Mowat-Wilson syndrome is a rare genetic condition characterized by intellectual disability, structural anomalies, and dysmorphic features. It is caused by haploinsufficiency of the ZEB2 gene in chromosome 2q22.3. Over 180 distinct mutations in ZEB2 have been reported, including nonsense and missense point mutations, deletions, and large chromosomal rearrangements. We report on a 14-year-old female with a clinical diagnosis of Mowat-Wilson syndrome. Chromosomal microarray identified a novel de novo 69-kb duplication containing exons 1 and 2 of the ZEB2 gene. Sequence analysis identified no other variants in this gene. This is the first report of a partial duplication of the ZEB2 gene resulting in Mowat-Wilson syndrome.

• Mowat-Wilson syndrome is caused by heterozygous mutation of ZEB2.

• Over 180 different disease-causing mutations have been reported, including point mutations, deletions, and large chromosomal rearrangements.

• We present the first reported case of Mowat-Wilson syndrome caused by a partial duplication of ZEB2.

• This case expands the types of mutations that can cause Mowat-Wilson syndrome and highlights the significance of partial gene duplications in the pathogenesis of genetic conditions.

Mowat-Wilson syndrome (MWS; OMIM 235730) is a rare genetic syndrome seen in 1:50,000-70,000 individuals [Mowat and Wilson, 2010]. First described by Mowat et al. in 1998, it is characterized by intellectual disability, structural anomalies, and dysmorphic features [Mowat et al., 1998; Adam et al., 2013]. Intellectual disability is typically in the moderate to severe range, expressive speech is particularly impaired, and about 70-75% of affected individuals have seizures [Wilson et al., 2003; Garavelli et al., 2009; Cordelli et al., 2013]. Common structural abnormalities include Hirschsprung disease and defects of the heart, genitourinary system, eyes, and brain (anomalies of corpus callosum, hippocampal abnormalities, enlargement of cerebral ventricles, white matter abnormalities, large basal ganglia, cortical and cerebellar malformations) [Adam et al., 2006; Garavelli and Mainardi, 2007; Ariss et al., 2012; Bourchany et al., 2015; Coyle and Puri, 2015; Garavelli et al., 2016]. Distinctive facial features include hypertelorism, broad medial eyebrows, low-hanging columella, prominent or pointed chin, and uplifted earlobes with a central depression [Zweier et al., 2005]. Additionally, individuals with this condition may have growth delay, microcephaly, and musculoskeletal anomalies [Mowat et al., 2003; Adam et al., 2006; Cordelli et al., 2013]. Birth weight and length are typically normal.

MWS is due to dominantly expressed, heterozygous mutations of the zinc finger E box-binding homeobox 2 gene (ZEB2, also called ZFHX1B or SIP1) located on chromosome 2q22.3 [Cacheux et al., 2001; Wakamatsu et al., 2001]. ZEB2 has 10 exons (exon 1 is noncoding) and is about 165 kb in size [Wakamatsu et al., 2001]. Mutations leading to MWS are thought to result in truncated or absent protein products [Mowat et al., 2003].

More than 180 different disease-causing mutations of ZEB2 have been reported [Ghoumid et al., 2013]. Loss-of-function point mutations account for 70-80% of MWS diagnoses, while deletions (ranging in size from exonic to multiple contiguous genes) represent another 15-20% [Garavelli and Mainardi, 2007; Saunders et al., 2009]. In rare cases (up to 2% of affected individuals), large chromosomal rearrangements, such as translocations, are identified [Adam et al., 2007]. A few individuals with MWS who harbor missense mutations in ZEB2 have been reported [Heinritz et al., 2006; Dastot-Le Moal et al., 2007; Ghoumid et al., 2013; Wenger et al., 2014]. There is also a small proportion of individuals who have a clinical diagnosis of MWS but in whom a ZEB2 mutation is not identified.

We present a case of a 14-year-old female with a de novo 69-kb duplication encompassing part of ZEB2. Specifically, the duplication contained exons 1 and 2. She presented with developmental delays, multiple congenital anomalies, and a clinical diagnosis of MWS based on her medical history, physical features, and clinical presentation. She represents the first published case of an individual with a novel partial duplication of ZEB2 resulting in MWS.

Clinical Report

Our patient was first evaluated genetically at age 14 years at which time she presented with multiple health problems, intellectual disability, and a clinical diagnosis of MWS made at 2 years of age. At age 14 years, her height was 148 cm (<3rd centile), weight was 41.4 kg (5th centile), and her head circumference was 49.3 cm (<3rd centile). Her BMI was 18.9. Congenital anomalies present at birth included cardiac defects (atrial septal defect, patent ductus arteriosus, and a bicuspid aortic valve), midline cutaneous pigment abnormalities, and a sacral dimple. Breast and bottle feeding were unsuccessful, so a nasogastric tube and, later, a G-tube were placed. Shortly after birth, she was diagnosed with global developmental delay and Hirschsprung disease. She has had 2 pull-through surgeries and one colostomy with 47 cm of her colon removed. She currently utilizes laxatives and a predigestive formula for treatment. She has a neurogenic bladder, kidney reflux, incontinence, and a tethered cord (for which surgery was recently performed). A head MRI showed agenesis of the corpus callosum and 3 frontal lobe cysts which diminished in size on repeated brain imaging studies. Seizures developed at age 2 years and are currently medically controlled. At age 10 years, she began consuming food by mouth but did not chew normally. She drinks liquid from a straw.

At age 14 years, her inner canthal distance was 3.7 cm (>97th centile); outer canthal distance was 8.8 cm (40th centile), and her ear length was 5.5 cm (20th centile). Hand length was 15.7 cm (3rd centile) and middle finger length was 6.6 cm (5th centile). She had a square face with medial flare to her eyebrows, a depressed and wide nasal root, hypertelorism with downslanting palpebral fissures, ptosis, strabismus, a short prominent nose with a broad nasal tip, prominent vertical philtral ridges with a short philtrum, full everted upper and lower lips with thinning of upper lip laterally, and downturned corners of the mouth with prognathism. Her ears were posteriorly rotated and fleshy with attached lobes and mild uplifting of the right earlobe (see Fig. 1). The pupils were round and equal in size without scleral icterus. There was a normal range of motion of the neck. The cardiopulmonary system was within normal limits without a heart murmur, rhythm irregularity, or abnormal breath sounds. Her abdomen was soft with no masses or hernias. Major joints had a normal range of motion, but she displayed increased reflexes and tone of both upper and lower body segments as well as poor coordination with a wide-based gait. She walked at age 5 years. She bruises easily and has a history of low platelet counts, first identified at age 10 years. She did not require transfusions but continues to have low platelet count. Cutis marmorata was noted. She was diagnosed with osteopenia and treated for hypothyroidism since age 10 years. She had not begun menstrual cycles.

A 3-generation pedigree found our patient's 20-year-old full sister and father are alive and well, and her mother has a history of arthritis, a positive antinuclear antibody titer, irritable bowel syndrome, and melanoma. There is a positive family history of hemophilia type A in a paternal uncle. Consanguinity was denied. Family history is otherwise unremarkable, and no other family members have MWS.

Chromosomal Microarray Analysis

Genetic testing was performed by Lineagen, Inc. (Salt Lake City, UT, USA) using a custom 2.8M probe whole genome chromosome microarray built upon the Affymetrix CytoScanHD® platform (Affymetrix, Santa Clara, CA, USA) with 88,435 additional probes designed to optimize testing for individuals with developmental delay/intellectual disability and/or autism spectrum disorder. The microarray contains both copy number and SNP probes. Coverage for ZEB2 included 380 probes with an average probe spacing <500 bp. The Affymetrix Chromosome Analysis Suite (ChAS) software was used for copy number detection using standard protocols (Affymetrix). The proband's whole genome was evaluated by chromosomal microarray analysis (CMA) for clinical diagnostic testing. Parental DNA was subsequently submitted, and only the region of interest was evaluated by CMA on a research basis.

ZEB2 Gene Sequence Analysis

This patient's genomic DNA specimen was used for next-generation sequencing. DNA was sheared into small fragments that were end-ligated oligo-adapters, target-enriched for coding exons along with 10 bp of flanking intronic sequence in ZEB2 (RefSeq NM_014795.3), and amplified by PCR and sequenced using bridge amplification. This targeted gene sequencing strategy interrogates the coding regions and splice site junctions of ZEB2 only.

CMA identified a novel 69-kb duplication of 2q22.3 including ZEB2, arr[hg19] 2q22.3(145,218,807-145,287,401)×3, encompassing exons 1 and 2 as well as intron 1 and part of intron 2 (see Fig. 2, 3). ZEB2 was the only gene that was partially included in this duplication. Next-generation sequencing of ZEB2, performed to rule out the presence of sequence variants contributing to the patient's phenotype, identified no ZEB2 sequence variants. Parental CMA assessing only the region of interest on chromosome 2 did not identify any CNVs, indicating that the patient's duplication is de novo.

A variety of mutations have been described in individuals with MWS. The majority of individuals with MWS have point mutations within ZEB2 that cause protein truncation and, therefore, loss of function [Dastot-Le Moal et al., 2007]. Within this cohort, germline mosaicism has been suggested and low-level somatic mosaicism reported. The estimated recurrence is 1% when parents test negative for a familial mutation [Zweier et al., 2005]. Additionally, another 15-20% of individuals with MWS have exonic or complete gene deletions of ZEB2[Adam et al., 2013].

Most features seen in our patient, including microcephaly, seizures, distinctive facies, and Hirschsprung disease, are consistent with loss or altered function of ZEB2. However, chromosomal microarray cannot in itself determine if a partial duplication as observed in this case has disrupted gene function. Interpretation of duplications is always challenging as they may not alter gene function or could lead to increased dosage, gene disruption, or novel function due to gene fusion [Newman et al., 2015]. This has been demonstrated to be true for duplications of ZEB2 as well.

Phenotypic consequences from increased dosage of ZEB2 have been suggested (see Fig. 4; Table 1). Yuan et al. [2015] reported a boy with a de novo 2.9-Mb triplication/duplication involving chromosome 2q22.3 which encompassed the entire ZEB2 gene as well as GTDC1, TEX41, and part of ARHGAP15. This boy presented with hypotonia, cognitive impairment, behavioral abnormalities, dysmorphic features, and an atrial septal heart defect. While his severe speech impairment and open-mouth appearance are commonly reported in those with MWS, his facial features were significantly different than those seen in MWS.

A duplication of an overlapping 2.1-Mb region was recently reported by Mak et al. [2016]. Just as the duplication reported by Yuan et al. [2015], this duplication fully included ZEB2 as well as GTDC1, TEX41, and part of ARHGAP15. This 32-year-old woman presented with tetralogy of Fallot, patent foramen ovale, mild intellectual disability, adjustment disorder, dysmorphic features, and a hypernasal voice. Notably, her dysmorphic features were not similar to those seen in MWS.

Seven other individuals with copy number gains involving part or all of ZEB2 were identified within the DECIPHER ( [Firth et al., 2009; Swaminathan et al., 2012] and ISCA (International Standards for Cytogenomic Arrays) clinical genomic resource databases ( Direct comparisons between these previous reports and the current patient may be difficult as most of the previous duplications are much larger and contained additional genes. Six of these gains were de novo and 1 did not have parental testing information available. Six of these individuals had clinical features that overlapped with those seen in MWS (intellectual disability, developmental delay, dysmorphic features, multiple congenital anomalies, and seizures). One patient presented only with hearing impairment (DECIPHER 238286).

Also, 1 patient (nssv578831) found in the ISCA database had a reportedly pathogenic de novo 203-kb duplication which included exons 1 and 2 and intron 1 with part of intron 2 within ZEB2 (larger but similar duplication region as seen in our patient; see Fig. 3). This patient was reported to have seizures. It is unknown if the patient had or developed any other features of MWS that were not reported in the ISCA database.

Additionally, Jiang et al. [2011] described 4 unrelated individuals with Hirschsprung disease who had 2q22.3 duplications (ranging from 1.42-1.99 kb in size) involving part of exon 1 and all of exon 2. Three of these individuals had additional physical anomalies, but 1 individual had a mutation in RET and 2 had a SOX2 duplication with a mutation in RET that likely contributed to their clinical features. These reports indicate that partial duplications of ZEB2 likely contribute to pathogenesis of Hirschsprung disease, one of the core features of MWS.

All of these previously reported cases demonstrate that both partial and full duplications of ZEB2 can be pathogenic, resulting in abnormal clinical features. Previous reports of partial duplications of ZEB2 have been identified in individuals with some clinical features of MWS, but none of these previously reported individuals were actually specifically diagnosed of MWS or presented with all of the key features of this condition.

Causative partial gene duplications have been reported in several other conditions that are typically caused by point mutations. These include neurofibromatosis type 1, Rubinstein-Taybi syndrome, Cornelia de Lange syndrome, and Kleefstra syndrome [Roelfsema et al., 2005; Russo et al., 2012; Nemethova et al., 2013; Schwaibold et al., 2014]. These cases provide evidence that a partial duplication of ZEB2 could similarly result in a haploinsufficient condition.

In conclusion, our patient represents the first report of a partial duplication of ZEB2 in an individual with an established clinical diagnosis of MWS. This highlights the significance of partial gene duplications in the pathogenesis of genetic conditions and expands the types of mutations that can cause MWS.

The authors thank the patient and her parents for their participation in this publication. We also thank Pat Rushton for providing ChAS images, Megan Martin for her guidance on the manuscript, and Karen Ho for useful discussions and manuscript review. We acknowledge support from the National Institute of Child Health and Human Development (NICHD) grant HD02528.

Informed consent was obtained for this report from the parents of the patient in accordance with institutional standards for the protection of human research subjects.

The authors have no conflicts of interest to declare.

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