Abstract
ObjectiveTo identify the pathogenic mechanism of the c.244G>T mutation in NR5A1 gene found in a Chinese patient with 46, XY disorders of sex development (DSD). Subjects and methods: Genomic DNA was extracted from a Chinese 46, XY DSD patient. Targeted next-generation and Sanger sequencing were performed to investigate and validate the gene mutation causing 46, XY DSD, respectively. In silico tools were used to predict the pathogenicity of the variant. Dual luciferase reporter gene assay and minigene splicing reporter assay were used to identify the pathogenicity of the variant.ResultsA novel heterozygous variant, c.244G>T (p.Ala82Ser), in NR5A1 gene was detected in the 46, XY DSD patient. Four of five silico tools predicting pathogenicity of missense variants indicated that the variant was pathogenic. However, in vitro functional study showed that p.Ala82Ser did not affect the transcriptional activity of NR5A1. In silico tools predicting the potential splicing loci revealed that c.244G>T led to aberrant splicing of NR5A1 RNA. Minigene splicing reporter assay confirmed that c.244G>T resulted in the deletion of exon2 or deletion of 19 nucleotides in 3′ end of exon2.ConclusionsMutation of c.244G>T in NR5A1 results in 46, XY DSD by inducing abnormal splicing of NR5A1 RNA instead of amino acid substitution of NR5A1.
Highlights
Four of five silico tools predicting pathogenicity of missense variants indicated that the variant was pathogenic
In silico tools predicting the potential splicing loci revealed that c.244G>T led to aberrant splicing of NR5A1 RNA
Minigene splicing reporter assay confirmed that c.244G>T resulted in the deletion of exon2 or deletion of 19 nucleotides in 3′ end of exon2
Summary
Patient and genetic analysis A 16-year-old girl was referred to our hospital because of clitoris hypertrophy and primary amenorrhea. At the age of 12 years, hirsutism and deepening of the voice were noted She was admitted to the local hospital for karyotype testing, which showed a 46, XY karyotype. Next-generation sequencing revealed a novel heterozygous NR5A1 variant, c.244G>T, which had led to the change of the amino acid at the position 82 from Ala to Ser; Sanger sequencing confirmed the variant (Fig. 1). In silico analysis of NR5A1 variants The amino acid, Ala, is highly conserved (Fig. 2). Sanger sequencing of the longest product showed that the wild type sequence came from the normal splicing (Fig. 4b). The sequencing of the medium product showed a loss of 19 nucleotide base pairs (GTGGGGATGCGCCTGGAAG) in 3′ end of exon (Fig. 4c), whereas the shortest product revealed a skipping of exon (Fig. 4d)
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