Sphingosine 1-phosphate (S1P), a metabolic product of cell membrane sphingolipids, is bound to extracellular chaperones, is enriched in circulatory fluids, and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, postnatal organ function, and disease. S1PRs regulate essential processes such as adaptive immune cell trafficking, vascular development, and homeostasis. Moreover, S1PR signaling is a driver of multiple diseases. The past decade has witnessed an exponential growth in this field, in part because of multidisciplinary research focused on this lipid mediator and the application of S1PR-targeted drugs in clinical medicine. This has revealed fundamental principles of lysophospholipid mediator signaling that not only clarify the complex and wide ranging actions of S1P but also guide the development of therapeutics and translational directions in immunological, cardiovascular, neurological, inflammatory, and fibrotic diseases.

Sphingosine 1-phosphate: Lipid signaling in pathology and therapy

For many years, linkage analysis was the primary tool used for the genetic mapping of Mendelian and complex traits with familial aggregation. Linkage analysis was largely supplanted by the wide adoption of genome-wide association studies (GWASs). However, with the recent increased use of whole-genome sequencing (WGS), linkage analysis is again emerging as an important and powerful analysis method for the identification of genes involved in disease aetiology, often in conjunction with WGS filtering approaches. Here, we review the principles of linkage analysis and provide practical guidelines for carrying out linkage studies using WGS data.

Genetic linkage analysis in the age of whole-genome sequencing.

Genotyping-by-sequencing (GBS) is becoming an attractive alternative to array-based methods for genotyping individuals for a large number of single nucleotide polymorphisms (SNPs). Costs can be lowered by reducing the mean sequencing depth, but this results in genotype calls of lower quality. A common analysis strategy is to filter SNPs to just those with sufficient depth, thereby greatly reducing the number of SNPs available. We investigate methods for estimating relatedness using GBS data, including results of low depth, using theoretical calculation, simulation and application to a real data set. We show that unbiased estimates of relatedness can be obtained by using only those SNPs with genotype calls in both individuals. The expected value of this estimator is independent of the SNP depth in each individual, under a model of genotype calling that includes the special case of the two alleles being read at random. In contrast, the estimator of self-relatedness does depend on the SNP depth, and we provide a modification to provide unbiased estimates of self-relatedness. We refer to these methods of estimation as kinship using GBS with depth adjustment (KGD). The estimators can be calculated using matrix methods, which allow efficient computation. Simulation results were consistent with the methods being unbiased, and suggest that the optimal sequencing depth is around 2–4 for relatedness between individuals and 5–10 for self-relatedness. Application to a real data set revealed that some SNP filtering may still be necessary, for the exclusion of SNPs which did not behave in a Mendelian fashion. A simple graphical method (a ‘fin plot’) is given to illustrate this issue and to guide filtering parameters. We provide a method which gives unbiased estimates of relatedness, based on SNPs assayed by GBS, which accounts for the depth (including zero depth) of the genotype calls. This allows GBS to be applied at read depths which can be chosen to optimise the information obtained. SNPs with excess heterozygosity, often due to (partial) polyploidy or other duplications can be filtered based on a simple graphical method.

/pdf/construction-of-relatedness-matrices-using-genotyping-by-3bdba1z0on.pdf

Construction of relatedness matrices using genotyping-by-sequencing data

To interpret genetic variants discovered from next-generation sequencing, integration of heterogeneous information is vital for success. This article describes a framework named PERCH (Polymorphism Evaluation, Ranking, and Classification for a Heritable trait), available at http://BJFengLab.org/. It can prioritize disease genes by quantitatively unifying a new deleteriousness measure called BayesDel, an improved assessment of the biological relevance of genes to the disease, a modified linkage analysis, a novel rare-variant association test, and a converted variant call quality score. It supports data that contain various combinations of extended pedigrees, trios, and case-controls, and allows for a reduced penetrance, an elevated phenocopy rate, liability classes, and covariates. BayesDel is more accurate than PolyPhen2, SIFT, FATHMM, LRT, Mutation Taster, Mutation Assessor, PhyloP, GERP++, SiPhy, CADD, MetaLR, and MetaSVM. The overall approach is faster and more powerful than the existing quantitative method pVAAST, as shown by the simulations of challenging situations in finding the missing heritability of a complex disease. This framework can also classify variants of unknown significance (variants of uncertain significance) by quantitatively integrating allele frequencies, deleteriousness, association, and co-segregation. PERCH is a versatile tool for gene prioritization in gene discovery research and variant classification in clinical genetic testing.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/humu.23158

PERCH: A Unified Framework for Disease Gene Prioritization

Vertebrates are endowed with a closed circulatory system, the evolution of which required novel structural and regulatory changes. Furthermore, immune cell trafficking paradigms adapted to the barriers imposed by the closed circulatory system. How did such changes occur mechanistically? We propose that spatial compartmentalization of the lipid mediator sphingosine 1-phosphate (S1P) may be one such mechanism. In vertebrates, S1P is spatially compartmentalized in the blood and lymphatic circulation, thus comprising a sharp S1P gradient across the endothelial barrier. Circulatory S1P has critical roles in maturation and homeostasis of the vascular system as well as in immune cell trafficking. Physiological functions of S1P are tightly linked to shear stress, the key biophysical stimulus from blood flow. Thus, circulatory S1P confinement could be a primordial strategy of vertebrates in the development of a closed circulatory system. This review discusses the cellular and molecular basis of the S1P gradients and aims to interpret its physiological significance as a key feature of the closed circulatory system.

/pdf/vascular-and-immunobiology-of-the-circulatory-sphingosine-1-gauik9o2zr.pdf

Vascular and Immunobiology of the Circulatory Sphingosine 1-Phosphate Gradient.

The sphingosine-1-phosphate receptors (S1PRs) are a well-studied class of transmembrane G protein-coupled sphingolipid receptors that mediate multiple cellular processes. However, S1PRs have not been previously reported to be involved in the genetic etiology of human traits. S1PR2 lies within the autosomal-recessive nonsyndromic hearing impairment (ARNSHI) locus DFNB68 on 19p13.2. From exome sequence data we identified two pathogenic S1PR2 variants, c.323G>C (p.Arg108Pro) and c.419A>G (p.Tyr140Cys). Each of these variants co-segregates with congenital profound hearing impairment in consanguineous Pakistani families with maximum LOD scores of 6.4 for family DEM4154 and 3.3 for family PKDF1400. Neither S1PR2 missense variant was reported among ∼120,000 chromosomes in the Exome Aggregation Consortium database, in 76 unrelated Pakistani exomes, or in 720 Pakistani control chromosomes. Both DNA variants affect highly conserved residues of S1PR2 and are predicted to be damaging by multiple bioinformatics tools. Molecular modeling predicts that these variants affect binding of sphingosine-1-phosphate (p.Arg108Pro) and G protein docking (p.Tyr140Cys). In the previously reported S1pr2−/− mice, stria vascularis abnormalities, organ of Corti degeneration, and profound hearing loss were observed. Additionally, hair cell defects were seen in both knockout mice and morphant zebrafish. Family PKDF1400 presents with ARNSHI, which is consistent with the lack of gross malformations in S1pr2−/− mice, whereas family DEM4154 has lower limb malformations in addition to hearing loss. Our findings suggest the possibility of developing therapies against hair cell damage (e.g., from ototoxic drugs) through targeted stimulation of S1PR2.

Autosomal-Recessive Hearing Impairment Due to Rare Missense Variants within S1PR2

Recent advances in next-generation sequencing (NGS) make it possible to directly sequence genomes and exomes of individuals with Mendelian diseases and screen sequence data for causal variants. With the reduction in cost of NGS, DNA samples from entire families can be sequenced and linkage analysis can be performed directly using NGS data. Inspired by 'burden' tests, which are used for complex trait rare variant association studies, we developed the collapsed haplotype pattern (CHP) method for linkage analysis. Using data from several deafness genes we demonstrate that the CHP method is substantially more powerful than analyzing individual variants. Unlike applying NGS data filtering approaches, the CHP method provides statistical evidence of a gene's involvement in disease etiology and is also less likely to exclude causal variants in the presence of phenocopies and/or reduced penetrance. The CHP method was implemented in the SEQLinkage software package, which can perform linkage analysis on NGS data or can generate data compatible with many linkage analysis programs, reviving them for use in NGS era.

/pdf/collapsed-haplotype-pattern-method-for-linkage-analysis-of-4c97oqnigf.pdf

Collapsed haplotype pattern method for linkage analysis of next-generation sequence data

Background Woolly hair (WH) is a hair abnormality that is primarily characterised by tightly curled hair with abnormal growth

Methods In two unrelated consanguineous Pakistani families with non-syndromic autosomal recessive (AR) WH, homozygosity mapping and linkage analysis identified a locus within 17q211–q22, which contains the type I keratin gene cluster A DNA sample from an affected individual from each family underwent exome sequencing

Results A homozygous missense variant c950T>C (p(Leu317Pro)) within KRT25 segregated with ARWH in both families, and has a combined maximum two-point LOD score of 79 at ϴ=0 The KRT25 variant is predicted to result in disruption of the second α-helical rod domain and the entire protein structure, thus possibly interfering with heterodimerisation of K25 with type II keratins within the inner root sheath (IRS) of the hair follicle and the medulla of the hair shaft

Conclusions Our findings implicate a novel gene involved in human hair abnormality, and are consistent with the curled, fragile hair found in mice with Krt25 mutations, and further support the role of IRS-specific type I keratins in hair follicle development and maintenance of hair texture

https://jmg.bmj.com/content/jmedgenet/early/2015/07/09/jmedgenet-2015-103255.full.pdf

Hang Dai

Papers

Autosomal-Recessive Hearing Impairment Due to Rare Missense Variants within S1PR2

Collapsed haplotype pattern method for linkage analysis of next-generation sequence data

A homozygous missense variant in type I keratin KRT25 causes autosomal recessive woolly hair