JMSCR Volume||2||Issue||7||Page 1626-1646||July 2014 2014 www.jmscr.igmpublication.org Impact Factor 1.1147 ISSN (e)-2347-176x Disorders of Sex Development: Molecular Versus Clinical and Cytogenic Studies and Their Correlation with Diagnostic Outcome and Sex of Rearing in A Local Community Authors Manal Mohammed Kadhim (PhD) 1, Mohammed Joudi Aboud (F.IC.M.S) 2 1Medical Microbiology Department , College of Medicine, Al-Qadisiya University, Iraq E-mail: [email protected] 2Pediatric Surgery Unit , Maternity and Child Teaching Hospital, Al-Qadisiya, Iraq E-mail: [email protected] Corresponding Author Mohammed Joudi Aboud (F.I.C.M.S) Pediatric Surgery Unit , Maternity and Child Teaching Hospital, Al-Qadisiya, Iraq Email: [email protected] ABSTRACT BACKGROUND: This study aimed to support clinical professionals in the initial evaluation and diagnosis of children with suspected DSD , to provide a framework to standardize laboratory and clinical practice, and to acquire more knowledge on the molecular mechanism of sex determination . METHODS: This was a single center, prospective case review. A total of 42 patients with DSD were referred to our Pediatric Surgery Unit at the Maternity and Child Teaching Hospital . We examined Barr bodies and karyotype for all patients. PCR amplification was also performed for the detection of SRY and ALT1 gene loci on Y and X chromosomes, respectively. Statistical analyses were performed using SPSS version 20 computer software. RESULTS: A pure 46XX karyotype was identified in 35.7% of cases, while a pure 46XY karyotype was identified in 50% of cases. The SRY locus identified by PCR was tested for its validity in predicting a pure 46XY DSD karyotype. The test was 100% sensitive and 85.7% specific. Among the group of male social sex, the mean internal masculinization score was significantly higher in those with a pure 46XY karyotype (5.9) compared with those with a pure 46XX and those with a mixed karyotype (3.5) . CONCLUSIONS: The management of children with DSD and a lack of diagnostic facilities still remain major challenges in Iraq. Multicenter studies, including larger population sizes, using molecular genetic analyses to detect the actual incidence of genital anomalies and DSD in our Mohammed Joudi Aboud et al JMSCR Volume 2 Issue 7 July 2014 Page 1626 JMSCR Volume||2||Issue||7||Page 1626-1646||July 2014 2014 community, are required to serve these patients and their families. Key words : DSD, polymerase chain reaction, karyotype, diagnostic outcome . BACKGROUND Disorders of sex development (DSDs) are serious This leads to Sertoli cell differentiation, with conditions with a worldwide incidence of 1:5000 subsequent production of Mullerian inhibiting live births. However, in Iraq, there are no accurate substance and regression of the Mullerian ducts data on DSDs. DSD is an important topic in [12,13]. Careful genetic analysis of cases with Islamic countries because of the strong abnormal sexual development, presenting with psychological pressure they impose, such as chromosomal translocations or stigmatization and social exclusion, leading to deletions/duplications, has resulted in the suicide in some cases. The generic term DSD was identification of many genes playing a role in sex proposed and defined as congenital conditions in determination [14]. Polymerase chain reaction which development of chromosomal, gonadal, or (PCR)-based sex determination identified by the anatomical sex is atypical [1-4]. This presence (male) or absence (female) of the SRY nomenclature, which is recommended instead of gene has already been described. This method the word “intersex”, referring primarily to external needs to have an internal control to verify the genital ambiguity, may be confusing in the absence of the SRY gene from the failure of PCR clinical evaluation of patients. amplification [15]. In this study, we used the DSD can serve as an "experiment of ATL1 locus as an internal control. The ATL1 is nature" to decipher the genetic and hormonal the sequence in the FMR1 (fragile X mental components of sex-specific gene expression in retardation 1) gene located on the long arm of the humans. Genetic and hormonal influences on sex X chromosome [16]. extend beyond the reproductive tract, modifying The main objective of this study was to body proportions, hair distribution, gender support clinical professionals in the initial identity, sex-specific behavior, and many other evaluation and diagnosis of children with features that also affect DSD patients clinically in suspected disorders of sex development. We also multiple ways [5-7]. Improved understanding of aimed to provide a framework to standardize the architecture of sex-specific gene expression laboratory and clinical practice, and to review our may also contribute to better clinical assessment experience to provide a simple PCR-based of DSD patients. genomic DNA diagnostic method [17]. We The presence or absence of the Y evaluated the clinical variant, different patterns of chromosome (SRY gene in particular) determines presentation, methods of diagnosis of DSD and to the sex in mammals. The sex-determining region acquire more knowledge on the molecular of the human Y chromosome (SRY) encodes a mechanism of sex determination in Al Qadisiya testes-determining factor, which initiates male sex governorate, Iraq. determination [8-11]. However, this does not exclude the possible effects of other sex genes on METHODS the Y chromosome. If these genes exist, they must be located adjacent to the pseudoautosomal This was a single center, prospective case review. boundary, close to SRY [8]. SRY is hypothesized A total of 42 patients with DSD were referred to our Pediatric Surgery Unit at The Maternity and to function as a transcription factor, triggering a st cascade of gene interactions that induce the Child Teaching Hospital from the 1 of January bipotential fetal gonads to develop into testes. 2008 to the end of May 2012, and all their record Mohammed Joudi Aboud et al JMSCR Volume 2 Issue 7 July 2014 Page 1627 JMSCR Volume||2||Issue||7||Page 1626-1646||July 2014 2014 were analyzed. Diagnosis was set according to min) for initial DNA denaturation, followed by the appearance of external genitalia, cytogenetic 35 cycles of 94°C (15 sec) for DNA denaturation, analysis, and hormonal status Ascertainment of 65°C (20 sec) for primer annealing, and 72°C (20 the gonadal sex and was made based on the sec) for primer extension. A final extension of the inability to determine the sex of the child on a cycle was performed at 72°C for 10 min for the physical examination. The severity of the SRY gene and the same PCR protocol was ambiguity (according to Sinnecker et al.’s performed with a change in annealing classification and Ahmed et al.’s external temperature to 60°C for the ALT1 gene. The masculinization score [EMS]) [18-19] , penile amplified PCR products were separated on 2.5% length (z score according to Gabrich et al.) and agarose gels and stained with ethidium bromide. the degree of severity of virilization of female They were visualized under ultra violet external genitalia were determined by applying transillumination for the presence of a band size the Prader classification [20-21] . A thorough of 254 bp for SRY (Y chromosome) and a band clinical examination, including associated size of 300 bp for ATL1 (X chromosome). The anomalies or dysmorphic features, was A100bp DNA ladder (Bioneer) was included in performed. Routine investigations were requested each run. The sequences of oligonucleotide for all the patients. Barr bodies in buccal smear primers were 5’-CAT GAA CGC ATT CAT cells, karyotype (cytogenic) using two techniques CGT GTG GTC-3’ and 5’-CTG CGG GAA GCA with lymphocyte culture, chromosomal analysis AAC TGC AAT TCT T-3’ for SRY, and 5’-CCC from venous blood sampling, and 20 metaphase TGA TGA AGA ACT TGT ATC TC-3’ and 5’- spreads were studied on the basis of the trypsin- GAA ATT ACA CAC ATA GGT GGC ACT- 3’ Giemsa banding (GTG) technique ( used in for ATL1 [22]. Patients were usually co-managed limited patients ) because such an approach is by pediatricians, clinical immunologists, only available in a special center in the capital pathologists, and radiologists. Written informed and it is expensive. Accordingly and for selected consent was obtained from all the parents for patients we performed an abdominopelvic publication of this article and accompanying ultrasound scan, an abdominopelvic MRI and CT images. The study was approved and authorized scan study, and a voiding cystourethrogram. We by the statistics and archives unit at Al Qadisiya measured serum electrolytes and performed Health Office, the Ministry of Health, Iraq. Data hormonal assays for all patients. Gonadal were translated into a computerized database. biopsies were carried out in selected cases . Expert statistical advice was sought. Statistical We performed PCR amplification for the analyses were performed using SPSS version 20 detection of SRY and ALT1 gene loci on the Y software. and X chromosomes, respectively, in all of the patients at the Medical Microbiology and Clinical RESULTS Immunology Department of Al Qadisiya University. DNA extraction purification was A total of 42 patients met the criteria for DSD. performed by a genomic DNA mini kit from There was a wide variation in age at presentation, whole blood (Geneaid, USA). Each 35-ul PCR ranging from 1 month to 19 years. There was no reaction comprised 5 ul of template DNA obvious or statistically significant difference in solution, 1.5 ul of 25 pmol each of SRY and the mean age between males and females (7 and ATL1 primers, 20 ul of PCR preMix (Bioneer, 6.8 years, respectively) (Table 1). A social label USA), and 12 ul of nuclease-free water. All PCR of male sex was observed in 52.4% of the study reactions were performed in a thermal cycler sample (Table 2).