DNA hypomethylation at ALOX12 is associated with persistent wheezing in childhood.

RATIONALE: Epigenetic changes may play a role in the occurrence of asthma-related phenotypes. OBJECTIVES: To identify epigenetic marks in terms of DNA methylation of asthma-related phenotypes in childhood, and to assess the effect of prenatal exposures and genetic variation on these epigenetic marks. METHODS: Data came from two cohorts embedded in the Infancia y Medio Ambiente (INMA) PROJECT: Menorca (n = 122) and Sabadell (n = 236). Wheezing phenotypes were defined at age 4-6 years. Cytosine-guanine (CpG) dinucleotide site DNA methylation differences associated with wheezing phenotypes were screened in children of the Menorca study using the Illumina GoldenGate Panel I. Findings were validated and replicated using pyrosequencing. Information on maternal smoking and folate supplement use was obtained through questionnaires. Dichlorodiphenyldichloroethylene was measured in cord blood or maternal serum. Genotypes were extracted from genome-wide data. MEASUREMENT AND MAIN RESULTS: Screening identified lower DNA methylation at a CpG site in the arachidonate 12-lipoxygenase (ALOX12) gene in children having persistent wheezing compared with those never wheezed (P = 0.003). DNA hypomethylation at ALOX12 loci was associated with higher risk of persistent wheezing in the Menorca study (odds ratio per 1% methylation decrease, 1.13; 95% confidence interval, 0.99-1.29; P = 0.077) and in the Sabadell study (odds ratio, 1.16; 95% confidence interval, 1.03-1.37; P = 0.017). Higher levels of prenatal dichlorodiphenyldichloroethylene were associated with DNA hypomethylation of ALOX12 in the Menorca study (P = 0.033), but not in the Sabadell study (P = 0.377). ALOX12 DNA methylation was strongly determined by underlying genetic polymorphisms. CONCLUSIONS: DNA methylation of ALOX12 may be an epigenetic biomarker for the risk of asthma-related phenotypes.

Familial hemiplegic migraine: linkage to chromosome 14q32 in a Spanish kindred.

We sought to map the disease-causing gene in a large Spanish kindred with familial hemiplegic migraine (FHM). Patients were classified according to the ICHD-II criteria. After ruling out linkage to known migraine genetic loci, a single nucleotide polymorphism-based, 0.62-cM density genome-wide scan was performed. Among 13 affected subjects, FHM was the prevailing migraine phenotype in six, migraine with aura in four and migraine without aura in three. Linkage analysis revealed a disease locus in a 4.15-Mb region on 14q32 with a maximum two-point logarithm of odds (LOD) score of 3.1 and a multipoint parametric LOD score of 3.8. This genomic region does not overlap with the reported migraine loci on 14q21-22. Sequence analysis of three candidate genes in the region, SLC24A4, ATXN3 and ITPK1, failed to show disease-causing mutations in our patients. Genetic heterogeneity in FHM may be greater than previously suspected.

Citizen science charts two major "stomatotypes" in the oral microbiome of adolescents and reveals links with habits and drinking water composition.

BACKGROUND: The oral cavity comprises a rich and diverse microbiome, which plays important roles in health and disease. Previous studies have mostly focused on adult populations or in very young children, whereas the adolescent oral microbiome remains poorly studied. Here, we used a citizen science approach and 16S profiling to assess the oral microbiome of 1500 adolescents around Spain and its relationships with lifestyle, diet, hygiene, and socioeconomic and environmental parameters. RESULTS: Our results provide a detailed snapshot of the adolescent oral microbiome and how it varies with lifestyle and other factors. In addition to hygiene and dietary habits, we found that the composition of tap water was related to important changes in the abundance of several bacterial genera. This points to an important role of drinking water in shaping the oral microbiota, which has been so far poorly explored. Overall, the microbiome samples of our study can be clustered into two broad compositional patterns (stomatotypes), driven mostly by Neisseria and Prevotella, respectively. These patterns show striking similarities with those found in unrelated populations. CONCLUSIONS: We hypothesize that these stomatotypes represent two possible global optimal equilibria in the oral microbiome that reflect underlying constraints of the human oral niche. As such, they should be found across a variety of geographical regions, lifestyles, and ages.

A De Novo Nonsense Mutation in MAGEL2 in a Patient Initially Diagnosed as Opitz-C: Similarities Between Schaaf-Yang and Opitz-C Syndromes.

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes.

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms.

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."

Genomic analysis of the blood attributed to Louis XVI (1754-1793), king of France.

A pyrographically decorated gourd, dated to the French Revolution period, has been alleged to contain a handkerchief dipped into the blood of the French king Louis XVI (1754-1793) after his beheading but recent analyses of living males from two Bourbon branches cast doubts on its authenticity. We sequenced the complete genome of the DNA contained in the gourd at low coverage (~2.5×) with coding sequences enriched at a higher ~7.3× coverage. We found that the ancestry of the gourd's genome does not seem compatible with Louis XVI's known ancestry. From a functional perspective, we did not find an excess of alleles contributing to height despite being described as the tallest person in Court. In addition, the eye colour prediction supported brown eyes, while Louis XVI had blue eyes. This is the first draft genome generated from a person who lived in a recent historical period; however, our results suggest that this sample may not correspond to the alleged king.

HumMeth27QCReport: an R package for quality control and primary analysis of Illumina Infinium methylation data.

BACKGROUND: The study of the human DNA methylome has gained particular interest in the last few years. Researchers can nowadays investigate the potential role of DNA methylation in common disorders by taking advantage of new high-throughput technologies. Among these, Illumina Infinium assays can interrogate the methylation levels of hundreds of thousands of CpG sites, offering an ideal solution for genome-wide methylation profiling. However, like for other high-throughput technologies, the main bottleneck remains at the stage of data analysis rather than data production. FINDINGS: We have developed HumMeth27QCReport, an R package devoted to researchers wanting to quickly analyse their Illumina Infinium methylation arrays. This package automates quality control steps by generating a report including sample-independent and sample-dependent quality plots, and performs primary analysis of raw methylation calls by computing data normalization, statistics, and sample similarities. This package is available at CRAN repository, and can be integrated in any Galaxy instance through the implementation of ad-hoc scripts accessible at Galaxy Tool Shed. CONCLUSIONS: Our package provides users of the Illumina Infinium Methylation assays with a simplified, automated, open-source quality control and primary analysis of their methylation data. Moreover, to enhance its use by experimental researchers, the tool is being distributed along with the scripts necessary for its implementation in the Galaxy workbench. Finally, although it was originally developed for HumanMethylation27, we proved its compatibility with data generated with the HumanMethylation450 Bead Chip.

Design and evaluation of a panel of single-nucleotide polymorphisms in microRNA genomic regions for association studies in human disease.

MicroRNAs (miRNA) are recognized posttranscriptional gene repressors involved in the control of almost every biological process. Allelic variants in these regions may be an important source of phenotypic diversity and contribute to disease susceptibility. We analyzed the genomic organization of 325 human miRNAs (release 7.1, miRBase) to construct a panel of 768 single-nucleotide polymorphisms (SNPs) covering approximately 1 Mb of genomic DNA, including 131 isolated miRNAs (40%) and 194 miRNAs arranged in 48 miRNA clusters, as well as their 5-kb flanking regions. Of these miRNAs, 37% were inside known protein-coding genes, which were significantly associated with biological functions regarding neurological, psychological or nutritional disorders. SNP coverage analysis revealed a lower SNP density in miRNAs compared with the average of the genome, with only 24 SNPs located in the 325 miRNAs studied. Further genotyping of 340 unrelated Spanish individuals showed that more than half of the SNPs in miRNAs were either rare or monomorphic, in agreement with the reported selective constraint on human miRNAs. A comparison of the minor allele frequencies between Spanish and HapMap population samples confirmed the applicability of this SNP panel to the study of complex disorders among the Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 in the CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showing geographical allelic frequency variation among the four HapMap populations, probably because of differences in natural selection. The designed miRNA SNP panel could help to identify still hidden links between miRNAs and human disease.

Analysis of nonsense-mediated mRNA decay in mutant alleles identified in Spanish Gaucher disease patients.

Most of the mutations described in the GBA gene as responsible for Gaucher disease are missense mutations. Nevertheless, other alterations, including nonsense and frameshift mutations, have been reported. These mutations generate premature termination codons (PTC) that could trigger the degradation of mRNA through a mechanism known as nonsense-mediated decay (NMD). It has been established that NMD requires the presence of at least one intron downstream of the PTC, and that this PTC should be at least 50-55 nucleotides upstream of the 3'-most exon-exon junction. In this study, we analyse four GBA truncating mutations - c.108G > A (W(-4)X; HGVS recommended nomenclature: p.W36X), c.886C > T (R257X; HGVS: p.R296X), c.1098_1099insA and c.1451_1452delAC - found in Spanish Gaucher disease patients in order to determine whether they undergo mRNA decay and, if so, whether this occurs via the NMD pathway. RT-PCR showed a clear reduction of RNA for three of the alleles: W(-4)X, R257X and c.1098_1099insA. After treatment with cycloheximide (CHX), a known inhibitor of both protein synthesis and NMD, two of the mutant alleles, R257X and c.1098_1099insA, showed a partial recovery of the amount of mRNA. The third mutation, W(-4)X, did not show any significant CHX-induced recovery, while allele c.1451_1452delAC did not show mRNA decay at all. Real-time PCR confirmed these results and allowed the decay and recovery to be quantified. Finally, the protein truncation test was performed to detect the corresponding proteins. Expected products for alleles R257X, c.1451_1452delAC and c.1098_1099insA, but not for W(-4)X, were observed.

Expression and functional characterization of human mutant sulfamidase in insect cells.

Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo syndrome) is an autosomal recessive lysosomal disorder caused by the deficiency of sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. The molecular defects of 26 unrelated Spanish MPS IIIA patients were recently reported by our group. Here we describe the heterologous expression, using a baculovirus system, of the cDNAs corresponding to eight out of the 14 mutant alleles present in this patient group and the characterization of the corresponding mutant enzymes. In particular, we expressed the following alleles: p.S66W, p.R74H, p.Q85R, p.R206P, p.L386R, p.R433W, p.R433Q, and c.1079delC (previously named as c.1091delC), and the two variants of the polymorphism p.R456H. The expression of the mutant alleles and the characterization of the corresponding enzymes revealed that their activity was severely compromised. Only mutations p.S66W and p.R206P retained low levels of residual activity. However, Western blot analysis showed in all cases the presence of the expected two forms of the sulfamidase, the precursor and the mature proteins, indicating a normal processing of the mutant enzyme.