Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal.

BACKGROUND: The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents. METHODS AND FINDINGS: We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937-1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974-1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988-1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe. CONCLUSIONS: In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.

Human chromosome 11 DNA sequence and analysis including novel gene identification.

Chromosome 11, although average in size, is one of the most gene- and disease-rich chromosomes in the human genome. Initial gene annotation indicates an average gene density of 11.6 genes per megabase, including 1,524 protein-coding genes, some of which were identified using novel methods, and 765 pseudogenes. One-quarter of the protein-coding genes shows overlap with other genes. Of the 856 olfactory receptor genes in the human genome, more than 40% are located in 28 single- and multi-gene clusters along this chromosome. Out of the 171 disorders currently attributed to the chromosome, 86 remain for which the underlying molecular basis is not yet known, including several mendelian traits, cancer and susceptibility loci. The high-quality data presented here--nearly 134.5 million base pairs representing 99.8% coverage of the euchromatic sequence--provide scientists with a solid foundation for understanding the genetic basis of these disorders and other biological phenomena.

DNA sequence and analysis of human chromosome 8.

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

Analysis of the DNA sequence and duplication history of human chromosome 15.

Here we present a finished sequence of human chromosome 15, together with a high-quality gene catalogue. As chromosome 15 is one of seven human chromosomes with a high rate of segmental duplication, we have carried out a detailed analysis of the duplication structure of the chromosome. Segmental duplications in chromosome 15 are largely clustered in two regions, on proximal and distal 15q; the proximal region is notable because recombination among the segmental duplications can result in deletions causing Prader-Willi and Angelman syndromes. Sequence analysis shows that the proximal and distal regions of 15q share extensive ancient similarity. Using a simple approach, we have been able to reconstruct many of the events by which the current duplication structure arose. We find that most of the intrachromosomal duplications seem to share a common ancestry. Finally, we demonstrate that some remaining gaps in the genome sequence are probably due to structural polymorphisms between haplotypes; this may explain a significant fraction of the gaps remaining in the human genome.

DNA sequence and analysis of human chromosome 18.

Chromosome 18 appears to have the lowest gene density of any human chromosome and is one of only three chromosomes for which trisomic individuals survive to term. There are also a number of genetic disorders stemming from chromosome 18 trisomy and aneuploidy. Here we report the finished sequence and gene annotation of human chromosome 18, which will allow a better understanding of the normal and disease biology of this chromosome. Despite the low density of protein-coding genes on chromosome 18, we find that the proportion of non-protein-coding sequences evolutionarily conserved among mammals is close to the genome-wide average. Extending this analysis to the entire human genome, we find that the density of conserved non-protein-coding sequences is largely uncorrelated with gene density. This has important implications for the nature and roles of non-protein-coding sequence elements.

Suture constructs for rehabilitation using early active motion after tendon transfer surgery.

The purpose of this study was to better define an ideal tendon transfer suture construct to allow for early active range of motion. A side-to-side tendon construct was used to test suture technique (cross stich vs. Krackow stitch), number of suture throws, and calibre of suture. A minimum load to failure of 100 N was used to comfortably allow early motion while minimizing rupture risk. All constructs tested, except the 4-0 Krackow construct, were strong enough to withstand 100 N of load. The choice of suture should be based on surgeon preference, patient compliance, and specific surgery, and 3-0 non-absorbable suture may be more suitable for tendon transfers from a yield force standpoint.

Lift-off Test Results After Lesser Tuberosity Osteotomy Versus Subscapularis Peel in Primary Total Shoulder Arthroplasty.

BACKGROUND: The ideal method for management of the subscapularis tendon during anatomic total shoulder arthroplasty (TSA) remains controversial. METHODS: In a retrospective cohort study, primary anatomic TSA procedures performed with either a subscapularis peel or a lesser tuberosity osteotomy from 2002 to 2010 were reviewed at a minimum 1-year follow-up. The primary outcome measure was the performance of a normal lift-off test postoperatively. Multivariate logistic regression analysis was performed to determine if other covariates besides surgical technique correlated with an abnormal lift-off test result. RESULTS: Ninety TSA procedures were evaluated. Forty-six procedures were performed with subscapularis peel, and 44 were performed with lesser tuberosity osteotomy. Mean follow-up was 4 years. In the subscapularis peel group, 32 of 46 shoulders (69.6%) had a normal lift-off test, compared with 40 of 44 shoulders (90.9%) in the lesser tuberosity osteotomy group (P = 0.01). The results of multivariate logistic regression suggested that lesser tuberosity osteotomy was associated with a normal postoperative lift-off test 4.5 times more often than was subscapularis peel. CONCLUSIONS: Our study suggests that the use of lesser tuberosity osteotomy as the surgical approach for anatomic TSA is a reliable option that provides the patient with a better chance of maintaining subscapularis function postoperatively than the subscapularis peel does. LEVEL OF EVIDENCE: Level III retrospective cohort study.

An improved canine genome and a comprehensive catalogue of coding genes and non-coding transcripts.

The domestic dog, Canis familiaris, is a well-established model system for mapping trait and disease loci. While the original draft sequence was of good quality, gaps were abundant particularly in promoter regions of the genome, negatively impacting the annotation and study of candidate genes. Here, we present an improved genome build, canFam3.1, which includes 85 MB of novel sequence and now covers 99.8% of the euchromatic portion of the genome. We also present multiple RNA-Sequencing data sets from 10 different canine tissues to catalog ∼175,000 expressed loci. While about 90% of the coding genes previously annotated by EnsEMBL have measurable expression in at least one sample, the number of transcript isoforms detected by our data expands the EnsEMBL annotations by a factor of four. Syntenic comparison with the human genome revealed an additional ∼3,000 loci that are characterized as protein coding in human and were also expressed in the dog, suggesting that those were previously not annotated in the EnsEMBL canine gene set. In addition to ∼20,700 high-confidence protein coding loci, we found ∼4,600 antisense transcripts overlapping exons of protein coding genes, ∼7,200 intergenic multi-exon transcripts without coding potential, likely candidates for long intergenic non-coding RNAs (lincRNAs) and ∼11,000 transcripts were reported by two different library construction methods but did not fit any of the above categories. Of the lincRNAs, about 6,000 have no annotated orthologs in human or mouse. Functional analysis of two novel transcripts with shRNA in a mouse kidney cell line altered cell morphology and motility. All in all, we provide a much-improved annotation of the canine genome and suggest regulatory functions for several of the novel non-coding transcripts.

High quality draft genome sequence of Segniliparus rugosus CDC 945(T)= (ATCC BAA-974(T)).

Segniliparus rugosus represents one of two species in the genus Segniliparus, the sole genus in the family Segniliparaceae. A unique and interesting feature of this family is the presence of extremely long carbon-chain length mycolic acids bound in the cell wall. S. rugosus is also a medically important species because it is an opportunistic pathogen associated with mammalian lung disease. This report represents the second species in the genus to have its genome sequenced. The 3,567,567 bp long genome with 3,516 protein-coding and 49 RNA genes is part of the NIH Roadmap for Medical Research, Human Microbiome Project.

Comparative functional genomics of the fission yeasts.

The fission yeast clade--comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus--occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

A sequence-based map of the nine genes of the human interleukin-1 cluster.

Six novel genes encoding proteins with the interleukin (IL)-1 fold have been identified recently. The classical family members are involved in inflammatory signaling. Previous work has placed the novel genes close to or within the same cluster as IL1A, IL1B, and IL1RN, which occupy an approximately 400-kb interval on chromosome 2. We have combined the incomplete public database sequence with our own sequence to generate a reference sequence and map that encompass all of the novel genes, allowing determination of the gene structures, precise localization of exons, and determination of distances between conventional SNP and microsatellite markers. Gene order from centromere to telomere is IL1A-IL1B-IL1F7-IL1F9-IL1F6-IL1F8-IL1F5-IL1F10-IL1RN, of which only IL1A, IL1B, and IL1F8 are transcribed towards the centromere. The gene order relates to the evolutionary relationship between the genes. Key features of exon boundaries are conserved. There is no evidence for other IL-1 family members within the cluster.

The complete genome and proteome of Mycoplasma mobile.

Although often considered "minimal" organisms, mycoplasmas show a wide range of diversity with respect to host environment, phenotypic traits, and pathogenicity. Here we report the complete genomic sequence and proteogenomic map for the piscine mycoplasma Mycoplasma mobile, noted for its robust gliding motility. For the first time, proteomic data are used in the primary annotation of a new genome, providing validation of expression for many of the predicted proteins. Several novel features were discovered including a long repeating unit of DNA of approximately 2435 bp present in five complete copies that are shown to code for nearly identical yet uniquely expressed proteins. M. mobile has among the lowest DNA GC contents (24.9%) and most reduced set of tRNAs of any organism yet reported (28). Numerous instances of tandem duplication as well as lateral gene transfer are evident in the genome. The multiple available complete genome sequences for other motile and immotile mycoplasmas enabled us to use comparative genomic and phylogenetic methods to suggest several candidate genes that might be involved in motility. The results of these analyses leave open the possibility that gliding motility might have arisen independently more than once in the mycoplasma lineage.

A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait.

Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high-bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted beta-propeller module of the low-density lipoprotein receptor-related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.

Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness.

Asthma is a common respiratory disorder characterized by recurrent episodes of coughing, wheezing and breathlessness. Although environmental factors such as allergen exposure are risk factors in the development of asthma, both twin and family studies point to a strong genetic component. To date, linkage studies have identified more than a dozen genomic regions linked to asthma. In this study, we performed a genome-wide scan on 460 Caucasian families and identified a locus on chromosome 20p13 that was linked to asthma (log(10) of the likelihood ratio (LOD), 2.94) and bronchial hyperresponsiveness (LOD, 3.93). A survey of 135 polymorphisms in 23 genes identified the ADAM33 gene as being significantly associated with asthma using case-control, transmission disequilibrium and haplotype analyses (P = 0.04 0.000003). ADAM proteins are membrane-anchored metalloproteases with diverse functions, which include the shedding of cell-surface proteins such as cytokines and cytokine receptors. The identification and characterization of ADAM33, a putative asthma susceptibility gene identified by positional cloning in an outbred population, should provide insights into the pathogenesis and natural history of this common disease.