A rare case of Waardenburg syndrome with unilateral hearing loss caused by nonsense variant c.772C>T (p.Arg259*) in the MITF gene in Yakut patient from the Eastern Siberia (Sakha Republic, Russia).

Waardenburg syndrome (WS) is an orphan genetic disease with autosomal dominant pattern of inheritance characterised by varying degrees of hearing loss accompanied by skin, hair and iris pigmentation abnormalities. Four types of WS differing in phenotypic characteristics are now described. We performed a Sanger sequencing of coding regions of genes PAX3, MITF, SOX10 and SNAI2 in the patient with WS from a Yakut family living in the Sakha Republic. No changes were found in the PAX3, SOX10 and SNAI2 coding regions while a previously reported heterozygous transition c.772C>T (p.Arg259*) in exon 8 of the MITF gene was found in this patient. This patient presents rare phenotype of WS type 2: congenital unilateral hearing loss, unilateral heterochromia of irises, and absence of skin/hair depigmentation and dystopia canthorum. Audiological variability in WS type 2, caused by the c.772C>T (p.Arg259*) variant in the MITF gene, outlines the importance of molecular analysis and careful genotype-phenotype comparisons in order to optimally inform patients about the risk of hearing loss. The results of this study confirm the association of pathogenic variants in the MITF gene with WS type 2 and expanded data on the variability of audiological features of the WS.

Introduction

Waardenburg syndrome (WS) is an orphan genetic disease with an autosomal dominant pattern of inheritance, leading to hearing loss accompanied by skin, hair and iris pigmentation abnormalities [1-4]. The incidence of WS is estimated as 1 on 42,000 among Caucasian populations, or 2–5% among patients with congenital deafness, and 0.9–2.8% among adults with hearing impairment [2,3,5-7]. Four types of WS differing in phenotypic characteristics are now described. The WS type 1 (MIM 193,500) and the WS type 2 (MIM 193,510) share similar main symptoms (pigmentation and hearing abnormalities). However, the patients with WS type 1 additionally have a dystopia canthorum (lateral displacement of the inner canthi of the eyes with the normal interpupillary distance) while this feature is absent in patients with WS type 2. The WS type 3 or Waardenburg–Klein syndrome (MIM 148,820) includes upper limbs anomalies in addition to main WS symptoms. The WS type 4 (also known as Waardenburg-Shah syndrome, MIM 277,580) has main WS features accompanied by Hirschprung disease [1-13]. Detailed and correct characterisation of identified WS cases is very important since phenotypical features of the WS often vary even among members of one family [1-13]. Mutations in the РАХ3, MITF, SOX10 and SNAI2 genes are known as main causes for WS [9,10]. Pathogenic variations in any of these genes disrupt normal development of melanocytes, altering pigmentation of skin, hair, eyes and lead to sensorineural hearing impairment [1-13].

In this report, we present a rare case of Waardenburg syndrome with unilateral hearing loss caused by nonsense variant c.772C>T (p.Arg259*) in the MITF gene in a Yakut patient from the Sakha Republic (Eastern Siberia, Russia).

Materials and methods

Patient

The case of this study was 17-year-old male patient with WS phenotype (II:4 in Figure 1) from a Yakut family living in the Sakha Republic (Eastern Siberia, Russia). His father (I:2) had the WS type 2 features (iris heterochromia with unilateral hearing loss) while the probands’ mother (I:1) had profound bilateral hearing loss and no signs of WS, and his brother (II:3) had normal hearing (Figure 1(a)).

Figure 1.

The case of the WS type 2 with variant c.772C>T (p.Arg259*) in the MITF gene

DNA-extraction

Isolation of genomic DNA was performed by standard phenol-chloroform extraction from the venous blood leukocytes and was quantified by the P330 spectrophotometer (Implen, Germany). The DNA was subsequently stored at 20°C until further use (the DNA Biobank “Genome of Yakutia”, BRK 0556–2017-0003).

PCR and Sanger sequencing

For mutation analysis we performed a Sanger sequencing of 25 fragments of the protein coding regions of the PAX3 (9 exons), MITF (9 exons), SOX10 (4 exons) and SNAI2 (3 exons) genes. The amplification reaction was set in a total volume of 25 µl containing Taq polymerase (Sileks, Russia), x10 Mg2+ PCR buffer (Sileks, Russia) and Betaine (Sigma-Aldrich, Inc., USA) with previously described primers [10] (Supplementary Table). The reaction was performed for 30 cycles: denaturation at 95°C for 30 sec, primer annealing at 56°C to 60°C for 30 sec and elongation at 72°C for 30 sec on a T200 thermal cycler (Bio-Rad, USA). The PCR products sequencing with the same primers was performed on an ABI Prism 3130XL automatic sequencer (Applied Biosystems, USA) with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The obtained nucleotide sequences of the investigated DNA fragments were compared to reference sequences of the PAX3, MITF, SOX10 and SNAI2 genes from the GenBank database.

Case report

No changes were found in the PAX3, SOX10 and SNAI2 coding regions, while a c.772C>T transition in exon 8 of the MITF gene (Reference sequence: NM_000248.3) was found in heterozygous state in proband II:4 (Figure 1(d)). The c.772C>T transition leads to premature stop-codon (p.Arg259*) terminating translation of melanocyte inducing transcription factor (MITF). Description of phenotype of proband II:4 is presented in Table 1. This patient had no dystopia canthorum (index W = 1.66 cm) (Figure 1(b)). Thus, this patient is more likely to present phenotypic features of WS type 2: iris heterochromia (right eye is dark brown, left – diamond blue) (Figure 1(b)) and sensorineural hearing loss. His hearing loss was unilateral: profound (118 dB) – in the left ear, normal hearing (15 dB) – in the right ear (Figure 1(c)). The patient is a student in a regular school and has no limitations in communication, because he uses both oral speech and sign language (Table 1).

Table 1.

Phenotype of observed patient with Waardenburg syndrome

Phenotypic features	Proband II:4
Gender	Male
Age	17 years old
Ethnicity	Yakut
Type of inheritance	AD
Hearing loss	Unilateral
Degree of hearing loss	Profound on the left (>90 dB in speech range), normal hearing on the right
Communication	Speech/Sign language
Eyes	Unilateral iris heterochromia (right eye is dark brown, left – diamond blue with some brown pigmentation
Skin	Normal pigmentation
Hair	Normal pigmentation
W index	1.66 cm
WS type	2

Age is stated as on the moment of examination (May 2015); speech range (0.5, 1.0, 2.0, 4.0 kHz); dB – decibels; AD – autosomal dominant; W index – dystopia canthorum index: value over 1.95 cm signs on presence of dystopia canthorum; WS type – Waardenburg syndrome type.

Close relatives of proband II:4 were not thoroughly clinically examined. Nevertheless, his mother (I:1) has profound bilateral hearing loss and no signs of WS while his father (I:2) has the WS type 2 features: iris heterochromia and unilateral hearing loss (profound hearing loss in one ear, normal hearing in another ear) and his brother had normal hearing (Figure 1(a)).

Discussion

The MITF gene (MIM 156,845) is located on chromosome 3 and consists of 9 exons. This gene encodes a transcription factor MITF (melanocyte inducing transcription factor) involved in melanocytes development [14,15]. MITF is a basic helix-loop-helix (hHLH)-leucine zipper protein that plays an important role in the development of various cell types, including neural crest-derived melanocytes and optic cup-derived retinal pigment epithelial cells [16]. According to the ClinVar database (March 2019), there are ~80 variants in MITF associated with WS and Tietz syndrome (MIM 103,500) [17], one variant associated with skin melanoma and renal cell carcinoma (MIM 614,456), and other (~30) variants in MITF have unknown or no clinical significance. In our study proband II:4 had no features of Tietz syndrome and no skin pigmentation impairment or melanoma. The c.772C>T (p.Arg259*) variant in the MITF gene is not reported in the 1000 Genomes Project, the dbSNP, the ExAC (the Exome Aggregation Consortium) and the ClinVar database. However, we found information about c.772C>T (p.Arg259*) in the HGMD (Human Genome Mutation Database) [18] with reference to Nobukuni et al. [14]. In this study, the c.772C>T (p.Arg259*) variant was revealed in a family with 13 individuals having WS type 2 phenotype [14]. All affected members of this family have a typical WS type 2 anomalies with sensorineural hearing loss, heterochromia iriditis, white forelock and early greying. None of the family members showed dystopia canthorum [14]. However, in the paper of Nobukuni et al. [14], detailed audiological features of these 13 patients were not described.

In our study patient with the c.772C>T (p.Arg259*) variant in the MITF gene presents rare WS type 2 phenotype: congenital unilateral hearing loss (profound deafness on the left side, normal hearing on the right side), unilateral iris heterochromia (right eye is dark brown, left – diamond blue) and absent of dystopia canthorum (index W = 1.66 cm.) (Table 1). Meta-analysis of the WS cases (417 patients) in different regions of the world showed that pathogenic variants in MITF were detected in 28% out of all examined WS cases and in 89.6% – among patients with the WS type 2 [9]. Moreover, almost 90% of patients with WS type 2 having pathogenic MITF variants presented bilateral sensorineural hearing loss [9].

In conclusion, the results of this study confirm the association of pathogenic variants in the MITF gene with the WS type 2 and enrich current information on the clinical manifestation of this rare syndrome worldwide. In addition, obtained data provide important targeted information to genetic counselling of families affected by the WS type 2 and will be useful for medical practitioners working with such patients.

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