Gene Mapping And Mutation Detection Of A Family With Hypospadias

Hypospadias is a congenital malformation disease of male urinary and reproductive system, urethra opening in the penis, scrotum or perineum. The main clinical features are ectopic urethral orifice, penile curvature and penis foreskin dorsal accumulation. Base on the different openings, hypospadias can be sorted to glandular hypospadias, penial hypospadias, penoscrotal hypospadias, scrotal hypospadias and perineal hypospadias. At present, the incidence of hypospadias is 9.15/104, the etiology is not very clear. It is very important to reveal the causative genes and elucidate the pathogenesis of the hypospadias by study genetic hypospadias family.Currently the causative genes of hypospadias are SRY,SOX9,WT-1,SRD5A2,AR,MAMLD1 and MID1 etc. A Han family from Hubei Provience with X-linked hypospadias have identified and characterized. The major clinical character of the proband was breast development, no Adam's apple and sparse beard, except hypospadias. In order to study the cause of the family, molecular genetics analysis was carried out. According to the family structure; the genetic form is X chromosome linked genetic disease. Linkage with known genes of X-linked hypospadias, linkage was identified with markers DXS991 and DXS986. Haplotype analysis defined the causative gene between DXS991 and DXS986. The AR gene located between the two markers in the X chromosome. This indicated that AR gene was probably the cause of the disease. Mutational analysis of all exons and exon-intron boundaries of AR in the proband was carried out using direct DNA sequence analysis. A novel mutation of a C to T transition at nucleotide 2084 of AR (c.2084C>T) was found. This mutation leads to a substitution of a highly conserved amino acid residue Proline with a Leucine residue at 695 (p.P695L). Becasuse of this mutation, a restriction site Taq I was occurred, detection of mutation c.2084C>T was further confirmed by RFLP analysis. The RFLP analysis showed that the presence of the hemizygous mutant allele in amle patients, both wild type and mutant alleles in female carriers, and homozygous and hemizygous wild type alleles in normal family members and 200 normal controls (100 males and 100 femals). The p.P695L mutation is located in the ligand binding domain (LBD) of AR. The p.P695L mutation occurs at residue that is evolutionary highly conserved from fish to human. The ability of AR protein binding with the androgen was probably affected by the p.P695L mutation, resulting in the reduced AR promoting activation of the direct target genes. This indicated that the mutation c.2084C>T (p.Pro695Leu) of AR gene might be the cause of the hypospadias in the family.