Genomic rearrangements and diseases

Copy number variations (CNVs) are major contributors of genomic imbalances disorders. On the short arm of chromosome 16, CNVs of the distal 220 kb BP2-BP3 region show mirror effect on BMI and head size, and association with autism and schizophrenia, as previously reported for the proximal 600 kb BP4-BP5 deletion and duplication. These two CNVs-prone regions at 16p11.2 are also reciprocally engaged in complex chromatin looping, successfully confirmed by 4C-seq, FISH, Hi-C and concomitant expression changes, and are chromatin interactors of other loci linked to autism and/or mirror phenotypes of BMI and head circumference, for example the 2p15 cytoband. Zebrafish modeling of the BP2-BP3 duplication revealed that the overexpression of the linker for activation of T cells (LAT) induces a reduction in dividing cells in the brain and number of post-mitotic neurons in the anterior forebrain, and of intertectal axonal tracts, resulting in microcephaly, and suggested this gene as major contributor in the BP2-BP3 CNVs neurodevelopmental phenotypes. KCTD13, MVP, and MAPK3, three genes mapping within the BP4-BP5 locus and major driver and modifiers, respectively, of the head circumference phenotype linked to that region, and LAT act in additive manner to increase the severity of the microcephaly phenotype, supporting the presence of genetic interaction, in addition to proximity in 3D nuclear space, between these two loci. Smith-Magenis syndrome (SMS) is a developmental disability/multiple congenital anomaly disorder resulting from deletion at 17p11.2 that includes the RAI1 gene or a nucleotide variant in that gene. We investigated a cohort of 15 individuals with a clinical suspicion of SMS, who showed negative deletion and mutational analysis in RAI1. Potentially deleterious variants were identified in eight of these subjects using WES in KMT2D, ZEB2, MAP2K2, GLDC, CASK, MECP2, KDM5C and POGZ. Analyses of coexpression, biomedical text mining, transcriptome profiling of Rai1-/- mice and chromosome contacts suggest that these genes and RAI1 are part of the same disease network. Our 4C-seq results from 16p11.2 and 17p11.2 studies indicate that chromosomal contacts' maps can be exploited to uncover functionally and clinically related genes. These findings also encourage the integration of the results obtained from various genomic approaches to unravel complex disorders and CNVs. Abstract (French) « Structure du génôme et pathologies» La variation du nombre de copies (en anglais, Copy Number Variation, CNV) est un des contributeurs principaux à la pathogenèse des syndromes génétiques rares, mais aussi des maladies multifactorielles fréquentes. Sur le bras court du chromosome 16, les CNVs de la région distale BP2-BP3 de longueur 220 kb conduisent à un effet miroir entre sous- poids et obésité sévère, et micro- et macrocéphalies, et ils sont aussi associés avec l'autisme et la schizophrénie. Des phénotypes similaires ont été observés précédemment sur la même bande chromosomique (16p11.2) pour des délétions et duplications proximales dans la région BP4-BP5 (600 kb). Ces régions BP2-BP3 et BP4- BP5 présentent des contacts chromatiniens réciproques, confirmés avec succès par différentes techniques (4C-seq, FISH, co-régulation dans l'expression des gènes, et des données Hi-C). Elles décrivent aussi des interactions au niveau de la chromatine avec d'autres loci liés à l'autisme et/ou aux phénotypes miroir de l'IMC (Indice de Masse Corporelle) et de la circonférence de la tête, par exemple avec la bande chromosomique 2p15. La modélisation de la duplication de la région BP2-BP3 dans le poisson-zèbre a révélé que la surexpression du gene LAT (en anglais, Linker for Activation of T-cells) diminue la prolifération des cellules dans le cerveau et des neurones post-mitotiques dans le cerveau antérieur et le nombre des axones entre les tecta optiques, au début du développement embryonaire. Dans les stades suivants du développement, nous observons une microcéphalie des poissons. Tous ces éléments indiquent que ce gène est le contributeur essentiel des phénotypes neuro-développementaux des CNVs de la région BP2-BP3. KCTD13, MVP et MAPK3 sont situés dans la région BP4-BP5 et, sont, respectivement, un gène principal et deux gènes modificateurs des anomalies de la taille de la tête liée à cette région. Ces trois gènes et LAT augmentent ensemble de manière additive la gravité de la microcéphalie, en soutenant la présence d'une interaction, pas seulement dans l'espace 3D nucléaire, mais aussi génétique entre les deux loci. Le syndrome de Smith-Magenis (SMS) se caractérise par un retard mental, des dysmorphies, des troubles du comportement et du sommeil très sévères, dues à une microdélétion dans la bande 17p11.2 du chromosome 17, qui comprend le gène RAI1 ou une mutation de ce gène. Nous avons étudié une cohorte de quinze personnes avec un diagnostic de SMS, mais n'ayant pas de délétion ou mutation du gène RAI1. Par le séquençage de l'exome, des mutations potentiellement délétères ont été identifiées chez huit de ces sujets dans les gènes KMT2D, ZEB2, MAP2K2, GLDC, CASK, MECP2, KDM5C et POGZ. Les analyses de la co-expression des gènes, des données de text mining, du profilage du transcriptome des souris Rai1-/- et des contacts chromatiniens font penser que ces gènes et RAI1 font partie du même « disease network ». Les résultats de 4C-seq obtenus par les études des bandes 16p11.2 et 17p11.2 indiquent que les contacts chromosomiques peuvent être exploitées pour découvrir des gènes liés d'un point de vue fonctionnel et clinique. Ces résultats encouragent également l'intégration des données obtenues à partir de différentes approches génomiques pour démêler des troubles complexes et les larges CNVs

Identification of a RAI1-associated disease network through integration of exome sequencing, transcriptomics, and 3D genomics.

Smith-Magenis syndrome (SMS) is a developmental disability/multiple congenital anomaly disorder resulting from haploinsufficiency of RAI1. It is characterized by distinctive facial features, brachydactyly, sleep disturbances, and stereotypic behaviors. We investigated a cohort of 15 individuals with a clinical suspicion of SMS who showed neither deletion in the SMS critical region nor damaging variants in RAI1 using whole exome sequencing. A combination of network analysis (co-expression and biomedical text mining), transcriptomics, and circularized chromatin conformation capture (4C-seq) was applied to verify whether modified genes are part of the same disease network as known SMS-causing genes. Potentially deleterious variants were identified in nine of these individuals using whole-exome sequencing. Eight of these changes affect KMT2D, ZEB2, MAP2K2, GLDC, CASK, MECP2, KDM5C, and POGZ, known to be associated with Kabuki syndrome 1, Mowat-Wilson syndrome, cardiofaciocutaneous syndrome, glycine encephalopathy, mental retardation and microcephaly with pontine and cerebellar hypoplasia, X-linked mental retardation 13, X-linked mental retardation Claes-Jensen type, and White-Sutton syndrome, respectively. The ninth individual carries a de novo variant in JAKMIP1, a regulator of neuronal translation that was recently found deleted in a patient with autism spectrum disorder. Analyses of co-expression and biomedical text mining suggest that these pathologies and SMS are part of the same disease network. Further support for this hypothesis was obtained from transcriptome profiling that showed that the expression levels of both Zeb2 and Map2k2 are perturbed in Rai1 (-/-) mice. As an orthogonal approach to potentially contributory disease gene variants, we used chromatin conformation capture to reveal chromatin contacts between RAI1 and the loci flanking ZEB2 and GLDC, as well as between RAI1 and human orthologs of the genes that show perturbed expression in our Rai1 (-/-) mouse model. These holistic studies of RAI1 and its interactions allow insights into SMS and other disorders associated with intellectual disability and behavioral abnormalities. Our findings support a pan-genomic approach to the molecular diagnosis of a distinctive disorder

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.Molecular Psychiatry advance online publication, 31 May 2016; doi:10.1038/mp.2016.84

The Immune Signaling Adaptor LAT Contributes to the Neuroanatomical Phenotype of 16p11.2 BP2-BP3 CNVs

Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%-1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes.PMC563023

Identification of a RAI1-associated disease network through integration of exome sequencing, transcriptomics, and 3D genomics

Abstract Background Smith-Magenis syndrome (SMS) is a developmental disability/multiple congenital anomaly disorder resulting from haploinsufficiency of RAI1. It is characterized by distinctive facial features, brachydactyly, sleep disturbances, and stereotypic behaviors. Methods We investigated a cohort of 15 individuals with a clinical suspicion of SMS who showed neither deletion in the SMS critical region nor damaging variants in RAI1 using whole exome sequencing. A combination of network analysis (co-expression and biomedical text mining), transcriptomics, and circularized chromatin conformation capture (4C-seq) was applied to verify whether modified genes are part of the same disease network as known SMS-causing genes. Results Potentially deleterious variants were identified in nine of these individuals using whole-exome sequencing. Eight of these changes affect KMT2D, ZEB2, MAP2K2, GLDC, CASK, MECP2, KDM5C, and POGZ, known to be associated with Kabuki syndrome 1, Mowat-Wilson syndrome, cardiofaciocutaneous syndrome, glycine encephalopathy, mental retardation and microcephaly with pontine and cerebellar hypoplasia, X-linked mental retardation 13, X-linked mental retardation Claes-Jensen type, and White-Sutton syndrome, respectively. The ninth individual carries a de novo variant in JAKMIP1, a regulator of neuronal translation that was recently found deleted in a patient with autism spectrum disorder. Analyses of co-expression and biomedical text mining suggest that these pathologies and SMS are part of the same disease network. Further support for this hypothesis was obtained from transcriptome profiling that showed that the expression levels of both Zeb2 and Map2k2 are perturbed in Rai1 –/– mice. As an orthogonal approach to potentially contributory disease gene variants, we used chromatin conformation capture to reveal chromatin contacts between RAI1 and the loci flanking ZEB2 and GLDC, as well as between RAI1 and human orthologs of the genes that show perturbed expression in our Rai1 –/– mouse model. Conclusions These holistic studies of RAI1 and its interactions allow insights into SMS and other disorders associated with intellectual disability and behavioral abnormalities. Our findings support a pan-genomic approach to the molecular diagnosis of a distinctive disorder