Simultaneous genotyping of multiple polymorphisms in human serotonin transporter gene and detection of novel allelic variants

The serotonin transporter, called SLC6A4, SERT or 5-HTT, modulates neurotransmission by removal of serotonin from the synapse of serotonergic neurons, facilitating serotonin reuptake into the presynaptic terminus. Selective serotonin reuptake inhibitors block the action of the serotonin transporter and are used to treat depression and other neuropsychiatric disorders. Three polymorphisms in the 5-HTT gene have been implicated in treatment response and neuropsychiatric disorders. A 44-bp promoter ins/del polymorphism (5-HTTLPR) produces primarily long and/or short alleles due to either 14 (short) or 16 (long) repeats of variably conserved 20–23 bp units. Also implicated, a 17–18 bp variable number tandem repeat found in intron2 (StIn2) is expressed as triallelic content with 9, 10, or 12 repeats (StIn2.9, StIn2.10 or StIn2.12). Finally, a single nucleotide polymorphism rs25531 located within the promoter polymorphic-linked region alters the function of the long promoter allele. We developed a PCR-based fragment analysis assay, which is analyzed on an ABI sequencer, whereby we are able to detect all three genotypes simultaneously. Using this technique, we identified novel sequences, which demonstrate promoter repeat regions containing (1) a 17 repeat with rs25531 A/G polymorphism, (2) two with 18-repeat units, (3) one with 20-repeat units and (4) a 24-repeat sequence. The novel repeats were confirmed by direct sequencing of gel-purified amplicons

DNA methylation in peripheral tissues and left-handedness

This is the final version. Available on open access from Nature Research vis the DOI in this recordData availability: The HumanMethylation450 BeadChip data from the NTR are available as part of the Biobank-based Integrative Omics Studies (BIOS) Consortium in the European Genome-phenome Archive (EGA), under the accession code EGAD00010000887 (https://ega-archive.org/datasets/EGAD00010000887). The Infinium MethylationEPIC from NTR are available from the Netherlands Twin Register on reasonable request (https://tweelingenregister.vu.nl/information_for_researchers/working-with-ntr-data). DNA methylation data from ALSPAC are available at ALSPAC and can be provided on request. The study website contains details of all the data that is available through a fully searchable data dictionary and variable search tool (http://www.bristol.ac.uk/alspac/researchers/our-data). The code used to perform the primary and secondary analyses is available at https://github.com/MRCIEU/handedness-ewas. The pipeline for the DNA methylation array analysis developed by the Biobank-based Integrative Omics Study (BIOS) consortium are available here: https://molepi.github.io/DNAmArray_workflow/. EWAS summary statistics for the top 100 CpGs are given in Supplemental Tables 6–11 and 15–29. The full EWAS summary statistics from the meta-analysis with basic and adjusted model are provided in Supplemental Tables 32 and 33. The full summary statistics for all other analyses are available upon request from the corresponding author.Handedness has low heritability and epigenetic mechanisms have been proposed as an etiological mechanism. To examine this hypothesis, we performed an epigenome-wide association study of left-handedness. In a meta-analysis of 3914 adults of whole-blood DNA methylation, we observed that CpG sites located in proximity of handedness-associated genetic variants were more strongly associated with left-handedness than other CpG sites (P = 0.04), but did not identify any differentially methylated positions. In longitudinal analyses of DNA methylation in peripheral blood and buccal cells from children (N = 1737), we observed moderately stable associations across age (correlation range [0.355-0.578]), but inconsistent across tissues (correlation range [- 0.384 to 0.318]). We conclude that DNA methylation in peripheral tissues captures little of the variance in handedness. Future investigations should consider other more targeted sources of tissue, such as the brain

De novo single-nucleotide and copy number variation in discordant monozygotic twins reveals disease-related genes

Recent studies have demonstrated genetic differences between monozygotic (MZ) twins. To test the hypothesis that early post-twinning mutational events associate with phenotypic discordance, we investigated a cohort of 13 twin pairs (n = 26) discordant for various clinical phenotypes using whole-exome sequencing and screened for copy number variation (CNV). We identified a de novo variant in PLCB1, a gene involved in the hydrolysis of lipid phosphorus in milk from dairy cows, associated with lactase non-persistence, and a variant in the mitochondrial complex I gene MT-ND5 associated with amyotrophic lateral sclerosis (ALS). We also found somatic variants in multiple genes (TMEM225B, KBTBD3, TUBGCP4, TFIP11) in another MZ twin pair discordant for ALS. Based on the assumption that discordance between twins could be explained by a common variant with variable penetrance or expressivity, we screened the twin samples for known pathogenic variants that are shared and identified a rare deletion overlapping ARHGAP11B, in the twin pair manifesting with either schizotypal personality disorder or schizophrenia. Parent-offspring trio analysis was implemented for two twin pairs to assess potential association of variants of parental origin with susceptibility to disease. We identified a de novo variant in RASD2 shared by 8-year-old male twins with a suspected diagnosis of autism spectrum disorder (ASD) manifesting as different traits. A de novo CNV duplication was also identified in these twins overlapping CD38, a gene previously implicated in ASD. In twins discordant for Tourette's syndrome, a paternally inherited stop loss variant was detected in AADAC, a known candidate gene for the disorder

Identical twins carry a persistent epigenetic signature of early genome programming

The mechanisms underlying how monozygotic (or identical) twins arise are yet to be determined. Here, the authors investigate this in an epigenome-wide association study, showing that monozygotic twinning has a characteristic DNA methylation signature in adult somatic tissues.Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin.Molecular Epidemiolog

Low prevalence of male microchimerism in women with Mayer-Rokitansky-Küster-Hauser syndrome

STUDY QUESTION: Is there an increased prevalence of male microchimerism in women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, as evidence of fetal exposure to blood and anti-Müllerian hormone (AMH) from a (vanished) male co-twin resulting in regression of the Müllerian duct derivatives? SUMMARY ANSWER: Predominant absence of male microchimerism in adult women with MRKH syndrome does not support our hypothesis that intrauterine blood exchange with a (vanished) male co-twin is the pathophysiological mechanism. WHAT IS KNOWN ALREADY: The etiology of MRKH is unclear. Research on the phenotype analogous condition in cattle (freemartinism) has yielded the hypothesis that Müllerian duct development is inhibited by exposure to AMH in utero. In cattle, the male co-twin has been identified as the source for AMH, which is transferred via placental blood exchange. In human twins, a similar exchange of cellular material has been documented by detection of chimerism, but it is unknown whether this has clinical consequences. STUDY DESIGN, SIZE, DURATION: An observational case-control study was performed to compare the presence of male microchimerism in women with MRKH syndrome and control women. Through recruitment via the Dutch patients' association of women with MRKH (comprising 300 members who were informed by email or regular mail), we enrolled 96 patients between January 2017 and July 2017. The control group consisted of 100 women who reported never having been pregnant. PARTICIPANTS/MATERIALS, SETTING, METHODS: After written informed consent, peripheral blood samples were obtained by venipuncture, and genomic DNA was extracted. Male microchimerism was detected by Y-chromosome-specific real-time quantitative PCR, with use of DYS14 marker. Possible other sources for microchimerism, for example older brothers, were evaluated using questionnaire data. MAIN RESULTS AND THE ROLE OF CHANCE: The final analysis included 194 women: 95 women with MRKH syndrome with a mean age of 40.9 years and 99 control women with a mean age of 30.2 years. In total, 54 women (56.8%) were identified as having typical MRKH syndrome, and 41 women (43.2%) were identified as having atypical MRKH syndrome (when extra-genital malformations were present). The prevalence of male microchimerism was significantly higher in the control group than in the MRKH group (17.2% versus 5.3%, P = 0.009). After correcting for age, women in the control group were 5.8 times more likely to have male microchimerism (odds ratio 5.84 (CI 1.59-21.47), P = 0.008). The mean concentration of male microchimerism in the positive samples was 56.0 male genome equivalent per 1 000 000 cells. The prevalence of male microchimerism was similar in women with typical MRKH syndrome and atypical MRKH syndrome (5.6% versus 4.9%, P = 0.884). There were no differences between women with or without microchimerism in occurrence of alternative sources of XY cells, such as older brothers, previous blood transfusion, or history of sexual intercourse. LIMITATIONS, REASON FOR CAUTION: We are not able to draw definitive conclusions regarding the occurrence of AMH exchange during embryologic development in women with MRKH syndrome. Our subject population includes all adult women and therefore is reliant on long-term prevalence of microchimerism. Moreover, we have only tested blood, and, theoretically, the cells may have grafted anywhere in the body during development. It must also be considered that the exchange of AMH may occur without the transfusion of XY cells and therefore cannot be discovered by chimerism detection. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to test the theory that freemartinism causes the MRKH syndrome in humans. The study aimed to test the presence of male microchimerism in women with MRKH syndrome as a reflection of early fetal exposure to blood and AMH from a male (vanished) co-twin. We found that male microchimerism was only present in 5.3% of the women with MRKH syndrome, a significantly lower percentage than in the control group (17.2%). Our results do not provide evidence for an increased male microchimerism in adult women with MRKH as a product of intrauterine blood exchange. However, the significant difference in favor of the control group is of interest to the ongoing discussion on microchimeric cell transfer and the possible sources of XY cells. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: Dutch trial register, NTR5961

附記・奥付

The methylome is subject to genetic and environmental effects. Their impact may depend on sex and age, resulting in sex- and age-related physiological variation and disease susceptibility. Here we estimate the total heritability of DNA methylation levels in whole blood and estimate the variance explained by common single nucleotide polymorphisms at 411,169 sites in 2,603 individuals from twin families, to establish a catalogue of between-individual variation in DNA methylation. Heritability estimates vary across the genome (mean=19%) and interaction analyses reveal thousands of sites with sex-specific heritability as well as sites where the environmental variance increases with age. Integration with previously published data illustrates the impact of genome and environment across the lifespan at methylation sites associated with metabolic traits, smoking and ageing. These findings demonstrate that our catalogue holds valuable information on locations in the genome where methylation variation between people may reflect disease-relevant environmental exposures or genetic variation

A Molecular Survey of S. Mutans and P. Gingivalis Oral Microbial Burden in Human Saliva Using Relative Endpoint Polymerase Chain Reaction (RE-PCR) within the Population of a Nevada Dental School Revealed Disparities among Minorities

Background: The University of Nevada, Las Vegas School of Dental Medicine recently opened an orthodontic treatment clinic to address the needs of the racially and ethnically diverse population of Southern Nevada, primarily focusing on the treatment and care of low-income and minority patients. Although orthodontic treatment and therapy has been shown to induce changes in the oral cavity, much of this evidence was collected from traditional White, teenage orthodontic clinic populations. The primary goal of this study was to describe the microbial burden of the cariogenic and periodontal pathogens, Streptococcus mutans and Porphyromonas gingivalis within the UNLV-SDM patient population.Methods: Representative saliva samples were collected from healthy adult patients for DNA isolation. Relative endpoint polymerase chain reaction (RE-PCR) was performed to ascertain the presence and relative microbial burden of these oral pathogens.Results: Nearly one quarter (13/56) or 23.3% of these patients had elevated levels of S. mutans, while (10/56) and 17.8% of these samples were found to have elevated levels of P. gingivalis, - with (90%) of P. gingivalis-positive samples from minority patients (X-2 = 17.921, d.f. = 1; p \u3c 0.0001).Conclusions: These findings of elevated P. gingivalis levels, primarily among minority patients, may suggest underlying oral health practices contributing to adverse oral health conditions within this population. Oral health knowledge and practices among minority patients may be strongly influenced by other factors, including education and socioeconomic status, suggesting additional research may be needed to accurately determine the most appropriate standards for care and oral health education within this patient population