Background Genome wide association studies (GWAS) have been hugely successful in identifying susceptibility loci for autoimmune diseases. One interesting outcome of GWAS is the observation that many of the loci are shared across these diseases. The regions identified now require more detailed fine-mapping to localize the association signal and identify putative functional variants. The Immunochip consortium was established to pool confirmed loci from 12 diseases onto a custom genotyping chip investigating ∼200 established autoimmune susceptibility loci. For each locus, all known genetic variation from multiple sources was included. Juvenile idiopathic arthritis (JIA) is the most common arthritic disease of childhood. Candidate gene studies have identified a number of common autoimmune genes that confer susceptibility to JIA, however JIA has been less well studied using large-scale approaches. Objectives To use the Immunochip to fine-map previously associated regions and to identifying novel loci for JIA. Methods Genotyping was performed using the Immunochip, in a large cohort from the UK, US and Germany comprising 1749 JIA oligoarthritis and RF negative polyarthritis cases and 8854 controls. All raw genotyping data was combined for clustering and QC. SNPs failed QC based on a call rate <98% and/or cluster separation score <0.4. Samples failed QC based on a call rate <98%. Outliers of mean heterozygosity, related individuals and ancestral outliers were removed. Final sample size after QC was 1609 cases and 7153 controls. Analysis was performed using logistic regression adjusting for the top 5 principal components in PLINK 1.07. Results Initial analysis has not only confirmed previously associated JIA loci (HLA, PTPN22, IL2, STAT4, PTPN2 and SH2B3/ATXN2) but has strengthened their association, such that all now reach genome-wide significance. A number of novel loci have been identified, some of which showed weak evidence previously, such as IL2RA, IL7R and IRF1, and others which have never been associated with JIA, such as RUNX1, FAS and ANKRD55. These will require validation in independent cohorts. Conclusions The Immunochip project enables cost-effective fine-mapping of autoimmune loci in diseases such as JIA. This preliminary analysis has confirmed and strengthened the association of a number of previously associated genes as well as the identification of novel susceptibility loci for JIA. Further analysis of this data will help characterize all associated variants and identify the likely causal variants for future functional studies. Acknowledgements:Childhood Arthritis Prospective Study (CAPS), Childhood Arthritis Response to Medication Study (SPARKS-CHARMS), BSPAR study group, UK JIA Genetics Consortium (UKJIAGC), Cincinnati Registry for Juvenile Arthritis Genetics (CRJAG), Consortium for Juvenile Arthritis Genetics (CJAG), and USA-Juvenile Arthritis Genetics Cohort (USA-JAGC). Disclosure of Interest None Declared

Background: Osteoarthritis (OA) affects tens of millions of people worldwide. Twin studies and human GWAS have shown that there is a strong genetic component to the disease, however, to date relatively few genes have been linked to OA onset and progression. Using zebrafish, a genetically tractable organism, we aim to identify novel genes relevant to OA. Methods: We undertook a forward genetic screen of 600 zebrafish families, screening for cartilage and bone phenotypes by Alcian blue (cartilage) and Alizarin Red (bone) staining. We identified 5 families with phenotypes resembling OA, including progressive loss of joint mobility, cartilage matrix breakdown and osteophyte (bony spur) formation. We are mapping the causative genes using recombination distance based mapping techniques. Results: We have identified one of the causative genes as chst11 (also known as C4ST1) a gene involved in the metabolism of chondroitin sulphate. Chst11 has also been implicated in human OA. Loss of chst11 leads to misassembly of the cartilage matrix and changes to chondrocyte cell behaviour, such as altered cell proliferation and premature chondrocyte hypertrophy. Additionally loss of chst11 leads to increased osteoblast differentiation in vivo. Conclusions: Identification of a gene from a zebrafish forward genetic screen that has also been implicated in human OA pathogenesis acts as a proof of principle that zebrafish display sufficient similarities in their skeletal system to be a useful model in osteoarthritis. Zebrafish are not only genetically tractable, but are also transparent and use of fluorescent transgenic reporter lines allows us to track gene expression in the living fish in real time. We have generated numerous transgenic reporters marking chondrocytes at various stages of differentiation, as well as osteoblasts and important signalling molecules such as indian hedgehog, these allow us to track changes in the joint in real time at a level impossible in other model organisms. In conclusion zebrafish can form a useful complement to existing models of OA. Disclosure statement: The author has declared no conflicts of interest.

Objectives: Homozygous C1q deficiency is an extremely rare condition and strongly associated with systemic lupus erythematosus. To assess and characterize C1q deficiency in an African-American lupus pedigree, C1q genomic region was evaluated in the lupus cases and family members. Methods: Genomic DNA from patient was obtained and C1q A, B and C gene cluster was sequenced using next generation sequencing method. The identified mutation was further confirmed by direct Sanger sequencing method in the patient and all blood relatives. C1q levels in serum were measured using sandwich ELISA method. Results: In an African-American patient with lupus and C1q deficiency, we identified and confirmed a novel homozygote start codon mutation in C1qA gene that changes amino acid methionine to arginine at position 1. The Met1Arg mutation prevents protein translation (Met1Arg). Mutation analyses of the patient’s family members also revealed the Met1Arg homozygote mutation in her deceased brother who also had lupus with absence of total complement activity consistent with a recessive pattern of inheritance. Conclusion: The identification of new mutation in C1qA gene that disrupts the start codon (ATG to AGG (Met1Arg)) has not been reported previously and it expands the knowledge and importance of the C1q gene in the pathogenesis of lupus especially in the high-risk African-American population.

Background: Osteoarthritis (OA) affects tens of millions of people worldwide. Twin studies and human GWAS have shown that there is a strong genetic component to the disease, however, to date relatively few genes have been linked to OA onset and progression. Using zebrafish, a genetically tractable organism, we aim to identify novel genes relevant to OA. Methods: We undertook a forward genetic screen of 600 zebrafish families, screening for cartilage and bone phenotypes by Alcian blue (cartilage) and Alizarin Red (bone) staining. We identified 5 families with phenotypes resembling OA, including progressive loss of joint mobility, cartilage matrix breakdown and osteophyte (bony spur) formation. We are mapping the causative genes using recombination distance based mapping techniques. Results: We have identified one of the causative genes as chst11 (also known as C4ST1) a gene involved in the metabolism of chondroitin sulphate. Chst11 has also been implicated in human OA. Loss of chst11 leads to misassembly of the cartilage matrix and changes to chondrocyte cell behaviour, such as altered cell proliferation and premature chondrocyte hypertrophy. Additionally loss of chst11 leads to increased osteoblast differentiation in vivo. Conclusions: Identification of a gene from a zebrafish forward genetic screen that has also been implicated in human OA pathogenesis acts as a proof of principle that zebrafish display sufficient similarities in their skeletal system to be a useful model in osteoarthritis. Zebrafish are not only genetically tractable, but are also transparent and use of fluorescent transgenic reporter lines allows us to track gene expression in the living fish in real time. We have generated numerous transgenic reporters marking chondrocytes at various stages of differentiation, as well as osteoblasts and important signalling molecules such as indian hedgehog, these allow us to track changes in the joint in real time at a level impossible in other model organisms. In conclusion zebrafish can form a useful complement to existing models of OA. Disclosure statement: The author has declared no conflicts of interest.

Rubinstein–Taybi syndrome (RTS) is a rare multiple congenital anomaly/intellectual impairment syndrome. Loss of function in CREBBP or EP300 genes has been found in about 50% of patients with RTS. Genotype–phenotype correlations were investigated in 93 patients meeting diagnostic criteria for RTS during 2 international RTS family conferences. Mutation analysis of CREBBP was performed on all 31 coding exons and exon–intron junctions; a subset of patients had FISH analysis for large deletions. A total of 64 different variations were observed in the DNA sequence, and determined to be definitive mutations in 52 patients (56%). Mutations detected included: 10 missense mutations; 36 truncating or splice‐site mutations; and 6 large deletions detectable by FISH. Fourteen patients had synonymous changes of unknown significance. The majority of mutations affected the HAT domain of CREBBP or predicted termination of the protein before the HAT region. Extensive phenotypic data were collected on each patient and analyzed to determine correlations with mutation types, that is, truncating, large deletions, single amino acid substitutions, or no CREBBP mutation. All four groups displayed the characteristic facial and thumb dysmorphology. Growth retardation in height and weight was seen more frequently in patients with no CREBBP mutation; seizure disorder was more frequent in those with CREBBP mutations. Degree of mental retardation was similar in all groups, although there was a trend toward lower IQ and autistic features in patients with large deletions. Similarity in phenotype between the groups implies that the several genes involved in causing RTS likely have effects through the same pathway. © 2008 Wiley‐Liss, Inc.

We agree with Parr et al. that our finding of linkage to 21q in relative pairs affected with autism and regression needs to be replicated. However, it is our hypothesis that regression represents a modification of disease presentation that may not be present in all relatives affected by autism. The goal of our study was to identify markers that segregated with regression. Thus, it was important to examine only those pairs concordant for regression. In this study, Parr and colleagues examined 86 autism affected relative pairs in which at least one had a history of regression, but only 12 of those pairs were concordant. They found no evidence of linkage, and this concurs with our analysis of the larger AGRE data set of affected relative pairs in which one or both had a history of regression (n=136). Defining the phenotype as autism in this group, we found only nominal evidence of linkage on chromosome 21. It is our hope that the large consortiums for genetic studies of autism will accrue large enough samples to conduct an analysis of affected relative pairs concordant for autism and regression that would replicate our study.

Autism is a pervasive developmental disorder with a strong genetic component. While candidate regions of the genome have been identified, location of genes conferring susceptibility to autism has been hindered by the heterogeneity within this clinically defined disorder, and the likely contribution of many genes of weak effect. Subsetting samples on the basis of distinct, nondiagnostic clinical features has been recommended to decrease sample heterogeneity. In this study, linkage analysis was performed on a subset of families in the database of the Autism Genetic Resource Exchange (AGRE). This set of autism-affected relative pairs (n=34) was also concordant for a history of developmental regression as measured by the Autism Diagnostic Interview—Revised (ADI-R). In this sample, a maximum multipoint LOD score of 3.4 under the dominant mode of inheritance and an NPL score of 3.0 (P=1.3 × 10−3) were observed on chromosome 21 near D21S1437. On chromosome 7 near D7S483 a LOD score of 2.0 under the dominant mode of inheritance and an NPL score of 3.7 (P=7.9 × 10−5) were observed. Genetic elements in these regions of 21q and 7q are likely to confer susceptibility to autism or modify the disease presentation in a subgroup of children characterized by a history of developmental regression.

Although there is considerable evidence for a strong genetic component to idiopathic autism, several genome-wide screens for susceptibility genes have been carried out with limited concordance of linked loci, reflecting numerous genes of weak effect and/or sample heterogeneity. In the current study, linkage analysis was carried out in a sample of 62 autism-affected relative pairs with more severe obsessive–compulsive behaviors, selected from a larger (n=115) set of autism-affected relative pairs as a means of reducing sample heterogeneity. Obsessive–compulsive behaviors were assessed using the Autism Diagnostic Interview-Revised (ADI-R). In the sample with more severe obsessive–compulsive behaviors, multipoint NPL scores above 2 were observed on chromosomes 1, 4, 5, 6, 10, 11 and 19, with the strongest evidence for linkage on chromosome 1 at the marker D1S1656, where the multipoint NPL score was 3.06, and the two-point NPL score was 3.21. In follow-up analyses, analyzing the subset of families (n=35) where the patients had the most severe obsessive–compulsive behaviors generated a multipoint NPL score of 2.76, and a two-point NPL score of 2.79, indicating that the bulk of evidence for linkage was derived from the families most severely affected with obsessive–compulsive behaviors. The data suggest that there is an autism susceptibility gene on chromosome 1 and provide further support for the presence of autism susceptibility genes on chromosomes 6 and 19.

OBJECTIVETo determine the genotypic and phenotypic correlations of hearing impairment (HI) in a midwestern US population related to autosomal recessive nonsyndromic hearing loss locus 1 (DFNB1).DESIGNA retrospective review.SETTINGTertiary care children's hospital.PATIENTSA total of 160 consecutive children diagnosed with idiopathic sensorineural hearing loss.MAIN OUTCOME MEASURESGJB2 genotype and audiometric phenotype.RESULTSThe prevalence of subjects with HI having biallelic GJB2-related mutations was 15.3% (24/157). Of these 24 patients, 9 (38%) were homozygous 35delG, 6 (25%) had other biallelic nonsense mutations, and 9 (38%) had a missense mutation of at least 1 allele. The allelic prevalence of 35delG was 8.6% (27/314) in the study population and 48% (23/48) in the DFNB1 group. The M34T allele mutation was next most prevalent at 2.2% (7/314) in the study population and 10% (5/48) in the DFNB1 group. Severe to profound HI occurred in 59% of DFNB1 subjects. Genotypes with biallelic nonsense mutations had a high risk of severe to profound HI (88%). DFNB1-related HI was usually bilateral, symmetric, nonprogressive, and had flat audiograms. However, asymmetric HI (22%), sloping audiograms (26%), and even borderline-normal hearing in 1 ear was observed, and these were associated with the presence of at least 1 missense mutation. Two novel mutations, K15T and L90V, were identified. A subject presenting to our clinic with severe to profound HI had a 40% risk of biallelic GJB2 mutation.CONCLUSIONSOur population represents a consecutively enrolled clinic population with sensorineural hearing loss. In our DFNB1-related HI cohort, the 35delG mutation and severe to profound HI rates were lower than previously reported. Our missense mutation and M34T allelic prevalence rates were higher than expected and were associated with a less severe hearing loss. The presence of biallelic nonsense mutations was associated with severe to profound hearing loss in nearly 90% of cases. Mild asymmetric HI and sloping audiograms were more often associated with missense mutations.

A sequencing protocol for the acid beta-glucosidase (GCase) gene (GBA) was developed using a long-range PCR template. This protocol has an advantage of greater DNA yields over similar strategies. Seven Gaucher's disease patients had four novel and five other rare alleles. A non-pseudogene in-frame deletion (g.2600-2602delTAC) and a new complex mutation (null allele) were identified in Gaucher's disease type 1, i.e. the g.2600-2602delTAC deletion is associated with the non-neuronopathic variant. An F251L allele was found in a baby with the collodion skin phenotype. Three mutant alleles were identified in a single primary family with type 3. The patients' father at 45 years is healthy and is heteroallelic for the G202R and E326K alleles. Family studies indicated that E326K is in trans to G202R and L444P, and that isolated E326K is non-pathogenic in this family. A rare mutation R257Q was identified in a type 2 patient, providing an association with neuronopathic disease. A genotype L444P/L444P was noted in a 22-year-old non-neuronopathic patient. Complete gene sequencing showed a new complex allele consisting of L444P and g.7741T > C in the 3' UTR. Three additional complex alleles also involved the 3' UTR. Complete gene characterization in Gaucher's disease should allow greater insights into the correlation of specific alleles with phenotype.

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.

The autoimmune thyroid diseases (AITD), encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT), occur in genetically susceptible individuals. In order to identify the AITD susceptibility genes, we have studied DNA markers in the regions of 8 candidate genes: (1) the HLA region, (2) the TSH receptor, (3) thyroid peroxidase, (4) thyroglobulin, (5) IDDM-4, (6) IDDM-5, (7) Immunoglobulin heavy chain gene and (8) CTLA-4. One hundred and seven subjects from 19 informative families were studied, 14 subjects had GD and 32 subjects had HT. LOD scores were maximized assuming both dominant and recessive modes of inheritance. No linkage was found for any marker in patients with HT. In patients with GD, negative LOD scores were obtained for all the candidate genes, except for markers in the TSH receptor region on chromosome 14q31. Positive LOD scores were found for several markers on 14q31. Marker D14S81 gave the highest score (Z max = 2.05, theta = 0.01) assuming a dominant mode of inheritance and a penetrance of 0.8. These data confirm our previous observations of a lack of a necessary disease locus for AITD in the HLA gene region. Further, the data suggest the presence of an important susceptibility gene on 14q31 but at a considerable distance from the TSH receptor gene.

It has recently been proposed that afferent fibers from skeletal muscle could sense the state of the microvascular circulation, linking ventilation to the degree of peripheral perfusion or vascular distension (Huszczuk et al., Respir. Physiol., 91: 207–226, 1993). Ventilatory and circulatory responses to manipulation of peripheral vascular pressures in the hind limbs of anaestetized (sodium thiopental) sheep were examined. Inflatable balloons were placed at the caudal ends of the abdominal aorta and the vena cava (Vc). Aortic (Ao) occlusion induced a consistent normocapnic decrease in minute ventilation (V̇e). In contrast, V̇e increased significantly during vena cava obstruction, leading to hypocapnia. Small changes in systemic blood pressure were observed (+ 7 mmHg for Ao occlusion and −12 mmHg during Vc obstruction). Moreover, inflation of the caval balloon superimposed on a previously established Ao occlusion, preventing venous drainage of an astomotic inflow, resulted in a significant rise in distal vascular pressures with trivial changes in systolic blood pressure. This led to a gradual rise of V̇e, despite further reduction of the CO2 flux to the lungs. The subsequent deflation of the aortic balloon, exposing the hindlimb vasculature to aortic pressure, resulted in an even more profound hypocapnic hyperpnea. The concurrent arterial blood pressure changes were too small to possibly involve the ventilatory component of the arterial baroreflex. We therefore hypothesize, that perfusion-related afferent signals within the muscles could contribute to respiratory homeostasis by maintaining ventilation of the lungs commensurate with the circulatory state of the muscular apparatus.