Mobile genes in the human microbiome are structured from global to individual scales.

Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts.

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases.

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.

The 1.4-Mb CMT1A duplication/HNPP deletion genomic region reveals unique genome architectural features and provides insights into the recent evolution of new genes.

Duplication and deletion of the 1.4-Mb region in 17p12 that is delimited by two 24-kb low copy number repeats (CMT1A-REPs) represent frequent genomic rearrangements resulting in two common inherited peripheral neuropathies, Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsy (HNPP). CMT1A and HNPP exemplify a paradigm for genomic disorders wherein unique genome architectural features result in susceptibility to DNA rearrangements that cause disease. A gene within the 1.4-Mb region, PMP22, is responsible for these disorders through a gene-dosage effect in the heterozygous duplication or deletion. However, the genomic structure of the 1.4-Mb region, including other genes contained within the rearranged genomic segment, remains essentially uncharacterized. To delineate genomic structural features, investigate higher-order genomic architecture, and identify genes in this region, we constructed PAC and BAC contigs and determined the complete nucleotide sequence. This CMT1A/HNPP genomic segment contains 1,421,129 bp of DNA. A low copy number repeat (LCR) was identified, with one copy inside and two copies outside of the 1.4-Mb region. Comparison between physical and genetic maps revealed a striking difference in recombination rates between the sexes with a lower recombination frequency in males (0.67 cM/Mb) versus females (5.5 cM/Mb). Hypothetically, this low recombination frequency in males may enable a chromosomal misalignment at proximal and distal CMT1A-REPs and promote unequal crossing over, which occurs 10 times more frequently in male meiosis. In addition to three previously described genes, five new genes (TEKT3, HS3ST3B1, NPD008/CGI-148, CDRT1, and CDRT15) and 13 predicted genes were identified. Most of these predicted genes are expressed only in embryonic stages. Analyses of the genomic region adjacent to proximal CMT1A-REP indicated an evolutionary mechanism for the formation of proximal CMT1A-REP and the creation of novel genes by DNA rearrangement during primate speciation.

Analysis of the cat eye syndrome critical region in humans and the region of conserved synteny in mice: a search for candidate genes at or near the human chromosome 22 pericentromere.

We have sequenced a 1.1-Mb region of human chromosome 22q containing the dosage-sensitive gene(s) responsible for cat eye syndrome (CES) as well as the 450-kb homologous region on mouse chromosome 6. Fourteen putative genes were identified within or adjacent to the human CES critical region (CESCR), including three known genes (IL-17R, ATP6E, and BID) and nine novel genes, based on EST identity. Two putative genes (CECR3 and CECR9) were identified, in the absence of EST hits, by comparing segments of human and mouse genomic sequence around two solitary amplified exons, thus showing the utility of comparative genomic sequence analysis in identifying transcripts. Of the 14 genes, 10 were confirmed to be present in the mouse genomic sequence in the same order and orientation as in human. Absent from the mouse region of conserved synteny are CECR1, a promising CES candidate gene from the center of the contig, neighboring CECR4, and CECR7 and CECR8, which are located in the gene-poor proximal 400 kb of the contig. This latter proximal region, located approximately 1 Mb from the centromere, shows abundant duplicated gene fragments typical of pericentromeric DNA. The margin of this region also delineates the boundary of conserved synteny between the CESCR and mouse chromosome 6. Because the proximal CESCR appears abundant in duplicated segments and, therefore, is likely to be gene poor, we consider the putative genes identified in the distal CESCR to represent the majority of candidate genes for involvement in CES.

Initial sequencing and analysis of the human genome.

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

Sequence and comparative analysis of the mouse 1-megabase region orthologous to the human 11p15 imprinted domain.

A major barrier to conceptual advances in understanding the mechanisms and regulation of imprinting of a genomic region is our relatively poor understanding of the overall organization of genes and of the potentially important cis-acting regulatory sequences that lie in the nonexonic segments that make up 97% of the genome. Interspecies sequence comparison offers an effective approach to identify sequence from conserved functional elements. In this article we describe the successful use of this approach in comparing a approximately 1-Mb imprinted genomic domain on mouse chromosome 7 to its orthologous region on human 11p15.5. Within the region, we identified 112 exons of known genes as well as a novel gene identified uniquely in the mouse region, termed Msuit, that was found to be imprinted. In addition to these coding elements, we identified 33 CpG islands and 49 orthologous nonexonic, nonisland sequences that met our criteria as being conserved, and making up 4.1% of the total sequence. These conserved noncoding sequence elements were generally clustered near imprinted genes and the majority were between Igf2 and H19 or within Kvlqt1. Finally, the location of CpG islands provided evidence that suggested a two-island rule for imprinted genes. This study provides the first global view of the architecture of an entire imprinted domain and provides candidate sequence elements for subsequent functional analyses.

An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y.

In humans, deletion of any one of three Y-chromosomal regions- AZFa, AZFb or AZFc-disrupts spermatogenesis, causing infertility in otherwise healthy men. Although candidate genes have been identified in all three regions, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): high-resolution physical and transcript map of the candidate region in chromosome region 13q11.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is a neurodegenerative disease frequent in northeastern Québec. In a previous study, we localized the disease gene to chromosome region 13q11 by identifying excess sharing of a marker allele in patients followed by linkage analysis and haplotyping. To create a detailed physical map of this region, we screened CEPH mega-YACs with 41 chromosome 13 sequence-tagged-sites (STSs) known to map to 13q11-q12. The YAC contig, composed of 27 clones, extends on the genetic map from D13S175 to D13S221, an estimated distance of at least 19.3 cM. A high-resolution BAC and PAC map that includes the ARSACS critical region flanked by D13S1275 and D13S292 was constructed. These YAC and BAC/PAC maps allowed the accurate placement of 29 genes and ESTs previously mapped to the proximal region of chromosome 13q. We confirmed the position of two candidate genes within the critical region and mapped the other 27 genes and ESTs to nearby intervals. Six BAC/PAC clones form a contig between D13S232 and D13S787 for sequencing within the ARSACS critical region.

Perfect conserved linkage across the entire mouse chromosome 10 region homologous to human chromosome 21.

The distal end of human Chromosome (HSA) 21 from PDXK to the telomere shows perfect conserved linkage with mouse Chromosome (MMU) 10. This region is bounded on the proximal side by a segment of homology to HSA22q11.2, and on the distal side by a region of homology with HSA19p13.1. A high-resolution PAC-based physical map is described that spans 2.8 Mb, including the entire 2.1 Mb from Pdxk to Prmt2 corresponding to HSA21. Thirty-four expressed sequences are mapped, three of which were not mapped previously in any species and nine more that are mapped in mouse for the first time. These genes confirm and extend the conserved linkage between MMU10 and HSA21. The ordered PACs and dense STS map provide a clone resource for biological experiments, for rapid and accurate mapping, and for genomic sequencing. The new genes identified here may be involved in Down syndrome (DS) or in several genetic diseases that map to this conserved region of HSA21.

Human genome anatomy: BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine.

Human genome sequencing is accelerating rapidly. Multiple genome maps link this sequence to problems in biology and clinical medicine. Because each map represents a different aspect of the structure, content, and behavior of human chromosomes, these fundamental properties must be integrated with the genome to understand disease genes, cancer instability, and human evolution. Cytogenetic maps use 400-850 visible band landmarks and are the primary means for defining prenatal defects and novel cancer breakpoints, thereby providing simultaneous examination of the entire genome. Recent genetic, physical, and transcript maps use PCR-based landmarks called sequence-tagged sites (STSs). We have integrated these genome maps by anchoring the human cytogenetic to the STS-based genetic and physical maps with 1021 STS-BAC pairs at an average spacing of approximately 1 per 3 Mb. These integration points are represented by 872 unique STSs, including 642 polymorphic markers and 957 bacterial artificial chromosomes (BACs), each of which was localized on high resolution fluorescent banded chromosomes. These BACs constitute a resource that bridges map levels and provides the tools to seamlessly translate questions raised by genomic change seen at the chromosomal level into answers based at the molecular level. We show how the BACs provide molecular links for understanding human genomic duplications, meiosis, and evolution, as well as reagents for conducting genome-wide prenatal diagnosis at the molecular level and for detecting gene candidates associated with novel cancer breakpoints.

A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant.

Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery. In conjuction with recent biochemical studies, this report demonstrates the importance of this gene family in vertebrate embryonic development.

A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Integration of cytogenetic with recombinational and physical maps of mouse chromosome 16.

To link the cytogenetic map for mouse chromosome 16 (Chr 16) to the more detailed recombinational and physical maps, multiple probes were mapped by fluorescence in situ hybridization (FISH). Sixteen large insert clones (YACs, BACs, and PACs) containing markers that have been assigned to the Chr 16 recombinational map were localized to a cytogenetic band or subband by high-resolution FISH. This linkage of the cytogenetic and recombinational maps provides a useful tool for assigning new probe locations and for defining breakpoints in mice with chromosomal rearrangements. A direct application of these probes is demonstrated by identifying mice trisomic for distal Chr 16 using FISH analysis of interphase nuclei.

An integrated somatic cell hybrid, YAC, and BAC map of the Rmc1 region of mouse chromosome 1.

Rmc1, the cellular receptor for the polytropic class of murine retroviruses, determines the tissue tropism of the virus and therefore plays a critical role in the pathogenesis of polytropic virus-induced leukemia. Previously we reported the physical mapping of this gene to a 5-cM region of mouse chromosome 1 and the construction of a yeast artificial chromosome (YAC) contig across this region. In this report we describe the refinement of the Rmc1 candidate region to approximately 600 kb and the generation of an integrated somatic cell hybrid, YAC, and bacterial artificial chromosome contig spanning the region. A number of genes and loci were physically ordered along the chromosome, including a recently identified candidate for Rmc1.

Campomelic dysplasia translocation breakpoints are scattered over 1 Mb proximal to SOX9: evidence for an extended control region.

Campomelic dysplasia (CD), a skeletal malformation syndrome with or without XY sex reversal, is usually caused by mutations within the SOX9 gene on distal 17q. Several CD translocation and inversion cases have been described with breakpoints outside the coding region, mapping to locations >130 kb proximal to SOX9. Such cases are generally less severely affected than cases with SOX9 coding-region mutations, as is borne out by three new translocation cases that we present. We have cloned the region extending 1.2 Mb upstream of the SOX9 gene in overlapping bacterial-artificial-chromosome and P1-artificial-chromosome clones and have established a restriction map with rare-cutter enzymes. With sequence-tagged-site-content mapping in somatic-cell hybrids, as well as with FISH, we have precisely mapped the breakpoints of the three new and of three previously described CD cases. The six CD breakpoints map to an interval that is 140-950 kb proximal to the SOX9 gene. With exon trapping, we could isolate five potential exons from the YAC 946E12 that spans the region, four of which could be placed in the contig in the vicinity of the breakpoints. They show the same transcriptional orientation, but only two have an open reading frame (ORF). We failed to detect expression of these fragments in several human and mouse cDNA libraries, as well as on northern blots. Genomic sequence totaling 1,063 kb from the SOX9 5'-flanking region was determined and was analyzed by the gene-prediction program GENSCAN and by a search of dbEST and other databases. No genes or transcripts could be identified. Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.

Mouse molecular cytogenetic resource: 157 BACs link the chromosomal and genetic maps.

We have established a collection of strong molecular cytogenetic markers that span the mouse autosomes and X chromosome at an average spacing of one per 19 Mb and identify 127 distinct band landmarks. In addition, this Mouse Molecular Cytogenetic Resource relates the ends of the genetic maps to their chromosomal locations. The resource consists of 157 bacterial artificial chromosome (BAC) clones, each of which identifies specific mouse chromosome bands or band borders, and 42 of which are linked to genetic markers that define the centromeric and telomeric ends of the Whitehead/MIT recombinational maps. In addition, 108 randomly selected and 6 STS-linked BACs have been assigned to single chromosome bands. We have also developed a high-resolution fluorescent reverse-banding technique for mouse chromosomes that allows simultaneous localization of probes by fluorescence in situ hybridization (FISH) with respect to the cytogenetic landmarks. This approach integrates studies of the entire mouse genome. Moreover, these reagents will simplify gene mapping and analyses of genomic fragments in fetal and adult mouse models. As shown with the MMU16 telomeric marker for the trisomy 16 mouse model of Down syndrome, these clones can obviate the need for metaphase analyses. The potential contribution of this resource and associated methods extends well beyond mapping and includes clues to understanding mouse chromosomes and their rearrangements in cancers and evolution. Finally it will facilitate the development of an integrated view of the mouse genome by providing anchor points from the genetic to the cytogenetic and functional maps of the mouse as we attempt to understand mutations, their biological consequences, and gene function.