Juan C. Perin

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

High-resolution mapping and analysis of copy number variations in the human genome: A data resource for clinical and research applications

We present a database of copy number variations (CNVs) detected in 2026 disease-free individuals, using high-density, SNP-based oligonucleotide microarrays. This large cohort, comprised mainly of Caucasians (65.2%) and African-Americans (34.2%), was analyzed for CNVs in a single study using a uniform array platform and computational process. We have catalogued and characterized 54,462 individual CNVs, 77.8% of which were identified in multiple unrelated individuals. These nonunique CNVs mapped to 3272 distinct regions of genomic variation spanning 5.9% of the genome; 51.5% of these were previously unreported, and >85% are rare. Our annotation and analysis confirmed and extended previously reported correlations between CNVs and several genomic features such as repetitive DNA elements, segmental duplications, and genes. We demonstrate the utility of this data set in distinguishing CNVs with pathologic significance from normal variants. Together, this analysis and annotation provides a useful resource to assist with the assessment of CNVs in the contexts of human variation, disease susceptibility, and clinical molecular diagnostics.

Genomic Analysis Using High-Density Single Nucleotide Polymorphism-Based Oligonucleotide Arrays and Multiplex Ligation-Dependent Probe Amplification Provides a Comprehensive Analysis of INI1/SMARCB1 in Malignant Rhabdoid Tumors

PURPOSE A high-resolution genomic profiling and comprehensive targeted analysis of INI1/SMARCB1 of a large series of pediatric rhabdoid tumors was done. The aim was to identify regions of copy number change and loss of heterozygosity (LOH) that might pinpoint additional loci involved in the development or progression of rhabdoid tumors and define the spectrum of genomic alterations of INI1 in this malignancy. EXPERIMENTAL DESIGN A multiplatform approach using Illumina single nucleotide polymorphism-based oligonucleotide arrays, multiplex ligation-dependent probe amplification, fluorescence in situ hybridization, and coding sequence analysis was used to characterize genome-wide copy number changes, LOH, and genomic alterations of INI1/SMARCB1 in a series of pediatric rhabdoid tumors. RESULTS The biallelic alterations of INI1 that led to inactivation were elucidated in 50 of 51 tumors. INI1 inactivation was shown by a variety of mechanisms, including deletions, mutations, and LOH. The results from the array studies highlighted the complexity of rearrangements of chromosome 22 compared with the low frequency of alterations involving the other chromosomes. CONCLUSIONS The results from the genome-wide single nucleotide polymorphism array analysis suggest that INI1 is the primary tumor suppressor gene involved in the development of rhabdoid tumors with no second locus identified. In addition, we did not identify hotspots for the breakpoints in sporadic tumors with deletions of chromosome 22q11.2. By employing a multimodality approach, the wide spectrum of alterations of INI1 can be identified in the majority of patients, which increases the clinical utility of molecular diagnostic testing.

Rare structural variation of synapse and neurotransmission genes in autism.

Autism spectrum disorders (ASDs) comprise a constellation of highly heritable neuropsychiatric disorders. Genome-wide studies of autistic individuals have implicated numerous minor risk alleles but few common variants, suggesting a complex genetic model with many contributing loci. To assess commonality of biological function among rare risk alleles, we compared functional knowledge of genes overlapping inherited structural variants in idiopathic ASD subjects relative to healthy controls. In this study we show that biological processes associated with synapse function and neurotransmission are significantly enriched, with replication, in ASD subjects versus controls. Analysis of phenotypes observed for mouse models of copy-variant genes established significant and replicated enrichment of observable phenotypes consistent with ASD behaviors. Most functional terms retained significance after excluding previously reported ASD loci. These results implicate several new variants that involve synaptic function and glutamatergic signaling processes as important contributors of ASD pathophysiology and suggest a sizable pool of additional potential ASD risk loci.

NMNAT1 mutations cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss. Two-thirds of LCA cases are caused by mutations in 17 known disease-associated genes (Retinal Information Network (RetNet)). Using exome sequencing we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 that is likely to be disease causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in the kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD+) biosynthesis. Functional studies showed that the p.Val9Met alteration decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated families with LCA identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease in humans and indicate that NMNAT1 mutations cause LCA.

Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming

Significance Mitochondria generate signals that regulate nuclear gene expression via retrograde signaling, but this phenomenon is rendered more complex by the quantitative differences in the percentage of mutant and normal mtDNAs that can exist within patient cells. This study demonstrates that depending upon its relative cytoplasmic levels, a single mtDNA point mutation can cause a discrete set of cellular transcriptional responses within cells of the same nuclear background. This qualitative regulation of nuclear gene expression by quantitative changes in mtDNA mutant levels challenges the traditional “single mutation–single disease” concept and provides an alternative perspective on the molecular basis of complex metabolic and degenerative diseases, cancer, and aging. Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNALeu(UUR) 3243A>G mutation and harbor ∼10–30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50–90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90–100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20–30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50–90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

CNV Workshop: an integrated platform for high-throughput copy number variation discovery and clinical diagnostics

BackgroundRecent studies have shown that copy number variations (CNVs) are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. The increasing availability of high-resolution genome surveillance platforms provides opportunity for rapidly assessing research and clinical samples for CNV content, as well as for determining the potential pathogenicity of identified variants. However, few informatics tools for accurate and efficient CNV detection and assessment currently exist.ResultsWe developed a suite of software tools and resources (CNV Workshop) for automated, genome-wide CNV detection from a variety of SNP array platforms. CNV Workshop includes three major components: detection, annotation, and presentation of structural variants from genome array data. CNV detection utilizes a robust and genotype-specific extension of the Circular Binary Segmentation algorithm, and the use of additional detection algorithms is supported. Predicted CNVs are captured in a MySQL database that supports cohort-based projects and incorporates a secure user authentication layer and user/admin roles. To assist with determination of pathogenicity, detected CNVs are also annotated automatically for gene content, known disease loci, and gene-based literature references. Results are easily queried, sorted, filtered, and visualized via a web-based presentation layer that includes a GBrowse-based graphical representation of CNV content and relevant public data, integration with the UCSC Genome Browser, and tabular displays of genomic attributes for each CNV.ConclusionsTo our knowledge, CNV Workshop represents the first cohesive and convenient platform for detection, annotation, and assessment of the biological and clinical significance of structural variants. CNV Workshop has been successfully utilized for assessment of genomic variation in healthy individuals and disease cohorts and is an ideal platform for coordinating multiple associated projects.Availability and ImplementationAvailable on the web at: http://sourceforge.net/projects/cnv

Genome-Wide Analysis of Interferon Regulatory Factor I Binding in Primary Human Monocytes

IRF1 is a transcription factor that participates in interferon signaling. Previous studies of IRF1 binding have utilized in vitro assays. We used ChIP-seq in human monocytes to better define the recognition motif for IRF1. The newly identified 18bp motif (RAAASNGAAAGTGAAASY) is a refinement of the 13bp IRF1 motif commonly used. We utilized the 18bp consensus motif and identified 345 potential target genes. To compare the 18bp motif with the 13bp motif, we compared putative gene targets. Only 56 potential gene targets were defined by both consensus motifs. To compare biological effects of interferon on the 13bp and the 18bp consensus targets, we mined expression data from cells exposed to interferons or transfected with IRF1. In all cases, the 18bp consensus motif was more strongly associated with transcriptional responses than the 13bp motif. Therefore, the new 18bp consensus motif appears to have a greater association with biological activities of IRF1.

Efficient digest of high-throughput sequencing data in a reproducible report

BackgroundHigh-throughput sequencing (HTS) technologies are spearheading the accelerated development of biomedical research. Processing and summarizing the large amount of data generated by HTS presents a non-trivial challenge to bioinformatics. A commonly adopted standard is to store sequencing reads aligned to a reference genome in SAM (Sequence Alignment/Map) or BAM (Binary Alignment/Map) files. Quality control of SAM/BAM files is a critical checkpoint before downstream analysis. The goal of the current project is to facilitate and standardize this process.ResultsWe developed bamchop, a robust program to efficiently summarize key statistical metrics of HTS data stored in BAM files, and to visually present the results in a formatted report. The report documents information about various aspects of HTS data, such as sequencing quality, mapping to a reference genome, sequencing coverage, and base frequency. Bamchop uses the R language and Bioconductor packages to calculate statistical matrices and the Sweave utility and associated LaTeX markup for documentation. Bamchops efficiency and robustness were tested on BAM files generated by local sequencing facilities and the 1000 Genomes Project. Source code, instruction and example reports of bamchop are freely available from https://github.com/CBMi-BiG/bamchop.ConclusionsBamchop enables biomedical researchers to quickly and rigorously evaluate HTS data by providing a convenient synopsis and user-friendly reports.

Cytokine-Induced Monocyte Characteristics in SLE

Monocytes in SLE have been described as having aberrant behavior in a number of assays. We examined gene expression and used a genome-wide approach to study the posttranslational histone mark, H4 acetylation, to examine epigenetic changes in SLE monocytes. We compared SLE monocyte gene expression and H4 acetylation with three types of cytokine-treated monocytes to understand which cytokine effects predominated in SLE monocytes. We found that γ-interferon and α-interferon both replicated a broad range of the gene expression changes seen in SLE monocytes. H4 acetylation in SLE monocytes was overall higher than in controls and there was less correlation of H4ac with cytokine-treated cells than when gene expression was compared. A set of chemokine genes had downregulated expression and H4ac. Therefore, there are significant clusters of aberrantly expressed genes in SLE which are strongly associated with altered H4ac, suggesting that these cells have experienced durable changes to their epigenome.