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Abdominal aortic aneurysm (AAA) is a multifactorial condition. The transforming growth factor β (TGF-β) pathway regulates vascular remodeling and mutations in its receptor genes, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysm (TAA). The TGF-β pathway may be involved in aneurysm development in general. We performed an association study by analyzing all the common genetic variants in TGFBR1 and TGFBR2 using tag single nucleotide polymorphisms (SNPs) in a Dutch AAA case–control population in a two-stage genotyping approach. In stage 1, analyzing 376 cases and 648 controls, three of the four TGFBR1 SNPs and nine of the 28 TGFBR2 SNPs had a P<0.07. Genotyping of these SNPs in an independent cohort of 360 cases and 376 controls in stage 2 confirmed association (P<0.05) for the same allele of one SNP in TGFBR1 and two SNPs in TGFBR2. Joint analysis of the 736 cases and 1024 controls showed statistically significant associations of these SNPs, which sustained after proper correction for multiple testing (TGFBR1 rs1626340 OR 1.32 95% CI 1.11–1.56 P=0.001 and TGFBR2 rs1036095 OR 1.32 95% CI 1.12–1.54 P=0.001 and rs4522809 OR 1.28 95% CI 1.12–1.46 P=0.0004). We conclude that genetic variations in TGFBR1 and TGFBR2 associate with AAA in the Dutch population. This suggests that AAA may develop partly by similar defects as TAA, which in the future may provide novel therapeutic options. [ABSTRACT FROM AUTHOR]

Background: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models., Results: pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors., Conclusion: Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important., (© 2023. The Author(s).)

Background: SCN1A is one of the most important epilepsy-related genes, with pathogenic variants leading to a range of phenotypes with varying disease severity. Different modifying factors have been hypothesized to influence SCN1A-related phenotypes. We investigate the presence of rare and more common variants in epilepsy-related genes as potential modifiers of SCN1A-related disease severity., Methods: 87 patients with SCN1A-related epilepsy were investigated. Whole-exome sequencing was performed by the Beijing Genomics Institute (BGI). Functional variants in 422 genes associated with epilepsy and/or neuronal excitability were investigated. Differences in proportions of variants between the epilepsy genes and four control gene sets were calculated, and compared to the proportions of variants in the same genes in the ExAC database., Results: Statistically significant excesses of variants in epilepsy genes were observed in the complete cohort and in the combined group of mildly and severely affected patients, particularly for variants with minor allele frequencies of <0.05. Patients with extreme phenotypes showed much greater excesses of epilepsy gene variants than patients with intermediate phenotypes., Conclusion: Our results indicate that relatively common variants in epilepsy genes, which would not necessarily be classified as pathogenic, may play a large role in modulating SCN1A phenotypes. They may modify the phenotypes of both severely and mildly affected patients. Our results may be a first step toward meaningful testing of modifier gene variants in regular diagnostics for individual patients, to provide a better estimation of disease severity for newly diagnosed patients., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: Pathogenic variants in SCN1A cause variable epilepsy disorders with different disease severities. We here investigate whether common variation in the promoter region of the unaffected SCN1A allele could reduce normal expression, leading to a decreased residual function of Nav1.1, and therefore to more severe clinical outcomes in patients affected by pathogenic SCN1A variants., Methods: Five different SCN1A promoter-haplotypes were functionally assessed in SH-SY5Y cells using Firefly and Renilla luciferase assays. The SCN1A promoter region was analyzed in a cohort of 143 participants with SCN1A pathogenic variants. Differences in clinical features and outcomes between participants with and without common variants in the SCN1A promoter-region of their unaffected allele were investigated., Results: All non-wildtype haplotypes showed a significant reduction in luciferase expression, compared to the wildtype promoter-region (65%-80%, p = 0.039-0.0023). No statistically significant differences in clinical outcomes were observed between patients with and without common promoter variants. However, patients with a wildtype promoter-haplotype on their unaffected SCN1A allele showed a nonsignificant trend for milder phenotypes., Conclusion: The nonsignificant observed trends in our study warrant replication studies in larger cohorts to explore the potential modifying role of these common SCN1A promoter-haplotypes., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: Dravet syndrome is a severe genetic encephalopathy, caused by pathogenic variants in SCN1A. Low-grade parental mosaicism occurs in a substantial proportion of families (7%-13%) and has important implications for recurrence risks. However, parental mosaicism can remain undetected by methods regularly used in diagnostics. In this study, we use single-molecule molecular inversion probes (smMIP), a technique with high sensitivity for detecting low-grade mosaic variants and high cost-effectiveness, to investigate the incidence of parental mosaicism of SCN1A variants in a cohort of 90 families and assess the feasibility of this technique., Methods: Deep sequencing of SCN1A was performed using smMIPs. False positive rates for each of the proband's pathogenic variants were determined in 145 unrelated samples. If parents showed corresponding variant alleles at a significantly higher rate than the established noise ratio, mosaicism was confirmed by droplet digital PCR (ddPCR)., Results: Sequence coverage of at least 100× at the location of the corresponding pathogenic variant was reached for 80 parent couples. The variant ratio was significantly higher than the established noise ratio in eight parent couples, of which four (5%) were regarded as true mosaics, based on ddPCR results. The false positive rate of smMIP analysis without ddPCR was therefore 50%. Three of these variants had previously been considered de novo in the proband by Sanger sequencing., Conclusion: smMIP technology combined withnext generation sequencing (NGS) performs better than Sanger sequencing in the detection of parental mosaicism. Because parental mosaicism has important implications for genetic counselling and recurrence risks, we stress the importance of implementing high-sensitivity NGS-based assays in standard diagnostics., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Phenotypes caused by de novo SCN1A pathogenic variants are very variable, ranging from severely affected patients with Dravet syndrome to much milder genetic epilepsy febrile seizures plus cases. The most important determinant of disease severity is the type of variant, with variants that cause a complete loss of function of the SCN1A protein (α-subunit of the neuronal sodium channel Nav1.1) being detected almost exclusively in Dravet syndrome patients. However, even within Dravet syndrome disease severity ranges greatly, and consequently other disease modifiers must exist. A better prediction of disease severity is very much needed in daily practice to improve counseling, stressing the importance of identifying modifying factors in this patient group. We evaluated 128 participants with de novo, pathogenic SCN1A variants to investigate whether mosaicism, caused by postzygotic mutation, is a major modifier in SCN1A-related epilepsy., Methods: Mosaicism was investigated by reanalysis of the pathogenic SCN1A variants using single molecule molecular inversion probes and next generation sequencing with high coverage. Allelic ratios of pathogenic variants were used to determine whether mosaicism was likely. Selected mosaic variants were confirmed by droplet digital polymerase chain reaction and sequencing of different tissues. Developmental outcome was classified based on available data on intelligence quotient and school functioning/education., Results: Mosaicism was present for 7.5% of de novo pathogenic SCN1A variants in symptomatic patients. Mosaic participants were less severely affected than nonmosaic participants if only participants with truncating variants are considered (distribution of developmental outcome scores, Mann-Whitney U, P = .023)., Significance: Postzygotic mutation is a common phenomenon in SCN1A-related epilepsies. Participants with mosaicism have on average milder phenotypes, suggesting that mosaicism can be a major modifier of SCN1A-related diseases. Detection of mosaicism has important implications for genetic counseling and can be achieved by deep sequencing of unique reads., (© 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.)

Background: Mutations in the KIAA2022 gene have been reported in male patients with X-linked intellectual disability, and related female carriers were unaffected. Here, we report 14 female patients who carry a heterozygous de novo KIAA2022 mutation and share a phenotype characterised by intellectual disability and epilepsy., Methods: Reported females were selected for genetic testing because of substantial developmental problems and/or epilepsy. X-inactivation and expression studies were performed when possible., Results: All mutations were predicted to result in a frameshift or premature stop. 12 out of 14 patients had intractable epilepsy with myoclonic and/or absence seizures, and generalised in 11. Thirteen patients had mild to severe intellectual disability. This female phenotype partially overlaps with the reported male phenotype which consists of more severe intellectual disability, microcephaly, growth retardation, facial dysmorphisms and, less frequently, epilepsy. One female patient showed completely skewed X-inactivation, complete absence of RNA expression in blood and a phenotype similar to male patients. In the six other tested patients, X-inactivation was random, confirmed by a non-significant twofold to threefold decrease of RNA expression in blood, consistent with the expected mosaicism between cells expressing mutant or normal KIAA2022 alleles., Conclusions: Heterozygous loss of KIAA2022 expression is a cause of intellectual disability in females. Compared with its hemizygous male counterpart, the heterozygous female disease has less severe intellectual disability, but is more often associated with a severe and intractable myoclonic epilepsy., Competing Interests: KLH and ST are employed by and receive a salary from Ambry Genetics. BAM was supported by Genome Canada and the Ontario Brain Institute. BM, SB and SS are funded by the Epilepsy Society and Wellcome Trust. Part of this work was undertaken at University College London Hospitals, which received a proportion of funding from the NIHR Biomedical Research Centres funding scheme., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Coronary Artery Disease (CAD) remains the leading cause of mortality worldwide. Mortality rates associated with CAD have shown an exceptional increase particularly in fast developing economies like the Kingdom of Saudi Arabia (KSA). Over the past twenty years, CAD has become the leading cause of death in KSA and has reached epidemic proportions. This rise is undoubtedly caused by fast urbanization that is associated with a life-style that promotes CAD. However, the question remains whether genetics play a significant role and whether genetic susceptibility is increased in KSA compared to the well-studied Western European populations. Therefore, we performed an Exome-wide association study (EWAS) in 832 patients and 1,076 controls of Saudi Arabian origin to test whether population specific, strong genetic risk factors for CAD exist, or whether the polygenic risk score for known genetic risk factors for CAD, lipids, and Type 2 Diabetes show evidence for an enriched genetic burden. Our results do not show significant associations for a single genetic locus. However, the heritability estimate for CAD for this population was high (h(2) = 0.53, S.E. = 0.1, p = 4e(-12)) and we observed a significant association of the polygenic risk score for CAD that demonstrates that the population of KSA, at least in part, shares the genetic risk associated to CAD in Western populations.

Birdshot chorioretinopathy (BSCR) is a rare form of autoimmune uveitis that can lead to severe visual impairment. Intriguingly, >95% of cases carry the HLA-A29 allele, which defines the strongest documented HLA association for a human disease. We have conducted a genome-wide association study in 96 Dutch and 27 Spanish cases, and 398 unrelated Dutch and 380 Spanish controls. Fine-mapping the primary MHC association through high-resolution imputation at classical HLA loci, identified HLA-A*29:02 as the principal MHC association (odds ratio (OR) = 157.5, 95% CI 91.6-272.6, P = 6.6 × 10(-74)). We also identified two novel susceptibility loci at 5q15 near ERAP2 (rs7705093; OR = 2.3, 95% CI 1.7-3.1, for the T allele, P = 8.6 × 10(-8)) and at 14q32.31 in the TECPR2 gene (rs150571175; OR = 6.1, 95% CI 3.2-11.7, for the A allele, P = 3.2 × 10(-8)). The association near ERAP2 was confirmed in an independent British case-control samples (combined meta-analysis P = 1.7 × 10(-9)). Functional analyses revealed that the risk allele of the polymorphism near ERAP2 is strongly associated with high mRNA and protein expression of ERAP2 in B cells. This study further defined an extremely strong MHC risk component in BSCR, and detected evidence for a novel disease mechanism that affects peptide processing in the endoplasmic reticulum., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Recently, de novo SCN8A missense mutations have been identified as a rare dominant cause of epileptic encephalopathies (EIEE13). Functional studies on the first described case demonstrated gain-of-function effects of the mutation. We describe a novel de novo mutation of SCN8A in a patient with epileptic encephalopathy, and functional characterization of the mutant protein., Design: Whole exome sequencing was used to discover the variant. We generated a mutant cDNA, transfected HEK293 cells, and performed Western blotting to assess protein stability. To study channel functional properties, patch-clamp experiments were carried out in transfected neuronal ND7/23 cells., Results: The proband exhibited seizure onset at 6 months of age, diffuse brain atrophy, and more profound developmental impairment than the original case. The mutation p.Arg233Gly in the voltage sensing transmembrane segment D1S4 was present in the proband and absent in both parents. This mutation results in a temperature-sensitive reduction in protein expression as well as reduced sodium current amplitude and density and a relative increased response to a slow ramp stimulus, though this did not result in an absolute increased current at physiological temperatures., Conclusion: The new de novo SCN8A mutation is clearly deleterious, resulting in an unstable protein with reduced channel activity. This differs from the gain-of-function attributes of the first SCN8A mutation in epileptic encephalopathy, pointing to heterogeneity of mechanisms. Since Nav1.6 is expressed in both excitatory and inhibitory neurons, a differential effect of a loss-of-function of Nav1.6 Arg223Gly on inhibitory interneurons may underlie the epilepsy phenotype in this patient., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A genetic contribution to a broad range of epilepsies has been postulated, and particularly copy number variations (CNVs) have emerged as significant genetic risk factors. However, the role of CNVs in patients with epilepsies with complex phenotypes is not known. Therefore, we investigated the role of CNVs in patients with unclassified epilepsies and complex phenotypes. A total of 222 patients from three European countries, including patients with structural lesions on magnetic resonance imaging (MRI), dysmorphic features, and multiple congenital anomalies, were clinically evaluated and screened for CNVs. MRI findings including acquired or developmental lesions and patient characteristics were subdivided and analyzed in subgroups. MRI data were available for 88.3% of patients, of whom 41.6% had abnormal MRI findings. Eighty-eight rare CNVs were discovered in 71 out of 222 patients (31.9%). Segregation of all identified variants could be assessed in 42 patients, 11 of which were de novo. The frequency of all structural variants and de novo variants was not statistically different between patients with or without MRI abnormalities or MRI subcategories. Patients with dysmorphic features were more likely to carry a rare CNV. Genome-wide screening methods for rare CNVs may provide clues for the genetic etiology in patients with a broader range of epilepsies than previously anticipated, including in patients with various brain anomalies detectable by MRI. Performing genome-wide screens for rare CNVs can be a valuable contribution to the routine diagnostic workup in patients with a broad range of childhood epilepsies.

Intra-tumor heterogeneity is a hallmark of many cancers and may lead to therapy resistance or interfere with personalized treatment strategies. Here, we combined topographic mapping of somatic breakpoints and transcriptional profiling to probe intra-tumor heterogeneity of treatment-naïve stage IIIC/IV epithelial ovarian cancer. We observed that most substantial differences in genomic rearrangement landscapes occurred between metastases in the omentum and peritoneum versus tumor sites in the ovaries. Several cancer genes such as NF1, CDKN2A, and FANCD2 were affected by lesion-specific breakpoints. Furthermore, the intra-tumor variability involved different mutational hallmarks including lesion-specific kataegis (local mutation shower coinciding with genomic breakpoints), rearrangement classes, and coding mutations. In one extreme case, we identified two independent TP53 mutations in ovary tumors and omentum/peritoneum metastases, respectively. Examination of gene expression dynamics revealed up-regulation of key cancer pathways including WNT, integrin, chemokine, and Hedgehog signaling in only subsets of tumor samples from the same patient. Finally, we took advantage of the multilevel tumor analysis to understand the effects of genomic breakpoints on qualitative and quantitative gene expression changes. We show that intra-tumor gene expression differences are caused by site-specific genomic alterations, including formation of in-frame fusion genes. These data highlight the plasticity of ovarian cancer genomes, which may contribute to their strong capacity to adapt to changing environmental conditions and give rise to the high rate of recurrent disease following standard treatment regimes.

In a 24-year-old man with mild intellectual disability, congenital heart defects and obesity, we identified up to 4 small supernumerary marker chromosomes (sSMCs) in blood metaphases. The ring-shaped sSMCs were derived from chromosomes 11, 12 and X as well as a fourth, unidentified chromosome. In interphase nuclei of epithelial cells from the urinary tract and buccal mucosa, the presence of the r(11), r(12) and r(X) was confirmed by FISH. Using Illumina Infinium 317K SNP-arrays, we detected 3 copies of the pericentromeric regions of chromosomes 11, 12 and X. The r(X) was present in 84-89% of cells in the various tissues examined, lacks the XIST gene, but contains FAM123B, a potential dosage-sensitive candidate gene for congenital cardiac abnormalities, and ARHGEF9, a candidate gene for intellectual disability. ARHGEF9 encodes collybistin (CB), which is required for localization of the inhibitory receptor-anchoring protein gephyrin and for formation and maintenance of postsynaptic GABAA and glycine receptors. We propose that the 2-fold increase in dosage of ARHGEF9 disturbs the stoichiometry of CB with its interacting proteins at inhibitory postsynapses. SNP alleles and short tandem repeat markers on the r(11) and r(X) were compatible with a maternal origin of both sSMCs through a meiosis II error. The sSMCs may have resulted from predivision chromatid nondisjunction, leading to anaphase lagging, followed by incomplete degradation of the supernumerary chromosomes.

Systemic sclerosis (SSc) is complex autoimmune disease affecting the connective tissue; influenced by genetic and environmental components. Recently, we performed the first successful genome-wide association study (GWAS) of SSc. Here, we perform a large replication study to better dissect the genetic component of SSc. We selected 768 polymorphisms from the previous GWAS and genotyped them in seven replication cohorts from Europe. Overall significance was calculated for replicated significant SNPs by meta-analysis of the replication cohorts and replication-GWAS cohorts (3237 cases and 6097 controls). Six SNPs in regions not previously associated with SSc were selected for validation in another five independent cohorts, up to a total of 5270 SSc patients and 8326 controls. We found evidence for replication and overall genome-wide significance for one novel SSc genetic risk locus: CSK [P-value = 5.04 × 10(-12), odds ratio (OR) = 1.20]. Additionally, we found suggestive association in the loci PSD3 (P-value = 3.18 × 10(-7), OR = 1.36) and NFKB1 (P-value = 1.03 × 10(-6), OR = 1.14). Additionally, we strengthened the evidence for previously confirmed associations. This study significantly increases the number of known putative genetic risk factors for SSc, including the genes CSK, PSD3 and NFKB1, and further confirms six previously described ones.

Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient's phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diagnosis of the index patient using ADOS-G and ADI-R was performed, and the Social Responsiveness Scale (SRS) was administered to the index patients, both parents, and all available siblings. CNVs were identified using SNP arrays and confirmed by FISH or array CGH. To evaluate the clinical significance of CNVs, we analyzed three families with multiple affected children (multiplex) and six families with a single affected child (simplex) in which at least one child carried a CNV with a brain-transcribed gene. CNVs containing genes that participate in pathways previously implicated in ASD, such as the phosphoinositol signaling pathway (PIK3CA, GIRDIN), contactin-based networks of cell communication (CNTN6), and microcephalin (MCPH1) were found not to co-segregate with ASD phenotypes. In one family, a loss of CNTN5 co-segregated with disease. This indicates that most CNVs may by themselves not be sufficient to cause ASD, but still may contribute to the phenotype by additive or epistatic interactions with inherited (transmitted) mutations or non-genetic factors. Our study extends the scope of genome-wide CNV profiling beyond de novo CNVs in sporadic patients and may aid in uncovering missing heritability in genome-wide screening studies of complex psychiatric disorders.

Background: Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear., Results: We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner., Conclusions: We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10(-12), OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10(-6), OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10(-7), OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10(-61), OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10(-76), OR = 8.84), and in NOTCH4 with ACA P = 8.84×10(-21), OR = 0.55) and ATA (P = 1.14×10(-8), OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc., Competing Interests: The authors have declared that no competing interests exist.

Background: HIV-1 infected macrophages play an important role in rendering resting T cells permissive for infection, in spreading HIV-1 to T cells, and in the pathogenesis of AIDS dementia. During highly active anti-retroviral treatment (HAART), macrophages keep producing virus because tissue penetration of antiretrovirals is suboptimal and the efficacy of some is reduced. Thus, to cure HIV-1 infection with antiretrovirals we will also need to efficiently inhibit viral replication in macrophages. The majority of the current drugs block the action of viral enzymes, whereas there is an abundance of yet unidentified host factors that could be targeted. We here present results from a genome-wide association study identifying novel genetic polymorphisms that affect in vitro HIV-1 replication in macrophages., Methodology/principal Findings: Monocyte-derived macrophages from 393 blood donors were infected with HIV-1 and viral replication was determined using Gag p24 antigen levels. Genomic DNA from individuals with macrophages that had relatively low (n = 96) or high (n = 96) p24 production was used for SNP genotyping with the Illumina 610 Quad beadchip. A total of 494,656 SNPs that passed quality control were tested for association with HIV-1 replication in macrophages, using linear regression. We found a strong association between in vitro HIV-1 replication in monocyte-derived macrophages and SNP rs12483205 in DYRK1A (p = 2.16 × 10(-5)). While the association was not genome-wide significant (p<1 × 10(-7)), we could replicate this association using monocyte-derived macrophages from an independent group of 31 individuals (p = 0.0034). Combined analysis of the initial and replication cohort increased the strength of the association (p = 4.84 × 10(-6)). In addition, we found this SNP to be associated with HIV-1 disease progression in vivo in two independent cohort studies (p = 0.035 and p = 0.0048)., Conclusions/significance: These findings suggest that the kinase DYRK1A is involved in the replication of HIV-1, in vitro in macrophages as well as in vivo.

Background: AIDS develops typically after 7-11 years of untreated HIV-1 infection, with extremes of very rapid disease progression (<2 years) and long-term non-progression (>15 years). To reveal additional host genetic factors that may impact on the clinical course of HIV-1 infection, we designed a genome-wide association study (GWAS) in 404 participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS., Methods: The association of SNP genotypes with the clinical course of HIV-1 infection was tested in Cox regression survival analyses using AIDS-diagnosis and AIDS-related death as endpoints., Results: Multiple, not previously identified SNPs, were identified to be strongly associated with disease progression after HIV-1 infection, albeit not genome-wide significant. However, three independent SNPs in the top ten associations between SNP genotypes and time between seroconversion and AIDS-diagnosis, and one from the top ten associations between SNP genotypes and time between seroconversion and AIDS-related death, had P-values smaller than 0.05 in the French Genomics of Resistance to Immunodeficiency Virus cohort on disease progression., Conclusions: Our study emphasizes that the use of different phenotypes in GWAS may be useful to unravel the full spectrum of host genetic factors that may be associated with the clinical course of HIV-1 infection.

Structural genome aberrations are frequently associated with highly variable congenital phenotypes involving mental retardation and developmental delay. Although some of these aberrations may result in recognizable phenotypes, a high degree of phenotypic variability often complicates a comprehensive clinical and genetic diagnosis. We describe four patients with overlapping deletions in chromosomal region 1q44, who show developmental delay, in particular of expressive speech, seizures, hypotonia, CNS anomalies, including variable thickness of the abnormal corpus callosum in three of them. High resolution oligonucleotide and SNP array-based segmental aneuploidy profiling showed that these three patients share a 0.440 Mb interstitial deletion, which does not overlap with previously published consensus regions of 1q44 deletions. Two copies of AKT3 and ZNF238, two previously proposed dosage sensitive candidate genes for microcephaly and agenesis of the corpus callosum, were retained in two of our patients. The deletion shared by our patients encompassed the FAM36A, HNRPU, EFCAB2 and KIF26B genes. Since HNRPU is involved in the regulation of embryonic brain development, this represents a novel plausible candidate gene for the combination of developmental delay, speech delay, hypotonia, hypo- or agenesis of the corpus callosum, and seizures in patients with 1q44 deletions. Since only one of the two patients with deletions including the ZNF124 gene showed a vermis hypoplasia, mere hemizygosity for this gene is not sufficient to cause this anomaly. Moreover, to reconcile the variability in the corpus callosum thickness, additional mechanisms, such as unmasking of hemizygous mutations, position effects and possible interactions with other loci need consideration., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

High resolution genomic copy-number analysis has shown that inherited and de novo copy-number variations contribute significantly to autism pathology, and that identification of small chromosomal aberrations related to autism will expedite the discovery of risk genes involved. Here, we report a microduplication of chromosome 15q11.2, spanning only four genes, co-segregating with autism in a Dutch pedigree, identified by SNP microarray analysis, and independently confirmed by FISH and MLPA analysis. Quantitative RT-PCR analysis revealed over 70% increase in peripheral blood mRNA levels for the four genes present in the duplicated region in patients, and RNA in situ hybridization on mouse embryonic and adult brain sections revealed that two of the four genes, CYFIP1 and NIPA1, were highly expressed in the developing mouse brain. These findings point towards a contribution of microduplications at chromosome 15q11.2 to autism, and highlight CYFIP1 and NIPA1 as autism risk genes functioning in axonogenesis and synaptogenesis. Thereby, these findings further implicate defects in dosage-sensitive molecular control of neuronal connectivity in autism. However, the prevalence of this microduplication in patient samples was statistically not significantly different from control samples (0.94% in patients vs. 0.42% controls, P = 0.247), which suggests that our findings should be interpreted with caution and indicates the need for studies that include large numbers of control subjects to ascertain the impact of these changes on a population scale.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify new SSc susceptibility loci, we conducted the first genome-wide association study in a population of European ancestry including a total of 2,296 individuals with SSc and 5,171 controls. Analysis of 279,621 autosomal SNPs followed by replication testing in an independent case-control set of European ancestry (2,753 individuals with SSc (cases) and 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22-23, rs2056626, P = 2.09 x 10(-7) in the discovery samples, P = 3.39 x 10(-9) in the combined analysis). Additionally, we confirm and firmly establish the role of the MHC (P = 2.31 x 10(-18)), IRF5 (P = 1.86 x 10(-13)) and STAT4 (P = 3.37 x 10(-9)) gene regions as SSc genetic risk factors.

Abdominal aortic aneurysm (AAA) is a multifactorial condition. The transforming growth factor beta (TGF-beta) pathway regulates vascular remodeling and mutations in its receptor genes, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysm (TAA). The TGF-beta pathway may be involved in aneurysm development in general. We performed an association study by analyzing all the common genetic variants in TGFBR1 and TGFBR2 using tag single nucleotide polymorphisms (SNPs) in a Dutch AAA case-control population in a two-stage genotyping approach. In stage 1, analyzing 376 cases and 648 controls, three of the four TGFBR1 SNPs and nine of the 28 TGFBR2 SNPs had a P<0.07. Genotyping of these SNPs in an independent cohort of 360 cases and 376 controls in stage 2 confirmed association (P<0.05) for the same allele of one SNP in TGFBR1 and two SNPs in TGFBR2. Joint analysis of the 736 cases and 1024 controls showed statistically significant associations of these SNPs, which sustained after proper correction for multiple testing (TGFBR1 rs1626340 OR 1.32 95% CI 1.11-1.56 P=0.001 and TGFBR2 rs1036095 OR 1.32 95% CI 1.12-1.54 P=0.001 and rs4522809 OR 1.28 95% CI 1.12-1.46 P=0.0004). We conclude that genetic variations in TGFBR1 and TGFBR2 associate with AAA in the Dutch population. This suggests that AAA may develop partly by similar defects as TAA, which in the future may provide novel therapeutic options.

To determine the phenotypic significance of copy number changes (CNCs) in the human genome, we performed genome-wide segmental aneuploidy profiling by BAC-based array-CGH of 278 unrelated patients with multiple congenital abnormalities and mental retardation (MCAMR) and in 48 unaffected family members. In 20 patients, we found de novo CNCs composed of multiple consecutive probes. Of the 125 probes making up these probably pathogenic CNCs, 14 were also found as single CNCs in other patients and 5 in healthy individuals. Thus, these CNCs are not by themselves pathogenic. Almost one out of five patients and almost one out of six healthy individuals in our study cohort carried a gain or a loss for any one of the recently discovered microdeletion/microduplication loci, whereas seven patients and one healthy individual showed losses or gains for at least two different loci. The pathogenic burden resulting from these CNCs may be limited as they were found with similar frequencies among patients and healthy individuals (P=0.165; Fischer's exact test), and several individuals showed CNCs at multiple loci. CNCs occurring specifically in our study cohort were enriched for components of the glutamate receptor family (GRIA2, GRIA4, GRIK2 and GRIK4) and genes encoding proteins involved in guiding cell localization during development (ATP1A2, GIRK3, GRIA2, KCNJ3, KCNJ10, KCNK17 and KCNK5). This indicates that disease cohort-specific compilations of CNCs may aid in identifying loci, genes and biological processes that contribute to the phenotype of patients.

The recent identification of copy-number variation in the human genome has opened up new avenues for the discovery of positional candidate genes underlying complex genetic disorders, especially in the field of psychiatric disease. One major challenge that remains is pinpointing the susceptibility genes in the multitude of disease-associated loci. This challenge may be tackled by reconstruction of functional gene-networks from the genes residing in these loci. We applied this approach to autism spectrum disorder (ASD), and identified the copy-number changes in the DNA of 105 ASD patients and 267 healthy individuals with Illumina Humanhap300 Beadchips. Subsequently, we used a human reconstructed gene-network, Prioritizer, to rank candidate genes in the segmental gains and losses in our autism cohort. This analysis highlighted several candidate genes already known to be mutated in cognitive and neuropsychiatric disorders, including RAI1, BRD1, and LARGE. In addition, the LARGE gene was part of a sub-network of seven genes functioning in glycobiology, present in seven copy-number changes specifically identified in autism patients with limited co-morbidity. Three of these seven copy-number changes were de novo in the patients. In autism patients with a complex phenotype and healthy controls no such sub-network was identified. An independent systematic analysis of 13 published autism susceptibility loci supports the involvement of genes related to glycobiology as we also identified the same or similar genes from those loci. Our findings suggest that the occurrence of genomic gains and losses of genes associated with glycobiology are important contributors to the development of ASD.

We report on a patient with developmental delay and several facial characteristics reminiscent of Wolf-Hirschhorn syndrome, who carries a terminal 4p16.3 deletion of minimally 1.691 Mb and maximally 1.698 Mb. This deletion contains the FGFRL1 gene, but does not include the WHSC1 gene. Given its expression pattern and its involvement in bone and cartilage formation during embryonic development, the FGFRL1 gene represents a plausible candidate gene for part of the facial characteristics of Wolf-Hirshhorn syndrome in 4p16.3 deletion patients.

The known genetic variants determining plasma HDL cholesterol (HDL-C) levels explain only part of its variation. Three hundred eighty-four single nucleotide polymorphisms (SNPs) across 251 genes based on pathways potentially relevant to HDL-C metabolism were selected and genotyped in 3,575 subjects from the Doetinchem cohort, which was examined thrice over 11 years. Three hundred fifty-three SNPs in 239 genes passed the quality-control criteria. Seven SNPs [rs1800777 and rs5882 in cholesteryl ester transfer protein (CETP); rs3208305, rs328, and rs268 in LPL; rs1800588 in LIPC; rs2229741 in NRIP1] were associated with plasma HDL-C levels with false discovery rate (FDR) adjusted q values (FDR&#95;q) < 0.05. Five other SNPs (rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, rs6060717 near SCAND1, and rs3213451 in MBTPS2 in women) were associated with plasma HDL-C levels with FDR&#95;q between 0.05 and 0.2. Two less well replicated associations (rs3135506 in APOA5 and rs1800961 in HNF4A) known from the literature were also observed, but their significance disappeared after adjustment for multiple testing (P = 0.008, FDR&#95;q = 0.221 for rs3135506; P = 0.018, FDR&#95;q = 0.338 for rs1800961, respectively). In addition to replication of previous results for candidate genes (CETP, LPL, LIPC, HNF4A, and APOA5), we found interesting new candidate SNPs (rs2229741 in NRIP1, rs3213451 in MBTPS2, rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, and rs6060717 near SCAND1) for plasma HDL-C levels that should be evaluated further.

The two main phenotypes of inflammatory bowel disease (IBD)--Crohn's disease (CD) and ulcerative colitis (UC)--are chronic intestinal inflammatory disorders with a complex genetic background. Using a three-stage design, we performed a functional candidate-gene analysis of innate immune pathway in IBD. In phase I, we typed 354 SNPs from 85 innate immunity genes in 520 Dutch IBD patients (284 CD, 236 UC) and 808 controls. In phase II, ten autosomal SNPs showing association at p < 0.006 in phase I were replicated in a second cohort of 545 IBD patients (326 CD, 219 UC) and 360 controls. In phase III, four SNPs with p < 0.01 in the combined phase I and phase II analysis were genotyped in an additional 786 IBD samples (452 CD, 334 UC) and 768 independent controls. Joint analysis of 1851 IBD patients (1062 CD, 789 UC) and 1936 controls demonstrated strong association to the IL18RAP rs917997 SNP for both CD and UC (p(IBD) 1.9 x 10(-8); OR 1.35). Association in CD is independently supported by the Crohn's disease dataset of the Wellcome Trust Case Control Consortium (imputed SNP rs917997, p = 9.19 x 10(-4)). In addition, an association of the CARD9 rs10870077 SNP to CD and UC was observed (p(IBD) = 3.25 x 10(-5); OR 1.21). Both genes are located in extended haplotype blocks on 2q11-2q12 and 9q34.3, respectively. Our results indicate two IBD loci and further support the importance of the innate immune system in the predisposition to both CD and UC.

We report on an 8(1)/(2)-year-old girl with severe pre- and postnatal growth retardation, congenital heart malformation, facial asymmetry, oculocutaneous albinism without misrouting and subluxation of the radial heads. Her intelligence was in the low normal range. By GTG-banding a deletion of band 15q26 was found. Array-CGH, using a 3783 BAC array, revealed a segmental monosomy of the 15(q26.2-->qter) region, which was narrowed down to a 6.87Mb deletion by using the Illumina Infinium 317 K SNP array system, and subsequently confirmed by fluorescence in situ hybridisation (FISH) analysis. The deletion appeared to have arisen de novo. The IGF1R (insulin-like growth factor 1 receptor) and the NR2F2 genes were situated within, but the OCA2 (oculocutaneous albinism II) gene (formerly called the P gene) was located outside the deleted region. Clinical findings in our patient were compared with previously reported cases carrying terminal deletions of 15q26.2. This allowed us to expand the clinical phenotype of terminal 15q26.2 deletions and to indicate candidate genes for several phenotypic features.

Background: Amyotrophic lateral sclerosis (ALS) is a devastating disease characterised by progressive degeneration of motor neurons in the brain and spinal cord. ALS is thought to be multifactorial, with both environmental and genetic causes. Our aim was to identify genetic variants that predispose for sporadic ALS., Methods: We did a three-stage genome-wide association study in 461 patients with ALS and 450 controls from The Netherlands, using Illumina 300K single-nucleotide polymorphism (SNP) chips. The SNPs that were most strongly associated with ALS were analysed in a further 876 patients and 906 controls in independent sample series from The Netherlands, Belgium, and Sweden. We also investigated the possible pathological functions of associated genes using expression data from whole blood of patients with sporadic ALS and of control individuals who were included in the genome-wide association study., Findings: A genetic variant in the inositol 1,4,5-triphosphate receptor 2 gene (ITPR2) was associated with ALS (p=0.012 after Bonferroni correction). Combined analysis of all samples (1337 patients and 1356 controls) confirmed this association (p=3.28x10(-6), odds ratio 1.58, 95% CI 1.30-1.91). ITPR2 expression was greater in the peripheral blood of 126 ALS patients than in that of 126 healthy controls (p=0.00016)., Interpretation: Genetic variation in ITPR2 is a susceptibility factor for ALS. ITPR2 is a strong candidate susceptibility gene for ALS because it is involved in glutamate-mediated neurotransmission, is one of the main regulators of intracellular calcium concentrations, and has an important role in apoptosis.

Background: Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infection in infants. Only a proportion of children infected with RSV require hospitalization. Because known risk factors for severe disease, such as premature birth, cannot fully explain differences in disease severity, genetic factors have been implicated., Methods: To study the complexity of RSV susceptibility and to identify the genes and biological pathways involved in its development, we performed a genetic association study involving 470 children hospitalized for RSV bronchiolitis, their parents, and 1008 random, population controls. We analyzed 384 single-nucleotide polymorphisms (SNPs) in 220 candidate genes involved in airway mucosal responses, innate immunity, chemotaxis, adaptive immunity, and allergic asthma., Results: SNPs in the innate immune genes VDR (rs10735810; P=.0017), JUN (rs11688; P=.0093), IFNA5 (rs10757212; P=.0093), and NOS2 (rs1060826; P=.0031) demonstrated the strongest association with bronchiolitis. Apart from association at the allele level, these 4 SNPs also demonstrated association at the genotype level (P=.0056, P=.0285, P=.0372, and P=.0117 for the SNPs in VDR, JUN, IFNA5, and NOS2, respectively). The role of innate immunity as a process was reinforced by association of the whole group of innate immune SNPs when the global test for groups of genes was applied (P=.046)., Conclusion: SNPs in innate immune genes are important in determining susceptibility to RSV bronchiolitis.

The gene family of serine protease inhibitors of the Kazal type (SPINK) are functional and positional candidate genes for celiac disease (CD). Our aim was to assess the gut mucosal gene expression and genetic association of SPINK1, -2, -4, and -5 in the Dutch CD population. Gene expression was determined for all four SPINK genes by quantitative reverse-transcription polymerase chain reaction in duodenal biopsy samples from untreated (n=15) and diet-treated patients (n=31) and controls (n=16). Genetic association of the four SPINK genes was tested within a total of 18 haplotype tagging SNPs, one coding SNP, 310 patients, and 180 controls. The SPINK4 study cohort was further expanded to include 479 CD cases and 540 controls. SPINK4 DNA sequence analysis was performed on six members of a multigeneration CD family to detect possible point mutations or deletions. SPINK4 showed differential gene expression, which was at its highest in untreated patients and dropped sharply upon commencement of a gluten-free diet. Genetic association tests for all four SPINK genes were negative, including SPINK4 in the extended case/control cohort. No SPINK4 mutations or deletions were observed in the multigeneration CD family with linkage to chromosome 9p21-13 nor was the coding SNP disease-specific. SPINK4 exhibits CD pathology-related differential gene expression, likely derived from altered goblet cell activity. All of the four SPINK genes tested do not contribute to the genetic risk for CD in the Dutch population.

Pooling of DNA samples instead of individual genotyping can speed up genetic association studies. However, for microsatellite markers, the electrophoretic pattern of DNA pools can be complex, and procedures for deriving allele frequencies are often confounded by PCR-induced stutter artefacts. We have developed a mathematical procedure to remove stutter noise and accurately determine allele frequencies in pools. A stutter correction model can be reliably derived from one standard 'training set' of the same 10 individual DNA samples for each marker, which can also include heterozygous patterns with partially overlapping peaks. Compared with earlier methods, this reduces the number of genotypes needed in the training set considerably, and allows standardization of analyses for different markers. Moreover, the use of a procedure that fits all data simultaneously makes the method less sensitive to aberrant data. The model was tested with 34 markers, 18 of which were newly defined from human sequence data. Allele frequencies derived from stutter-corrected DNA pool patterns were compared with the summed individual genotyping results of all the individuals in the pools (n = 109 and n = 64). We show that the model is robust and accurately extracts allele frequencies from pooled DNA samples for 32 of the 34 microsatellite markers tested. Finally, we performed a case-control study in celiac disease and found that weakly associated disease alleles, identified by individual genotyping, were only detectable in pools after stutter correction. This efficient method for correcting stutter artefacts in microsatellite markers enables large-scale genetic association studies using DNA pools to be performed.