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Mowat–Wilson syndrome detected by using high resolution microarray
Gene 532 (2013) 307–309
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Mowat–Wilson syndrome detected by using high resolution microarray Jae Young Park a,1, Eun Hae Cho b,1, Eun Hee Lee b, You Sun Kang b, Kyung Ran Jun c, Yun Jung Hur a,⁎ a b c
Department of Pediatrics, Inje University College of Medicine, Haeundae Paik Hospital, Busan, South Korea Green Cross Reference Laboratory, Yong-In City, Kyunggi-do, South Korea Department of Laboratory Medicine, Inje University College of Medicine, Haeundae Paik Hospital, Busan, South Korea
a r t i c l e
i n f o
Article history: Accepted 18 July 2013 Available online 9 September 2013 Keywords: Mowat–Wilson syndrome Microarray ZEB2
a b s t r a c t Individuals with Mowat–Wilson syndrome (MWS; OMIM#235730) have characteristic facial features, a variety of congenital anomalies such as Hirschsprung disease, and intellectual disabilities caused by mutation or deletion of ZEB2 gene. This deletion or cytogenetic abnormality has been reported primarily from Europe, Australia and the United States, but not in Korea. Here we report a patient with characteristic facial features of MWS, developmental delay and spasticity. High resolution microarray analysis revealed 0.9 Mb deletion of 2q22.3 involving two genes: ZEB2 and GTDC1. This case shows the important role of high resolution microarray in patients with unexplained psychomotor retardation and/or facial dysmorphism. Knowledge about the most striking clinical signs and implementation of effective molecular tests like microarray could significantly increase the detection rate of new cases of MWS in Korea. This is the first reported case of MWS in Korea. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mowat–Wilson syndrome (MWS) is rare, little-known congenital disorder characterized by severe intellectual disability with seizures, specific facial dimorphisms and multiple congenital abnormalities. Other conditions that can occur in the broad spectrum include facial dysmorphism, structural anomalies such as Hirschsprung disease, genitourinary anomalies, congenital heart defects, agenesis or hypogenesis of corpus callosum, eye defect, moderate to severe intellectual disability, severe speech impairment, seizures, growth retardation with microcephaly, and chronic constipation in those without Hirschsprung disease. About 200 cases of Mowat–Wilson syndrome (MWS) have been reported, primarily from Europe, Australia and the United States, (Garavelli et al., 2009; Mowat et al., 1998) but it has not yet been reported in Korea. Because this syndrome is little-known among healthcare professionals, and has a broad-spectrum of congenital abnormalities, it is likely to be under-diagnosed until the affected person is older or even an adult. Early recognition can lead to confirmative diagnosis and prompt medical or surgical treatments with appropriate care and specialized services.
Abbreviations: MWS, Mowat–Wilson syndrome; ZEB2, Zinc finger e-box protein 2; GTDC1, glycosyltransferase-like domain containing 1. ⁎ Corresponding author at: Department of Pediatrics, Haeundae Paik Hospital, Inje University College of Medicine, 1435, Jwa-dong, Haeundae-gu, Busan 612-862, South Korea. Tel.: + 82 51 797 2006; fax: + 82 51 797 0032. E-mail address: [email protected] (Y.J. Hur). 1 Co-first author. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.07.067
Here, we report the first case of MWS in Korea detected by using high resolution microarray with unexplained psychomotor retardation and/or facial dysmorphism. 2. Clinical report A nine-month old girl was referred to our clinic for the evaluation of facial dysmorphism and developmental delay. She was born, with birth weight 2.49 kg by cesarean section at 38 weeks of gestation. The baby's height, head and chest circumferences at birth were not recorded. Her mother has been healthy and took no drugs during pregnancy nor did she have any genetic or neurologic disease in family history. At the age of 9 months, the height was 71.4 cm (25–50th percentile), weight was 8.1 kg (10–25th percentile) and the head circumference was 41.0 cm (b 3th percentile). The patient had facial dysmorphism including triangular lip, micrognathia, prominent columella, medially flared and broad eyebrows, ocular hypertelorism, depressed epicanthal fold, down slanted eyelids, depressed nasal bridge, deep set eyes, prominent chin, uplifted ear lobes with a central depression and full or everted lower lip. Her head control was good but she made poor eye contact with the investigator, and showed increased deep tendon reflexes and extensor tone. Development was markedly delayed, with average 50 scores with motor, language and cognitive functions at the age of 9 months, and the same results at the age of 15 months in Bayley Scales of Infant Development test. At the age of 21 months, she had failure to thrive with microcephaly, height, 86.7 cm (50–75th percentile), weight, 9.7 kg (b 3th percentile) and the head circumference, 44.0 cm (b3th percentile). Brain magnetic resonance image showed abnormal size of corpus callosum and echocardiogram showed very small secundum atrial septal defect sized about 1.8 mm. Mild bilateral pelviectasia and
308
J.Y. Park et al. / Gene 532 (2013) 307–309
mild hydronephrosis were observed in genitourinary tract but the patient did not present any clinical symptoms. Esotropia and astigmatism were observed in ophthalmologic evaluation. She had chronic constipation without Hirschsprung disease. She had attack of seizures associated with febrile illness twice. At the age of 30 months, she had the first attack of afebrile seizure so EEG was performed revealing abnormal tracing due to high voltage, slow and disorganized background rhythm. These findings suggested a mild degree of diffuse encephalopathy. She did not take any antiepileptic drug since she did not have any recurrent seizures with or without febrile illness. Ophthalmologic examination showed the progressive astigmatism and esotropia, finally diagnosed as acquired so that surgery may be needed to prevent worsening. Now she is undergoing rehabilitation without any medication. Sequence analysis detects mutations in approximately 81% of individuals; FISH or microarray detects large deletions encompassing all or part of ZEB2 in approximately 15% of persons; chromosomal rearrangements that disrupt the ZEB2 gene cause MWS in approximately 2% of individuals; and an additional 2% has intermediate-sized deletions that can be detected by techniques such as quantitative PCR, MLPA and gene-specific high resolution microarray (Adam et al., 2008). In this case, it was hard to diagnose the patient as clinical ground, karyotype analysis was performed to rule out cytogenetic abnormalities. The karyotype was 46,XX. High resolution microarray analysis using cytogenetics whole genome 2.7 M array (Affymetrix, CA, USA) was performed to rule out submicroscopic abnormalities. The test was performed according to the manufacturer's instructions and data were analyzed using software chromosome analysis suite (Affymetrix, CA, USA) according to the human genome assembly 19. The results revealed an interstitial.0.9-Mb deletion at 2q22.3, involving genes ZEB2 and GTDC1 (Fig. 1). The final karyotype revealed as 46, XX, arr 2q22.3q22.3 (144,670,286–145,576,689)×1. We recommended a parental study to find the origin but they refused. Karyotype analysis for the patient's elder sister who has a normal phenotype was done.
3. Discussion Patients with MWS show a distinct facial phenotype, intellectual disability and constellation of additional clinical signs, Hirschsprung disease, congenital heart disease, brain anomalies, microcephaly and seizures. It is a single gene disorder, caused by heterozygous mutation or deletion mutations of the ZEB2 gene, residing within the 2q21 region in most patients. Even though patients with MWS have distinctive facial appearance and intellectual disability, other clinical signs appear in various spectra for each patient. Deletion size in ZEB2 gene and presented clinical manifestations could be correlated. Large deletions (11 Mb and 5 Mb) have been reported for spasticity, and neonatal seizure disorder. Small deletions (300 kb and 700 kb) have been reported for anomalies of the corpus callosum (Table 1: Hoffer et al., 2007; Zweier et al., 2003). Regardless of deletion size, Hirschsprung disease, hypotonia, and congenital heart disease are present with varying ages of onset (Hoffer et al., 2007; Zweier et al., 2003). In our case, the patient had a small deletion but did not have anomalies of the corpus callosum and Hirschsprung disease. Instead, she had spasticity and three attacks of febrile and afebrile seizures. At this point, the manifestation of corelated disease may not be influenced by deletion size. However, deletion sizes and breakpoints in MWS syndrome have a wide spectrum of variation so it is important to rule out a true microdeletion with recurring breakpoints. This variability of symptoms makes MWS under-diagnosed (Garavelli et al., 2009). Characterizing the phenotypic variation of MWS at different deletion sizes can improve both the clinical diagnosis and the clinical management of each condition. The application of high resolution microarray study can improve the yield of diagnostic test. Recently the development of resolution technique and probe selection has made the microarray useful tool for diagnosing MWS. Several MWS cases with a large deletion which can be detected by microarray have been reported (Hoffer et al., 2007; Kluk et al., 2011). The deletion sizes and breakpoints in patients vary widely from
Fig. 1. The analysis with affymetrix cytogenetics whole genome 2.7 M array showed 0.9-Mb deletion of 2q22.3 involving two genes: ZEB2 and GTDC1.
J.Y. Park et al. / Gene 532 (2013) 307–309 Table 1 Phenotype compare to deletion size of previously reported cases with ours in MWS. Phenotype
Deletion size Large (N = 4, 11 Mb, 6 Mb, 5 Mb)
Spasticity Neonatal seizure Agenesis of the corpus callosum Microcephaly Mental retardation Hirschsprung disease Hypotonia Congenital heart defects Genitourinary anomalies a
Small (N = 3, 0.3 Mb, 0.7 Mb, 0.9 Mba)
50% 25% 0%
33% 0% 66%
100% 100% 50% 50% 75% 25%
66% 100% 33% 33% 66% 100%
309
results from ZEB2 haplo-insufficiency rather than being a contiguous gene syndrome (Kluk et al., 2011). This is the first reported case of MWS in Korea. Knowledge about the most striking clinical signs and implementation of effective molecular tests like high resolution microarray in patients with unexplained psychomotor retardation and/or facial dysmorphism will increase the detection rate of new cases of MWS in Korea. Moreover, detection of new cases in MWS will allow us to analyze correlations between deletion size and clinical manifestations. Finally it will help us understand the variability of congenital anomalies of MWS and lead us to timely diagnosis. Conflicts of interest No potential conflict of interest relevant to this article was reported.
our case
References 300 kb to at least 11 Mb. The usage of microarray offers a genome-wide analysis allowing detection of large deletions involving ZEB2 as well as large deletions/duplications of other loci. Microarray analysis in our case showed 0.9 Mb deletion involving two genes ZEB2 and GTDC1. GTDC1 is glycosyltransferase-like domain containing 1. No disease associated mutation and deletion and copy number variation at Databases of Genomic Variation have been reported. Whether the involvement of GTDC1 influences on this patient's phenotype is uncertain. However, by a previous report, the clinical phenotype does not appear to significantly change according to the underlying genotype. Almost all patients, regardless of the specific underlying ZEB2 abnormality (i.e, small mutations or large deletions), have the facial gestalt and moderate to severe intellectual disability, supporting the concept that this syndrome
Adam, M.P., Bean, L.J.H., Miller, V.R., 2008. Mowat–Wilson syndrome. In: Pagon, R.A., Bird, T.D., Dolan, C.R., et al. (Eds.), GeneReviews™ ([Internet]. Accessed 2008 Feb 11. Available from http://www.ncbi.nih.gov/books/NBK1412). Garavelli, L., et al., 2009. Mowat–Wilson syndrome: facial phenotype changing with age: study of 19 Italian patients and review of the literature. Am. J. Med. Genet. A 149A (3), 417–426. Hoffer, M.J., et al., 2007. A 6 Mb deletion in band 2q22 due to a complex chromosome rearrangement associated with severe psychomotor retardation, microcephaly and distinctive dysmorphic facial features. Eur. J. Med. Genet. 50 (2), 149–154. Kluk, M.J., et al., 2011. Avoiding pitfalls in molecular genetic testing: case studies of highresolution array comparative genomic hybridization testing in the definitive diagnosis of Mowat–Wilson syndrome. J. Mol. Diagn. 13 (3), 363–367. Mowat, D.R., et al., 1998. Hirschsprung disease, microcephaly, intellectual disability, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22–q23. J. Med. Genet. 35 (8), 617–623. Zweier, C., et al., 2003. Caracterisation of deletions of the ZFHX1B region and genotypephenotype analysis in Mowat-Wilson syndrome. J. Med. Genet. 40 (8), 601–605.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Mowat–Wilson syndrome detected by using high resolution microarray Jae Young Park a,1, Eun Hae Cho b,1, Eun Hee Lee b, You Sun Kang b, Kyung Ran Jun c, Yun Jung Hur a,⁎ a b c
Department of Pediatrics, Inje University College of Medicine, Haeundae Paik Hospital, Busan, South Korea Green Cross Reference Laboratory, Yong-In City, Kyunggi-do, South Korea Department of Laboratory Medicine, Inje University College of Medicine, Haeundae Paik Hospital, Busan, South Korea
a r t i c l e
i n f o
Article history: Accepted 18 July 2013 Available online 9 September 2013 Keywords: Mowat–Wilson syndrome Microarray ZEB2
a b s t r a c t Individuals with Mowat–Wilson syndrome (MWS; OMIM#235730) have characteristic facial features, a variety of congenital anomalies such as Hirschsprung disease, and intellectual disabilities caused by mutation or deletion of ZEB2 gene. This deletion or cytogenetic abnormality has been reported primarily from Europe, Australia and the United States, but not in Korea. Here we report a patient with characteristic facial features of MWS, developmental delay and spasticity. High resolution microarray analysis revealed 0.9 Mb deletion of 2q22.3 involving two genes: ZEB2 and GTDC1. This case shows the important role of high resolution microarray in patients with unexplained psychomotor retardation and/or facial dysmorphism. Knowledge about the most striking clinical signs and implementation of effective molecular tests like microarray could significantly increase the detection rate of new cases of MWS in Korea. This is the first reported case of MWS in Korea. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mowat–Wilson syndrome (MWS) is rare, little-known congenital disorder characterized by severe intellectual disability with seizures, specific facial dimorphisms and multiple congenital abnormalities. Other conditions that can occur in the broad spectrum include facial dysmorphism, structural anomalies such as Hirschsprung disease, genitourinary anomalies, congenital heart defects, agenesis or hypogenesis of corpus callosum, eye defect, moderate to severe intellectual disability, severe speech impairment, seizures, growth retardation with microcephaly, and chronic constipation in those without Hirschsprung disease. About 200 cases of Mowat–Wilson syndrome (MWS) have been reported, primarily from Europe, Australia and the United States, (Garavelli et al., 2009; Mowat et al., 1998) but it has not yet been reported in Korea. Because this syndrome is little-known among healthcare professionals, and has a broad-spectrum of congenital abnormalities, it is likely to be under-diagnosed until the affected person is older or even an adult. Early recognition can lead to confirmative diagnosis and prompt medical or surgical treatments with appropriate care and specialized services.
Abbreviations: MWS, Mowat–Wilson syndrome; ZEB2, Zinc finger e-box protein 2; GTDC1, glycosyltransferase-like domain containing 1. ⁎ Corresponding author at: Department of Pediatrics, Haeundae Paik Hospital, Inje University College of Medicine, 1435, Jwa-dong, Haeundae-gu, Busan 612-862, South Korea. Tel.: + 82 51 797 2006; fax: + 82 51 797 0032. E-mail address: [email protected] (Y.J. Hur). 1 Co-first author. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.07.067
Here, we report the first case of MWS in Korea detected by using high resolution microarray with unexplained psychomotor retardation and/or facial dysmorphism. 2. Clinical report A nine-month old girl was referred to our clinic for the evaluation of facial dysmorphism and developmental delay. She was born, with birth weight 2.49 kg by cesarean section at 38 weeks of gestation. The baby's height, head and chest circumferences at birth were not recorded. Her mother has been healthy and took no drugs during pregnancy nor did she have any genetic or neurologic disease in family history. At the age of 9 months, the height was 71.4 cm (25–50th percentile), weight was 8.1 kg (10–25th percentile) and the head circumference was 41.0 cm (b 3th percentile). The patient had facial dysmorphism including triangular lip, micrognathia, prominent columella, medially flared and broad eyebrows, ocular hypertelorism, depressed epicanthal fold, down slanted eyelids, depressed nasal bridge, deep set eyes, prominent chin, uplifted ear lobes with a central depression and full or everted lower lip. Her head control was good but she made poor eye contact with the investigator, and showed increased deep tendon reflexes and extensor tone. Development was markedly delayed, with average 50 scores with motor, language and cognitive functions at the age of 9 months, and the same results at the age of 15 months in Bayley Scales of Infant Development test. At the age of 21 months, she had failure to thrive with microcephaly, height, 86.7 cm (50–75th percentile), weight, 9.7 kg (b 3th percentile) and the head circumference, 44.0 cm (b3th percentile). Brain magnetic resonance image showed abnormal size of corpus callosum and echocardiogram showed very small secundum atrial septal defect sized about 1.8 mm. Mild bilateral pelviectasia and
308
J.Y. Park et al. / Gene 532 (2013) 307–309
mild hydronephrosis were observed in genitourinary tract but the patient did not present any clinical symptoms. Esotropia and astigmatism were observed in ophthalmologic evaluation. She had chronic constipation without Hirschsprung disease. She had attack of seizures associated with febrile illness twice. At the age of 30 months, she had the first attack of afebrile seizure so EEG was performed revealing abnormal tracing due to high voltage, slow and disorganized background rhythm. These findings suggested a mild degree of diffuse encephalopathy. She did not take any antiepileptic drug since she did not have any recurrent seizures with or without febrile illness. Ophthalmologic examination showed the progressive astigmatism and esotropia, finally diagnosed as acquired so that surgery may be needed to prevent worsening. Now she is undergoing rehabilitation without any medication. Sequence analysis detects mutations in approximately 81% of individuals; FISH or microarray detects large deletions encompassing all or part of ZEB2 in approximately 15% of persons; chromosomal rearrangements that disrupt the ZEB2 gene cause MWS in approximately 2% of individuals; and an additional 2% has intermediate-sized deletions that can be detected by techniques such as quantitative PCR, MLPA and gene-specific high resolution microarray (Adam et al., 2008). In this case, it was hard to diagnose the patient as clinical ground, karyotype analysis was performed to rule out cytogenetic abnormalities. The karyotype was 46,XX. High resolution microarray analysis using cytogenetics whole genome 2.7 M array (Affymetrix, CA, USA) was performed to rule out submicroscopic abnormalities. The test was performed according to the manufacturer's instructions and data were analyzed using software chromosome analysis suite (Affymetrix, CA, USA) according to the human genome assembly 19. The results revealed an interstitial.0.9-Mb deletion at 2q22.3, involving genes ZEB2 and GTDC1 (Fig. 1). The final karyotype revealed as 46, XX, arr 2q22.3q22.3 (144,670,286–145,576,689)×1. We recommended a parental study to find the origin but they refused. Karyotype analysis for the patient's elder sister who has a normal phenotype was done.
3. Discussion Patients with MWS show a distinct facial phenotype, intellectual disability and constellation of additional clinical signs, Hirschsprung disease, congenital heart disease, brain anomalies, microcephaly and seizures. It is a single gene disorder, caused by heterozygous mutation or deletion mutations of the ZEB2 gene, residing within the 2q21 region in most patients. Even though patients with MWS have distinctive facial appearance and intellectual disability, other clinical signs appear in various spectra for each patient. Deletion size in ZEB2 gene and presented clinical manifestations could be correlated. Large deletions (11 Mb and 5 Mb) have been reported for spasticity, and neonatal seizure disorder. Small deletions (300 kb and 700 kb) have been reported for anomalies of the corpus callosum (Table 1: Hoffer et al., 2007; Zweier et al., 2003). Regardless of deletion size, Hirschsprung disease, hypotonia, and congenital heart disease are present with varying ages of onset (Hoffer et al., 2007; Zweier et al., 2003). In our case, the patient had a small deletion but did not have anomalies of the corpus callosum and Hirschsprung disease. Instead, she had spasticity and three attacks of febrile and afebrile seizures. At this point, the manifestation of corelated disease may not be influenced by deletion size. However, deletion sizes and breakpoints in MWS syndrome have a wide spectrum of variation so it is important to rule out a true microdeletion with recurring breakpoints. This variability of symptoms makes MWS under-diagnosed (Garavelli et al., 2009). Characterizing the phenotypic variation of MWS at different deletion sizes can improve both the clinical diagnosis and the clinical management of each condition. The application of high resolution microarray study can improve the yield of diagnostic test. Recently the development of resolution technique and probe selection has made the microarray useful tool for diagnosing MWS. Several MWS cases with a large deletion which can be detected by microarray have been reported (Hoffer et al., 2007; Kluk et al., 2011). The deletion sizes and breakpoints in patients vary widely from
Fig. 1. The analysis with affymetrix cytogenetics whole genome 2.7 M array showed 0.9-Mb deletion of 2q22.3 involving two genes: ZEB2 and GTDC1.
J.Y. Park et al. / Gene 532 (2013) 307–309 Table 1 Phenotype compare to deletion size of previously reported cases with ours in MWS. Phenotype
Deletion size Large (N = 4, 11 Mb, 6 Mb, 5 Mb)
Spasticity Neonatal seizure Agenesis of the corpus callosum Microcephaly Mental retardation Hirschsprung disease Hypotonia Congenital heart defects Genitourinary anomalies a
Small (N = 3, 0.3 Mb, 0.7 Mb, 0.9 Mba)
50% 25% 0%
33% 0% 66%
100% 100% 50% 50% 75% 25%
66% 100% 33% 33% 66% 100%
309
results from ZEB2 haplo-insufficiency rather than being a contiguous gene syndrome (Kluk et al., 2011). This is the first reported case of MWS in Korea. Knowledge about the most striking clinical signs and implementation of effective molecular tests like high resolution microarray in patients with unexplained psychomotor retardation and/or facial dysmorphism will increase the detection rate of new cases of MWS in Korea. Moreover, detection of new cases in MWS will allow us to analyze correlations between deletion size and clinical manifestations. Finally it will help us understand the variability of congenital anomalies of MWS and lead us to timely diagnosis. Conflicts of interest No potential conflict of interest relevant to this article was reported.
our case
References 300 kb to at least 11 Mb. The usage of microarray offers a genome-wide analysis allowing detection of large deletions involving ZEB2 as well as large deletions/duplications of other loci. Microarray analysis in our case showed 0.9 Mb deletion involving two genes ZEB2 and GTDC1. GTDC1 is glycosyltransferase-like domain containing 1. No disease associated mutation and deletion and copy number variation at Databases of Genomic Variation have been reported. Whether the involvement of GTDC1 influences on this patient's phenotype is uncertain. However, by a previous report, the clinical phenotype does not appear to significantly change according to the underlying genotype. Almost all patients, regardless of the specific underlying ZEB2 abnormality (i.e, small mutations or large deletions), have the facial gestalt and moderate to severe intellectual disability, supporting the concept that this syndrome
Adam, M.P., Bean, L.J.H., Miller, V.R., 2008. Mowat–Wilson syndrome. In: Pagon, R.A., Bird, T.D., Dolan, C.R., et al. (Eds.), GeneReviews™ ([Internet]. Accessed 2008 Feb 11. Available from http://www.ncbi.nih.gov/books/NBK1412). Garavelli, L., et al., 2009. Mowat–Wilson syndrome: facial phenotype changing with age: study of 19 Italian patients and review of the literature. Am. J. Med. Genet. A 149A (3), 417–426. Hoffer, M.J., et al., 2007. A 6 Mb deletion in band 2q22 due to a complex chromosome rearrangement associated with severe psychomotor retardation, microcephaly and distinctive dysmorphic facial features. Eur. J. Med. Genet. 50 (2), 149–154. Kluk, M.J., et al., 2011. Avoiding pitfalls in molecular genetic testing: case studies of highresolution array comparative genomic hybridization testing in the definitive diagnosis of Mowat–Wilson syndrome. J. Mol. Diagn. 13 (3), 363–367. Mowat, D.R., et al., 1998. Hirschsprung disease, microcephaly, intellectual disability, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22–q23. J. Med. Genet. 35 (8), 617–623. Zweier, C., et al., 2003. Caracterisation of deletions of the ZFHX1B region and genotypephenotype analysis in Mowat-Wilson syndrome. J. Med. Genet. 40 (8), 601–605.