Characteristics of a spina bifida population including North American Caucasian and Hispanic individuals.

BACKGROUND: Meningomyelocele (MM) is a common human birth defect. MM is a disorder of neural development caused by contributions from genes and environmental factors that result in the NTD and lead to a spectrum of physical and neurocognitive phenotypes. METHODS: A multidisciplinary approach has been taken to develop a comprehensive understanding of MM through collaborative efforts from investigators specializing in genetics, development, brain imaging, and neurocognitive outcome. Patients have been recruited from five different sites: Houston and the Texas-Mexico border area; Toronto, Canada; Los Angeles, California; and Lexington, Kentucky. Genetic risk factors for MM have been assessed by genotyping and association testing using the transmission disequilibrium test. RESULTS: A total of 509 affected child/parent trios and 309 affected child/parent duos have been enrolled to date for genetic association studies. Subsets of the patients have also been enrolled for studies assessing development, brain imaging, and neurocognitive outcomes. The study recruited two major ethnic groups, with 45.9% Hispanics of Mexican descent and 36.2% North American Caucasians of European descent. The remaining patients are African-American, South and Central American, Native American, and Asian. Studies of this group of patients have already discovered distinct corpus callosum morphology and neurocognitive deficits that associate with MM. We have identified maternal MTHFR 667T allele as a risk factor for MM. In addition, we also found that several genes for glucose transport and metabolism are potential risk factors for MM. CONCLUSIONS: The enrolled patient population provides a valuable resource for elucidating the disease characteristics and mechanisms for MM development.

Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems1,2. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene3–9, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulin’s effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.

Gene by BMI Interactions Influencing C-Reactive Protein Levels in European-Americans

C-Reactive Protein (CRP) is a biomarker indicating tissue damage, inflammation, and infection. High-sensitivity CRP (hsCRP) is an emerging biomarker often used to estimate an individual’s risk for future coronary heart disease (CHD). hsCRP levels falling below 1.00 mg/l indicate a low risk for developing CHD, levels ranging between 1.00 mg/l and 3.00 mg/l indicate an elevated risk, and levels exceeding 3.00 mg/l indicate high risk. Multiple Genome-Wide Association Studies (GWAS) have identified a number of genetic polymorphisms which influence CRP levels. SNPs implicated in such studies have been found in or near genes of interest including: CRP, APOE, APOC, IL-6, HNF1A, LEPR, and GCKR. A strong positive correlation has also been found to exist between CRP levels and BMI, a known risk factor for CHD and a state of chronic inflammation. We conducted a series of analyses designed to identify loci which interact with BMI to influence CRP levels in a subsample of European-Americans in the ARIC cohort. In a stratified GWA analysis, 15 genetic regions were identified as having significantly (p-value < 2.00*10-3) distinct effects on hsCRP levels between the two obesity strata: lean (18.50 kg/m2 < BMI < 24.99 kg/m2) and obese (BMI ≥ 30.00 kg/m2). A GWA analysis performed on all individuals combined (i.e. not a priori stratified for obesity status) with the inclusion of an additional parameter for BMI by gene interaction, identified 11 regions which interact with BMI to influence hsCRP levels. Two regions containing the genes GJA5 and GJA8 (on chromosome 1) and FBXO11 (on chromosome 2) were identified in both methods of analysis suggesting that these genes possibly interact with BMI to influence hsCRP levels. We speculate that atrial fibrillation (AF), age-related cataracts and the TGF-β pathway may be the biological processes influenced by the interaction of GJA5, GJA8 and FBXO11, respectively, with BMI to cause changes in hsCRP levels. Future studies should focus on the influence of gene x bmi interaction on AF, age-related cataracts and TGF-β.

THE EXPRESSION PROFILE OF A GWAS DETECTED GENE (NINJ2) IN THE BRAIN OF A MOUSE MODEL OF ISCHEMIC STROKE

Stroke is the third leading cause of death and a major debilitating disease in the United States. Multiple factors, including genetic factors, contribute to the development of the disease. Genome-wide association studies (GWAS) have contributed to the identification of genetic loci influencing risk for complex diseases, such as stroke. In 2010, a GWAS of incident stroke was performed in four large prospective cohorts from the USA and Europe and identified an association of two Single Nucleotide Polymorphisms (SNPs) on chromosome 12p13 with a greater risk of ischemic stroke in individuals of European and African-American ancestry. These SNPs are located 11 Kb upstream of the nerve injury-induced gene 2, Ninjurin2 (NINJ2), suggesting that this gene may be involved in stroke pathogenesis. NINJ2 is a cell adhesion molecule induced in the distal glial cells from a sciatic-nerve injury at 7-days after injury. In an effort to ascribe a possible role of NINJ2 in stroke, we have assessed changes in the level of gene and protein expression of NINJ2 following a time-course from a transiently induced middle cerebral artery ischemic stroke in mice brains. We report an increase in the gene expression of NINJ2 in the ischemic and peri-infarct (ipsilateral) cortical tissues at 7 and 14-days after stroke. We also report an increase in the protein expression of NINJ2 in the cortex of both the ipsilateral and contralateral cortical tissues at the same time-points. We conclude that the expression of NINJ2 is regulated by an ischemic stroke in the cortex and is consistent with NINJ2 being involved in the recovery time-points of stroke.

IL28B expression depends on a novel TT/-G polymorphism which improves HCV clearance prediction

Approximately 3% of the world population is chronically infected with the hepatitis C virus (HCV), with potential development of cirrhosis and hepatocellular carcinoma. Despite the availability of new antiviral agents, treatment remains suboptimal. Genome-wide association studies (GWAS) identified rs12979860, a polymorphism nearby IL28B, as an important predictor of HCV clearance. We report the identification of a novel TT/-G polymorphism in the CpG region upstream of IL28B, which is a better predictor of HCV clearance than rs12979860. By using peripheral blood mononuclear cells (PBMCs) from individuals carrying different allelic combinations of the TT/-G and rs12979860 polymorphisms, we show that induction of IL28B and IFN-γ–inducible protein 10 (IP-10) mRNA relies on TT/-G, but not rs12979860, making TT/-G the only functional variant identified so far. This novel step in understanding the genetic regulation of IL28B may have important implications for clinical practice, as the use of TT/G genotyping instead of rs12979860 would improve patient management.

New generation genomic platforms in investigation of complex diseases and BEN.

(Full text is available at http://www.manu.edu.mk/prilozi). New generation genomic platforms enable us to decipher the complex genetic basis of complex diseases and Balkan Endemic Nephropathy (BEN) at a high-throughput basis. They give valuable information about predisposing Single Nucleotide Polymorphisms (SNPs), Copy Number Variations (CNVs) or Loss of Heterozygosity (LOH) (using SNP-array) and about disease-causing mutations along the whole sequence of candidate-genes (using Next Generation Sequencing). This information could be used for screening of individuals in risk families and moving the main medicine stream to the prevention. They also might have an impact on more effective treatment. Here we discuss these genomic platforms and report some applications of SNP-array technology in a case with familial nephrotic syndrome. Key words: complex diseases, genome wide association studies, SNP, genomic arrays, next generation sequ-encing.

Two Loci on Chromosome 5 Are Associated with Serum IgE Levels in Labrador Retrievers

Crosslinking of immunoglobulin E antibodies (IgE) bound at the surface of mast cells and subsequent mediator release is considered the most important trigger for allergic reactions. Therefore, the genetic control of IgE levels is studied in the context of allergic diseases, such as asthma, atopic rhinitis, or atopic dermatitis (AD). We performed genome-wide association studies in 161 Labrador Retrievers with regard to total and allergen-specific immunoglobulin E (IgE) levels. We identified a genome-wide significant association on CFA 5 with the antigen-specific IgE responsiveness to Acarus siro. We detected a second genome-wide significant association with respect to the antigen-specific IgE responsiveness to Tyrophagus putrescentiae at a different locus on chromosome 5. A. siro and T. putrescentiae both belong to the family Acaridae and represent so-called storage or forage mites. These forage mites are discussed as major allergen sources in canine AD. No obvious candidate gene for the regulation of IgE levels is located under the two association signals. Therefore our studies offer a chance of identifying a novel mechanism controlling the host's IgE response.

A mutation in the FAM83G gene in dogs with hereditary footpad hyperkeratosis (HFH)

Hereditary footpad hyperkeratosis (HFH) represents a palmoplantar hyperkeratosis, which is inherited as a monogenic autosomal recessive trait in several dog breeds, such as e.g. Kromfohrländer and Irish Terriers. We performed genome-wide association studies (GWAS) in both breeds. In Kromfohrländer we obtained a single strong association signal on chromosome 5 (p(raw) = 1.0×10(-13)) using 13 HFH cases and 29 controls. The association signal replicated in an independent cohort of Irish Terriers with 10 cases and 21 controls (p(raw) = 6.9×10(-10)). The analysis of shared haplotypes among the combined Kromfohrländer and Irish Terrier cases defined a critical interval of 611 kb with 13 predicted genes. We re-sequenced the genome of one affected Kromfohrländer at 23.5× coverage. The comparison of the sequence data with 46 genomes of non-affected dogs from other breeds revealed a single private non-synonymous variant in the critical interval with respect to the reference genome assembly. The variant is a missense variant (c.155G>C) in the FAM83G gene encoding a protein with largely unknown function. It is predicted to change an evolutionary conserved arginine into a proline residue (p.R52P). We genotyped this variant in a larger cohort of dogs and found perfect association with the HFH phenotype. We further studied the clinical and histopathological alterations in the epidermis in vivo. Affected dogs show a moderate to severe orthokeratotic hyperplasia of the palmoplantar epidermis. Thus, our data provide the first evidence that FAM83G has an essential role for maintaining the integrity of the palmoplantar epidermis.

The brown coat colour of Coppernecked goats is associated with a non-synonymous variant at the TYRP1 locus on chromosome 8

The recent development of a goat SNP genotyping microarray enables genome-wide association studies in this important livestock species. We investigated the genetic basis of the black and brown coat colour in Valais Blacknecked and Coppernecked goats. A genome-wide association analysis using goat SNP50 BeadChip genotypes of 22 cases and 23 controls allowed us to map the locus for the brown coat colour to goat chromosome 8. The TYRP1 gene is located within the associated chromosomal region, and TYRP1 variants cause similar coat colour phenotypes in different species. We thus considered TYRP1 as a strong positional and functional candidate. We resequenced the caprine TYRP1 gene by Sanger and Illumina sequencing and identified two non-synonymous variants, p.Ile478Thr and p.Gly496Asp, that might have a functional impact on the TYRP1 protein. However, based on the obtained pedigree and genotype data, the brown coat colour in these goats is not due to a single recessive loss-of-function allele. Surprisingly, the genotype distribution and the pedigree data suggest that the (496) Asp allele might possibly act in a dominant manner. The (496) Asp allele was present in 77 of 81 investigated Coppernecked goats and did not occur in black goats. This strongly suggests heterogeneity underlying the brown coat colour in Coppernecked goats. Functional experiments or targeted matings will be required to verify the unexpected preliminary findings.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

Disease-modifying genes and monogenic disorders: experience in cystic fibrosis

The mechanisms responsible for the determination of phenotypes are still not well understood; however, it has become apparent that modifier genes must play a considerable role in the phenotypic heterogeneity of Mendelian disorders. Significant advances in genetic technologies and molecular medicine allow huge amounts of information to be generated from individual samples within a reasonable time frame. This review focuses on the role of modifier genes using the example of cystic fibrosis, the most common lethal autosomal recessive disorder in the white population, and discusses the advantages and limitations of candidate gene approaches versus genome-wide association studies. Moreover, the implications of modifier gene research for other monogenic disorders, as well as its significance for diagnostic, prognostic, and therapeutic approaches are summarized. Increasing insight into modifying mechanisms opens up new perspectives, dispelling the idea of genetic disorders being caused by one single gene.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥20 g/day for ≥5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Variants in the Mannose-binding Lectin Gene MBL2 do not Associate With Sepsis Susceptibility or Survival in a Large European Cohort.

BACKGROUND  Sepsis is an increasingly common condition, which continues to be associated with unacceptably high mortality. A large number of association studies have investigated susceptibility to, or mortality from, sepsis for variants in the functionally important immune-related gene MBL2. These studies have largely been underpowered and contradictory. METHODS  We genotyped and analyzed 4 important MBL2 single nucleotide polymorphisms (SNPs; rs5030737, rs1800450, rs1800451, and rs7096206) in 1839 European community-acquired pneumonia (CAP) and peritonitis sepsis cases, and 477 controls from the United Kingdom. We analyzed the following predefined subgroups and outcomes: 28-day and 6 month mortality from sepsis due to CAP or peritonitis combined, 28-day mortality from CAP sepsis, peritonitis sepsis, pneumococcal sepsis or sepsis in younger patients, and susceptibility to CAP sepsis or pneumococcal sepsis in the United Kingdom. RESULTS  There were no significant associations (all P-values were greater than .05 after correction for multiple testing) between MBL2 genotypes and any of our predefined analyses. CONCLUSIONS  In this large, well-defined cohort of immune competent adult patients, no associations between MBL2 genotype and sepsis susceptibility or outcome were identified.