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To investigate the genetic regulation of platelet (PLT) levels we carried out a whole-genome association analysis in 6,528 Sardinians from the general population of the Lanusei valley. We found 6 variants significantly influencing PLT levels, including a novel rare missense mutation (p.Pro27Ser) in the GP1BB protein that is associated with PLT reduction (P=1.17×10−16). This mutation is rare in the SardiNIA population cohort (frequency of 0.45%), even rarer in the rest of the Sardinian island (frequency of 0.16%), and not reported elsewhere. Notably, GP1BB is involved in Bernard-Soulier syndrome (BSS), a rare autosomal recessive bleeding disorder caused by a defect in the platelet GPIb-IX-V protein complex. Consistently, the 57 identified individuals heterozygous for the p.P27S mutation showed mild thrombocytopenia, morphologically enlarged platelets (P=2.13×10−10), and reduced expression of two GPIb-IX-V-complex components: GPIbα (−26.51%, P=3.66×10−8) and GPIX (−24.69%, P=2.66×10−6). Molecular modeling infers a corresponding reduction in the stability of GP1BB. These observations predict that in homozygosity as well as in individuals carrying specific compound heterozygous configurations, this variant likely causes BSS.

International Parkinson’s Disease Genomics Consortium (IPDGC), UK Brain Expression Consortium (UKBEC)., Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved understanding of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, interrogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain transcriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/., Mina Ryten, David Zhang, and Karishma D’Sa were supported by the UK Medical Research Council (MRC) through the award of Tenure-track Clinician Scientist Fellowship to Mina Ryten (MR/N008324/1). Sebastian Guelfi was supported by Alzheimer’s Research UK through the award of a PhD Fellowship (ARUK-PhD2014-16). Regina Reynolds was supported through the award of a Leonard Wolfson Doctoral Training Fellowship in Neurodegeneration.

Dystonia is a neurological disorder characterized by sustained or intermittent muscle contractions causing abnormal movements and postures, often occurring in absence of any structural brain abnormality. Psychiatric comorbidities, including anxiety, depression, obsessive-compulsive disorder and schizophrenia, are frequent in dystonia patients. While mutations in a fast-growing number of genes have been linked to Mendelian forms of dystonia, the cellular, anatomical, and molecular basis remains unknown for most genetic forms of dystonia, as does its genetic and biological relationship to neuropsychiatric disorders. Here we applied an unbiased systems-biology approach to explore the cellular specificity of all currently known dystonia-associated genes, predict their functional relationships, and test whether dystonia and neuropsychiatric disorders share a genetic relationship. To determine the cellular specificity of dystonia-associated genes in the brain, single-nuclear transcriptomic data derived from mouse brain was used together with expression-weighted cell-type enrichment. To identify functional relationships amongst dystonia-associated genes, we determined the enrichment of these genes in co-expression networks constructed from ten human brain regions. Stratified linkage-disequilibrium score regression was used to test whether co-expression modules enriched for dystonia-associated genes significantly contribute to the heritability of anxiety, major depressive disorder, obsessive-compulsive disorder, schizophrenia, and Parkinson’s disease. Dystonia-associated genes were significantly enriched in adult nigral dopaminergic neurons and striatal medium spiny neurons. Furthermore, four of the 220 gene co-expression modules tested were significantly enriched for the dystonia-associated genes. The identified modules were derived from the substantia nigra, putamen, frontal cortex, and white matter, and were all significantly enriched for genes associated with synaptic function. Finally, we demonstrated significant enrichments of the heritability of depression, obsessive-compulsive disorder and schizophrenia, but not anxiety and Parkinson’s disease, within the putamen and white matter modules. In conclusion, multiple dystonia-associated genes interact and contribute to pathogenesis likely through dysregulation of synaptic signalling in striatal medium spiny neurons, adult nigral dopaminergic neurons and frontal cortical neurons. Furthermore, the enrichment of the heritability of psychiatric disorders in the co-expression modules enriched for dystonia-associated genes indicates that psychiatric symptoms associated with dystonia are likely to be intrinsic to its pathophysiology.

Dystonia is a neurological disorder characterized by sustained or intermittent muscle contractions causing abnormal movements and postures, often occurring in absence of any structural brain abnormality. Psychiatric comorbidities, including anxiety, depression, obsessive-compulsive disorder and schizophrenia, are frequent in patients with dystonia. While mutations in a fast-growing number of genes have been linked to Mendelian forms of dystonia, the cellular, anatomical, and molecular basis remains unknown for most genetic forms of dystonia, as does its genetic and biological relationship to neuropsychiatric disorders. Here we applied an unbiased systems-biology approach to explore the cellular specificity of all currently known dystonia-associated genes, predict their functional relationships, and test whether dystonia and neuropsychiatric disorders share a genetic relationship. To determine the cellular specificity of dystonia-associated genes in the brain, single-nuclear transcriptomic data derived from mouse brain was used together with expression-weighted cell-type enrichment. To identify functional relationships among dystonia-associated genes, we determined the enrichment of these genes in co-expression networks constructed from 10 human brain regions. Stratified linkage-disequilibrium score regression was used to test whether co-expression modules enriched for dystonia-associated genes significantly contribute to the heritability of anxiety, major depressive disorder, obsessive-compulsive disorder, schizophrenia, and Parkinson’s disease. Dystonia-associated genes were significantly enriched in adult nigral dopaminergic neurons and striatal medium spiny neurons. Furthermore, 4 of 220 gene co-expression modules tested were significantly enriched for the dystonia-associated genes. The identified modules were derived from the substantia nigra, putamen, frontal cortex, and white matter, and were all significantly enriched for genes associated with synaptic function. Finally, we demonstrate significant enrichments of the heritability of major depressive disorder, obsessive-compulsive disorder and schizophrenia within the putamen, frontal cortex and white matter modules, and nominal enrichment of the heritability of Parkinson’s disease within the substantia nigra module. In conclusion, multiple dystonia-associated genes interact and contribute to pathogenesis likely through dysregulation of synaptic signalling in striatal medium spiny neurons, adult nigral dopaminergic neurons and frontal cortical neurons. Furthermore, the enrichment of the heritability of psychiatric disorders in the co-expression modules enriched for dystonia-associated genes indicates that psychiatric symptoms associated with dystonia are likely to be intrinsic to its pathophysiology., See Busch and Klein (doi:10.1093/brain/awaa253) for a scientific commentary on this article. Mencacci et al. show that multiple dystonia genes are functionally related and likely contribute to modulation of synaptic signalling in striatal, dopaminergic and frontal cortical neurons. They also demonstrate a genetic relationship between dystonia and psychiatric disorders including depression, OCD and schizophrenia.

Background Weighted Gene Co-expression Network Analysis (WGCNA) is a widely used R software package for the generation of gene co-expression networks (GCN). WGCNA generates both a GCN and a derived partitioning of clusters of genes (modules). We propose k-means clustering as an additional processing step to conventional WGCNA, which we have implemented in the R package km2gcn (k-means to gene co-expression network, https://github.com/juanbot/km2gcn ). Results We assessed our method on networks created from UKBEC data (10 different human brain tissues), on networks created from GTEx data (42 human tissues, including 13 brain tissues), and on simulated networks derived from GTEx data. We observed substantially improved module properties, including: (1) few or zero misplaced genes; (2) increased counts of replicable clusters in alternate tissues (x3.1 on average); (3) improved enrichment of Gene Ontology terms (seen in 48/52 GCNs) (4) improved cell type enrichment signals (seen in 21/23 brain GCNs); and (5) more accurate partitions in simulated data according to a range of similarity indices. Conclusions The results obtained from our investigations indicate that our k-means method, applied as an adjunct to standard WGCNA, results in better network partitions. These improved partitions enable more fruitful downstream analyses, as gene modules are more biologically meaningful.

Rare variants in TREM2 cause susceptibility to late-onset Alzheimer's disease. Here we use microarray-based expression data generated from 101 neuropathologically normal individuals and covering 10 brain regions, including the hippocampus, to understand TREM2 biology in human brain. Using network analysis, we detect a highly preserved TREM2-containing module in human brain, show that it relates to microglia, and demonstrate that TREM2 is a hub gene in 5 brain regions, including the hippocampus, suggesting that it can drive module function. Using enrichment analysis we show significant overrepresentation of genes implicated in the adaptive and innate immune system. Inspection of genes with the highest connectivity to TREM2 suggests that it plays a key role in mediating changes in the microglial cytoskeleton necessary not only for phagocytosis, but also migration. Most importantly, we show that the TREM2-containing module is significantly enriched for genes genetically implicated in Alzheimer's disease, multiple sclerosis, and motor neuron disease, implying that these diseases share common pathways centered on microglia and that among the genes identified are possible new disease-relevant genes.

Background Weighted Gene Co-expression Network Analysis (WGCNA) is a widely used R software package for the generation of gene co-expression networks (GCN). WGCNA generates both a GCN and a derived partitioning of clusters of genes (modules). We propose k-means clustering as an additional processing step to conventional WGCNA, which we have implemented in the R package km2gcn (k-means to gene co-expression network, https://github.com/juanbot/km2gcn). Results We assessed our method on networks created from UKBEC data (10 different human brain tissues), on networks created from GTEx data (42 human tissues, including 13 brain tissues), and on simulated networks derived from GTEx data. We observed substantially improved module properties, including: (1) few or zero misplaced genes; (2) increased counts of replicable clusters in alternate tissues (x3.1 on average); (3) improved enrichment of Gene Ontology terms (seen in 48/52 GCNs) (4) improved cell type enrichment signals (seen in 21/23 brain GCNs); and (5) more accurate partitions in simulated data according to a range of similarity indices. Conclusions The results obtained from our investigations indicate that our k-means method, applied as an adjunct to standard WGCNA, results in better network partitions. These improved partitions enable more fruitful downstream analyses, as gene modules are more biologically meaningful. Electronic supplementary material The online version of this article (doi:10.1186/s12918-017-0420-6) contains supplementary material, which is available to authorized users.

Objective: A meta-analysis of genome-wide linkage studies allows us to summarize the extensive information available from family-based studies, as the field moves into genome-wide association studies. Methods: Here we apply the genome scan meta-analysis (GSMA) method, a rank-based, model-free approach, to combine results across eight independent genome-wide linkages performed on celiac disease (CD), including 554 families with over 1,500 affected individuals. We also investigate the agreement between signals we identified from this meta-analysis of linkage studies and those identified from genome-wide association analysis using a hypergeometric distribution. Results: Not surprisingly, the most significant result was obtained in the HLA region. Outside the HLA region, suggestive evidence for linkage was obtained at the telomeric region of chromosome 10 (10q26.12-qter; p = 0.00366), and on chromosome 8 (8q22.2-q24.21; p = 0.00491). Testing signals of association and linkage within bins showed no significant evidence for co-localization of results. Conclusion: This meta-analysis allowed us to pool the results from available genome-wide linkage studies and to identify novel regions potentially harboring predisposing genetic variation contributing to CD. This study also shows that linkage and association studies may identify different types of disease-predisposing variants.

Autoimmune diseases are chronic disorders initiated by a loss of immunologic tolerance to self-antigens. They cluster within families, and patients may be diagnosed with more than one disease, suggesting pleiotropic genes are involved in the aetiology of different diseases. To identify potential loci, which confer susceptibility to autoimmunity independent of disease phenotype, we pooled results from genome-wide linkage studies, using the genome scan meta-analysis method (GSMA). The meta-analysis included 42 independent studies for 11 autoimmune diseases, using 7350 families with 18 291 affected individuals. In addition to the HLA region, which showed highly significant genome-wide evidence for linkage, we obtained suggestive evidence for linkage on chromosome 16, with peak evidence at 10.0–19.8 Mb. This region may harbour a pleiotropic gene (or genes) conferring risk for several diseases, although no such gene has been identified through association studies. We did not identify evidence for linkage at several genes known to confer increased risk to different autoimmune diseases (PTPN22, CTLA4), even in subgroups of diseases consistently found to be associated with these genes. The relative risks conferred by variants in these genes are modest (

Two functional polymorphisms, a 44 bp insertion/deletion polymorphism in the 5′ regulatory region and a variable number of tandem repeat polymorphisms in the second intron of the serotonin transporter gene (5-HTT), were previously identified and suggested to modulate transcription. The current study was designed to determine the contribution of these polymorphisms in the 5-HTT gene to susceptibility to temporal lobe epilepsy (TLE). Two hundred and seventy six patients with TLE, and 309 age- and sex-matched healthy controls from Calabria (Southern Italy) were studied. Patients and controls were genotyped using the WAVE TM DNA Fragment Analysis System for the insertion/deletion polymorphism in the promoter region (5-HTTLPR), and the GENESCAN TM System for the variable number tandem repeat (VNTR) in the second intron of the 5-HTT gene (5-HTTVNTR). The program UNPHASED was used to compare genotype, allele and haplotype frequencies between cases and controls, including age and gender as covariates in the model. No significant differences between cases and controls were observed for 5-HTTLPR, but a significant association was obtained for the 5-HTTVNTR polymorphism, both modeling genotypes (P-value = 0.0145) or alleles (P-value = 0.0086). Patients with TLE showed lower frequencies of the 10 repeat at 5-HTTVNTR than the controls (26.2% in patients versus 40.8% in controls). The frequency of homozygous individuals for the 10 allele was observed to be lower among patients than the controls (5.2% of patients were 10/10 versus 18.8% of controls). Haplotype analysis did not increase the evidence for association. These results suggest that the serotonin transporter gene may play a role in the etiology of TLE.

Many genome-wide linkage studies in multiple sclerosis (MS) have been performed, but results are disappointing, with linkage confirmed only in the HLA region. We combined results from all available, non-overlapping genome-wide linkage studies in MS using the Genome Search Meta-Analysis method (GSMA). The GSMA is a rank-based analysis, which assesses the strongest evidence for linkage within bins of traditionally 30 cM width on the autosomes and X chromosome. Genome-wide evidence for linkage was confirmed on chromosome 6p (HLA region; P=0.00004). Suggestive evidence for linkage was found on chromosomes 10q (P=0.0077), 18p (P=0.0054) and 20p (P=0.0079). To explore how these results could be affected by bin definition, we analysed the data using different bin widths (20 and 40 cM) and using a shifted 30 cM bin by moving bin boundaries by 15 cM. Consistently significant results were obtained for the 6p region. The regions on 10q and 18p provided suggestive evidence for linkage in some analyses, and, interestingly, a region on 6q, that showed only nominal significance in the original analysis, yielded increased, suggestive significance in two of the additional analyses. These regions may provide targets to focus candidate gene studies or to prioritise results from genome-wide association studies.

Background: The Genome Search Meta-Analysis (GSMA) method enables researchers to pool results across genome-wide linkage studies, to increase the power to detect linkage. Results from individual studies must be extracted, with the maximum evidence for linkage placed into bins, usually of 30 cM width, and ranked within the study. Ranks are then summed across studies, with high summed ranks potentially showing evidence for linkage in the meta-analysis. Objectives: In this paper we study the properties of the GSMA method considering two different issues: (1) data binning from genome-wide results when indexed markers or graphs are available, based on either predefined boundary markers, or equal-length bins; (2) the use of selected instead of genome-wide results, using simulation to estimate power and type I error rates of GSMA. This is relevant when published papers show only summary results (e.g. with NPL score >1). Results: Using digitizing software to extract linkage statistics from graphs and assigning equal bin length is accurate, with the resulting ranking of bins similar to those defined through boundary markers. Simulation results show that power can fall substantially when genome-wide results are not available, particularly when only results from a single marker are available in a linked region. However there is no increase in false positive findings. Conclusions: The GSMA method is robust across different bin definitions and methods of data presentation and extraction. Using studies based on only the top ranked bins does not produce false positive results, but lacks power to detect genes conferring a modest increase in risk. Therefore, we advise that effort should be made to obtain genome-wide results from investigators or from published papers to avoid limiting the utility of the GSMA.

Recently, a major locus on chromosome 7q was found in association with the taste sensitivity to phenylthiocarbamide (PTC) in humans. This region contains the TAS2R38 gene that encodes a member of the TAS2R bitter taste receptor family. Three SNPs within this gene demonstrated a strong association with taster status in Utah families and in an additional sample of 85 unrelated individuals. We studied a small isolated village in eastern Sardinia and carried out a genome-wide scan to map the genetic basis of PTC perception in this population. We performed both qualitative and quantitative PTC-taste linkage analysis. Qualitative analysis was carried out by defining a cut-off from the bimodal distribution of the trait and classifying subjects as tasters and non-tasters (75 and 25%, respectively). Linkage analysis on 131 subjects belonging to a unique large multi-generation pedigree comprising 239 subjects confirmed significant evidence for linkage at 7q35 also in our population. Haplotype analyses of the three SNPs inside the PTC gene allowed us to identify only two haplotypes that were associated with the non-taster phenotype (80% AVI homozygous) and to taster phenotype (40% PAV homozygous and 56% PAV/AVI heterozygous). Sex, age and haplotype effect explained 77.2 % of the total variance in PTC sensitivity.

Importance The core clinical and neuropathological feature of the autosomal dominant spinocerebellar ataxias (SCAs) is cerebellar degeneration. Mutations in the known genes explain only 50% to 60% of SCA cases. To date, no effective treatments exist, and the knowledge of drug-treatable molecular pathways is limited. The examination of overlapping mechanisms and the interpretation of how ataxia genes interact will be important in the discovery of potential disease-modifying agents. Objectives To address the possible relationships among known SCA genes, predict their functions, identify overlapping pathways, and provide a framework for candidate gene discovery using whole-transcriptome expression data. Design, Setting, and Participants We have used a systems biology approach based on whole-transcriptome gene expression analysis. As part of the United Kingdom Brain Expression Consortium, we analyzed the expression profile of 788 brain samples obtained from 101 neuropathologically healthy individuals (10 distinct brain regions each). Weighted gene coexpression network analysis was used to cluster 24 SCA genes into gene coexpression modules in an unsupervised manner. The overrepresentation of SCA transcripts in modules identified in the cerebellum was assessed. Enrichment analysis was performed to infer the functions and molecular pathways of genes in biologically relevant modules. Main Outcomes and Measures Molecular functions and mechanisms implicating SCA genes, as well as lists of relevant coexpressed genes as potential candidates for novel SCA causative or modifier genes. Results Two cerebellar gene coexpression modules were statistically enriched in SCA transcripts ( P = .021 for the tan module and P = 2.87 × 10 −5 for the light yellow module) and contained established granule and Purkinje cell markers, respectively. One module includes genes involved in the ubiquitin-proteasome system and contains SCA genes usually associated with a complex phenotype, while the other module encloses many genes important for calcium homeostasis and signaling and contains SCA genes associated mostly with pure ataxia. Conclusions and Relevance Using normal gene expression in the human brain, we identified significant cell types and pathways in SCA pathogenesis. The overrepresentation of genes involved in calcium homeostasis and signaling may indicate an important target for therapy in the future. Furthermore, the gene networks provide new candidate genes for ataxias or novel genes that may be critical for cerebellar function.

Background Multiple sclerosis (MS) is a common disease of the central nervous system and a major cause of disability amongst young adults. Genome-wide association studies have identified many novel susceptibility loci including rs2248359. We hypothesized that genotypes of this locus could increase the risk of MS by regulating expression of neighboring gene, CYP24A1 which encodes the enzyme responsible for initiating degradation of 1,25-dihydroxyvitamin D3. Methods We investigated this hypothesis using paired gene expression and genotyping data from three independent datasets of neurologically healthy adults of European descent. The UK Brain Expression Consortium (UKBEC) consists of post-mortem samples across 10 brain regions originating from 134 individuals (1231 samples total). The North American Brain Expression Consortium (NABEC) consists of cerebellum and frontal cortex samples from 304 individuals (605 samples total). The brain dataset from Heinzen and colleagues consists of prefrontal cortex samples from 93 individuals. Additionally, we used gene network analysis to analyze UKBEC expression data to understand CYP24A1 function in human brain. Findings The risk allele, rs2248359-C, is strongly associated with increased expression of CYP24A1 in frontal cortex (p-value=1.45×10−13), but not white matter. This association was replicated using data from NABEC (p-value=7.2×10−6) and Heinzen and colleagues (p-value=1.2×10−4). Network analysis shows a significant enrichment of terms related to immune response in eight out of the 10 brain regions. Interpretation The known MS risk allele rs2248359-C increases CYP24A1 expression in human brain providing a genetic link between MS and vitamin D metabolism, and predicting that the physiologically active form of vitamin D3 is protective. Vitamin D3's involvement in MS may relate to its immunomodulatory functions in human brain. Funding Medical Research Council UK; King Faisal Specialist Hospital and Research Centre, Saudi Arabia; Intramural Research Program of the National Institute on Aging, National Institutes of Health, USA., Highlights • SNP associated with MS is also associated with cortical CYP24A1 expression. • CYP24A1 is the vitamin D metabolizing enzyme. • This implies that those who have low brain levels of vitamin D for genetic reasons have increased risk of MS. • This is consistent with epidemiological data on vitamin D and MS.

Renal stone formation is a common multifactorial disorder, of unknown etiology, with an established genetic contribution. Lifetime risk for nephrolithiasis is approximately 10% in Western populations, and uric acid stones account for 5%-10% of all stones, depending on climatic, dietary, and ethnic differences. We studied a small, isolated founder population in Sardinia, characterized by an increased prevalence of uric acid stones, and performed a genomewide search in a deep-rooted pedigree comprising many members who formed uric acid renal stones. The pedigree was created by tracing common ancestors of affected individuals through a genealogical database based on archival records kept by the parish church since 1640. This genealogical information was used as the basis for the study strategy, involving screening for alleles shared among affected individuals, originating from common ancestors, and utilization of large pedigrees to obtain greater power for linkage detection. We performed multistep linkage and allele-sharing analyses. In the initial stage, 382 markers were typed in 14 closely related affected subjects; interesting regions were subsequently investigated in the whole sample. We identified two chromosomal regions that may harbor loci with susceptibility genes for uric acid stones. The strongest evidence was observed on 10q21-q22, where a LOD score of 3.07 was obtained for D10S1652 under an affected-only dominant model, and a LOD score of 3.90 was obtained using a dominant pseudomarker assignment. The localization was supported also by multipoint allele-sharing statistics and by haplotype analysis of familial cases and of unrelated affected subjects collected from the isolate. In the second region on 20q13.1-13.3, multipoint nonparametric scores yielded suggestive evidence in a approximately 20-cM region, and further analysis is needed to confirm and fine-map this putative locus. Replication studies are required to investigate the involvement of these regions in the genetic contribution to uric acid stone formation.

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous disorder characterised by progressive spasticity of the lower limbs. Beside ‘pure’ forms of HSP, ‘complicated’ forms are reported, where spasticity occurs associated with additional symptoms. We recently described an Italian family with a complicated dominant form of HSP (SPG9) and we mapped the gene responsible to 10q23.3–q24.2, in a 12 cM interval between markers D10S564 and D10S603. The phenotypic manifestations in our family are reminiscent of those already described in a smaller British pedigree. We typed individuals from this British family using markers located in the SPG9 critical interval and haplotype reconstruction showed the disorder co-segregating with SPG9. To characterise the SPG9 region better, we constructed a contig of 22 YACs, assigned it to 18 polymorphic markers and positioned 54 ESTs. Furthermore, we searched for ESTs containing a trinucleotide repeat sequence, since anticipation of symptoms was reported in both families. Finally, analysis of a muscle biopsy specimen from one patient was normal, suggesting that, contrary to SPG7, mitochondrial disturbance could not be a primary feature of SPG9.

SummaryWe have recently observed a large pedigree with a new rare autosomal dominant spastic paraparesis. In three subsequent generations, 13 affected individuals presented with bilateral cataracts, gastroesophageal reflux with persistent vomiting, and spastic paraparesis with amyotrophy. Bilateral cataracts occurred in all affected individuals, with the exception of one patient who presented with a chorioretinal dystrophy, whereas clinical signs of spastic paraparesis showed a variable expressivity. Using a genomewide mapping approach, we mapped the disorder to the long arm of chromosome 10 on band q23.3-q24.2, in a 12-cM chromosomal region where additional neurologic disorders have been localized. The spectrum of phenotypic manifestations in this family is reminiscent of a smaller pedigree, reported recently, confirming the possibility of a new syndrome. Finally, the anticipation of symptoms suggests that an unstable trinucleotide repeat may be responsible for the condition.

Although several studies have demonstrated familial aggregation of nonsyndromic cleft palate (CP), the mode of inheritance still remains uncertain. We report the results of complex segregation analysis performed in families of 357 consecutive newborns affected with nonsyndromic CP (i.e., CP not a component feature of malformation syndrome, sequence or association), and registered in the North East Italy and Emilia Romagna congenital malformation registries in the period 1981-1993. This sample, based on a large number of consecutive births, in a well-defined geographical area, with quality control to detect associated anomalies and malformation syndromes, is independent of the number of affected subjects in the family and of CP severity, fitness, and survival. We have analyzed, using the mixed model, the whole sample of nonsyndromic CP, including isolated (i.e., without other anomalies) CP (CPI) and CP associated with at least one other anomaly (CPA), for which a diagnosis of malformation syndrome was not possible. When nonsyndromic CP (including CPA) are considered in the analysis, there is no heterogeneity between CPA and CPI nor between CP including hard palate (CPH) and CP of the soft palate only (CPS). POINTER and COMDS programs cannot discriminate between alternative genetic models; only the hypothesis of non-genetic transmission is rejected. The COMDS analysis two-locus model, which indicates that a modifier locus (or loci) operates in addition to a single major locus (SML), does not show evidence of better fit than SML, polygenic, and multifactorial models. When the severity parameter (defined as CPH and CPS) is added, CPI and CPA show heterogeneity. Eventually, when the analysis is limited to CPI and includes information on severity, a recessive SML, with low penetrance and determining CPH, provides a significant best fit. To have defined a genetic model for CPI and provided evidence for SML inheritance suggests that genetic linkage studies could be implemented. This conclusion is in agreement with previous studies which showed a significant association between alleles of transforming growth factor alpha and CP only in humans, and that single recessive genes may play a crucial role during palatogenesis in mice as well as in Brittany spaniels. Application of the candidate genes to human CPH families could reveal whether these genes are involved.

In contrast to large GWA studies based on thousands of individuals and large meta-analyses combining GWAS results, we analyzed a small case/control sample for uric acid nephrolithiasis. Our cohort of closely related individuals is derived from a small, genetically isolated village in Sardinia, with well-characterized genealogical data linking the extant population up to the 16th century. It is expected that the number of risk alleles involved in complex disorders is smaller in isolated founder populations than in more diverse populations, and the power to detect association with complex traits may be increased when related, homogeneous affected individuals are selected, as they are more likely to be enriched with and share specific risk variants than are unrelated, affected individuals from the general population. When related individuals are included in an association study, correlations among relatives must be accurately taken into account to ensure validity of the results. A recently proposed association method uses an empirical genotypic covariance matrix estimated from genome-screen data to allow for additional population structure and cryptic relatedness that may not be captured by the genealogical data. We apply the method to our data, and we also investigate the properties of the method, as well as other association methods, in our highly inbred population, as previous applications were to outbred samples. The more promising regions identified in our initial study in the genetic isolate were then further investigated in an independent sample collected from the Italian population. Among the loci that showed association in this study, we observed evidence of a possible involvement of the region encompassing the gene LRRC16A, already associated to serum uric acid levels in a large meta-analysis of 14 GWAS, suggesting that this locus might lead a pathway for uric acid metabolism that may be involved in gout as well as in nephrolithiasis., Author Summary There are a number of factors that contribute to renal stone formation, including diet and obesity, specific drugs, other diseases, climate changes, metabolic disorders, and genetic predisposition. In this article, we focus on identifying genomic regions that may be involved with nephrolithiasis associated with a uric acid component. We analyze data from a genetic isolate in Sardinia to take advantage of the potential improvement in power to detect association with complex traits when related, homogeneous affected individuals are selected. To take into account the correlations among our related sample of cases and controls, we applied a recently proposed method that corrects for both known and unknown population and pedigree structure using genome-wide data. In simulation studies for outbred populations with related individuals and population structure, the method has been demonstrated to provide a substantial improvement over a number of existing methods in terms of power and type 1 error. We investigate the properties of this new method, as well as other association methods, in our inbred sample. To our knowledge, this is the first application of this recently proposed method to a founder population. This study is also the first genome-wide association study carried out for uric acid nephrolithiasis.

Asthma is caused by a heterogeneous combination of environmental and genetic factors. In the context of GA2LEN (Global Allergy and Asthma European Network), we carried out meta-analyses of almost all genome-wide linkage screens conducted to date in 20 independent populations from different ethnic origins (>= 3024 families with >= 10 027 subjects) for asthma, atopic asthma, bronchial hyper-responsiveness and five atopy-related traits (total immunoglobulin E level, positive skin test response (SPT) to at least one allergen or to House Dust Mite, quantitative score of SPT (SPTQ) and eosinophils (EOS)). We used the genome scan meta-analysis method to assess evidence for linkage within bins of traditionally 30-cM width, and explored the manner in which these results were affected by bin definition. Meta-analyses were conducted in all studies and repeated in families of European ancestry. Genome-wide evidence for linkage was detected for asthma in two regions (2p21-p14 and 6p21) in European families ascertained through two asthmatic sibs. With regard to atopy phenotypes, four regions reached genome-wide significance: 3p25.3-q24 in all families for SPT and three other regions in European families (2q32-q34 for EOS, 5q23-q33 for SPTQ and 17q12-q24 for SPT). Tests of heterogeneity showed consistent evidence of linkage of SPTQ to 3p11-3q21, whereas between-study heterogeneity was detected for asthma in 2p22-p13 and 6p21, and for atopic asthma in 1q23-q25. This large-scale meta-analysis provides an important resource of information that can be used to prioritize further fine-mapping studies and also be integrated with genome-wide association studies to increase power and better interpret the outcomes of these studies. European Journal of Human Genetics (2010) 18, 700-706; doi: 10.1038/ejhg.2009.224; published online 13 January 2010

artículo (arbitrado) -- Universidad de Costa Rica. Centro de Investigación en Biología Celular y Molecular, 2008. A genome scan meta analysis (GSMA) was carried out on 32 independent genome-wide linkage scan analyses that included 3255 pedigrees with 7413 genotyped cases affected with schizophrenia (SCZ) or related disorders. The primary GSMA divided the autosomes into 120 bins, rank-ordered the bins within each study according to the most positive linkage result in each bin, summed these ranks (weighted for study size) for each bin across studies and determined the empirical probability of a given summed rank (PSR) by simulation. Suggestive evidence for linkage was observed in two single bins, on chromosomes 5q (142–168 Mb) and 2q (103–134 Mb). Genome-wide evidence for linkage was detected on chromosome 2q (119– 152 Mb) when bin boundaries were shifted to the middle of the previous bins. The primary analysis met empirical criteria for ‘aggregate’ genome-wide significance, indicating that some or all of 10 bins are likely to contain loci linked to SCZ, including regions of chromosomes 1, 2q, 3q, 4q, 5q, 8p and 10q. In a secondary analysis of 22 studies of European-ancestry samples, suggestive evidence for linkage was observed on chromosome 8p (16–33 Mb). Although the newer genome-wide association methodology has greater power to detect weak associations to single common DNA sequence variants, linkage analysis can detect diverse genetic effects that segregate in families, including multiple rare variants within one locus or several weakly associated loci in the same region. Therefore, the regions supported by this meta-analysis deserve close attention in future studies. The work reported here was supported by: Medical Research Council (UK) Grants G0400960 (CML) and G9309834 (MO); National Institute of Mental Health Grants 7R01MH062276 (to DFL [Aust/US], CL [France/La Re´union], MO [Cardiff] and DW [Bonn]), 5R01MH068922 (to PG [ENH]), 5R01MH068921 (to AEP [Johns Hopkins]), 5R01MH068881 (to BR [VCU/Ireland]), MH-41953 (to KSK [VCU/Ireland]); MH63356 and MH80299 (to WB [Palau]); MH58586 (to JMS [Aust/US]); MH 56242 (to VLM [US/Sweden]), MH61399 (EMW, MK [Kosrae, South Africa]), NIMH Grant MH062440, Canadian Institutes of Health Research Grants MOP-53216 and MOP- 12155, a National Alliance for Research on Schizophrenia and Depression Distinguished Investigator Award and Canada Research Chair in Schizophrenia Genetics (LMB, ASB [Canada]); Australian National Health and Medical Research Council Grants 910234, 941087, and 971095 (to BJM [Aust/US]), MRC project Grant G880473N, The European Science Foundation, SANE, the Iceland Department of Health, the General Hospital Reykjavik, the Joseph Levy Charitable Foundation, the Wellcome Trust Grant 055379, The Priory Hospital, the Neuroscience Research Charitable Trust, the University of Iceland and the Icelandic Science Council (HMDG [UCL]); Warner-Lambert, Parke-Davis Pharmaceuticals Company and NIMH Grant R01- MH44245 (LEL [US/International]); the Deutsche Forschungsgemeinschaft (HWM [Kiel]; MA WM, SGS, DBW [Indonesia]); the German Israeli Foundation for Scientific Research (BL; DBW); Mammalian Genotyping Service HV48141 (DBW [Indonesia]; CREST of JST (Japan Science and Technology Agency) TA [Japan]; Pfizer, Inc. and the SANE Foundation (LEL [Costa Rica]); the Israel Science Foundation, US. Israel Binational Science Foundation, the National Alliance for Research on Schizophrenia and Depression, and the Harry Stern Family Foundation (BL and YK [Israel]); the VA Merit Review Program (AF); recruitment of the NIMH Genetics Initiative sample was supported by NIMH Grants 5 UO1MH46318, UO1MH46289 and UO1MH46276; the Taiwan Schizophrenia Linkage Study was supported by NIMH Grant 1R01 MH59624-01 and Grant NHRI-90-8825PP, NHRI -EX91,92-9113PP from the National Health Research Institute, Taiwan, and support from the Genomic Medicine Research Program of Psychiatric Disorders, National Taiwan University Hospital; the VA Linkage Study was supported by funds from the Department of Veterans Affairs Cooperative Studies Program; the US/Mexico/Central America study was supported by a collaborative NIMH grant (‘Genetics of Schizophrenia in Latino Populations’) (MH60881 and MH60875) to ME [University of Texas Health Science Center at San Antonio], R Mendoza [University of California at Los Angeles-Harbor], HR [University of Costa Rica, San Jose, Costa Rica], A Ontiveros [Instituto de Informacion de Investigacion en Salud Mental, Monterrey, Mexico], HN [Medical and Family Research Group, Carracci SC, Mexico City, Mexico], and R Munoz [Family Health Centers of San Diego, CA]. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)

Familial late-onset Alzheimer's disease (LOAD) shows high heritability. However, with the exception of ApoE, no well-replicated susceptibility genes have been identified to date. Several genome-wide linkage studies have nominated potential susceptibility loci but results are inconsistent, with individual scans showing few significant LOD scores. We have pooled linkage results from five independent genome scans and used the genome search meta-analysis (GSMA) method to analyse these data. The combined sample results in 2206 affected individuals and 785 families from Caucasian and Caribbean Hispanic ethnicities. The Caucasian samples included subjects from the US, the Netherlands and Sweden. Genome-wide suggestive evidence for linkage was observed on chromosomes 1p13.3-q31.1, 7pter-p21.1 and 8p22-p21.1 in the weighted GSMA analysis. The chromosome 8p region achieved the lowest summed rank p-value of 0.001. We also identified seven loci with nominally significant evidence for linkage to 3q12.3-q22.1, 6p21.1-q15, 7p14.1-q21.11, 17q24.3-qter and 19p13.3-qter. The GSMA finding suggests that these loci may harbour susceptibility genes for LOAD. We have also developed a web resource (http://alzres.iop.kcl.ac.uk/) to present additional GSMA analyses with different study selection criteria, facilitate the reanalysis of genome-wide linkage data and provide open access to the GSMA data.

Purpose Mutations in the genes encoding the alfa 2 , alfa 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR) play a causative role in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Moreover, variations in the promoter of the corticotropic-releasing hormone gene ( CRH ) were also associated with ADNFLE. Here, we investigated whether nine brain-expressed genes ( CHRNA2 , CHRNA3 , CHRNA4 , CHRNA5 , CHRNA6 , CHRNA7 , CHRNB2 , CHRNB3 , CHRNB4 ), encoding distinct nAChR subunits, and CRH are associated with the disease in three distinct ADNFLE families from Southern Italy. Methods There were 14 living affected individuals (9 women), ranging in age from 14 to 57 years, pertaining to three unrelated families. Age at onset of seizures clustered around 9 years of age (range from 7 and 16 years, mean: 9.1 years±3.8). All affected individuals manifested nocturnal partial seizures of frontal lobe origin, which were well controlled by medications. Exon 5 of CHRNA4 and CHRNB2 genes, harboring all the known mutations, was sequenced in the probands. Then, we performed a linkage study on 13 affected and 26 non-affected individuals belonging to the three families with microsatellite markers and an intragenic polymorphisms encompassing the chromosome localization of the nAChR subunit genes and of the CRH gene. Results Mutational and linkage analyses allowed us to exclude the involvement of all known nAChR subunit genes and of the CRH gene in ADNFLE in our families. Conclusion Our results further illustrate the considerable genetic heterogeneity for such a syndrome, despite the quite homogeneous clinical picture. It is therefore reasonable to hypothesize that at least another gene not belonging to the nAChR gene family, in addition to CRH , is involved in the pathogenesis of ADNFLE.

Two functional polymorphisms, a 44bp insertion/deletion polymorphism in the 5' regulatory region and a variable number of tandem repeat polymorphisms in the second intron of the serotonin transporter gene (5-HTT), were previously identified and suggested to modulate transcription. The current study was designed to determine the contribution of these polymorphisms in the 5-HTT gene to susceptibility to temporal lobe epilepsy (TLE). Two hundred and seventy six patients with TLE, and 309 age- and sex-matched healthy controls from Calabria (Southern Italy) were studied. Patients and controls were genotyped using the WAVE TM DNA Fragment Analysis System for the insertion/deletion polymorphism in the promoter region (5-HTTLPR), and the GENESCAN TM System for the variable number tandem repeat (VNTR) in the second intron of the 5-HTT gene (5-HTTVNTR). The program UNPHASED was used to compare genotype, allele and haplotype frequencies between cases and controls, including age and gender as covariates in the model. No significant differences between cases and controls were observed for 5-HTTLPR, but a significant association was obtained for the 5-HTTVNTR polymorphism, both modeling genotypes (P-value=0.0145) or alleles (P-value=0.0086). Patients with TLE showed lower frequencies of the 10 repeat at 5-HTTVNTR than the controls (26.2% in patients versus 40.8% in controls). The frequency of homozygous individuals for the 10 allele was observed to be lower among patients than the controls (5.2% of patients were 10/10 versus 18.8% of controls). Haplotype analysis did not increase the evidence for association. These results suggest that the serotonin transporter gene may play a role in the etiology of TLE.

A genetic contribution to the development of systemic lupus erythematosus (SLE) is well established. Several genome-wide linkage scans have identified a number of putative susceptibility loci for SLE, some of which have been replicated in independent samples. This study aimed to identify the regions showing the most consistent evidence for linkage by applying the genome scan meta-analysis (GSMA) method. The study identified two genome-wide suggestive regions on 6p21.1–q15 and 20p11–q13.13 (P-value=0.0056 and P-value=0.0044, respectively) and a region with P-value

Genetic mapping by linkage analysis has been an invaluable tool in the positional strategy to identify the molecular basis of many rare Mendelian disorders. With the attention of the scientific and medical community shifting towards the analysis of more common, complex traits, it has become necessary to develop new approaches that take into account the complexity of the genetic basis of these disorders and their possible interaction with other, nongenetic factors. Linkage disequilibrium studies are now becoming increasingly popular thanks to the advent of genotyping platforms that allow genome-wide searching for association between hundreds of thousands of random polymorphisms and disease phenotypes in large samples of unrelated individuals. Moreover, the definition of the disease phenotype itself is being reconsidered to include quantitative traits that may better define the underlying biologic mechanisms for many pathologic conditions. This article will review classic and new approaches to genetic mapping by linkage and association analysis and discuss the directions this field is likely to take in the near future.

We have recently mapped a new rare form of spastic paraplegia complicated by bilateral cataracts, gastroesophageal reflux with persistent vomiting, and amyotrophy to chromosome 10q23.3-q24.2. This locus, named SPG9, is located in an interval spanning about 12 cM of genomic DNA, between markers D10S536 and D10S603, where different neurological disorders have been mapped. In particular, a gene for partial epilepsy has been assigned to a 3 cM interval between markers D10S185 and D10S577, which is completely included in the SPG9 critical region. A few families affected with spastic paraplegia and epilepsy have been reported; in the present study, we tested a pedigree with concurrence of spastic paraplegia, epilepsy, and mental retardation inherited as an autosomal dominant trait, using markers located in the SPG9 interval. Haplotype reconstruction excluded the linkage to 10q23.3-q24.2. In addition, the seven different loci so far reported to be associated with autosomal dominant pure forms of spastic paraplegia have been tested and excluded by linkage analysis and haplotype reconstruction, including SPG4 on chromosome 2p22-p21, where a familial form of spastic paraplegia associated with dementia and epilepsy has been mapped. These data confirm genetic heterogeneity in familial spastic paraplegia with epilepsy and suggest a specific locus for the family here analyzed.

Recent studies indicate that, whereas the Sardinian population as a whole is comparable to outbred populations for linkage disequilibrium (LD) mapping of common variants, LD in Sardinian sub-isolates is more extended, making these populations particularly suitable for this approach. To evaluate the extent of LD between microsatellite markers, we compared different sub-populations within Sardinia selected on the basis of their geographical position and isolation: two small isolated villages (Talana, Urzulei), two larger but remote areas (Ogliastra, Nuoro province) and a cohort of samples representing the wider Sardinian population. LD analysis was carried out by using six microsatellite markers that are located on Xq13.3 and that have been extensively studied in different populations. We found different extents and patterns of LD in the sub-population samples depending on their degree of isolation and demographic history. All LD measurements and haplotype analyses indicate that there is a decreasing trend from Talana (the most inbred population, LD up to 9.5-11.5 Mb) to the more outbred Sardinian population (LD only for intervals

We describe a Sicilian family in which optic disc drusen, angioid streaks, and mottled fundus--without dermatological signs of pseudoxanthoma elasticum (PXE)--are present in various combinations and segregate as an autosomal dominant trait. Since these ocular manifestations can be part of the clinical signs of PXE, we examined the possible involvement of a mutation in the ABCC6 gene, which is known to be responsible for PXE.Linkage analysis was performed with both intragenic and flanking markers. We used marker D16B9722 and a single-nucleotide polymorphism located in exon 15 of the ABCC6 gene. LOD score values were calculated on the assumption of a gene frequency of 0.0001 and both complete penetrance and reduced penetrance (90%), with theta values between 0.0 and 0.4.LOD score values excluded the involvement of the ABCC6 gene.The dominant transmission of optic disc drusen, mottled fundus, and angioid streaks in this family is not due to alterations in the ABCC6 gene.

Genetic isolates represent exceptional resources for the mapping of complex traits but not all isolates are similar. We have selected a genetic and cultural isolate, the village of Talana from an isolated area of Sardinia, and propose that this population is suitable for the mapping of complex traits. A wealth of historical and archive data allowed the reconstruction of the demographic and genealogical history of the village. Key features of the population, which has grown slowly with no significant immigration, were defined by using a combination of historical, demographic and genetic studies. The genealogy of each Talana inhabitant was reconstructed and the main maternal and paternal lineages of the village were defined. Haplotype and phylogenetic analyses of the Y chromosome and characterisation of mitochondrial DNA haplogroups were used to determine the number of ancestral village founders. The extent of linkage disequilibrium (LD) was evaluated by the analysis of several microsatellites in chromosomal region Xq13.3, which was previously used to asses the extension of LD. Genealogical reconstructions were confirmed and reinforced by the genetic analyses, since some lineages were found to have merged prior to the beginning of the archival records, suggesting an even smaller number of founders than initially predicted. About 80% of the present-day population appears to derive from eight paternal and eleven maternal ancestral lineages. LD was found to span, on average, a 5-Mb region in Xq13.3. This suggests the possibility of identifying identical-by-descent regions associated with complex traits in a genome-wide search by using a low-density marker map. The present study emphasises the importance of combining genetic studies with genealogical and historical information.

Inflammatory bowel disease (IBD) is a chronic relapsing disorder affecting the gastro-intestinal tract and is subdivided into two main subtypes: Crohn's disease (CD) and ulcerative colitis (UC). Although the aetiology of IBD is unknown, a strong genetic susceptibility is suggested and different candidate regions have been identified for both CD and UC. The IBD1 region on chromosome 16 has been confirmed to be important for susceptibility to CD, whereas conflicting evidence has been obtained for UC. We performed a combined linkage and segregation analysis in the identified IBD1 region on a sample of 82 extended families with IBD using a parametric method implemented in the computer program COMDS. This approach allows simultaneous evaluation of linkage while estimating the mode of inheritance and to include severity of the trait to characterise the CD and UC phenotypes. Our results are consistent with the presence of a major gene in the IBD1 region close to D16S408 involved in both UC and CD. Furthermore, our data support evidence that a single mutation in the gene leads more frequently to UC, whereas inheritance of two mutant alleles results in the more severe CD. In our study the IBD1 locus was found to have a major role in IBD predisposition in the Italian population.

Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive malformations. ARM patients show different degrees of sacral hypodevelopment while the hemisacrum is characteristic of the Currarino syndrome (CS). Cases of CS present an association of ARM, hemisacrum and presacral mass. A gene responsible for CS has recently been mapped in 7q36. Among the genes localized in this critical region, sonic hedgehog (SHH) was thought to represent a candidate gene for CS as well as for ARM with different levels of sacral hypodevelopment according to its role in the differentiation of midline mesoderm. By linkage analysis we confirmed the critical region in one large family with recurrence of CS. In addition. the screening of SHH in 7 CS and in 15 sporadic ARM patients with sacral hypodevelopment allowed us to exclude its role in the pathogenesis of these disorders.

Background Improved characterisation of the risk factors for rheumatoid arthritis raises the possibility that they could be combined to identify individuals at high risk of disease in whom preventive strategies can be evaluated. Our aim was to develop a prediction model capable of identifying such individuals with sufficient accuracy to enable the assessment of preventive treatments. Methods Our prediction model combines odds ratios for 15 HLA-DRB1 alleles, 31 non-HLA single nucleotide polymorphisms (SNPs), and smoking status to classify an individual's risk of seropositive rheumatoid arthritis. Our novel modelling technique employs confidence intervals to classify disease risks using a computer-simulated population. We developed several models (HLA, HLA-10/20/31 SNP, HLA-smoking models) to evaluate the impact of different factors on prediction. The ability of each model to discriminate between rheumatoid arthritis and controls was evaluated in two European cohorts: the Wellcome Trust Case Control Consortium (WTCCC: 1542 cases, 1226 controls) and the UK Rheumatoid Arthritis Genetics Consortium (UKRAG: 2623 cases, 1503 controls). Findings HLA-DRB1 alleles conferred most prediction: the WTCCC HLA-only model classified 50% antibodies to citrullinated protein antigens (ACPA)-positive rheumatoid arthritis versus 17% controls as high risk and 60% controls versus 25% ACPA-positive rheumatoid arthritis as reduced risk. Adding smoking information improved prediction (p=0·00033); SNPs provided no significant benefits. The highest area under the curve was 0·81 (95% CI 0·78–0·85). Only a minority had substantially elevated risks of rheumatoid arthritis: 6·9% ACPA-positive cases and 0·31% controls in WTCCC had an odds ratio for rheumatoid arthritis of more than 20 when evaluated with the HLA-31 SNP model. Interpretation Combined information on HLA-DRB1 alleles and smoking provides informative risk prediction for rheumatoid arthritis. Since only a minority of individuals are at substantially elevated risks, modelling may be best focused in a-priori high-risk groups such as those with family histories of rheumatoid arthritis. Further work is needed to define risk factors with large effect sizes for incorporation within our modelling framework. Funding Arthritis Research UK.

SummaryAlthough astigmatism is a frequent refractive error, its mode of inheritance remains uncertain. Complex segregation analysis was performed, by the POINTER and COMDS programs, with data from a geographically well-defined sample of 125 nuclear families of individuals affected by astigmatism. POINTER could not distinguish between alternative genetic models, and only the hypothesis of no familial transmission could be rejected. After inclusion of the severity parameter, COMDS results defined a genetic model for corneal astigmatism and provided evidence for single-major-locus inheritance. These results suggest that genetic linkage studies could be implemented and that they should be limited to multiplex families with severely affected individuals.

Uric acid nephrolithiasis (UAN) is a common disease with an established genetic component that presents a complex mode of inheritance. While studying an ancient founder population in Talana, a village in Sardinia, we recently identified a susceptibility locus of approximately 2.5 cM for UAN on 10q21-q22 in a relatively small sample that was carefully selected through genealogical information. To refine the critical region and to identify the susceptibility gene, we extended our analysis to severely affected subjects from the same village. We confirm the involvement of this region in UAN through identical-by-descent sharing and autozygosity mapping, and we refine the critical region to an interval of approximately 67 kb associated with UAN by linkage-disequilibrium mapping. After inspecting the genomic sequences available in public databases, we determined that a novel gene overlaps this interval. This gene is divided into 15 exons, spanning a region of approximately 300 kb and generating at least four different proteins (407, 333, 462, and 216 amino acids). Interestingly, the last isoform was completely included in the 67-kb associated interval. Computer-assisted analysis of this isoform revealed at least one membrane-spanning domain and several N- and O-glycosylation consensus sites at N-termini, suggesting that it could be an integral membrane protein. Mutational analysis shows that a coding nucleotide variant (Ala62Thr), causing a missense in exon 12, is in strong association with UAN (P=.0051). Moreover, Ala62Thr modifies predicted protein secondary structure, suggesting that it may have a role in UAN etiology. The present study underscores the value of our small, genealogically well-characterized, isolated population as a model for the identification of susceptibility genes underlying complex diseases. Indeed, using a relatively small sample of affected and unaffected subjects, we identified a candidate gene for multifactorial UAN.

Essential hypertension (EH) is a complex disorder that results from the interaction of a number of susceptibility genes and environmental factors. We studied an isolated Sardinian village (Talana) in which the prevalence of hypertension is comparable to that in most Western populations. Talana exhibits features, such as slow demographic growth, high inbreeding, a low number of founders, stable lifestyle and culture, and accurate genealogical records, that make it suitable for the study of complex disorders. Clinical assessment of the entire adult population (N= approximately 1,000) identified approximately 100 hypertensive subjects. For our study, we selected the individuals with the most-severe EH (i.e., diastolic blood pressure100 mm Hg), belonging to a single deep-rooted pedigree (12 generations), whose common ancestors lived in the 17th century. We performed a three-stage genomewide search using 36 affected individuals, by means of parametric linkage and allele-sharing approaches. LOD scores1 were observed on chromosomes 1, 2, 13, 15, 17, and 19 (stage I). The most striking result was found in a 7.57-cM region on chromosome 2p24-p25. All five nonparametric linkage statistics estimated by the SimWalk2 program lie above the significance threshold of P.008 for the whole region. Similar significance was obtained for 2p24-25 when parametric linkage (LOD score 1.99) and linkage disequilibrium mapping (P=.00006) were used, suggesting that a hypertension-susceptibility locus is located between D2S2278 and D2S168. This finding is strengthened by a recent report of linkage with marker D2S168 in a hypertensive sib-pair sample from China.