Variants in congenital hypogonadotrophic hypogonadism genes identified in an Indonesian cohort of 46,XY under-virilised boys.

BACKGROUND: Congenital hypogonadotrophic hypogonadism (CHH) and Kallmann syndrome (KS) are caused by disruption to the hypothalamic-pituitary-gonadal (H-P-G) axis. In particular, reduced production, secretion or action of gonadotrophin-releasing hormone (GnRH) is often responsible. Various genes, many of which play a role in the development and function of the GnRH neurons, have been implicated in these disorders. Clinically, CHH and KS are heterogeneous; however, in 46,XY patients, they can be characterised by under-virilisation phenotypes such as cryptorchidism and micropenis or delayed puberty. In rare cases, hypospadias may also be present. RESULTS: Here, we describe genetic mutational analysis of CHH genes in Indonesian 46,XY disorder of sex development patients with under-virilisation. We present 11 male patients with varying degrees of under-virilisation who have rare variants in known CHH genes. Interestingly, many of these patients had hypospadias. CONCLUSIONS: We postulate that variants in CHH genes, in particular PROKR2, PROK2, WDR11 and FGFR1 with CHD7, may contribute to under-virilisation phenotypes including hypospadias in Indonesia.

Comprehensive genotyping reveals RPE65 as the most frequently mutated gene in Leber congenital amaurosis in Denmark.

Leber congenital amaurosis (LCA) represents the most severe form of inherited retinal dystrophies with an onset during the first year of life. Currently, 21 genes are known to be associated with LCA and recurrent mutations have been observed in AIPL1, CEP290, CRB1 and GUCY2D. In addition, sequence analysis of LRAT and RPE65 may be important in view of treatments that are emerging for patients carrying variants in these genes. Screening of the aforementioned variants and genes was performed in 64 Danish LCA probands. Upon the identification of heterozygous variants, Sanger sequencing was performed of the relevant genes to identify the second allele. In combination with prior arrayed primer extension analysis, this led to the identification of two variants in 42 of 86 cases (49%). Remarkably, biallelic RPE65 variants were identified in 16% of the cases, and one novel variant, p.(D110G), was found in seven RPE65 alleles. We also collected all previously published RPE65 variants, identified in 914 alleles of 539 patients with LCA or early-onset retinitis pigmentosa, and deposited them in the RPE65 Leiden Open Variation Database (LOVD). The in silico pathogenicity assessment of the missense and noncanonical splice site variants, as well as an analysis of their frequency in ~60 000 control individuals, rendered 864 of the alleles to affect function or probably affect function. This comprehensive database can now be used to select patients eligible for gene augmentation or retinoid supplementation therapies.

Combination of Aspartate Aminotranferase and Tumor Necrosis Factor-α as Non Invasive Diagnostic Tools for Non Alcoholic Steatohepatitis (NASH).

AIM: to develop a non-invasive diagnostic test for non-alcoholic steatohepatitis NASH. METHODS: this is a cross-sectional study on non-alcoholic fatty liver disease (NAFLD) subjects. Sample was taken by consecutive sampling method. Diagnostic criteria of NAFLD were confirmed by liver biopsy. Clinical variables include metabolic syndrome, aspartate aminotransferase (AST), alanine aminotransferase (ALT), adiponectine, TNF-, insulin, homeostatic model assessment insulin resistance (HOMA-IR) index and liver biopsy. Patients were divided into two groups based on their liver biopsy, group 1: Non-NASH (NAFLD activity score <3) and group 2: NASH (NAFLD activity score of >4). Statistical analyses were performed using Student's t-test, Mann Whitney U, chi-square, the ROC curve, sensitivity and specificity test. RESULTS: fifty NAFLD patients were recruited, 30 males and 20 females. Among these patients, 12 (24%) had type 2 diabetes, 36 (72%) had metabolic syndrome, the remaining 2 (4%) did not fulfilled metabolic syndrome. Liver biopsy confirmed 21 (42%) non- NASH and 29 (58%) NASH respectively. Level of AST and ALT, plasma level of adiponectine and TNF- were statistically different between two groups. The AST level (>25 U/L) in combination with TNF- (>3.28 pg/cc) demonstrated a good diagnostic accuracy for NASH (Accuracy 82%, Sensitivity 76%, Specificity 90%, PPV 92%, and NPV 73%). CONCLUSION: the combined diagnostic tests of AST and TNF- plasma levels demonstrated a good accuracy for the detection of NASH among NAFLD patients. This combination test can be used as a noninvasive method to diagnose NASH.

Body image and sexuality in Indonesian adults with a disorder of sex development (DSD).

In Indonesia, disorders of sex development (DSDs) are not well recognized and medical care for affected individuals is scarce. Consequently, many patients live with ambiguous genitalia and appearance. We compared reported outcomes on body image, sexual functioning, and sexual orientation of 39 adults with DSDs (aged 18 to 41) and 39 healthy controls matched for gender, age, and residential setting (urban, suburban, rural). Differences in gender and treatment status (treated or untreated) were also explored. On body image, adults with DSDs reported dissatisfaction with sex-related body parts. Compared to the matched controls, women with DSDs reported greater sexual distress, and men with DSDs reported lower erectile and ejaculation frequencies, and more dissatisfaction with sexual life but not with sexual desire and activities. Men with DSDs who had undergone genital surgery reported higher erectile and ejaculation frequencies than untreated men. More women than men in the DSDs group reported a nonexclusive heterosexual orientation. DSDs and infertility had a great impact on sexuality. Fear of ostracism complicated DSD acceptance. Findings were compared to those of Western studies. Based on these results, education about DSDs and their psychosexual consequences may help reduce the sexual distress and problems in adults with DSDs and improve quality of life.

Novel compound heterozygous NMNAT1 variants associated with Leber congenital amaurosis.

PURPOSE: The gene encoding nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) was recently found to be mutated in a subset of patients with Leber congenital amaurosis (LCA) with macular atrophy. The aim of this study was to determine the occurrence and frequency of NMNAT1 mutations and associated phenotypes in different types of inherited retinal dystrophies. METHODS: DNA samples of 161 patients with LCA without genetic diagnosis were analyzed for variants in NMNAT1 using Sanger sequencing. Variants in exon 5 of NMNAT1, which harbors the majority of the previously identified mutations, were screened in 532 additional patients with retinal dystrophies. This cohort encompassed 108 persons with isolated or autosomal recessive cone-rod dystrophy (CRD), 271 with isolated or autosomal recessive retinitis pigmentosa (RP), and 49 with autosomal dominant RP, as well as 104 persons with LCA in whom the causative mutation was previously identified. RESULTS: Compound heterozygous alterations were found in six patients with LCA and in one person with early-onset RP. All except one carried the common p.E257K variant on one allele. Macular atrophy was absent in one patient, who carried this variant in combination with a truncating mutation on the other allele. The p.E257K alteration was also found in a heterozygous state in five individuals with LCA and one with RP while no mutation was detected on the other allele. Two individuals with LCA carried other NMNAT1 variants in a heterozygous state, whereas no NMNAT1 variants in exon 5 were identified in individuals with CRD. The p.E257K variant was found to be enriched in a heterozygous state in individuals with LCA (0.94%) compared to Caucasian controls (0.18%), although the difference was statistically insignificant (p=0.12). CONCLUSIONS: Although macular atrophy can occur in LCA and CRD, no NMNAT1 mutations were found in the latter cohort. NMNAT1 variants were also not found in a large group of patients with sporadic or autosomal recessive RP. The enrichment of p.E257K in a heterozygous state in patients with LCA versus controls suggests that this allele could act as a modifier in other genetic subtypes of LCA.

Virilization due to androgen hypersecretion in a patient with ovarian leydig cell tumor: diagnostic and psychosocial implications.

Virilization due to hyperandrogenism in women causes male signs and symptoms such as swelling of the clitoris, deepening of the voice, facial hair and increase in body hair. Virilization is caused by less than 0.5% of all ovarian tumors. Here we report a case of virilizing Leydig cell tumor of the left ovary in a 36 year old woman. Misinterpretation of symptoms, conflicting medical information and advice from previous doctors had confused the patient. We performed a diagnostic evaluation including clinical, hormonal parameters, imaging, anatomical pathology examinations, and psychological assessment. Blood analysis showed a high testosterone level. The presence of an ovarian tumor was confirmed by laparoscopy. Since the patient refused ovariectomy, a biopsy of the left ovary was performed. Pathology showed a Leydig cell tumor without histological signs of malignancy. In spite of extensive explanation and psychological counseling, cultural barriers prevented appropriate treatment. An ovarian Leydig cell tumor should always be considered for a woman in the reproductive age with symptoms of virilization. The diagnosis is suspected on the basis of an ovarian mass on examination and further investigation and should be proven by biopsy.

p.Ser252Trp and p.Pro253Arg mutations in FGFR2 gene causing Apert syndrome: the first clinical and molecular report of Indonesian patients.

Apert syndrome (AS) is a rare autosomal dominant disorder characterised by craniosynostosis and limb malformations, and is associated with congenital heart disease and other systemic malformations, including intellectual disability. We report two Indonesian patients with AS, in whom molecular analysis detected p.Ser252Trp (c.755C>G) and p.Pro253Arg (c.758C>G) mutations in the fibroblast growth factor receptor 2 (FGFR2) gene, respectively. Although the syndrome has been frequently described, this is the first clinical report of AS confirmed by molecular analysis in Indonesia. The difference in severity of clinical features in the two patients may be consistent with a genotype-phenotype correlation of the FGFR2mutation. The management of individuals with AS is best achieved within a multidisciplinary setting. However, in most developing countries, early intervention may be delayed due to late diagnosis, a lack of facilities and financial constraints. This report underpins the benefits of early diagnosis for AS management.

Gonadal malignancy in 13 consecutive collected patients with disorders of sex development (DSD) from Semarang (Indonesia).

AIMS: Caucasian patients with disorders of sex development (DSD) are at a high risk of developing germ cell cancer (GCC). GCC is prominent in young adults in Western countries, while the incidence is significantly lower in Asia. So far, the risk of GCC in Asian DSD patients is unknown. METHODS AND RESULTS: A detailed study of gonad histology , morphology and immunohistochemistry (OCT3/4, testis-specific protein Y-encoded, VASA, SCF/KITLG, SOX9, FOXL2) of 16 Indonesian DSD patients was undertaken. 13 cases could be analysed, including ovarian tissue (n=3), streak gonad (n=1), undifferentiated gonad (n=1) and testicular tissue (n=8), diagnosed as 46, XX (n=1), 46, XY (n=7) and sex chromosome DSD (n=5). The precursor lesion gonadoblastoma or carcinoma in situ, or GCC was diagnosed in four cases (30.8%; three 46, XY and one sex chromosome DSD ). A hormone producing ovarian Leydig cell tumour was identified in a 46, XX patient, supposed to be a late onset congenital adrenal hyperplasia. CONCLUSIONS: In spite of the significantly lower risk of GCC in the general Asian population, DSD is a dominant risk factor. The study demonstrates the power of immunohistochemical markers for (early) diagnosis. This knowledge will deepen understanding of the pathobiology of GCC and clinical handling of patients with DSD, globally.

Immune-mediated disorders among women carriers of fragile X premutation alleles.

The relative risk of immune-mediated disorders (IMDs) among women carriers of premutation alleles is estimated by a survey for IMDs among 344 carrier women (age 19-81 years; mean 46.35 and SD 12.60) and 72 controls (age 18-87 years; mean 52.40 and SD 15.40). One hundred fifty four (44.77%) women carrier had at least one IMD, as did 20 controls (27.78%). Among women carriers, autoimmune thyroid disorder was the most common (24.4%), then fibromyalgia (10.2%), irritable bowel syndrome (IBS; 9.9%), Raynaud's phenomenon (7.6%), rheumatoid arthritis (RA; 3.8%), Sjögren syndrome (2.6%), systemic lupus erythematosus (SLE; 2.03%), multiple sclerosis (1.74%). Of 55 carriers age 40 or older with FXTAS, 72.73% had at least one IMD, compared to 46.54% of those without FXTAS (n = 159), and 31.58% of controls (n = 57). The estimated odds ratio (OR) for IMD is 2.6 (95% CI 1.2-5.6, P = 0.015) for women with FXTAS relative to those without FXTAS; the likelihood of IMD in carriers without or with FXTAS was also significantly higher than for controls (OR 2.1, 95% CI 1.1-4.2, P = 0.034; OR 5.5, 95% CI 2.4-12.5, P < 0.001, respectively). Similarly, the odds of having an IMD among carriers with FXPOI is about 2.4 times higher when compared to carriers without FXPOI (95% CI 1.1-5.0; P = 0.021). The likelihood of IMD in carriers with or without FXPOI is greater (OR 2.4, 95% CI 1.1-5.0; P = 0.021) compared to that of controls.

Severe dengue is associated with consumption of von Willebrand factor and its cleaving enzyme ADAMTS-13.

BACKGROUND: Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition. METHODS AND PRINCIPAL FINDINGS: In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease. CONCLUSION: These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.

Application of the new classification on patients with a disorder of sex development in indonesia.

Disorder of sex development (DSD) patients in Indonesia most often do not receive a proper diagnostic evaluation and treatment. This study intended to categorize 88 Indonesian patients in accordance with the new consensus DSD algorithm. Diagnostic evaluation including clinical, hormonal, genetic, imaging, surgical, and histological parameters was performed. Fifty-three patients were raised as males, and 34 as females. Of 22 patients with 46, XX DSD, 15 had congenital adrenal hyperplasia, while in one patient, an ovarian Leydig cell tumor was found. In all 58 46, XY DSD patients, 29 were suspected of a disorder of androgen action (12 with an androgen receptor mutation), and in 9, gonadal dysgenesis was found and, in 20, severe hypospadias e.c.i. Implementation of the current consensus statement in a resource-poor environment is very difficult. The aim of the diagnostic workup in developing countries should be to end up with an evidence-based diagnosis. This is essential to improve treatment and thereby to improve the patients' quality of life.

Molecular genetic analysis of retinitis pigmentosa in Indonesia using genome-wide homozygosity mapping.

PURPOSE: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous retinal disorder. Despite tremendous knowledge about the genes involved in RP, little is known about the genetic causes of RP in Indonesia. Here, we aim to identify the molecular genetic causes underlying RP in a small cohort of Indonesian patients, using genome-wide homozygosity mapping. METHODS: DNA samples from affected and healthy individuals from 14 Indonesian families segregating autosomal recessive, X-linked, or isolated RP were collected. Homozygosity mapping was conducted using Illumina 6k or Affymetrix 5.0 single nucleotide polymorphism (SNP) arrays. Known autosomal recessive RP (arRP) genes residing in homozygous regions and X-linked RP genes were sequenced for mutations. RESULTS: In ten out of the 14 families, homozygous regions were identified that contained genes known to be involved in the pathogenesis of RP. Sequence analysis of these genes revealed seven novel homozygous mutations in ATP-binding cassette, sub-family A, member 4 (ABCA4), crumbs homolog 1 (CRB1), eyes shut homolog (Drosophila) (EYS), c-mer proto-oncogene tyrosine kinase (MERTK), nuclear receptor subfamily 2, group E, member 3 (NR2E3) and phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A), all segregating in the respective families. No mutations were identified in the X-linked genes retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (X-linked recessive; RP2). CONCLUSIONS: Homozygosity mapping is a powerful tool to identify the genetic defects underlying RP in the Indonesian population. Compared to studies involving patients from other populations, the same genes appear to be implicated in the etiology of recessive RP in Indonesia, although all mutations that were discovered are novel and as such may be unique for this population.

Gonadoblastoma arising in undifferentiated gonadal tissue within dysgenetic gonads.

PURPOSE: The purpose of the study was to define the histological origin of gonadoblastomas, allowing the identification of high-risk patients. EXPERIMENTAL DESIGN: Sixty paraffin-embedded gonadectomy or gonadal biopsy samples of 43 patients with gonadal dysgenesis were selected from our archives. We studied the morphology and immunohistochemical properties of the germ cells in 40 samples without neoplastic transformation and compared these findings with the morphological and immunohistochemical characteristics of 20 samples containing gonadoblastoma/dysgerminoma. RESULTS: The overall incidence of germ cell tumors in our patient series was 35%. In dysgenetic gonads without germ cell neoplasia, besides the presence of areas with testicular and/or ovarian differentiation, areas of undifferentiated gonadal tissue were identified in 13 of 40 samples (32.5%). A subpopulation of germ cells within these undifferentiated areas stained positive for octamer binding transcription factor (OCT)3/4, the stem cell factor receptor, placental-like alkaline phosphatase, and testis-specific protein-Y encoded. Gonadoblastoma germ cells display identical staining results. Moreover, in gonads containing gonadoblastoma, adjacent to this lesion, areas of undifferentiated gonadal tissue with identical immunohistochemical characteristics were identified in 10 of 20 samples (50%). No adjacent tissue was available in five cases, whereas in the five remaining cases, it consisted of streak tissue. In three cases, an accumulation of OCT3/4-positive germ cells in the proximity of the malignant lesions was found, suggesting clonal expansion and final organization into gonadoblastoma nests. CONCLUSIONS: Based on these observations, we hypothesize that gonadoblastomas originate from surviving OCT3/4-positive germ cells in areas of undifferentiated gonadal tissue within the dysgenetic gonad. Supportive evidence was obtained that carcinoma in situ arises in regions with testicular differentiation.