrative Assistant 913-222-8630 dnorthup@kellencompany.com

Association Business

Letter from the President Greetings Fellow Genetic Technology Professionals!

This will be my last missive as president of AGT. I will hand over the reins of this distinguished professional organization to Jason Yuhas at the close of the annual conference in St. Louis in June. I am extremely honored to have served in this capacity for the past two years. I wish that I could say that I left AGT in better shape than I found it, but here lies the frustration. Despite the fact that the field of genetic medicine has become more and more relevant in the search for the cause of birth defects, what gene mutations may lead to acquired diseases, what constitutes a gene, and what other influences may lead to “genetic” diseases without changing the actual nucleotide sequence, the membership in AGT has steadily decreased. That the Association of Genetic Technologists started as the Association of Cytogenetic Technologists may have given the impression that technologists in other genetic technologies were not welcome or that their need for information would not be met was identified as a possible stumbling block right away.

The content of the Journal of the AGT and the annual conference was changed to offer all genetic technologies updates on interesting topics, as well as the opportunity to broaden the scope of their knowledge, and to network with others at the technologist level. I have always said that no one knows more about how the genetic technologies work, and how to make them work better, than the folks at the technologist level. Having the opportunity to discuss techniques, problems, answers, and ideas with people who understand what you do is probably the best aspect of attending an annual AGT conference.

I must say that we have made some strides attracting molecular technologists, as our meeting content is as close to 50:50 cytogenetics/molecular, with a bit of biochemical thrown in for good measure, as it can be. I hope with time and positive communication that we will truly benefit all genetic technologists. I know that there are many opportunities for online continuing education, but face-to-face is still the best, even better than the stellar webinar series provided by AGT! I am also aware that AGT membership costs money, and fewer and fewer institutions are underwriting these costs.

My mantra is, “Whose career is it? Your institutions’, or yours?” People who are considered to the in a professional position, and who consider themselves to be professionals, should demonstrate their professionalism by belonging to professional organizations. I daresay you will be happy to never hear this from me again, and I will be happy to rid myself of the frustration for the need to say it.

As I leave the board of directors of AGT, I want to thank all of the people who have helped me along the way.

I truly couldn’t have done it without you, and, perhaps, I should have done a better job…. I leave the president position to Jason Yuhas, my faithful shadow. Good luck, my friend. Denise Juroske-Short did a stellar job keeping a tight rein on expenses. You were my rock, Denise! Sally Kochmar provided excellent and timely educational content, and a superb webinar series. Ride Sally Kochmar! Ephrem Chin revolutionized our social media presence and offered invaluable insight into how others view AGT. You made a difference, my friend! What to say about Jennifer Sanmann and Tina Mendiola? Only the best for our organization!! If the meeting turns out as good as it looks on paper, we are in for a real treat!! Jen, thank you for your talent and your professionalism. Tina, best of luck! I look forward to your meeting.

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Association Business

I also want to thank the members of the Council of Representatives, a most valuable group of folks. Peter Hu, Amy Groszbach, Hilary Blair, Helen Bixenman, and Jun Gu. You represented us exceptionally well. I am proud to have worked with you, and I wish you all the best.

I must mention the Foundation for Genetic Technologists, under the direction of Robin Vandergon. Thank you for providing us with the monetary backing we needed. Your efforts have allowed us to provide excellent speakers for our memberâ&#x20AC;&#x2122;s enjoyment.

Monica, Christie, Dede, and everyone else at Kellen, thank you so much for your assistance to me. It was a pleasure working with you.

Last, but definitely not least, I was to thank Mark Terry, our editor, for not chastising me for needing to be noodged all the time. I did enjoy writing my column!

I guess that is all for me. I will treasure my time on the AGT board of directors and council of representatives. I look forward to attending more AGT conferences and to volunteer my time for whatever is needed in the future.

Cheers!

Pat Dowling, AGT President

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Foundation for Genetic Technology 2016-2017 Board of Trustees Voting Members President Robin Vandergon NeoGenomics 30 E River Park Place West, Ste. 400 Fresno, CA 93720 559-392-0512 cell 559-433-6601 Fax rvandergon@neogenomics.com Vice President, AGT Representative, Grants Committee Chair Patricia LeMay 301-22 Spring Street Red Bank, NJ 07701 plemay1945@aol.com Secretary DeNesha Criswell NeoGenomics 618 Grassmere Park Drive, Unit 20 Nashville, TN 37211

Treasurer, Chair Capital Management Committee Tara Ellingham MUSC-Childrenâ&#x20AC;&#x2122;s Hospital Cytogenetics Lab 165 Ashley Ave., Suite 309 Charleston, SC 29425 843-792-6873 ellingha@musc.edu tellingham@hotmail.com

Public Member, Corporate Compliance Officer Bob Gasparini Consultant 12701 Commonwealth Dr. Ft. Myers, FL 33913 239-357-4237 bgasparini@neogenomics.com

Non Voting Members

AGT Representative, Awards & Scholarship Chair Denise Juroske Short 219 Timberland Trail Ln. Lake City, TN 37769 832-878-6119 dmj4565@gmail.com

Advisor, AGT President Patricia K. Dowling Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 PDowling@pathlinelabs.com

Public Member, Chair FGT Fundraising Jeff Sanford MetaSystems Group, Inc. 70 Bridge St., Ste. 100 Newton, MA 02458 617-924-9950 jsanford@metasystems.org

Ex-Officio, AGT Education Director Sally J. Kochmar Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

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43

Regular Members. Regular membership shall be available to persons who are professionally interested in the field of genetics.

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Student Members. Student membership shall be available to persons who are pursuing a full or part-time course of study at an educational institution or school and who are interested in pursuing a career in the field of genetics.

Emeritus Members. Emeritus membership shall be available to persons who are retired from or inactive in the field of genetics.

Collaborative Members. Collaborative membership shall be available to persons who currently hold membership in any other health-related national organization and who have never been members of ACT/AGT.

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Journal Hard Copy Order: Although The Journal of Genetic Technologists is available online to ALL MEMBERS, only North American members can elect to receive a hard copy via regular mail for an additional fee of $100. This fee covers four issues. If you are a North American resident and would like a hard copy of the Journal, please remit the additional fee with your membership application by checking the box and adding the amount to your total payment.  $100 Please note: AGT does not accept purchase orders and does not bill/invoice for services.

Mail application form and appropriate fee for membership in correct U.S currency. Money order or check in U.S. funds drawn on a U.S. bank only. CHECKS DRAWN ON INTERNATIONAL BANKS WILL NOT BE ACCEPTED. Make checks payable to Association of Genetic Technologists. For your convenience, you may pay by credit card. Applications received after September 15 are applied toward the next membership year. NOTE: Membership expires on December 31 of each year.

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The AGT Cytogenetics Laboratory Manual, 4th Edition

[Please note: Note: The 4th Edition of AGT's Cytogenetics Laboratory Manual is currently in production.

Preorder publication now through the publisher (available April 2017)

The Cytogenetics Symposia, 2nd Edition

The AGT Molecular Biology Techniques Review Guide Select method of delivery:

 Dropbox (no shipping cost)  Secure Document Hyperlink (no shipping cost) The Dynamics of Chromosome Spreading Video – CD featuring Jack Spurbeck

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AGT Executive Office, 4400 College Boulevard, Suite 220, Overland Park, KS 66211 Fax (913) 222-8606  Email: agt-info@kellencompany.com  Website: www.agt-info.org

Please allow 2-4 weeks for shipping.

• Explore our degree program in Cytogenetic Technology including oncampus part-time enrollment, oncampus full-time enrollment, and hybrid online enrollment • Explore our Internet-Based Review Course in Clinical Cytogenetics with ongoing enrollment • Explore our Annual Comprehensive Review Course in Clinical Cytogenetics to prepare for ASCP-BOC (CG) exam For more information contact Jun Gu, M.D., Ph.D., CG (ASCP) Program Director/Associate Professor jungu@mdanderson.org 1-800-551-9503

The Journal of the Association of Genetic Technologists The Journal of the Association of Genetic Technologists is a peer-reviewed journal, and scientific materials for publication containing original research will be reviewed by independent referees. Manuscripts that require revision or that contain major editorial changes will be returned to the senior author of the article. Materials submitted will not be retained following publication nor will photographs, disks, or hard copies of manuscripts be returned to authors. Rejected manuscripts will not normally be returned, although an effort will be made to return original photographs and prints.

Manuscript content is the responsibility of the author(s). All articles published, including editorials, letters, book reviews, invited articles, Brain Ticklers, columns, and reviews, represent the opinions of the authors and do not reflect the official policy of AGT or the institution with which the author is affiliated unless specified by the author. AGT, its members, and the editor of The Journal of the Association of Genetic Technologists make no warranty and assume no liability with respect to the information contained herein.

Information For Authors The Journal of the Association of Genetic Technologists is pleased to consider manuscripts that describe experience with cytogenetics, molecular genetics, or biochemical genetics and the application of these disciplines.

Submitted manuscripts must be typed, preferably double-spaced, using a 12 point font and 1” margins. In addition to the original, three copies of the manuscript and camera-ready illustrations must be submitted to the editor-in-chief. Items to be italicized or enhanced (bold, underlined) should be clearly indicated. The conversion factor for print equivalency is as follows: two double-spaced typed pages equal approximately a one-half typeset page.

Authors may supply the material on a 3½” disk, preferably in Microsoft Word, WordPerfect, or ASCII format, along with the hard copy. Macintosh disks are also acceptable, but conversion costs will be assessed accordingly to AGT and a delay in processing may occur. Materials may alternatively be supplied to the editor via email at the address shown on inside front cover. Email submission is preferred.

Illustrations must be original photographs, computer-generated digitized files (preferably saved as a .tif, .eps, or .bmp file), or black and white line drawings, professionally prepared. The cost of separating and printing color photographs or illustrations will be charged to the author. Photographs must be properly identified on the back, including the author’s name, title of article, and top direction. A ball point pen should not be used for labeling. The affixing of a typewritten label to the illustration or table will prevent damage.

Notation & References Authors’ titles must be accompanied by a position description of less than 15 words, which will be printed with the article.

Textual citations to the referenced literature should be parenthetically noted by author’s surname followed by year of publication, and arranged chronologically and then alphabetically, as demonstrated in the following example: (Lese and Ledbetter, 1998; Reilly, 1998a; Morgan et al., 1999).

In situations with more than two authors, the first author’s surname should be followed with et al. When references are made to more than one paper published in the same year by the same author, a lower case a, b, etc. should be appended to the date of publication and should be included in both textual citations and the reference list.

References should be listed completely at the end of the paper in alphabetical order by surname of first author, and then by year of publication.

When more than one publication appears with the same first author, listings will be alphabetized by the first varying co-author. Irrespective of the number of authors, et al. should not be used in the reference list. Journal titles should be abbreviated according to Index Medicus and book titles should be italicized. Use the following format for references: Journal Article Brothman AR, Zhu XL, Maxell T, Cui J, Derbler DA. Advances in the cytogenetics of prostate cancer. J Assoc Genet Technol. 1999;25(1):1-6.

Book Chapter Barch MJ and Lawce HJ. The cell and cell division. In: Barch MJ, Knutsen T, Spurbeck JL (eds). The AGT Cytogenetics Laboratory Manual, 3rd ed. Philadelphia: Lippincott-Raven; 1997:1-18.

Book Mark HFL. Medical Cytogenetics. New York: Marcel Dekker; 2000.

All references should be complete. Accuracy is the responsibility of the authors. Only published articles and those in press may be included in the reference list. If necessary, unpublished data and submitted manuscripts should be cited parenthetically within the text.

Reprint Orders Reprints of articles can be purchased by authors at cost within two years after publication. On the order request, specify the journal’s volume and issue numbers, year of publication, page numbers, article title, author(s), and quantity requested. Include the contact name(s), address(es) and phone number(s) to be used for either shipping purposes or related questions. Payment should accompany the order. Checks must be made payable to AGT. Minimum order is 50 copies.

Reprints are produced on 60# white offset paper, saddle-stitched (unless under four pages), and will appear exactly as they do in the journal.

Price is based on article length, quantity ordered, and color requirements. Orders are not processed until payment is received. Once payment is received, allow four weeks for printing and shipping. Prices quoted include shipping by UPS ground; expedited shipping is available at an additional charge. Journal copies can be purchased by AGT members for $25/each, if copies are available.

Please forward reprint orders or questions regarding price quotations to the AGT Executive Office (see inside front cover for address).

ISSN 1523-7834