Detection of Molecular Variations at Androgen Receptor Gene in 46,XY Differences in Sex Development Cases.

Introduction: One of the common causes of 46,XY differences in sex development (DSD) cases is androgen insensitivity syndrome. This X-linked recessive inherited condition is associated with pathological variations of the AR gene, leading to defects in androgen action. Affected 46,XY infants or individuals experience variable degrees of undervirilization and those with severe form will have female-like external genitalia. Therefore, they were more likely assigned and reared as females. The confirmatory molecular test is often needed due to similar clinical manifestations with other conditions causing 46,XY DSD. Since in our country, the molecular test for the AR gene is lacking, the study is conducted as a preliminary study to elaborate on the possibility of developing a molecular test for the AR gene in 46,XY DSD cases. Methods: Archived DNAs of 13 46,XY DSD cases were analyzed using polymerase chain reaction and direct sequencing for molecular defects in the AR gene. Clinical and hormonal data were collected and analyzed. Results: The study successfully amplified and visualized the eight exons of the AR gene and revealed two subjects carrying AR gene variants at exon 7. In the first case, 1.2-year-old boy carried heterozygous p.Gln825Arg, which has never been reported elsewhere, and the second subject, a 2.1-year-old girl with heterozygous p.Arg841His. Both subjects presented with severe undervirilization of external genitalia with external genitalia masculinization scores (EMS) of 1.5 and 3. Conclusion: In this series, two of 13 46,XY DSD cases carried variants at the AR gene, resulting in complete androgen insensitivity syndrome.

Significant increase in anti-SARS-CoV-2 antibodies after administration of heterologous mRNA-based vaccine booster in individuals receiving two doses of inactivated COVID-19 vaccine: A single-center study in healthcare workers in Jakarta, Indonesia.

BACKGROUND: Vaccine plays an important role in breaking SARS-CoV-2 transmission and accelerating the path to pandemic recovery. Currently, there is still limited data on heterologous COVID-19 booster vaccination efficacy and effectiveness in Indonesia. METHODS: Antibody response was retrospectively analyzed from 156 serum collected from healthcare workers that have received mRNA-1273 vaccine as the booster against SARS-CoV-2. These individuals had previously received the full two doses of inactivated anti-SARS-CoV-2 vaccine. Serological analysis was performed to measure total antibody, as well as IgA and IgG antibodies specific to spike (S) protein using ECLIA and ELISA methods. RESULTS: A significant increase in total, IgA, and IgG antibody titers was reported in vaccine receiving a third heterologous booster dose of mRNA-based COVID-19 vaccine following two doses of inactivated type. CONCLUSION: The third heterologous booster dose of vaccine may be beneficial to individuals with or without previous history of SARS-CoV-2 infection.

Accuracy of Urinary Etiocholanolone/Androsterone Ratio as Alternative to Serum Testosterone/Dihydrotestosterone Ratio for Diagnosis of 5 Alpha-reductase Type 2 Deficiency Patients and Carriers in Indonesia.

BACKGROUND: The 5 Alpha-reductase type 2 deficiency (5ARD2) is an inherited condition, which clinically presents as variable degrees of under virilization in affected 46,XY individuals. In the diagnostic pathway of 5ARD2, the testosterone/dihydrotestosterone (T/DHT) ratio is broadly employed before molecular analysis of the SRD5A2 gene. However, due to cost-benefit considerations, the DHT test in our country is routinely lacking in clinical settings; therefore, we considered applying the urinary etiocholanolone/androsterone (Et/An) ratio as an alternative test. OBJECTIVES: We aimed to determine the diagnostic value of the urinary Et/An ratio versus the T/DHT ratio in diagnosing 5ARD2 patients and carriers. METHODS: Sixty-six suspected 5ARD2 46,XY disorders of sex development (DSD) individuals and 95 family members were recruited in the study. Their clinical manifestations, T/DHT and urinary Et/An ratios, and SRD5A2 genes were analyzed. Using molecular analysis of the SRD5A2 gene as the gold standard, we compared the accuracy of both ratios in diagnosing 5ARD2 patients and carriers with receiver operating characteristic (ROC) curve analysis. RESULTS: Thirty-seven patients were confirmed molecularly to have 5ARD2, and the rest (n = 29) were assessed as normal controls, while in the carrier group, 53 were molecularly confirmed as carriers and 42 as controls. The AUCs (areas under the curve) of the T/DHT and urinary Et/An ratios were 57.7% (95% CI 43.0 - 72.4%, P > 0.05) and 79.7% (95% CI 69.0 - 90.4%, P < 0.001), respectively, in diagnosing 5ARD2 patients and 54.1% (95% CI 42.4 - 65.8%, P > 0.05) and 75.1% (95% CI 65.1 - 85.1%, P < 0.001), respectively, in diagnosing carriers. The cutoff value of the urinary Et/An ratio was set at ≥ 0.95 for detecting 5ARD2 patients and ≥ 0.99 for detecting carriers. CONCLUSIONS: The testosterone/DHT ratio was inaccurate in diagnosing 5ARD2 patients. When molecular analysis for the SRD5A2 gene is lacking, the urinary Et/An ratio may be a useful test to diagnose 5ARD2 patients and carriers.

Characterising SRD5A2 Gene Variants in 37 Indonesian Patients with 5-Alpha-Reductase Type 2 Deficiency.

The 5-alpha-reductase type 2 deficiency (5ARD2) is an autosomal recessive condition associated with impairment in the conversion of testosterone to dihydrotestosterone. This condition leads to undervirilisation in 46,XY individuals. To date, there have been more than 100 variations identified in the gene responsible for 5ARD2 development (steroid 5-alpha-reductase 2, SRD5A2). However, few studies have examined the molecular characterisation of Indonesian 5ARD2 cases. In the current study, we analysed 37 subjects diagnosed with 46,XY DSD (disorders of sex development) with confirmed variations in the SRD5A2 gene. We examined results from testosterone/dihydrotestosterone (T/DHT) and urinary etiocholanolone/androsterone (Et/An) ratios, as well as from molecular and clinical analyses. Twelve variants in the SRD5A2 gene were identified, and 6 of which were novel, namely, c.34-38delGinsCCAGC, p.Arg50His, p.Tyr136 ∗ , p.Gly191Arg, p.Phe194Ile, and p.Ile253Val variants. Moreover, we determined that 20 individuals contained harmful mutations, while the remaining 17 variants were benign. Those containing harmful mutations exhibited more severe phenotypes with median external genitalia masculinisation scores (EMS) of 3 (1.5-9) and were more likely to be diagnosed at a later age, reared as female, and virilised at pubertal age. In addition, the respective sensitivities for detecting severe 5ARD2 cases using T/DHT (cutoff: 10) and urinary Et/An ratios (cutoff: 0.95) were 85% and 90%, whereas mild cases were only identified with 64.7% and 47.1% sensitivity, respectively. Although we were unable to identify clear correlations between genotypic and phenotypic characteristics in this study, we clearly showed that individuals who were homozygous or compound heterozygous for any of the harmful mutations were more likely to exhibit classic 5ARD2 phenotypes, lower EMS, female assignment at birth, and virilisation during puberty. These results serve to inform the development of improved clinical and molecular 5ARD2 diagnostic approaches, specifically in Indonesian patients.

Loss of DMRT1 gene in a Mos 45,XY,-9[8]/46,XY,r(9)[29]/47,XY,+idic r(9)× 2[1]/46,XY,idic r(9)[1]/46,XY[1] female presenting with short stature.

BACKGROUND: A 46,XY sex reversal syndrome is characterized by discordant genetic and phenotypic sex, leading to normal external female genitalia, undeveloped gonads and presence of Müllerian structures in an otherwise 46,XY individual. Chromosome 9pter aberrations, such as ring chromosome have been reported to cause 46,XY disorders of sex development (DSD), due to involvement of DMRT1 gene located at the 9p24.3 region. CASE PRESENTATION: This study presents a unique case of a 12-year-old female with mos 46,XY, (r)9[31]/45,XY,-9[9] karyotype, presenting with intellectual disability and short stature, mimicking Turner syndrome. Re-karyotyping was performed using standard GTL-banding technique. Further cytogenetic study using standard metaphase fluorescent in situ hybridization (FISH) technique was applied to cultured lymphocytes from peripheral blood, hybridized using green control probe specific to 9q21 loci, and red DMRT1 probe specific to 9p24.3 loci. Cytogenetics and FISH analysis revealed mos 45,XY,-9[8]/46,XY,r(9)[29]/47,XY,+idic r(9)× 2[1]/46,XY,idic r(9)[1]/46,XY[1] and haploinsufficiency of DMRT1 gene in most cells. CGH array revealed a deletion around 1.25 Mb at 9p24.3 loci [arr 9p24.3(204,193-1,457,665)× 1] and three duplications around 13 Mb [9p24.3p22.3(1,477,660-14,506,754)× 3] near the breakage point that formed the ring chromosome 9. CONCLUSIONS: The clinical presentation of the subject that mimics Turner syndrome highlights the importance of cytogenetic analysis to detect the possibility of ring chromosome 9. Sex reversal due to haploinsufficiency of DMRT1 gene in ring chromosome 9 structures is exceedingly rare with only a handful of cases ever reported. This finding further highlights the importance of DMRT1 gene in sex determination and differentiation in males. More research is required to pinpoint the exact mechanism that underlies sex reversal caused by DMRT1 haploinsufficiency.

Exceptional complex chromosomal rearrangements in three generations.

We report an exceptional complex chromosomal rearrangement (CCR) found in three individuals in a family that involves 4 chromosomes with 5 breakpoints. The CCR was ascertained in a phenotypically abnormal newborn with additional chromosomal material on the short arm of chromosome 4. Maternal karyotyping indicated that the mother carried an apparently balanced CCR involving chromosomes 4, 6, 11, and 18. Maternal transmission of the derivative chromosome 4 resulted in partial trisomy for chromosomes 6q and 18q and a partial monosomy of chromosome 4p in the proband. Further family studies found that the maternal grandmother carried the same apparently balanced CCR as the proband's mother, which was confirmed using the whole chromosome painting (WCP) FISH. High resolution whole genome microarray analysis of DNA from the proband's mother found no evidence for copy number imbalance in the vicinity of the CCR translocation breakpoints, or elsewhere in the genome, providing evidence that the mother's and grandmother's CCRs were balanced at a molecular level. This structural rearrangement can be categorized as an exceptional CCR due to its complexity and is a rare example of an exceptional CCR being transmitted in balanced and/or unbalanced form across three generations.

Chromosome abnormalities in Indonesian patients with short stature.

BACKGROUND: Short stature is associated with several disorders including wide variations of chromosomal disorders and single gene disorders. The objective of this report is to present the cytogenetic findings in Indonesian patients with short stature. METHODS: G-banding and interphase/metaphase FISH were performed on short stature patients with and without other clinical features who were referred by clinicians all over Indonesia to our laboratory during the year 2003-2009. RESULTS: The results of chromosomal analysis of ninety seven patients (mean age: 10.7 years old) were collected. The group of patients with other clinical features showed sex chromosome abnormalities in 45% (18/40) and autosomal abnormalities in 10% (4/40), whereas those with short stature only, 42.1% (24/57) had sex chromosome abnormalities and 1.75% (1/57) had autosomal abnormalities. The autosomal chromosomal abnormalities involved mostly subtelomeric regions. Results discrepancies between karyotype and FISH were found in 10 patients, including detection of low-level monosomy X mosaicism in 6 patients with normal karyotype, and detection of mosaic aneuploidy chromosome 18 in 1 patient with 45,XX,rob(13;14)(q10;q10).Statistical analysis showed no significant association between the groups and the type of chromosomal abnormalities. CONCLUSION: Chromosome abnormalities account for about 50% of the short stature patients. Wide variations of both sex and autosomal chromosomes abnormalities were detected in the study. Since three out of five patients had autosomal structural abnormalities involving the subtelomeric regions, thus in the future, subtelomeric FISH or even a more sensitive method such as genomic/SNP microarray is needed to confirm deletions of subtelomeric regions of chromosome 9, 11 and 18. Low-level mosaicism in normal karyotype patients indicates interphase FISH need to be routinely carried out in short stature patients as an adjunct to karyotyping.

Diversity of sex chromosome abnormalities in a cohort of 95 Indonesian patients with monosomy X.

BACKGROUND: Monosomy × or 45,X is a cytogenetic characteristic for Turner syndrome. This chromosome anomaly is encountered in around 50% of cases, but wide variations of other anomalies have been found. This report is to describe the cytogenetic characteristics of 45,X individuals. To the best of our knowledge, there were no large series of 45,X cases has been reported from Indonesia. RESULTS: Ninety five cases with 45,X cell line found, of which 60 were detected by karyotyping, 4 by FISH for sex chromosomes, and 31 by both karyotyping and FISH. Using karyotyping 37 out of 91 cases(40.6%) were identified as 45,X individuals, while cases who underwent FISH only 4 out of 35 cases (11.4%) showed 45,X result, resulting in total of 39 45,X cases (41.1%), and the rest 56 (58.9%) cases are mosaic. Among these cases, 21 out of 95 (22.1%) have Y or part of Y as the second or third sex chromosome in their additional cell lines. Result discrepancies revealed in 22 out of 31 cases who underwent both FISH and karyotyping, of which 7 showed normal 46,XX or 46,XY karyotypes, but by FISH, additional monosomy × cell line was found. Most of the cases were referred at the age of puberty (8-13 years old) or after that (14-18 years old), 31 and 21 cases respectively, and there were 14 cases were sent in adulthood. CONCLUSION: Wide variations of sex chromosome aberrations have been detected using the combination of conventional cytogenetic and FISH, including detection of low level of mosaicism and Y-chromosome fragments. Result discrepancies using both techniques were found in 22/31 cases, and in order to obtain a more details of sex chromosome constitution of individuals with 45,X cell line both FISH and karyotyping should be carried out simultaneously.