A novel loss of function mutation in exon 10 of the FSH receptor gene causing hypergonadotrophic hypogonadism: clinical and molecular characteristics

BACKGROUND: Inactivating mutations of the FSH receptor (FSHR) are a rare cause of hypergonadotrophic hypogonadism in women. Only one patient with primary amenorrhoea due to an FSHR gene mutation has been reported outside of Finland, where the prevalence of Ala189Val mutations is particularly high. METHODS AND RESULTS: Here, we describe the clinical, molecular genetic and functional characteristics associated with a novel inactivating mutation in exon 10 of the FSHR gene identified in a patient who presented with primary amenorrhoea at 17 years of age. The C to G transversion found at nucleotide 1043 causes a Pro348Arg substitution in the extracellular region of the FSHR and results in a mutant FSHR that is completely inactive in functional studies and that does not bind FSH. The proband exhibits apparent homozygosity for this recessive mutation. Her father is heterozygous for the mutation while analysis of exon 10 of the FSHR gene from her mother revealed only wild-type sequence. Chromosome painting was used to exclude deletions or rearrangements of 2p, and microsatellite markers did not show paternal uniparental isodisomy for this region. These findings suggest that the proband is hemizygous, with an inherited or de-novo microdeletion, or alternatively a de-novo gene conversion, of the accompanying FSHR allele. CONCLUSIONS: This case confirms the importance of the FSHR in female pubertal development and reproduction, and supports a relationship between phenotype and function for FSHR mutations.     

Cardiac drift during prolonged exercise with echocardiographic evidence of reduced diastolic function of the heart

This study examined whether, in 16 male subjects, a continuous increase in heart rate (HR) during 4 h of ergometry cycling relates to cardiac fatigue or cardiomyocyte damage. Serum cardiac troponin T (cTnT) was determined and echocardiographic assessment was carried out prior to and after 2 h of exercise, within 15 min of completing exercise and after 24 h. Left ventricular contractile function (end-systolic blood pressure–volume relationship [SBP/ESV]) and diastolic filling (ratio of early to late peak left ventricular filling velocities [E:A]) were calculated. During exercise HR was 132±5 beats min^–1 after 2 h and increased to 141±5 beats min^–1 (mean ± SD; P<0.05), but there was no evidence of altered LV contractile function (SBP/ESV 39.0±5.1 mmHg cm^–1 to 36.5±5.2 mmHg cm^–1 and SBP/ESV was not correlated to maximal oxygen uptake (r²=0.363). In contrast, E:A decreased (1.82±0.32 to 1.48±0.30; P<0.05) and returned towards baseline after 24 h (1.78±0.28), and individual changes were correlated to maximal oxygen uptake (r²=0.61; P<0.05). Low levels of cTnT were detected in two subjects after 4 h of exercise that had normalised by 24 h of recovery. During prolonged exercise cardiovascular drift occurred with echocardiographic signs of a reduced diastolic function of the heart, especially in those subjects with a high maximal oxygen uptake.     

Nuclear and cytoplasmic bcl-2 expression in endometrial hyperplasia and adenocarcinoma

The bcl-2 proto-oncogene, which inhibits programmed cell death (apoptosis), has recently been found to be cyclically expressed in human endometrium. In order to investigate its role in endometrial hyperlasia and neoplasia, bcl-2 expression was studied in 25 cases of endometrial carcinoma and 20 cases of endometrial hyperplasia (eight simple, two complex, and ten atypical hyperplasias). Uniform intense cytoplasmic bcl-2 expression was found in all cases of non-atypical hyperplasia, and less strong positivity in eight out of ten cases of atypical hyperplasia. In well-differentiated carcinomas, nine out of ten showed weak to moderate bcl-2 expression, whereas six out of seven poorly differentiated carcinomas were bcl-2-negative. Moderately differentiated tumours were an intermediate group, with six out of eight being positive. Widespread localization of bcl-2 protein to the chromosomes of dividing cells was also demonstrated, regardless of cytoplasmic bcl-2 expression, with rare staining of interphase nuclei. Our findings suggest a role for bcl-2 in the natural history of endometrial neoplasia and studies are needed to determine its usefulness as a prognostic marker. The finding of bcl-2 localization to chromosomes has important implications for its mode and site of action.     

Replication of the RNA segments of a bipartite viral genome is coordinated by a transactivating subgenomic RNA

The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments.     

Millon Clinical Multiaxial Inventory-III subtypes of opioid dependence: validity and matching to behavioral therapies

The concurrent and predictive validity of 2 different methods of Millon Clinical Multiaxial Inventory-III subtyping (protocol sorting, cluster analysis) was evaluated in 125 recently detoxified opioid-dependent outpatients in a 12-week randomized clinical trial. Participants received naltrexone and relapse prevention group counseling and were assigned to 1 of 3 intervention conditions: (a) no-incentive vouchers, (b) incentive vouchers alone, or (c) incentive vouchers plus relationship counseling. Affective disturbance was the most common Axis I protocol-sorted subtype (66%), antisocial-narcissistic was the most common Axis II subtype (46%), and cluster analysis suggested that a 2-cluster solution (high vs. low psychiatric severity) was optimal. Predictive validity analyses indicated less symptom improvement for the higher problem subtypes, and patient treatment matching analyses indicated that some subtypes had better outcomes in the no-incentive voucher conditions.     

Effects of adenoviral-mediated gene transfer of interleukin-10, interleukin-4, and transforming growth factor-beta on the survival of axotomized retinal ganglion cells

Nitric oxide, synthesized by reactive microglia and astrocytes has been implicated in promoting neuronal degeneration observed in many diseases and insults of the central nervous system. We have recently shown that inducible nitric oxide synthase is expressed by retinal glial cells following optic nerve transection and that inhibition of nitric oxide synthesis enhances the survival of injured retinal ganglion cells. Anti-inflammatory cytokines including interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-beta) have been shown to prevent inducible nitric oxide synthase expression, and inhibit nitric oxide synthesis by microglia and astrocytes in culture. In the present study, we examined the effects of adenoviral mediated gene transfer of anti-inflammatory cytokines on the survival of axotomized retinal ganglion cells. Intraocular administration of adenoviral vectors encoding interleukin-10 (Ad.IL-10) and interleukin-4 (Ad.IL-4) enhanced the survival of axotomized retinal ganglion cells at 14 days after axotomy. Adenoviral vectors encoding TGF-beta (Ad.TGF-beta) had no effect on retinal ganglion cell survival. Separate animals were pretreated by injection of Ad.IL-10 or Ad.IL-4 into the superior colliculus (s.c.), the major target of ganglion cells, 7 days prior to axotomy. S.c. administration of Ad.IL-10 or Ad.IL-4 significantly increased ganglion cell survival compared with intraocular injection. IL-10 and IL-4 gene transfer also reduced the density of infiltrating ED1 positive monocytes in the nerve fiber layer at 14 days postaxotomy. Ad.TGF-beta increased the density of ED1 positive monocytes infiltrating the nerve fiber layer after axotomy. Vectors encoding IL-10 or IL-4 also decreased nitrotyrosine immunoreactivity in the inner retina at 7 days postaxotomy, suggesting that these cytokines protect retinal ganglion cells from peroxynitrite formation that results from nitric oxide synthesis by activated glial cells. The present study has implications for the treatment of CNS injury and diseases that involve reactive microglia and astrocytes. Our results suggest that interleukin-10 and interleukin-4 may help prevent neurodegeneration caused by the activation of glial cells after CNS injury.     

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.     

Daily versus thrice-weekly interferon alfa-2b plus ribavirin for the treatment of chronic hepatitis C in HIV-infected persons: a multicenter randomized controlled trial

Among HIV-infected persons, chronic hepatitis C virus (HCV) infection causes substantial morbidity and mortality. However, few studies have evaluated the safety and efficacy of interferon alfa (IFN) and ribavirin (RBV) therapy in co-infected persons. Accordingly, a randomized, controlled, open-label, multicenter trial was conducted to establish the safety, tolerability, and efficacy of IFN alfa-2b 3 mIU daily plus RBV 800 mg/d compared with IFN alfa-2b 3 mIU thrice weekly (TIW) plus RBV 800 mg/d in HCV treatment-naive, HIV-infected subjects with compensated liver disease and stable HIV disease. The primary endpoint was sustained virologic response (SVR), defined as an undetectable HCV RNA level 24 weeks after discontinuation of HCV therapy. At study entry, subjects in both groups were similar with respect to age, gender, HCV genotype, and HIV disease status. Of 180 randomized subjects, 162 received at least 1 dose of study medication, constituting the modified intention-to-treat population. After 12 weeks of therapy, 122 (75%) had serum HCV RNA levels assessed; of these subjects, early virologic response (undetectable HCV RNA or >2 log10 decrease from baseline) was observed in 33 (42%) and 13 (16%) of subjects taking daily and TIW IFN, respectively (P < 0.001). SVR was observed in 15 (19.0%) and 7 (8.4%) of subjects taking daily and TIW IFN, respectively (P = 0.05). Adverse events were similar in both groups. However, while no deaths or opportunistic infections were observed, nearly 30% of subjects stopped treatment due to adverse events and 7 subjects experienced a serious adverse event. In conclusion, SVR was achieved in 19% of HIV/HCV coinfected subjects treated with daily IFN plus RBV, but the effectiveness of therapy was substantially diminished by relatively high rates of treatment-related toxicity.     

Different specificities of an HLA-DRw6 haplotype detected by alloreactive T lymphocytes

DRw6 has been difficult to define serologically. In the present experiments we have developed T cell lines in order to characterize the components of a DRw6 haplotype. This was accomplished by priming T cells with allogeneic mononuclear cells mismatched for DRw6, Dw6, and MT2. Subsequently, three sublines with distinct reactivity patterns were derived by limiting dilution. The specificities detected by these sublines included: (a) a specificity found on a subset of cells positive for DRw6 which was inhibited by monoclonal antibodies against DS(DC), the human homologue of the murine IA-encoded molecules, (b) another DRw6-associated specificity blocked by an MT2-like antibody, and (c) an MT2-like specificity blocked by monoclonal antibodies reactive with a different MT2-associated determinant. These results show that more than one IE-like, as well as the DS/DC (IA-like) molecules, carry distinctive antigenic epitopes that can be recognized by allogeneic T cells. Primed T cell lines may be useful for a better definition of certain haplotypes which are at present difficult to characterize with serological reagents alone.