When information can save lives: the duty to warn relatives about sudden cardiac death and environmental risks.

In certain cases of sudden death, forensic experts may discover during an investigation or autopsy that family members of the deceased are also at risk of harm-from genetic disease, for instance. But do they have a duty to warn them? Looking at similar duties of physicians and researchers to warn third parties of risk suggests they do.

In vitro whole-genome analysis identifies a susceptibility locus for HIV-1.

Advances in large-scale analysis of human genomic variability provide unprecedented opportunities to study the genetic basis of susceptibility to infectious agents. We report here the use of an in vitro system for the identification of a locus on HSA8q24.3 associated with cellular susceptibility to HIV-1. This locus was mapped through quantitative linkage analysis using cell lines from multigeneration families, validated in vitro, and followed up by two independent association studies in HIV-positive individuals. Single nucleotide polymorphism rs2572886, which is associated with cellular susceptibility to HIV-1 in lymphoblastoid B cells and in primary T cells, was also associated with accelerated disease progression in one of two cohorts of HIV-1-infected patients. Biological analysis suggests a role of the rs2572886 region in the regulation of the LY6 family of glycosyl-phosphatidyl-inositol (GPI)-anchored proteins. Genetic analysis of in vitro cellular phenotypes provides an attractive approach for the discovery of susceptibility loci to infectious agents.

Genetics of normal and pathological sleep in humans.

The complexity of sleep-wake regulation, in addition to the many environmental influences, includes genetic predisposing factors, which begin to be discovered. Most of the current progress in the study of sleep genetics comes from animal models (dogs, mice, and drosophila). Multiple approaches using both animal models and different genetic techniques are needed to follow the segregation and ultimately to identify 'sleep genes' and molecular bases of sleep disorders. Recent progress in molecular genetics and the development of detailed human genome map have already led to the identification of genetic factors in several complex disorders. Only a few genes are known for which a mutation causes a sleep disorder. However, single gene disorders are rare and most common disorders are complex in terms of their genetic susceptibility, environmental factors, gene-gene, and gene-environment interactions. We review here the current progress in the genetics of normal and pathological sleep and suggest a few future perspectives.

Novel calmodulin mutations associated with congenital arrhythmia susceptibility.

BACKGROUND: Genetic predisposition to life-threatening cardiac arrhythmias such as congenital long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) represent treatable causes of sudden cardiac death in young adults and children. Recently, mutations in calmodulin (CALM1, CALM2) have been associated with severe forms of LQTS and CPVT, with life-threatening arrhythmias occurring very early in life. Additional mutation-positive cases are needed to discern genotype-phenotype correlations associated with calmodulin mutations. METHODS AND RESULTS: We used conventional and next-generation sequencing approaches, including exome analysis, in genotype-negative LQTS probands. We identified 5 novel de novo missense mutations in CALM2 in 3 subjects with LQTS (p.N98S, p.N98I, p.D134H) and 2 subjects with clinical features of both LQTS and CPVT (p.D132E, p.Q136P). Age of onset of major symptoms (syncope or cardiac arrest) ranged from 1 to 9 years. Three of 5 probands had cardiac arrest and 1 of these subjects did not survive. The clinical severity among subjects in this series was generally less than that originally reported for CALM1 and CALM2 associated with recurrent cardiac arrest during infancy. Four of 5 probands responded to β-blocker therapy, whereas 1 subject with mutation p.Q136P died suddenly during exertion despite this treatment. Mutations affect conserved residues located within Ca(2+)-binding loops III (p.N98S, p.N98I) or IV (p.D132E, p.D134H, p.Q136P) and caused reduced Ca(2+)-binding affinity. CONCLUSIONS: CALM2 mutations can be associated with LQTS and with overlapping features of LQTS and CPVT.

The role of microRNAs in the development of type 2 diabets

Pancreatic ß cells are highly specialized endocrine cells located within the islets of Langerhans in the pancreas. Their main role is to produce and secrete insulin, the hormone essential for the regulation of glucose homeostasis and body's metabolism. Diabetes mellitus develops when the amount of insulin released by ß cells is not sufficient to cover the metabolic demand. In type 1 diabetes (5-10% of diagnoses) insulin deficiency is caused by the autoimmune destruction of pancreatic ß cells. Type 2 diabetes (90% of diagnoses) results from a genetic predisposition and from the presence of adverse environmental conditions. The combination of these factors reduces insulin sensitivity of peripheral target tissues, causes impairment in ß-cell function and can lead to partial loss of ß cells. The development of novel therapeutic strategies for the treatment of diabetes necessitates the comprehension of the cellular processes involved in dysfunction and loss of ß cells. My thesis was focused on the involvement in the physiopathological processes leading to the development of diabetes of a class of small regulatory RNA molecules, called microRNAs (miRNAs) that post- transcriptionally regulate gene expression. Global miRNA profiling in pancreatic islets of two animal models of diabetes, the db/db mice and mice that were fed a high fat diet (HFD), characterized by obesity and insulin resistance, led us to identify two groups of miRNAs displaying expression changes under pre-diabetic and diabetic conditions. Among the miRNAs already upregulated in pre-diabetic db/db mice and HFD mice, miR- 132 was found to have beneficial effects on pancreatic ß cell function and survival. Indeed, mimicking the upregulation of miR-132 in primary pancreatic islet cells and ß-cell lines improved glucose- induced insulin secretion and favored survival of the cells upon exposure to pro-apoptotic stimuli such as palmitate and cytokines. MiR-132 was found to exert its action by enhancing the expression of MafA, a transcription factor essential for ß-cell function, survival and identity. On the other hand, up-regulation of miR-199a-5p and miR-199a-3p was detectable only in the islets of diabetic db/db mice and resulted in impaired insulin secretion and sensitization of the cells to apoptosis. MiR-199a- 5p was found to decrease insulin secretion by inducing the expression of granuphilin, a potent inhibitor of ß cell exocytosis. In contrast, miR-199a-3p was demonstrated to directly target and reduce the expression of two key ß-cell genes, mTOR and cMET, resulting in impaired ß-cell adaptation to metabolic demands and loss by apoptosis. Our findings suggest that miRNAs are important players in the onset of type 2 diabetes. MiRNA expression is adjusted in pancreatic ß cells exposed to a diabetogenic environment. These changes initially concern miRNAs responsible for adaptive processes aimed at compensating the onset of insulin resistance, but later such changes can be overlapped by modifications in the level of several additional miRNAs that favor ß-cell failure and the onset of type 2 diabetes.

Autoreactive T cells bypass negative selection and respond to self-antigen stimulation during infection.

Central and peripheral tolerance prevent autoimmunity by deleting the most aggressive CD8(+) T cells but they spare cells that react weakly to tissue-restricted antigen (TRA). To reveal the functional characteristics of these spared cells, we generated a transgenic mouse expressing the TCR of a TRA-specific T cell that had escaped negative selection. Interestingly, the isolated TCR matches the affinity/avidity threshold for negatively selecting T cells, and when developing transgenic cells are exposed to their TRA in the thymus, only a fraction of them are eliminated but significant numbers enter the periphery. In contrast to high avidity cells, low avidity T cells persist in the antigen-positive periphery with no signs of anergy, unresponsiveness, or prior activation. Upon activation during an infection they cause autoimmunity and form memory cells. Unexpectedly, peptide ligands that are weaker in stimulating the transgenic T cells than the thymic threshold ligand also induce profound activation in the periphery. Thus, the peripheral T cell activation threshold during an infection is below that of negative selection for TRA. These results demonstrate the existence of a level of self-reactivity to TRA to which the thymus confers no protection and illustrate that organ damage can occur without genetic predisposition to autoimmunity.

Male-pattern baldness susceptibility locus at 20p11.

We conducted a genome-wide association study for androgenic alopecia in 1,125 men and identified a newly associated locus at chromosome 20p11.22, confirmed in three independent cohorts (n = 1,650; OR = 1.60, P = 1.1 x 10(-14) for rs1160312). The one man in seven who harbors risk alleles at both 20p11.22 and AR (encoding the androgen receptor) has a sevenfold-increased odds of androgenic alopecia (OR = 7.12, P = 3.7 x 10(-15)).

Suggestive linkage to chromosome 1q for bone mineral apparent density in Brazilian sister adolescents.

OBJECTIVE: To investigate linkage to chromosome 1q and 11q region for lumbar spine, femoral neck and total body BMD and volumetric BMD in Brazilian sister adolescents aged 10-20-year-old and 57 mothers. METHODS: We evaluated 161 sister pairs (n=329) aged 10-20 years old and 57 of their mothers in this study. Physical traits and lifestyle factors were collected as covariates for lumbar spine (LS), femoral neck (FN) and total body (TB) BMD and bone mineral apparent density (BMAD). We selected nine microsatellite markers in chromosome 1q region (spanning nearly 33cM) and eight in chromosome 11q region (spanning nearly 34cM) to perform linkage analysis. RESULTS: The highest LOD score values obtained from our data were in sister pairs LS BMAD analysis. Their values were: 1.32 (P<0.006), 2.61 (P<0.0002) and 2.44 (P<0.0004) in D1S218, D1S2640 and D1S2623 markers, respectively. No significant LOD score was found with LS and FN BMD/BMAD in chromosome 11q region. Only TB BMD showed significant linkage higher than 1.0 for chromosome 11q region in the markers D11S4191 and D11S937. DISCUSSION/CONCLUSIONS: Our results provided suggestive linkage for LS BMAD at D1S2640 marker in adolescent sister pairs and suggest a possible candidate gene (LHX4) related to adolescent LS BMAD in this region. These results reinforce chromosome 1q21-23 as a candidate region to harbor one or more bone formation/maintenance gene. In the other hand, it did not repeat for chromosome 11q12-13 in our population.

Molecular genetics and treatment of narcolepsy.

Narcolepsy is a neurological disorder characterized by excessive daytime sleepiness and cataplexy. The hypocretin/orexin deficiency is likely to be the key to its pathophysiology in most of cases although the cause of human narcolepsy remains elusive. Acting on a specific genetic background, an autoimmune process targeting hypocretin neurons in response to yet unknown environmental factors is the most probable hypothesis in most cases of human narcolepsy with cataplexy. Although narcolepsy presents one of the tightest associations with a specific human leukocyte antigen (HLA) (DQB1*0602), there is strong evidence that non-HLA genes also confer susceptibility. In addition to a point mutation in the prepro-hypocretin gene discovered in an atypical case, a few polymorphisms in monoaminergic and immune-related genes have been reported associated with narcolepsy. The treatment of narcolepsy has evolved significantly over the last few years. Available treatments include stimulants for hypersomnia with the quite recent widespread use of modafinil, antidepressants for cataplexy, and gamma-hydroxybutyrate for both symptoms. Recent pilot open trials with intravenous immunoglobulins appear an effective treatment of cataplexy if applied at early stages of narcolepsy. Finally, the discovery of hypocretin deficiency might open up new treatment perspectives.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa.

PURPOSE: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. METHODS: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. RESULTS: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. CONCLUSIONS: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

An integrated encyclopedia of DNA elements in the human genome.

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research.

Alterations of the 5'untranslated region of SLC16A12 lead to age-related cataract.

PURPOSE. Knowledge of genetic factors predisposing to age-related cataract is very limited. The aim of this study was to identify DNA sequences that either lead to or predispose for this disease. METHODS. The candidate gene SLC16A12, which encodes a solute carrier of the monocarboxylate transporter family, was sequenced in 484 patients with cataract (134 with juvenile cataract, 350 with age-related cataract) and 190 control subjects. Expression studies included luciferase reporter assay and RT-PCR experiments. RESULTS. One patient with age-related cataract showed a novel heterozygous mutation (c.-17A>G) in the 5'untranslated region (5'UTR). This mutation is in cis with the minor G-allele of the single nucleotide polymorphism (SNP) rs3740030 (c.-42T/G), also within the 5'UTR. Using a luciferase reporter assay system, a construct with the patient's haplotype caused a significant upregulation of luciferase activity. In comparison, the SNP G-allele alone promoted less activity, but that amount was still significantly higher than the amount of the common T-allele. Analysis of SLC16A12 transcripts in surrogate tissue demonstrated striking allele-specific differences causing 5'UTR heterogeneity with respect to sequence and quantity. These differences in gene expression were mirrored in an allele-specific predisposition to age-related cataract, as determined in a Swiss population (odds ratio approximately 2.2; confidence intervals, 1.23-4.3). CONCLUSIONS. The monocarboxylate transporter SLC16A12 may contribute to age-related cataract. Sequences within the 5'UTR modulate translational efficiency with pathogenic consequences.

Biomedical risk assessment as an aid for smoking cessation.

BACKGROUND: A possible strategy for increasing smoking cessation rates could be to provide smokers who have contact with healthcare systems with feedback on the biomedical or potential future effects of smoking, e.g. measurement of exhaled carbon monoxide (CO), lung function, or genetic susceptibility to lung cancer. OBJECTIVES: To determine the efficacy of biomedical risk assessment provided in addition to various levels of counselling, as a contributing aid to smoking cessation. SEARCH METHODS: For the most recent update, we searched the Cochrane Collaboration Tobacco Addiction Group Specialized Register in July 2012 for studies added since the last update in 2009. SELECTION CRITERIA: Inclusion criteria were: a randomized controlled trial design; subjects participating in smoking cessation interventions; interventions based on a biomedical test to increase motivation to quit; control groups receiving all other components of intervention; an outcome of smoking cessation rate at least six months after the start of the intervention. DATA COLLECTION AND ANALYSIS: Two assessors independently conducted data extraction on each paper, with disagreements resolved by consensus. Results were expressed as a relative risk (RR) for smoking cessation with 95% confidence intervals (CI). Where appropriate, a pooled effect was estimated using a Mantel-Haenszel fixed-effect method. MAIN RESULTS: We included 15 trials using a variety of biomedical tests. Two pairs of trials had sufficiently similar recruitment, setting and interventions to calculate a pooled effect; there was no evidence that carbon monoxide (CO) measurement in primary care (RR 1.06, 95% CI 0.85 to 1.32) or spirometry in primary care (RR 1.18, 95% CI 0.77 to 1.81) increased cessation rates. We did not pool the other 11 trials due to the presence of substantial clinical heterogeneity. Of the remaining 11 trials, two trials detected statistically significant benefits: one trial in primary care detected a significant benefit of lung age feedback after spirometry (RR 2.12, 95% CI 1.24 to 3.62) and one trial that used ultrasonography of carotid and femoral arteries and photographs of plaques detected a benefit (RR 2.77, 95% CI 1.04 to 7.41) but enrolled a population of light smokers and was judged to be at unclear risk of bias in two domains. Nine further trials did not detect significant effects. One of these tested CO feedback alone and CO combined with genetic susceptibility as two different interventions; none of the three possible comparisons detected significant effects. One trial used CO measurement, one used ultrasonography of carotid arteries and two tested for genetic markers. The four remaining trials used a combination of CO and spirometry feedback in different settings. AUTHORS' CONCLUSIONS: There is little evidence about the effects of most types of biomedical tests for risk assessment on smoking cessation. Of the fifteen included studies, only two detected a significant effect of the intervention. Spirometry combined with an interpretation of the results in terms of 'lung age' had a significant effect in a single good quality trial but the evidence is not optimal. A trial of carotid plaque screening using ultrasound also detected a significant effect, but a second larger study of a similar feedback mechanism did not detect evidence of an effect. Only two pairs of studies were similar enough in terms of recruitment, setting, and intervention to allow meta-analyses; neither of these found evidence of an effect. Mixed quality evidence does not support the hypothesis that other types of biomedical risk assessment increase smoking cessation in comparison to standard treatment. There is insufficient evidence with which to evaluate the hypothesis that multiple types of assessment are more effective than single forms of assessment.

Common variants near MC4R are associated with fat mass, weight and risk of obesity.

To identify common variants influencing body mass index (BMI), we analyzed genome-wide association data from 16,876 individuals of European descent. After previously reported variants in FTO, the strongest association signal (rs17782313, P = 2.9 x 10(-6)) mapped 188 kb downstream of MC4R (melanocortin-4 receptor), mutations of which are the leading cause of monogenic severe childhood-onset obesity. We confirmed the BMI association in 60,352 adults (per-allele effect = 0.05 Z-score units; P = 2.8 x 10(-15)) and 5,988 children aged 7-11 (0.13 Z-score units; P = 1.5 x 10(-8)). In case-control analyses (n = 10,583), the odds for severe childhood obesity reached 1.30 (P = 8.0 x 10(-11)). Furthermore, we observed overtransmission of the risk allele to obese offspring in 660 families (P (pedigree disequilibrium test average; PDT-avg) = 2.4 x 10(-4)). The SNP location and patterns of phenotypic associations are consistent with effects mediated through altered MC4R function. Our findings establish that common variants near MC4R influence fat mass, weight and obesity risk at the population level and reinforce the need for large-scale data integration to identify variants influencing continuous biomedical traits.