Kallmann syndrome gene on the X and Y chromosomes: implications for evolutionary divergence of human sex chromosomes.

The recently identified gene for X-linked Kallmann syndrome (hypogonadotropic hypogonadism and anosmia) has a closely related homologue on the Y chromosome. The X and Y copies of this gene are located in a large region of X/Y homology, on Xp22.3 and Yq11.2, respectively. Comparison of the structure of the X-linked Kallmann syndrome gene and its Y homologue shed light on the evolutionary history of this region of the human sex chromosomes. Our data show that the Y homologue is not functional. Comparative analysis of X/Y sequence identity at several loci on Xp22.3 and Yq11.2 suggests that the homology between these two regions is the result of a complex series of events which occurred in the recent evolution of sex chromosomes.     

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.     

Diversity of microRNAs in human and chimpanzee brain

We used massively parallel sequencing to compare the microRNA (miRNA) content of human and chimpanzee brains, and we identified 447 new miRNA genes. Many of the new miRNAs are not conserved beyond primates, indicating their recent origin, and some miRNAs seem species specific, whereas others are expanded in one species through duplication events. These data suggest that evolution of miRNAs is an ongoing process and that along with ancient, highly conserved miRNAs, there are a number of emerging miRNAs.     

Comparative genomic analysis of three Leishmania species that cause diverse human disease

Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only approximately 200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader-associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.     

Y chromosome sequence variation and the history of human populations

Binary polymorphisms associated with the non-recombining region of the human Y chromosome (NRY) preserve the paternal genetic legacy of our species that has persisted to the present, permitting inference of human evolution, population affinity and demographic history. We used denaturing high-performance liquid chromatography (DHPLC; ref. 2) to identify 160 of the 166 bi-allelic and 1 tri-allelic site that formed a parsimonious genealogy of 116 haplotypes, several of which display distinct population affinities based on the analysis of 1062 globally representative individuals. A minority of contemporary East Africans and Khoisan represent the descendants of the most ancestral patrilineages of anatomically modern humans that left Africa between 35,000 and 89,000 years ago.     

Dysregulation of the TSC-mTOR pathway in human disease

The mammalian target of rapamycin (mTOR) has a central role in the regulation of cell growth. mTOR receives input from multiple signaling pathways, including growth factors and nutrients, to stimulate protein synthesis by phosphorylating key translation regulators such as ribosomal S6 kinase and eukaryote initiation factor 4E binding protein 1. High levels of dysregulated mTOR activity are associated with several hamartoma syndromes, including tuberous sclerosis complex, the PTEN-related hamartoma syndromes and Peutz-Jeghers syndrome. These disorders are all caused by mutations in tumor-suppressor genes that negatively regulate mTOR. Here we discuss the emerging evidence for a functional relationship between the mTOR signaling pathway and several genetic diseases, and we present evidence supporting a model in which dysregulation of mTOR may be a common molecular basis, not only for hamartoma syndromes, but also for other cellular hypertrophic disorders.     

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.     

Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene.

Aniridia is a semidominant disorder in which development of the iris, lens, cornea and retina is disturbed. The mouse mutation Small eye (Sey), which has been proposed as a model for aniridia, results from defects in Pax-6, a gene containing paired-box and homeobox motifs that is specifically expressed in the developing eye and brain. To test the role of PAX6 in aniridia, we isolated human cDNA clones and determined the intron-exon structure of this gene. PAX6 spans 22 kilobases and is divided into 14 exons. Analysis of DNA from 10 unrelated aniridia patients revealed intragenic mutations in three familial and one sporadic case. These findings indicate that the human aniridia and murine Small eye phenotypes arise from homologous defects in PAX6.     

Isolation of chromosome 21-specific yeast artificial chromosomes from a total human genome library.

A new approach for the isolation of chromosome-specific subsets from a human genomic yeast artificial chromosome (YAC) library is described. It is based on the hybridization with an Alu polymerase chain reaction (PCR) probe. We screened a 1.5 genome equivalent YAC library of megabase insert size with Alu PCR products amplified from hybrid cell lines containing human chromosome 21, and identified a subset of 63 clones representative of this chromosome. The majority of clones were assigned to chromosome 21 by the presence of specific STSs and in situ hybridization. Twenty-nine of 36 STSs that we tested were detected in the subset, and a contig spanning 20 centimorgans in the genetic map and containing 8 STSs in 4 YACs was identified. The proposed approach can greatly speed efforts to construct physical maps of the human genome.     

Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.

Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9). Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe. The chromodomain of mouse HP1 (and Swi6 in S. pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly. We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin. In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length. Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome. These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.     

Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions

Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single- and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination-derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.     

A novel GC-rich human macrosatellite VNTR in Xq24 is differentially methylated on active and inactive X chromosomes.

A new X chromosome-specific repetitive sequence, a 3 kilobase HindIII clone with a base composition of 63% C+G, has been isolated. The sequence is organized as a hypervariable tandem repeat cluster ranging in size from 150-350 kilobases, with outlying single copies. This locus, designated DXZ4 and mapped to chromosome band Xq24, may consist of as many as 50 variable-length alleles. It represents a class of variable number of tandem repeat polymorphism which may be termed 'macrosatellite'. The cluster is highly methylated on the active X chromosome and hypomethylated on the inactive X.     

G protein-coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth

Hypotrichosis simplex is a group of nonsyndromic human alopecias. We mapped an autosomal recessive form of this disorder to chromosome 13q14.11-13q21.33, and identified homozygous truncating mutations in P2RY5, which encodes an orphan G protein-coupled receptor. Furthermore, we identified oleoyl-L-alpha-lysophosphatidic acid (LPA), a bioactive lipid, as a ligand for P2Y5 in reporter gene and radioligand binding experiments. Homology and studies of signaling transduction pathways suggest that P2Y5 is a member of a subgroup of LPA receptors, which also includes LPA4 and LPA5. Our study is the first to implicate a G protein-coupled receptor as essential for and specific to the maintenance of human hair growth. This finding may provide opportunities for new therapeutic approaches to the treatment of hair loss in humans.     

Comparative analysis of the genome sequences of Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica

Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are closely related Gram-negative beta-proteobacteria that colonize the respiratory tracts of mammals. B. pertussis is a strict human pathogen of recent evolutionary origin and is the primary etiologic agent of whooping cough. B. parapertussis can also cause whooping cough, and B. bronchiseptica causes chronic respiratory infections in a wide range of animals. We sequenced the genomes of B. bronchiseptica RB50 (5,338,400 bp; 5,007 predicted genes), B. parapertussis 12822 (4,773,551 bp; 4,404 genes) and B. pertussis Tohama I (4,086,186 bp; 3,816 genes). Our analysis indicates that B. parapertussis and B. pertussis are independent derivatives of B. bronchiseptica-like ancestors. During the evolution of these two host-restricted species there was large-scale gene loss and inactivation; host adaptation seems to be a consequence of loss, not gain, of function, and differences in virulence may be related to loss of regulatory or control functions.     

Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection

Many human Y-chromosomal deletions are thought to severely impair reproductive fitness, which precludes their transmission to the next generation and thus ensures their rarity in the population. Here we report a 1.6-Mb deletion that persists over generations and is sufficiently common to be considered a polymorphism. We hypothesized that this deletion might affect spermatogenesis because it removes almost half of the Y chromosome's AZFc region, a gene-rich segment that is critical for sperm production. An association study established that this deletion, called gr/gr, is a significant risk factor for spermatogenic failure. The gr/gr deletion has far lower penetrance with respect to spermatogenic failure than previously characterized Y-chromosomal deletions; it is often transmitted from father to son. By studying the distribution of gr/gr-deleted chromosomes across the branches of the Y chromosome's genealogical tree, we determined that this deletion arose independently at least 14 times in human history. We suggest that the existence of this deletion as a polymorphism reflects a balance between haploid selection, which culls gr/gr-deleted Y chromosomes from the population, and homologous recombination, which continues to generate new gr/gr deletions.     

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.     

Conditional and joint multiple-SNP analysis of GWAS summary statistics identifies additional variants influencing complex traits

We present an approximate conditional and joint association analysis that can use summary-level statistics from a meta-analysis of genome-wide association studies (GWAS) and estimated linkage disequilibrium (LD) from a reference sample with individual-level genotype data. Using this method, we analyzed meta-analysis summary data from the GIANT Consortium for height and body mass index (BMI), with the LD structure estimated from genotype data in two independent cohorts. We identified 36 loci with multiple associated variants for height (38 leading and 49 additional SNPs, 87 in total) via a genome-wide SNP selection procedure. The 49 new SNPs explain approximately 1.3% of variance, nearly doubling the heritability explained at the 36 loci. We did not find any locus showing multiple associated SNPs for BMI. The method we present is computationally fast and is also applicable to case-control data, which we demonstrate in an example from meta-analysis of type 2 diabetes by the DIAGRAM Consortium.