A study of splicing mutations in disorders of sex development

Flavia Leme De Calais,Diana Baralle,Andréa Trevas Maciel-Guerra,Lindsay D Smith,Gil Guerra-Junior,Maricilda Palandi De Mello,Michela Raponi

doi:10.1038/s41598-017-16296-3

Flavia Leme De Calais, Diana Baralle + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-16296-3

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Abstract

The presence of splicing sequence variants in genes responsible for sex development in humans may compromise correct biosynthesis of proteins involved in the normal development of gonads and external genitalia. In a cohort of Brazilian patients, we identified mutations in HSD17B3 and SRD5A2 which are both required for human sexual differentiation. A number of these mutations occurred within regions potentially critical for splicing regulation. Minigenes were used to validate the functional effect of mutations in both genes. We evaluated the c.277 + 2 T > G mutation in HSD17B3, and the c.544 G > A, c.548-44 T > G and c.278delG mutations in SRD5A2. We demonstrated that these mutations altered the splicing pattern of these genes. In a genomic era these results illustrate, and remind us, that sequence variants within exon-intron boundaries, which are primarily identified for diagnostic purposes and have unknown pathogenicity, need to be assessed with regards to their impact not only on protein expression, but also on mRNA splicing.

Highlights

Discovered in the late 1970s, the splicing process is a crucial step in the biogenesis of eukaryotic mRNA1,2
To functionally validate whether the identified sequence variants disrupted splicing, minigenes containing the mutation of interest or the WT sequence were constructed
G > A SRD5A2 mutation is located in the last nucleotide of exon 3 (5′ splice site) (Fig. 3)

Summary

Introduction

Discovered in the late 1970s, the splicing process is a crucial step in the biogenesis of eukaryotic mRNA1,2. Cis-acting mutations can disrupt the use of essential splice sites resulting in loss of gene expression or activation of alternative splice sites, which force the expression of alternative mRNA isoforms. Alterations in the spliceosome machinery and point mutations located at intron-exon boundaries can result in severe changes to the splicing processe and impact on the appearance of disease[11]. In the present study we have evaluated the effect of mutations within two genes critical for human sexual development: HSD17B3 and SRD5A2. These genes are involved in the biosynthesis of steroid hormones, testosterone (T) and dihydrotestosterone (DHT) respectively.

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