Chromatin three-dimensional interactions mediate genetic effects on gene expression.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

Intellectual developmental disorder with cardiac arrhythmia syndrome in a child with compound heterozygous GNB5 variants.

Identification of a novel compound heterozygous of GNB5 in a patient with intellectual developmental disorder with cardiac arrhytmia (IDDCA), from non-consaguineous family. Three-dimensional modelling and in silico predictions suggest that GNB5 variants are causative of the phenotype, extending the number of IDDCA patients so far identified.

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.

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.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

[Screening for anti-glycolipid antibody profiles from patients with immune-mediated peripheral neuropathies by Dotzen Ganglio Profile Antibodies]. / Les anticorps anti-glycolipides dans le diagnostic des neuropathies périphériques auto-immunes par la technique Dotzen Ganglio Profile Antibodies.

The presence of anti-glycolipid specific antibodies have been found to be associated with acute and chronic immune-mediated peripheral neuropathies. Recently a number of anti-glycolipid antibody assays have became commercially available. In this study we established specific anti-glycolipid antibody profiles in a series of sera by the Dotzen Ganglio Profile antibodies. This kit screens for the simultaneous detection of ten anti-glycolipid antibodies against GM3, GM2, GM1, GD3, GD1a, GD1b, GT1a, GT1b, GQ1b gangliosides and sulfatides of the IgM and IgG classes. Sera from 89 patients with acute and chronic neuropathies were selected in a well-characterized cohort of banked sera with anti-glycolipid antibody profiles identified by in-house immunodot assay. Serum from 52 clinical variants of Guillain-Barré syndrome with IgG autoantibody profiles and 37 chronic acquired peripheral neuropathy with IgM autoantibody profiles were tested. The assay correctly identified with good agreement 50 of 52 IgG antibody profiles and 32 of 37 IgM antibody profiles. The assay compared well with in-house immunodot assay. It is easy to screen 10 crossreacting glycolipid antibodies to establish specific antibody profiles to define different subgroups of immune-mediated peripheral neuropathies for classification and immune management.

Two high throughput technologies to detect segmental aneuploidies identify new Williams-Beuren syndrome patients with atypical deletions.

OBJECTIVE: To develop and compare two new technologies for diagnosing a contiguous gene syndrome, the Williams-Beuren syndrome (WBS). METHODS: The first proposed method, named paralogous sequence quantification (PSQ), is based on the use of paralogous sequences located on different chromosomes and quantification of specific mismatches present at these loci using pyrosequencing technology. The second exploits quantitative real time polymerase chain reaction (QPCR) to assess the relative quantity of an analysed locus. RESULTS: A correct and unambiguous diagnosis was obtained for 100% of the analysed samples with either technique (n = 165 and n = 155, respectively). These methods allowed the identification of two patients with atypical deletions in a cohort of 182 WBS patients. Both patients presented with mild facial anomalies, mild mental retardation with impaired visuospatial cognition, supravalvar aortic stenosis, and normal growth indices. These observations are consistent with the involvement of GTF2IRD1 or GTF2I in some of the WBS facial features. CONCLUSIONS: Both PSQ and QPCR are robust, easy to interpret, and simple to set up. They represent a competitive alternative for the diagnosis of segmental aneuploidies in clinical laboratories. They have advantages over fluorescence in situ hybridisation or microsatellites/SNP genotyping for detecting short segmental aneuploidies as the former is costly and labour intensive while the latter depends on the informativeness of the polymorphisms.

From PREDs and open reading frames to cDNA isolation: revisiting the human chromosome 21 transcription map.

A supernumerary copy of human chromosome 21 (HC21) causes Down syndrome. To understand the molecular pathogenesis of Down syndrome, it is necessary to identify all HC21 genes. The first annotation of the sequence of 21q confirmed 127 genes, and predicted an additional 98 previously unknown "anonymous" genes (predictions (PREDs) and open reading frames (C21orfs)), which were foreseen by exon prediction programs and/or spliced expressed sequence tags. These putative gene models still need to be confirmed as bona fide transcripts. Here we report the characterization and expression pattern of the putative transcripts C21orf7, C21orf11, C21orf15, C21orf18, C21orf19, C21orf22, C21orf42, C21orf50, C21orf51, C21orf57, and C21orf58, the GC-rich sequence DNA-binding factor candidate GCFC (also known as C21orf66), PRED12, PRED31, PRED34, PRED44, PRED54, and PRED56. Our analysis showed that most of the C21orfs originally defined by matching spliced expressed sequence tags were correctly predicted, whereas many of the PREDs, defined solely by computer prediction, do not correspond to genuine genes. Four of the six PREDs were incorrectly predicted: PRED44 and C21orf11 are portions of the same transcript, PRED31 is a pseudogene, and PRED54 and PRED56 were wrongly predicted. In contrast, PRED12 (now called C21orf68) and PRED34 (C21orf63) are now confirmed transcripts. We identified three new genes, C21orf67, C21orf69, and C21orf70, not previously predicted by any programs. This revision of the HC21 transcriptome has consequences for the entire genome regarding the quality of previous annotations and the total number of transcripts. It also provides new candidates for genes involved in Down syndrome and other genetic disorders that map to HC21.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

The tripartite motif family identifies cell compartments.

A functional genomic approach, based on systematic data gathering, was used to characterize a family of proteins containing a tripartite motif (TRIM). A total of 37 TRIM genes/proteins were studied, 21 of which were novel. The results demonstrate that TRIM proteins share a common function: by means of homo-multimerization they identify specific cell compartments.

WBSCR14, a gene mapping to the Williams--Beuren syndrome deleted region, is a new member of the Mlx transcription factor network.

Williams-Beuren syndrome (WBS) is a developmental disorder associated with haploinsufficiency of multiple genes at 7q11.23. Here, we report the functional characterization of WBS critical region gene 14 (WBSCR14), a gene contained in the WBS commonly deleted region. It encodes a basic-helix--loop--helix leucine zipper (bHLHZip) transcription factor of the Myc/Max/Mad superfamily. WBSCR14 is expressed in multiple tissues, including regions of the brain and the intestinal tract. WBSCR14 forms heterodimers with the bHLHZip protein Mlx to bind the DNA sequence CACGTG. Like Max, Mlx has no intrinsic transcriptional activity, but its association with Mad1, Mad4, Mnt or WBSCR14 can repress E-box-dependent transcription. Preliminary results suggest a possible role of WBSCR14 in growth control. Our data support the view that the Max-like bHLHZip protein, Mlx, is a key element of a transcription factor network. We thus suggest that WBSCR14 may contribute to some aspects of the WBS pathology.

The murine orthologue of the Golgi-localized TPTE protein provides clues to the evolutionary history of the human TPTE gene family.

The human TPTE gene encodes a testis-specific protein that contains four potential transmembrane domains and a protein tyrosine phosphatase motif, and shows homology to the tumor suppressor PTEN/MMAC1. Chromosomal mapping revealed multiple copies of the TPTE gene present on the acrocentric chromosomes 13, 15, 21 and 22, and the Y chromosome. Zooblot analysis suggests that mice may possess only one copy of TPTE. In the present study, we report the isolation and initial characterization of the full-length cDNA of the mouse homologue Tpte. At least three different mRNA transcripts ( Tpte.a, b, c) are produced via alternative splicing, encoding predicted proteins that would contain four potential transmembrane domains and a protein tyrosine phosphatase motif. Transfection of a 5'EGFP-TPTE fusion protein in Hela cells revealed an intracellular localization within the Golgi apparatus. Tpte was mapped by radiation hybrid to a region of mouse chromosome 8 that shows conserved synteny with human 13q14.2-q21 between NEK3 and SGT1. This region of the human genome was found to contain a partial, highly diverged copy of TPTE that is likely to represent the ancestral copy from which the other copies of TPTE arose through duplication events. The Y chromosome copy of TPTE is a pseudogene and is not therefore involved in the testis expression of this gene family.

Mlx, a new Max-like bHLHZip family member: the center stage of a novel transcription factors regulatory pathway?

The Myc proto-oncogene family members have been identified as the cellular homologs of the transforming oncogene of avian retroviruses. They encode central regulators of mammalian cell proliferation and apoptosis, and they associate with the bHLHZip protein Max to bind specific DNA sequences and regulate the expression of genes important for cell cycle progression. The other family members, Mad1, Mxi1, Mad3, Mad4 and Rox (Mnt) antagonize their activities. The Mads and Rox compete with Myc in heterodimerizing with Max and in binding to the same specific target sequences. These Mads:Max and Rox:Max dimers repress transcription through binding to the mSIN3 corepressor protein and by tethering histone deacetylase-containing complexes to the DNA. In a screen for Rox interactors we isolated Mlx, a bHLHZip protein previously identified in a screen for Mad1 interactors. In the present work we extend the known dimerization partners of Mlx by demonstrating its ability to interact with Rox. Moreover, we show that contrary to previous reports Mlx is able to homodimerize and to bind E-box sequences at low concentration levels. The possible role of Mlx in an emerging regulatory pathway and acting parallel to the Max driven network is discussed.

Evidence for interaction between human PRUNE and nm23-H1 NDPKinase.

We have isolated a human and murine homologue of the Drosophila prune gene through dbEST searches. The gene is ubiquitously expressed in human adult tissues, while in mouse developing embryos a high level of expression is confined to the nervous system particularly in the dorsal root ganglia, cranial nerves, and neural retina. The gene is composed of eight exons and is located in the 1q21.3 chromosomal region. A pseudogene has been sequenced and mapped to chromosomal region 13q12. PRUNE protein retains the four characteristic domains of DHH phosphoesterases. The synergism between prune and awdK-pn in Drosophila has led various authors to propose an interaction between these genes. However, such an interaction has never been supported by biochemical data. By using interaction-mating and in vitro co-immunoprecipitation experiments, we show for the first time the ability of human PRUNE to interact with the human homologue of awd protein (nm23-H1). In contrast, PRUNE is impaired in its interaction with nm-23-H1-S120G mutant, a gain-of-function mutation associated with advanced neuroblastoma stages. Consistently, PRUNE and nm23-H1 proteins partially colocalize in the cytoplasm. The data presented are consistent with the view that PRUNE acts as a negative regulator of the nm23-H1 protein. We discuss how PRUNE regulates nm23-H1 protein and postulate possible implications of PRUNE in neuroblastoma progression.

Molecular cloning and characterization of a novel retinoblastoma-binding protein.

We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein. This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain. Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals. Rim mRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines. The Rim gene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2.

The human ROX gene: genomic structure and mutation analysis in human breast tumors.

We have recently isolated a human gene, ROX, encoding a new member of the basic helix-loop-helix leucine zipper protein family. ROX is capable of heterodimerizing with Max and acts as a transcriptional repressor in an E-box-driven reporter gene system, while it was found to activate transcription in HeLa cells. ROX expression levels vary during the cell cycle, being down-regulated in proliferating cells. These biological properties of ROX suggest a possible involvement of this gene in cell proliferation and differentiation. The ROX gene maps to chromosome 17p13.3, a region frequently deleted in human malignancies. Here we report the genomic structure of the human ROX gene, which is composed of six exons and spans a genomic region of less than 40 kb. In an attempt to identify possible inactivating mutations in the ROX gene in human breast cancer, we performed a single-strand conformation polymorphism analysis of its coding region in 16 sporadic breast carcinomas showing loss of heterozygosity in the 17p13.3 region. No mutations were found in this analysis. Five nucleotide polymorphisms were identified in the ROX gene, three of which caused an amino acid substitution. These nucleotide changes were present in the peripheral blood DNAs of both the patients and the control individuals. In vitro translated assays did not show a significant decrease in the ability of the ROX mutant proteins to bind DNA or to repress transcription of a driven reporter gene in HEK293 cells. Despite experimental evidence that ROX might act as a tumor suppressor gene, our data suggest that mutations in the coding region of ROX are uncommon in human breast tumorigenesis.

Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor.

Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. We report the isolation, characterization and mapping of a human gene and its mouse homolog encoding a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, we demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. We demonstrate that repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia.