Mutation analysis of mitogen activated protein kinase 1 gene in Indian cases of 46,XY disorder of sex development

Abstract

Background: Determination of sex is the result of cascade of molecular events that cause undifferentiated bipotential gonad to develop as a testis or an ovary. A series of genes such as SRY, steroidogenic factor-1 (SF1), AR, SRD5 α, Desert hedgehog (DHH) etc., have been reported to have a significant role in development of sex in the fetus and secondary sexual characteristics at the time of puberty. Recently, mitogen activated protein kinase kinase kinase 1 (MAP3K1) gene was found to be associated with 46, XY disorders of sex development (DSD).

Aim: The present study is focused to identify mutations in MAP3K1 gene in the cohort of 10 Indian patients with 46,XY DSD including one family with two affected sisters. These patients were already screened for SRY, SF1 and DHH gene, but no mutation was observed in any of these genes.

Materials and methods: The entire coding regions of MAP3K1 were amplified and sequenced using the gene specific primers.

Results and discussions: Sequence analysis of MAP3K1 gene has revealed four variants including one missense, two silent and one deletion mutation. The missense mutation p.D806N was observed in four patients with hypospadias. Two patients showed the presence of silent mutation p.Q1028Q present in exon 14. Another silent mutation p.T428T was observed in a patient with gonadal dysgenesis. We have also observed one deletion mutation p. 942insT present in two patients. The pathogenicity of the missense mutation p.D806N was carried out using in-silico approach. Sequence homology analysis has revealed that the aspartate at 806 was found to be well-conserved across species, indicated the importance of this residue. The score for polyphen analysis of this mutation was found to be 0.999 indicating to be pathogenic mutation. Since, p.D806N mutation was found to be important residue; it might contribute to sexual development. We have reported the presence of mutations/polymorphism in MAP3K1 gene. All the mutations were found to be polymorphism upon comparing to single nucleotide polymorphism database. However, in-silico analysis of the missense mutation revealed to be a pathogenic mutation.

Keywords: Disorders of sex development; mitogen activated protein kinase kinase kinase 1; mutation.

Figure 1

Deoxyribonucleic acid sequence chromatogram showing the presence of mutations. (a) p.D806N mutation in exon 14 of mitogen activated protein kinase kinase kinase 1 (MAP3K1) gene. This is a heterozygous mutation indicated by an arrow showing presence of two overlapping peaks corresponding to two nucleotides A (wild type) and G (mutant) (b) p.Q1028Q mutation in exon 14 of MAP3K1 (c) p.T428T heterozygous mutation in exon 6 of MAP3K1 (d) p.T942ins mutation in exon 14 of MAP3K1 showing insertion of CAA nucleotide triplet. The chromatogram for this mutation has been reverse complemented for which the frameshift has been depicted toward left

Figure 2

Multiple sequence alignment with the homologues from different species showing the extent of conservation at the mutation site. (a) A multiple sequence alignment of p.D806N mutation showing the conserved aspartate (D) across species (highlighted by red shaded box) (b) WebLogo showing the conservation of p.D806N mutation showing the conserved aspartate (D) residue indicated by highlighted area