Lifetime risk and genetic predisposition to post-traumatic OA of the knee in the UK Biobank.

OBJECTIVE: Acute knee injury is associated with post-traumatic OA (PTOA). Very little is known about the genome-wide associations of PTOA when compared with idiopathic OA (iOA). Our objective was to describe the development of knee OA after a knee injury and its genetic associations in UK Biobank (UKB). DESIGN: Clinically significant structural knee injuries in those ≤50 years were identified from electronic health records and self-reported data in 502,409 UKB participants. Time-to-first knee osteoarthritis (OA) code was compared in injured cases and age-/sex-matched non-injured controls using Cox Proportional Hazards models. A time-to-OA genome-wide association study (GWAS) sought evidence for PTOA risk variants 6 months to 20 years following injury. Evidence for associations of two iOA polygenic risk scores (PRS) was sought. RESULTS: Of 4233 knee injury cases, 1896 (44.8%) were female (mean age at injury 34.1 years [SD 10.4]). Over a median of 30.2 (IQR 19.5-45.4) years, 1096 (25.9%) of injured cases developed knee OA. The overall hazards ratio (HR) for knee OA after injury was 1.81 (1.70,1.93), P = 8.9 × 10-74. Female sex and increasing age at injury were associated with knee OA following injury (HR 1.15 [1.02,1.30];1.07 [1,07,1.07] respectively). OA risk was highest in the first 5 years after injury (HR 3.26 [2.67,3.98]), persisting for 40 years. In 3074 knee injury cases included in the time-to-OA GWAS, no variants reached genome-wide significance. iOA PRS was not associated with time-to-OA (HR 0.43 [0.02,8.41]). CONCLUSIONS: Increasing age at injury and female sex appear to be associated with future development of PTOA in UKB, the risk of which was greatest in the 5 years after injury. Further international efforts towards a better-powered meta-analysis will definitively elucidate genetic similarities and differences of PTOA and iOA.

Correction: Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa.

The fortieth author's name was listed incorrectly. The correct presentation is A Keski-Rahkonen.

Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa.

Anorexia nervosa (AN) is a complex neuropsychiatric disorder presenting with dangerously low body weight, and a deep and persistent fear of gaining weight. To date, only one genome-wide significant locus associated with AN has been identified. We performed an exome-chip based genome-wide association studies (GWAS) in 2158 cases from nine populations of European origin and 15 485 ancestrally matched controls. Unlike previous studies, this GWAS also probed association in low-frequency and rare variants. Sixteen independent variants were taken forward for in silico and de novo replication (11 common and 5 rare). No findings reached genome-wide significance. Two notable common variants were identified: rs10791286, an intronic variant in OPCML (P=9.89 × 10-6), and rs7700147, an intergenic variant (P=2.93 × 10-5). No low-frequency variant associations were identified at genome-wide significance, although the study was well-powered to detect low-frequency variants with large effect sizes, suggesting that there may be no AN loci in this genomic search space with large effect sizes.

A genome-wide association study of anorexia nervosa.

Anorexia nervosa (AN) is a complex and heritable eating disorder characterized by dangerously low body weight. Neither candidate gene studies nor an initial genome-wide association study (GWAS) have yielded significant and replicated results. We performed a GWAS in 2907 cases with AN from 14 countries (15 sites) and 14 860 ancestrally matched controls as part of the Genetic Consortium for AN (GCAN) and the Wellcome Trust Case Control Consortium 3 (WTCCC3). Individual association analyses were conducted in each stratum and meta-analyzed across all 15 discovery data sets. Seventy-six (72 independent) single nucleotide polymorphisms were taken forward for in silico (two data sets) or de novo (13 data sets) replication genotyping in 2677 independent AN cases and 8629 European ancestry controls along with 458 AN cases and 421 controls from Japan. The final global meta-analysis across discovery and replication data sets comprised 5551 AN cases and 21 080 controls. AN subtype analyses (1606 AN restricting; 1445 AN binge-purge) were performed. No findings reached genome-wide significance. Two intronic variants were suggestively associated: rs9839776 (P=3.01 × 10(-7)) in SOX2OT and rs17030795 (P=5.84 × 10(-6)) in PPP3CA. Two additional signals were specific to Europeans: rs1523921 (P=5.76 × 10(-)(6)) between CUL3 and FAM124B and rs1886797 (P=8.05 × 10(-)(6)) near SPATA13. Comparing discovery with replication results, 76% of the effects were in the same direction, an observation highly unlikely to be due to chance (P=4 × 10(-6)), strongly suggesting that true findings exist but our sample, the largest yet reported, was underpowered for their detection. The accrual of large genotyped AN case-control samples should be an immediate priority for the field.

Evidence of cis-acting regulatory variation in PTPN22 in patients with rheumatoid arthritis.

OBJECTIVES: To assess whether there are cis-regulatory polymorphisms that regulate protein tyrosine phosphatase, non-receptor type 22 (PTPN22) expression in rheumatoid arthritis (RA). METHODS: RNA was extracted from positively selected CD56+, CD8+, and CD4+ mononuclear cells and the 'residual' cells from 12 RA patients heterozygous for the PTPN22 C1858T single nucleotide polymorphism (SNP) (rs2476601). Relative allelic expression was measured by single base extension (SBE) assay. RESULTS: There was relative differential allelic expression (DAE ≥ 20%) in eight patients (p < 10(-5)); seven patients demonstrated DAE in more than one cell type; four patients had statistically significant differences between these cell populations (p(corrected) < 0.05). CONCLUSIONS: We have demonstrated significant differences in expression of PTPN22 alleles in RA patients, indicating the probable existence of cis-acting regulatory elements.

Insights into the genetic architecture of osteoarthritis from stage 1 of the arcOGEN study.

OBJECTIVES: The genetic aetiology of osteoarthritis has not yet been elucidated. To enable a well-powered genome-wide association study (GWAS) for osteoarthritis, the authors have formed the arcOGEN Consortium, a UK-wide collaborative effort aiming to scan genome-wide over 7500 osteoarthritis cases in a two-stage genome-wide association scan. Here the authors report the findings of the stage 1 interim analysis. METHODS: The authors have performed a genome-wide association scan for knee and hip osteoarthritis in 3177 cases and 4894 population-based controls from the UK. Replication of promising signals was carried out in silico in five further scans (44,449 individuals), and de novo in 14 534 independent samples, all of European descent. RESULTS: None of the association signals the authors identified reach genome-wide levels of statistical significance, therefore stressing the need for corroboration in sample sets of a larger size. Application of analytical approaches to examine the allelic architecture of disease to the stage 1 genome-wide association scan data suggests that osteoarthritis is a highly polygenic disease with multiple risk variants conferring small effects. CONCLUSIONS: Identifying loci conferring susceptibility to osteoarthritis will require large-scale sample sizes and well-defined phenotypes to minimise heterogeneity.

Is the thrifty genotype hypothesis supported by evidence based on confirmed type 2 diabetes- and obesity-susceptibility variants?

AIMS/HYPOTHESIS: According to the thrifty genotype hypothesis, the high prevalence of type 2 diabetes and obesity is a consequence of genetic variants that have undergone positive selection during historical periods of erratic food supply. The recent expansion in the number of validated type 2 diabetes- and obesity-susceptibility loci, coupled with access to empirical data, enables us to look for evidence in support (or otherwise) of the thrifty genotype hypothesis using proven loci. METHODS: We employed a range of tests to obtain complementary views of the evidence for selection: we determined whether the risk allele at associated 'index' single-nucleotide polymorphisms is derived or ancestral, calculated the integrated haplotype score (iHS) and assessed the population differentiation statistic fixation index (F (ST)) for 17 type 2 diabetes and 13 obesity loci. RESULTS: We found no evidence for significant differences for the derived/ancestral allele test. None of the studied loci showed strong evidence for selection based on the iHS score. We find a high F (ST) for rs7901695 at TCF7L2, the largest type 2 diabetes effect size found to date. CONCLUSIONS/INTERPRETATION: Our results provide some evidence for selection at specific loci, but there are no consistent patterns of selection that provide conclusive confirmation of the thrifty genotype hypothesis. Discovery of more signals and more causal variants for type 2 diabetes and obesity is likely to allow more detailed examination of these issues.

Genetic evidence for a family-based Scandinavian settlement of Shetland and Orkney during the Viking periods.

The Viking age witnessed the expansion of Scandinavian invaders across much of northwestern Europe. While Scandinavian settlements had an enduring cultural impact on North Atlantic populations, the nature and extent of their genetic legacy in places such as Shetland and Orkney is not clear. In order to explore this question further, we have made an extensive survey of both Y-chromosomal and mitochondrial DNA (mtDNA) variation in the North Atlantic region. Our findings indicate an overall Scandinavian ancestry of approximately 44% for Shetland and approximately 30% for Orkney, with approximately equal contributions from Scandinavian male and female subjects in both cases. This contrasts with the situation for the Western Isles, where the overall Scandinavian ancestry is less ( approximately 15%) and where there is a disproportionately high contribution from Scandinavian males. In line with previous studies, we find that Iceland exhibits both the greatest overall amount of Scandinavian ancestry (55%) and the greatest discrepancy between Scandinavian male and female components. Our results suggest that while areas close to Scandinavia, such as Orkney and Shetland, may have been settled primarily by Scandinavian family groups, lone Scandinavian males, who later established families with female subjects from the British Isles, may have been prominent in areas more distant from their homeland.

SNP haplotypes in the angiotensin I-converting enzyme (ACE) gene: analysis of Nigerian family data using gamete competition models.

Gamete competition models were used to explore the relationships between 13 ACE gene polymorphisms and plasma ACE concentration in a set of Nigerian families. Several markers in the 5' and 3' regions of the gene were significantly associated with ACE concentration (P < 10(-4)). Multi-locus genotypes comprising different combinations of markers from the 5' UTR and the 3' region of the gene were also analysed; in addition to G2350A, in the 3' region, two markers from the 5' UTR (A-5466C and A-240T) were found to be associated with ACE concentration. These results are consistent with reports that have suggested the presence of at least two ACE-linked QTLs, and demonstrate the utility of gamete competition models in the exploratory investigation of the relationship between a quantitative trait and multiple variants in a small genomic region.

A radiation hybrid transcript map of the mouse genome.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid-based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation-placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

Linkage and association analysis of angiotensin I-converting enzyme (ACE)-gene polymorphisms with ACE concentration and blood pressure.

Considerable effort has been expended to determine whether the gene for angiotensin I-converting enzyme (ACE) confers susceptibility to cardiovascular disease. In this study, we genotyped 13 polymorphisms in the ACE gene in 1,343 Nigerians from 332 families. To localize the genetic effect, we first performed linkage and association analysis of all the markers with ACE concentration. In multipoint variance-component analysis, this region was strongly linked to ACE concentration (maximum LOD score 7.5). Likewise, most of the polymorphisms in the ACE gene were significantly associated with ACE (P<.0013). The two most highly associated polymorphisms, ACE4 and ACE8, accounted for 6% and 19% of the variance in ACE, respectively. A two-locus additive model with an additive x additive interaction of these polymorphisms explained most of the ACE variation associated with this region. We next analyzed the relationship between these two polymorphisms (ACE4 and ACE8) and blood pressure (BP). Although no evidence of linkage was detected, significant association was found for both systolic and diastolic BP when a two-locus additive model developed for ACE concentration was used. Further analyses demonstrated that an epistasis model provided the best fit to the BP variation. In conclusion, we found that the two polymorphisms explaining the greatest variation in ACE concentration are significantly associated with BP, through interaction, in this African population sample. Our study also demonstrates that greater statistical power can be anticipated with association analysis versus linkage, when markers in strong linkage disequilibrium with a trait locus have been identified. Furthermore, allelic interaction may play an important role in the dissection of complex traits such as BP.

Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses.

EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85%) of the 34 probands: in 22 of these (76%), the mutation was in EXT1; seven patients (24%) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95% and 93% respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP.

Human islets of Langerhans express the delta(C) isoform of calcium/calmodulin-dependent protein kinase II.

BACKGROUND: There is considerable evidence that calcium/calmodulin-dependent protein kinase II (CaM kinase II) plays a key role in insulin secretion and the enzyme provides a candidate gene for Type 2 diabetes. Since several isoforms of the enzyme exist, it is essential to define which are expressed by the beta-cell. METHODS: A human islet cDNA library in lambdaZAPII was screened with a probe for the 5'-end of human gamma CaM kinase II. Since this region is very homologous between the different isoforms, it is expected that isoforms other than gamma would be detected. From each of the six positive clones obtained, DNA was prepared and subjected to PCR using primers spanning the variable region in which the main variability of CaM kinase II isoforms resides. PCR products were purified and sequenced in both directions. The beta-cell line MIN6 was screened for CaM kinase II delta by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blotting. RESULTS: The sequences of five of the human islet PCR products indicated that the clones corresponded to the gamma(B) isoform whose expression in human islets we have previously documented. The other PCR product, however, gave a sequence containing the variable domains II and VII characteristic of CaM kinase II delta. This sequence and the absence of other domains in this region identified the clone as CaM kinase II delta(C). The expression of CaM kinase II delta in MIN6 beta-cells was confirmed by RT-PCR and by Western blotting. CONCLUSIONS: Human islets of Langerhans express the delta(C) isoform of CaM kinase II.

Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3)

The disease non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is characterized by abnormally high blood glucose resulting from a relative deficiency of insulin. It affects about 2% of the world's population and treatment of diabetes and its complications are an increasing health-care burden. Genetic factors are important in the aetiology of NIDDM, and linkage studies are starting to localize some of the genes that influence the development of this disorder. Maturity-onset diabetes of the young (MODY), a single-gene disorder responsible for 2-5% of NIDDM, is characterized by autosomal dominant inheritance and an age of onset of 25 years or younger. MODY genes have been localized to chromosomes 7, 12 and 20 (refs 5, 7, 8) and clinical studies indicate that mutations in these genes are associated with abnormal patterns of glucose-stimulated insulin secretion. The gene on chromosome 7 (MODY2) encodes the glycolytic enzyme glucokinases which plays a key role in generating the metabolic signal for insulin secretion and in integrating hepatic glucose uptake. Here we show that subjects with the MODY3-form of NIDDM have mutations in the gene encoding hepatocyte nuclear factor-1alpha (HNF-1alpha, which is encoded by the gene TCF1). HNF-1alpha is a transcription factor that helps in the tissue-specific regulation of the expression of several liver genes and also functions as a weak transactivator of the rat insulin-I gene.

Woodchuck hepatitis virus: experimental infection and natural occurrence.

Sera from 588 woodchucks were assayed for woodchuck hepatitis virus (WHV) markers using hepatitis B virus (HBV) reagents which have cross-reactivity with WHV markers. Twenty per cent of these woodchucks, trapped in Delaware, Maryland and Pennsylvania, had WHsAg; 50% of these had DNA polymerase. There are areas of high and low endemicity within these states. Female woodchucks may have a higher incidence of WHV markers than do males. Woodchuck hepatitis surface antigen (WHsAg) and anti-WHc often occur together but less commonly than HBsAg and anti-HBc do in human HBV infection. Experimental infection of woodchucks with WHV produced a prolonged infection (up to 40 weeks). WHsAg and DNA polymerase appeared to be more reliable indicators of infectivity than anti-WHc, woodchuck hepatitis e antigen (WHeAg) or anti-WHe. WHeAg was not detected throughout this period of infection, while anti-WHe appeared late in two of three experimentally infected animals. Four male and four female woodchucks which developed primary hepatocellular carcinoma in captivity were analyzed for WHV markers throughout their period of confinement. Seven were WHsAg and anti-WHc positive when captured. The animal that was free of WHV markers on capture converted to the WHsAg and anti-WHc positive state prior to the development of primary hepatocellular carcinoma. One primary hepatocellular carcinoma animal produced WHeAg and none anti-WHs or anti-WHe.

The zoapatle IV--toxicological and clinical studies.

The zoapatle aqueous crude extract has been used in Mexico for the last 5 centuries for the induction of labor, treatment of post-partum bleeding problems, and as a menses inducer. Today, it is sold in street markets, and its long documented history of use by humans could be taken as indirect evidence of a lack of toxicity. Rigorous pharmacological and clinical studies described here, fully confirm the empirical observations.

PIP: For the last 5 centuries in Mexico the zoapatle aqueous crude extract (ZACE) has been used for the induction of labor, treatment of postpartum bleeding problems, and as a menses inducer. Despite widespread and continuous use of ZACE on the part of the Mexican population and the absence of documented side effects or toxicological phenomenon associated with its use, it was decided to undertake acute and subacute toxicological studies prior to clinical studies in volunteer subjects in Mexico and Sweden. Plant specimens for the studies were collected in different batches at several locations near the National University of Mexico. The material was properly identified at the university's botanical herbarium. ZACE was prepared by boiling 100 g of dry leaves in 400 ml of distilled water for 20-30 minutes, filtered through gauze, and the final volume obtained by using a rotor evaporator 50-60 degrees Centigrade with light negative pressure, to 100 ml. The ZACE concentration was 1 g dry leaves/ml. The extract was tested in an "in vitro" guinea pig uterine strip assay. For the 1st series of studies in Mexico, 60 female Sprague-Dawley rats, 8 weeks old, and 6 mongrel female dogs, were used. 25 rats were used as control and 35 tested. All animals were maintained in a controlled laboratory condition with 12/12 hour light/darkness cycle. 2 dogs served as control, and 4 were treated. All experimental animals received, through a rubber cannula, 5 ml/KG of ZACE daily. Control animals received distilled water. Each animal was carefully observed for 30 minutes after fluid intake. At the end of the study, the rats were sacrificed with ether, and the dogs with phenobarbital. Complete anatomapathological examinations were performed. 6 control and 8 treated rats died during the study, and all deaths were associated with accidental placement of the rubber cannula. Behavioral changes were observed in neither rats nor dogs throughout the study. No changes were recorded in body weights. No macro nor microscopic alteration was found in either group of rats. Results of hematological examinations did not differ between the 2 groups. It was concluded that ZACE is devoid of acute and subacute toxicity. In addition to the clinical observations reported by Gallegos in Mexico, where 10 normally menstruating volunteer women received 15 or 30 gm/day of zoapatle dry leaves extract orally for 3-9 consecutive days without clinical evidence of any side effects, clinical investigations were conducted by Landgren et al. in Sweden. 6 women in early pregnancy were given zoapatle; 6 women in early pregnancy served as a control group. A significant dilation of the cervix was found in each of the subjects treated with zoapatle decocts. A menstrual like cramp was reported by all and bleeding occurred in 4 of 6 subjects. No gastrointestinal symptoms were observed. No pain or bleeding nor significant dilatation of the cervix was evident prior to surgery. Immediately prior to vacuum extraction, cervical dilatation was less than 5 mm in all subjects. In sum, ZACE was devoid of toxic complication or side effects.