Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain.

The levels of a specific mitochondrial DNA deletion (mtDNA4977) measured in 12 brain regions of 6 normal adults 39 to 82 years old exhibited striking variation among anatomical locations. Comparisons of the same region among individuals showed an increase of mtDNA4977 with age. The three regions with the highest levels, caudate, putamen and substantia nigra, are characterized by a high dopamine metabolism. The breakdown of dopamine by mitochondrial MAO produces H2O2 which can lead to oxygen radical formation. We suggest that mtDNA4977 may be the "tip of the iceberg" of the spectrum of somatic mutations produced by oxidative damage.     

High-throughput oncogene mutation profiling in human cancer

Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.     

Conserved noncoding sequences are selectively constrained and not mutation cold spots

Noncoding genetic variants are likely to influence human biology and disease, but recognizing functional noncoding variants is difficult. Approximately 3% of noncoding sequence is conserved among distantly related mammals, suggesting that these evolutionarily conserved noncoding regions (CNCs) are selectively constrained and contain functional variation. However, CNCs could also merely represent regions with lower local mutation rates. Here we address this issue and show that CNCs are selectively constrained in humans by analyzing HapMap genotype data. Specifically, new (derived) alleles of SNPs within CNCs are rarer than new alleles in nonconserved regions (P = 3 x 10(-18)), indicating that evolutionary pressure has suppressed CNC-derived allele frequencies. Intronic CNCs and CNCs near genes show greater allele frequency shifts, with magnitudes comparable to those for missense variants. Thus, conserved noncoding variants are more likely to be functional. Allele frequency distributions highlight selectively constrained genomic regions that should be intensively surveyed for functionally important variation.     

Genome-wide analysis of single-nucleotide polymorphisms in human expressed sequences

Single-nucleotide polymorphisms (SNPs) have been explored as a high-resolution marker set for accelerating the mapping of disease genes. Here we report 48,196 candidate SNPs detected by statistical analysis of human expressed sequence tags (ESTs), associated primarily with coding regions of genes. We used Bayesian inference to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimaeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing, sequencing error rates, context-sensitivity and cDNA library origin. Three separate validations-comparison with 54 genes screened for SNPs independently, verification of HLA-A polymorphisms and restriction fragment length polymorphism (RFLP) testing-verified 70%, 89% and 71% of our predicted SNPs, respectively. Our method detects tenfold more true HLA-A SNPs than previous analyses of the EST data. We found SNPs in a large fraction of known disease genes, including some disease-causing mutations (for example, the HbS sickle-cell mutation). Our comprehensive analysis of human coding region polymorphism provides a public resource for mapping of disease genes (available at http://www.bioinformatics.ucla.edu/snp).     

CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer

Aberrant DNA methylation of CpG islands has been widely observed in human colorectal tumors and is associated with gene silencing when it occurs in promoter areas. A subset of colorectal tumors has an exceptionally high frequency of methylation of some CpG islands, leading to the suggestion of a distinct trait referred to as 'CpG island methylator phenotype', or 'CIMP'. However, the existence of CIMP has been challenged. To resolve this continuing controversy, we conducted a systematic, stepwise screen of 195 CpG island methylation markers using MethyLight technology, involving 295 primary human colorectal tumors and 16,785 separate quantitative analyses. We found that CIMP-positive (CIMP+) tumors convincingly represent a distinct subset, encompassing almost all cases of tumors with BRAF mutation (odds ratio = 203). Sporadic cases of mismatch repair deficiency occur almost exclusively as a consequence of CIMP-associated methylation of MLH1 . We propose a robust new marker panel to classify CIMP+ tumors.     

MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability

DNA mismatch repair is important because of its role in maintaining genomic integrity and its association with hereditary non-polyposis colon cancer (HNPCC). To identify new human mismatch repair proteins, we probed nuclear extracts with the conserved carboxy-terminal MLH1 interaction domain. Here we describe the cloning and complete genomic sequence of MLH3, which encodes a new DNA mismatch repair protein that interacts with MLH1. MLH3 is more similar to mismatch repair proteins from yeast, plants, worms and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans. Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability. Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility I (Ccs1). Although we were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumours from congenic Ccs1 mice exhibit microsatellite instability. Functional redundancy among Mlh3, Pms1 and Pms2 may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.     

Great ape DNA sequences reveal a reduced diversity and an expansion in humans

The extent of DNA sequence variation of chimpanzees is several-fold greater than that of humans. It is unclear, however, if humans or chimpanzees are exceptional among primates in having low and high amounts of DNA sequence diversity, respectively. To address this, we have determined approximately 10,000 bp of noncoding DNA sequences at Xq13.3 (which has been extensively studied in both humans and chimpanzees) from 10 western lowland gorillas (Gorilla gorilla gorilla) and 1 mountain gorilla (Gorilla gorilla beringei; that is, from 2 of the 3 currently recognized gorilla subspecies), as well as 8 Bornean (Pongo pygmaeus pygmaeus) and 6 Sumatran (Pongo pygmaeus abelii) orang-utans, representing both currently recognized orang-utan subspecies. We show that humans differ from the great apes in having a low level of genetic variation and a signal of population expansion.     

Variation in genome-wide mutation rates within and between human families

J.B.S. Haldane proposed in 1947 that the male germline may be more mutagenic than the female germline. Diverse studies have supported Haldane's contention of a higher average mutation rate in the male germline in a variety of mammals, including humans. Here we present, to our knowledge, the first direct comparative analysis of male and female germline mutation rates from the complete genome sequences of two parent-offspring trios. Through extensive validation, we identified 49 and 35 germline de novo mutations (DNMs) in two trio offspring, as well as 1,586 non-germline DNMs arising either somatically or in the cell lines from which the DNA was derived. Most strikingly, in one family, we observed that 92% of germline DNMs were from the paternal germline, whereas, in contrast, in the other family, 64% of DNMs were from the maternal germline. These observations suggest considerable variation in mutation rates within and between families.     

Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation.

Mutations of mitochondrial DNA (mtDNA) are an important cause of genetic disease. We describe a family with an unusual homoplasmic mutation that resulted in six neonatal deaths and one surviving child with Leigh syndrome. The mother is clinically normal, but a severe biochemical and molecular genetic defect was present in both a fatally affected child and the mother. This family highlights the role of homoplasmic mt-tRNA mutations in genetic disease.     

Bayesian inference of ancient human demography from individual genome sequences

Whole-genome sequences provide a rich source of information about human evolution. Here we describe an effort to estimate key evolutionary parameters based on the whole-genome sequences of six individuals from diverse human populations. We used a Bayesian, coalescent-based approach to obtain information about ancestral population sizes, divergence times and migration rates from inferred genealogies at many neutrally evolving loci across the genome. We introduce new methods for accommodating gene flow between populations and integrating over possible phasings of diploid genotypes. We also describe a custom pipeline for genotype inference to mitigate biases from heterogeneous sequencing technologies and coverage levels. Our analysis indicates that the San population of southern Africa diverged from other human populations approximately 108-157 thousand years ago, that Eurasians diverged from an ancestral African population 38-64 thousand years ago, and that the effective population size of the ancestors of all modern humans was ～9,000.     

High mutation rates have driven extensive structural polymorphism among human Y chromosomes

Although much structural polymorphism in the human genome has been catalogued, the kinetics of underlying change remain largely unexplored. Because human Y chromosomes are clonally inherited, it has been possible to capture their detailed relationships in a robust, worldwide genealogical tree. Examination of structural variation across this tree opens avenues for investigating rates of underlying mutations. We selected one Y chromosome from each of 47 branches of this tree and searched for large-scale variation. Four chromosomal regions showed extensive variation resulting from numerous large-scale mutations. Within the tree encompassed by the studied chromosomes, the distal-Yq heterochromatin changed length > or = 12 times, the TSPY gene array changed length > or = 23 times, the 3.6-Mb IR3/IR3 region changed orientation > or = 12 times and the AZFc region was rearranged > or = 20 times. After determining the total time spanned by all branches of this tree (approximately 1.3 million years or 52,000 generations), we converted these mutation counts to lower bounds on rates: > or = 2.3 x 10(-4), > or = 4.4 x 10(-4), > or = 2.3 x 10(-4) and > or = 3.8 x 10(-4) large-scale mutations per father-to-son Y transmission, respectively. Thus, high mutation rates have driven extensive structural polymorphism among human Y chromosomes. At the same time, we found limited variation in the copy number of Y-linked genes, which raises the possibility of selective constraints.     

Identification of foreign gene sequences by transcript filtering against the human genome.

We have developed a computational subtraction approach to detect microbial causes for putative infectious diseases by filtering a set of human tissue-derived sequences against the human genome. We demonstrate the potential of this method by identifying sequences from known pathogens in established expressed-sequence tag libraries.     

A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition

Disruption of histone acetylation patterns is a common feature of cancer cells, but very little is known about its genetic basis. We have identified truncating mutations in one of the primary human histone deacetylases, HDAC2, in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. The presence of the HDAC2 frameshift mutation causes a loss of HDAC2 protein expression and enzymatic activity and renders these cells more resistant to the usual antiproliferative and proapoptotic effects of histone deacetylase inhibitors. As such drugs may serve as therapeutic agents for cancer, our findings support the use of HDAC2 mutational status in future pharmacogenetic treatment of these individuals.     

Targeted breakage of a human chromosome mediated by cloned human telomeric DNA.

Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.     

Defective colour vision associated with a missense mutation in the human green visual pigment gene.

All red/green colour vision defects described so far have been associated with gross rearrangements within the red/green opsin gene array (Xq28). We now describe a male with severe deuteranomaly without such a rearrangement. A substitution of a highly conserved cysteine by arginine at position 203 in the green opsins presumably accounted for his colour vision defect. Surprisingly, this mutation was fairly common (2%) in the population but apparently was not always expressed. In analogy with nonexpression of some 5'green-red hybrid genes in persons with normal colour vision, we suggest that failure of manifestation occurs when the mutant gene is located at a distal (3') position among several green opsin genes. This mutation might also predispose to certain X-linked retinal dystrophies.     

A mutation of human cytochrome c enhances the intrinsic apoptotic pathway but causes only thrombocytopenia

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.     

Meiotic instability of human minisatellite CEB1 in yeast requires DNA double-strand breaks.

Minisatellites are tandemly repeated DNA sequences of 10-100-bp units. Some minisatellite loci are highly unstable in the human germ line, and structural analysis of mutant alleles has suggested that repeat instability results from a recombination-based process. To provide insights into the molecular mechanism of human minisatellite instability, we developed Saccharomyces cerevisiae strains carrying alleles of the most unstable human minisatellite locus, CEB1 (ref. 2). We observed that CEB1 is destabilized in meiosis, resulting in a variety of intra- and inter-allelic gains or losses of repeat units, similar to rearrangements described in humans. Using mutations affecting the initiation of recombination (spo11) or mismatch repair (msh2 pms1 ), we demonstrate that meiotic destabilization depends on the initiation of homologous recombination at nearby DNA double-strand break (DSBs) sites and involves a 'rearranged heteroduplex' intermediate. Most of the human and yeast data can be explained and unified in the context of DSB repair models.