Nonparametric Linkage Analysis Research Articles

FBLN2 is associated with Goldenhar syndrome and is essential for cranial neural crest cell development.

Goldenhar syndrome, a rare craniofacial malformation, is characterized by developmental anomalies in the first and second pharyngeal arches. Its etiology is considered to be heterogenous, including both genetic and environmental factors that remain largely unknown. To further elucidate the genetic cause in a five-generation Goldenhar syndrome pedigree and exploit the whole-exome sequencing (WES) data of this pedigree, we generated collapsed haplotype pattern markers based on WES and employed rare variant nonparametric linkage analysis. FBLN2 was identified as a candidate gene via analysis of WES data across the significant linkage region. A fbln2 knockout zebrafish line was established by CRISPR/Cas9 to examine the gene's role in craniofacial cartilage development. fbln2 was expressed specifically in the mandible during the zebrafish early development, while fbln2 knockout zebrafish exhibited craniofacial malformations with abnormal chondrocyte morphologies. Functional studies revealed that fbln2 knockout caused abnormal chondrogenic differentiation, apoptosis, and proliferation of cranial neural crest cells (CNCCs), and downregulated the bone morphogenic protein (BMP) signaling pathway in the zebrafish model. This study demonstrates the role of FBLN2 in CNCC development and BMP pathway regulation, and highlights FBLN2 as a candidate gene for Goldenhar syndrome, which may have implications for the selection of potential screening targets and the development of treatments for conditions like microtia-atresia.

Whole genome sequencing identifies candidate genes for familial essential tremor and reveals biological pathways implicated in essential tremor aetiology.

SummaryBackgroundEssential tremor (ET), one of the most common neurological disorders, has a phenotypically heterogeneous presentation characterized by bilateral kinetic tremor of the arms and, in some patients, tremor involving other body regions (e.g., head, voice). Genetic studies suggest that ET is genetically heterogeneous.MethodsWe analyzed whole genome sequence data (WGS) generated on 104 multi-generational white families with European ancestry affected by ET. Genome-wide parametric linkage and association scans were analyzed using adjusted logistic regression models through the application of the Pseudomarker software. To investigate the additional contribution of rare variants in familial ET, we also performed an aggregate variant non-parametric linkage (NPL) analysis using the collapsed haplotype method implemented in CHP-NPL software.FindingsParametric linkage analysis of common variants identified several loci with significant evidence of linkage (HLOD ≥3.6). Among the gene regions within the strongest ET linkage peaks were BTC (4q13.3, HLOD=4.53), N6AMT1 (21q21.3, HLOD=4.31), PCDH9 (13q21.32, HLOD=4.21), EYA1 (8q13.3, HLOD=4.04), RBFOX1 (16p13.3, HLOD=4.02), MAPT (17q21.31, HLOD=3.99) and SCARB2 (4q21.1, HLOD=3.65). CHP-NPL analysis identified fifteen additional genes with evidence of significant linkage (LOD ≥3.8). These genes include TUBB2A, VPS33B, STEAP1B, SPINK5, ZRANB1, TBC1D3C, PDPR, NPY4R, ETS2, ZNF736, SPATA21, ARL17A, PZP, BLK and CCDC94. In one ET family contributing to the linkage peak on chromosome 16p13.3, we identified a likely pathogenic heterozygous canonical splice acceptor variant in exon 2 of RBFOX1 (ENST00000547372; c.4-2A>G), that co-segregated with the ET phenotype in the family.InterpretationLinkage and association analyses of WGS identified several novel ET candidate genes, which are implicated in four major pathways that include 1) the epidermal growth factor receptor-phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha-AKT serine/threonine kinase 1 (EGFR-PI3K-AKT) and Mitogen-activated protein Kinase 1 (ERK) pathways, 2) Reactive oxygen species (ROS) and DNA repair, 3) gamma-aminobutyric acid-ergic (GABAergic) system and 4) RNA binding and regulation of RNA processes. Our study provides evidence for a possible overlap in the genetic architecture of ET, neurological disease, cancer and aging. The genes and pathways identified can be prioritized in future genetic and functional studies.FundingNational Institutes of Health, NINDS, NS073872 (USA) and NIA AG058131(USA).

Linkage analysis identifies novel loci in early-onset Alzheimer disease in non-Hispanic white families.

Early-onset Alzheimer disease (EOAD) is a highly genetic dementia typically defined as Alzheimer disease dementia with an age of onset of 65 years or younger. While known mutations in genes such as APP, PSEN1, and PSEN2 account for a small portion of EOAD, approximately 90% of all EOAD cases lack these known mutations. Since little is known about causes and course of EOAD, more research must be completed to identify the factors that contribute EOAD risk. To that end, we present here a genetic linkage analysis in multiplex EOAD families to identify loci likely to impact EOAD risk. Linkage analysis was performed on 21 non-Hispanic white families (197 family members) with two or more individuals with EOAD (AAO ≤ 65 years). Genome-wide array data were used to type single-nucleotide polymorphisms. Merlin was used to perform parametric linkage analysis, non-parametric linkage analysis, and two-point parametric affected only analysis. The two-point parametric analysis identified 5 EOAD linkage regions with a HLOD score of 3.5 or greater (5q31.1[HLOD=5.18], 6p25.2 [HLOD=5.71], 9q31.1 [HLOD=3.65], 11q23.3 [HLOD=9.11], 15q24.3 [HLOD=5.86]). Regions 5q31.1, 6p25.2, 11q23.3, 15q24.3 generated HLOD scores greater than 4.0. Several Alzheimer disease (AD) associated genes were identified near these regions including PPT2, AGER, BACE1. Using a suggestive linkage threshold of LOD ≥ 2.20, P = 7.4×10-4 , the parametric multi-point analysis identified one linkage region at 4.p16.3 (LOD=2.35). The 1-LOD unit interval for this region identified PDE5A, which has potential as a therapeutic target in several neurological diseases. Our non-parametric linkage analysis identified 3 linkage regions with a LOD score equal to or exceeding the suggestive threshold of 2.20 (3p21.1 [LOD=2.21], 3p14.3 [LOD=2.28], 3p14.1[LOD=2.22]). The APPL1 gene, which has been linked to APP-induced dysfunction, was located within the 1-LOD unit interval of these regions. Several novel linkage EOAD regions have been identified that where not previously identified in known LOAD regions. The identification of these regions may provide information to establish EOAD as a separate disease from Late-onset Alzheimer disease as well as provide therapeutic targets for AD.

Germline RBBP6 Mutations In Myeloproliferative Neoplasms

The pathogenesis of myeloproliferative neoplasms (MPN) is determined by the acquisition of somatic mutations in several genes, such as JAK2, MPL, and TET2. Familial clustering of MPN is observed in 5-10% of cases following predominantly autosomal dominant inheritance with incomplete penetrance. Familial MPN patients carry somatically acquired mutations in JAK2, MPL and other genes similar to sporadic cases. It is thought that the germline mutations in familial cases of MPN predispose to acquisition of somatic mutations which in turn drive the disease. No gene has been implicated in familial MPN predisposition so far. The JAK2haplotype predisposing to MPN at population level does not explain familial MPN cases.We studied a family with five MPN cases in four generations displaying an autosomal dominant pattern of inheritance with reduced penetrance. DNA was available from three MPN cases in the family. One of the patients carried a JAK2-V617F mutation and a deletion on chromosome 22q, while an MPL-W515L mutation was detected in another affected member of the family. The third patient did not have JAK2 or MPLmutations, but carried three chromosomal aberrations on chromosomes 2p, 7q and 15q. Chromosomal aberrations were detected using Affymetrix SNP 6.0 microarrays. The genotypic data from SNP 6.0 arrays was used to perform high-resolution nonparametric linkage analysis. In order to identify the germline mutation predisposing to MPN in the family we performed whole exome sequencing of the three affected members using Illumina next-generation sequencing technology. The mutations identified from exome sequencing were combined with the linkage analysis data.We identified a germline mutation in the RBBP6 gene that segregates with the MPN phenotype in the studied pedigree. Further screening in familial and sporadic MPN cases identified several other germline RBBP6mutations in 5% of the familial and 0.6% of the sporadic MPN cases. Familial MPN cases mostly had the diagnosis of primary myelofibrosis. All the detected mutations cluster in putative p53-binding region of the protein, suggesting a possible link of the mutations and p53 function.The functional effect of the mutations was assessed in a doxycycline-inducible HEK-flp-in cell line system. Upon doxycyline induction, the wild-type and mutant RBBP6 constructs were over-expressed and the consequent functional effect of mutation was assessed using RNA sequencing and global proteomic analyses. The combination of the two datasets yielded 109 genes/proteins that were significantly changed in both the RNA sequencing and the global proteomic results when comparing wild-type to mutant RBBP6, 87 of which were downregulated by the mutation, while 22 were upregulated. Several important cell cycle and apoptosis regulators were found among the deregulated genes, including TP53, BAX, BID and USP7 (downregulated) and CDK6 (upregulated). KEGG pathway analysis of the downregulated genes showed significant enrichment for the p53 and the apoptosis pathways.We have identified germline mutations in RBBP6 that predispose to the development of MPN and are enriched in the families. Functional studies have shown that the mutations likely affect the p53 pathway and apoptosis and thus increase the risk of acquiring somatic mutations that lead to MPN. Our study implicates RBBP6 in MPN susceptibility and suggests that RBBP6 is an important cancer-associated gene. Disclosures:No relevant conflicts of interest to declare.

'Essential Tremor' Phenotype in FMR1 Premutation/Gray Zone Sibling Series: Exploring Possible Genetic Modifiers.

Fragile X-associated tremor/ataxia syndrome (FXTAS) occurs in carriers of fragile X mental retardation 1 (FMR1) X-linked small CGG expansion (gray zone [GZ] and premutation [PM]) alleles, containing 41-200 repeats. Major features comprise kinetic tremor, gait ataxia, cognitive decline and cerebellar peduncular white matter lesions, but atypical/incomplete FXTAS may occur. We explored the possibility of polygenic effects modifying the FXTAS spectrum phenotypes. We used three motor scales and selected cognitive tests in a series of three males and three females from a single sibship carrying PM or GZ alleles (44 to 75 repeats). The molecular profiles from these siblings were determined by genomewide association study with single-nucleotide polymorphism (SNP) genotyping by Illumina Global Screening Array. Nonparametric linkage analysis was applied and Parkinson's disease (PD) polygenic risk scores (PRSs) were calculated for all the siblings, based on 107 known risk variants. All male and female siblings manifested similar kinetic tremor phenotypes. In contrast to FXTAS, they showed negligible gait ataxia, and few white matter lesions on MRI. Cognitive functioning was unaffected. Suggestive evidence of linkage to a broad region of the short arm of chromosome 10 was obtained, and median PD PRS for the sibship fell within the top 30% of a sample of over 500,000 UK and Australian controls. The genomewide study results are suggestive of modifying effects of genetic risk loci linked to PD, on the neurological phenotype of FMR1-CGG small expansion carriers, resulting in an oligosymptomatic kinetic tremor seen in FXTAS spectrum, but also consistent with essential tremor.

Obelisc: an identical-by-descent mapping tool based on SNP streak.

MotivationGenetic linkage analysis has made a huge contribution to the genetic mapping of Mendelian diseases. However, most previously available linkage analysis methods have limited applicability. Since parametric linkage analysis requires predefined model of inheritance with a fixed set of parameters, it is inapplicable without fully structured pedigree information. Furthermore, the analytical results are dependent on the specification of model parameters. While non-parametric linkage analysis can avoid these problems, the runs of homozygosity (ROH) mapping, a widely used non-parametric linkage analysis method, can only deal with recessive inheritance. The implementation of non-parametric linkage analyses capable of dealing with both dominant and recessive inheritance has been required.ResultsWe have developed the Obelisc (Observational linkage scan), a flexibly applicable user-friendly non-parametric linkage analysis tool, which also provides an intuitive visualization of the analytical results. Obelisc is based on the SNP streak approach, which does not require any predefined inheritance model with parameters. In contrast to the ROH mapping, the SNP streak approach is applicable to both dominant and recessive traits. To illustrate the performance of Obelisc, we generated a pseudo-pedigree from the publicly available BioBank Japan Project genome-wide genotype dataset (n > 180 000). By applying Obelisc to this pseudo-pedigree, we successfully identified the regions with inherited identical-by-descent haplotypes shared among the members of the pseudo-pedigree, which was validated by the population-based haplotype phasing approach.Availability and implementationObelisc is feely available at https://github.com/qsonehara/Obelisc as a python package with example datasets.Supplementary information Supplementary data are available at Bioinformatics online.

Genome-Wide Linkage Analysis of the Risk of Contracting a Bloodstream Infection in 47 Pedigrees Followed for 23 Years Assembled From a Population-Based Cohort (the HUNT Study).

Bloodstream infection is an important cause of death worldwide. The main objective of this study was to identify genetic loci linked to risk of contracting a bloodstream infection. Genome-wide linkage analysis. Population-based, Norwegian cohort, followed between 1995 and 2017. Among 69,423 genotyped subjects, there were 47 families with two or more second-degree relatives with bloodstream infection in the follow-up period. There were 365 subjects in these families, of which 110 were affected. None. The cohort was genotyped using Illumina HumanCoreExome (Illumina, San Diego, CA) arrays. Before linkage analysis, single-nucleotide polymorphisms were pruned and clumped. In nonparametric linkage analysis using an exponential model, we found three loci with a suggestive linkage to bloodstream infection, all on chromosome 4, at 46.6 centimorgan (logarithm of odds, 2.3), 57.7 centimorgan (logarithm of odds, 3.2), and 70.0 centimorgan (logarithm of odds, 2.1). At the peak of the lead region are three genes: TLR10, TLR1, and TLR6. Variations in the TLR10/1/6 locus appear to be linked with the risk of contracting a bloodstream infection.

Mapping of developmental dysplasia of the hip to two novel regions at 8q23-q24 and 12p12.

Developmental dysplasia of the hip (DDH), previously known as congenital hip dislocation, is a frequently disabling condition characterized by premature arthritis later in life. Genetic factors play a key role in the aetiology of DDH. In the present study, a genome-wide linkage scan with the Affymetrix 10K GeneChip was performed on a four-generation Chinese family, which included 19 healthy members and 5 patients. Parametric and non-parametric multipoint linkage analyses were carried out with Genespring GT v.2.0 software, and the logarithm of odds (LOD) score and nonparametric linkage (NPL) score were calculated. Parametric linkage analysis was performed, assuming an autosomal recessive trait with full penetrance and Affymetrix 'Asian' allele frequencies. The strongest evidence for linkage was found on chromosome 8q23-24, with a peak LOD score of 2.658 (θ=0), covering 2.377 Mb from single nucleotide polymorphisms (SNPs) rs724717 to rs720132. This interval included nine additional successive SNPs: rs1566071, rs1902121, rs756404, rs702768, rs777813, rs2033995, rs147959, rs2884367 and rs1898287. The same region also yielded the highest NPL score of 2.883 (P=0.0156) from the non-parametric multipoint linkage analysis. Additionally, the second highest NPL score of 2.727 (P=0.0156) and LOD score of 2.528 (θ=0) were obtained on chromosome 12p12 for three consecutive markers (rs1919980, rs763853 and rs725124). This region overlapped a narrow distance of 0.642 Mb. Notably, in addition to these two regions; no significant linkage was identified for other chromosomal regions (with LOD and NPL scores >2.0). For the first time, at least for this pedigree, the evidence in the present study showed that DDH is mapped to two novel regions at 8q23-q24 and 12p12.

Evidence for gene-smoking interactions for hearing loss and deafness in Japanese American families

BackgroundThis study investigated the relationship between smoking and hearing loss and deafness (HLD) and whether the relationship is modified by genetic variation. Data for these analyses was from the subset of Japanese American families collected as part of the American Diabetes Association Genetics of Non-insulin Dependent Diabetes Mellitus study. Logistic regression with generalized estimating equations assessed the relationship between HLD and smoking. Nonparametric linkage analysis identified genetic regions harboring HLD susceptibility genes and ordered subset analysis was used to identify regions showing evidence for gene-smoking interactions. Genetic variants within these candidate regions were then each tested for interaction with smoking using logistic regression models. ResultsAfter adjusting for age, sex, diabetes status and smoking duration, for each pack of cigarettes smoked per day, risk of HLD increased 4.58 times (odds ratio (OR) = 4.58; 95% Confidence Interval (CI): (1.40,15.03)), and ever smokers were over 5 times more likely than nonsmokers to report HLD (OR = 5.22; 95% CI: (1.24, 22.03)). Suggestive evidence for linkage for HLD was observed in multiple genomic regions (Chromosomes 5p15, 8p23 and 17q21), and additional suggestive regions were identified when considering interactions with smoking status (Chromosomes 7p21, 11q23, 12q32, 15q26, and 20q13) and packs-per-day (Chromosome 8q21). ConclusionsTo our knowledge this was the first report of possible gene-by-smoking interactions in HLD using family data. Additional work, including independent replication, is needed to understand the basis of these findings. HLD are important public health issues and understanding the contributions of genetic and environmental factors may inform public health messages and policies.

A Rare Variant Nonparametric Linkage Method for Nuclear and Extended Pedigrees with Application to Late-Onset Alzheimer Disease via WGS Data

To analyze family-based whole-genome sequence (WGS) data for complex traits, we developed a rare variant (RV) non-parametric linkage (NPL) analysis method, which has advantages over association methods. The RV-NPL differs from the NPL in that RVs are analyzed, and allele sharing among affected relative-pairs is estimated only for minor alleles. Analyzing families can increase power because causal variants with familial aggregation usually have larger effect sizes than those underlying sporadic diseases. Differing from association analysis, for NPL only affected individuals are analyzed, which can increase power, since unaffected family members can be susceptibility variant carriers. RV-NPL is robust to population substructure and admixture, inclusion of nonpathogenic variants, as well as allelic and locus heterogeneity and can readily be applied outside of coding regions. In contrast to analyzing common variants using NPL, where loci localize to large genomic regions (e.g., >50 Mb), mapped regions are well defined for RV-NPL. Using simulation studies, we demonstrate that RV-NPL is substantially more powerful than applying traditional NPL methods to analyze RVs. The RV-NPL was applied to analyze 107 late-onset Alzheimer disease (LOAD) pedigrees of Caribbean Hispanic and European ancestry with WGS data, and statistically significant linkage (LOD ≥ 3.8) was found with RVs in PSMF1 and PTPN21 which have been shown to be involved in LOAD etiology. Additionally, nominally significant linkage was observed with RVs in ABCA7, ACE, EPHA1, and SORL1, genes that were previously reported to be associated with LOAD. RV-NPL is an ideal method to elucidate the genetic etiology of complex familial diseases.

Biallelic expansion of an intronic repeat in RFC1 is a common cause of late-onset ataxia.

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG)11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia.

Whole-exome sequencing in a Japanese pedigree implicates a rare non-synonymous single-nucleotide variant in BEST3 as a candidate for mandibular prognathism.

Mandibular prognathism is a phenotype of facial deformity seen in populations around the world, but with higher incidence among East Asian populations. Five genome-wide nonparametric linkage analyses and a genome-wide association study to identify susceptibility loci of the phenotype have shown inconsistent results. To explore variants related to mandibular prognathism, we undertook whole-exome sequencing in a Japanese pedigree. The pedigree was ascertained as mandibular prognathism. The pedigree comprised 15 individuals from 4 generations. Four affected individuals across 2 generations and 5 unaffected individuals were chosen for whole-exome sequencing. Five non-synonymous single-nucleotide variants (SNVs) of UBASH3B, OR6M1, OR8D4, OR8B4, and BEST3 genes were detected in all 4 affected individuals, but in none of the 5 unaffected individuals. A non-synonymous SNV of the BEST3 gene, Chr12(GRCh37):g.70048878G>T, NM_032735.2:c.1816C>A, p.(L606I), was identified as rare missense variant. BEST3 is located on chromosome 12q15 and encodes bestrophin 3 from the bestrophin family of anion channels. The 4 other non-synonymous SNVs of UBASH3B, OR6M1, OR8D4, and OR8B4 were not considered plausible candidates for mandibular prognathism. Our whole-exome sequencing implicates a rare non-synonymous SNV of BEST3 as a candidate for mandibular prognathism in the Japanese pedigree.

Evidence of linkage to chromosome 5p13.2-q11.1 in a large inbred family with genetic generalized epilepsy.

The clinical genetics of genetic generalized epilepsy suggests complex inheritance; large pedigrees, with multiple affected individuals, are rare exceptions. We studied a large consanguineous family from Turkey where extensive electroclinical phenotyping revealed a familial phenotype most closely resembling juvenile myoclonic epilepsy. For a subject to be considered affected (n = 14), a diagnostic electroencephalogram was required. Seizure onset ranged between 6 and 19 years (mean = 12 years). Thirteen of 14 experienced myoclonic jerks; in 11, this was associated with eyelid blinking, and in 10 it was interspersed with absences. Generalized tonic-clonic seizures were seen in 11. One individual had generalized tonic-clonic seizures alone. Electroencephalograms demonstrated generalized polyspike and wave discharges that were not associated with photoparoxysmal response. Intellect was normal. Nineteen family members were subsequently chosen for nonparametric multipoint linkage analyses, which identified a 39.5 Mb region on chromosome 5 (P < 0.0001). Iterative analysis, including discovery of a subtly affected individual, narrowed the critical region to 15.4 Mb and possibly to 5.5 Mb. Homozygous versus heterozygous state of the refined 5p13.2-q11.1 haplotype was not associated with phenotypic severity or onset age, suggesting that one versus two pathogenic variants may result in similar phenotypes. Whole exome sequencing (n = 3) failed to detect any rare, protein-coding variants within the highly significant linkage region that includes HCN1 as a promising candidate.

Novel caries loci in children and adults implicated by genome-wide analysis of families

BackgroundDental caries is a common chronic disease among children and adults alike, posing a substantial health burden. Caries is affected by multiple genetic and environmental factors, and prior studies have found that a substantial proportion of caries susceptibility is genetically inherited.MethodsTo identify such genetic factors, we conducted a genome-wide linkage scan in 464 extended families with 2616 individuals from Iowa, Pennsylvania and West Virginia for three dental caries phenotypes: (1) PRIM: dichotomized as zero versus one or more affected primary teeth, (2) QTOT1: age-adjusted quantitative caries measure for both primary and permanent dentitions including pre-cavitated lesions, and (3) QTOT2: age-adjusted quantitative caries excluding pre-cavitated lesions. Genotyping was conducted for approximately 600,000 SNPs on an Illumina platform, pruned to 127,511 uncorrelated SNPs for the analyses reported here.ResultsMultipoint non-parametric linkage analyses generated peak LOD scores exceeding 2.0 for eight genomic regions, but no LOD scores above 3.0 were observed. The maximum LOD score for each of the three traits was 2.90 at 1q25.3 for PRIM, 2.38 at 6q25.3 for QTOT1, and 2.76 at 5q23.3 for QTOT2. Some overlap in linkage regions was observed among the phenotypes. Genes with a potential role in dental caries in the eight chromosomal regions include CACNA1E, LAMC2, ALMS1, STAMBP, GXYLT2, SLC12A2, MEGF10, TMEM181, ARID1B, and, as well as genes in several immune gene families. Our results are also concordant with previous findings from association analyses on chromosomes 11 and 19.ConclusionsThese multipoint linkage results provide evidence in favor of novel chromosomal regions, while also supporting earlier association findings for these data. Understanding the genetic etiology of dental caries will allow designing personalized treatment plans based on an individual’s genetic risk of disease.

Primary Biliary Cholangitis in British Columbia First Nations: Clinical features and discovery of novel genetic susceptibility loci.

Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterized by destruction of intrahepatic bile ducts, portal inflammation and cirrhosis. Although rare in most populations, it is prevalent and often familial in British Columbia First Nations. We hypothesized that major genetic factors increased the risk in First Nations. In all, 44 individuals with Primary Biliary Cholangitis and 61 unaffected relatives from 32 First Nations families participated. Family history and co-morbidities were documented. Medical records were reviewed and available biopsies were re-reviewed by our team pathologist. Genotyping was performed on DNA from 36 affected persons and 27 unaffected relatives using the Affymetrix Human Mapping 500K Array Set. MERLIN software was used to carry out multipoint parametric and nonparametric linkage analysis. Candidate genes were identified and entered into InnateDB and KEGG software to identify potential pathways affecting pathogenesis. In all, 34% of families were multiplex. Fifty per cent of cases and 33% of unaffected relatives reported other autoimmune disease. Three genomic regions (9q21, 17p13 and 19p13) produced LOD scores of 2.3 or greater suggestive of linkage, but no single linkage peak reached statistical significance. Candidate genes identified in the three regions suggested involvement of IL17, NFκB, IL6, JAK-STAT, IFNγ and TGFβ immune signalling pathways. Specifically, four genes-ACT1, PIN1, DNMT1 and NTN1-emerged as having roles in these pathways that may influence Primary Biliary Cholangitis pathogenesis. Our whole genome linkage study results reflect the multifactorial nature of Primary Biliary Cholangitis, support previous studies suggesting signalling pathway involvement and identify new candidate genes for consideration.

Mutation analysis of a Chinese pedigree of Leber hereditary optic neuropathy with only male patients

Background Leber hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by a bilateral acute or subacute painless central visual loss in young adults, predominately in males.So far no one theory can completely explain all clinical manifestations of LHON. Objective This study was to investigate whether there is a linkage between X-chromosomal and mitochondrial mutation in the inheritance of a Chinese LHON pedigree with only male patients. Methods This study was approved by Ethic Committee of Affiliated First Hospital of Zhengzhou University and followed by Declaration of Helsinki.A Chinese LHON pedigree was included in Anyang city from January 2008 to August 2016.Periphery blood of 5-10 ml was collected from 4 sufferers, 13 maternal members and 10 non-maternal members for DNA extraction and PCR sequencing.The gene scanning and genotyping analysis were performed by ABI-PRISM 3100 genetic analyzer and Genotyper 3.7 software, and linkage analysis was carried out with Linkage software for the calculation of logarithm of odds (LOD). Mitochondrial DNA (mtDNA) sequence, fluorescence-based Genescan for X- chromosomal sequence were analyzed in the propositus and haplotype was evaluated. Results A total of 5 generations and 71 families were included in the pedigree, with 6 male sufferers, 30 maternal members and 41 non-maternal members.The visual acuity was ≤0.10, and the central visual field defection, the optic nerve flushing was found in the acute phase, different levels of the optic nerve fibers atrophy were found in the chronic phase; visual evoked potential (VEP) amplitude was low and peak latency were found in the male patients, and no any ocular abnormality was seen in the maternal members, meeting a maternally inherited characteristics, with the penetrance of 20%.The three primary mutations were not been found in this family by PCR sequencing.mtDNA sequencing appeared 31 variation of loci in the proband, including a known G3635A mutation, as well as an unknown ND5 A12340G missense mutation and ND4 T11809C synonymous mutation as well as 28 polymorphism of locus, and the proband was mitochondrial haplotype F1.The maternal families were mutation carriers of G3635A and A12340G loci, while the same mutation was not found in the normal family members and 107 controls.The maximum two point parametric LOD score was 1.46(θ=0.0) for marker DXS1060, located at Xp22.3, and the two-point and multipoint non-parametric linkage analysis were significant (all at P>0.05). Conclusions The ND5 A12340G and ND1 G3635A mutations coexist in this LHON family, and the ND5 A12340G mutation is a newly reported mutation.There is no evidence for an X-linked modifiers loci in this Chinese LHON family. Key words: Leber hereditary optic neuropathy/genetics; X-linked modifiers locus; Microsatellite marker; Parametric linkage analysis; Non-parametric linkage analysis

Genome-Wide Linkage Analysis of Large Multiple Multigenerational Families Identifies Novel Genetic Loci for Coronary Artery Disease

Coronary artery disease (CAD) is the leading cause of death, and genetic factors contribute significantly to risk of CAD. This study aims to identify new CAD genetic loci through a large-scale linkage analysis of 24 large and multigenerational families with 433 family members (GeneQuest II). All family members were genotyped with markers spaced by every 10 cM and a model-free nonparametric linkage (NPL-all) analysis was carried out. Two highly significant CAD loci were identified on chromosome 17q21.2 (NPL score of 6.20) and 7p22.2 (NPL score of 5.19). We also identified four loci with significant NPL scores between 4.09 and 4.99 on 2q33.3, 3q29, 5q13.2 and 9q22.33. Similar analyses in individual families confirmed the six significant CAD loci and identified seven new highly significant linkages on 9p24.2, 9q34.2, 12q13.13, 15q26.1, 17q22, 20p12.3, and 22q12.1, and two significant loci on 2q11.2 and 11q14.1. Two loci on 3q29 and 9q22.33 were also successfully replicated in our previous linkage analysis of 428 nuclear families. Moreover, two published risk variants, SNP rs46522 in UBE2Z and SNP rs6725887 in WDR12 by GWAS, were found within the 17q21.2 and 2q33.3 loci. These studies lay a foundation for future identification of causative variants and genes for CAD.

Genetic Markers for Adolescent Idiopathic Scoliosis on Chromosome 19p13.3 among Saudi Arabian Girls.

Study DesignProspective case-controlled study.PurposeThis study aimed to assess genetic influence in Saudi Arabian children with adolescent idiopathic scoliosis (AIS).Overview of LiteratureThe genetic locus linked to chromosome 19p for idiopathic scoliosis has been described. A pilot study conducted at King Fahd Hospital of the University, Al-Khobar showed that three microsatellite markers (D19S216, D19S894, and DS1034) of chromosome 19p13.3 were significant in Saudi Arabian females compared with healthy subjects.MethodsA total of 100 unrelated Saudi Arabian girls treated for AIS, their parents, healthy siblings, and healthy subjects were recruited for genetic analysis of markers on chromosome 19p13.3. After informed consent was obtained from their parents, blood samples were collected and parametric and nonparametric linkage analyses were performed using GENEHUNTER ver. 2.1. Multipoint linkage analysis was used to specify an autosomal dominant trait with a gene frequency of 0.01 and an estimated penetrance of 80% at the genotypic and allelic levels.ResultsFive hundred blood samples were collected and analyzed for microsatellite markers (D19S216, D19S894, and DS1034) of chromosome 19p13.3. Comparison among patients, family members, and healthy subjects revealed no significant association between markers and scoliosis at the genotypic level: D19S216 (p=0.21), D19S894 (p=0.37), and DS1034 (p=0.25). However, at the allelic level, a statistically significant association was observed for marker DS1034 (p=0.008), and marker D19S216 showed significance between fathers and patients (p<0.001) compared with patients and mothers. The other two markers, D19S216 (p=0.25) and D19S894 (p=0.17), showed no significant association between patients and mothers.ConclusionsAt the allelic level, marker DS1034 was significantly associated with AIS patients and their fathers. This allelic marker on chromosome 19p13.3 appears to be important in AIS etiology.

A Genome-Wide SNP Linkage Analysis Suggests a Susceptibility Locus on 6p21 for Ankylosing Spondylitis and Inflammatory Back Pain Trait.

ObjectivesTo screen susceptibility loci for ankylosing spondylitis (AS) using an affected-only linkage analysis based on high-density single nucleotide polymorphisms (SNPs) in a genome-wide manner.Patients and MethodsAS patients from ten families with Cantonese origin of China were enrolled in the study. Blood samples were genotyped using genomic DNA derived from peripheral blood leukocytes by Illumina HumanHap 610-Quad SNP Chip. Genotype data were generated using the Illumina BeadStudio 3.2 software. PLINK package was used to remove non-autosomal SNPs and to further eliminate markers of typing errors. An affected-only linkage analysis was carried out using both non-parametric and parametric linkage analyses, as implemented in MERLIN.ResultSeventy-eight AS patients (48 males and 30 females, mean age: 39±16 years) were enrolled in the study. The mean age of onset was 23±10 years and mean duration of disease was 16.7±12.2 years. Iritis (2/76, 2.86%), dactylitis (5/78, 6.41%), hip joint involvement (9/78, 11.54%), peripheral arthritis (22/78, 28.21%), inflammatory back pain (IBP) (69/78, 88.46%) and HLA-B27 positivity (70/78, 89.74%) were observed in these patients. Using non-parameter linkage analysis, we found one susceptibility locus for AS, IBP and HLA-B27 in 6p21 respectively, spanning about 13.5Mb, 20.9Mb and 21.2Mb, respectively No significant results were found in the other clinical trait groups including dactylitis, hip involved and arthritis. The identical susceptibility locus region spanning above 9.44Mb was detected in AS IBP and HLA-B27 by the parametric linkage analysis.ConclusionOur genome-wide SNP linkage analysis in ten families with ankylosing spondylitis suggests a susceptibility locus on 6p21 in AS, which is a risk locus for IBP in AS patients.

Admixture mapping of serum vitamin D and parathyroid hormone concentrations in the African American—Diabetes Heart Study

Vitamin D and intact parathyroid hormone (iPTH) concentrations differ between individuals of African and European descent and may play a role in observed racial differences in bone mineral density (BMD). These findings suggest that mapping by admixture linkage disequilibrium (MALD) may be informative for identifying genetic variants contributing to these ethnic disparities. Admixture mapping was performed for serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, vitamin D-binding protein (VDBP), bioavailable vitamin D, and iPTH concentrations and computed tomography measured thoracic and lumbar vertebral volumetric BMD in 552 unrelated African Americans with type 2 diabetes from the African American—Diabetes Heart Study. Genotyping was performed using a custom Illumina ancestry informative marker (AIM) panel. For each AIM, the probability of inheriting 0, 1, or 2 copies of a European-derived allele was determined. Non-parametric linkage analysis was performed by testing for association between each AIM using these probabilities among phenotypes, accounting for global ancestry, age, and gender. Fine-mapping of MALD peaks was facilitated by genome-wide association study (GWAS) data. VDBP levels were significantly linked in proximity to the protein coding locus (rs7689609, LOD=11.05). Two loci exhibited significant linkage signals for 1,25-dihydroxyvitamin D on 13q21.2 (rs1622710, LOD=3.20) and 12q13.2 (rs11171526, LOD=3.10). iPTH was significantly linked on 9q31.3 (rs7854368, LOD=3.14). Fine-mapping with GWAS data revealed significant known (rs7041 with VDBP, P=1.38×10−82) and novel (rs12741813 and rs10863774 with VDBP, P<6.43×10−5) loci with plausible biological roles. Admixture mapping in combination with fine-mapping has focused efforts to identify loci contributing to ethnic differences in vitamin D-related traits.