Nephrolithiasis is a prevalent condition with a high morbidity. Although dozens of monogenic causes have been identified, the fraction of single-gene disease has not been well studied. To determine the percentage of cases that can be molecularly explained by mutations in 1 of 30 known kidney stone genes, we conducted a high-throughput mutation analysis in a cohort of consecutively recruited patients from typical kidney stone clinics. The cohort comprised 272 genetically unresolved individuals (106 children and 166 adults) from 268 families with nephrolithiasis (n=256) or isolated nephrocalcinosis (n=16). We detected 50 likely causative mutations in 14 of 30 analyzed genes, leading to a molecular diagnosis in 14.9% (40 of 268) of all cases; 20 of 50 detected mutations were novel (40%). The cystinuria gene SLC7A9 (n=19) was most frequently mutated. The percentage of monogenic cases was notably high in both the adult (11.4%) and pediatric cohorts (20.8%). Recessive causes were more frequent among children, whereas dominant disease occurred more abundantly in adults. Our study provides an in-depth analysis of monogenic causes of kidney stone disease. We suggest that knowledge of the molecular cause of nephrolithiasis and nephrocalcinosis may have practical implications and might facilitate personalized treatment.

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997–109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

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Background and objectives Cystinuria is a rare inherited renal stone disease. Mutations in the amino acid exchanger System b 0,+ , the two subunits of which are encoded by SLC3A1 and SLC7A9, predominantly underlie this disease. The work analyzed the epidemiology of cystinuria and the influence of mutations in these two genes on disease severity in a United Kingdom cohort.Design, setting, participants, & measurements Prevalent patients were studied from 2012 to 2014 in the northeast and southwest of the United Kingdom. Clinical phenotypes were defined, and genetic analysis of SLC3A1 and SLC7A9 combining Sanger sequencing and multiplex ligation probe-dependent amplification was performed.Results In total, 76 patients (42 men and 34 women) were studied. All subjects had proven cystine stones. Median age of presentation (first stone episode) was 24 years old, but 21% of patients presented after 40 years old. Patients had varied clinical courses, with 37% of patients having $10 stone episodes; 70% had evidence of CKD, and 9% had reached ESRD as a result of cystinuria and its complications. Patients with cystinuria received a variety of different therapies, with no obvious treatment consensus. Notably, 20% of patients had staghorn calculi, with associated impaired renal function in 80% of these patients. Genetic analysis revealed that biallelic mutations were present in either SLC3A1 (n=27) or SLC7A9 (n=20); 22 patients had only one mutated allele detected (SLC3A1 in five patients and SLC7A9 in 17 patients). In total, 37 different mutant variant alleles were identified, including 12 novel mutations; 22% of mutations were caused by large gene rearrangements. No genotypephenotype association was detected in this cohort.Conclusions Patients with cystinuria in the United Kingdom often present atypically with staghorn calculi at $40 years old and commonly develop significant renal impairment. There is no association of clinical course with genotype. Treatments directed toward reducing stone burden need to be rationalized and developed to optimize patient care.

Objective: Osteoarthritis (OA) is polygenic with over 90 independent genome-wide association loci so far reported. A key next step is the identification of target genes and the molecular mechanisms through which this genetic risk operates. The majority of OA risk-conferring alleles are predicted to act by modulating gene expression. DNA methylation at CpG dinucleotides may be a functional conduit through which this occurs and is detectable by mapping methylation quantitative trait loci, or mQTLs. This approach can therefore provide functional insight into OA risk and will prioritize genes for subsequent investigation. That was our goal, with a focus on the largest set of OA loci yet to be reported. Method: We investigated DNA methylation, genotype and RNA sequencing data derived from the cartilage of patients who had undergone arthroplasty and combined this with in silico analyses of expression quantitative trait loci, epigenomes and chromatin interactions. Results: We investigated 42 OA risk loci and in ten of these we identified 24 CpGs in which methylation correlated with genotype (false discovery rate (FDR) P-values ranging from 0.049 to 1.73x10 À25). In silico analyses of these mQTLs prioritised genes and regulatory elements at the majority of the ten loci, with COLGALT2 (encoding a collagen galactosyltransferase), COL11A2 (encoding a polypeptide chain of type XI collagen) and WWP2 (encoding a ubiquitin ligase active during chondrogenesis) emerging as particularly compelling target genes. Conclusion: We have highlighted the pivotal role of DNA methylation as a link between genetic risk and OA and prioritized genes for further investigation.

Objective To identify methylation quantitative trait loci (mQTLs) correlating with osteoarthritis (OA) risk alleles and to undertake mechanistic characterization as a means of target gene prioritization. Methods We used genome‐wide genotyping and cartilage DNA methylation array data in a discovery screen of novel OA risk loci. This was followed by methylation, gene expression analysis, and genotyping studies in additional cartilage samples, accompanied by in silico analyses. Results We identified 4 novel OA mQTLs. The most significant mQTL contained 9 CpG sites where methylation correlated with OA risk genotype, with 5 of the CpG sites having P values <1 × 10−10. The 9 CpG sites reside in an interval of only 7.7 kb within the PLEC gene and form 2 distinct clusters. We were able to prioritize PLEC and the adjacent gene GRINA as independent targets of the OA risk. We identified PLEC and GRINA expression QTLs operating in cartilage, as well as methylation‐expression QTLs operating on the 2 genes. GRINA and PLEC also demonstrated differential expression between OA hip and non‐OA hip cartilage. Conclusion PLEC encodes plectin, a cytoskeletal protein that maintains tissue integrity by regulating intracellular signaling in response to mechanical stimuli. GRINA encodes the ionotropic glutamate receptor TMBIM3 (transmembrane BAX inhibitor 1 motif–containing protein family member 3), which regulates cell survival. Based on our results, we hypothesize that in a joint predisposed to OA, expression of these genes alters in order to combat aberrant biomechanics, and that this is epigenetically regulated. However, carriage of the OA risk–conferring allele at this locus hinders this response and contributes to disease development.

Osteoarthritis (OA) is a common, multifactorial and polygenic skeletal disease that, in its severest form, requires joint replacement surgery to restore mobility and to relieve chronic pain. Using tissues from the articulating joints of 260 patients with OA and a range of in vitro experiments, including CRISPR-Cas9, we have characterized an intergenic regulatory element. Here, genotype at an OA risk locus correlates with differential DNA methylation, with altered gene expression of both a transcriptional regulator (RUNX2), and a chromatin remodelling protein (SUPT3H). RUNX2 is a strong candidate for OA susceptibility, with its encoded protein being essential for skeletogenesis and healthy joint function. The OA risk locus includes single nucleotide polymorphisms (SNPs) located within and flanking the differentially methylated region (DMR). The OA association SNP, rs10948172, demonstrates particularly strong correlation with methylation, and two intergenic SNPs falling within the DMR (rs62435998 and rs62435999) demonstrate genetic and epigenetic effects on the regulatory activity of this region. We therefore posit that the OA signal mediates its effect by modulating the methylation of the regulatory element, which then impacts on gene expression, with RUNX2 being the principal target. Our study highlights the interplay between DNA methylation, OA genetic risk and the downstream regulation of genes critical to normal joint function.

To date, genome-wide association studies have implicated at least 35 loci in osteoarthritis but, due to linkage disequilibrium, the specific variants underlying these associations and the mechanisms by which they contribute to disease risk have yet to be pinpointed. Here, we functionally test 1,605 single nucleotide variants associated with osteoarthritis for regulatory activity using a massively parallel reporter assay. We identify six single nucleotide polymorphisms (SNPs) with differential regulatory activity between the major and minor alleles. We show that the most significant SNP, rs4730222, exhibits differential nuclear protein binding in electrophoretic mobility shift assays and drives increased expression of an alternative isoform of HBP1 in a heterozygote chondrosarcoma cell line, in a CRISPR-edited osteosarcoma cell line, and in chondrocytes derived from osteoarthritis patients. This study provides a framework for prioritization of GWAS variants and highlights a role of HBP1 and Wnt signaling in osteoarthritis pathogenesis.