Case of Conradi–Hünermann–Happle syndrome due to a nonsense mutation of c.245G>A (p.W82*)

Abstract

rs984225803) variant located in the promoter region of SLC45A2. 3 In addition, four of the 110 normal Japanese control individuals (3.4%) harbored the heterozygous promoter variant, suggesting that this variant may not only be the cause of OCA4, but also one of the genetic factors contributing to color variation in the Japanese population. 3 Here, we further analyzed the genotype of the variant in more normally pigmented Japanese females (n = 424) whose skin tone (melanin index, MI) had been already investigated, 4 and patients with suspected OCA. The protocol for the study was approved by the ethics committee of the Yamagata University Faculty of Medicine. The subjects provided written informed consent for participation in this study. The identification of the genotype was performed by TaqMan allelic discrimination real-time polymerase chain reaction (PCR) using Custom TaqMan SNP genotyping assays (Applied Biosystems, Carlsbad, CA, USA).Seventeen of the 424 subjects (4%; allele frequency, 2%) harbored the heterozygous deletion mutation, which was consistent with the previous study. 3 The subjects with heterozygous deletion variant exhibited lower MI values compared with the subjects without the variant (mean AE standard deviation 0.738 AE 0.128 and 0.782 AE 0.155, respectively); however, the difference was not statistically significant (two-sided Wilcoxon-Mann-Whitley test; Fig. S1); this may be due to the low number of subjects with the deletion variant. While none of the 424 subjects were homozygous for the deletion variant, we detected two unrelated OCA-suspected patients harboring the homozygous deletion variant. Patients 1 and 2 were girls who were 20 and 6 months old, respectively (Fig. 1a,b). They had blond-to-brown hair, light skin tone and slight photophobia. None of them showed nystagmus, amblyopia or other complications. The result of their TaqMan SNP genotyping assay was confirmed by Sanger sequencing using the following primers: forward, 5 0 -TGAGAGAGAAGCTGTCATGTGTAA-3 0 and reverse, 5 0 -GACCTACGCTGAGCAGGACT-3 0 . PCR amplicons were sequenced using an internal primer (5 0 -GCCAGGTGTGATC ACGTT-3 0 ) (Fig. 1c). To assess the effects of the homozygous deletion variant of the two patients, we further performed targeted resequencing using the Ion AmpliSeq Custom Panel (Thermo Fisher Scientific, Fremont, CA, USA), which was designed by an Ion AmpliSeq Designer (Thermo Fisher Scientific) to screen the genes responsible for genetic pigmentation disorders, including all subtypes of albinism known in humans (20 genes for non-syndromic and syndromic OCA and one gene for ocular albinism). The amplified DNA libraries were sequenced by Ion personal genome machine (Thermo Fisher Scientific), followed by analysis of the sequence data using Ion Reporter 4.0 (Thermo Fisher Scientific). As a result, no other pathological variants were detected in the two patients. Hence, we concluded that the homozygous 4-bp deletion variant (rs984225803) caused the OCA-like phenotype in the patients.