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.2011.01.008

Consultations in Molecular DiagnosticsAvoiding Pitfalls in Molecular Genetic Testing

Case Studies of High-Resolution Array ComparativeGenomic Hybridization Testing in the Definitive Diagnosis of

Mowat-Wilson Syndrome

Michael Joseph Kluk,*† Yu An,*‡ Philip James,*David Coulter,* David Harris,* Bai-Lin Wu,*†‡ andYiping Shen*†‡

From the Departments of Laboratory Medicine, Neurology and

Genetics,* Children’s 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,

Shanghai, China

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, 13:363–367; 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 Children’s 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, Children’s Hospital Boston, 300 Longwood Ave., Boston,

MA 02115.E-mail: yiping.shen@childrens.harvard.edu.

363

ere obt

364 Kluk et alJMD May 2011, Vol. 13, No. 3

percentile) 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 manufacturer’sprotocol. 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). Agilentscanner–captured 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 accessed

in the

ating the ZEB2 deletions that would be provided by

to the sh

aCGH Diagnosis of Mowat-Wilson Syndrome 365JMD May 2011, Vol. 13, No. 3

knowledge 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 oligonucleotide

microarray 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 shows

ay datathe CGH

viewed extensively.1–3

366 Kluk et alJMD May 2011, Vol. 13, No. 3

The 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 do not appear to occur in low copy repeatregions, are variable.13,15,16 At present, the mechanismof deletion appears unclear but, interestingly, the deletionmost often involves the paternal allele.13,16

Techniques capable of assessing ZEB2 intragenic orwhole gene deletion include targeted techniques suchas fluorescent in situ hybridization,11,15 multiplex liga-tion-dependent probe amplification (MLPA; P169-B1Hirschsprung-1 Assay; MRC-Holland, Amsterdam, theNetherlands),14 quantitative PCR (qPCR),11,13,16 andarray CGH.11 The application of array CGH in the as-sessment of patients with a variety of different geneticsyndromes has been described previously and thistechnique currently is used routinely in many molecularlaboratories. Over the past 10 years, array CGH tech-nology has evolved with ever-increasing resolution andgenome coverage. There have been several recentcomprehensive reviews describing the use of arrayCGH in various settings (including prenatal test-ing).17–19 The increasing use of high-resolution arrayCGH (capable of detecting large deletions of ZEB2) orMLPA [capable of detecting smaller deletions (ie, sin-gle exon deletions) of ZEB2] will help to identify dele-tions that evaded earlier generation tests and, there-fore, will reveal the true frequency of all ZEB2 deletiontypes in the pathogenesis of Mowat-Wilson syndrome.It is important to emphasize that although MLPA andqPCR are capable of detecting deletions/duplicationsof single exons within ZEB2 if multiple probes covereach exon, such resolution is not possible with the244K Agilent Technologies oligonucleotide array CGHassay. To obtain exon level resolution, target gene-specific exon CGH array can be custom designed ashas been shown before.20 Nevertheless, array CGH iscapable of detecting large deletions/duplications in ZEB2and can assess many other genes simultaneously becauseit is a genome-wide platform. On the other hand, MLPA andqPCR assays are capable of detecting single-exon dele-tions/duplications but they are designed to assess only fewtarget genes simultaneously.

Given the differences in the various testing platforms, itis most appropriate to use a stepwise approach in diag-nostic testing for suspected cases of Mowat-Wilson syn-drome.14 One possible cost-efficient testing strategy forpatients with a clear, high clinical suspicion of Mowat-Wilson syndrome would include initial Sanger sequenc-ing of the ZEB2 coding region exons (especially of exon8) and adjacent splice sites that, if negative, could befollowed by MLPA or qPCR for deletion/duplication analy-sis of ZEB2 exons. If still negative, those tests could befollowed by array CGH for genome-wide analysis and, ifindicated, Sanger sequencing of KIAA1279 to rule outGoldberg-Shprintzen megacolon syndrome. However,when the clinical diagnosis is less certain, as in the casesdescribed herein, high-resolution array CGH testing maybe appropriate as a second-tier test even though it ismore expensive than MLPA and could miss small to in-termediate deletions (eg, single-exon deletions) if thearray oligonucleotide probe coverage is suboptimal; de-

spite these issues, because array CGH offers a genome-

aCGH Diagnosis of Mowat-Wilson Syndrome 367JMD May 2011, Vol. 13, No. 3

wide analysis capable of detecting large deletions involv-ing ZEB2 as well as large deletions/duplications of otherloci, it may be indicated as a second-tier test in caseswith a broad differential diagnosis. In such cases, thetesting algorithm could begin with Sanger sequencing ofthe ZEB2 coding region exons (especially of exon 8) that,if negative, could be followed by genome-wide high-resolution array CGH to detect large deletions in ZEB2 orother genes. However, if the array CGH testing is nega-tive and there is still a clinical suspicion of Mowat-Wilsonsyndrome, MLPA or qPCR should be performed to as-sess for the presence of small (eg, single exon) deletionsof ZEB2. Most importantly, during the testing process,there should be clear communication between the mo-lecular pathologist/cytogeneticist and the clinician withrespect to the nature of the genetic abnormalities under-lying each clinical entity in the differential diagnosis (ie,point mutation versus small deletion versus large dele-tion) and with regard to the limitations of each molecularassay performed.

Acknowledgment

We thank the members of the Genetic Diagnostic Labo-ratory at Children’s Hospital Boston for support.

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