Rare de novo and transmitted copy-number variation in autistic spectrum disorders

To explore the genetic contribution to autistic spectrum disorders (ASDs), we have studied genomic copy-number variation in a large cohort of families with a single affected child and at least one unaffected sibling. We confirm a major contribution from de novo deletions and duplications but also find evidence of a role for inherited "ultrarare" duplications. Our results show that, relative to males, females have greater resistance to autism from genetic causes, which raises the question of the fate of female carriers. By analysis of the proportion and number of recurrent loci, we set a lower bound for distinct target loci at several hundred. We find many new candidate regions, adding substantially to the list of potential gene targets, and confirm several loci previously observed. The functions of the genes in the regions of de novo variation point to a great diversity of genetic causes but also suggest functional convergence.     

Functional copy-number alterations in cancer

Understanding the molecular basis of cancer requires characterization of its genetic defects. DNA microarray technologies can provide detailed raw data about chromosomal aberrations in tumor samples. Computational analysis is needed (1) to deduce from raw array data actual amplification or deletion events for chromosomal fragments and (2) to distinguish causal chromosomal alterations from functionally neutral ones. We present a comprehensive computational approach, RAE, designed to robustly map chromosomal alterations in tumor samples and assess their functional importance in cancer. To demonstrate the methodology, we experimentally profile copy number changes in a clinically aggressive subtype of soft-tissue sarcoma, pleomorphic liposarcoma, and computationally derive a portrait of candidate oncogenic alterations and their target genes. Many affected genes are known to be involved in sarcomagenesis; others are novel, including mediators of adipocyte differentiation, and may include valuable therapeutic targets. Taken together, we present a statistically robust methodology applicable to high-resolution genomic data to assess the extent and function of copy-number alterations in cancer.     

Segmental duplications and copy-number variation in the human genome

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders.     

Identification of germline genomic copy number variation in familial pancreatic cancer

Adenocarcinoma of the pancreas is a significant cause of cancer mortality, and up to 10?% of cases appear to be familial. Heritable genomic copy number variants (CNVs) can modulate gene expression and predispose to disease. Here, we identify candidate predisposition genes for familial pancreatic cancer (FPC) by analyzing germline losses or gains present in one or more high-risk patients and absent in a large control group. A total of 120 FPC cases and 1,194 controls were genotyped on the Affymetrix 500K array, and 36 cases and 2,357 controls were genotyped on the Affymetrix 6.0 array. Detection of CNVs was performed by multiple computational algorithms and partially validated by quantitative PCR. We found no significant difference in the germline CNV profiles of cases and controls. A total of 93 non-redundant FPC-specific CNVs (53 losses and 40 gains) were identified in 50 cases, each CNV present in a single individual. FPC-specific CNVs overlapped the coding region of 88 RefSeq genes. Several of these genes have been reported to be differentially expressed and/or affected by copy number alterations in pancreatic adenocarcinoma. Further investigation in high-risk subjects may elucidate the role of one or more of these genes in genetic predisposition to pancreatic cancer.     

Tandem repeat copy-number variation in protein-coding regions of human genes

Background  

Tandem repeat variation in protein-coding regions will alter protein length and may introduce frameshifts. Tandem repeat variants are associated with variation in pathogenicity in bacteria and with human disease. We characterized tandem repeat polymorphism in human proteins, using the UniGene database, and tested whether these were associated with host defense roles.     

Tandem repeat copy-number variation in protein-coding regions of human genes

Background

Tandem repeat variation in protein-coding regions will alter protein length and may introduce frameshifts. Tandem repeat variants are associated with variation in pathogenicity in bacteria and with human disease. We characterized tandem repeat polymorphism in human proteins, using the UniGene database, and tested whether these were associated with host defense roles.

Results

Protein-coding tandem repeat copy-number polymorphisms were detected in 249 tandem repeats found in 218 UniGene clusters; observed length differences ranged from 2 to 144 nucleotides, with unit copy lengths ranging from 2 to 57. This corresponded to 1.59% (218/13,749) of proteins investigated carrying detectable polymorphisms in the copy-number of protein-coding tandem repeats. We found no evidence that tandem repeat copy-number polymorphism was significantly elevated in defense-response proteins (p = 0.882). An association with the Gene Ontology term 'protein-binding' remained significant after covariate adjustment and correction for multiple testing. Combining this analysis with previous experimental evaluations of tandem repeat polymorphism, we estimate the approximate mean frequency of tandem repeat polymorphisms in human proteins to be 6%. Because 13.9% of the polymorphisms were not a multiple of three nucleotides, up to 1% of proteins may contain frameshifting tandem repeat polymorphisms.

Conclusion

Around 1 in 20 human proteins are likely to contain tandem repeat copy-number polymorphisms within coding regions. Such polymorphisms are not more frequent among defense-response proteins; their prevalence among protein-binding proteins may reflect lower selective constraints on their structural modification. The impact of frameshifting and longer copy-number variants on protein function and disease merits further investigation.     

Rare germline copy number variants in colorectal cancer predisposition characterized by exome sequencing analysis

正Colorectal cancer(CRC)is one of the most common neoplasms and an important cause of mortality worldwide(http://globocan.iarc.fr/).Approximately 35%of the variation in CRC susceptibility is likely due to heritable factors(Lichtenstein et al.,2000).Genetic variations in the human genome include single nucleotide variants(SNVs),short insertions and deletions,and larger structural vari-     

The fine-scale and complex architecture of human copy-number variation

Despite considerable excitement over the potential functional significance of copy-number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at approximately 1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1020 of 1153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of currently known common human CNVs is likely smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with interindividual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously uncharacterized complexity.     

Adaptive evolution of UGT2B17 copy-number variation

The human UGT2B17 gene varies in copy number from zero to two per individual and also differs in mean number between populations from Africa, Europe, and East Asia. We show that such a high degree of geographical variation is unusual and investigate its evolutionary history. This required first reinterpreting the reference sequence in this region of the genome, which is misassembled from the two different alleles separated by an artifactual gap. A corrected assembly identifies the polymorphism as a 117 kb deletion arising by nonallelic homologous recombination between ~4.9 kb segmental duplications and allows the deletion breakpoint to be identified. We resequenced ~12 kb of DNA spanning the breakpoint in 91 humans from three HapMap and one extended HapMap populations and one chimpanzee. Diversity was unusually high and the time to the most recent common ancestor was estimated at ~2.4 or ~3.0 million years by two different methods, with evidence of balancing selection in Europe. In contrast, diversity was low in East Asia where a single haplotype predominated, suggesting positive selection for the deletion in this part of the world.     

Rare de novo methylation within the transposable element activator (Ac) in transgenic tobacco plants

Summary The single glucoamylase gene (SGA1) of the yeast Saccharomyces cerevisiae is expressed exclusively during the sporulation phase of the life cycle. Enzymatic studies and nucleic acid sequence comparisons have shown that the SGA1 glucoamylase is closely related to the secreted enzymes of S. cerevisiae var. diastaticus. The latter are encoded by any of three unlinked STA genes, which have been proposed to derive from the ancestral SGA1 form by genomic rearrangement. We show that the regulation of SGA1 is distinct from that of the other members of the STA gene family. SGA1 expression did not respond to STA10, the primary determinant of glucoamylase expression from STA2. Unlike STA2, SGA1 was not regulated directly by the mating type locus. Expression of SGA1 depended on the function of the MAT products in supporting sporulation and not on the formation of haploid progeny spores or on the composition of the mating type locus per se. We conclude that the STA genes acquired regulation by STA10 and MAT by the genomic rearrangements that led to their formation. This regulation is thus distinct from that of the ancestral SGA1 gene.     

The de novo DNA methyltransferase DNMT3A in development and cancer

DNA methylation, one of the best-characterized epigenetic modifications, plays essential roles in development, aging and diseases. The de novo DNA methyltransferase DNMT3A is responsible for the establishment of de novo genomic DNA methylation patterns and, as such, involved in normal development as well as in many diseases including cancer. In recent years, our understanding of this important protein has made significant progress, which was facilitated by stunning development in the analysis of the DNA methylome of multiple organs and cell types. In this review, recent developments in the characterization of DNMT3A were discussed with special emphasis on the roles of DNMT3A in development and cancer.     

Extensive copy-number variation of the human olfactory receptor gene family

As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of large genomic regions violate the assumptions of statistical methods that are commonly used in the assessment of gene enrichment. Using more appropriate methods, we provide evidence that OR enrichment in CNVs is not due to positive selection but is because of OR preponderance in segmentally duplicated regions, which are known to be frequently copy-number variable, and because purifying selection against CNVs is lower in OR-containing regions than in regions containing essential genes. We also combine multiplex ligation-dependent probe amplification (MLPA) and PCR to assay the copy numbers of 37 candidate CNV ORs in a panel of ~50 human individuals. We confirm copy-number variation of 18 ORs but find no variation in this human-diversity panel for 16 other ORs, highlighting the caveat that reported intervals often overrepresent true CNVs. The copy-number variation we describe is likely to underpin significant variation in olfactory abilities among human individuals. Finally, we show that both homology-based and homology-independent processes have played a recent role in remodeling the OR family.     

Prevention of de novo prostate cancer by immunization with tumor-derived vaccines

Since advanced prostate cancer is difficult to treat, we have chosen a very different approach: the development of vaccines to prevent initial de novo tumor formation. To test the hypothesis that prostate cancer can be prevented by vaccination, Lobund–Wistar (LW) rats were vaccinated subcutaneously with complete Freunds adjuvant (CFA) plus glutaraldehyde-fixed (GFT) whole cell or potassium thiocyanate extract (PTE) preparations derived from in vivo tumors, or with media and CFA (media-vaccinated). Rats were vaccinated each month substituting incomplete Freunds adjuvant for CFA, from age 3 to 12 months, and methylnitrosourea (30 mg/kg) was administered intravenously at 4 months of age. Groups of 30 GFT cell–vaccinated rats showed a 90% reduction, and PTE-vaccinated rats, a 50% reduction in the occurrence of de novo prostate tumors compared with media-vaccinated controls. When splenocytes from vaccinated rats were incubated with tumor cells prior to subcutaneous implantation, PTE-vaccinated rats showed a 80% reduction, and GFT cell–vaccinated rats showed a 40% reduction in the occurrence of tumors, demonstrating a role for the spleen in the protective response. The inflammatory responses in tumors from GFT cell–vaccinated rats and PTE-vaccinated rats were distinguished by an influx of eosinophils compared with the responses in tumors from media-vaccinated rats. These results demonstrate the possibility that prostate cancer can be prevented by immunization with vaccines based on whole tumor–derived vaccines.     

An algorithm for inferring complex haplotypes in a region of copy-number variation

Recent studies have extensively examined the large-scale genetic variants in the human genome known as copy-number variations (CNVs), and the universality of CNVs in normal individuals, along with their functional importance, has been increasingly recognized. However, the absence of a method to accurately infer alleles or haplotypes within a CNV region from high-throughput experimental data hampers the finer analyses of CNV properties and applications to disease-association studies. Here we developed an algorithm to infer complex haplotypes within a CNV region by using data obtained from high-throughput experimental platforms. We applied this algorithm to experimental data and estimated the population frequencies of haplotypes that can yield information on both sequences and numbers of DNA copies. These results suggested that the analysis of such complex haplotypes is essential for accurately detecting genetic differences within a CNV region between population groups.     

Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.