Germline ERBB3 mutation in familial non-small-cell lung carcinoma: expanding ErbB's role in oncogenesis.

Lung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable, with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. Germline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and subcellular localization were assessed, and cellular proliferation, pAkt/Akt and pERK levels determined. A novel germline variant in ERBB3 (c.1946 T > G: p.Iso649Arg), coding for receptor tyrosine-protein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80 kDa form (which enhances proliferation) compared with the full-length (180 kDa) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localization pre- and post-epidermal growth factor stimulation; however, epidermal growth factor receptor (EGFR) levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared with wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 min post-stimulation compared with wtErbB3 transfection, demonstrating altered signalling pathway activation. Cumulatively, these results support this mutation as tumorogenic. This is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

Proteome profiling of salivary small extracellular vesicles in glioblastoma patients.

BACKGROUND: Extracellular vesicles (EVs) play a critical role in intercellular communication under physiological and pathological conditions, including cancer. EVs cargo reflects their cell of origin, suggesting their utility as biomarkers. EVs are detected in several biofluids, and their ability to cross the blood-brain barrier has highlighted their potential as prognostic and diagnostic biomarkers in gliomas, including glioblastoma (GBM). Studies have demonstrated the potential clinical utility of plasma-derived EVs in glioma. However, little is known about the clinical utility of saliva-derived EVs in GBM. METHODS: Small EVs were isolated from whole mouth saliva of GBM patients pre- and postoperatively. Isolation was performed using differential centrifugation and/or ultracentrifugation. EVs were characterized by concentration, size, morphology, and EVs cell-surface protein markers. Protein cargo in EVs was profiled using mass spectrometry. RESULTS: There were no statistically significant differences in size and concentration of EVs derived from pre- and post GBM patients' saliva samples. A higher number of proteins were detected in preoperative samples compared to postoperative samples. The authors found four highly abundant proteins (aldolase A, 14-3-3 protein ε, enoyl CoA hydratase 1, and transmembrane protease serine 11B) in preoperative saliva samples from GBM patients with poor outcomes. Functional enrichment analysis of pre- and postoperative saliva samples showed significant enrichment of several pathways, including those related to the immune system, cell cycle and programmed cell death. CONCLUSIONS: This study, for the first time, demonstrates the feasibility of isolating and characterizing small EVs from pre- and postoperative saliva samples from GBM patients. Preliminary findings encourage further large cohort validation studies on salivary small EVs to evaluate prognosis in GBM.

Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice.

The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.

Genome-wide association study in Turkish and Iranian populations identify rare familial Mediterranean fever gene (MEFV) polymorphisms associated with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a highly heritable immune-mediated arthritis common in Turkish and Iranian populations. Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease most common in people of Mediterranean origin. MEFV, an FMF-associated gene, is also a candidate gene for AS. We aimed to identify AS susceptibility loci and also examine the association between MEFV and AS in Turkish and Iranian cohorts. We performed genome-wide association studies in 1001 Turkish AS patients and 1011 Turkish controls, and 479 Iranian AS patients and 830 Iranian controls. Serum IL-1ß, IL-17 and IL-23 cytokine levels were quantified in Turkish samples. An association of major effect was observed with a novel rare coding variant in MEFV in the Turkish cohort (rs61752717, M694V, OR = 5.3, P = 7.63×10(-12)), Iranian cohort (OR = 2.9, P = 0.042), and combined dataset (OR = 5.1, P = 1.65×10(-13)). 99.6% of Turkish AS cases, and 96% of those carrying MEFV rs61752717 variants, did not have FMF. In Turkish subjects, the association of rs61752717 was particularly strong in HLA-B27-negative cases (OR = 7.8, P = 8.93×10(-15)), but also positive in HLA-B27-positive cases (OR = 4.3, P = 7.69×10(-8)). Serum IL-1ß, IL-17 and IL-23 levels were higher in AS cases than controls. Among AS cases, serum IL-1ß and IL-23 levels were increased in MEFV 694V carriers compared with non-carriers. Our data suggest that FMF and AS have overlapping aetiopathogenic mechanisms. Functionally important MEFV mutations, such as M694V, lead to dysregulated inflammasome function and excessive IL-1ß function. As IL-1 inhibition is effective in FMF, AS cases carrying FMF-associated MEFV variants may benefit from such therapy.

Polygenic Risk Scores have high diagnostic capacity in ankylosing spondylitis.

OBJECTIVE: We sought to test the hypothesis that Polygenic Risk Scores (PRSs) have strong capacity to discriminate cases of ankylosing spondylitis (AS) from healthy controls and individuals in the community with chronic back pain. METHODS: PRSs were developed and validated in individuals of European and East Asian ethnicity, using data from genome-wide association studies in 15 585 AS cases and 20 452 controls. The discriminatory values of PRSs in these populations were compared with other widely used diagnostic tests, including C-reactive protein (CRP), HLA-B27 and sacroiliac MRI. RESULTS: In people of European descent, PRS had high discriminatory capacity with area under the curve (AUC) in receiver operator characteristic analysis of 0.924. This was significantly better than for HLA-B27 testing alone (AUC=0.869), MRI (AUC=0.885) or C-reactive protein (AUC=0.700). PRS developed and validated in individuals of East Asian descent performed similarly (AUC=0.948). Assuming a prior probability of AS of 10% such as in patients with chronic back pain under 45 years of age, compared with HLA-B27 testing alone, PRS provides higher positive values for 35% of patients and negative predictive values for 67.5% of patients. For PRS, in people of European descent, the maximum positive predictive value was 78.2% and negative predictive value was 100%, whereas for HLA-B27, these values were 51.9% and 97.9%, respectively. CONCLUSIONS: PRS have higher discriminatory capacity for AS than CRP, sacroiliac MRI or HLA-B27 status alone. For optimal performance, PRS should be developed for use in the specific ethnic groups to which they are to be applied.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture.

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Correction: Defining the genetic susceptibility to cervical neoplasia-A genome-wide association study.

[This corrects the article DOI: 10.1371/journal.pgen.1006866.].

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Defining the genetic susceptibility to cervical neoplasia-A genome-wide association study.

A small percentage of women with cervical HPV infection progress to cervical neoplasia, and the risk factors determining progression are incompletely understood. We sought to define the genetic loci involved in cervical neoplasia and to assess its heritability using unbiased unrelated case/control statistical approaches. We demonstrated strong association of cervical neoplasia with risk and protective HLA haplotypes that are determined by the amino-acids carried at positions 13 and 71 in pocket 4 of HLA-DRB1 and position 156 in HLA-B. Furthermore, 36% (standard error 2.4%) of liability of HPV-associated cervical pre-cancer and cancer is determined by common genetic variants. Women in the highest 10% of genetic risk scores have approximately >7.1% risk, and those in the highest 5% have approximately >21.6% risk, of developing cervical neoplasia. Future studies should examine genetic risk prediction in assessing the risk of cervical neoplasia further, in combination with other screening methods.

Comprehensive genetic screening: The prevalence of maturity-onset diabetes of the young gene variants in a population-based childhood diabetes cohort.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is caused by autosomal dominant mutations in one of 13 confirmed genes. Estimates of MODY prevalence vary widely, as genetic screening is usually restricted based on clinical features, even in population studies. We aimed to determine prevalence of MODY variants in a large and unselected pediatric diabetes cohort. METHODS: MODY variants were assessed using massively parallel sequencing in the population-based diabetes cohort (n = 1363) of the sole tertiary pediatric diabetes service for Western Australia (population 2.6 million). All individuals were screened, irrespective of clinical features. MODY variants were also assessed in a control cohort (n = 993). RESULTS: DNA and signed consent were available for 821 children. Seventeen children had pathogenic/likely pathogenic variants in MODY genes, two diagnosed with type 2 diabetes, four diagnosed with antibody-negative type 1 diabetes (T1DM), three diagnosed with antibody-positive T1DM, and eight previously diagnosed with MODY. Prevalence of MODY variants in the sequenced cohort was 2.1%, compared to 0.3% of controls. CONCLUSIONS: This is the first comprehensive study of MODY variants in an unselected population-based pediatric diabetes cohort. The observed prevalence, increasing access to rapid and affordable genetic screening, and significant clinical implications suggest that genetic screening for MODY could be considered for all children with diabetes, irrespective of other clinical features.

HLA and KIR Associations of Cervical Neoplasia.

Background: Cervical cancer is the fourth most common cancer in women, and we recently reported human leukocyte antigen (HLA) alleles showing strong associations with cervical neoplasia risk and protection. HLA ligands are recognized by killer immunoglobulin-like receptors (KIRs) expressed on a range of immune cell subsets, governing their proinflammatory activity. We hypothesized that the inheritance of particular HLA-KIR combinations would increase cervical neoplasia risk. Methods: Here, we used HLA and KIR dosages imputed from single-nucleotide polymorphism genotype data from 2143 cervical neoplasia cases and 13858 healthy controls of European decent. Results: The following 4 novel HLA alleles were identified in association with cervical neoplasia, owing to their linkage disequilibrium with known cervical neoplasia-associated HLA-DRB1 alleles: HLA-DRB3*9901 (odds ratio [OR], 1.24; P = 2.49 × 10-9), HLA-DRB5*0101 (OR, 1.29; P = 2.26 × 10-8), HLA-DRB5*9901 (OR, 0.77; P = 1.90 × 10-9), and HLA-DRB3*0301 (OR, 0.63; P = 4.06 × 10-5). We also found that homozygosity of HLA-C1 group alleles is a protective factor for human papillomavirus type 16 (HPV16)-related cervical neoplasia (C1/C1; OR, 0.79; P = .005). This protective association was restricted to carriers of either KIR2DL2 (OR, 0.67; P = .00045) or KIR2DS2 (OR, 0.69; P = .0006). Conclusions: Our findings suggest that HLA-C1 group alleles play a role in protecting against HPV16-related cervical neoplasia, mainly through a KIR-mediated mechanism.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects.

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition.

Identification of a novel FGFRL1 MicroRNA target site polymorphism for bone mineral density in meta-analyses of genome-wide association studies.

MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNA target sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)- and femoral neck (FN)-bone mineral density (BMD). In stage I, 41 102 poly-miRTSs were meta-analyzed in seven cohorts with a genome-wide significance (GWS) α = 0.05/41 102 = 1.22 × 10(-6). By applying α = 5 × 10(-5) (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P = 7.67 × 10(-6) and 1.58 × 10(-5)) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P = 5.08 × 10(-3)) at α = 0.10/11 = 9.09 × 10(-3). PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P = 7.55 × 10(-6)) at α = 0.05/2 = 0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P = 8.87 × 10(-12)). Dual-luciferase reporter assays demonstrated that FGFRL1 3' untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation.

Homozygous variant in C21orf2 in a case of Jeune syndrome with severe thoracic involvement: Extending the phenotypic spectrum.

We previously reported exome sequencing in a short-rib thoracic dystrophy (SRTD) cohort, in whom recessive mutations were identified in SRTD-associated genes in 10 of 11 cases. A heterozygous stop mutation in the known SRTD gene WDR60 was identified in the remaining case; no novel candidate gene/s were suggested by homozygous/compound heterozygous analysis. This case was thus considered unsolved. Re-analysis following an analysis pipeline update identified a homozygous mutation in C21orf2 (c.218G > C; p.Arg73Pro). This homozygous variant was previously removed at the quality control stage by the default GATK parameter "in-breeding co-efficient." C21orf2 was recently associated with both Jeune asphyxiating thoracic dystrophy (JATD) and axial spondylometaphyseal dysplasia (axial SMD); this particular mutation was reported in homozygous and compound heterozygous state in both conditions. Our case has phenotypic features of both JATD and axial SMD; and the extent of thoracic involvement appears more severe than in other C21orf2-positive cases. Identification of a homozygous C21orf2 mutation in this case emphasizes the value of exome sequencing for simultaneously screening known genes and identifying novel genes. Additionally, it highlights the importance of re-interrogating data both as novel gene associations are identified and as analysis pipelines are refined. Finally, the severity of thoracic restriction in this case adds to the phenotypic spectrum attributable to C21orf2 mutations.

Mutations in LTBP3 cause acromicric dysplasia and geleophysic dysplasia.

BACKGROUND: Acromelic dysplasias are a group of disorders characterised by short stature, brachydactyly, limited joint extension and thickened skin and comprises acromicric dysplasia (AD), geleophysic dysplasia (GD), Myhre syndrome and Weill-Marchesani syndrome. Mutations in several genes have been identified for these disorders (including latent transforming growth factor ß (TGF-ß)-binding protein-2 (LTBP2), ADAMTS10, ADAMSTS17 and fibrillin-1 (FBN1) for Weill-Marchesani syndrome, ADAMTSL2 for recessive GD and FBN1 for AD and dominant GD), encoding proteins involved in the microfibrillar network. However, not all cases have mutations in these genes. METHODS: Individuals negative for mutations in known acromelic dysplasia genes underwent whole exome sequencing. RESULTS: A heterozygous missense mutation (exon 14: c.2087C>G: p.Ser696Cys) in latent transforming growth factor ß (TGF-ß)-binding protein-3 (LTBP3) was identified in a dominant AD family. Two distinct de novo heterozygous LTPB3 mutations were also identified in two unrelated GD individuals who had died in early childhood from respiratory failure-a donor splice site mutation (exon 12 c.1846+5G>A) and a stop-loss mutation (exon 28: c.3912A>T: p.1304*Cysext*12). CONCLUSIONS: The constellation of features in these AD and GD cases, including postnatal growth retardation of long bones and lung involvement, is reminiscent of the null ltbp3 mice phenotype. We conclude that LTBP3 is a novel component of the microfibrillar network involved in the acromelic dysplasia spectrum.

Fryns Syndrome Associated with Recessive Mutations in PIGN in two Separate Families.

Fryns syndrome is an autosomal recessive condition characterized by congenital diaphragmatic hernia (CDH), dysmorphic facial features, distal digital hypoplasia, and other associated malformations, and is the most common syndromic form of CDH. No gene has been associated with this condition. Whole-exome sequence data from two siblings and three unrelated individuals with Fryns syndrome were filtered for rare, good quality, coding mutations fitting a recessive inheritance model. Compound heterozygous mutations in PIGN were identified in the siblings, with appropriate parental segregation: a novel STOP mutation (c.1966C>T: p.Glu656X) and a rare (minor allele frequency <0.001) donor splice site mutation (c.1674+1G>C) causing skipping of exon 18 and utilization of a cryptic acceptor site in exon 19. A further novel homozygous STOP mutation in PIGN (c.694A>T: p.Lys232X) was detected in one unrelated case. All three variants affected highly conserved bases. The two remaining cases were negative for PIGN mutations. Mutations in PIGN have been reported in cases with multiple congenital anomalies, including one case with syndromic CDH. Fryns syndrome can be caused by recessive mutations in PIGN. Whether PIGN affects other syndromic and non-syndromic forms of CDH warrants investigation.

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density.

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10(-8)) level: 14q24.2 (rs227425, P-value 3.98 × 10(-13), SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10(-9), CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n = 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis.

Genetic determinants of heel bone properties: genome-wide association meta-analysis and replication in the GEFOS/GENOMOS consortium.

Quantitative ultrasound of the heel captures heel bone properties that independently predict fracture risk and, with bone mineral density (BMD) assessed by X-ray (DXA), may be convenient alternatives for evaluating osteoporosis and fracture risk. We performed a meta-analysis of genome-wide association (GWA) studies to assess the genetic determinants of heel broadband ultrasound attenuation (BUA; n = 14 260), velocity of sound (VOS; n = 15 514) and BMD (n = 4566) in 13 discovery cohorts. Independent replication involved seven cohorts with GWA data (in silico n = 11 452) and new genotyping in 15 cohorts (de novo n = 24 902). In combined random effects, meta-analysis of the discovery and replication cohorts, nine single nucleotide polymorphisms (SNPs) had genome-wide significant (P < 5 × 10(-8)) associations with heel bone properties. Alongside SNPs within or near previously identified osteoporosis susceptibility genes including ESR1 (6q25.1: rs4869739, rs3020331, rs2982552), SPTBN1 (2p16.2: rs11898505), RSPO3 (6q22.33: rs7741021), WNT16 (7q31.31: rs2908007), DKK1 (10q21.1: rs7902708) and GPATCH1 (19q13.11: rs10416265), we identified a new locus on chromosome 11q14.2 (rs597319 close to TMEM135, a gene recently linked to osteoblastogenesis and longevity) significantly associated with both BUA and VOS (P < 8.23 × 10(-14)). In meta-analyses involving 25 cohorts with up to 14 985 fracture cases, six of 10 SNPs associated with heel bone properties at P < 5 × 10(-6) also had the expected direction of association with any fracture (P < 0.05), including three SNPs with P < 0.005: 6q22.33 (rs7741021), 7q31.31 (rs2908007) and 10q21.1 (rs7902708). In conclusion, this GWA study reveals the effect of several genes common to central DXA-derived BMD and heel ultrasound/DXA measures and points to a new genetic locus with potential implications for better understanding of osteoporosis pathophysiology.

Short-rib polydactyly and Jeune syndromes are caused by mutations in WDR60.

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis.

Mutations in the gene encoding IFT dynein complex component WDR34 cause Jeune asphyxiating thoracic dystrophy.

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.