An exploration of linkage fine-mapping on sequences from case-control studies

Linkage analysis maps genetic loci for a heritable trait by identifying genomic regions with excess relatedness among individuals with similar trait values. Analysis may be conducted on related individuals from families, or on samples of unrelated individuals from a population. For allelically heterogeneous traits, population-based linkage analysis can be more powerful than genotypic-association analysis. Here, we focus on linkage analysis in a population sample, but use sequences rather than individuals as our unit of observation. Earlier investigations of sequence-based linkage mapping relied on known sequence relatedness, whereas we infer relatedness from the sequence data. We propose two ways to associate similarity in relatedness of sequences with similarity in their trait values and compare the resulting linkage methods to two genotypic- association methods. We also introduce a procedure to label case sequences as potential carriers or non-carriers of causal variants after an association has been found. This post-hoc labeling of case sequences is based on inferred relatedness to other case sequences. Our simulation results indicate that methods based on sequence-relatedness improve localization and perform as well as genotypic-association methods for detecting rare causal variants. Sequence-based linkage analysis therefore has potential to fine-map allelically heterogeneous disease traits.

Interferon inducible pseudouridine modification in human mRNA by quantitative nanopore profiling

Pseudouridine (Ψ) is an abundant mRNA modification in mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome-wide mapping. We develop a nanopore native RNA sequencing method for quantitative Ψ prediction (NanoPsu) that utilizes native content training, machine learning modeling, and single-read linkage analysis. Biologically, we find interferon inducible Ψ modifications in interferon-stimulated gene transcripts which are consistent with a role of Ψ in enabling efficacy of mRNA vaccines.

Expanding the phenotypic spectrum of mutations in LRP2: a novel candidate gene of non-syndromic familial comitant strabismus

Background Comitant strabismus (CS) is a heterogeneous disorder that is a major contributing factor to unilateral childhood-onset visual impairment. Studies have confirmed that genetic factors play an important role in the development of CS. The aim of this study was to identify the genetic cause of non-syndromic familial CS. Methods Fourteen unrelated CS families were recruited for the study. Twelve affected and 2 unaffected individuals from a large four-generation family (CS08) were selected to perform whole genome-wide linkage analysis. Parallel whole-exome sequencing (WES) was conducted in the same family (9 patients and 1 unaffected member) and 31 additional CS cases from 13 other unrelated families. Sanger sequencing was used to determine whether any of the remaining variants co-segregated with the disease phenotype in the corresponding family. Results Based on linkage analysis, CS in family CS08 mapped to a novel region of 34.17 centimorgan (cM) on chromosome 2q22.3-2q32.1 between markers D2S151 and D2S364, with a maximum log odds (LOD) score of 3.54 (theta = 0) at D2S142. Parallel WES identified a heterozygous variant, LRP2 c.335 A > G (p.Q112R), located in such a linkage interval that completely co-segregated with the disease in the family. Furthermore, another novel heterozygous variant (c.7274A > G, p.D2425G) in LRP2 that co-segregated was detected in 2 additional affected individuals from another unrelated family by WES. Both variants are predicted to be damaging by PolyPhen-2, SIFT and MutationTaster, and were absent in 100 ethnically matched normal controls. Conclusion LRP2 is a novel candidate genetic cause of non-syndromic familial CS.

Identification of a Novel KRT9 Frameshift Mutation in a Chinese Pedigree with Epidermolytic Palmoplantar Keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis caused by variants of keratin 9 (KRT9) or KRT1 gene. In this study causative gene mapping in a Chinese EPPK family was performed with Two-point linkage analysis and haplotyping. Positive linkage results were obtained on 17q (Zmax=2.06, θmax=0.0) at D17S799, which indicated KRT9 to be the most responsible gene for the family. Subsequently, direct sequencing identified a novel frameshift mutation caused by a 5bp deletion (∆GGAGG) in KRT9 in all affected individuals but not in the unaffected members or the 50 unrelated controls. The frameshift changed the encoding of the following nine amino acids and resulted in a readthrough translation in exon 7. The data revealed that the novel frameshift mutation in KRT9 was responsible for the Chinese EPPK pedigree. The researchers’ findings broaden the spectrum of KRT9 variants and provide further evidence for the highly genetic heterogeneity of EPPK.

New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements

BackgroundAuriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (PLCB4, GNAI3 and EDN1) and some cases remain unsolved. Here we studied a large unsolved four-generation family.MethodsWe performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells.ResultsThis study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includes HDAC9, which is located over 200 kb telomeric to the top candidate gene TWIST1. Notably, Capture-C analysis revealed multiple cis interactions between the TWIST1 promoter and possible regulatory elements within the duplicated region. Modelling of the disease revealed an increased expression of HDAC9 and its neighbouring gene, TWIST1, in neural crest cells. We also identified decreased migration of iPSC-derived neural crest cells together with dysregulation of osteogenic differentiation in iPSC-affected mesenchymal stem cells.ConclusionOur findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of TWIST1 expression during craniofacial development can contribute to the phenotype.

LAMA2 and LOXL4 are candidate FSGS genes

Background Focal and segmental glomerulosclerosis (FSGS) is a histologic pattern of injury that characterizes a wide spectrum of diseases. Many genetic causes have been identified in FSGS but even in families with comprehensive testing, a significant proportion remain unexplained. Methods In a family with adult-onset autosomal dominant FSGS, linkage analysis was performed in 11 family members followed by whole exome sequencing (WES) in 3 affected relatives to identify candidate genes. Results Pathogenic variants in known nephropathy genes were excluded. Subsequently, linkage analysis was performed and narrowed the disease gene(s) to within 3% of the genome. WES identified 5 heterozygous rare variants, which were sequenced in 11 relatives where DNA was available. Two of these variants, in LAMA2 and LOXL4, remained as candidates after segregation analysis and encode extracellular matrix proteins of the glomerulus. Renal biopsies showed classic segmental sclerosis/hyalinosis lesion on a background of mild mesangial hypercellularity. Examination of basement membranes with electron microscopy showed regions of dense mesangial matrix in one individual and wider glomerular basement membrane (GBM) thickness in two individuals compared to historic control averages. Conclusions Based on our findings, we postulate that the additive effect of digenic inheritance of heterozygous variants in LAMA2 and LOXL4 leads to adult-onset FSGS. Limitations to our study includes the absence of functional characterization to support pathogenicity. Alternatively, identification of additional FSGS cases with suspected deleterious variants in LAMA2 and LOXL4 will provide more evidence for disease causality. Thus, our report will be of benefit to the renal community as sequencing in renal disease becomes more widespread.

QTL Mapping And Candidate Gene Mining of Flag Leaf Size Traits In Japonica Rice Based On Linkage Mapping And Genome-Wide Association Study

As one of the most important part of the ideal plant type of japonica rice, leaf shape affects the photosynthesis and carbohydrate accumulation directly. Mining and using new leaf shape related genes/QTLs can further enrich the theory of molecular breeding and accelerate the breeding process of japonica rice. In the present study, 2 RILs and a natural population with 295 japonica rice varieties were used to map QTLs for flag leaf length (FL), flag leaf width (FW) and flag leaf area (FLA) by linkage analysis and genome-wide association study (GWAS) through 2 years. A total of 64 QTLs were detected by 2 ways, and pleiotropic QTLs qFL2 (Chr2_33,332,579) and qFL10 (Chr10_10,107,835; Chr10_10,230,100) consisted of overlapping QTLs mapped by linkage analysis and GWAS through 2 years were identified. The candidate genes LOC_Os02g54254, LOC_Os02g54550, LOC_Os10g20160, LOC_Os10g20240, LOC_Os10g20260 were obtained, filtered by linkage disequilibrium (LD), and haplotype analysis. LOC_Os10g20160 (SD-RLK-45) showed outstanding characteristics in quantitative real-time PCR (qRT-PCR) analysis in leaf development period, belongs to S-domain receptor-like protein kinases gene and probably to be a main gene regulating flag leaf width of japonica rice. The results of this study provide valuable resources for mining the main genes/QTLs of japonica rice leaf development and molecular breeding of japonica rice ideal leaf shape.