Genetic complexity of diagnostically unresolved Ehlers-Danlos syndrome.

BACKGROUND: The Ehlers-Danlos syndromes (EDS) are heritable disorders of connective tissue (HDCT), reclassified in the 2017 nosology into 13 subtypes. The genetic basis for hypermobile Ehlers-Danlos syndrome (hEDS) remains unknown. METHODS: Whole exome sequencing (WES) was undertaken on 174 EDS patients recruited from a national diagnostic service for complex EDS and a specialist clinic for hEDS. Patients had already undergone expert phenotyping, laboratory investigation and gene sequencing, but were without a genetic diagnosis. Filtered WES data were reviewed for genes underlying Mendelian disorders and loci reported in EDS linkage, transcriptome and genome-wide association studies (GWAS). A genetic burden analysis (Minor Allele Frequency (MAF) <0.05) incorporating 248 Avon Longitudinal Study of Parents and Children (ALSPAC) controls sequenced as part of the UK10K study was undertaken using TASER methodology. RESULTS: Heterozygous pathogenic (P) or likely pathogenic (LP) variants were identified in known EDS and Loeys-Dietz (LDS) genes. Multiple variants of uncertain significance where segregation and functional analysis may enable reclassification were found in genes associated with EDS, LDS, heritable thoracic aortic disease (HTAD), Mendelian disorders with EDS symptomatology and syndromes with EDS-like features. Genetic burden analysis revealed a number of novel loci, although none reached the threshold for genome-wide significance. Variants with biological plausibility were found in genes and pathways not currently associated with EDS or HTAD. CONCLUSIONS: We demonstrate the clinical utility of large panel-based sequencing and WES for patients with complex EDS in distinguishing rare EDS subtypes, LDS and related syndromes. Although many of the P and LP variants reported in this cohort would be identified with current panel testing, they were not at the time of this study, highlighting the use of extended panels and WES as a clinical tool for complex EDS. Our results are consistent with the complex genetic architecture of EDS and suggest a number of novel hEDS and HTAD candidate genes and pathways.

A genetic risk score and diabetes predict development of alcohol-related cirrhosis in drinkers.

BACKGROUND & AIMS: Only a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk. METHODS: Three cohorts (GenomALC-1: n = 1,690, GenomALC-2: n = 3,037, UK Biobank: relevant n = 6,898) with a history of heavy alcohol consumption (≥80 g/day (men), ≥50 g/day (women), for ≥10 years) were included. Cases were participants with alcohol-related cirrhosis. Controls had a history of similar alcohol consumption but no evidence of liver disease. Risk scores were computed from up to 8 genetic loci identified previously as associated with alcohol-related cirrhosis and 3 clinical risk factors. Score performance for the stratification of alcohol-related cirrhosis risk was assessed and compared across the alcohol-related liver disease spectrum, including hepatocellular carcinoma (HCC). RESULTS: A combination of 3 single nucleotide polymorphisms (SNPs) (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) and diabetes status best discriminated cirrhosis risk. The odds ratios (ORs) and (95% CIs) between the lowest (Q1) and highest (Q5) score quintiles of the 3-SNP score, based on independent allelic effect size estimates, were 5.99 (4.18-8.60) (GenomALC-1), 2.81 (2.03-3.89) (GenomALC-2), and 3.10 (2.32-4.14) (UK Biobank). Patients with diabetes and high risk scores had ORs of 14.7 (7.69-28.1) (GenomALC-1) and 17.1 (11.3-25.7) (UK Biobank) compared to those without diabetes and with low risk scores. Patients with cirrhosis and HCC had significantly higher mean risk scores than patients with cirrhosis alone (0.76 ± 0.06 vs. 0.61 ± 0.02, p = 0.007). Score performance was not significantly enhanced by information on additional genetic risk variants, body mass index or coffee consumption. CONCLUSIONS: A risk score based on 3 genetic risk variants and diabetes status enables the stratification of heavy drinkers based on their risk of cirrhosis, allowing for the provision of earlier preventative interventions. LAY SUMMARY: Excessive chronic drinking leads to cirrhosis in some people, but so far there is no way to identify those at high risk of developing this debilitating disease. We developed a genetic risk score that can identify patients at high risk. The risk of cirrhosis is increased >10-fold with just two risk factors - diabetes and a high genetic risk score. Risk assessment using this test could enable the early and personalised management of this disease in high-risk patients.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Genome-wide Association Study and Meta-analysis on Alcohol-Associated Liver Cirrhosis Identifies Genetic Risk Factors.

BACKGROUND AND AIMS: Only a minority of heavy drinkers progress to alcohol-associated cirrhosis (ALC). The aim of this study was to identify common genetic variants that underlie risk for ALC. APPROACH AND RESULTS: We analyzed data from 1,128 subjects of European ancestry with ALC and 614 heavy-drinking subjects without known liver disease from Australia, the United States, the United Kingdom, and three countries in Europe. A genome-wide association study (GWAS) was performed, adjusting for principal components and clinical covariates (alcohol use, age, sex, body mass index, and diabetes). We validated our GWAS findings using UK Biobank. We then performed a meta-analysis combining data from our study, the UK Biobank, and a previously published GWAS. Our GWAS found genome-wide significant risk association of rs738409 in patatin-like phospholipase domain containing 3 (PNPLA3) (odds ratio [OR] = 2.19 [G allele], P = 4.93 × 10-17 ) and rs4607179 near HSD17B13 (OR = 0.57 [C allele], P = 1.09 × 10-10 ) with ALC. Conditional analysis accounting for the PNPLA3 and HSD17B13 loci identified a protective association at rs374702773 in Fas-associated factor family member 2 (FAF2) (OR = 0.61 [del(T) allele], P = 2.56 × 10-8 ) for ALC. This association was replicated in the UK Biobank using conditional analysis (OR = 0.79, P = 0.001). Meta-analysis (without conditioning) confirmed genome-wide significance for the identified FAF2 locus as well as PNPLA3 and HSD17B13. Two other previously known loci (SERPINA1 and SUGP1/TM6SF2) were also genome-wide significant in the meta-analysis. GeneOntology pathway analysis identified lipid droplets as the target for several identified genes. In conclusion, our GWAS identified a locus at FAF2 associated with reduced risk of ALC among heavy drinkers. Like the PNPLA3 and HSD17B13 gene products, the FAF2 product has been localized to fat droplets in hepatocytes. CONCLUSIONS: Our genetic findings implicate lipid droplets in the biological pathway(s) underlying ALC.

An international genome-wide meta-analysis of primary biliary cholangitis: Novel risk loci and candidate drugs.

BACKGROUNDS & AIMS: Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intrahepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening. METHODS: We combined new and existing genotype data for 10,516 cases and 20,772 controls from 5 European and 2 East Asian cohorts. RESULTS: We identified 56 genome-wide significant loci (20 novel) including 46 in European, 13 in Asian, and 41 in combined cohorts; and a 57th genome-wide significant locus (also novel) in conditional analysis of the European cohorts. Candidate genes at newly identified loci include FCRL3, INAVA, PRDM1, IRF7, CCR6, CD226, and IL12RB1, which each play key roles in immunity. Pathway analysis reiterated the likely importance of pattern recognition receptor and TNF signalling, JAK-STAT signalling, and differentiation of T helper (TH)1 and TH17 cells in the pathogenesis of this disease. Drug efficacy screening identified several medications predicted to be therapeutic in PBC, some of which are well-established in the treatment of other autoimmune disorders. CONCLUSIONS: This study has identified additional risk loci for PBC, provided a hierarchy of agents that could be trialled in this condition, and emphasised the value of genetic and genomic approaches to drug discovery in complex disorders. LAY SUMMARY: Primary biliary cholangitis (PBC) is a chronic liver disease that eventually leads to cirrhosis. In this study, we analysed genetic information from 10,516 people with PBC and 20,772 healthy individuals recruited in Canada, China, Italy, Japan, the UK, or the USA. We identified several genetic regions associated with PBC. Each of these regions contains several genes. For each region, we used diverse sources of evidence to help us choose the gene most likely to be involved in causing PBC. We used these 'candidate genes' to help us identify medications that are currently used for treatment of other conditions, which might also be useful for treatment of PBC.

Amino acid residues in five separate HLA genes can explain most of the known associations between the MHC and primary biliary cholangitis.

Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterised by progressive destruction of intrahepatic bile ducts. The strongest genetic association is with HLA-DQA1*04:01, but at least three additional independent HLA haplotypes contribute to susceptibility. We used dense single nucleotide polymorphism (SNP) data in 2861 PBC cases and 8514 controls to impute classical HLA alleles and amino acid polymorphisms using state-of-the-art methodologies. We then demonstrated through stepwise regression that association in the HLA region can be largely explained by variation at five separate amino acid positions. Three-dimensional modelling of protein structures and calculation of electrostatic potentials for the implicated HLA alleles/amino acid substitutions demonstrated a correlation between the electrostatic potential of pocket P6 in HLA-DP molecules and the HLA-DPB1 alleles/amino acid substitutions conferring PBC susceptibility/protection, highlighting potential new avenues for future functional investigation.

Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically characterised cohort☆.

BACKGROUND & AIMS: Genetic factors associated with non-alcoholic fatty liver disease (NAFLD) remain incompletely understood. To date, most genome-wide association studies (GWASs) have adopted radiologically assessed hepatic triglyceride content as the reference phenotype and so cannot address steatohepatitis or fibrosis. We describe a GWAS encompassing the full spectrum of histologically characterised NAFLD. METHODS: The GWAS involved 1,483 European NAFLD cases and 17,781 genetically matched controls. A replication cohort of 559 NAFLD cases and 945 controls was genotyped to confirm signals showing genome-wide or close to genome-wide significance. RESULTS: Case-control analysis identified signals showing p values ≤5 × 10-8 at 4 locations (chromosome [chr] 2 GCKR/C2ORF16; chr4 HSD17B13; chr19 TM6SF2; chr22 PNPLA3) together with 2 other signals with p <1 × 10-7 (chr1 near LEPR and chr8 near IDO2/TC1). Case-only analysis of quantitative traits showed that the PNPLA3 signal (rs738409) had genome-wide significance for steatosis, fibrosis and NAFLD activity score and a new signal (PYGO1 rs62021874) had close to genome-wide significance for steatosis (p = 8.2 × 10-8). Subgroup case-control analysis for NASH confirmed the PNPLA3 signal. The chr1 LEPR single nucleotide polymorphism also showed genome-wide significance for this phenotype. Considering the subgroup with advanced fibrosis (≥F3), the signals on chr2, chr19 and chr22 maintained their genome-wide significance. Except for GCKR/C2ORF16, the genome-wide significance signals were replicated. CONCLUSIONS: This study confirms PNPLA3 as a risk factor for the full histological spectrum of NAFLD at genome-wide significance levels, with important contributions from TM6SF2 and HSD17B13. PYGO1 is a novel steatosis modifier, suggesting that Wnt signalling pathways may be relevant in NAFLD pathogenesis. LAY SUMMARY: Non-alcoholic fatty liver disease is a common disease where excessive fat accumulates in the liver and may result in cirrhosis. To understand who is at risk of developing this disease and suffering liver damage, we undertook a genetic study to compare the genetic profiles of people suffering from fatty liver disease with genetic profiles seen in the general population. We found that particular sequences in 4 different areas of the human genome were seen at different frequencies in the fatty liver disease cases. These sequences may help predict an individual's risk of developing advanced disease. Some genes where these sequences are located may also be good targets for future drug treatments.

Methylation quantitative trait locus analysis of osteoarthritis links epigenetics with genetic risk.

Osteoarthritis (OA) is a common, painful and debilitating disease of articulating joints resulting from the age-associated loss of cartilage. Well-powered genetic studies have identified a number of DNA polymorphisms that are associated with OA susceptibility. Like most complex trait loci, these OA loci are thought to influence disease susceptibility through the regulation of gene expression, so-called expression quantitative loci, or eQTLs. One mechanism through which eQTLs act is epigenetic, by modulating DNA methylation. In such cases, there are quantitative differences in DNA methylation between the two alleles of the causal polymorphism, with the association signal referred to as a methylation quantitative trait locus, or meQTL. In this study, we aimed to investigate whether the OA susceptibility loci identified to date are functioning as meQTLs by integrating genotype data with whole genome methylation data of cartilage DNA. We investigated potential genotype-methylation correlations within a 1.0-1.5 Mb region surrounding each of 16 OA-associated single-nucleotide polymorphisms (SNPs) in 99 cartilage samples and identified four that function as meQTLs. Three of these replicated in an additional cohort of up to 62 OA patients. These observations suggest that OA susceptibility loci regulate the level of DNA methylation in cis and provide a mechanistic explanation as to how these loci impact upon OA susceptibility, further increasing our understanding of the role of genetics and epigenetics in this common disease.

Genome-wide association study identifies loci on 12q24 and 13q32 associated with tetralogy of Fallot.

We conducted a genome-wide association study to search for risk alleles associated with Tetralogy of Fallot (TOF), using a northern European discovery set of 835 cases and 5159 controls. A region on chromosome 12q24 was associated (P = 1.4 × 10(-7)) and replicated convincingly (P = 3.9 × 10(-5)) in 798 cases and 2931 controls [per allele odds ratio (OR) = 1.27 in replication cohort, P = 7.7 × 10(-11) in combined populations]. Single nucleotide polymorphisms in the glypican 5 gene on chromosome 13q32 were also associated (P = 1.7 × 10(-7)) and replicated convincingly (P = 1.2 × 10(-5)) in 789 cases and 2927 controls (per allele OR = 1.31 in replication cohort, P = 3.03 × 10(-11) in combined populations). Four additional regions on chromosomes 10, 15 and 16 showed suggestive association accompanied by nominal replication. This study, the first genome-wide association study of a congenital heart malformation phenotype, provides evidence that common genetic variation influences the risk of TOF.

Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease.

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10(-5)). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ~5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ~4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias.

Phenotype-specific effect of chromosome 1q21.1 rearrangements and GJA5 duplications in 2436 congenital heart disease patients and 6760 controls.

Recurrent rearrangements of chromosome 1q21.1 that occur via non-allelic homologous recombination have been associated with variable phenotypes exhibiting incomplete penetrance, including congenital heart disease (CHD). However, the gene or genes within the ~1 Mb critical region responsible for each of the associated phenotypes remains unknown. We examined the 1q21.1 locus in 948 patients with tetralogy of Fallot (TOF), 1488 patients with other forms of CHD and 6760 ethnically matched controls using single nucleotide polymorphism genotyping arrays (Illumina 660W and Affymetrix 6.0) and multiplex ligation-dependent probe amplification. We found that duplication of 1q21.1 was more common in cases of TOF than in controls [odds ratio (OR) 30.9, 95% confidence interval (CI) 8.9-107.6); P = 2.2 × 10(-7)], but deletion was not. In contrast, deletion of 1q21.1 was more common in cases of non-TOF CHD than in controls [OR 5.5 (95% CI 1.4-22.0); P = 0.04] while duplication was not. We also detected rare (n = 3) 100-200 kb duplications within the critical region of 1q21.1 in cases of TOF. These small duplications encompassed a single gene in common, GJA5, and were enriched in cases of TOF in comparison to controls [OR = 10.7 (95% CI 1.8-64.3), P = 0.01]. These findings show that duplication and deletion at chromosome 1q21.1 exhibit a degree of phenotypic specificity in CHD, and implicate GJA5 as the gene responsible for the CHD phenotypes observed with copy number imbalances at this locus.

A polygenic risk score for alcohol-associated cirrhosis among heavy drinkers with European ancestry.

BACKGROUND: Polygenic Risk Scores (PRS) based on results from genome-wide association studies offer the prospect of risk stratification for many common and complex diseases. We developed a PRS for alcohol-associated cirrhosis by comparing single-nucleotide polymorphisms among patients with alcohol-associated cirrhosis (ALC) versus drinkers who did not have evidence of liver fibrosis/cirrhosis. METHODS: Using a data-driven approach, a PRS for ALC was generated using a meta-genome-wide association study of ALC (N=4305) and an independent cohort of heavy drinkers with ALC and without significant liver disease (N=3037). It was validated in 2 additional independent cohorts from the UK Biobank with diagnosed ALC (N=467) and high-risk drinking controls (N=8981) and participants in the Indiana Biobank Liver cohort with alcohol-associated liver disease (N=121) and controls without liver disease (N=3239). RESULTS: A 20-single-nucleotide polymorphisms PRS for ALC (PRSALC) was generated that stratified risk for ALC comparing the top and bottom deciles of PRS in the 2 validation cohorts (ORs: 2.83 [95% CI: 1.82 -4.39] in UK Biobank; 4.40 [1.56 -12.44] in Indiana Biobank Liver cohort). Furthermore, PRSALC improved the prediction of ALC risk when added to the models of clinically known predictors of ALC risk. It also stratified the risk for metabolic dysfunction -associated steatotic liver disease -cirrhosis (3.94 [2.23 -6.95]) in the Indiana Biobank Liver cohort -based exploratory analysis. CONCLUSIONS: PRSALC incorporates 20 single-nucleotide polymorphisms, predicts increased risk for ALC, and improves risk stratification for ALC compared with the models that only include clinical risk factors. This new score has the potential for early detection of heavy drinking patients who are at high risk for ALC.

Genotype/phenotype correlations in complement factor H deficiency arising from uniparental isodisomy.

We report a male infant who presented at 8 months of age with atypical hemolytic uremic syndrome (aHUS) responsive to plasma therapy. Investigation showed him to have complement factor H (CFH) deficiency associated with a homozygous CFH mutation (c.2880delT [p.Phe960fs]). Mutation screening of the child's parents revealed that the father was heterozygous for this change but that it was not present in his mother. Chromosome 1 uniparental isodisomy of paternal origin was confirmed by genotyping chromosome 1 SNPs. CD46 SNP genotyping was undertaken in this individual and another patient with CFH deficiency associated with chromosome 1 uniparental isodisomy. This showed a homozygous aHUS risk haplotype (CD46GGAAC) in the patient with aHUS and a homozygous glomerulonephritis risk haplotype (CD46AAGGT) in the patient with endocapillary glomerulonephritis. We also showed that FHL-1 (factor H-like protein 1) was present in the patient with aHUS and absent in the patient with glomerulonephritis. This study emphasizes that modifiers such as CD46 and FHL-1 may determine the kidney phenotype of patients who present with homozygous CFH deficiency.

Erratum: Author Correction: The influence of HLA genotype on the development of metal hypersensitivity following joint replacement.

[This corrects the article DOI: 10.1038/s43856-022-00137-0.].

The influence of HLA genotype on the development of metal hypersensitivity following joint replacement.

Background: Over five million joint replacements are performed across the world each year. Cobalt chrome (CoCr) components are used in most of these procedures. Some patients develop delayed-type hypersensitivity (DTH) responses to CoCr implants, resulting in tissue damage and revision surgery. DTH is unpredictable and genetic links have yet to be definitively established. Methods: At a single site, we carried out an initial investigation to identify HLA alleles associated with development of DTH following metal-on-metal hip arthroplasty. We then recruited patients from other centres to train and validate an algorithm incorporating patient age, gender, HLA genotype, and blood metal concentrations to predict the development of DTH. Accuracy of the modelling was assessed using performance metrics including time-dependent receiver operator curves. Results: Using next-generation sequencing, here we determine the HLA genotypes of 606 patients. 176 of these patients had experienced failure of their prostheses; the remaining 430 remain asymptomatic at a mean follow up of twelve years. We demonstrate that the development of DTH is associated with patient age, gender, the magnitude of metal exposure, and the presence of certain HLA class II alleles. We show that the predictive algorithm developed from this investigation performs to an accuracy suitable for clinical use, with weighted mean survival probability errors of 1.8% and 3.1% for pre-operative and post-operative models respectively. Conclusions: The development of DTH following joint replacement appears to be determined by the interaction between implant wear and a patient's genotype. The algorithm described in this paper may improve implant selection and help direct patient surveillance following surgery. Further consideration should be given towards understanding patient-specific responses to different biomaterials.