Osteoporosis in Klinefelter's syndrome.

Hypogonadism represents one of the most important causes of male osteoporosis. Testosterone regulates male bone metabolism both indirectly by aromatization to estrogens and directly through the androgen receptor (AR) on osteoblasts, promoting periosteal bone formation during puberty and reducing bone resorption during adult life. Early onset of testosterone deficiency, as observed in Klinefelter's syndrome (KS), is an important risk factor for precocious osteoporosis. Osteoporosis is present in up to 40% of subjects with KS and has usually been attributed to low testosterone levels. However, reduced bone mass might be present also in KS men with normal testosterone levels and testosterone replacement therapy does not always restore bone density in KS patients. Possible new determinants for osteoporosis in KS might be related to the AR function and insulin-like factor 3 (INSL3) levels. The CAG length and inactivation pattern of the AR in KS have been related to osteoporosis, but definitive proof is lacking. INSL3 has an anabolic role on bone metabolism by acting on osteoblasts and INSL3 levels are low in KS. Therefore, low INSL3 concentrations might represent a possible new pathogenic mechanism for reduced bone mass in KS.

Detailed functional studies on androgen receptor mild mutations demonstrate their association with male infertility.

CONTEXT: Mutations in the androgen receptor (AR) gene can cause the androgen insensitivity syndrome (AIS). For complete and severe partial AIS, well-characterized in vitro functional assays can be used for genotype-phenotype correlation; however, for mild forms of AIS, as associated with male infertility, experimental evidence is scarce or lacking. In particular, optimal in vitro functional tests informative about the genotype-phenotype relation have not been described. OBJECTIVE: The objective of this study was to investigate the association among genotype and phenotype for AR mutations found in infertile males by conventional functional assays and additional in-depth studies performed with several gene reporters. DESIGN: To this aim, we selected four AR missense mutations associated with isolated male infertility (L547F and two novel mutations A474V and S650G) or partial AIS (Y571H). After introduction of the specific mutations in AR expression plasmid, we performed classical in vitro studies (Western immunoblotting, electrophoretic mobility shift assay, hormone-response curves) and transactivation assays with different reporter constructs (MMTV, Sc-ARU-TK, TAT-GRE- 2X, Slp-ARU-TK and PEM). RESULTS AND CONCLUSIONS: Our results showed that standard functional tests provide sufficient information only for severe AR mutations, whereas for AR mutations found in mild AIS patients with male infertility, only an extensive analysis with different in vitro systems, and in particular with PEM promoter, can give information on the functionality of the AR and therefore on the pathogenicity of the mutations and on genotype-phenotype correlation.

Insulin-like factor 3 gene mutations in testicular dysgenesis syndrome: clinical and functional characterization.

Insulin-like factor 3 (INSL3) plays a crucial role in testicular descent. Genetic ablation of Insl3 or its G protein-coupled receptor, leucine-rich repeat-containing G-protein-coupled receptor (Lgr8), causes cryptorchidism in mice. Mutation analyses of INSL3 in humans showed an association with cryptorchidism but led to non-conclusive data about a causative role. In this study, we explored the hypothesis that mutations in INSL3 may be associated with the signs of testicular dysgenesis syndrome (TDS). We screened for mutations in INSL3 gene in 967 subjects with a history of maldescended testes and/or infertility and/or testicular cancer and in 450 controls. Furthermore, we carried out in vitro functional analysis of three novel mutations by analysis of INSL3-dependent cAMP increase in cells expressing LGR8. We found six INSL3 mutations in 18 of 967 patients (1.9%) and no mutations in controls. Prevalence of mutations was similar in the different groups of patients (cryptorchidism and/or infertility and/testicular cancer). Three mutations were novel findings (R4H, W69R, and R72K); however, their analysis showed normal cAMP increase after the activation of LGR8 receptor. In conclusion, we found a significant association of INSL3 gene mutations in men presenting one or more signs of TDS syndrome. However, a causative role for some of these mutations is not clearly supported by functional analyses. Although a role for mutations of INSL3 and LGR8 genes in cryptorchidism is reasonable, additional studies are needed to establish an association between the disruption of INSL3 pathway and higher risk of infertility or testicular cancer.

Circulating endothelial progenitor cells and endothelial function after chronic Tadalafil treatment in subjects with erectile dysfunction.

We evaluated the effect of a chronic treatment with Tadalafil on progenitor cells (PCs) number and endothelial function in patients with erectile dysfunction (ED) with or without cardiovascular risk factors. Twenty-six subjects with ED and 23 aged matched controls were studied. All subjects underwent blood tests, International Index of Erectile Function (IIEF-5), Nocturnal Penile Tumescence Rigidity Monitoring test (NPTRM), brachial artery flow-mediated dilation (FMD) and PCs count. International index of erectile function, FMD and PC count were re-evaluated in all subjects at the end of Tadalafil and placebo treatment. With respect to controls patients had lower basal FMD (P < 0.05) and basal PCs (P < 0.05). Treatment with Tadalafil determined a significant increase in PCs (P < 0.001) and FMD (P < 0.001) with respect to basal level. Positive correlation was found between basal FMD and PCs (P < 0.05) and between basal FMD and PCs increase after Tadalafil treatment (P < 0.05). Tadalafil promotes a mobilization of PCs and improves endothelial function in ED patients.

Molecular analysis of the androgen receptor gene in testicular cancer.

Testicular cancer (TC) is the most common solid tumour in white males aged 20-34 years, and its incidence has doubled over the past 40 years. Some risk factors for TC have been proposed, such as cryptorchidism, infertility and testicular dysgenesis. However, the causes of TC remain still largely unknown. Recently a genetic basis for TC has been proposed, but specific genetic alterations have not been identified. The risk of TC is markedly increased in subjects with androgen insensitivity and some authors have suggested that mutations in the androgen receptor (AR) gene or disorders of CAG and GGC repeats could be related to TC. However, definitive data have not been produced. In this study, we analysed the AR gene for mutations and CAG and GGC triplets in exon 1 in 123 patients affected by TC. In three patients (2.3%) we found a mutation in the AR gene, two of which represent a novel mutation. Evaluation of CAG and GGC repeat numbers showed no difference with respect to controls when these variables were analysed separately. However, when joint distributions of CAG and GGC were considered, we found that the combination CAG=20/GGC=17 was significantly more frequent in TC patients (8.1%) with respect to controls (1.7%, P<0.05). Furthermore, we observed that in TC subjects, differently from controls, the joint analysis of CAG and GGC showed a statistically significant dependence among these variable repeats. In conclusion, our data show for the first time a high prevalence of AR gene mutations in patients affected by TC and suggest that some CAG/GGC combinations might be more frequently associated with an increased risk of TC.

Somatic mosaicism in von Hippel-Lindau Disease.

von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome predisposing to the development of retinal and central nervous system haemangioblastomas, pheochromocytomas, renal and pancreatic cancer. In the course of a molecular analysis conducted to detect germline mutations of this gene in von Hippel-Lindau patients and individuals affected by sporadic tumors, we have identified a case of somatic mosaicism in the asymptomatic mother of a VHL patient who was subsequently diagnosed with pheochromocytoma. This is the first report providing molecular evidence of somatic mosaicism in von Hippel-Lindau disease. Mosaicism could provide some genetic explanation for the clinical heterogeneity and variable severity of the VHL phenotype, and should be considered, as a possible event when evaluating sporadic cases of VHL or patients with isolated VHL-related tumors. Hum Mutat 15:114, 2000.

Cx26 deafness: mutation analysis and clinical variability.

Mutations in the gap junction protein connexin 26 (Cx26) gene (GJB2) seem to account for many cases of congenital sensorineural hearing impairment, the reported prevalence being 34-50% in autosomal recessive cases and 10-37% in sporadic cases. The hearing impairment in these patients has been described as severe or profound. We have studied 53 unrelated subjects with congenital non-syndromic sensorineural hearing impairment in order to evaluate the prevalence and type of Cx26 mutations and establish better genotype-phenotype correlation. Mutations in the Cx26 gene were found in 53% of the subjects tested, 35.3% of the autosomal recessive and 60% of the sporadic cases in our series. Three new mutations were identified. The hearing deficit varied from mild to profound even in 35delG homozygotes within the same family. No evidence of progression of the impairment was found. Alterations of the Cx26 gene account for a large proportion of cases of congenital non-syndromic sensorineural deafness, so it seems appropriate to extend the molecular analysis even to subjects with mild or moderate prelingual hearing impairment of unknown cause.

Molecular diagnosis of inherited diseases.

The importance of the interaction between basic science and clinical practice has long been known but it has become even more evident in the past few decades with the impressive rate of development in the field of molecular genetics. This short article reviews molecular diagnosis of two different diseases for which scientific progress has immediately been translated into a dramatic improvement of the quality of medical care: the Fragile X Syndrome, paradigm of the new mutational mechanism of the unstable triplet repeats, and von Hippel-Lindau disease, a recent acquisition in the growing number of familial cancer syndromes.

Molecular genetics applied to clinical practice: the Cx26 hearing impairment.

Mutations in the Cx26/GJB2 gene account for a large proportion of pre-lingual hearing impairment with a prevalence up to 50% in autosomal recessive cases and a still undefined prevalence in sporadic cases. Ninety-four subjects affected by non-syndromal sensorineural hearing impairment (NSHI) were enrolled in the study. The patients had either a family history of childhood hearing deficit or represented sporadic cases. The risk of an acquired cause of the deficit has been carefully excluded. Audiological characteristics were investigated. Cx26 mutations were found in 50% of subjects. Seventy-three per cent of mutations in this gene were 35delG, with significant geographical variations. In 7% of the putative Cx26 alleles no mutations were detected either in the coding region or in the non-coding exon 1. Cx26 hearing impairment involves all frequencies, is of variable severity, and is very rarely progressive and most frequently symmetrical between the two ears. The high occurrence of this type of pre-lingual hearing impairment argues for modification of the protocols used to investigate the aetiology of childhood hearing impairment. Early screening for Cx26 mutations in all patients with non-syndromal familial and sporadic permanent childhood hearing impairment seems justified.

The guanine triphosphatase (GTPase) activating protein (GAP)-related domain of the neurofibromatosis type 1 gene is not mutated in neural crest-derived sporadic tumours.

We conducted a mutation analysis of the most conserved region of the neurofibromatosis type 1 (NF1) gene, the guanine triphosphatase (GTPase) activating protein (GAP)-related domain (NF1 GRD), to which the function of tumour suppressor is attributed. Sixty primary neuroectodermal tumours were analysed. The rationale for the study was based on the likelihood of finding structural alterations resulting in loss of function of this region in tumours of neuroepithelial tissues, where the activity of neurofibromin seems to be crucial in regulating the mechanisms of signal transduction and cell transformation mediated by p21 ras. Following analysis of the whole NF1 GRD sequence, no mutations were identified in the tumours analysed. We conclude that the loss of NF1 gene tumour suppressor function, that might lead or contribute to the development of malignancies in neuroectodermal tissues, is not due to structural abnormalities of the region of the gene which interacts with p21 ras.

Amplification of the Xq28 FRAXE repeats: extreme phenotype variability?

We report on a new case of FRAXE mutation identified through the screening of a population of FRAXA-negative mentally retarded individuals. The index case, a 4-year-old boy with distinct minor anomalies and mental retardation with severe verbal impairment, his older brother, referred to as normal, and the mother have undergone careful clinical and molecular evaluation. The molecular defect, characterized by standard Southern blot analysis, is represented by a hypermethylated "full mutation" in the 2 boys and by a unique, altered, presumably unmethylated, band in the mother, which is interpreted as a "premutation." The cytogenetic analysis failed to detect a folate-sensitive Xq27-28 fragile site in either "fully mutated" individual. The phenotype and intellectual performance of the 15-year-old brother of the propositus appeared completely normal. Our propositus shares some traits with previously described FRAXE-mutated subjects, suggesting an association with the Xq28 molecular defect; nevertheless, we find it difficult to reconcile the molecular identity and phenotypic difference in these mutated members of the same family. This could be a case of extreme phenotypic variability or a result of a more complicated molecular mechanism.