Genetic control of serum 25(OH)D levels and its association with ethnicity.

BACKGROUND: Identified DNA variants associated with serum 25-hydroxyvitamin vitamin D (25[OH]D) concentration may provide mechanistic insights into the vitamin D metabolic pathway in individuals. Our aim was to further characterise participants and their serum 25(OH)D concentration at baseline using candidate single nucleotide polymorphism (SNP) genotyping. METHODS: 5110 participants, aged 50-84 years, were recruited from the community. Blood samples were collected at baseline to measure serum 25(OH)D by liquid chromatography mass spectrometry and the participants were genotyped for four markers close to or within genes in the vitamin D metabolic pathway known to be associated with differences in 25(OH)D. The markers and their associated genes were rs12785878 (DHCR7), rs10741657 (CYP2R1), rs4588 (DBP) and rs2228570 (VDR). RESULTS: All four markers had significantly different genotype distributions and minor allele frequencies between the four self-determined ethnicities (European/Other, Maori, Pacific, and South Asian). For example, the frequency in each ethnic group of the G allele for the marker rs12785878 was 0.26, 0.71, 0.89, and 0.78 respectively. Using multivariable regression in the full cohort, three out of four markers were significantly associated with baseline concentrations of 25(OH)D (mean differences: 2.9-10.9 nmol/L). Collectively, the four markers explained 8.4% of the variation in 25(OH)D concentrations. CONCLUSION: Significant ethnic variations exist in the distribution of alleles associated with serum 25(OH)D concentration, particularly rs12785878, in a multi-ethnic community sample from New Zealand.

Proof of concept for multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer.

Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×-19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.

A Multi-Omic Huntington's Disease Transgenic Sheep-Model Database for Investigating Disease Pathogenesis.

BACKGROUND: The pathological mechanism of cellular dysfunction and death in Huntington's disease (HD) is not well defined. Our transgenic HD sheep model (OVT73) was generated to investigate these mechanisms and for therapeutic testing. One particular cohort of animals has undergone focused investigation resulting in a large interrelated multi-omic dataset, with statistically significant changes observed comparing OVT73 and control 'omic' profiles and reported in literature. OBJECTIVE: Here we make this dataset publicly available for the advancement of HD pathogenic mechanism discovery. METHODS: To enable investigation in a user-friendly format, we integrated seven multi-omic datasets from a cohort of 5-year-old OVT73 (n = 6) and control (n = 6) sheep into a single database utilising the programming language R. It includes high-throughput transcriptomic, metabolomic and proteomic data from blood, brain, and other tissues. RESULTS: We present the 'multi-omic' HD sheep database as a queryable web-based platform that can be used by the wider HD research community (https://hdsheep.cer.auckland.ac.nz/). The database is supported with a suite of simple automated statistical analysis functions for rapid exploratory analyses. We present examples of its use that validates the integrity relative to results previously reported. The data may also be downloaded for user determined analysis. CONCLUSION: We propose the use of this online database as a hypothesis generator and method to confirm/refute findings made from patient samples and alternate model systems, to expand our understanding of HD pathogenesis. Importantly, additional tissue samples are available for further investigation of this cohort.

Castration delays epigenetic aging and feminizes DNA methylation at androgen-regulated loci.

In mammals, females generally live longer than males. Nevertheless, the mechanisms underpinning sex-dependent longevity are currently unclear. Epigenetic clocks are powerful biological biomarkers capable of precisely estimating chronological age and identifying novel factors influencing the aging rate using only DNA methylation data. In this study, we developed the first epigenetic clock for domesticated sheep (Ovis aries), which can predict chronological age with a median absolute error of 5.1 months. We have discovered that castrated male sheep have a decelerated aging rate compared to intact males, mediated at least in part by the removal of androgens. Furthermore, we identified several androgen-sensitive CpG dinucleotides that become progressively hypomethylated with age in intact males, but remain stable in castrated males and females. Comparable sex-specific methylation differences in MKLN1 also exist in bat skin and a range of mouse tissues that have high androgen receptor expression, indicating that it may drive androgen-dependent hypomethylation in divergent mammalian species. In characterizing these sites, we identify biologically plausible mechanisms explaining how androgens drive male-accelerated aging.

Rotation sensitivity and shot-noise-limited detection in an exceptional-point coupled-ring gyroscope.

A theoretical study is performed of the sensitivity and quantum-noise limit of a passive coupled-ring optical gyroscope operated at and detuned from its exceptional point (EP) and interrogated with a practical conventional readout system. When tuned to its EP, the Sagnac frequency splitting is proportional to the square root of the applied rotation rate, but the signal generated by the sensor is shown to be proportional to the applied rotation rate. The sensitivity is never larger, and the minimum detectable rotation rate in the quantum-noise limit never lower, than that of a standard single-ring gyro of the same radius and loss, even when the coupled-ring gyro is tuned exactly to its EP. As pointed out elsewhere for other EP sensors, in this particular passive sensor at least, there is no sensitivity or resolution benefit in operating at an EP.

Enhanced rotation sensing and exceptional points in a parity-time-symmetric coupled-ring gyroscope.

Enhancement in rotation sensitivity is achieved in a parity-time-symmetric gyroscope consisting of a ring with gain coupled to a lossy ring, operated below laser threshold and in the vicinity of its exceptional point (EP). An external laser and a conventional readout system are used to measure the large rotation-induced shifts in resonance frequency known to occur in this device. A complete model of the rotation sensitivity is derived that accounts for gain saturation caused by the large circulating power. Compared to a single-ring gyro, the sensitivity is enhanced by a factor of ∼300 when the inter-ring coupling is tuned to its EP value κEP, and ∼2400 when it is decreased from κEP, even though the Sagnac frequency shift is then much smaller. ∼40% of this 2400-fold enhancement is assigned to a new sensing mechanism where rotation alters the gain saturation. These results show that this compact gyro has a far greater sensitivity than a conventional ring gyro, and that this improvement arises mostly from the gain compensating the loss, as opposed to the enhanced Sagnac frequency shift from the EP. This gyro is also shown to be much more stable against gain fluctuations than a single-ring gyro with gain.

DNA methylation study of Huntington's disease and motor progression in patients and in animal models.

Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.