Identification of allele-specific KIV-2 repeats and impact on Lp(a) measurements for cardiovascular disease risk.

The abundance of Lp(a) protein holds significant implications for the risk of cardiovascular disease (CVD), which is directly impacted by the copy number (CN) of KIV-2, a 5.5 kbp sub-region. KIV-2 is highly polymorphic in the population and accurate analysis is challenging. In this study, we present the DRAGEN KIV-2 CN caller, which utilizes short reads. Data across 166 WGS show that the caller has high accuracy, compared to optical mapping and can further phase ~50% of the samples. We compared KIV-2 CN numbers to 24 previously postulated KIV-2 relevant SNVs, revealing that many are ineffective predictors of KIV-2 copy number. Population studies, including USA-based cohorts, showed distinct KIV-2 CN, distributions for European-, African-, and Hispanic-American populations and further underscored the limitations of SNV predictors. We demonstrate that the CN estimates correlate significantly with the available Lp(a) protein levels and that phasing is highly important.

Challenges of Francisella classification exemplified by an atypical clinical isolate.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains.

Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis.

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

High-Quality Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain OR96-0246.

The bacterial pathogen Francisella tularensis was recently renewed as a tier-one select agent. F. tularensis subsp. tularensis (type A) and holarctica (type B) are of clinical relevance. Here, we report the complete genome of a virulent F. tularensis type B strain and describe its usefulness in comparative genomics.

Whole-exome sequencing points to considerable genetic heterogeneity of cerebral palsy.

Cerebral palsy (CP) is a common, clinically heterogeneous group of disorders affecting movement and posture. Its prevalence has changed little in 50 years and the causes remain largely unknown. The genetic contribution to CP causation has been predicted to be ~2%. We performed whole-exome sequencing of 183 cases with CP including both parents (98 cases) or one parent (67 cases) and 18 singleton cases (no parental DNA). We identified and validated 61 de novo protein-altering variants in 43 out of 98 (44%) case-parent trios. Initial prioritization of variants for causality was by mutation type, whether they were known or predicted to be deleterious and whether they occurred in known disease genes whose clinical spectrum overlaps CP. Further, prioritization used two multidimensional frameworks-the Residual Variation Intolerance Score and the Combined Annotation-dependent Depletion score. Ten de novo mutations in three previously identified disease genes (TUBA1A (n=2), SCN8A (n=1) and KDM5C (n=1)) and in six novel candidate CP genes (AGAP1, JHDM1D, MAST1, NAA35, RFX2 and WIPI2) were predicted to be potentially pathogenic for CP. In addition, we identified four predicted pathogenic, hemizygous variants on chromosome X in two known disease genes, L1CAM and PAK3, and in two novel candidate CP genes, CD99L2 and TENM1. In total, 14% of CP cases, by strict criteria, had a potentially disease-causing gene variant. Half were in novel genes. The genetic heterogeneity highlights the complexity of the genetic contribution to CP. Function and pathway studies are required to establish the causative role of these putative pathogenic CP genes.

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray.

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8(+) T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20-30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5-10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Next-generation sequencing study finds an excess of rare, coding single-nucleotide variants of ADAMTS13 in patients with deep vein thrombosis.

BACKGROUND: The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Rare single-nucleotide variants (SNVs) of the coding areas of hemostatic genes may explain part of this missing heritability. The ADAMTS13 and VWF genes encode two interconnected proteins with fundamental hemostatic functions, the disruption of which may result in thrombosis. OBJECTIVES: To study the distribution and burden of rare coding SNVs of ADAMTS13 and VWF found by sequencing in cases and controls of DVT. PATIENTS/METHODS: The protein-coding areas of 186 hemostatic/proinflammatory genes were sequenced by next-generation technology in 94 thrombophilia-negative patients with DVT and 98 controls. Gene-specific information on ADAMTS13 and VWF was used to study the association between DVT and rare coding SNVs of the two genes. RESULTS: More than 70 billion base pairs of raw sequence data were produced to sequence the 700-kb target area with a median redundancy of × 45 in 192 individuals. Most of the 4366 SNVs identified were rare and non-synonymous, indicating pathogenetic potential. Rare (frequency of < 1%) and low-frequency (< 5%) coding SNVs of ADAMTS13 were associated with DVT (prevalence 17% vs. 4%; odds ratio [OR] 4.8 and 95% confidence interval [CI] 1.6-15.0 for rare coding; prevalence 36% vs. 23%, OR 1.9 and 95% CI 1.0-3.5 for low-frequency coding). Patients with rare coding SNVs of ADAMTS13 had lower plasma levels of ADAMTS-13 activity than patients without them. SNVs of VWF were not associated with DVT. CONCLUSIONS: We found an excess of rare coding SNVs of the ADAMTS13 gene in patients with DVT.

CRHR1 genotypes, neural circuits and the diathesis for anxiety and depression.

The corticotrophin-releasing hormone (CRH) system integrates the stress response and is associated with stress-related psychopathology. Previous reports have identified interactions between childhood trauma and sequence variation in the CRH receptor 1 gene (CRHR1) that increase risk for affective disorders. However, the underlying mechanisms that connect variation in CRHR1 to psychopathology are unknown. To explore potential mechanisms, we used a validated rhesus macaque model to investigate association between genetic variation in CRHR1, anxious temperament (AT) and brain metabolic activity. In young rhesus monkeys, AT is analogous to the childhood risk phenotype that predicts the development of human anxiety and depressive disorders. Regional brain metabolism was assessed with (18)F-labeled fluoro-2-deoxyglucose (FDG) positron emission tomography in 236 young, normally reared macaques that were also characterized for AT. We show that single nucleotide polymorphisms (SNPs) affecting exon 6 of CRHR1 influence both AT and metabolic activity in the anterior hippocampus and amygdala, components of the neural circuit underlying AT. We also find evidence for association between SNPs in CRHR1 and metabolism in the intraparietal sulcus and precuneus. These translational data suggest that genetic variation in CRHR1 affects the risk for affective disorders by influencing the function of the neural circuit underlying AT and that differences in gene expression or the protein sequence involving exon 6 may be important. These results suggest that variation in CRHR1 may influence brain function before any childhood adversity and may be a diathesis for the interaction between CRHR1 genotypes and childhood trauma reported to affect human psychopathology.

Comparative sequence analysis of a gene-rich cluster at human chromosome 12p13 and its syntenic region in mouse chromosome 6.

The Human Genome Project has created a formidable challenge: the extraction of biological information from extensive amounts of raw sequence. With the increasing availability of genomic sequence from other species, one approach to extracting coding and regulatory element information is through cross-species sequence comparison. To assess the strengths and weaknesses of this methodology for large-scale sequence analysis, 227 kb of mouse sequence syntenic to a gene-rich cluster on human chromosome 12p13 was obtained. Primarily through percent identity plots (PIPs) of SIM comparative sequence alignments, the sequence of coding regions, putative alternative exons, conserved noncoding regions, and correlation in repetitive element insertions were easily determined. The analysis demonstrated that the number, order, and orientation of all 17 genes are conserved between the two species, whereas two human pseudogenes are absent in mouse. In addition, apart from MIRs, no direct correlation of distribution or position of the majority of repetitive elements between the two species is seen. Finally, in examining the synonymous and nonsynonymous substitution rates in the conserved genes, a large variation in nonsynonymous rates is observed indicating that the genes in this region are diverging at different rates. This study indicates the utility and strength of large-scale cross-species sequence comparisons in the extraction of biological information from raw sequence, especially when combined with other computational tools such as GRAIL and BLAST.

Large-scale comparative sequence analysis of the human and murine Bruton's tyrosine kinase loci reveals conserved regulatory domains.

Large-scale genomic DNA sequencing of orthologous and paralogous loci in different species should contribute to a basic understanding of the evolution of both the protein-coding regions and noncoding regulatory elements. We compared 93 kb of human sequence to 89 kb of mouse sequence in the Bruton's tyrosine kinase (BTK) region. In addition to showing the conservation of both position and orientation of the five functionally unrelated genes in the region (BTK, alpha-D-galactosidase A, L44L, FTP-3, and FCI-12), the comparison revealed conservation of clusters of noncoding sequence flanking the first exon of each gene. Furthermore, in the sequence comparison at the BTK locus, the conservation of clusters of noncoding sequence extends throughout the locus; the noncoding sequence is more highly conserved in the BTK locus in comparison to the flanking loci. This suggests a correlation with the complex developmental regulation of expression of btk. To determine whether a highly conserved 3.5-kb segment flanking the first exon of BTK contains transcriptional regulatory signals, we tested various portions of the segment for promoter and expression activity in several appropriate cell lines. The results demonstrate the contribution of the conserved region flanking the first exon to the cell lineage-specific expression pattern of btk. These data show the usefulness of large scale sequence comparisons to focus investigation on regions of noncoding sequence that play essential roles in complex gene regulation.

Large-scale concatenation cDNA sequencing.

A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

Large-scale sequencing in human chromosome 12p13: experimental and computational gene structure determination.

The detailed genomic organization of a gene-dense region at human chromosome 12p13, spanning 223 kb of contiguous sequence, was determined. This region is composed of 20 genes and several other expressed sequences. Experimental tools including RT-PCR and cDNA sequencing, combined with gene prediction programs, were utilized in the analysis of the sequence. Various computer software programs were employed for sequence similarity searches and functional predictions. The high number of genes with diverse functions and complex transcriptional patterns make this region ideal for addressing challenges of gene discovery and genomic characterization amenable to large-scale sequence analysis.

Molecular and phenotypic variation in patients with severe Hunter syndrome.

Severe Hunter syndrome is a fatal X-linked lysosomal storage disorder caused by iduronate-2-sulphatase (IDS) deficiency. Patients with complete deletion of the IDS locus often have atypical phenotypes including ptosis, obstructive sleep apnoea, and the occurrence of seizures. We have used genomic DNA sequencing to identify several new genes in the IDS region. DNA deletion patients with atypical symptoms have been analysed to determine whether these atypical symptoms could be due to involvement of these other loci. The occurrence of seizures in two individuals correlated with a deletion extending proximal of IDS, up to and including part of the FMR2 locus. Other (non-seizure) symptoms were associated with distal deletions. In addition, a group of patients with no variant symptoms, and a characteristic rearrangement involving a recombination between the IDS gene and an adjacent IDS pseudogene (IDS psi), showed normal expression of loci distal to IDS. Together, these results identify FMR2 as a candidate gene for seizures, when mutated along with IDS.

Method for 96-well M13 DNA template preparations for large-scale sequencing.

Efficient preparation of DNA templates is an important step in large-scale DNA sequencing. The ensure high-quality sequence data, we have prepared M13 phage DNA templates using a glass fiber-filtration method. We present the adaptation of this protocol to a 96-well format using commercially available filter plates. Two variations are described: one using polyethylene glycol precipitation and a second where the phage particles are disrupted before filtration, thus eliminating the need for precipitation. Using either of these protocols, 96 templates can be prepared in less than 2 h. Sufficient DNA for 1-2 dye primer sequencing reactions is routinely obtained from 1 mL of culture, and the resulting sequence data are of high quality.

A gene-rich cluster between the CD4 and triosephosphate isomerase genes at human chromosome 12p13.

The genomic sequence of the human CD4 gene and its neighboring region, located at chromosome 12p13, was generated using the large-scale shotgun sequencing strategy. A total of 117 kb of genomic sequence and approximately 11 kb of cDNA sequence were obtained. Six genes, including CD4, triosephosphate isomerase, B3 subunit of G proteins (GNB3), and ubiquitin isopeptidase T (ISOT), with known functions, and two new genes with unknown functions were identified. Using a battery of strategies, the exon/intron boundaries, splice variants, and tissue expression patterns of the genes were determined. Various computer software was utilized for analyses of the DNA and amino acid sequences. The results of the analyses and sequence-based strategies for gene identification are discussed.

A recombination hotspot responsible for two inherited peripheral neuropathies is located near a mariner transposon-like element.

The Charcot-Marie Tooth disease type 1A (CMT1A) duplication and hereditary neuropathy with liability to pressure palsies (HNPP) deletion are reciprocal products of an unequal crossing-over event between misaligned flanking CMT1A-REP repeats. The molecular aetiology of this apparently homologous recombination event was examined by sequencing the crossover region. Through the detection of novel junction fragments from the recombinant CMT1A-REPs in both CMT1A and HNPP patients, a 1.7-kb recombination hotspot within the approximately 30-kb CMT1A-REPs was identified. This hotspot is 98% identical between CMT1A-REPs indicating that sequence identity is not likely the sole factor involved in promoting crossover events. Sequence analysis revealed a mariner transposon-like element (MITE) near the hotspot which we hypothesize could mediate strand exchange events via cleavage by a transposase at or near the 3' end of the element.