Locus and allelic heterogeneity and phenotypic variability in Waardenburg syndrome

1 INTRODUCTION

Waardenburg syndrome (WS) is a neurocristopathy.1 It is a clinically and genetically heterogeneous disorder. The characteristic clinical features include sensorineural hearing loss (SNHL), heterochromia iridis and pigmentary abnormalities of hair and skin.2 Prevalence of WS is estimated to be 1 in 42 000 and it accounts for approximately 2% to 5% of congenital hearing loss population.3, 4

Clinically WS is classified into four subtypes (WS1-4). WS1 (MIM #193500) and WS3 (MIM #148820) are characterized by common findings of telecanthus, sensorineural hearing loss, heterochromia iridis and hypopigmentation of skin and/or hair. In addition, WS3 has musculoskeletal abnormalities of the upper limbs. WS2 clinically differs from WS1 by the absence of telecanthus. WS4 or Shah-WS is associated with Hirschsprung disease (HD).

WS1 is caused by heterozygous variants in PAX3 (MIM *606597). Heterozygous as well as biallelic variants in PAX3 have been reported for WS3. Heterozygous variants in MITF (MIM *156845), SOX10 (MIM *602229) cause WS2 (MIM #193510, MIM #611584). Homozygous and less frequently heterozygous variants in EDNRB and EDN3 cause WS4A (MIM #277580) and WS4B (MIM #613265), respectively. Heterozygous variants in SOX10 have been reported to cause WS4C (MIM #613266).5

WS is characterized by significant intra- and inter-familial phenotypic variability and reduced penetrance.5 We herein provide novel phenotypic and genetic insights into various subtypes of WS.

2 MATERIALS AND METHODS

Subjects presenting with SNHL and heterochromia iridis or hypopigmentation of hair/skin with or without additional abnormalities were included in the study. Sixteen subjects from fourteen families were recruited. Genetics testing was performed by Sanger sequencing or whole-exome sequencing (WES). Copy number variations (CNV) analysis was performed using ExomeDepth algorithm. Quantitative polymerase chain reaction (qPCR) was used to validate CNV detected by ExomeDepth algorithm. Detailed methodology including molecular testing strategy is described in Appendix S1, Supporting Information. Written informed consents for the use of photographs and research findings were obtained from all participants. The study was approved by Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee.

3 RESULTS AND DISCUSSION

In the present study, we report 15 subjects clinically diagnosed with WS and one subject with congenital NSHL. We identified 11 single nucleotide variants (SNV) and one CNV in genes known to cause WS. Clinical diagnosis, pathogenic variants, in silico prediction, allele frequency and final molecular diagnosis are provided in Table 1. Detailed clinical summary findings, pedigree of the families and molecular findings of respective families are provided in Appendix S1. We observed phenotypic variability, non-penetrance and novel molecular insights among WS families.

WS1 is caused by pathogenic variants in PAX3, which encodes for paired box transcription factor.5 In our cohort, we identified two pathogenic variants in PAX3 in two families. A novel missense variant, c.256A > T [p.(Ile86Phe)] in PAX3 in heterozygous state was identified in similarly affected siblings (II-1, II-2) in family 8. The variant was inherited from clinically asymptomatic father (I-1) who harbored the variant in a gonosomal mosaic state. This molecular mechanism has not been previously reported in PAX3 related WS. In family 9, proband (III-1), was found to have a novel nonsense variant, c.1230C > G [p.(Tyr410Ter)] in heterozygous state in PAX3. His father (II-6) was also heterozygous for the same variant and had telecanthus and premature graying of hair. He did not have hearing loss or heterochromia iridis suggesting variable phenotypic expressivity as reported previously for PAX3 related WS.5-7

WS2A is caused by pathogenic variants in MITF, which encodes for a basic helix-loop-helix leucine zipper (bHLH-Zip) protein.8 Heterozygous variants in MITF are known to cause WS/Tietz syndrome.5 In our cohort, we noted five subjects from four families with pathogenic variants in MITF. In family 3, a novel nonsense variant, c.979G > T [p.(Glu327Ter)] occurring in the HLH domain of MITF protein was observed in the proband III-1) and similarly affected mother (II-2). In family 10, a previously reported canonical splice site variant, c.1013 + 1G > A was observed in proband (III-1) and affected sibling (III-2). However, mother (II-2) harbored the same variant was non-penetrant for the phenotype.

We identified a novel missense variant, c.1021C > G [p.(Arg341Gly)] in MITF in homozygous state in the proband (III-1) of family 5. Clinical features in him were suggestive of WS2/Tietz syndrome. Previously, biallelic variants in MITF have been associated with a severe phenotype, COMMAD syndrome (MIM #617306) characterized by microphthalmia, coloboma, macrocephaly, sensorineural hearing loss, osteopetrosis, albinism and facial dysmorphism. The severity seen in these individuals was attributed to the dominant negative effect of variants present in the DNA binding domain of MITF.9 However, the variant in the proband (III-1) was located in bHLH domain of the MITF protein which is involved in dimerization of MITF protein.8

Pathogenic variants in MITF are also reported to cause NSHL.10 In the proband (III-8) of family 14, we identified a known pathogenic nonsense variant, c.1066C > T [p.(Arg356Ter)] in MITF. Targeted sequencing showed presence of this variant in heterozygous state in II-9 with unilateral hearing loss, III-5 with bilateral hearing loss and also in other unaffected members (III-7, III-6, and II-8). This variant was previously reported in a family with WS2.11

Pathogenic SNVs and CNVs (small and large heterozygous deletions) in SOX10 are known to cause both WS2E and WS4C.5 Two novel variants, c.1169C > G and c.415G > T in heterozygous state in SOX10 were identified in two probands (II-2 and III-1) of family 7 and 12 diagnosed with WS2, respectively. Variants in SOX10 have a high de novo rate as reported by recent study.12 However, because of non-availability of parental samples segregation analysis could not be performed.

In family 6, proband (V-1) with clinical diagnosis of WS4 had a de novo heterozygous deletion of approximately 0.178 Mb spanning 11 genes and 1 pseudogene (H1F0, GCAT, GALR3, ANKRD54, MIR658, MIR659, EIF3L, MICALL1, C22orf23, POLR2F, SOX10 and RNU6-900P). None of the above mentioned genes, except SOX10 are known to contribute to the phenotype. Previous studies have shown CNVs can range from single exon deletion of SOX10 to 6 Mb deletion around the SOX10 locus causing WS2E or WS4C.13, 14

WS4A and WS4B are caused by pathogenic variants in EDNRB and EDN3, respectively. EDNRB and EDN3 encode for endothelin receptor type B and its ligand endothelin, respectively. In Family 1, proband (II-3) was diagnosed with WS4. We identified a novel missense variant, c.332G > T [p.(Cys111Phe)] in homozygous state in EDN3. The variant occurs within the endothelin domain of the EDN3 protein, which is essential for binding to its receptor, EDNRB.

In family 4, a novel variant, c.327C > A [p.(Cys109Ter)] in EDNRB was observed in heterozygous state in proband (II-1) and father (I-1). Proband showed features suggestive of WS1 including hearing loss, heterochromia irides and telecanthus (Figure S4) without HD. Father was clinically asymptomatic and non-penetrant. Another novel variant, c.673G > A [p.(Gly225Ser)] in EDNRB was observed in proband (III-1) of family 13 in homozygous state. He had features of WS2 without any evidence of HD. His asymptomatic parents were heterozygous carriers for the variant. Incomplete penetrance has been observed among few families with heterozygous variants in EDNRB.15 Homozygous variants in EDNRB have been reported with WS1.16 Heterozygous variants in EDNRB have been described in individuals with WS without HD and also with isolated HD.15, 17 Although variants in EDNRB have been associated with WS4, these findings further validate that both heterozygous as well as homozygous variants in EDNRB can cause other subtypes of WS.

In our cohort, two individuals fulfilling diagnostic criteria for WS did not have any pathogenic variants in genes known to cause WS. In family 11, proband (III-2) was noted to have a novel pathogenic variant, c.1608C > G [p.(Tyr536Ter)] in homozygous state in ADGRV1 (MIM *602851). ADGRV1 is known to cause Usher syndrome 2C (MIM #605472). It is clinically characterized by SNHL and progressive retinitis pigmentosa. ADGRV1 variants are also infrequently associated with NSHL according to Deafness Variation Database. Our subject had SNHL, heterochromia iridis and premature graying of hair and albinotic fundus without any evidence of retinitis pigmentosa. In addition, he had a missense variant c.575C > A (p.Ser192Tyr) in homozygous state in TYR which is associated with reduced tyrosinase enzyme activity. This variant has a minor allele frequency of 0.2545 in gnomAD Exomes. However, Indian Genome Variation Consortium genotyped this variant in 1871 normal healthy Indians and found this variant to be under represented among Dravidians as compared to Indo-Europeans.18 It has also been reported as cause of oculocutaneous albinism type 1 in a family with three affected siblings.19 Therefore, this variant is the probably cause of hypopigmentation noted in him.

In Family 2, proband (IV-1) had a known pathogenic variant, c.71G > A [p.(Trp24Ter)] in homozygous state in GJB2 (MIM *121011).20 Biallelic pathogenic variants in GJB2 causes non-syndromic hearing loss, DFNB1A (MIM #220290). This variant explains only the hearing loss phenotype observed in the proband but not the heterochromia iridis, telecanthus and synophrys (Figure S2) seen in her.

To summarize, our cohort demonstrates intra- and inter-familial phenotypic variability and non-penetrance in families with WS. We report gonosomal mosaicism in WS1 and biallelic variants in MITF and EDNRB causing WS2. We observed NSHL because of a pathogenic variant in MITF. Blended phenotype of NSHL and albinism mimicked WS. A phenocopy of WS1 was seen in a subject with a pathogenic variant in GJB2. With these, our study adds to the spectrum of molecular and phenotypic findings and further understanding of WS.

CONFLICTS OF INTEREST

Authors declare no conflict of interest.