Vincent Raymond

Recurrent Mutation of the Gene Encoding sequestosome 1 (SQSTM1/p62) in Paget Disease of Bone

Paget disease of bone (PDB) is a common disorder characterized by focal and disorganized increases of bone turnover. Genetic factors are important in the pathogenesis of PDB. We and others recently mapped the third locus associated with the disorder, PDB3, at 5q35-qter. In the present study, by use of 24 French Canadian families and 112 unrelated subjects with PDB, the PDB3 locus was confined to approximately 300 kb. Within this interval, two disease-related haplotype signatures were observed in 11 families and 18 unrelated patients. This region encoded the ubiquitin-binding protein sequestosome 1 (SQSTM1/p62), which is a candidate gene for PDB because of its association with the NF-kappaB pathway. Screening SQSTM1/p62 for mutations led to the identification of a recurrent nonconservative change (P392L) flanking the ubiquitin-associated domain (UBA) (position 394-440) of the protein that was not present in 291 control individuals. Our data demonstrate that two independent mutational events at the same position in SQSTM1/p62 caused PDB in a high proportion of French Canadian patients.

HOMOZYGOTES CARRYING AN AUTOSOMAL DOMINANT TIGR MUTATION DO NOT MANIFEST GLAUCOMA

nature genetics volume 19 august 1998 319 Autosomal dominant disorders typically result in more severe clinical manifestations in mutant homozygotes than in heterozygotes1. Huntington disease is the only reported non-neoplastic human pathology in which no phenotypic variance has been detected between these two types of carriers, and where the mutant allele is truly dominant over its wild-type counterpart2. Primary openangle glaucoma (POAG), a leading cause of blindness worldwide, is characterized by progressive degeneration of the optic nerve and is usually associated with intraocular hypertension3 (OHT). Several loci involved in glaucoma have been localized4,5. Recently, mutations in the trabecular meshwork-inducible glucocorticoid response (TIGR) gene, also known as myocilin6, mapping to the GLC1A locus at 1q23−q25, were identified in families affected by autosomal dominant POAG (refs 7−10). We investigated a FrenchCanadian family, pedigree GV-001, in which POAG was transmitted as an autosomal dominant trait linked to the GLC1A locus11. The large size of this kindred and its relative isolation in eastern Québec allowed us to assess the effects of a TIGR mutation present in double dosage in four adult homozygotes. These individuals were asymptomatic for the disorder, with POAG affecting only the heterozygotes. The pedigree currently contains 622 individuals, of which 83 manifested either juvenile-onset (JOAG), a subset of POAG that has an early age at onset3, or adultonset POAG. Ten individuals also displayed OHT, which often preceded POAG by several years11. A marriage in branch GV-510 between two affected seconddegree cousins, mother V-1 and her spouse, father V-2 (Fig. 1a), resulted in 10 children, now 33−50 years of age. It was expected that these siblings would carry two wild-type copies of TIGR in a proportion of approximately 25%, and approximately 75% would harbour at least one mutant allele. It was also anticipated that a high proportion of these adults would be affected. Phenotypic evaluation showed only two sibs to be affected: son VI-3 and daughter VI-4 (Fig. 1a). The other eight sibs disHomozygotes carrying an autosomal dominant TIGR mutation do not manifest glaucoma

Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia

Background Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. Methods We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. Results Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. Conclusions Based on our genetic results, TTC7A is the likely causal gene for MIA.

LHRH rapidly stimulates phosphatidylinositol metabolism in enriched gonadotrophs

The effects of luteinizing hormone-releasing hormone (LHRH) and human pancreatic growth hormone-releasing factor (hpGRF(1-40)-NH2) on phospholipid metabolism were studied in rat anterior pituitary cells in primary culture. In a 4-fold enriched population of gonadotrophs, 30 nM LHRH increased 32Pi incorporation into phosphatidic acid (PA) as early as 1 min after its addition. Phosphatidylinositol (PI) labeling was increased 1 min later. The stimulatory action of LHRH was observed in both phospholipids up to 100 min, the last time interval studied. The decapeptide did not affect 32Pi labeling of phosphatidylcholine (PC), lysoPC, phosphatidylethanolamine or phosphatidylserine. Dose-response studies performed after 25 min of incubation showed an ED50 value of LHRH action at approximately 1 nM for PI labeling. In contrast, the addition of 0.1 microM GRF to anterior pituitary cells enhanced 32Pi incorporation only into PC after a 60 min incubation period. The present data suggest that stimulation of acidic phospholipid metabolism, particularly an increase in PA-PI turnover, may represent an early event in the mechanism of action of LHRH but not GRF in the anterior pituitary gland.

Evidence for association and linkage between atopy, airway hyper-responsiveness, and the β subunit Glu237Gly variant of the high-affinity receptor for immunoglobulin E in the French-Canadian population

Abstract. Following detection of linkage between atopy and chromosome 11q13 markers, association between this disorder and variants of the β subunit of the high-affinity receptor for immunoglobulin E (FcεRI-β, a candidate gene for asthma-related conditions co-localizing within the same region) was reported in Australian, British and Japanese populations. Investigations in several other ethnic groups failed to replicate these observations. Due to the complexity of defining intermediate phenotypes related to asthma, detection of such associations may have been hampered by clinical misclassifications. To assess whether the FcεRI-β gene was involved in atopy and/or airway hyperresponsiveness (AHR) in the French-Canadian population, we conducted a case-control study in 200 subjects using strict criteria for asthma and related conditions. The Ile181Leu and Glu237Gly FcεRI-β sequence variants were tested exploiting two amplification refractory mutation systems. No association was detected between atopy or AHR and the Ile181Leu FcεRI-β variant. However, a strong association was observed between atopy and the Glu237Gly FcεRI-β variant (odds ratio=12.25). Four large Eastern Québec families (n=106 subjects) were also recruited to perform a genetic linkage study. We observed suggestive evidence of linkage between atopy and the Glu237Gly FcεRI-β variant (Zmax=2.30). This study is the first to detect the presence of an association between atopy and the Glu237Gly FcεRI-β variant in French-Canadians. Our data suggest that a susceptibility locus for atopy is located on chromosome 11q13 in this population.

Glaucoma-associated WDR36 Variants Encode Functional Defects in a Yeast Model System

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. POAG is associated with a characteristic progression of changes to ocular morphology and degeneration at the optic nerve head with the loss of visual fields. Physical mapping efforts identified genomic loci in which to search for causative POAG gene mutations. WDR36, at locus GLC1G, was initially identified as a gene with a low frequency of non-synonymous sequence variations that were exclusive to adult-onset POAG patients. It has since been shown that rare WDR36 sequence variants are also present in the normal population at similarly low frequencies. The lack of a consistent genotype:phenotype correlation prompted us to investigate the functional consequences of WDR36 sequence variations. WDR36 is involved in rRNA processing, a critical step in ribosome biogenesis, and is very similar to yeast Utp21p which is a member of the small subunit (SSU) processome complex responsible for maturation of 18S rRNA. We, therefore, developed a yeast model system to test the functional and phenotypic consequences of POAG-associated sequence variants introduced into UTP21. Alone, the POAG variants did not produce any significant defects in cell viability or rRNA processing. However, when combined with disruption of STI1 (which synthetically interacts with UTP21), 5 of the 11 tested variants had increased or decreased cell viability which corresponded to reduced or elevated levels of pre-rRNA, respectively. These results demonstrate that, in the correct genetic background, WDR36 sequence variants can lead to an altered cellular phenotype, supporting the theory that WDR36 participates in polygenic forms of glaucoma.

Stimulation by prostaglandin F2α of phosphatidic acid-phosphatidylinositol turnover in rat luteal cells

Prostaglandin F2 alpha (PGF 2 alpha) causes a rapid and marked increase of [32P]-orthophosphate incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min after PGF 2 alpha addition into PA and PI, respectively, and by 10 min has reached a 2-fold stimulation over control in both lipid moieties. The labeling of other phospholipids is not affected. PGF 2 alpha exerts its stimulatory effect at an ED50 value of approximately 200 and 60 nM on PI and PA labeling, respectively. By contrast, human chorionic gonadotropin has no effect alone and does not interfere with the PGF 2 alpha-induced stimulation of PA-PI labeling. The striking similarity between the effects of PGF 2 alpha and LHRH on PA-PI labeling suggests that the two agents may exert their direct action on the corpus luteum via a common intracellular mechanism involving acidic phospholipid metabolism.

PITX2 Is Involved in Stress Response in Cultured Human Trabecular Meshwork Cells through Regulation of SLC13A3

PURPOSE Mutations of the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) and glaucoma. In this study, the authors investigated genes directly regulated by the PITX2 transcription factor to gain insight into the mechanisms underlying these disorders. METHODS RNA from nonpigmented ciliary epithelium cells transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses. Data were analyzed using dCHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of activated PITX2 were subjected to in silico and biochemical analyses to validate them as direct regulatory targets. One target gene was further characterized by studying the effect of its knockdown in a cell model of oxidative stress, and its expression in zebrafish embryos was analyzed by in situ hybridization. RESULTS Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as 1 of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression, as demonstrated by luciferase reporter and chromatin immunoprecipitation assays. Reduction of PITX2 or SLC13A3 levels by small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork cells exposed to hydrogen peroxide. Zebrafish slc13a3 is expressed in anterior ocular regions in a pattern similar to that of pitx2. CONCLUSIONS The results indicate that SLC13A3 is a direct downstream target of PITX2 transcriptional regulation and that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.

How pragmatic or explanatory is the randomized, controlled trial? The application and enhancement of the PRECIS tool to the evaluation of a smoking cessation trial

BackgroundNumerous explanatory randomized trials support the efficacy of chronic disease interventions, including smoking cessation treatments. However, there is often inadequate adoption of these interventions for various reasons, one being the limitation of generalizability of the explanatory studies in real-world settings. Randomized controlled trials can be rated as more explanatory versus pragmatic along 10 dimensions. Pragmatic randomized clinical trials generate more realistic estimates of effectiveness with greater relevance to clinical practice and for health resource allocation decisions. However, there is no clear method to scale each dimension during the trial design phase to ensure that the design matches the intended purpose of the study.MethodsWe designed a pragmatic, randomized, controlled study to maximize external validity by addressing several barriers to smoking cessation therapy in ambulatory care. We analyzed our design and methods using the recently published ‘Pragmatic–Explanatory Continuum Indicatory Summary (PRECIS)’ tool, a qualitative method to assess trial design across 10 domains. We added a 20-point numerical rating scale and a modified Delphi process to improve consensus in rating these domains.ResultsAfter two rounds of review, there was consensus on all 10 domains of study design. No single domain was scored as either fully pragmatic or fully explanatory; but overall, the study scored high on pragmatism.ConclusionsThis addition to the PRECIS tool may assist other trial designers working with interdisciplinary co-investigators to rate their study design while building consensus.

High-fat meal-induced changes in the duodenum mucosa transcriptome.

In order to identify the potential peripheral signals of appetite and satiety from duodenum, we have performed a transcriptomic study in the mucosa after high‐fat (HF) and low‐fat (LF) meal ingestion. After fasting, one group of mice was killed and the others were fed ad libitum with HF or LF diet, and killed 30 min, 1 h, and 3 h after the beginning of the meal. The duodenum mucosa was sampled, and the serial analysis of gene expression (SAGE) method was performed. The mRNA regulations were confirmed by real‐time PCR. Energy, protein, and fat intakes were higher in the HF than in the LF group. Gene expression profile revealed 118 characterized or partially characterized differentially expressed transcripts. The HF meal delayed the expressions of peptidases compared to the LF groups. Most of mRNAs related to fat absorption, including apolipoprotein A‐IV (Apoa4), were decreased in HF1h group, whereas plasma triglyceride (TG) levels were comparable between HF and LF groups. Noteworthy, these downregulations were concomitant to a break in fat intake 1 h after HF meal. At the same time, the HF meal induced transcripts related to cell growth and organization, whereas transcripts involved in cell defense were repressed. Moreover, we have identified fat‐responsive transcripts. This study has characterized the molecular responses of duodenum mucosa after HF or LF meal ingestion. Characterization of novel fat‐specific candidates whose relations with feeding behavior have never been reported may contribute to the development of new therapeutic targets for appetite and satiety controls.