Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes.

Genetic analysis of an adult patient with an unusual course of ketosis-prone diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the 5'-untranslated region (UTR) of PDX1, a ß-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm (DE) stage. Metabolomics analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the DE to the pancreatic progenitor (PP) stage, and RNA sequencing showed that defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR/Cas9 manipulation of the PDX1 variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the PDX1 5'-UTR, leading to decreased PDX1 expression at the early PP stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical ß-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset ß-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multiomics analysis of patient-specific stem cells for clinical discovery.

Short Communication: Lack of Support for Socially Connected HIV-1 Transmission Among Young Adult Black Men Who Have Sex with Men.

We explore the phylogenetic relationships among HIV sequences sampled from young adult black men who have sex with men (YAB-MSM), who are connected through peer referral/social ties and who attend common venues. Using 196 viral sequences sampled from the peripheral blood mononuclear cells of 10 individuals, our preliminary phylogenetic results indicate that these socially connected YAB-MSM are infected with distantly related viruses and provide no evidence for viral transmission between network members. Our results suggest that HIV-prevention strategies that target young adult MSM should extend beyond their network members and local community.

Human adipose tissue as a reservoir for memory CD4+ T cells and HIV.

OBJECTIVE: The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. DESIGN: We examined memory CD4(+) T cells and HIV DNA in adipose tissue-stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4(+) T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4(+) T cells. METHODS: AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4(+) T-cell activation and HIV production were measured by flow cytometry and ELISA. RESULTS: AT-SVF CD3(+) T cells were activated (>60% CD69(+)) memory CD4(+) and CD8(+) T cells in uninfected and HIV-infected persons, but the AT-SVF CD4(+)/CD8(+) ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4(+) T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-α1ß1. Adipocytes also enhanced T-cell viability. CONCLUSION: Adipose tissues of ART-treated patients harbour activated memory CD4(+) T cells and HIV DNA. Adipocytes promote CD4(+) T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.

Association of TCF7L2 variation with single islet autoantibody expression in children with type 1 diabetes.

BACKGROUND: The transcription factor 7-like 2 (TCF7L2) gene has the strongest genetic association with type 2 diabetes. TCF7L2 also associates with latent autoimmune diabetes in adults, which often presents with a single islet autoantibody, but not with classical type 1 diabetes. METHODS: We aimed to test if TCF7L2 is associated with single islet autoantibody expression in pediatric type 1 diabetes. We studied 71 prospectively recruited children who had newly diagnosed type 1 diabetes and evidence of islet autoimmunity, that is, expressed ≥1 islet autoantibody to insulin, glutamic acid decarboxylase 65, islet cell autoantigen 512, or zinc transporter 8. TCF7L2 rs7903146 alleles were identified. Data at diagnosis were cross-sectionally analyzed. RESULTS: We found that 21.1% of the children with autoimmune type 1 diabetes expressed a single islet autoantibody. The distribution of TCF7L2 rs7903146 genotypes in children with a single autoantibody (n=15) was 40% CC, 26.7% CT and 33.3% TT, compared with children with ≥2 islet autoantibodies (50% CC, 42.9% CT and 7.1% TT, p=0.024). Furthermore, compared with children with ≥2 autoantibodies, single-autoantibody children had characteristics reflecting milder autoimmune destruction of ß-cells. Restricting to lean children (body mass index<85th centile; n=36), 45.5% of those expressing a single autoantibody were rs7903146 TT homozygotes, compared with 0% of those with ≥2 autoantibodies (p<0.0001). CONCLUSION: These results suggest that, in children with only mild islet autoimmunity, mechanisms associated with TCF7L2 genetic variation contribute to diabetogenesis, and this contribution is larger in the absence of obesity.

Untangling the influences of unmodeled evolutionary processes on phylogenetic signal in a forensically important HIV-1 transmission cluster.

Stochastic models of sequence evolution have been developed to reflect many biologically important processes, allowing for accurate phylogenetic reconstruction when an appropriate model is selected. However, commonly used models do not incorporate several potentially important biological processes. Spurious phylogenetic inference may result if these processes play an important role in the evolution of a dataset yet are not incorporated into assumed models. Few studies have attempted to assess the relative importance of multiple processes in producing spurious inferences. The application of phylogenetic methods to infer the source of HIV-1 transmission clusters depends upon accurate phylogenetic results, yet there are several relevant unmodeled biological processes (e.g., recombination and convergence) that may cause complications. Here, through analyses of HIV-1 env sequences from a small, forensically important transmission cluster, we tease apart the impact of these processes and present evidence suggesting that convergent evolution and high rates of insertions and deletions (causing alignment uncertainty) led to spurious phylogenetic signal with forensic relevance. Previous analyses show paraphyly of HIV-1 lineages sampled from an individual who, based on non-phylogenetic evidence, had never acted as a source of infection for others in this transmission cluster. If true, this pattern calls into question assumptions underlying phylogenetic approaches to source and recipient identification. By systematically assessing the contribution of different unmodeled processes, we demonstrate that removal of sites likely influenced by strong positive selection both reduces the alignment-wide signal supporting paraphyly of viruses sampled from this individual and eliminates support for the effects of recombination. Additionally, the removal of ambiguously aligned sites alters strongly supported relationships among viruses sampled from different individuals. These observations highlight the need to jointly consider multiple unmodeled evolutionary processes and motivate a phylogenomic perspective when inferring viral transmission histories.

Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators.

Recent developments of unique nucleotide probes have expanded our understanding of DNA polymerase function, providing many benefits to techniques involving next-generation sequencing (NGS) technologies. The cyclic reversible termination (CRT) method depends on efficient base-selective incorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3'-O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3'-OH unblocked nucleotides, called Lightning Terminators™, which have a terminating 2-nitrobenzyl moiety attached to hydroxymethylated nucleobases. A key structural feature of this photocleavable group displays a 'molecular tuning' effect with respect to single-base termination and improved nucleotide fidelity. Using Therminator DNA polymerase, we demonstrate that these 3'-OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3'-O-amino-TTP and 3'-O-azidomethyl-TTP. Lightning Terminators show maximum incorporation rates (k(pol)) that range from 35 to 45 nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k(pol)/K(D)) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improved nucleotide selectivity is a significant reduction in k(pol) by >1000-fold over TTP misincorporation. These studies highlight the importance of structure-function relationships of modified nucleotides in dictating polymerase performance.

Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates.

We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.

Source identification in two criminal cases using phylogenetic analysis of HIV-1 DNA sequences.

Phylogenetic analysis has been widely used to test the a priori hypothesis of epidemiological clustering in suspected transmission chains of HIV-1. Among studies showing strong support for relatedness between HIV samples obtained from infected individuals, evidence for the direction of transmission between epidemiologically related pairs has been lacking. During transmission of HIV, a genetic bottleneck occurs, resulting in the paraphyly of source viruses with respect to those of the recipient. This paraphyly establishes the direction of transmission, from which the source can then be inferred. Here, we present methods and results from two criminal cases, State of Washington v Anthony Eugene Whitfield, case number 04-1-0617-5 (Superior Court of the State of Washington, Thurston County, 2004) and State of Texas v Philippe Padieu, case numbers 219-82276-07, 219-82277-07, 219-82278-07, 219-82279-07, 219-82280-07, and 219-82705-07 (219th Judicial District Court, Collin County, TX, 2009), which provided evidence that direction can be established from blinded case samples. The observed paraphyly from each case study led to the identification of an inferred source (i.e., index case), whose identity was revealed at trial to be that of the defendant.

Sequencing technologies - the next generation.

Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

A-beta-subtype of ketosis-prone diabetes is not predominantly a monogenic diabetic syndrome.

OBJECTIVE: Ketosis-prone diabetes (KPD) is an emerging syndrome that encompasses several distinct phenotypic subgroups that share a predisposition to diabetic ketoacidosis. We investigated whether the A-beta- subgroup of KPD, characterized by complete insulin dependence, absent beta-cell functional reserve, lack of islet cell autoantibodies, and strong family history of type 2 diabetes, represents a monogenic form of diabetes. RESEARCH DESIGN AND METHODS: Over 8 years, 37 patients with an A-beta- phenotype were identified in our longitudinally followed cohort of KPD patients. Seven genes, including hepatocyte nuclear factor 4A (HNF4A), glucokinase (GCK), HNF1A, pancreas duodenal homeobox 1 (PDX1), HNF1B, neurogenic differentiation 1 (NEUROD1), and PAX4, were directly sequenced in all patients. Selected gene regions were also sequenced in healthy, unrelated ethnically matched control subjects, consisting of 84 African American, 96 Caucasian, and 95 Hispanic subjects. RESULTS: The majority (70%) of the A-beta- KPD patients had no significant causal polymorphisms in either the proximal promoter or coding regions of the seven genes. The combination of six potentially significant low-frequency, heterozygous sequence variants in HNF-1 alpha (A174V or G574S), PDX1 (putative 5'-untranslated region CCAAT box, P33T, or P239Q), or PAX4 (R133W) were found in 27% (10/37) of patients, with one additional patient revealing two variants, PDX1 P33T and PAX4 R133W. The A174V variant has not been previously reported. CONCLUSIONS: Despite its well-circumscribed, robust, and distinctive phenotype of severe, nonautoimmune-mediated beta-cell dysfunction, A-beta- KPD is most likely not a predominantly monogenic diabetic syndrome. Several A-beta- KPD patients have low-frequency variants in HNF1A, PDX1, or PAX4 genes, which may be of functional significance in their pathophysiology.

Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates.

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N(6)-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N(6)-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N(6)-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology.

We have designed fluorescent "through-bond energy-transfer cassettes" that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1-4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein- and rhodamine-derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high-throughput DNA sequencing), and they exhibit faster energy-transfer rates than a similar through-space energy-transfer cassette. Specifically, energy-transfer rates in these cassettes is around 6-7 ps, in contrast to a similar through-space energy-transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein-derived donors. This was confirmed by bulk fluorescent measurements, and in single-molecule-detection studies. Modification of a commercial automated DNA-sequencing apparatus to detect the emissions of these four energy-transfer cassettes enabled single-color dye-primer sequencing.

The DNA sequence, annotation and analysis of human chromosome 3.

After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

The finished DNA sequence of human chromosome 12.

Human chromosome 12 contains more than 1,400 coding genes and 487 loci that have been directly implicated in human disease. The q arm of chromosome 12 contains one of the largest blocks of linkage disequilibrium found in the human genome. Here we present the finished sequence of human chromosome 12, which has been finished to high quality and spans approximately 132 megabases, representing approximately 4.5% of the human genome. Alignment of the human chromosome 12 sequence across vertebrates reveals the origin of individual segments in chicken, and a unique history of rearrangement through rodent and primate lineages. The rate of base substitutions in recent evolutionary history shows an overall slowing in hominids compared with primates and rodents.

l7Rn6 encodes a novel protein required for clara cell function in mouse lung development.

The highly secretory Clara cells play a pivotal role in protecting the lung against inflammation and oxidative stress. This study reports the positional cloning of a novel protein required for Clara cell physiology in mouse lung development. The perinatal lethal N-ethyl-N-nitrosourea-induced l7Rn6(4234SB) allele contained a nonsense mutation in the previously hypothetical gene NM_026304 on chromosome 7. Whereas l7Rn6 mRNA levels were indistinguishable from wild type, l7Rn6(4234SB) homozygotes exhibited decreased expression of the truncated protein, suggesting protein instability. During late gestation, l7Rn6 was widely expressed in the cytoplasm of lung epithelial cells, whereas perinatal expression was restricted to the bronchiolar epithelium. Homozygosity for the l7Rn6(4234SB) allele did not affect early steps in lung patterning, growth, or cellular differentiation. Rather, mutant lungs demonstrated severe emphysematous enlargement of the distal respiratory sacs at birth. Clara cell pathophysiology was evident from decreased cytoplasmic CCSP and SP-B protein levels, enlargement and disorganization of the Golgi complex, and formation of aberrant vesicular structures. Additional support for a role in the secretory pathway derived from l7Rn6 localization to the endoplasmic reticulum. Thus, l7Rn6 represents a novel protein required for organization and/or function of the secretory apparatus in Clara cells in mouse lung.

Emerging technologies in DNA sequencing.

Demand for DNA sequence information has never been greater, yet current Sanger technology is too costly, time consuming, and labor intensive to meet this ongoing demand. Applications span numerous research interests, including sequence variation studies, comparative genomics and evolution, forensics, and diagnostic and applied therapeutics. Several emerging technologies show promise of delivering next-generation solutions for fast and affordable genome sequencing. In this review article, the DNA polymerase-dependent strategies of Sanger sequencing, single nucleotide addition, and cyclic reversible termination are discussed to highlight recent advances and potential challenges these technologies face in their development for ultrafast DNA sequencing.

Color-blind fluorescence detection for four-color DNA sequencing.

We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission wavelength-dependent approaches for identifying fluorescent species, the removal of the spectral component in PME confers a number of advantages including higher and normalized signals from all dyes present in the assay, the elimination of spectral cross-talk between dyes, and higher signal collection efficiency. Base-calling is unambiguously determined once dye mobility corrections are made. These advantages translate into significantly enhanced signal quality as illustrated in the primary DNA sequencing data and provide a means for achieving accurate base-calling at lower reagent concentrations.