Chromosomal and SRY gene findings by FISH in patients with disorders of sexual development Hallazgos cromosómicos y del gen SRY por FISH en pacientes con trastornos del desarrollo sexual

Objective: Disorders of sexual development are a group of congenital diseases that affect the normal formation of genital structures. Within the pathophysiologic mechanisms described, there are genetic factors caused by chromosomal or sex-determining gene alterations. Therefore, chromosomal analysis is an essential priority in the diagnostic approach. Alterations in the chromosomes and the SRY gene as a cause of disorder of sexual development was analyzed herein. Material and methods: G or R-banding karyotype and FISH analyses for the SRY gene were performed in lymphocytes, gonadal tissue, and scrotal tissue in twelve cases, three cases, and one case, respectively. The clinical information was obtained from the patients’ medical reports. Results: In 9 (73%) cases, the assigned sex was male, and in 3 (27%) cases, it was female. Karyotype 46,XY was found in 8 (66%) cases, 46,XX in 2 (17%) cases, and mosaic karyotype in 2 (17%) cases with idic(Y). A single case of gonadal tissue showed mosaicism due to the presence of a tetraploid cell line. The most common clinical diagnosis was abnormal genital differentiation in 8 (67%) cases, followed by hypospadias in 5 (41.7%) cases. Conclusions: The results show the importance of applying different cytogenetic tests in making the diagnosis, and the need for a multidisciplinary team to address the disorder.


Introduction
Disorders of sexual development involve congenital alterations related to the formation of sexual characteristics. (1) Those abnormalities are caused by a mismatch between chromosomal, gonadal, and phenotypic sex, thus requiring multidisciplinary management for proper diagnosis, treatment, and follow-up. (1)(2)(3) Disorders of sexual development include a broad spectrum of phenotypes, and their prevalence is not clearly determined. DSD is estimated to account for approximately 7.5% of all birth defects, worldwide. (4) A prevalence of DSD of 0.76 per 4,500 births has been reported in Colombia. (5) The correct determination of dysmorphic sex and the achievement of that differentiation can be interrupted by genetic and/or non-genetic factors, capable of altering any of the molecular signals that define the specific sexual development of the sex organs or endocrine function. (3) Most of those phenotypic alterations are very complex to diagnose and difficult to treat, so they must be approached by a multidisciplinary team, with the appropriate experience and knowledge for managing those conditions. (6)(7)(8)   as well as in directing patient management. (2) Due to the difficulties in the complete cyto- banding was obtained through staining, according to a standardized laboratory protocol. (11) Karyotype analysis was performed in gonadal or fibroblast tissue in three cases, in which a biopsy was performed for pathologic studies, and a small section of that biopsy tissue was used for cell culture and karyotype analysis.
The tissue was cut into small fragments that were placed in a culture flask with RPMI and 20% SFB for fibroblast-like cell growth, monolayer confluence, and finally in-situ cell growth on a plate, to obtain metaphase chromosomes.
In each case, 50 metaphases from lymphocytes with 550 to 700 band resolution were evaluated, whereas in solid tissues, the resolution level was 300 to 450 bands; in cases with mosaicism and sufficient material for evaluation, reading was extended to 100 metaphases.  Table 1 shows the data on age, assigned sex, and clinical characteristics of the group of patients analyzed. The average age was 7 years, ranging from 2 months to 44 years. In 9 (73%) cases, the sex assigned at birth was male, and in 3 (27%) cases it was female. The most frequent clinical finding was abnormal genital differentiation in 8 cases (67%). Other relevant findings in our series were 9 (75%) cases of micropenis and 5 (41.6%) cases of hypospadias, as shown in Table 1.     In both cases, the FISH test detected a signal from the SRY gene at each end (SRY++) of the Y chromosomes, and absence of the heterochromatin region in Yq12 (DYZ1-) ( Figure 2A). The probe for the centromeric region showed two centromeres on the Y chromosome, revealing isodicentric chromosomes (DYZ3++) in both cases ( Figure 2B and 2C). The sex assigned at birth in those patients was male (case 3) and female (case 10) ( Table 1). Gonad karyotype and FISH analyses of cases 1 and 12 coincided with the chromosomal constitution of the blood karyotype.

Results
However, in case 1, a second tetraploid cell line was observed in both gonads (Table 1). In case 3, the karyotype analyses performed on the cells taken from the scrotal tissue biopsy confirmed the presence of mosaicism, inversely proportional to that found in blood (Table 1).  have been related to ovotesticular DSD in the absence of SRY, (10) and should be investigated in such cases. (9) In DSD due to chromosomal abnormalities, it is rare to find 45X/46,XY and 45,X/47,XYY mosaicism, and it presents an approximate incidence of 1.7/10,000 pregnancies. (18) It has been estimated that only 5%-10% of individuals with those anomalies have abnormal genital differentiation at birth, and the rest have a male phenotype. (19) Cases with mosaicism can vary from male phenotypes with cryptorchidism or hypospadias to female phenotypes with gonadal dysgenesis. The typical combination in those patients is asymmetry in testis development on one side, and gonadal striae with persistence of Müllerian derivatives on the opposite side. (18)  of Y-derived sequences should be monitored. (20,21) On the other hand, it has been suggested that duplication of the SRY gene in cases with 45,X/46,X,idic(Yp) mosaicism does not determine male or female sex differentiation, but that it possibly depends on the proportion of each cell line in gonadal tissue. (22) In our two cases, each one with a different assigned sex, the cell line distribution in the gonadal tissue was impossible to confirm; in the first case, gonadal tissue was not available for analysis, and the second case was lost to follow-up.

Discussion
Clinically, DSD are very complex and difficult to diagnose and treat. (6)(7)(8) In addition to that complexity, the patient's family environment is also strongly affected and has an effect on adequate management. Therefore, for compre-

Conclusion
Cytogenetic results allowed us to establish the cause of the phenotype in two cases, and they suggested that other genes or regulatory mechanisms were involved in two other cases.
The results show the importance of applying different cytogenetic tests in the diagnosis of