Waardenburg Syndrome: The Contribution of Next-Generation Sequencing to the Identification of Novel Causative Variants

Abstract

Waardenburg syndrome (WS) is characterized by hearing loss and pigmentary abnormalities of the eyes, hair, and skin. The condition is genetically heterogeneous, and is classified into four clinical types differentiated by the presence of dystopia canthorum in type 1 and its absence in type 2. Additionally, limb musculoskeletal abnormalities and Hirschsprung disease differentiate types 3 and 4, respectively. Genes PAX3, MITF, SOX10, KITLG, EDNRB, and EDN3 are already known to be associated with WS. In WS, a certain degree of molecularly undetected patients remains, especially in type 2. This study aims to pinpoint causative variants using different NGS approaches in a cohort of 26 Brazilian probands with possible/probable diagnosis of WS1 (8) or WS2 (18). DNA from the patients was first analyzed by exome sequencing. Seven of these families were submitted to trio analysis. For inconclusive cases, we applied a targeted NGS panel targeting WS/neurocristopathies genes. Causative variants were detected in 20 of the 26 probands analyzed, these being five in PAX3, eight in MITF, two in SOX10, four in EDNRB, and one in ACTG1 (type 2 Baraitser-Winter syndrome, BWS2). In conclusion, in our cohort of patients, the detection rate of the causative variant was 77%, confirming the superior detection power of NGS in genetically heterogeneous diseases.

Keywords: NGS; Waardenburg syndrome; neurocristopathy.

Figure 1

(A) Combination of methodologies and molecular strategy to diagnose a cohort of clinically suspected Waardenburg syndrome patients. The number of causative variants found in each gene through each methodology is written below each gene name. In (B) two plots represent the types of variants found.

Figure 2

Illustration of the PAX3 duplication in case LGH6. (A) Gel electrophoresis of the PCR product of the exon 6 of PAX3. (B) Sanger sequencing of the PCR product carrying the duplication. (C) Schematic representation of the duplication. Arrows indicate primer location.

Figure 3

Illustration of the EDNRB deletion in case LGH24 (A) Gel electrophoresis showing the different band patterns from the EDNRB exon 8 deletion carriers (V:3—LGH24, IV:5, and III:2) compared to non-carriers (IV:6, IV:7); PCR conditions of the products in this gel were not suitable for the 1.5 Kb fragment. (B) The Sanger sequencing of the PCR product with the deletion. (C) Schematic representation of the deletion. Arrows indicate primers location.