A Novel Dominant Mesomelic Dysplasia Associated with A 1.0-Mb Microduplication of HOXD Gene Cluster at 2q31.1
ABSTRACT: A three-generation family with four patients affected by a mesomelic dysplasia was investigated for genome-wide DNA copy number variation profiles. This revealed a microduplication of a 1.0-Mb chromosomal segment at 2q31.1 spanning nine Homeo box D (HOXD) genes that co-segregated with the phenotype. Quantitative PCR analysis of a gene within this duplicated region showed consistent results. A phenotypically similar condition, mesomelic dysplasia Kantaputra type (MDKa; MIM 156232), has also been mapped to this chromosomal region, raising the possibility that MDKa and the condition observed in this family may be allelic. Overall design: three-generation family including four affected individuals and two unaffected individuals
INSTRUMENT(S): Agilent-022060 SurePrint G3 Human CGH Microarray 4x180K (Probe Name version)
Project description:A three-generation family with four patients affected by a mesomelic dysplasia was investigated for genome-wide DNA copy number variation profiles. This revealed a microduplication of a 1.0-Mb chromosomal segment at 2q31.1 spanning nine Homeo box D (HOXD) genes that co-segregated with the phenotype. Quantitative PCR analysis of a gene within this duplicated region showed consistent results. A phenotypically similar condition, mesomelic dysplasia Kantaputra type (MDKa; MIM 156232), has also been mapped to this chromosomal region, raising the possibility that MDKa and the condition observed in this family may be allelic. three-generation family including four affected individuals and two unaffected individuals
Project description:Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex DUP-TRP/INV-DUP rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals – 16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology—or homeology—driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication, and potentially higher order amplification of a genomic interval, can occur in a manner consistent with rolling circle amplification as predicted by the microhomology mediated break induced replication (MMBIR) model. To determine size, genomic extent and gene content for each rearrangement, we used a customized tiling-path oligonucleotide microarray spanning the Xq22 chromosomal region to query the genomic DNA of 7 males with Pelizaeus-Merzbacher disease, and 5 unaffected or carrier/unaffected family members. A 4x44k Agilent Technologies (Santa Clara, CA) microarray was designed using the Agilent e-array website targeting the region of the genome encompassing the dosage-sensitive PLP1 gene.
Project description:Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches. Overall design: anti-H3k4me3 ChIP-seq; 12 samples. 3 Kansl1 +/- , 3 Del/+, 6 wts
Project description:Here we describe an interstitial pure duplication of 19p13.3 that was initially considered as a de novo alteration, in a patient with intellectual disability studied by array-CGH. The finding of the same chromosomal alteration in a first-degree cousin of this patient led us to investigate the presence of insertional translocations. An intrachromosomal insertional translocation was found in at least three generations. Three intellectually disabled patients with the same duplication and multiples abortions among translocation carrier family members were found. A review of other published cases has allowed us to find three other cases with a similar pure duplication and some clinical findings present in all patients as intrauterine growth retardation, microcephaly, motor and speech delay, moderate to severe intellectual disability and dysmorphic features. These findings allow us to suggest the existence of a new microduplication syndrome in chromosomal region 19p13.3. Whole genome array-CGH was performed on Agilent oligo-chip 44K (human genome CGH microarray G4410B from Agilent Technologies, Palo Alto, CA) as recommended. The patients’ DNA samples were tested against a pool of 10 sex-matched normal DNA samples, all of them (patients and normal controls) from our geographical area. Scanned images were quantified using Agilent Feature Extraction Software (v9.0). The results were analyzed using Agilent Genomic Workbench software. Annotations have been obtained from UCSC (http://genome.ucsc.edu) based on human genome build GRCh37/hg19.
Project description:Bypass of Ess1 (Bye1) is a nuclear protein with a domain resembling the central domain in the transcription elongation factor TFIIS. Bye1 binds with its TFIIS-like domain (TLD) to RNA polymerase (Pol) II. Using a Bye1-TAP strain ChIP-chip was performed to examine the genome-wide association of Bye1 with chromatin in vivo. Bye1 is recruited to chromatin via its TLD and occupies the 5'-region of active genes. A plant homeo domain (PHD) in Bye1 binds histone H3 tails with trimethylated lysine 4, and this interaction is enhanced by the presence of neighboring posttranslational modifications (PTMs) that mark active transcription and conversely is impaired by repressive PTMs. We identify putative human homologs of Bye1, the proteins PHD finger protein 3 and death-inducer obliterator, which are both implicated in cancer. These results establish Bye1 as the founding member of a unique family of chromatin transcription factors that link histones with active PTMs to transcribing Pol II.
Project description:Here we present a high-density in situ synthesized microarray for Ovis aries, named Aristaeus, designed by means of a pipeline of software instruments that, starting from non-annotated redundant EST sequences, selects oligonucleotides suitable for in situ generation on chip. The chip was tested by comparing the gene expression profiles of two sheep breeds with different phenotype, Sarda and Gentile di Puglia. We carried out microarray experiments on liver and udder tissues from lactating individuals and identified a relevant number of differentially expressed genes, all involved in metabolism pathways. The results are consistent with literature knowledge, while selected differential gene expressions have been confirmed by quantitative real-time polymerase chain reaction analyses. Tissue samples of liver were collected from 4 lactating individuals of two sheep (Ovis aries) breeds, Gentile di Puglia and Sarda. Biopsies of liver tissue were taken at second lactation stage (first record, stage 01: 6 days after lambing; second record, stage 02: 44 days after lambing) in both breeds. Tissues from liver were immersed in RNAlater (Sigma) immediately after biopsy and stored at -20°C. Samples were pooled by breed and then reverse labeled (cy5 and cy3), resulting in four raw data sets.
Project description:Down syndrome (DS) (MIM 190685) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the first genetic cause of mental retardation, the molecular bases of the syndrome are still largely unknown. We analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. RNA from six DS and six control LCLs was independently hybridized on the Affymetrix HU133 plus 2.0 oligonucleotide array (Affymetrix, Santa Clara, CA).The experimental design was a diseased versus control comparison. In a second approach, variability of gene expression among the DS and control group, respectively, was assessed by the analysis of the coefficient of variation (CV), calculated as the ratio between standard deviation and mean expression level for each gene among samples.
Project description:Purpose: To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1). Methods: Next-generation sequencing was performed on DNA samples from 4 affected and 4 unaffected members of a previously reported family with PPCD1 linked to chromosome 20 between D20S182 and D20S195. Custom capture probes were utilized for targeted region capture of the linked interval. Single nucleotide variants (SNVs) and insertions/deletions (indels) were identified using two bioinformatics pipelines and two annotation databases. Candidate variants met the following criteria: quality score ≥20, read depth ≥5X, heterozygous, novel or rare (minor allele frequency (MAF) ≤ 0.05), present in each affected individual and absent in each unaffected individual. Structural variants were detected with two different microarray platforms to identify indels of varying sizes. Results: Sequencing reads aligned to the linked region on chromosome 20, and high coverage was obtained across the sequenced region. The majority of identified variants were detected with both pipelines and annotation databases, although unique variants were identified. Twelve SNVs in 10 genes (2 synonymous variants and 10 noncoding variants) and 9 indels in 7 genes met the filtering criteria and were considered candidate variants for PPCD1. Conclusions: Next-generation sequencing of the PPCD1 interval has identified 17 genes containing novel or rare SNVs and indels that segregate with the affected phenotype in an affected family previously mapped to the PPCD1 locus. We anticipate that screening of these candidate genes in other families previously mapped to the PPCD1 locus will result in the identification of the genetic basis of PPCD1. Four affected and 4 unaffected individuals from a single family were analyzed for copy number variation within the PPCD1 disease locus. Array design and analysis is based on genome build hg19.
Project description:We here describe the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had 5’ΔpyrG upstream of the region targeted for tandem chromosomal duplication and 3’ΔpyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under non-selective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method. A. oryzae strain bearing a 210-kb targeted tandem chromosomal duplication, A. oryzae strain bearing a 700-kb targeted tandem chromosomal duplication, and A. oryzae RIB40 (wild type strain), were cultivated in Polypeptone-dextrin medium. After 3 days cultivation, genomic DNAs from the samples were extracted, and array CGH analysis was carried out to confirm the chromosomal duplications in the strains.
Project description:To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family. Reduced representation bisulfite sequencing was applied to genomic DNA from leukocytes of 6 family members and two unrelated individuals.