Philippe Couvert

A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%–1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including β-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

Stimulation of Cholesterol Efflux by LXR Agonists in Cholesterol-Loaded Human Macrophages Is ABCA1-Dependent but ABCG1-Independent

Objective—Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. Methods and Results—ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti–Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. Conclusion—Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.

Missense mutation in PAK3, R67C, causes X-linked nonspecific mental retardation

X-linked mental retardation is a very common condition that affects approximately 1 in 600 males. Despite recent progress, in most cases the molecular defects underlying this disorder remain unknown. Recently, a study using the candidate gene approach demonstrated the presence of mutations in PAK3 (p21-activating kinase) associated with nonspecific mental retardation. PAK3 is a member of the larger family of PAK genes. PAK proteins have been implicated as critical downstream effectors that link Rho-GTPases to the actin cytoskeleton and to MAP kinase cascades, including the c-Jun amino-terminal kinase (JNK) and p38. We screened 12 MRX pedigrees that map to a large region overlying Xq21-q24. Mutation screening of the whole coding region of the PAK3 gene was performed by using a combination of denaturing gradient gel electrophoresis and direct sequencing. We have identified a novel missense mutation in exon 2 of PAK3 gene (R67C) in MRX47. This confirms the involvement of PAK3 in MRX following the report of a nonsense mutation recently reported in MRX30. In the MRX47 family, all affected males show moderate to severe mental retardation. No seizures, statural growth deficiency, or minor facial or other abnormal physical features were observed. This mutation R67C is located in a conserved polybasic domain (AA 66-68) of the protein that is predicted to play a major role in the GTPases binding and stimulation of Pak activity.

Parental origin of de novo MECP2 mutations in Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder occurring almost exclusively in females as sporadic cases. Recently, DNA mutations in the MECP2 gene have been detected in approximately 70% of patients with RTT. To explain the sex-limited expression of RTT, it has been suggested that de novo X-linked mutations occur exclusively in male germ cells resulting therefore only in affected daughters. To test this hypothesis, we have analysed 19 families with RTT syndrome due to MECP2 molecular defects. In seven informative families we have found by DHPLC a nucleotide variant which could be used to differentiate between the maternal and the paternal allele. In each subject investigated from these families, we have amplified specifically each allele and sequenced allele-specific PCR products to identify the allele bearing the mutation as well as the parental origin of each X chromosome. This approach allowed us to determine the parental origin of de novo mutations in all informative families. In five cases, the de novo MECP2 mutations have a paternal origin and in the two other cases a maternal origin. In all transitions at CpG, the de novo mutation observed was of paternal origin. The high frequency of male germ-line transmission of the mutation (71% of RTT informative cases) is consistent with a predominant occurrence of the disease in females.

In vitro follicular growth affects oocyte imprinting establishment in mice

In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.

Association between a frequent allele of the gene encoding OATP1B1 and enhanced LDL-lowering response to fluvastatin therapy

INTRODUCTION Marked lowering of low-density-lipoprotein cholesterol (LDL-C) levels (< or =50%) with intensive statin therapy is associated with major reduction in cardiovascular risk, but is limited by a potential increase in adverse effects, thereby justifying optimization of LDL-C reduction with minimal risk. The organic anion transporting polypeptide-1B1 encoded by the SLCO1B1 gene is implicated as a major transporter in cellular uptake of statins, and notably fluvastatin. We postulated that genetic variation in SLCO1B1 might affect statin bioavailability, and might therefore influence drug response and potential adverse effects. MATERIALS & METHODS Elderly hypercholesterolemic subjects (n = 724), whose plasma lipid profile was determined before and 2 months after fluvastatin extended-release treatment (80 mg/day, n = 420), or placebo (n = 304), were genotyped for the most frequent nonsynonymous polymorphisms (SNP) in the SLCO1B1 gene (c.388A>G, c.463C>A and c.521T>C). RESULTS Due to linkage disequilibrium, only four alleles (*1b, *5, *14 and *15) of SLCO1B1 were detected in addition to the wild-type allele (*1a). The c.463A genotype, which was systematically associated with the c.388G SNP corresponding to the *14 allele was significantly associated with percentage LDL-C reduction from baseline (p = 0.005) and with mean post-treatment LDL-C values (p = 0.0005). Subjects homozygous for the c.463C genotype (n = 294) exhibited significantly less LDL-C reduction and higher post-treatment LDL-C levels (-31.5%, 138 mg/dl) relative to heterozygous C/A patients (-36.2%, 126 mg/dl; n = 111), and to homozygous A/A subjects (-41%, 115 mg/dl; n = 15). CONCLUSIONS These results reveal that OATP1B1 is implicated in the pharmacological action and efficacy of fluvastatin. Indeed, the common *14 allele, which is distinguished by the presence of the c.463C>A polymorphism, was associated with enhanced lipid-lowering efficacy in this study.

Adrenocortical Scavenger Receptor Class B Type I Deficiency Exacerbates Endotoxic Shock and Precipitates Sepsis-Induced Mortality in Mice

Scavenger receptor class B type I (SR-BI)–deficient mice display reduced survival to endotoxic shock and sepsis. The understanding of the mechanisms underlying SR-BI protection has been hampered by the large spectrum of SR-BI functions and ligands. It notably plays an important role in the liver in high-density lipoprotein metabolism, but it is also thought to participate in innate immunity as a pattern recognition receptor for bacterial endotoxins, such as LPS. In this study, we sought to determine the tissue-specific contribution of SR-BI in the hyperinflammatory response and high mortality rates observed in SR-BI−/− mice in endotoxicosis or sepsis. Restoring plasma levels of high-density lipoprotein, which are critical lipoproteins for LPS neutralization, did not improve acute outcomes of LPS injection in SR-BI−/− mice. Mice deficient for SR-BI in hepatocytes, endothelial cells, or myeloid cells were not more susceptible to LPS-induced death. However, if SR-BI ablation in hepatocytes led to a moderate increase in systemic inflammatory markers, SR-BI deficiency in myeloid cells was associated with an anti-inflammatory effect. Finally, mice deficient for SR-BI in the adrenal cortex, where the receptor provides lipoprotein-derived cholesterol, had impaired secretion of glucocorticoids in response to stress. When exposed to an endotoxin challenge, these mice exhibited an exacerbated systemic and local inflammatory response, reduced activation of atrophy genes in muscle, and high lethality rate. Furthermore, polymicrobial sepsis induced by cecal ligature and puncture resulted in early death of these animals. Our study clearly demonstrates that corticoadrenal SR-BI is a critical element of the hypothalamic-pituitary-adrenal axis to provide effective glucocorticoid-dependent host defense after an endotoxic shock or bacterial infection.

Determination of the gene structure of human oligophrenin-1 and identification of three novel polymorphisms by screening of DNA from 164 patients with non-specific X-linked mental retardation.

We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX). The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library. Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1. OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation. Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C). The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.

Lewis-Sumner Syndrome and Tangier Disease

OBJECTIVE To report unusual electrophysiologic data in a patient with Tangier disease in an effort to better understand the pathophysiologic features of the peripheral nerve lesions in this disease. DESIGN Case report. PATIENT A 15-year-old girl had subacute onset of asymmetric neuropathy with persistent conduction block, resembling Lewis-Sumner syndrome. MAIN OUTCOME MEASURES Electrophysiologic data in Tangier disease. RESULTS After initially unsuccessful treatment with intravenously administered immunoglobulins, the finding of an abnormal lipid profile led to the diagnosis of Tangier disease due to the R587W mutation in the adenotriphosphate-binding cassette transporter-1 gene (ABCA1) (OMIM 9q22-q31). CONCLUSIONS Conduction block, which is the electrophysiologic hallmark of focal demyelination, can be present in Tangier disease. It could be induced by focal nerve ischemia or by preferential lipid deposition in the paranodal regions of myelinated Schwann cells. The presence of a conduction block in Tangier disease may lead to a misdiagnosis of dysimmune neuropathy.

Extended-Release Niacin/Laropiprant Improves Overall Efficacy of Postprandial Reverse Cholesterol Transport

Objectives— Postprandial atherogenic lipoproteins, characterizing high-risk patients, correlate positively with cardiovascular events. Although the effect of niacin on fasting lipids is well established, its impact on atheroprotective reverse cholesterol transport (RCT) pathway and on functional features of circulating lipoproteins during the postprandial state remains indeterminate. Approach and Results— We evaluated RCT pathway during postprandial phase in dyslipidemic patients displaying a low high-density lipoprotein (HDL) cholesterol phenotype. Ten subjects on stable statin therapy received 1 g/20 mg extended-release niacin/laropiprant (ERN/LRPT) for 4 weeks followed by 2 g/40 mg ERN/LRPT for additional 8 weeks. At each experimental period, postprandial hypertriglyceridemia and major steps of RCT, including cholesterol efflux from human macrophages, cholesteryl ester transfer protein–mediated cholesteryl ester transfer, and hepatic HDL-cholesteryl ester selective uptake were evaluated. Equally, the capacity of postprandial HDL particles isolated from patients before and after ERN/LRPT treatment to mediate RCT to feces was evaluated in vivo in human apolipoprotein B/cholesteryl ester transfer protein double transgenic mouse model. Compared with baseline, ERN/LRPT significantly reduced postprandial hypertriglyceridemia (incremental area under the curve-triglyceride: −53%; P=0.02). Postprandial increase in endogenous plasma cholesteryl ester transfer protein activity was completely abolished after ERN/LRPT treatment. Despite a slight reduction in plasma cholesterol efflux capacity from human THP-1 macrophages, evaluation of global RCT efficacy by combining both ex vivo and in vivo approaches indicate that postprandial HDL particles formed under ERN/LRPT therapy displayed a greater capacity for HDL-mediated RCT to feces. Conclusions— ERN/LRPT treatment efficiently attenuates atherogenic postprandial lipemia and stimulates HDL-mediated cholesterol return to the liver and elimination into feces during postprandial phase, thus maintaining an efficient removal of cholesterol from the body.