Cytogenetic and Molecular Characterization of Disorders of Sex Development with special emphasis on SRY gene

Abstract

Abstract Introduction Disorders of sex development (DSD) refer to a group of congenital conditions in which the development of the chromosomal, gonadal and/or anatomical sex have been atypical. Worldwide, DSD has an incidence of 125000 live births, being more common in Jewish and Hispanic, but unfortunately there are no accurate records about the incidence of DSD in Sudan. Molecular studies of sex reversal patients by Sinclair and his group in 1990 had led to identification of the SRY gene and its role in male sex differentiation. Since that time and till now, the full spectrum of SRY gene mutations and the role of other autosomal genes were not fully understood. In the present study, we aimed to characterize the phenotype, and cytogenetic alterations in Sudanese patients with DSD. In addition to that, we aimed also to characterize SRY mutations associated with 46,XY DSD and to correlate the mutations with the clinical profile of the patients. Materials and methods A total of 60 patients referred with provisional diagnosis of DSD were enrolled in this study. Ten ml of venous blood were collected from each patient under complete aseptic conditions (Sml in sodium heparin container for cytogenetic analysis and another 5ml in EDTA container for DNA extraction). All patients signed a written consent that has been approved by Al Neelain University Ethical Committee prior the study. The lymphocyte culture, chromosomal analysis, and karyotype were perfonned according to the standard cytogenetic technique, and in accordance with ISCN guidelines (1995). Whereas genomic DNA was extracted using Guanidine Chloride method, detection of SRY gene was performed by polymerase chain reaction (PCR) using XES10 and XESl1 primers. Reaction products were electrophoresed on 1% agarose —TBE gels and visualized with gel documentation system. Amplified region was directly sequenced in 19-46,XY-DSD-patients to identify and characterize mutations in SRY gene. Data were analyzed using SPSS l3 software. Descriptive statistics (frequencies and percentages) were obtained for categorical variables. Results Of the sixty patients examined, 65% grown up as females and 35% as males; age ranged between one month and 45 years. Of the 60 patients, 63.3% were presented afler pubertal age while, 36.7% were accessible before puberty. Sixty percent of the patients have been referred due to their ambiguous genitalia, 30% due to primary amenorrhea, and the remaining l0% were due to other complains. The cytogenetic analyses showed that 46,XY karyotype was present in 61.7% of patients, 46XX karyotype in 31.7%, and abnormal karyotype (46,XX/46,XY; 47,XYY; and 45,XO/46,X_X) in 6.7%. In 61.7% of the patients, the sex of rearing was matching with correct genotype, whereas in 38.3% of the patients the results showed clashing between the sexes of rearing and the genotype. 33.3% of the patients had 46,XY karyotype and grown up as females, 75% of those have experienced female genital mutilation while 25% have not.

According to the patient’s phenotypes, the phenotype was in complete accordance with the genotype in only 18.4% of the adult patients. The physical examination showed absent secondary sexual characteristics in 42.6% of the adult patients. SRY gene analysis showed physical presence of the gene in 60% of the cases, of which 51.7% had 46,XY, 5.0% had 46,XX, and 3.3% had mosaic karyotype (46,XX/46,XY). Complete absence of the SRY gene was seen in 40% of the patients, of which 26.6% had 46,XX, and 10% had 46,XY, and 1.7% had 47,XYY or 45,XO/46,XX karyotype. About 61.66% of the patients were diagnosed as 46,XY DSD, of those, 5% were finally diagnosed as Swyer syndrome and 3.33% as ovotesticular DSD. The result showed that 33.33% of the patients had the diagnosis of 46,XX DSD, of those, 5% had final diagnosis of complete gonadal dysgenesis (CGD), 6.7% had diagnosis of complete gonadal dysgenesis turner (CGD, Turner), ), 1.7% tum to be sex reversed with SRY gene translocation. The remaining patient (1.7%) was ovotesticular DSD. 5% of the patients were diagnosed as sex chromosome DSD. DNA sequencing of the selected samples revealed apparent single base changes in 7 cases, 5 different mutations were obtained, the detected mutations were not reported before. Two cases showed Heterozygous T to G transversion, at codon 71, resulting in Alanine (A) replacing Serine (S) in case of the mutant allele, this mutation lies inside the HMG box. Four cases showed Heterozygous G to A transition, at codon 132, resulting in Valine (V) replacing Alanine (A) in case of the mutant allele, this mutation lies inside the HMG box. Three cases showed Heterozygous G to C transversion, at codon 7, resulting in Tryptophan (W) replacing Serine (S) in case of the mutant allele this mutation lies inside the HMG box. Three Cases showed an insertion of A, at codon 180, resulting in Arginine (R) replacing serine (S) (This mutation lies outside the HMG-box of the SRY gene), whereas 1 case showed a mutation of A to C at position 93 (This mutation lies outside the ORF of the SRY gene) resulting in Threonine (T) replacing Asparagine (N). Conclusion: The present study revealed that, both eytogenetic and PCR techniques are reliable and efficient techniques in diagnosis and workup of DSD patients when used in combination. Another interesting finding is that genotype/phenotype and sex of rearing are not always in accordance in patients with DSD. Massive heterogeneity of SRY gene mutations was observed in the present study, and the missing SRY gene mutations suggest important role of autosomal genes in DSD and should be looked up in future studies.