Distribution of the C9orf72 hexanucleotide repeat expansion in healthy subjects: a multicenter study promoted by the Italian IRCCS network of neuroscience and neurorehabilitation.

Introduction: High repeat expansion (HRE) alleles in C9orf72 have been linked to both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); ranges for intermediate allelic expansions have not been defined yet, and clinical interpretation of molecular data lacks a defined genotype-phenotype association. In this study, we provide results from a large multicenter epidemiological study reporting the distribution of C9orf72 repeats in healthy elderly from the Italian population. Methods: A total of 967 samples were collected from neurologically evaluated healthy individuals over 70 years of age in the 13 institutes participating in the RIN (IRCCS Network of Neuroscience and Neurorehabilitation) based in Italy. All samples were genotyped using the AmplideXPCR/CE C9orf72 Kit (Asuragen, Inc.), using standardized protocols that have been validated through blind proficiency testing. Results: All samples carried hexanucleotide G4C2 expansion alleles in the normal range. All samples were characterized by alleles with less than 25 repeats. In particular, 93.7% of samples showed a number of repeats ≤10, 99.9% ≤20 repeats, and 100% ≤25 repeats. Conclusion: This study describes the distribution of hexanucleotide G4C2 expansion alleles in an Italian healthy population, providing a definition of alleles associated with the neurological healthy phenotype. Moreover, this study provides an effective model of federation between institutes, highlighting the importance of sharing genomic data and standardizing analysis techniques, promoting translational research. Data derived from the study may improve genetic counseling and future studies on ALS/FTD.

Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.

Metachromatic leukodystrophy (MLD) is a severe metabolic disorder caused by the deficient activity of arylsulfatase A due to ARSA gene mutations. According to the age of onset, MLD is classified into three forms: infantile, juvenile, and adult. In our study, we aimed to perform a genetic analysis for two siblings with juvenile MLD for a better characterization of the molecular mechanisms behind the disease. A consanguineous family including two MLD patients (PII.1 and PII.2) was enrolled in our study. The diagnosis was made based on the clinical and neuroimaging investigations. The sequencing of ARSA gene was performed followed by in silico analysis. Besides, the cis/trans distribution of the variants was verified through a PCR-RFLP. The ARSA gene sequencing revealed three known variants, two exonic c.1055A > G and c.1178C > G and an intronic one (c.1524 + 95A > G) in the 3'UTR region. All variants were present at heterozygous state in the two siblings and their mother. The assessment of the cis/trans distribution showed the presence of these variants in cis within the mother, while PII.2 and PII.2 present the c.1055A > G/c.1524 + 95A > G and the c.1178C > G in trans. Additionally, PII.1 harbored a de novo novel missense variant c.1119G > T, whose pathogenicity was supported by our predictive results. Our genetic findings, supported by a clinical examination, confirmed the affection of the mother by the adult MLD. Our results proved the implication of the variable distribution of the found variants in the age of MLD onset. Besides, we described a variable severity between the two siblings due to the de novo pathogenic variant. In conclusion, we identified a complex genotype of ARSA variants within two MLD siblings with a variable severity due to a de novo variant present in one of them. Our results allowed the establishment of an adult MLD diagnosis and highlighted the importance of an assessment of the trans/cis distribution in the cases of complex genotypes.

Polymorphisms in BMPRIB gene affect litter size in Chinese indigenous sheep breed.

The BMPRIB gene belongs to the TGF-ß superfamily and is considered to be a regulator of sheep reproductive performance. Single nucleotide polymorphisms (SNPs) of BMPRIB gene in the Small Tail Han, Hu, Mongolian, Oula, Gansu Alpine Fine-wool, Dorper and Australian White sheep were detected by Sanger sequencing. Five SNPs (rs427897187 G > A, rs418841713 A > G, rs159952533 T > C, rs429416173 C > A and rs403555643 A > G) of BMPRIB gene were identified. For rs427897187 G > A, further analysis revealed that genotype GG and GA had 0.26 (p < 0.05) and 0.33 (p < 0.05) litter size less than those with genotype AA in Oula sheep. For rs403555643 A > G, further analysis revealed that genotype GG and AG had 0.65 (p < 0.05) and 0.38 (p < 0.05) litter size more than those with genotype AA in Oula sheep, and genotype GG had 0.56 (p < 0.05) litter size more than those with genotype AA in Mongolian sheep. The results showed that rs427897187 G > A and rs403555643 A > G are potential molecular markers wich could improve litter size of Chinese indigenous sheep and be used in Chinese indigenous sheep breeding.

Crohn's Disease Susceptibility and Onset Are Strongly Related to Three NOD2 Gene Haplotypes.

The genetic background and the determinants influencing the disease form, course, and onset of inflammatory bowel disease (IBD) remain unresolved. We aimed to determine the NOD2 gene haplotypes and their relationship with IBD occurrence, clinical presentation, and onset, analyzing a cohort of 578 patients with IBD, including children, and 888 controls. Imaging or endoscopy with a histopathological confirmation was used to diagnose IBD. Genotyping was performed to assess the differences in genotypic and allelic frequencies. Linkage disequilibrium was analyzed, and associations between haplotypes and clinical data were evaluated. We emphasized the prevalence of risk alleles in all analyzed loci in patients with Crohn disease (CD). Interestingly, c.2722G>C and c.3019_3020insC alleles were also overrepresented in ulcerative colitis (UC). T-C-G-C-insC, T-C-G-T-insC, and T-T-G-T-wt haplotypes were correlated with the late-onset form of CD (OR = 23.01, 5.09, and 17.71, respectively), while T-T-G-T-wt and C-C-G-T-wt were prevalent only in CD children (OR = 29.36, and 12.93, respectively; p-value = 0.001). In conclusion, the presence of c.3019_3020insC along with c.802C>T occurred as the most fundamental contributing diplotype in late-onset CD form, while in CD children, the mutual allele in all predisposing haplotypes was the c.2798 + 158T. Identifying the unique, high-impact haplotypes supports further studies of the NOD2 gene, including haplotypic backgrounds.

Frequency and characterization of RHD variant alleles in a population of blood donors from southeastern Brazil: Comparison with other populations.

BACKGROUND: The correct determination of D antigen could help to avoid alloimmunization in pregnant women and patients receiving blood transfusions. However, there are limitations in the identification of D variants as the partial and weak D phenotypes make the determination of D antigen a great challenge in the transfusion routine.' STUDY DESIGN AND METHODS: The molecular characterization of D variants was performed on blood donors from southeastern Brazil with atypical D typing. Furthermore, the serological profile of all RHD variant alleles identified was analyzed using different Anti-D clones. The prevalence of RHD alleles and genotypes found was compared with those described in other countries and in other regions from Brazil. RESULTS: Atypical serologic D typing occurred in 0.79 % of blood donors. The majority of RHD variant alleles (88 %) were first characterized by multiplex PCR and PCR-SSP as RHD*weak partial 4 (47 %), followed by RHD*weak D type 3 (29.9 %), RHD*weak D type 2 (3.9 %) and RHD*weak D type 1 (3.1 %). Genomic DNA sequencing characterized the RHD*weak partial 4 variants found in RHD*DAR1.2 (weak 4.2.2) (22 %), RHD*DAR3 (weak 4.0.1) (2.4 %), RHD*DAR3.1 (weak 4.0) (22 %) and RHD*DAR4 (weak 4.1) (0.8 %). RHD variant alleles associated with partial D, such as, RHD*DAU-4 (1.6 %), RHD*DAU-5 (2.4 %), RHD*DAU-6 (1.6 %), RHD* DIII type 8 (1.6 %), RHD*DVII (3.9 %) and RHD* DMH (0.8 %) were also observed. CONCLUSION: The prevalence of RHD variant alleles observed in this cohort differ from those found in other populations, including Brazilians from other regions. RHD allele distribution in specific regions should be considered for implementation of algorithms and genotyping strategies aiming at a more effective and safe transfusion.

Exome-Wide Analysis of the DiscovEHR Cohort Reveals Novel Candidate Pharmacogenomic Variants for Clinical Pharmacogenomics.

Recent advances in next-generation sequencing technology have led to the production of an unprecedented volume of genomic data, thus further advancing our understanding of the role of genetic variation in clinical pharmacogenomics. In the present study, we used whole exome sequencing data from 50,726 participants, as derived from the DiscovEHR cohort, to identify pharmacogenomic variants of potential clinical relevance, according to their occurrence within the PharmGKB database. We further assessed the distribution of the identified rare and common pharmacogenomics variants amongst different GnomAD subpopulations. Overall, our findings show that the use of publicly available sequence data, such as the DiscovEHR dataset and GnomAD, provides an opportunity for a deeper understanding of genetic variation in pharmacogenes with direct implications in clinical pharmacogenomics.

Assessment of HLA-A, HLA-DR, and HLA-DQ alleles in patients with pemphigus vulgaris from eastern of Turkey.

BACKGROUND: Pemphigus vulgaris (PV) is a chronic progressive autoimmune bullous disease caused by the interaction of pathogenic factors, genetic, and environmental factors. HLA alleles, which are considered as protective factors against disease or predisposing factors, may be different in various populations and ethnic groups. AIMS: The purpose of this study is to examine the HLA-A, HLA-DR, and HLA-DQ alleles in patients that are diagnosed with PV in and around eastern of Turkey and to determine the alleles that create predisposition to disease or protect against the disease. PATIENTS/METHODS: Thirty patients diagnosed as PV with clinical, histopathological, and immunofluorescence findings and 30 healthy subjects were included in this study. The HLA-A, HLA-DR, and HLA-DQ typology in the DNA samples that were obtained from the blood samples of the groups was performed by using the PCR-SSP low-resolution gene panels. RESULTS: The HLA-A*03 allele was found to be significantly higher in patient group than the control group (P-value: .020). HLA-DRB1*04 and HLA-DRB1*14 alleles in PV patients were found to be significantly higher than the control group (P-value = .000). CONCLUSION: It was concluded that the HLA-DRB1*03, HLADQB1*02, and HLA-DQB1*06 alleles in and around eastern of Turkey showed protective effects against pemphigus vulgaris. It was also concluded that the HLA-A*03, HLA-DRB1*04, HLA-DRB1*14, HLA-DRB4, HLA-DQB1*03, and HLA-DQB1*05 alleles could cause predisposition to the disease.

Genetic diversity of Plasmodium knowlesi among human and long-tailed macaque populations in Peninsular Malaysia: The utility of microsatellite markers.

It has been discovered that Plasmodium knowlesi (P. knowlesi) is transmitted from macaque to man. Thus, the aim of the present study was to determine P. knowlesi genetic diversity in both human (n = 147) and long-tailed macaque (n = 26) samples from high- and low-endemicity localities. Genotyping was performed using seven neutral microsatellite loci markers. The size of the alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (HE), linkage disequilibrium (LD), and genetic differentiation (FST) were determined. In highly endemic P. knowlesi localities, the MOI for human and long-tailed macaque isolates was 1.04 and 1.15, respectively, while the Na was 11.14 and 7.86, respectively. Based on the allele frequency distribution for all loci, and with FST < 0.1, no genetic differentiation was seen between human and long-tailed macaque. In localities characterised by lower P. knowlesi endemicity, the MOI for human and long-tailed macaque isolates was 1.05 and 1.11, respectively, while the Na was 6.14 and 2.71, respectively. Further molecular analysis of the allele frequencies indicated that there was a significant genetic differentiation in human P. knowlesi isolates as compared to long-tailed macaque isolates, with a very low fixation index (FST = 0.016, p < .05) based on multiple loci analysis. Our results further indicate that, in Peninsular Malaysia, humans are mostly affected by P. knowlesi of a single genotype, while long-tailed macaque tend to acquire polyclonal infections, which supports the assumption that there is a higher rate of transmission among long-tailed macaque. Understanding the genetic diversity of P. knowlesi isolates can provide invaluable information for characterising patterns of the population structure and the migration rate of P. knowlesi in peninsular Malaysia.

Mutational Analysis Of Beta Thalassaemia By Multiplex Arms-Pcr In Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Beta thalassaemia is one of the commonest genetic conditions in the world. More than 200 different mutations have been reported in the beta globin chain genes. Notably, regional and ethnic variations in most common mutations in beta-thalassaemia have been identified. It is therefore imperative that region- and ethnicity- specific commonest mutations be identified for cost-effective molecular diagnosis of ß-thalassaemia mutations. The objective of this study was to determine the molecular mutations in ß-globin chain gene in patients with thalassemia in Khyber Pakhtunkhwa (KP) using multiplex- Amplification Refractory Mutation System (ARMS) PCR. METHODS: It was a cross sectional descriptive study. Blood samples from newly diagnosed ß thalassemia patients was collected and used as source for DNA isolation. ARMS PCR was performed for detection of mutations in ß-globin gene. SDS-PAGE was conducted for visualization of the amplicon. RESULTS: Prominent mutations were Fr 8-9 (+G), CD 5 (-CT) and Fr 41-42 (-TTCT). Congenital marriages and lack of awareness are largest contributing factor for increasing the disease burden. Organomegaly being a serious clinical complication which contributes to morbidity was proportional to age and disease progression. Fr 8-9 (+G) & CD 5 (- CT) were the most frequent mutation prevalent among different ethnic groups residing in KP. CONCLUSIONS: Multiplex-ARMS PCR is capable of assessing for multiple mutations in a single tube. Regional- and ethnic- variations in the commonest mutations in KP are noted. Any mutational diagnostic strategy should consider costs and genetic variations in a particular setting..

The mutation-drift balance in spatially structured populations.

In finite populations the action of neutral mutations is balanced by genetic drift, leading to a stationary distribution of alleles that displays a transition between two different behaviors. For small mutation rates most individuals will carry the same allele at equilibrium, whereas for high mutation rates of the alleles will be randomly distributed with frequencies close to one half for a biallelic gene. For well-mixed haploid populations the mutation threshold is µc=1/2N, where N is the population size. In this paper we study how spatial structure affects this mutation threshold. Specifically, we study the stationary allele distribution for populations placed on regular networks where connected nodes represent potential mating partners. We show that the mutation threshold is sensitive to spatial structure only if the number of potential mates is very small. In this limit, the mutation threshold decreases substantially, increasing the diversity of the population at considerably low mutation rates. Defining kc as the degree of the network for which the mutation threshold drops to half of its value in well-mixed populations we show that kc grows slowly as a function of the population size, following a power law. Our calculations and simulations are based on the Moran model and on a mapping between the Moran model with mutations and the voter model with opinion makers.

Validation of outlier loci through replication in independent data sets: a test on Arabis alpina.

Outlier detection and environmental association analysis are common methods to search for loci or genomic regions exhibiting signals of adaptation to environmental factors. However, a validation of outlier loci and corresponding allele distribution models through functional molecular biology or transplant/common garden experiments is rarely carried out. Here, we employ another method for validation, namely testing outlier loci in specifically designed, independent data sets. Previously, an outlier locus associated with three different habitat types had been detected in Arabis alpina. For the independent validation data set, we sampled 30 populations occurring in these three habitat types across five biogeographic regions of the Swiss Alps. The allele distribution model found in the original study could not be validated in the independent test data set: The outlier locus was no longer indicative of habitat-mediated selection. We propose several potential causes of this failure of validation, of which unaccounted genetic structure and technical issues in the original data set used to detect the outlier locus were most probable. Thus, our study shows that validating outlier loci and allele distribution models in independent data sets is a helpful tool in ecological genomics which, in the case of positive validation, adds confidence to outlier loci and their association with environmental factors or, in the case of failure of validation, helps to explain inconsistencies.

The HLA class II allele DQB1 0309 is associated with dilated cardiomyopathy.

Previous studies have shown weak associations between human dilated cardiomyopathy (DCM) and certain human leucocyte antigen (HLA) class II polymorphisms. Using a sequence-specific primer-PCR (SSP-PCR) technology, we compared the allelic distribution in the HLA-DQ and -DR locus in a cohort of German DCM patients (n=165) and DCM-free controls (n=79). With the exception of HLA-DQB1 0309, we found no significant differences between the two groups, even without adjustment for multiple testing. The HLA-DQB1 0309 allele, however, was detected more frequently in DCM patients as compared to controls (28.5% versus 10.1%, p=0.0010), leading to an odds ratio of 3.5 (95% confidence interval=1.5-9.1). The frequency of this allele was significantly higher in DCM patients without lymphocytic infiltrates in endomyocardial biopsies as compared to patients classified histologically as inflammatory DCM (33.1% versus 14.6%, p=0.028). There was no significant difference in the allelic HLA-DQB1 0309 distribution between DCM patients with and without viral genomes detected in the heart (24.2% versus 29.5%, p=0.668). In summary, the frequency of the HLA-DQB1 0309 allele is overrepresented in DCM patients, suggesting that carriers of this HLA class II variant are associated with an increased risk for developing DCM. Although Bonferroni adjustment was applied, controlled studies in larger samples of DCM patients and in different ethnic populations are warranted to confirm this observation and reveal the pathophysiological mechanisms behind this association.

Distribution of HLA-C alleles in Shandong Han population of China / 中华微生物学和免疫学杂志

Objective To analyze the distribution of HLA-C alleles in Shandong Han population of China.Methods One hundred and fifty unrelated potential donors,self-claimed as Han population from Shandong province,were selected from China Marrow Donor Program.Genotypes of HLA-C with the donors were identified by PCR-SBT.The frequencies of allele were calculated with direct counting method and the differences with other populations were analyzed with SPSS16.0 x2 software.Results A total of 25 alleles of HLA-C were observed and the most common alleles were C * 06:02 and C * 07:02 with the frequency of more than 10.00％.Moreover,there were 16 kinds of alleles with the frequency of more than 1.00％ accounting for 95.33％ of the total alleles.The distribution of HLA-C alleles in Shandong Han population was similar to that in northern Han population,but had some differences with that in southern Han population.In addition,the distribution of HLA-C alleles in Shandong Han population significantly differed from that of German/African American.Conclusion This study on the distribution of HLA-C alleles in Shandong Han population provides valuable references for further studies on the genetics of HLA,cross-match for organ transplantation and other genetic-associated diseases in this population.

Prenatal Population Screening for Fragile X Carrier and the Prevalence of Premutation Carriers in Korea

PURPOSE: Fragile X carrier detection before or at early pregnancy through a wide screening program may not only confer a risk of having offspring with Fragile X syndrome (FXS), but may also confer a risk for Fragile X-associated primary ovarian insufficiency and Fragile X-associated tremor/ataxia syndrome. However, prior to the implementation of such a program, the carrier prevalence in a population and the availability of effective screening test should be evaluated. The aim of our study was to determine the prevalence of premutation carriers and to evaluate the feasibility of screening test. MATERIALS AND METHODS: The blood samples were obtained from 8,641 pregnant women with no family history of mental retardation. We performed a three-primer CGG repeat primed (RP) PCR using the AmplideX(TM) FMR1 PCR kit (Asuragen, Inc. Austin, TX, USA). Samples showing full mutation alleles were reflexed to Southern blot analysis for methylation status and sizing. RESULTS: Among the 8,641 women, we found 8 premutation carriers (1:1,090, 0.09%) and 46 women with an intermediate allele (1:190, 0.53%). No woman was found to carry the fully mutated allele. All the detected alleles were within the CGG repeat range of 8-117. Among the 8,641 samples, 29 and 30 CGG repeats represent 66.6% of all cases. The CGG RP PCR method provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis. CONCLUSION: This is the first study that has focused on the prevalence of FXS premutation carriers and FMR1 allele distribution in normal pregnant women. These data have important implications for population-based fragile X carrier screening in Korea.

Prenatal Population Screening for Fragile X Carrier and the Prevalence of Premutation Carriers in Korea

PURPOSE: Fragile X carrier detection before or at early pregnancy through a wide screening program may not only confer a risk of having offspring with Fragile X syndrome (FXS), but may also confer a risk for Fragile X-associated primary ovarian insufficiency and Fragile X-associated tremor/ataxia syndrome. However, prior to the implementation of such a program, the carrier prevalence in a population and the availability of effective screening test should be evaluated. The aim of our study was to determine the prevalence of premutation carriers and to evaluate the feasibility of screening test. MATERIALS AND METHODS: The blood samples were obtained from 8,641 pregnant women with no family history of mental retardation. We performed a three-primer CGG repeat primed (RP) PCR using the AmplideX(TM) FMR1 PCR kit (Asuragen, Inc. Austin, TX, USA). Samples showing full mutation alleles were reflexed to Southern blot analysis for methylation status and sizing. RESULTS: Among the 8,641 women, we found 8 premutation carriers (1:1,090, 0.09%) and 46 women with an intermediate allele (1:190, 0.53%). No woman was found to carry the fully mutated allele. All the detected alleles were within the CGG repeat range of 8-117. Among the 8,641 samples, 29 and 30 CGG repeats represent 66.6% of all cases. The CGG RP PCR method provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis. CONCLUSION: This is the first study that has focused on the prevalence of FXS premutation carriers and FMR1 allele distribution in normal pregnant women. These data have important implications for population-based fragile X carrier screening in Korea.