Systematic data-querying of large pediatric biorepository identifies novel Ehlers-Danlos Syndrome variant

BACKGROUND: Ehlers Danlos Syndrome is a rare form of inherited connective tissue disorder, which primarily affects skin, joints, muscle, and blood cells. The current study aimed at finding the mutation that causing EDS type VII C also known as "Dermatosparaxis" in this family. METHODS: Through systematic data querying of the electronic medical records (EMRs) of over 80,000 individuals, we recently identified an EDS family that indicate an autosomal dominant inheritance. The family was consented for genomic analysis of their de-identified data. After a negative screen for known mutations, we performed whole genome sequencing on the male proband, his affected father, and unaffected mother. We filtered the list of non-synonymous variants that are common between the affected individuals. RESULTS: The analysis of non-synonymous variants lead to identifying a novel mutation in the ADAMTSL2 (p. Gly421Ser) gene in the affected individuals. Sanger sequencing confirmed the mutation. CONCLUSION: Our work is significant not only because it sheds new light on the pathophysiology of EDS for the affected family and the field at large, but also because it demonstrates the utility of unbiased large-scale clinical recruitment in deciphering the genetic etiology of rare mendelian diseases. With unbiased large-scale clinical recruitment we strive to sequence as many rare mendelian diseases as possible, and this work in EDS serves as a successful proof of concept to that effect

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9- sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo

Genomic copy number variation association study in Caucasian patients with nonsyndromic cryptorchidism

Copy number variation (CNV) is a potential contributing factor to many genetic diseases. Here we investigated the potential association of CNV with nonsyndromic cryptorchidism, the most common male congenital genitourinary defect, in a Caucasian population

Prevalence of Salmonella enterica and Listeria monocytogenes contamination in foods of animal origin in Italy.

The present survey collected and analyzed the results of routine testing for Salmonella enterica and Listeria monocytogenes on foods of animal origin submitted for official controls in Italy during 2001 to 2002. Salmonella was detected in 2.2% of 71,643 food samples examined, and the isolation rates ranged from 9.9% for raw poultry meat to less than 0.1% for dairy products. Isolation rates were also high in raw pork (4.9%) and processed meats (5.3%), which often involved pork. Low rates were observed in seafood (0.5%) and in ready-to-eat foods, such as grocery products (0.7%) and ice creams (0.1%). Serotyping showed that approximately 50% of the isolates belonged to the serotypes most commonly isolated from humans in Italy, thus confirming that most cases of human salmonellosis have a foodborne origin. Levels of L. monocytogenes were higher than what is accepted by the current regulation in 2.4% of 42,300 food samples. The positivity rates ranged from 10.3% in raw pork to none in eggs and egg products. Contamination rates were higher in other meat products (between 2 and 5%) and fish (6.5%) than in cheeses (1.1%) and other dairy products (0.6%). Routine control activities on the microbial contamination of foods can generate data with statistical and epidemiological value. Such data can be used as a basis for estimating the exposure of consumers to foodborne pathogens, following the trends of contamination over time, and evaluating the effects of control measures on the contamination of food

A Geologically Based Indoor-Radon Potential Map of Kentucky

We combined 71,930 short-term (median duration 4 days) home radon test results with 1:24,000-scale bedrock geologic map coverage of Kentucky to produce a statewide geologically based indoor-radon potential map. The test results were positively skewed with a mean of 266 Bq/m3, median of 122 Bq/m3, and 75th percentile of 289 Bq/m3. We identified 106 formations with ≥10 test results. Analysis of results from 20 predominantly monolithologic formations showed indoor-radon concentrations to be positively skewed on a formation-by-formation basis, with a proportional relationship between sample means and standard deviations. Limestone (median 170 Bq/m3) and dolostone (median 130 Bq/m3) tended to have higher indoor-radon concentrations than siltstones and sandstones (median 67 Bq/m3) or unlithified surficial deposits (median 63 Bq/m3). Individual shales had median values ranging from 67 to 189 Bq/m3; the median value for all shale values was 85 Bq/m3. Percentages of values falling above the U.S. Environmental Protection Agency (EPA) action level of 148 Bq/m3 were sandstone and siltstone: 24%, unlithified clastic: 21%, dolostone: 46%, limestone: 55%, and shale: 34%. Mississippian limestones, Ordovician limestones, and Devonian black shales had the highest indoor-radon potential values in Kentucky. Indoor-radon test mean values for the selected formations were also weakly, but statistically significantly, correlated with mean aeroradiometric uranium concentrations. To produce a map useful to nonspecialists, we classified each of the 106 formations into five radon-geologic classes on the basis of their 75th percentile radon concentrations. The statewide map is freely available through an interactive internet map service

Follow-Up Analysis of Genome-Wide Association Data Identifies Novel Loci for Type 1 Diabetes

OBJECTIVE—Two recent genome-wide association (GWA) studies have revealed novel loci for type 1 diabetes, a common multifactorial disease with a strong genetic component. To fully utilize the GWA data that we had obtained by genotyping 563 type 1 diabetes probands and 1,146 control subjects, as well as 483 case subject–parent trios, using the Illumina HumanHap550 BeadChip, we designed a full stage 2 study to capture other possible association signals

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

A "Candidate-Interactome" Aggregate Analysis of Genome-Wide Association Data in Multiple Sclerosis

Though difficult, the study of gene-environment interactions in multifactorial diseases is crucial for interpreting the relevance of non-heritable factors and prevents from overlooking genetic associations with small but measurable effects. We propose a “candidate interactome” (i.e. a group of genes whose products are known to physically interact with environmental factors that may be relevant for disease pathogenesis) analysis of genome-wide association data in multiple sclerosis. We looked for statistical enrichment of associations among interactomes that, at the current state of knowledge, may be representative of gene-environment interactions of potential, uncertain or unlikely relevance for multiple sclerosis pathogenesis: Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, HHV8-Kaposi sarcoma, H1N1-influenza, JC virus, human innate immunity interactome for type I interferon, autoimmune regulator, vitamin D receptor, aryl hydrocarbon receptor and a panel of proteins targeted by 70 innate immune-modulating viral open reading frames from 30 viral species. Interactomes were either obtained from the literature or were manually curated. The P values of all single nucleotide polymorphism mapping to a given interactome were obtained from the last genome-wide association study of the International Multiple Sclerosis Genetics Consortium & the Wellcome Trust Case Control Consortium, 2. The interaction between genotype and Epstein Barr virus emerges as relevant for multiple sclerosis etiology. However, in line with recent data on the coexistence of common and unique strategies used by viruses to perturb the human molecular system, also other viruses have a similar potential, though probably less relevant in epidemiological terms