Alice C. Young

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

Topographical and temporal diversity of the human skin microbiome.

The Close and Personal Biome Fortunately, our skin is readily accessible for ecological studies of the microbial communities that influence health and disease states. Grice et al. (p. 1190) present a metagenomic survey of body sites from 10 healthy human individuals sampled over time. Although, altogether 18 phyla were discovered, only a few predominated. The most diverse communities were found on the forearm and the least behind the ear, but between people the microorganisms living behind the knees, in the elbow, and behind the ear were most similar. This finding might have some bearing on the common occurrence of atopic dermatitis in these zones, although no similar relationship was discerned between skin microbial flora and psoriasis. The human skin provides a landscape of dry, damp, and greasy niches for a diversity of symbiotic microorganisms. Human skin is a large, heterogeneous organ that protects the body from pathogens while sustaining microorganisms that influence human health and disease. Our analysis of 16S ribosomal RNA gene sequences obtained from 20 distinct skin sites of healthy humans revealed that physiologically comparable sites harbor similar bacterial communities. The complexity and stability of the microbial community are dependent on the specific characteristics of the skin site. This topographical and temporal survey provides a baseline for studies that examine the role of bacterial communities in disease states and the microbial interdependencies required to maintain healthy skin.

Comparative analyses of multi-species sequences from targeted genomic regions

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum

Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.

The ClinSeq Project: Piloting large-scale genome sequencing for research in genomic medicine

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300-400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.

Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae

Single-molecule sequencing of bacteria at the NIH Clinical Center documents diverse plasmids encoding antibiotic resistance and their transfer between microbes. How Antibiotic Resistance Spreads Among Bacteria Antibiotic-resistant microbes are spreading at an alarming rate in health care facilities throughout the world. Conlan et al. use a new DNA sequencing method to take a close look at one way in which antibiotic resistance spreads. With single-molecule sequencing, the authors completely characterized individual plasmids, the circular bits of DNA that carry the genes for antibiotic resistance in bacteria. They focused on resistance to the carbapenems, a class of antibiotics that is often used for infections that do not respond to more conventional antimicrobial agents. By using this approach in their microbial surveillance program at the NIH Clinical Center, the authors found evidence that plasmids carrying carbapenemase genes moved from one microbial species to another within the hospital environment. They also used the technique to test hypotheses about patient-to-patient transmission and to characterize a previously undescribed carbapenemase-encoding plasmid carried by diverse bacterial species that could cause dangerous clinical infections. Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care–associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.

Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli. Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae. Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms. IMPORTANCE In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae. Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care. In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae. Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care.

Incidental Medical Information in Whole-Exome Sequencing

Genomic technologies, such as whole-exome sequencing, are a powerful tool in genetic research. Such testing yields a great deal of incidental medical information, or medical information not related to the primary research target. We describe the management of incidental medical information derived from whole-exome sequencing in the research context. We performed whole-exome sequencing on a monozygotic twin pair in which only 1 child was affected with congenital anomalies and applied an institutional review board–approved algorithm to determine what genetic information would be returned. Whole-exome sequencing identified 79 525 genetic variants in the twins. Here, we focus on novel variants. After filtering artifacts and excluding known single nucleotide polymorphisms and variants not predicted to be pathogenic, the twins had 32 novel variants in 32 genes that were felt to be likely to be associated with human disease. Eighteen of these novel variants were associated with recessive disease and 18 were associated with dominantly manifesting conditions (variants in some genes were potentially associated with both recessive and dominant conditions), but only 1 variant ultimately met our institutional review board–approved criteria for return of information to the research participants.

Gene-Based Sequencing Identifies Lipid-Influencing Variants with Ethnicity-Specific Effects in African Americans

Although a considerable proportion of serum lipids loci identified in European ancestry individuals (EA) replicate in African Americans (AA), interethnic differences in the distribution of serum lipids suggest that some genetic determinants differ by ethnicity. We conducted a comprehensive evaluation of five lipid candidate genes to identify variants with ethnicity-specific effects. We sequenced ABCA1, LCAT, LPL, PON1, and SERPINE1 in 48 AA individuals with extreme serum lipid concentrations (high HDLC/low TG or low HDLC/high TG). Identified variants were genotyped in the full population-based sample of AA (n = 1694) and tested for an association with serum lipids. rs328 (LPL) and correlated variants were associated with higher HDLC and lower TG. Interestingly, a stronger effect was observed on a “European” vs. “African” genetic background at this locus. To investigate this effect, we evaluated the region among West Africans (WA). For TG, the effect size among WA was the same in AA with only African local ancestry (2–3% lower TG), while the larger association among AA with local European ancestry matched previous reports in EA (10%). For HDLC, there was no association with rs328 in AA with only African local ancestry or in WA, while the association among AA with European local ancestry was much greater than what has been observed for EA (15 vs. ∼5 mg/dl), suggesting an interaction with an environmental or genetic factor that differs by ethnicity. Beyond this ancestry effect, the importance of African ancestry-focused, sequence-based work was also highlighted by serum lipid associations of variants that were in higher frequency (or present only) among those of African ancestry. By beginning our study with the sequence variation present in AA individuals, investigating local ancestry effects, and seeking replication in WA, we were able to comprehensively evaluate the role of a set of candidate genes in serum lipids in AA.

Personalized genomic medicine: Lessons from the exome

While genomic sequencing methods are powerful tools in the discovery of the genetic underpinnings of human disease, incidentally-revealed novel genomic risk factors may be equally important, both scientifically, and as relates to direct patient care. We performed whole-exome sequencing on a child with VACTERL association who suffered severe post-surgical neonatal pulmonary hypertension, and identified a potential novel genetic risk factor for this complication: a heterozygous mutation in CPSI. Newborn screening results from this patients monozygotic twin provided evidence that this mutation, in combination with an environmental trigger (in this case, surgery), may have resulted in pulmonary artery hypertension due to inadequate nitric oxide production. Identification of this genetic risk factor allows for targeted medical preventative measures in this patient as well as relatives with the same mutation, and illustrates the power of incidental medical information unearthed by whole-exome sequencing.