A population genetic approach to mapping neurological disorder genes using deep resequencing

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n  =  285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders

Novel VPS13B Mutations in Three Large Pakistani Cohen Syndrome Families Suggests a Baloch Variant with Autistic-Like Features.

BackgroundCohen Syndrome (COH1) is a rare autosomal recessive disorder, principally identified by ocular, neural and muscular deficits. We identified three large consanguineous Pakistani families with intellectual disability and in some cases with autistic traits.MethodsClinical assessments were performed in order to allow comparison of clinical features with other VPS13B mutations. Homozygosity mapping followed by whole exome sequencing and Sanger sequencing strategies were used to identify disease-related mutations.ResultsWe identified two novel homozygous deletion mutations in VPS13B, firstly a 1 bp deletion, NM_017890.4:c.6879delT; p.Phe2293Leufs*24, and secondly a deletion of exons 37-40, which co-segregate with affected status. In addition to COH1-related traits, autistic features were reported in a number of family members, contrasting with the "friendly" demeanour often associated with COH1. The c.6879delT mutation is present in two families from different regions of the country, but both from the Baloch sub-ethnic group, and with a shared haplotype, indicating a founder effect among the Baloch population.ConclusionWe suspect that the c.6879delT mutation may be a common cause of COH1 and similar phenotypes among the Baloch population. Additionally, most of the individuals with the c.6879delT mutation in these two families also present with autistic like traits, and suggests that this variant may lead to a distinct autistic-like COH1 subgroup

Analysis of common and rare VPS13C variants in late-onset Parkinson disease

Objective We aimed to study the role of coding VPS13C variants in a large cohort of patients with lateonset Parkinson disease (PD) (LOPD). Methods VPS13C and its untranslated regions were sequenced using targeted next-generation sequencing in 1,567 patients with PD and 1,667 controls from 3 cohorts. Association tests of rare potential homozygous and compound heterozygous variants and burden tests for rare heterozygous variants were performed. Common variants were analyzed using logistic regression adjusted for age and sex in each of the cohorts, followed by a meta-analysis. Results No biallelic carriers of rare VPS13C variants were found among patients, and 2 carriers of compound heterozygous variants were found in 2 controls. There was no statistically significant burden of rare (minor allele frequency [MAF] <1%) or very rare (MAF <0.1%) coding VPS13C variants in PD. A VPS13C haplotype including the p.R153H-p.I398I-p.I1132V-p.Q2376Q variants was nominally associated with a reduced risk for PD (meta-analysis of the tagging SNP p.I1132V [odds ratio = 0.48, 95% confidence interval = 0.28–0.82, p = 0.0052]). This haplotype was not in linkage disequilibrium with the known genome-wide association study top hit. Conclusions Our results do not support a role for rare heterozygous or biallelic VPS13C variants in LOPD. Additional genetic replication and functional studies are needed to examine the role of the haplotype identified here associated with reduced risk for PD

A Population Genetic Approach to Mapping Neurological Disorder Genes Using Deep Resequencing

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n = 285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders

HLA in isolated REM sleep behavior disorder and Lewy body dementia

peer reviewedSynucleinopathies-related disorders such as Lewy body dementia (LBD) and isolated/idiopathic REM sleep behavior disorder (iRBD) have been associated with neuroinflammation. In this study, we examined whether the human leukocyte antigen (HLA) locus plays a role in iRBD and LBD. In iRBD, HLA-DRB1*11:01 was the only allele passing FDR correction (OR = 1.57, 95 CI = 1.27–1.93, p = 2.70e-05). We also discovered associations between iRBD and HLA-DRB1 70D (OR = 1.26, 95\%CI = 1.12–1.41, p = 8.76e-05), 70Q (OR = 0.81, 95\%CI = 0.72–0.91, p = 3.65e-04) and 71R (OR = 1.21, 95\%CI = 1.08–1.35, p = 1.35e-03). Position 71 (pomnibus = 0.00102) and 70 (pomnibus = 0.00125) were associated with iRBD. Our results suggest that the HLA locus may have different roles across synucleinopathies

Exploring the fusion of metagenomic library and DNA microarray technologies

We explored the combination of metagenomic library and DNA microarray technologies into a single platform as a novel way to rapidly screen metagenomic libraries for genetic targets. In the "metagenomic microarray" system, metagenomic library clone DNA is printed on a microarray surface, and clones of interest are detected by hybridization to single-gene probes. This study represents the initial steps in the development of this technology. We constructed two 5,000-clone large-insert metagenomic libraries from two diesel-contaminated Arctic soil samples. We developed and optimized an automated fosmid purification protocol to rapidly-extract clone DNA in a high-throughput 96-well format. We then created a series of small prototype arrays to optimize various parameters of microarray printing and hybridization, to identify and resolve technical challenges, and to provide proof-of-principle of this novel application. Our results suggest that this method shows promise, but more experimentation must be done to establish the feasibility of this approach

A survey of the methods for the characterization of microbial consortia and communities

A survey of the available literature on methods most frequently used for the identification and characterization of microbial strains, communities, or consortia is presented. The advantages and disadvantages of the various methodologies were examined from several perspectives including technical, economic (time and cost), and regulatory. The methods fall into 3 broad categories: molecular biological, biochemical, and microbiological. Molecular biological methods comprise a broad range of techniques that are based on the analysis and differentiation of microbial DNA. This class of methods possesses several distinct advantages. Unlike most other commonly used methods, which require the production of secondary materials via the manipulation of microbial growth, molecular biological methods recover and test their source materials (DNA) directly from the microbial cells themselves, without the requirement for culturing. This eliminates both the time required for growth and the biases associated with cultured growth, which is unavoidably and artificially selective. The recovered nucleic acid can be cloned and sequenced directly or subpopulations can be specifically amplified using polymerase chain reaction (PCR), and subsequently cloned and sequenced. PCR technology, used extensively in forensic science, provides researchers with the unique ability to detect nucleic acids (DNA and RNA) in minute amounts, by amplifying a single target molecule by more than a million-fold. Molecular methods are highly sensitive and allow for a high degree of specificity, which, coupled with the ability to separate similar but distinct DNA molecules, means that a great deal of information can be gleaned from even very complex microbial communities. Biochemical methods are composed of a more varied set of methodologies. These techniques share a reliance on gas chromatography and mass spectrometry to separate and precisely identify a range of biomolecules, or else investigate biochemical properties of key cellular biomolecules. Like the molecular biological methods, some biochemical methods such as lipid analyses are also independent of cultured growth. However, many of these techniques are only capable of producing a profile that is characteristic of the microbial community as a whole, providing no information about individual members of the community. A subset of these methodologies are used to derive taxonomic information from a community sample; these rely on the identification of key subspecies of biomolecules that differ slightly but characteristically between species, genera, and higher biological groupings. However, when the consortium is already growing in chemically defined media (as is often the case with commercial products), the rapidity and relatively low costs of these procedures can mitigate concerns related to culturing biases. Microbiological methods are the most varied and the least useful for characterizing microbial consortia. These methods rely on traditional tools (cell counting, selective growth, and microscopic examination) to provide more general characteristics of the community as a whole, or else to narrow down and identify only a small subset of the members of that community. As with many of the biochemical methods, some of the microbiological methods can fairly rapidly and inexpensively create a community profile, which can be used to compare 2 or more entire consortia. However, for taxonomic identification of individual members, microbiological methods are useful only to screen for the presence of a few key predetermined species, whose preferred growth conditions and morphological characteristics are well defined and reproducible.R\ue9sum\ue9 : Nous pr\ue9sentons une \ue9tude sur l'ensemble de la litt\ue9rature disponible portant sur les m\ue9thodes les plus fr\ue9quentes utilis\ue9es dans l'identification et la caract\ue9risation de souches microbiennes, de communaut\ue9s ou de consortiums. Les avantages et les d\ue9savantages des diverses m\ue9thodologies ont \ue9t\ue9 examin\ue9s sous divers aspects, notamment au niveau technique, \ue9conomique (temps et co\ufbt) et r\ue9glementaire. Les m\ue9thodes peuvent \ueatre s\ue9par\ue9es en 3 cat\ue9gories g\ue9n\ue9rales: biologiques mol\ue9culaires, biochimiques, et microbiologiques. Les m\ue9thodes de biologie mol\ue9culaire comprennent un vaste \ue9ventail de techniques bas\ue9es sur l'analyse et la diff\ue9renciation d'ADN microbiens. Cette cat\ue9gorie de m\ue9thodes poss\ue8de plusieurs avantages distincts. Contrairement \ue0 la plupart des autres m\ue9thodes courantes n\ue9cessitant la production de mati\ue8res secondaires par le biais de la manipulation de la croissance microbienne, les m\ue9thodes de biologie mol\ue9culaire r\ue9cup\ue8rent et analysent leur mati\ue8res d'origine (l'ADN) directement \ue0 partir de cellules microbiennes sans avoir recours \ue0 la culture. Ceci \ue9limine \ue0 la fois le temps requis pour la croissance et les biais associ\ue9s avec la culture qui sont inexorablement et artificiellement s\ue9lectifs. L'acide nucl\ue9ique r\ue9cup\ue9r\ue9 peut \ueatre directement clon\ue9 et s\ue9quenc\ue9 ou des sous-populations peuvent \ueatre amplifi\ue9es sp\ue9cifiquement \ue0 l'aide de la r\ue9action de polym\ue9rase en cha\ueene (PCR) et clon\ue9es et s\ue9quenc\ue9es par la suite. La technologie du PCR, utilis\ue9e abondamment en m\ue9decine l\ue9gale, fournit aux chercheurs la capacit\ue9 unique de d\ue9tecter des acides nucl\ue9iques (ADN et ARN) en quantit\ue9s minimes en amplifiant plus d'un million de fois une seule mol\ue9cule cible. Les m\ue9thodes mol\ue9culaires sont hautement sensibles et permettent d'atteindre une degr\ue9 \ue9lev\ue9 de sp\ue9cificit\ue9 qui, combin\ue9 avec la capacit\ue9 de s\ue9parer des mol\ue9cules d'ADN semblables mais distinctes, signifie qu'une quantit\ue9 ph\ue9nom\ue9nale d'information peut \ueatre extraite des communaut\ue9s microbiennes les plus complexes. Les m\ue9thodes biochimiques comprennent un ensemble plus vari\ue9 de m\ue9thodologies. Ces techniques ont en commun une d\ue9pendance de la chromatographie en phase gazeuse et de la spectrom\ue9trie de masse afin de s\ue9parer et d'identifier pr\ue9cis\ue9ment un \ue9ventail de biomol\ue9cules ou encore d'\ue9tudier les propri\ue9t\ue9s biochimiques de mol\ue9cules biologiques cellulaires cl\ue9s. Comme les m\ue9thodes de biologie mol\ue9culaire, certaines m\ue9thodes biochimiques telles que l'analyse des lipides sont \ue9galement ind\ue9pendantes de la culture. Toutefois, plusieurs de ces techniques ne sont capables de g\ue9n\ue9rer qu'un profil propre \ue0 la communaut\ue9 microbienne en tant que telle, ne fournissant aucune information sur les membres individuels de la communaut\ue9. Une partie de ces m\ue9thodologies sont utilis\ue9es afin de g\ue9n\ue9rer des informations taxonomiques de l'\ue9chantillon d'une communaut\ue9; celles-ci d\ue9pendent de l'identification de sous-esp\ue8ces cl\ue9s de biomol\ue9cules divergeant l\ue9g\ue8rement mais sp\ue9cifiquement entre les esp\ue8ces, les genres et les groupements biologiques sup\ue9rieurs. Toutefois, lorsque le consortium se d\ue9veloppe d\ue9j\ue0 dans un milieu d\ue9fini chimiquement (ce qui est souvent le cas avec les produits commerciaux), la rapidit\ue9 et les co\ufbts relativement bas de ces proc\ue9dures peuvent temp\ue9rer les inqui\ue9tudes \ue0 propos des bais de culture. Les m\ue9thodes microbiologiques sont les plus vari\ue9es et les moins utiles \ue0 la caract\ue9risation des consortiums microbiens. Ces m\ue9thodes sont bas\ue9es sur des outils traditionnels (compte cellulaire, croissance s\ue9lective et examen microscopique) afin de fournir des caract\ue9ristiques plut\uf4t g\ue9n\ue9rales sur la communaut\ue9 enti\ue8re, ou encore afin de restreindre et d'identifier un petit sous-ensemble des membres de la communaut\ue9. Comme pour plusieurs des m\ue9thodes biochimiques, certaines des m\ue9thodes microbiologiques peuvent g\ue9n\ue9rer rapidement et \ue0 moindre co\ufbt un profil de communaut\ue9s qui pourra \ueatre utilis\ue9 pour comparer 2 ou plusieurs consortiums entiers. Toutefois, dans le cas de l'identification taxonomique de membres individuels, les m\ue9thodes microbiologiques ne sont utiles que pour rechercher la pr\ue9sence de quelques esp\ue8ces cl\ue9s pr\ue9d\ue9termin\ue9es dont les conditions de croissance pr\ue9f\ue9rentielles et les caract\ue9ristiques morphologiques sont bien d\ue9finies et reproductibles.NRC publication: Ye

Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15–30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens