Molecular Vision 2012; 18:2022-2032
Received 9 June 2012 | Accepted 17 July 2012 | Published 20 July 2012
1Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India; 2Department of Neurology, National Institute of Mental Health and Neuro Sciences, Bangalore, India; 3Department of Neuropathology, National Institute of Mental Health and Neuro Sciences, Bangalore, India; 4Centre for Cellular and Molecular Biology, Hyderabad, India
Correspondence to: Professor Arun Kumar, Ph.D., Department of Molecular Reproduction, Development & Genetics, Indian Institute of Science, Bangalore 560 012, India; Phone: 91-80-2293 2998; FAX: 91-80-2360 0999; email: firstname.lastname@example.org
Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS.
Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes.
Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD.
Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.
Waardenburg syndrome (WS) is a hereditary auditory-pigmentary syndrome characterized by pigmentary abnormalities of the hair, including a white forelock and premature graying, pigmentary changes of the iris such as heterochromia irides and brilliant blue eyes, lateral displacement of the medial canthi, and lacrimal points, congenital deafness, and intestinal abnormalities . The association of hearing loss, pigmentary abnormalities, and intestinal malformation results from an abnormal proliferation, survival, migration, or differentiation of neural crest derived melanocytes of skin and inner ear, glia, and neurons of the peripheral and enteric nervous system . The incidence of WS is around 1 in 42,000 in the general population. However, it occurs in 5%–6% of deaf individuals and is considered to be the most common autosomal-dominant form of syndromic deafness . It is clinically and genetically heterogeneous and is classified into four types (viz., type I to IV) based on the presence of variable clinical characteristics and additional symptoms. WS type I is characterized by the presence of dystopia canthorum (lateral displacement of inner canthi). WS type II is distinguished from type I by the absence of dystopia canthorum. WS type III patients have limb hypoplasia in addition to dystopia canthorum. WS type IV has an additional feature of Hirschsprung disease . Genetic analysis has identified at least eight loci for WS: WS1/WS3 on chromosome 2q36.1, WS2A on chromosome 3p14.1-p12.3, WS2B on chromosome 1p21-p13.3, WS2C on chromosome 8p23, WS2D on chromosome 8q11.21, WS2E/WS4C on chromosome 22q13.1, WS4A on chromosome 13q22.3, and WS4B on chromosome 20q13.2-q13.3 (OMIM). The genes for six loci are known: WS1/WS3-PAX3 (paired box 3), WS2A-MITF (micropthalmia-associated transcription factor), WS2D-SNAI2 (snail homolog 2, Drosophila), WS2E/WS4C-SOX10 (SRY-box containing gene 10), WS4A-EDNRB (endothelin receptor type B) and WS4B-EDN3 (endothelin 3) [4-12].
Duchenne muscular dystrophy (DMD) is the most common X-linked recessive disease. It is estimated to affect 1 in 3,500 newborn males worldwide  and caused by mutations in the DMD gene, encoding a cytoskeletal protein dystrophin. DMD is the largest human gene that spans >2,200 kb of DNA and is composed of 79 exons . Deletions account for approximately 65% of DMD mutations, duplications occur in approximately 6%–10% of cases and the remaining 30%–35% of mutations consist of small deletions, insertions, point mutations or splicing mutations, most of which introduce a premature stop codon [14-23].
Here we report on the genetic analysis of a consanguineous family from the south Indian state of Karnataka with members affected with two disorders: Waardenburg syndrome and Duchenne muscular dystrophy.
We ascertained a consanguineous family (Figure 1) with one individual (IV-3) affected with WS and DMD, and the other one (IV-2) affected with only WS from a south Indian state of Karnataka in the Department of Neurology, National Institute of Mental Health and Neuro Sciences, Bangalore. Both affected individuals were examined by Parayil Sankaran Bindu, Arun B. Taly and Sanjib Sinha. A detailed description of their clinical symptoms is given below.
Individual IV-3-- The propositus, a 9-year-old boy, was the third child born to consanguineous parents after an uneventful pregnancy and delivery. His developmental milestones were normal except for speech delay. A formal evaluation for the speech delay at the age of three years revealed profound bilateral sensorineural hearing loss. Since the age of six years, he developed gradually progressive difficulty in walking and getting up from sitting position. He had blue iris, faint white forelock, confluent eyebrows, bilateral calf hypertrophy, mild tendon Achilles contracture, exaggerated lumbar lordosis, and a waddling gait. He had symmetric weakness of proximal muscles of all four limbs and truncal muscles. All stretch reflexes were sluggish except for the ankle jerks. Gower’s sign was evident on getting up from the sitting position (Figure 2A). Ophthalmological evaluation showed retina with visible choroidal vessels and depigmented iridis (Figure 2B,G). Waardenburg index was 0.572, thus ruling out dystopia canthorum. Laboratory evaluation showed normal hemogram and routine biochemical parameters. Serum creatine phosphokinase was 3,218 IU/l (n<170 IU/l). Nerve conduction velocity studies that involved right median, ulnar and common peroneal, and sural nerve were normal. Electromyography of right biceps and vastus lateralis revealed a myopathic pattern. Audiometric evaluation showed profound sensorineural hearing loss and absence of bilateral brainstem auditory evoked responses. Magnetic resonance imaging of the brain showed normal myelination pattern. A left quadriceps biopsy revealed marked variation in fiber diameter with evidence of internal nuclei, occasional myophagocytosis, regenerating fiber and splitting, suggestive of muscular dystrophy. On immunohistochemistry with an anti-dystrophin antibody (Novocastra Laboratories, UK), there was absence of staining of dystrophin (Figure 2F). The above clinical symptoms suggested that this patient has both WS and DMD.
Individual IV-2-- The 11-year-old elder brother of the index patient had a history of Hirschsprung disease that required surgery at the age of 3 months. He had iris discoloration at birth but did not report any hearing problem till the age of 8 years. On examination, he had heterochromia of iris (Figure 2H) and a normal neurologic examination. Ophthalmological evaluation revealed hypopigmented iris and choroid with visualization of the choroidal vessels. Waardenburg index was 0.4059, thus ruling out dystopia canthorum. Audiometry revealed profound hearing loss on left side with absence of auditory brainstem evoked responses. Laboratory evaluation showed normal creatine kinase levels. Nerve conduction velocity studies and magnetic resonance imaging of the brain were normal. The above clinical symptoms suggested that he has only WS, and it could be WS type IV (WS4).
A detailed clinical examination of their parents, elder sister and paternal grandmother showed heterochromic iris only in the grandmother II-2 (Figure 2I).
Following informed consent, three-five milliliter of peripheral blood sample was collected in a Vacutainer EDTATM tube (Beckton-Dickinson, Franklin Lakes, NJ) from each individual for genomic DNA isolation using a Wizard® Genomic DNA Extraction Kit (Promega, Madison, WI). This research followed the tenets of the Declaration of Helsinki and the guidelines of the Indian Council of Medical Research, New Delhi. Although the clinical features in the affected individual IV-2 suggested this family to be of WS type IV, we still went ahead and selected two microsatellite markers from each of the eight WS loci (Table 1) and used them to genotype the family, according to Kumar et al. .
For mutational analysis, the entire coding region of the EDN3 gene (GenBank NM_000114.2) was amplified using primers that amplify all coding exons and their intron-exon junctions (Table 2). Primers were designed using the gene sequence from the UCSC Genome Bioinformatics site. Sequences and PCR conditions of these primers are provided in Table 2. Mutations were identified by sequencing the PCR products from an affected individual from the family on an ABIprism A370-automated sequencer (PE Biosystems, Foster City, CA). Once a mutation was identified, all members of the family were examined for the presence of the mutation by sequencing.
The mutation analysis of the DMD gene was performed using multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) technique and a kit from MRC-Holland, Amsterdam, the Netherlands according to the manufacturer’s recommendations. The kit contains specific probes for each of the 79 exons of the DMD gene on Xp21.2 and thus allows analysis of deletion and/or duplication of one or more of these exons. Briefly, 200 ng DNA was denatured and hybridized overnight at 60 °C with the probe mix. Samples were then treated with Ligase 65 for 15 min at 54 °C. The reactions were stopped by incubation at 98 °C for 5 min. Finally, PCR amplification was performed with the specific SALSA FAM PCR primers. Amplification products were run on an ABI PRISM 3730 Genetic Analyzer (PE Biosystems, Foster City, CA) and the obtained data were analyzed by using the Gene Marker 2.0 Software. Deletions of a probe’s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognizable by a 35%–50% reduction in relative peak area.
The visual inspection of the family suggested segregation of WS as an autosomal recessive trait (Figure 1). Haplotype analysis using microsatellite markers from each of the eight known WS loci suggested linkage of the family to the WS4B locus on chromosome 20q13.2-q13.3 (Figure 1 and Table 1), indicating that WS is caused by a mutation in the EDN3 gene. The genotypes of individuals for microsatellite markers from the other seven WS loci are given in Table 3. To determine the exact nature of the mutation, the entire coding region of EDN3 from the affected individual IV-2 was amplified using 5 sets of PCR primers (Table 2) and sequenced. The results revealed a novel homozygous substitution mutation, c.293C>T in exon 2, changing codon position 98 from threonine to methionine (p.T98M; Figure 3A). The affected sibling IV-3 was also homozygous for the mutation, both parents (III-1 and III-2) and paternal grandmother (II-2) were heterozygous and the normal sibling IV-1 was homozygous for the wild-type allele (Figure 3A). The 528 bp wild-type exon 2 amplicon contains a site for the restriction endonuclease Eco72I, cleaving it into two fragments of 345 bp and 183 bp. The mutation abolishes this restriction site, thus retaining the undigested 528 bp amplicon in affected indivuduals. We therefore developed a restriction fragment length polymorphism (RFLP) analysis to determine if the mutation was present in 50 normal controls. The analysis showed absence of the mutation in 50 ethnically matched controls, further suggesting that the c.293C>T change is a mutation (data not shown). The analysis also showed segregation of the mutation in the family (Figure 3B). In addition, the pathogenic nature of the mutation also comes from the fact that threonine residue at position 98 is conserved in different EDN3 orthologs from human to zebra fish (Figure 3C). According to the Mutation Taster, the mutation is predicted to be disease causing with a p-value (probability) of 0.52. We then used two other in silico methods, PolyPhen-2 and SIFT, to see the effect of this mutation on the protein function. The effect of this mutation on EDN3 was predicted to be probably damaging by PolyPhen-2 with a score of 1 (score ranges from 0 to a positive number, where 0 is neutral, and a high positive number is damaging). SIFT analysis predicted this mutation to be damaging and intolerant with a score of zero (score ranges from 0 to 1, where 0 is damaging and 1 is neutral).
Multiplex PCR and MLPA analyses of the affected individual IV-3 showed that he has a deletion from exons 49–51 in the DMD gene (Figure 4). MLPA analysis showed absence of the deletion in his mother (III-2), suggesting that it is a de novo mutation (data not shown). His sister (IV-1) also did not have this mutation (data not shown). This deletion causes a complete absence of the DMD protein in muscle tissues (Figure 2F) of the affected individual, and thus causing the phenotype.
A total of 10 mutations have been reported so far in the EDN3 gene in WS patients of different ethnic origins [2,9,25-30]. The novel mutation p.T98M reported here, along with two other mutations p.H112R and p.T98K, affect one of the 21 amino acids that constitute active peptide from amino acid positions 97–117 in endothelin-3 [2,29]. Further, with the mutation reported here, the total number of mutations in the EDN3 gene is 11 (Table 4).
Endothelins (EDNs) are a family of three active peptides EDN1, END2, and EDN3 that act as ligands and recognized by two G-protein coupled heptahelical receptors known as endothelin receptors, EDNRA and EDNRB. EDN3 preferentially binds the EDNRB . EDN3 is produced as preproendothelin-3 encoded by the EDN3 gene. The biologically active 21 amino acid long EDN3 is produced by proteolytic cleavage of preproendothelin-3 by endopeptidases to yield proendothelin-3, which is finally cleaved by endothelin converting enzyme-1 to produce the mature active EDN3 [32,33]. Studies have shown that EDN3/EDNRB interaction is required for the proper development of neural crest derived melanocytes and enteric neurons [34-36]. Mouse models for homozygous mutations in the EDNRB or EDN3 genes show pigmentation anomaly, aganglionic megacolon and cochlear disorder [31,37]. We suggest that the disease phenotype in the present family could be due to the absence of the active form of the protein as the mutation p.T98M might impair the EDN3 activity. Although both parents (III-1 and III-2) and grandmother (II-2) are heterozygous for the mutation (Figure 3), only the grandmother showed heterochromic iris (Figure 2I). However, it is not completely unexpected as heterozygous individuals for the EDN3 mutations have been reported to show a few clinical features of WS [25,29]. The manifestation of a few of the WS clinical symptoms in heterozygous individuals could be due to environmental factors, multigenic inheritance, modifier genes or stochastic events on cell fate or cell differentiation in early embryogenesis [25,27].
In summary, we report an interesting Indian family with members affected with two different disorders, WS and DMD. The WS is caused by a novel missense mutation, whereas DMD resulted due to a deletion. The present information will be useful to provide rapid prenatal diagnosis and genetic counseling for WS to the family and their relatives using a PCR-RFLP method developed during the study.
We thank the patients and their family members for their involvement in the study. We also thank Dr. Robert Church and two anonymous reviewers for their suggestions to improve the manuscript.