Avoiding Pitfalls in Molecular Genetic Testing
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The Journal of Molecular Diagnostics, Vol. 13, No. 3, May 2011
Copyright 2011 American Society for Investigative Pathology
and the Association for Molecular Pathology.
Published by Elsevier Inc. All rights reserved.
DOI: 10.1016/j.jmoldx.2011.01.008Consultations in Molecular DiagnosticsAvoiding Pitfalls in Molecular Genetic Testing
Case Studies of High-Resolution Array ComparativeGenomic Hybridization Testing in the Definitive Diagnosis of
Mowat-Wilson SyndromeMichael Joseph Kluk,* Yu An,* Philip James,*David Coulter,* David Harris,* Bai-Lin Wu,* andYiping Shen*
From the Departments of Laboratory Medicine, Neurology and
Genetics,* Childrens Hospital Boston, Boston, Massachusetts;
the Department of Pathology, Harvard Medical School,
Boston, Massachusetts; and the Institutes of Biomedical
Sciences and MOE Key Laboratory of Contemporary
Anthropology, School of Life Sciences, Fudan University,
The molecular testing options available for the diag-nosis of genetic disorders are numerous and include avariety of different assay platforms. The consultativeinput of molecular pathologists and cytogeneticists,working closely with the ordering clinicians, is oftenimportant for definitive diagnosis. Herein, we de-scribe two patients who had long histories of unex-plained signs and symptoms with a high clinical sus-picion of an underlying genetic etiology. Initialmolecular testing in both cases was negative, but theapplication of high-resolution array comparativegenomic hybridization technology lead to definitivediagnosis in both cases. We summarize the clinicalfindings and molecular testing in each case, discussthe differential diagnoses, and review the clinicaland pathological findings of Mowat-Wilson syn-drome. This report highlights the importance forthose involved in molecular testing to know thenature of the underlying genetic abnormalities as-sociated with the suspected diagnosis, to recognizethe limitations of each testing platform, and to per-sistently pursue repeat testing using high-resolu-tion technologies when indicated. This concept isapplicable to both germline and somatic moleculargenetic testing. (J Mol Diagn 2011, 13:363367; DOI:
10.1016/j.jmoldx.2011.01.008)The molecular testing options available for the diagnosisof genetic disorders are numerous and include a varietyof different assay platforms. Herein, we describe twopatients who had long histories of unexplained signs andsymptoms and a high clinical suspicion of an underlyinggenetic etiology.
The first patient, a boy now 13 years old, was born at37 weeks gestation. He has several congenital anoma-lies including short-segment Hirschsprung disease, ven-tricular septal defect, patent ductus arteriosus, bilateralrenal pelvis dilatation with hydronephrosis, vesicoureteralreflux, microcephaly, partial absence of corpus callosumand unilateral microphthalmia with optic nerve aplasia,and blindness of the left eye. Vision in his right eye isnormal. He also has significant developmental delay withautistic features (eg, essentially nonverbal, noninterac-tive) and a seizure disorder requiring medical treatment.
The physical examination was notable because hisoverall height and weight measurements were gener-ally low (third to fifth percentile; Figure 1A). He also hasmarked and persistent microcephaly (head circumfer-ence, 38.5 cm (fifth percentile) at 4.5 months; 43.3cm (third percentile) at 16 months; 44.8 cm (thirdpercentile) at 2 years 9 months; and 46 cm (third
Y.S. holds a Young Investigator Award from the Childrens Tumor Foun-dation and a Catalyst Award from Harvard Medical School. B.L.W. holdsa Fudan Scholar Research Award from Fudan University and a grant fromthe Chinese National 973 project on Population and Health (grant num-ber 2010CB529601) and a grant from the Science and Technology Coun-cil of Shanghai (grant number 09JC1402400). Y.A. holds a grant from theShanghai Natural Science Foundation (grant number 09ZR1404500).
M.J.K. and Y.A. are co-first authors and contributed equally to this work.B.L.W. and Y.S. are co-senior authors.
Accepted for publication January 14, 2011.
None of the authors disclosed any relevant financial relationships.
Address reprint requests to Yiping Shen, Ph.D., Department of Labo-ratory Medicine, Childrens Hospital Boston, 300 Longwood Ave., Boston,
MA 02115.E-mail: firstname.lastname@example.org.
364 Kluk et alJMD May 2011, Vol. 13, No. 3percentile) at 9 years 6 months). Imaging studies re-vealed microcephaly, partial absence of corpus callo-sum, and colpocephaly.
The second patient, a Caucasian girl now 11 years old,was born at full term. Overall, her clinical history andmanifestations are similar to those of the first patient. Shehas global developmental delay, congenital microceph-aly, congenital long-segment Hirschsprung disease, con-genital cataracts, and seizures. She has severe mentalretardation with markedly delayed gross and fine motorskills and cannot walk. Her receptive and expressivelanguage skills are rudimentary.
The physical examination was notable because heroverall height and weight measurements were consis-tently low (third to fifth percentile; Figure 1B). She alsohas marked and persistent microcephaly [head circum-ference, 45.6 cm at 6 years 5 months; 45.6 cm at 8 years3 months; 46.2 cm at 9 years 6 months; and 46 cm at 10years 9 months (all less than third percentile)].
As described later, the diagnostic work-up for thesepatients involved the use of several different testingplatforms; however, high-resolution array comparativegenomic hybridization (CGH) lead to definitive diagnosisin both cases. There are currently several assay plat-forms available for the diagnostic work-up of patients witha suspected diagnosis of Mowat-Wilson syndrome. In thisreport, we discuss the strengths and limitations of thevarious testing modalities and we propose two slightlydifferent testing strategies based on the level of clinical
Figure 1. A: The height and weight of the individual in case 1 were plottedin case 2 were plotted in the standard growth curve for girls. Growth charts wNovember 2010.suspicion for the diagnosis of Mowat-Wilson syndrome.Materials and Methods
The array CGH method involved testing of patients pe-ripheral blood genomic DNA with the high-resolutionwhole genome oligonucleotide array (244K; Agilent Tech-nologies, Santa Clara, CA) following the manufacturersprotocol. This array is a gene-centric (70% of the probesare intragenic), whole-genome cytogenetic array and isnot an exon-level or a ZEB2 gene-specific array. PatientDNA labeled with Cyanine 5 (Cy5) was compared with areference DNA labeled with Cyanine 3 (Cy3). Agilentscannercaptured images were quantified using FeatureExtraction software version 9.0. (Agilent Technologies)CGH analytic software version 3.4 subsequently wasused for data analysis using the Aberration DetectionMethod 2 algorithm. The criteria for detecting a deletioninclude a shift from the baseline Cy5/Cy3 signal value (0)to a value 0.6 and the changes must be present in atleast 5 consecutive probes. The median probe spacing inintragenic regions is 7.4 kb. At this resolution, single-exondeletions/duplications can be detected for some geneswith large exons that would contain at least 5 probes butdetection of single-exon deletions/duplications is notpossible for every gene with this array. The ZEB2 genecontains 10 exons. As shown in Figure 2, there are ap-proximately 17 probes spanning the ZEB2 gene in thisarray; this resolution would not detect a single-exon de-letion of ZEB2 and does not allow precise mapping of theZEB2 breakpoints. Any clues as to the mechanism medi-
standard growth curve for boys. B: The height and weight of the individualained from the CDC website: http://www.cdc.gov/growthcharts, last accessedin theating the ZEB2 deletions that would be provided by
to the sh
aCGH Diagnosis of Mowat-Wilson Syndrome 365JMD May 2011, Vol. 13, No. 3knowledge of the breakpoints awaits future study. How-ever, large deletions spanning several exons and/or in-trons of ZEB2 such as those described in the presentcases can be detected by this array.
Results and Discussion
Initial genetic testing of the first patient performed shortlyafter birth involved a metaphase karyotype that was nor-mal. Also, RET gene analysis performed at 16 months ofage (for Hirschsprung disease) was negative. At 10 yearsof age, a chromosomal microarray analysis using Spec-tral Genomics 1M BAC array (Houston, TX) detected nopathogenic copy number variant. At this time, Mowat-Wilson syndrome was suspected by the geneticist (D.H.),but ZEB2 sequence analysis was normal. At 13 years ofage, a high-resolution whole-genome oligonucleotidemicroarray revealed a deletion of approximately 348 kbon chromosome 2q22.2-q22.3 involving exons 4 to 10 ofZEB2 and part of GTDC1 (adjacent gene) (chromosomecoordinates: 144551411 to 144898926, Human GenomeBuild 18; Figure 2, top panel, case 1). No other clinicallysignificant genomic imbalances were detected. The par-tial deletion of ZEB2 confirmed the clinical diagnosis ofMowat-Wilson syndrome. Testing of the mother did notidentify the same variant and the father was not availablefor testing.
The initial genetic work-up for the second patientwas negative (metaphase karyotype, plasma aminoacid profile, and acylcarnitine profile). At 11 years of
Figure 2. Top and middle: custom tracks showing the oligonucleotide arr1; middle, case 2); the shaded regions are the deleted regions identified byThe relative positions of the ZEB2 and GTDC1 genes are shown with respectthe relative size and positions of the deletions.age, a high-resolution whole-genome oligonucleotidemicroarray revealed a deletion of approximately 923 kbon chromosome 2q22.3 involving exons 1 to 8 of ZEB2(chromosome coordinates: 144874055 to 145796638,Human Genome Build 18; Figure 2, middle panel, case2). The partial deletion of ZEB2 confirmed the sus-pected clinical diagnosis of Mowat-Wilson syndrome.No parental testing was performed.
These cases highlight the importance of high-resolu-tion array comparative genomic hybridization technol-ogy in the definitive diagnosis of Mowat-Wilson syn-drome in patients with a long history of an otherwiseunexplained constellation of signs and symptoms. Therecognition of the technical limitations of the prior testingmodalities (peripheral blood metaphase karyotype, earlygeneration chromosomal microarray, and ZEB2 Sangersequencing) was important because it prompted the pur-suit of additional testing with high-resolution array CGH,which effectively detected the large partial deletions ofZEB2 present in both cases. Array CGH technology,which is constantly undergoing improvement in probeselection and resolution, is an important testing modalityin the genetic work-up of older children and adult pa-tients, who either came to medical attention before arrayCGH testing was in widespread use or were tested withlow-resolution early generation CGH platforms (eg, BAC-based CGH). The effectiveness of genomic profilingtechnologies such as array CGH in patients with com-plex genetic disorders such as developmental delay/intellectual disability, autism spectrum disorders, andmonogenic Mendelian disorders previously was re-
that show the size and position of the deletions in the two cases (top, caseanalytic software. Each dot indicates an individual oligonucleotide probe.
aded deleted regions. Bottom: genome browser view of the data that showsay datathe CGHviewed extensively.13
366 Kluk et alJMD May 2011, Vol. 13, No. 3The clinical differential diagnosis in both cases includedthe following: i) Lenz microphthalmia syndrome (syndromicmicrophthalmia-1), an X-linked disorder (Xq27-q284) asso-ciated with unilateral eye anomaly, skeletal anomalies, var-ious urogenital and cardiovascular malformations,5 occa-sional microcephaly, mental retardation,6 and abnormalitiesof the corpus callosum; importantly, Lenz microphthalmia isnot associated with Hirschsprung disease; and ii) Gold-berg-Shprintzen megacolon syndrome, an autosomal-re-cessive disorder (10q21.3-q22.1) that has been associatedwith homozygous nonsense mutations in KIAA12797 andtypically manifests with Hirschsprung megacolon, micro-cephaly, eye anomalies, short stature, and learning prob-lems.8 Clearly, Goldberg-Shprintzen megacolon syndromeshows many overlapping clinical signs and symptoms withMowat-Wilson syndrome and would be difficult to rule outwithout appropriate molecular testing.
Mowat-Wilson syndrome is an autosomal-dominantdisorder associated with haploinsufficiency of ZEB2 (zincfinger E box-binding homeobox 2; also known as zincfinger homeobox 1B or Smad interacting protein 1; 2q22-q23) caused by de novo heterozygous nonsense muta-tions, frameshift mutations (small insertions or deletions),or large multiexon deletions.9,10 Patients with Mowat-Wil-son syndrome show a distinct facial phenotype (eg, hy-pertelorism, strabismus, elongated face, prominent andnarrow chin, elongated nasal tip, upturned lower earlobes, and broad eyebrows with medial flaring and in-creased medial separation), mental retardation, anoma-lies of the corpus callosum, Hirschsprung disease, sei-zure disorder, ocular anomalies, congenital heartdefects, and urogenital abnormalities.10,11 According toprior publications, there may be variability in the pres-ence and/or age of onset for some features (eg, micro-cephaly, short stature/growth parameters).10,12 Both ofour patients showed Hirschsprung disease, microceph-aly, eye abnormalities, delayed growth, significant devel-opmental delay/mental retardation, and seizures. The firstpatient additionally showed a cardiac anomaly, genito-urinary developmental anomaly, and partial absence ofthe corpus callosum, which were not reported in thesecond patient. At present, in Mowat-Wilson syndrome,the clinical phenotype does not appear to significantlychange according to the underlying genotype10,12; al-most all patients, regardless of the specific underlyingZEB2 abnormality (ie, small mutations or large deletions),have the facial gestalt and moderate to severe mentalretardation, supporting the concept that this syndromeresults from ZEB2 haploinsufficiency rather than it being acontiguous gene syndrome.10
Approximately 80% of the mutations described inZEB2 are nonsense mutations and frameshift mutations(small insertions or deletions)11,13,14 that are detectableby Sanger sequencing. Approximately 50% of such mu-tations are found in exon 8.13,14 Large intragenic dele-tions involving entire exons account for approximately15% to 20% of mutations in ZEB211,13,14 and are notdetected reliably by Sanger sequencing; indeed, suchcases may be under-represented in the literature. Unlikeclassic microdeletion syndromes, the intragenic dele-
tions in Mowat-Wilson syndrome vary significantly in size(partial exon to whole gene deletions) and the breakpointregions, which...