Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes.

Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts' maps could uncover functionally and clinically related genes.

New quality measure for SNP array based CNV detection.

MOTIVATION: Only a few large systematic studies have evaluated the impact of copy number variants (CNVs) on common diseases. Several million individuals have been genotyped on single nucleotide variation arrays, which could be used for genome-wide CNVs association studies. However, CNV calls remain prone to false positives and only empirical filtering strategies exist in the literature. To overcome this issue, we defined a new quality score (QS) estimating the probability of a CNV called by PennCNV to be confirmed by other software. RESULTS: Out-of-sample comparison showed that the correlation between the consensus CNV status and the QS is twice as high as it is for any previously proposed CNV filters. ROC curves displayed an AUC higher than 0.8 and simulations showed an increase up to 20% in statistical power when using QS in comparison to other filtering strategies. Superior performance was confirmed also for alternative consensus CNV definition and through improving known CNV-trait associations. AVAILABILITY AND IMPLEMENTATION: http://goo.gl/T6yuFM CONTACT: zoltan.kutalik@unil.ch or aurelien@mace@unil.chSupplementary information: Supplementary data are available at Bioinformatics online.

Individual differences in impulsive action and dopamine transporter function in rat orbitofrontal cortex.

Impulsivity, which can be subdivided into impulsive action and impulsive choice, is implicated as a factor underlying drug abuse vulnerability. Although previous research has shown that dopamine (DA) systems in prefrontal cortex are involved in impulsivity and substance abuse, it is not known if inherent variation in DA transporter (DAT) function contributes to impulsivity. The current study determined if individual differences in either impulsive action or impulsive choice are related to DAT function in orbitofrontal (OFC) and/or medial prefrontal cortex (mPFC). Rats were first tested both for impulsive action in a cued go/no-go task and for impulsive choice in a delay-discounting task. Following behavioral evaluation, in vitro [(3)H]DA uptake assays were performed in OFC and mPFC isolated from individual rats. Vmax in OFC, but not mPFC, was correlated with performance in the cued go/no-go task, with decreased OFC DAT function being associated with high impulsive action. In contrast, Vmax in OFC and mPFC was not correlated with performance in the delay-discounting task. The current results demonstrate that impulsive behavior in cued go/no-go performance is associated with decreased DAT function in OFC, suggesting that hyperdopaminergic tone in this prefrontal subregion mediates, at least in part, increased impulsive action.

16p11.2 Locus modulates response to satiety before the onset of obesity.

BACKGROUND: The 600 kb BP4-BP5 copy number variants (CNVs) at the 16p11.2 locus have been associated with a range of neurodevelopmental conditions including autism spectrum disorders and schizophrenia. The number of genomic copies in this region is inversely correlated with body mass index (BMI): the deletion is associated with a highly penetrant form of obesity (present in 50% of carriers by the age of 7 years and in 70% of adults), and the duplication with being underweight. Mechanisms underlying this energy imbalance remain unknown. OBJECTIVE: This study aims to investigate eating behavior, cognitive traits and their relationships with BMI in carriers of 16p11.2 CNVs. METHODS: We assessed individuals carrying a 16p11.2 deletion or duplication and their intrafamilial controls using food-related behavior questionnaires and cognitive measures. We also compared these carriers with cohorts of individuals presenting with obesity, binge eating disorder or bulimia. RESULTS: Response to satiety is gene dosage-dependent in pediatric CNV carriers. Altered satiety response is present in young deletion carriers before the onset of obesity. It remains altered in adolescent carriers and correlates with obesity. Adult deletion carriers exhibit eating behavior similar to that seen in a cohort of obesity without eating disorders such as bulimia or binge eating. None of the cognitive measures are associated with eating behavior or BMI. CONCLUSIONS: These findings suggest that abnormal satiety response is a strong contributor to the energy imbalance in 16p11.2 CNV carriers, and, akin to other genetic forms of obesity, altered satiety responsiveness in children precedes the increase in BMI observed later in adolescence.

The 16p11.2 locus modulates brain structures common to autism, schizophrenia and obesity.

Anatomical structures and mechanisms linking genes to neuropsychiatric disorders are not deciphered. Reciprocal copy number variants at the 16p11.2 BP4-BP5 locus offer a unique opportunity to study the intermediate phenotypes in carriers at high risk for autism spectrum disorder (ASD) or schizophrenia (SZ). We investigated the variation in brain anatomy in 16p11.2 deletion and duplication carriers. Beyond gene dosage effects on global brain metrics, we show that the number of genomic copies negatively correlated to the gray matter volume and white matter tissue properties in cortico-subcortical regions implicated in reward, language and social cognition. Despite the near absence of ASD or SZ diagnoses in our 16p11.2 cohort, the pattern of brain anatomy changes in carriers spatially overlaps with the well-established structural abnormalities in ASD and SZ. Using measures of peripheral mRNA levels, we confirm our genomic copy number findings. This combined molecular, neuroimaging and clinical approach, applied to larger datasets, will help interpret the relative contributions of genes to neuropsychiatric conditions by measuring their effect on local brain anatomy.

Genetic testing in patients with obesity.

The obesity epidemic is associated with the recent availability of highly palatable and inexpensive caloric food as well as important changes in lifestyle. Genetic factors, however, play a key role in regulating energy balance and numerous twin studies have estimated the BMI heritability between 40 and 70%. While common variants, identified through genome-wide association studies (GWAS) point toward new pathways, their effect size are too low to be of any use in the clinic. This review therefore concentrates on genes and genomic regions associated with very high risks of human obesity. Although there are no consensus guidelines, we review how the knowledge on these "causal factors" can be translated into the clinic for diagnostic purposes. We propose genetic workups guided by clinical manifestations in patients with severe early-onset obesity. While etiological diagnoses are unequivocal in a minority of patients, new genomic tools such as Comparative Genomic Hybridization (CGH) array, have allowed the identification of novel "causal" loci and next-generation sequencing brings the promise of accelerated pace for discoveries relevant to clinical practice.

JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.

AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. METHODS: Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. RESULTS: JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

Penetrance of marked cognitive impairment in older male carriers of the FMR1 gene premutation.

BACKGROUND: Male carriers of the FMR1 premutation are at risk of developing the fragile X-associated tremor/ataxia syndrome (FXTAS), a newly recognised and largely under-diagnosed late onset neurodegenerative disorder. Patients affected with FXTAS primarily present with cerebellar ataxia and intention tremor. Cognitive decline has also been associated with the premutation, but the lack of data on its penetrance is a growing concern for clinicians who provide genetic counselling. METHODS: The Mattis Dementia Rating Scale (MDRS) was administered in a double blind fashion to 74 men aged 50 years or more recruited from fragile X families (35 premutation carriers and 39 intrafamilial controls) regardless of their clinical manifestation. Based on previous publications, marked cognitive impairment was defined by a score

The Wnt receptor FZD1 mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

CNVs and genetic medicine (excitement and consequences of a rediscovery).

The extensive variability of individual human genomes contributes to phenotypic variability. Structural genomic variants, and copy number variants (CNVs) in particular, have recently been rediscovered as contributors to the genomic plasticity and evolution and as pathoetiologic elements for both monogenic and complex traits. Herein we review some of the consequences of CNVs in the context of human inherited diseases.

The c-Jun N-terminal kinase JNK participates in cytokine- and isolation stress-induced rat pancreatic islet apoptosis.

AIMS/HYPOTHESIS: The protocols used for the preparation of human pancreatic islets immediately induce a sustained and massive activation of the c-Jun-N-terminal kinase (JNK). JNK, which participates in apoptosis of insulin-secreting cells, is activated by mechanical stresses, as well as by exposure to pro-inflammatory cytokines. Here, we investigated whether the delivery of a protease-resistant JNK inhibitory peptide (D-JNKI) through a protein transduction system during pancreatic digestion might impair JNK signalling throughout the transplantation procedure. METHODS: Rat pancreases were treated with D-JNKI through the pancreatic duct and cells then isolated by enzymatic digestion. Protein extracts were prepared to determine JNK activity by kinase assays and total RNA was extracted to measure gene expressions by a Light-Cycler technique. Cell apoptosis rate was determined by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and by scoring cells displaying pycnotic nuclei. RESULTS: Our data establish that the peptide transduction system used here efficiently transfects islets, allowing for stable in vivo (up to 2 days) transfection of human islets transplanted under the kidney capsule. Further, D-JNKI decreases intracellular JNK signalling during isolation and following cytokine exposure in both human and rat islets, as measured by kinase assays and reduced c-fos expression; D-JNKI also confers protection against apoptosis induced during the rat islet preparation and subsequent to IL-1beta exposure. CONCLUSIONS/INTERPRETATION: JNK signalling participates in islet isolation- and IL-1beta-induced apoptosis in rat islets. Furthermore, the system we used might be more generally applicable for the persistent blockage (several days) of pro-apoptotic pathways in the transplanted islets; this days-long protection might potentially be an absolute prerequisite to help transplanted islets better survive the first wave of the non-specific inflammatory attack.

ATM haplotypes and breast cancer risk in Jewish high-risk women.

While genetic factors clearly play a role in conferring breast cancer risk, the contribution of ATM gene mutations to breast cancer is still unsettled. To shed light on this issue, ATM haplotypes were constructed using eight SNPs spanning the ATM gene region (142 kb) in ethnically diverse non-Ashkenazi Jewish controls (n=118) and high-risk (n=142) women. Of the 28 haplotypes noted, four were encountered in frequencies of 5% or more and accounted for 85% of all haplotypes. Subsequently, ATM haplotyping of high-risk, non-Ashkenazi Jews was performed on 66 women with breast cancer and 76 asymptomatic. One SNP (rs228589) was significantly more prevalent among breast cancer cases compared with controls (P=4 x 10(-9)), and one discriminative ATM haplotype was significantly more prevalent among breast cancer cases (33.3%) compared with controls (3.8%), (P< or =10(-10)). There was no significant difference in the SNP and haplotype distribution between asymptomatic high-risk and symptomatic women as a function of disease status. We conclude that a specific ATM SNP and a specific haplotype are associated with increased breast cancer risk in high-risk non-Ashkenazi Jews.

Confirmation of the origin of NISCH syndrome.

Neonatal ichthyosis-sclerosing cholangitis (NISCH) syndrome, a rare autosomal recessive ichthyosis syndrome characterized by scalp hypotrichosis, scarring alopecia, ichthyosis, and sclerosing cholangitis, was described for the first time in 2002. It is caused by a mutation in the gene coding for the tight junction protein claudin-1. Only four patients carrying the same mutation of the CLDN1 gene have been described until now. We report a patient presenting with the clinical characteristics of NISCH syndrome and carrying a novel mutation in the CLDN1 gene.

Genetic characterization of CHO production host DG44 and derivative recombinant cell lines.

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.

Circadian regulation of islet genes involved in insulin production and secretion.

Both transcription factors albumin site d-binding protein (DBP) and thyrotroph embryonic factor (TEF) are elements of the "cell-clock". Their circadian accumulation in suprachiasmatic nucleus (SCN) and peripheral tissues such as liver, kidney and lung is thought to participate in controlling circadian regulation of downstream genes. TEF and DBP control elements have never been investigated in the insulin-secreting cells, but impairment of the circadian rhythm of the beta-cells might be involved in the development of diabetic state as type 2 diabetics have lost daily temporal variations of insulin secretion. We investigated the expression pattern of TEF and DBP in insulin-secreting cells. TEF and DBP transcripts are expressed at extremely high levels in human pancreatic islets compared to other tissues, suggesting a potentially important circadian regulation of these cells. Both TEF and DPB accumulate in a circadian way in insulin-secreting cells after a serum shock known to restore circadian rhythms in cultured cells. In addition, the expression of islet-specific genes involved in glucose sensing (glucose transporter 2 (Glut2), glucokinase), insulin production (insulin) and secretion (migration inhibitory factor (MIF), somatostatin and syntaxin 1A) were modulated in the same daily rhythm as well. The circadian deregulation of these genes could therefore participate in the diabetic state development.

Genomic profiling of interpopulation diversity guides prioritization of candidate-genes for autoimmunity.

Autoimmune diseases seem to have strong genetic attributes, and are affected to some extent by shared susceptibility loci. The latter potentially amount to hundreds of candidate genes (CG), creating the need for a prioritization strategy in genetic association studies. To form such a strategy, 26 autoimmune-related CG were genotyped for a total of 72 single nucleotide polymorphisms (SNPs) in three distinct Israeli ethnic populations: Ashkenazi Jews, Sephardic Jews and Arabs. Four quantitative criteria reflecting population stratification were analyzed: allele frequencies, haplotype frequencies, the Fst statistic for homozygotes distribution and linkage disequilibrium extents. According to the consequent interpopulation genomic diversity profiles, the genes were classified into conserved, intermediate and diversified gene groups. Our results demonstrate a correlation between the biological role of autoimmune-related CG and their interpopulation diversity profiles as classified by the different analyses. Annotation analysis suggests that genes more readily influenced by environmental conditions, such as immunological mediators, are 'population specific'. Conversely, genes showing genetic conservation across all populations are characterized by apoptotic and cleaving functions. We suggest a research strategy by which CG association studies should focus first on likely conserved gene categories, to increase the likelihood of attaining significant results and promote the development of gene-based therapies.