Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant

Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R510stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G660R) that, surprisingly, does not affect the Ca2+ permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G660R and R510stop mutants and combinations with wild type TRPV6. We show that both the G660R and R510stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases.

Strict adherence to Mendel's First Law across a large sample of human sperm genomes

Mendel's Law of Segregation states that the offspring of a diploid, heterozygous parent will inherit either allele with equal probability. While the vast majority of loci adhere to this rule, research in model and non-model organisms has uncovered numerous exceptions whereby "selfish" alleles are disproportionately transmitted to the next generation. Evidence of such "transmission distortion" (TD) in humans remains equivocal in part because scans of human pedigrees have been under-powered to detect small effects. Recently published single-cell sequencing data from individual human sperm (n = 41,189; 969-3,377 cells from each of 25 donors) offer an opportunity to revisit this question with unprecedented statistical power, but require new methods tailored to extremely low-coverage data (~0.01× per cell). To this end, we developed a method, named rhapsodi, that leverages sparse gamete genotype data to phase the diploid genomes of the donor individuals, impute missing gamete genotypes, and discover meiotic recombination breakpoints, benchmarking its performance across a wide range of study designs. After applying rhapsodi to the sperm sequencing data, we then scanned the gametes for evidence of TD. Our results exhibited close concordance with binomial expectations under balanced transmission, in contrast to tenuous signals of TD that were previously reported in pedigree-based studies. Together, our work excludes the existence of even weak TD in this sample, while offering a powerful quantitative framework for testing this and related hypotheses in other cohorts and study systems.

Phenotyping of Klf14 mouse white adipose tissue enabled by whole slide segmentation with deep neural networks

White adipose tissue (WAT) plays a central role in metabolism, and multiple diseases and genetic mutations cause its remodeling, most notably obesity, which has reached pandemic levels. WAT is present in subcutaneous (SAT) and visceral (VAT) depots, and its main components are white adipocytes. Quantitative analysis of white adipocyte size and counts is of great interest to understand physiology and disease, due to intra- and inter-depot heterogeneity, as well as better prognosis for hypertrophy than hyperplasia, and for SAT expansion than VAT expansion. H&E histology of whole depot cross-sections provides excellent approximation of cell morphology and preserves spatial information. Previous studies have been limited to window subsampling of whole slides, and cell size analysis. In this paper, we present a deep learning pipeline that can segment overlapping white adipocytes on whole slides and filter out other cells. We also propose a statistical framework based on linear models to study WAT phenotypes with three interconnected levels (body weight BW, depot weight DW and quartile cell area). Applying it to find Klf14 phenotypes in mice using 147 whole slides of WAT H&E histology, we show sexual dimorphism, and different effects between depots, heterozygous parent of origin for the KO allele and genotype (WT vs. Het). In particular, whether variables are correlated (DW vs. BW and cell area vs. DW), and statistical differences between fitted linear models. We also find significant differences between hand-traced or window subsampling datasets and whole slide analysis. Finally, we provide heatmaps of cell size for all the slides, showing substantial spatial heterogeneity and local spatial correlations.

Maternal Transmission Ratio Distortion in Two Iberian Pig Varieties

Transmission ratio distortion (TRD) is defined as the allele transmission deviation from the heterozygous parent to the offspring from the expected Mendelian genotypic frequencies. Although TRD can be a confounding factor in genetic mapping studies, this phenomenon remains mostly unknown in pigs, particularly in traditional breeds (i.e., the Iberian pig). We aimed to describe the maternal TRD prevalence and its genomic distribution in two Iberian varieties. Genotypes from a total of 247 families (dam and offspring) of Entrepelado (n = 129) and Retinto (n = 118) Iberian varieties were analyzed. The offspring were sired by both ungenotyped purebred Retinto and Entrepelado Iberian boars, regardless of the dam variety used. After quality control, 16,246 single-nucleotide polymorphisms (SNPs) in the Entrepelado variety and 9744 SNPs in the Retinto variety were analyzed. Maternal TRD was evaluated by a likelihood ratio test under SNP-by-SNP, adapting a previous model solved by Bayesian inference. Results provided 68 maternal TRD loci (TRDLs) in the Entrepelado variety and 24 in the Retinto variety (q < 0.05), with mostly negative TRD values, increasing the transmission of the minor allele. In addition, both varieties shared ten common TRDLs. No strong evidence of biological effects was found in genes with TRDLs. However, some biological processes could be affected by TRDLs, such as embryogenesis at different levels and lipid metabolism. These findings could provide useful insight into the genetic mechanisms to improve the swine industry, particularly in traditional breeds.

Efficiency of SSR markers for determining the origin of melon plantlets derived through unfertilized ovary culture

The effects of temperature pre-treatment, thidiazuron, naphthaleneacetic acid, and 6-benzylaminopurine on in vitro gynogenic plant production from un-pollinated melon (Cucumis melo L.) ovaries were investigated. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. The temperature pre-treatment (4&deg;C) for 4 days increased embryo formation frequency (63.3%) significantly. Addition of thidiazuron (0.04 and 0.02 mg/l) in the induction medium significantly increased the number of responding ovaries (46.6%, 65.83%), respectively. The maximum number of plantlet regeneration (22.5%) was achieved by culturing the ovary derived embryos on Murashigue and Skoog medium (MS medium) supplement with 0.6 mg/l 6-benzylaminopurine. Spontaneous doubled haploids originated directly through embryogenesis were subjected to genetic analysis using SSR molecular marker with 23 primers pair for homozygosity. SSR markers with microsatellite CMGA172, confirmed that the alleles in the parental material were also present in the gynogenic plantlets, but amplified only two alleles as compared to four alleles of the heterozygous parent material at same locus. Therefore these regenerated plantlets were consider homozygous and produced through a process of gametophytic embryogenesis.

The GABA A-Receptor γ2 (GABRG2) Gene in obsessive-compulsive disorder

OBJECTIVE: The γ-aminobutyric acid type A (GABA A) system may be implicated in obsessive-compulsive disorder, based on its major role in modulation of anxiety and its function as the principal inhibitory neurotransmitter system in the cortex. In addition, glutamatergic/GABAergic mechanisms appear to play a role in the pathophysiology of obsessive-compulsive disorder, making the GABA A receptor-γ2 (GABργ2) gene a good candidate for susceptibility in this disorder. METHOD: 118 probands meeting DSM-IV criteria for primary obsessive-compulsive disorder and their available parents were recruited for participation in this study and informed consent was obtained. An NciI restriction site polymorphism in the second intron was genotyped and data was analyzed using the Transmission Disequilibrium Test. RESULTS: In total, 61 of the participating families were informative (i.e., with at least one heterozygous parent). No biases were observed in the transmission of either of the two alleles (χ2 = 0.016, 1 d.f., p = 0.898) to the affected probands in the total sample. CONCLUSION/DISCUSSION: While these results do not provide support for a major role for the GABA A receptor-γ2 in obsessive-compulsive disorder, further investigations of this gene in larger samples are warranted.

A Study of GluK1 Kainate Receptor Polymorphisms in Down Syndrome Reveals Allelic Non-Disjunction at 1173(C/T)

Mechanisms underlying Down syndrome (DS)-related mental retardation (MR) remain poorly understood. In trisomic offspring, non-disjunction may result in the reduction to homozygosity of a susceptibility allele inherited from a heterozygous parent. Accordingly, we sought evidence for allelic non-disjunction in the GluK1 gene that encodes the critical kainite-binding glutamate receptor subunit-5, maps to chromosome 21q22.1 in the DS critical region and is expressed in brain regions responsible for learning and memory. Three polymorphisms of GluK1 [522(A/C) rs363538; 1173(C/T) rs363430 and 2705(T/C) rs363504] were genotyped in 86 DS patient families by means of PCR-coupled RFLP assays and evaluated with respect to allele frequency, heterozygosity, linkage disequilibrium, stage and parental origin of allelic non-disjunction. We report that the distribution of allele frequencies is in Hardy-Weinberg equilibrium. Moderate heterozygosity (0.339) and a major allele frequency of 0.78 render the 1173(C/T) marker informative. Pair-wise comparisons reveal that 522(A/C)-1173(C/T) [χ2= 31.2,df= 1,p= 0.0001;D’ = 0.42] and 1173(C/T)-2705(T/C) [χ2= 18.3,df= 1,p= 0.0001;D’ = 0.34] are in significant linkage disequilibrium of weak magnitude. The estimated ratio of meiosis-I to meiosis-II errors arising from allelic non-disjunction of 1173(C/T) is 4:1 in maternal cases and 2:1 in paternal cases. Studies including additional markers and patient samples are warranted to further substantiate present findings.

Power calculations for the transmission/disequilibrium and affected sib pair tests using elementary probability methods

The transmission/disequilibrium test (TDT) and the affected sib pair test (ASP) both test for the association of a marker allele with some conditions. Here, we present methods for calculating the probability of detecting the association (power) for a study examining a fixed number of families for suitability for the study and for calculating the number of such families to be examined. Both calculations use a genetic model for the association. The model considered posits a bi-allelic marker locus that is linked to a bi-allelic disease locus with a possibly nonzero recombination fraction between the loci. The penetrance of the disease is an increasing function of the number of disease alleles. The TDT tests whether the transmission by a heterozygous parent of a particular allele at a marker locus to an affected offspring occurs with probability greater than 0·5. The ASP tests whether transmission of the same allele to two affected sibs occurs with probability greater than 0·5. In either case, evidence that the probability is greater than 0·5 is evidence for association between the marker and the disease. Study inclusion criteria (IC) can greatly affect the necessary sample size of a TDT or ASP study. IC considered by us include a randomly selected parent at least one parent or both parents required to be heterozygous. It also allows a specified minimum number of affected offspring to be required (TDT only). We use elementary probability calculations rather than complex mathematical manipulations or asymptotic methods (large sample size approximations) to compute power and requisite sample size for a proposed study. The advantages of these methods are simplicity and generality.