To date more than different mutations in the gene
To date, more than 50 different mutations in the gene have been described (Human Gene Mutation Database at the Institute of Medical Genetics in Cardiff: SRD5A2 Gene: http://www.hgmd.cf.ac.uk). Most of them are missense mutations but premature stop codons and deletions leading to non-functional or sub-functional proteins are also reported (Canto et al., 1997, Maimoun et al., 2011, Vilchis et al., 2008).
We report on seven Iraqi patients with genital ambiguity belonging to five families, with variable degree of incomplete virilization, harboring mutations in the SRD5A2 gene. This study was performed to report SRD5A2 deficiency-related DSD to the attention of clinical geneticists and child surgeons, and to help build up the appropriate strategies for clinical and surgical treatment of these patients. Furthermore, the possibility of a founder genetic effect and the presence of genetic modifiers are discussed.
Patients All examined subjects had normal male karyotype, cytogenetic analysis excluded structural rearrangements of X and Y chromosomes. All patients have consanguineous and healthy parents (Fig. 1). In all cases, written informed consent for molecular analysis was obtained from probands' parents before study enrollment.
Methods Genomic DNA was extracted from peripheral white blood ionomycin synthesis according to standard procedure. Exons 1-5 of the SRD5A2 gene and the intron-exon boundaries were amplified by PCR using previously described primers (Baldinotti et al., 2008). The CAG repeat region located in the androgen receptor (AR) gene was amplified using primers designed with Primer 3 software. Purified PCR products were bidirectionally sequenced using the BigDye terminator v3.1 Cycle Sequencing kit (Life Technologies, Italy) and were analyzed on a 3130Xl Genetic Analyzer (Applera). Nucleotide sequences were compared with the published sequences of the SRD5A2 gene (GenBank accession no. NG_008365.1) and AR gene (GenBank accession no. NG_009014.2). DNA mutation numbering is based on GenBank reference DNA sequence NM_000348.3 (www.hgvs.org/mutnomen).
Discussion We describe seven 46,XY patients of Iraqi origin with 5-α-reductase deficiency and mutations in the SRD5A2 gene. Steroid 5-α-reductase deficiency (SRD) is a rare autosomal recessive form of 46,XY disorder of sex development and it is responsible for incomplete virilization. Diagnosis of SRD is based not only on physical examination, but also on biochemical features and radiological findings. The diagnosis is usually supported by an increase in the T/DHT ratio after human chorionic gonadotropin (hCG) stimulation testing. However, SRD5A2 mutations have been reported in patients with a non-significant increase in this ratio, making DNA studies essential in order to obtain the correct diagnosis (Boudon et al., 1995, Maimoun et al., 2011, Mazen et al., 2003). SRD affects populations with a high rate of inbreeding as in our cohort, and subsequently, the majority of patients present homozygous mutations (Nordenskjöld et al., 1998, Wilson et al., 1993). Some of these are recurrent and reported in various ethnic groups, whereas others seemingly reflect a founder effect (Hafez et al., 2003, Mazen et al., 2003, Skordis et al., 2005). According to the reported data we found that in five patients belonging to three unrelated families, coming from the same ethnic group, a unique novel homozygous point mutation of the SRD5A2 gene was present. This suggests the presence of a common ancestor and a founder effect responsible for dissemination of this genetic abnormality in that population. Clinical presentation of SRD is extremely variable, ranging from male phenotype with hypospadias to female phenotype with bilateral testes, normal Wolffian structures and a blind vaginal pouch (expression of urogenital sinus defect) (Adiyaman et al., 2006, Boudon et al., 1995, Hafez et al., 2003, Mazen et al., 2003, Ocal et al., 2002, Skordis et al., 2005, Thigpen et al., 1992a, Thigpen et al., 1992b, Wilson et al., 1993). Generally, the latter are raised as females but the increase of serum testosterone and of 5 α-reductase type 1 enzyme activity at puberty, cause growth of musculature mass and deepening of the voice (Cohen-Kettenis, 2005, Imperato-McGinley et al., 1974, Mendez et al., 1995). Considering that a gender identity switch may have severe psychological effects, an early diagnosis and well-planned management are essential. The seven patients reported in this study were differently treated according to the social gender identity they developed in order to avoid psychological distress. Intrafamilial variability has been also reported in SRD. Sinnecker et al in 1996 reported SRD in two brothers with the same homozygous mutation (p.Arg227Gln) and different clinical severity: one brother had type 2 SRD while the other had a normal male development with only a small penis (Sinnecker et al., 1996). In addition, both patients had the same testosterone/dihydrotestosterone ratio. More recently Maimoun et al. (2011), reported two siblings with the same compound heterozygous mutations (p.Q126R/p.G203S) with difference in sex rearing: one sibling had normal clitoris and was raised as female, the second had microphallus and hypospadia and was raised as male (Maimoun et al., 2011). The described phenotypic variability may be due to residual 5-α-reductase type 2 activity and consequently due to mutation type, genetic modifiers, such as those influencing 5- α-reductase type 1 isoenzyme activity or AR activity and environmental factors. In the present study we confirmed the presence of various degrees of under-virilization in mutated patients, especially in two sibs. In family a, patient 1 presented micropenis, perineal hypospadias, vulva with separated vaginal and urethral openings, labioscrotal folds and cryptorchid testes while his brother presented only isolated hypospadias. Some studies have shown that longer AR(Gln)n are associated with isolated male infertility and moderate to severe undermasculinized genitalia (Castro-Nallar et al., 2010, Dowsing et al., 1999, Lim et al., 2000). Assuming that this polymorphism could act as a modifier factor, we decided to determine AR(Gln)n length in our patients. The obtained results do not seem to corroborate this hypothesis. In particular, we unexpectedly found the same number of repeats in patients 1 and 2, despite their phenotypic variance. However, the contribution of the polymorphism of the AR gene in the modulation of the severity of phenotype observed in undervirilized patients would deserve further investigation.