The unstable (CTG)$\sb{\rm n}$ trinucleotide repeat associated with myotonic dystrophy: What it does and where it came from

Myotonic dystrophy (DM), an autosomal dominant disorder mapping to human chromosome 19q13.3, is the most common neuromuscular disease in human adults.^ Following the identification of the mutation underlying the DM phenotype, an unstable (CTG)$\sb{n}$ trinucleotide repeat in the 3$\prime$ untranslated region (UTR) of a gene encoding a ser/thr protein kinase named DM protein kinase (DMPK), the study was targeted at two questions: (1) the identification of the disease-causing mechanism(s) of the unstable repeat, and at a more basic level, (2) the identification of the origin and the mechanism(s) involved in repeat instability. The first goal was to identify the pathophysiological mechanisms of the (CTG)$\sb{n}$ repeat.^ The normal repeat is transcribed but not translated; therefore, initial studies centered on the effect on RNA transcript levels. The vast majority of DM affecteds are heterozygous for the mutant expansion, so that the normal allele interferes with the analysis of the mutant allele. A quantitative allele-specific RT-PCR procedure was developed and applied to a spectrum of patient tissue samples and cell lines. Equal levels of unprocessed pre-mRNA were determined for the wild type (+) and disease (DM) alleles in skeletal muscle and cell lines of heterozygous DM patients, indicating that any nucleosome binding has no effect at the level of transcriptional initiation and transcription of the mutant DMPK locus. In contrast, processed mRNA levels from the DM allele were reduced relative to the + allele as the size of the expansion increased. The unstable repeat, therefore, impairs post-transcriptional processing of DM allele transcripts. This phenomenon has profound effects on overall DMPK locus steady-state transcript levels in cells missing a wild type allele and does not appear to be mediated by imprinting, decreased mRNA stability, generation of aberrant splice forms, or absence of polyadenylation of the mutant allele.^ In Caucasian DM subjects, the unstable repeat is in complete linkage disequlibrium with a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)$\sb{n}$ repeat in one of their DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)$\sb{n}$ repeat expansion from other alleles of the putative predisposing haplotype was found. Thus, the expanded (CTG)$\sb{n}$ repeat in this family was the result of an independent mutational event. Consequently, the origin of DM is unlikely the result of a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling. (Abstract shortened by UMI.) ^

Population and molecular evolution studies on the Melanocortin 1 receptor locus implicate its role in human pigmentation variation

Human pigmentation is a complex trait with the observed variation caused by the varied production of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) by the melanocytes. The melanocortin 1 receptor (MC1R), a G protein-coupled receptor expressed in the melanocytes, is a regulator eu- and phaeomelanin synthesis, and MC1R mutations causing skin and coat color changes are known in many mammals. To understand the role of MC1R in human pigmentation variation, I have sequenced the MC1R gene in 121 individuals sampled from world populations. In addition, I have sequenced the MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon to study the evolution of MC1R and to infer the ancestral human MC1R sequence. The ancestral MC1R sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. To further evaluate the role of MC1R variants in human pigmentation variation, I have combined these molecular evolution and population studies with functional assays on MC1R variants and primate MC1Rs. ^

DQB1 locus alone explains most of the risk and protection in narcolepsy with cataplexy in Europe

STUDY OBJECTIVE Prior research has identified five common genetic variants associated with narcolepsy with cataplexy in Caucasian patients. To replicate and/or extend these findings, we have tested HLA-DQB1, the previously identified 5 variants, and 10 other potential variants in a large European sample of narcolepsy with cataplexy subjects. DESIGN Retrospective case-control study. SETTING A recent study showed that over 76% of significant genome-wide association variants lie within DNase I hypersensitive sites (DHSs). From our previous GWAS, we identified 30 single nucleotide polymorphisms (SNPs) with P < 10(-4) mapping to DHSs. Ten SNPs tagging these sites, HLADQB1, and all previously reported SNPs significantly associated with narcolepsy were tested for replication. PATIENTS AND PARTICIPANTS For GWAS, 1,261 narcolepsy patients and 1,422 HLA-DQB1*06:02-matched controls were included. For HLA study, 1,218 patients and 3,541 controls were included. MEASUREMENTS AND RESULTS None of the top variants within DHSs were replicated. Out of the five previously reported SNPs, only rs2858884 within the HLA region (P < 2x10(-9)) and rs1154155 within the TRA locus (P < 2x10(-8)) replicated. DQB1 typing confirmed that DQB1*06:02 confers an extraordinary risk (odds ratio 251). Four protective alleles (DQB1*06:03, odds ratio 0.17, DQB1*05:01, odds ratio 0.56, DQB1*06:09 odds ratio 0.21, DQB1*02 odds ratio 0.76) were also identified. CONCLUSION An overwhelming portion of genetic risk for narcolepsy with cataplexy is found at DQB1 locus. Since DQB1*06:02 positive subjects are at 251-fold increase in risk for narcolepsy, and all recent cases of narcolepsy after H1N1 vaccination are positive for this allele, DQB1 genotyping may be relevant to public health policy.

Comparative genetic analyses point to HCP5 as susceptibility locus for HCV-associated hepatocellular carcinoma

BACKGROUND & AIMS: Recently, genetic variations in MICA (lead single nucleotide polymorphism [SNP] rs2596542) were identified by a genome-wide association study (GWAS) to be associated with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) in Japanese patients. In the present study, we sought to determine whether this SNP is predictive of HCC development in the Caucasian population as well. METHODS: An extended region around rs2596542 was genotyped in 1924 HCV-infected patients from the Swiss Hepatitis C Cohort Study (SCCS). Pair-wise correlation between key SNPs was calculated both in the Japanese and European populations (HapMap3: CEU and JPT). RESULTS: To our surprise, the minor allele A of rs2596542 in proximity of MICA appeared to have a protective impact on HCC development in Caucasians, which represents an inverse association as compared to the one observed in the Japanese population. Detailed fine-mapping analyses revealed a new SNP in HCP5 (rs2244546) upstream of MICA as strong predictor of HCV-related HCC in the SCCS (univariable p=0.027; multivariable p=0.0002, odds ratio=3.96, 95% confidence interval=1.90-8.27). This newly identified SNP had a similarly directed effect on HCC in both Caucasian and Japanese populations, suggesting that rs2244546 may better tag a putative true variant than the originally identified SNPs. CONCLUSIONS: Our data confirms the MICA/HCP5 region as susceptibility locus for HCV-related HCC and identifies rs2244546 in HCP5 as a novel tagging SNP. In addition, our data exemplify the need for conducting meta-analyses of cohorts of different ethnicities in order to fine map GWAS signals.

Localization of Epileptogenic Zone on Pre-surgical Intracranial EEG Recordings: Toward a Validation of Quantitative Signal Analysis Approaches

In patients diagnosed with pharmaco-resistant epilepsy, cerebral areas responsible for seizure generation can be defined by performing implantation of intracranial electrodes. The identification of the epileptogenic zone (EZ) is based on visual inspection of the intracranial electroencephalogram (IEEG) performed by highly qualified neurophysiologists. New computer-based quantitative EEG analyses have been developed in collaboration with the signal analysis community to expedite EZ detection. The aim of the present report is to compare different signal analysis approaches developed in four different European laboratories working in close collaboration with four European Epilepsy Centers. Computer-based signal analysis methods were retrospectively applied to IEEG recordings performed in four patients undergoing pre-surgical exploration of pharmaco-resistant epilepsy. The four methods elaborated by the different teams to identify the EZ are based either on frequency analysis, on nonlinear signal analysis, on connectivity measures or on statistical parametric mapping of epileptogenicity indices. All methods converge on the identification of EZ in patients that present with fast activity at seizure onset. When traditional visual inspection was not successful in detecting EZ on IEEG, the different signal analysis methods produced highly discordant results. Quantitative analysis of IEEG recordings complement clinical evaluation by contributing to the study of epileptogenic networks during seizures. We demonstrate that the degree of sensitivity of different computer-based methods to detect the EZ in respect to visual EEG inspection depends on the specific seizure pattern.

Classifying and mapping the urban transition in Vietnam

The urban transition almost always involves wrenching social adjustment as small agricultural communities are forced to adjust rapidly to industrial ways of life. Large-scale in-migration of young people, usually from poor regions, creates enormous demand and expectations for community and social services. One immediate problem planners face in approaching this challenge is how to define, differentiate, and map what is rural, urban, and transitional (i.e., peri-urban). This project established an urban classification for Vietnam by using national census and remote sensing data to identify and map the smallest administrative units for which data are collected as rural, peri-urban, urban, or urban core. We used both natural and human factors in the quantitative model: income from agriculture, land under agriculture and forests, houses with modern sanitation, and the Normalized Difference Vegetation Index. Model results suggest that in 2006, 71% of Vietnam's 10,891 communes were rural, 18% peri-urban, 3% urban, and 4% urban core. Of the communes our model classified as peri-urban, 61% were classified by the Vietnamese government as rural. More than 7% of Vietnam's land area can be classified as peri-urban and approximately 13% of its population (more than 11 million people) lives in peri-urban areas. We identified and mapped three types of peri-urban places: communes in the periphery of large towns and cities; communes along highways; and communes associated with provincial administration or home to industrial, energy, or natural resources projects (e.g., mining). We validated this classification based on ground observations, analyses of multi-temporal night-time lights data, and an examination of road networks. The model provides a method for rapidly assessing the rural–urban nature of places to assist planners in identifying rural areas undergoing rapid change with accompanying needs for investments in building, sanitation, road infrastructure, and government institutions.

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.

Genetic analysis of white facial and leg markings in the Swiss Franches-Montagnes Horse Breed

White markings and spotting patterns in animal species are thought to be a result of the domestication process. They often serve for the identification of individuals but sometimes are accompanied by complex pathological syndromes. In the Swiss Franches-Montagnes horse population, white markings increased vastly in size and occurrence during the past 30 years, although the breeding goal demands a horse with as little depigmented areas as possible. In order to improve selection and avoid more excessive depigmentation on the population level, we estimated population parameters and breeding values for white head and anterior and posterior leg markings. Heritabilities and genetic correlations for the traits were high (h(2) > 0.5). A strong positive correlation was found between the chestnut allele at the melanocortin-1-receptor gene locus and the extent of white markings. Segregation analysis revealed that our data fit best to a model including a polygenic effect and a biallelic locus with a dominant-recessive mode of inheritance. The recessive allele was found to be the white trait-increasing allele. Multilocus linkage disequilibrium analysis allowed the mapping of the putative major locus to a chromosomal region on ECA3q harboring the KIT gene.

Clinical Use of Quantitative Computed Tomography–Based Advanced Techniques in the Management of Osteoporosis in Adults: the 2015 ISCD Official Positions—Part III

The International Society for Clinical Densitometry (ISCD) has developed new official positions for the clinical use of computed tomography (CT) scans acquired without a calibration phantom, for example, CT scans obtained for other diagnosis such as colonography. This also addresses techniques suggested for opportunistic screening of osteoporosis. The ISCD task force for quantitative CT reviewed the evidence for clinical applications of these new techniques and presented a report with recommendations at the 2015 ISCD Position Development Conference. Here we discuss the agreed upon ISCD official positions with supporting medical evidence, rationale, controversy, and suggestions for further study. Advanced techniques summarized as statistical parameter mapping methods were also reviewed. Their future use is promising but the clinical application is premature. The clinical use of QCT of the hip is addressed in part I and of finite element analysis of the hip and spine in part II.

BIOCHEMICAL CHARACTERIZATION AND GENETIC MAPPING OF WILD TYPE AND MUTANT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ALLELES IN CHINESE HAMSTER OVARY CELLS

A UV-induced mutation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) was characterized in the CHO clone A24. The asymmetric 4-banded zymogram and an in vitro GAPD activity equal to that of wild type cells were not consistent with models of a mutant heterozygote producing equal amounts of wild type and either catalytically active or inactive mutant subunits that interacted randomly. Cumulative evidence indicated that the site of the mutation was the GAPD structural locus expressed in CHO wild type cells, and that the mutant allele coded for a subunit that differed from the wild type subunit in stability and kinetics. The evidence included the appearance of a fifth band, the putative mutant homotetramer, after addition of the substrate glyceraldehyde-3-phosphate (GAP) to the gel matrix; dilution experiments indicating stability differences between the subunits; experiments with subsaturating levels of GAP indicating differences in affinity for the substrate; GAPD zymograms of A24 x mouse hybrids that were consistent with the presence of two distinct A24 subunits; independent segregation of A24 wild type and mutant electrophoretic bands from the hybrids, which was inconsistent with models of mutation of a locus involved in posttranslational modification; the mapping of both wild type and mutant forms of GAPD to chromosome 8; and the failure to detect any evidence of posttranslational modification (of other A24 isozymes, or through mixing of homogenates of A24 and mouse).^ The extent of skewing of the zymogram toward the wild type band, and the unreduced in vitro activity were inconsistent with models based solely on differences in activity of the two subunits. Comparison of wild type homotetramer bands in wild type cells and A24 suggested the latter had a preponderance of wild type subunits over mutant subunits, and had more GAPD tetramers than did CHO controls.^ Two CHO linkages, GAPD-triose phosphate isomerase, and acid phosphatase 2-adenosine deaminase were reported provisionally, and several others were confirmed. ^

The genetic basis of thoracic aortic aneurysms and dissections: Genetic heterogeneity and mapping of TAAD1 and TAAD2 loci

Thoracic aortic aneurysms leading to aortic dissections (TAAD) are a major cause of morbidity and mortality in the United States. TAAD is a complication of some known genetic disorders, such as Marfan syndrome and Turner syndrome, but the majority of familial cases are not due to a known genetic syndrome. Previous studies by our group have established that nonsyndromic, familial TAAD is inherited in an autosomal dominant manner with decreased penetrance and variable expression. Using one large family with multiple members with TAAD for the genome wide scan, a major locus for familial TAAD was mapped to 5q13–14 (TAAD1). Nine out of 15 families studied were linked to this locus, establishing that TAAD1 was a major locus, and that there was genetic heterogeneity for the condition. Mapping of TAAD2 locus was accomplished using a single large family with multiple members with TAAD not linked to known loci of aneurysm formation. This established a second novel locus for familial TAAD on 3p24–25 (LOD score of 4.3), termed the TAAD2 locus. Two putative loci with suggestive LOD scores were mapped on 4q and 12q through a genome scan carried out using three families. TAAD phenotype in 12 families did not segregate with known loci, indicating further genetic heterogeneity. An STS-tagged BAC based contig was constructed for 7.8Mb and 25Mb critical interval of TAAD1 and TAAD2 respectively and characterized to identify the defective gene. The hypothesis that the defective genes responsible for the TAAD1 and TAAD2 encoded extracellular matrix (ECM) proteins, the major components of the elastic fiber system in the aortic media was tested. Four genes encoding ECM proteins, versican, thrombospondin-3, CRTL1, on TAAD1 and FBLN2 at TAAD2 were sequenced, but no disease-causing mutations were identified. Studies to identify the defective gene are initiated through the positional candidate gene approach using combination of bioinformatics and expression studies. The identification of the TAAD susceptibility genes will allow for presymptomatic diagnosis of individuals at risk for this life threatening disease. The identification of the molecular defects that contribute to TAAD will also further our understanding of the proteins that provide structural integrity to the aortic wall. ^

IR spectra from mapping of diamond sample JH7b

This data set contains 1851 infrared (IR) spectra, forming a single IR map of diamond sample JH7b. This data set is used to show the application of DiaMap, a computer routine written using PERL, to automatically process diamond IR spectra to obtain quantitative impurity data from them. Full abstract will be added after acceptance of publication.

Variance and covariance of actual relationship between relatives at one locus.

The relationship between pairs of individuals is an important topic in many areas of population and quantitative genetics. It is usually measured as the proportion of thegenome identical by descent shared by the pair and it can be inferred from pedigree information. But there is a variance in actual relationships as a consequence of Mendelian sampling, whose general formula has not been developed. The goal of this work is to develop this general formula for the one-locus situation,. We provide simple expressions for the variances and covariances of all actual relationships in an arbitrary complex pedigree. The proposed method relies on the use of the nine identity coefficients and the generalized relationship coefficients; formulas have been checked by computer simulation. Finally two examples for a short pedigree of dogs and a long pedigree of sheep are given.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.