Search

Haplotype-tagging SNP analyses were conducted to identify molecular genetic substrates of quantitative phenotypes derived from performance on a Continuous Performance Task (CPT). Three hundred sixty-four individuals were sampled from 152 families ascertained on the basis of at least one child having ADHD. Probands, their affected and unaffected siblings, and parents were administered a CPT. Four different components of performance were analyzed and tested for association with SNPs from 10 candidate genes involved in monoaminergic function. After correcting for multiple comparisons and controlling for multiple individuals from the same family, significant associations were identified between commission errors and SNPs in the DRD2 gene (rs2075654, rs1079596), and between reaction time variability and a SNP in the NET gene (rs3785155). These findings suggest that commission errors and reaction time variability are excellent candidates as ADHD endophenotypes based on previously published criteria. Results also shed light on the molecular genetic basis of specific processes that may underlie the disorder.

Expression of the serotonin transporter is affected by the genotype of the 5-HTTLPR (short and long forms) as well as the genotype of the SNP rs25531 within this region. Based on the combined genotypes for these polymorphisms, we designated each allele as a high or low expressing allele according to established expression levels—resulting in HiHi, HiLo, & LoLo genotype groups for analysis. We evaluated effects of gender and the promoter genotype on induction of negative affect by intravenous infusion of l-tryptophan (TRP). The protocol consisted of a day-1 sham saline infusion and a day-2 active TRP infusion. Models assessed 5-HTTLPR composite genotype and gender as predictors of change in ratings of negative emotion during TRP infusion. During sham infusion there were no significant changes from baseline in mood ratings. During TRP infusion all negative affect ratings increased significantly from baseline (P’s < .02). The genotype × gender interaction was a significant predictor of depression-dejection (P = .013), and trended towards predicting anger−hostility (P = .084). Males in the HiHi group had greater increases in negative affect during infusion, compared to all groups except LoLo females, who also showed increased negative affect.

Caregiving stress is associated with negative health outcomes. Neuroendocrine functioning may be a mediator of such outcomes. The MAOA gene regulates activity of neurotransmitters involved with neuroendocrine responses to stress. Differences in polymorphisms of this gene have been shown to influence susceptibility to stress. Therefore, we examined allelic variation in MAOA-uVNTR, a functional polymorphism of MAOA, as a moderator of chronic stress effects on urinary cortisol excretion in 74 males enrolled in a case/control study of caregivers for relatives with dementia. Mixed models analysis of variance were used to examine MAOA-uVNTR genotype (3 repeats vs. 3.5/4 repeats) as a moderator of the impact of stress (caregiver vs. non-caregiver) on the urinary excretion pattern (overnight, daytime, evening) of cortisol. Caregivers with MAOA-uVNTR alleles associated with less transcriptional activity (3-repeats) displayed a pattern of cortisol excretion -- a decrease from overnight to daytime -- that was suggestive of HPA axis blunting, as compared to non-caregivers and those caregivers with the more active alleles (3.5/4 repeats) (cortisol p.043). Individuals with less active MAOA-uVNTR alleles who are under chronic stress may be at increased risk for exhaustion of the HPA response to such stress.

BACKGROUND: NTDs are considered complex disorders that arise from an interaction between genetic and environmental factors. NTD family 8776 is a large multigenerational Caucasian family that provides a unique resource for the genetic analysis of NTDs. Previous linkage analysis using a genome-wide SNP screen in family 8776 with multipoint nonparametric mapping methods identified maximum LOD* scores of ∼3.0 mapping to 2q33.1–q35 and 7p21.1–pter. METHODS: We ascertained an additional nuclear branch of 8776 and conducted additional linkage analysis, fine mapping, and haplotyping. Expression data from lymphoblast cell lines were used to prioritize candidate genes within the minimum candidate intervals. Genomic copy number changes were evaluated using BAC tiling arrays and subtelomeric fluorescent in situ hybridization probes. RESULTS: Increased evidence for linkage was observed with LOD* scores of ∼3.3 for both regions. Haplotype analyses narrowed the minimum candidate intervals to a 20.3 Mb region in 2q33.1–q35 between markers rs1050347 and D2S434, and an 8.3 Mb region in 7p21.1–21.3 between a novel marker 7M0547 and rs28177. Within these candidate regions, 16 genes were screened for mutations; however, no obvious causative NTD mutation was identified. Evaluation of chromosomal aberrations using comparative genomic hybridization arrays, subtelomeric fluorescent in situ hybridization, and copy number variant detection techniques within the 2q and 7p regions did not detect any chromosomal abnormalities. CONCLUSIONS: This large NTD family has identified two genomic regions that may harbor NTD susceptibility genes. Ascertainment of another branch of family 8776 and additional fine mapping permitted a 9.1 Mb reduction of the NTD candidate interval on chromosome 7 and 37.3 Mb on chromosome 2 from previously published data. Identification of one or more NTD susceptibility genes in this family could provide insight into genes that may affect other NTD families. Birth Defects Research (Part A), 2008. © 2008 Wiley-Liss, Inc.

The Duffy glycoprotein (Fy) on red blood cells (RBCs) has been hypothesized to promote clearance of inflammatory cytokines, which may play a role in the pathogenesis of vasoocclusion in sickle cell disease (SCD). Persons with the African-type Fy(a-b-) phenotype--whose RBCs lack expression of Duffy--may less efficiently clear inflammatory cytokines. Therefore, the Duffy-negative genotype may be associated with more severe disease among patients with SCD.Genotyping was performed on blood samples from 249 adult patients with HbSS at the Duffy gene (FY) locus GATA site (rs2814778) that determines RBC expression of Duffy antigens. Patients with discordant genotype and phenotype data were excluded (n = 12). Differences in demographic, clinical and laboratory findings, end-organ damage, and overall disease severity were compared between FY+ and FY- patients.Of the 237 patients studied, 174 (73%) were FY-. FY+ patients had a higher mean white blood cell (WBC) count (13.2 x 10(9) +/- 4.1 x 10(9)/L vs. 11.8 x 10(9) +/- 3.3 x 10(9)/L; p = 0.03) and higher rates of treatment with hydroxyurea (72% vs. 49%; p = 0.002). In contrast, FY- status was strongly associated with chronic organ damage (85% of FY- patients vs. 65% of FY+ patients; p = 0.018) and proteinuria (32% vs. 12%; p = 0.02). These associations remained, even after controlling for the effects of age and sex.Duffy genotype may be a potential biomarker for the development of end-organ damage in SCD, particularly kidney dysfunction. The association of both WBC counts and hydroxyurea use with Duffy expression provides another avenue for investigation of the biologic role of this protein.

The monoamine oxidase-A (MAOA) gene plays a vital role in the metabolism of neurotransmitters, e.g, serotonin, norepinephrine, and dopamine. A polymorphism in the promoter region (MAOA-uVNTR) affects transcriptional efficiency. Allelic variation in MAOA-uVNTR has been associated with body mass index (BMI). We extended previous work by examining relations among this polymorphism and serum lipid levels.The sample consisted of 74 males enrolled in a study of caregivers for relatives with dementia. Regression models, adjusted for age, race, group status (caregiver/control), and cholesterol lowering medication (yes/no), were used to examine associations between high verses low MAOA-uVNTR activity alleles and total cholesterol, HDL, LDL, VLDL, LDL/HDL ratio, triglycerides, and BMI.Higher total cholesterol (p0.03), LDL/HDL ratio (p0.01), triglycerides (p0.02), and VLDL (p0.02) were associated with low activity MAOA-uVNTR alleles. HDL and LDL were modestly related to MAOA-uVNTR activity, however, they did not reach the conventional significance level (p0.07 and p0.10, respectively). BMI (p0.74) was unrelated to MAOA-uVNTR transcription.The present findings suggest that MAOA-uVNTR may influence lipid levels and individuals with less active alleles are at increased health risk.

Background Sickle cell disease patients are more likely than the general population to undergo surgery and usually do so at a younger age. Female sickle cell disease patients also have special gynecological and obstetric issues related to their disease. Methods We collected data through standardized clinical report forms, patient interviews, and medical records from 509 adult sickle cell disease patients. Logistic regression was used to estimate the association between multiple variables and each of the surgery types. We also determined the prevalence and outcomes of pregnancy in 284 women with sickle cell disease in this population. Results Almost 50% of patients aged 18-27 years had had a cholecystectomy. Mean corpuscular hemoglobin, total bilirubin, and lactate dehydrogenase were significantly higher in the postcholecystectomy group; 9.5% of 504 individuals had undergone splenectomy. Hematocrit, body mass index, and red blood cell count were significantly higher in the postsplenectomy group. Hip replacement had been performed in 9.2% of individuals, with the prevalence increasing as early as the fourth decade and continuing to increase through the sixth decade of life. A history of pregnancy was present in 190 women (67%). Of 410 pregnancies, only 53.9% resulted in live births, 16.6% were voluntarily terminated, and 29.5% were complicated by miscarriage, still birth, or ectopic implantation. Conclusions Sickle cell disease continues to have a strong effect on the mean age for common surgeries and impacts pregnancy outcomes. We conclude that this population has a unique surgical and obstetric history that should be further studied to provide insight into potentially more effective preventive approaches to end-organ damage.

Screening for pulmonary hypertension (pHTN) has not yet become routine in sickle cell disease (SCD), despite clinical evidence of its high prevalence and associated mortality. Our objectives are to identify clinical conditions and laboratory findings predictive of/or associated with pHTN. One hundred twenty-five adult outpatients with Hb SS, SC, SOArab, Sbeta(0), or Sbeta(+) thalassemia, who underwent echocardiography and/or right heart catheterization due to cardiorespiratory symptoms, were studied. pHTN was identified in 36% (28/77) of SS/Sbeta(0) and in 25% (12/48) of SC/SOArab/Sbeta(+) patients studied. In SS/Sbeta(0) patients, pHTN was associated with low hemoglobin, low GFR, increasing age, no history of treatment with hydroxyurea and a history of leg ulcers, with trends for associations with higher total bilirubin, LDH levels, systolic systemic blood pressure, history of avascular necrosis, seizures, and cerebrovascular events. Twelve (40%) of the SS/Sbeta(0) patients with pHTN had >or= 1+ proteinuria. (P

The short (s) variant of the serotonin transporter (5-HTT) gene linked functional polymorphic region (5-HTTLPR) is associated with depression. Stressful life events, gender, and race have been shown to moderate this association. We examined the relationship between 5-HTTLPR genotype and symptoms of depression in two samples. Study 1 = 288 participants from a study of caregiver stress; and Study 2 = 142 participants from a study examining psychosocial stressors, genetics, and health. Main effects of 5-HTTLPR on symptoms of depression were examined, along with moderation by stress (care-giving status or low childhood socioeconomic status (SES), gender, and race. The 5-HTTLPR × stress group × gender interaction was significant in both samples (P < 0.003, and P < 0.008, respectively). For females, the s allele, combined with caregiving stress (Study 1) or low childhood SES (Study 2), was associated with higher depression scores as compared to participants in the non-stressor group and those with the long (l) allele; whereas, in males, the l allele, combined with a stressor, was associated with higher depression scores as compared to those in the non-stressor group and those with the s allele. Findings from two independent samples suggest that the association of 5-HTTLPR with depression varies according to gender and stressful life events.

To examine the relationships among the variable number of tandem repeats in the monoamine oxidase-A linked polymorphic region allelic variation (MAOA-uVNTR) and the symptoms of depression and sleep quality. The monoamine oxidase-A (MAOA) gene, which plays a vital role in degradation of neurotransmitters such as serotonin, norepinephrine, and dopamine, contains a polymorphism in its promoter region (MAOA-uVNTR) that affects transcriptional efficiency. MAOA-uVNTR genotype has been associated with both psychological and physical measures.The sample consisted of 74 males enrolled in a case/control study of caregivers for relatives with dementia. Age- and race-adjusted linear regression models were used to examine the association between low versus high MAOA-uVNTR activity alleles, symptoms of depression (Center for Epidemiological Studies of Depression), and sleep quality ratings (Pittsburgh Sleep Quality Index).MAOA-uVNTR alleles associated with less transcriptional activity were related to increased symptoms of depression (p.04; Cohen's d = 0.52) and poorer sleep quality (p.04; Cohen's d = 0.31).Individuals with less active MAOA-uVNTR alleles may be at increased risk for depressive symptoms and poor sleep.

An association between multiple sclerosis (MS) and the human leukocyte antigen (HLA) complex, a dense cluster of genes on the short arm of chromosome 6, was first noted over 30 years ago. In Caucasian populations of Northern European descent, the DR15 haplotype (DRB1*1501-DQA1*0102-DQB1*0602) has been hypothesized to be the primary HLA genetic susceptibility factor for MS. However, studies of other populations have produced varying results. Thus, the authors reviewed the literature for articles on the association between the DR15 haplotype and MS. They identified 72 papers meeting the inclusion criteria: human genetic studies written in English that were published between 1993 and 2004 and that reported allele frequencies for HLA-DRB1*1501, HLA-DQA1*0102, or HLA-DQB1*0602 or the frequency of the DRB1*1501-DQA1*0102-DQB1*0602 haplotype. Most of the studies identified used a case-control design (n = 60), while the remainder used a family-based design (n = 22). In most of these papers, investigators reported a higher frequency of the DR15 haplotype and/or its component alleles among MS cases than among controls. However, the authors' confidence in these results is tempered by factors related to study design that may have biased the outcomes.

To test the hypothesis that allelic variation in 5HTT gene-linked polymorphic region (5-HTTLPR) genotype was associated with sleep quality (Pittsburgh Sleep Quality Index, PSQI) as a main effect and as moderated by the chronic stress of caregiving. Serotonin (5HT) is involved in sleep regulation and the 5HT transporter (5HTT) regulates 5HT function. A common 44-base pair deletion (s allele) polymorphism in the 5-HTTLPR is associated with reduced 5HTT transcription efficiency and 5HT uptake in vitro.Subjects were 142 adult primary caregivers for a spouse or parent with dementia and 146 noncaregiver controls. Subjects underwent genotyping and completed the PSQI.Variation in 5-HTTLPR genotype was not related to sleep quality as a main effect (p.36). However, there was a caregiver X 5-HTTLPR interaction (p.009), such that the s allele was associated with poorer sleep quality in caregivers as compared with controls.Findings suggest that the s allele may moderate sleep disturbance in response to chronic stress.

Corrigendum: A common variant mapping to CACNA1A is associated with susceptibility to exfoliation syndrome

Objective To examine the association between the 5-HTTLPR polymorphism of the serotonin transporter (SLC6A4) gene, combat exposure, and posttraumatic stress disorder (PTSD) diagnosis and among two samples of combat-exposed veterans. Method The first sample included 550 non-Hispanic Black (NHB) combat-exposed veterans. The second sample included 555 non-Hispanic White (NHW) combat-exposed veterans. Participants were genotyped for the 5-HTTLPR/rs25531 variants of the SLC6A4 gene. A structured clinical interview was used to diagnose PTSD. Combat and civilian trauma exposure were assessed with validated self-report instruments. Logistic regression was used to test for main effects of 5-HTTLPR on PTSD diagnosis as well as gene x environment (GxE) interactions after adjusting for sex, ancestry proportion scores, civilian trauma exposure, and combat exposure. Results Within the NHB sample, a significant additive effect was observed for 5-HTTLPR (OR = 1.502, p = .0025), such that the odds of having a current diagnosis of PTSD increased by 1.502 for each additional S’ allele. No evidence for an association between 5-HTTLPR and PTSD was observed in the NHW sample. In addition, no evidence for combat x 5-HTTLPR effects were observed in either sample. Conclusion The present study suggests that there may be an association between 5-HTTLPR genotype and PTSD diagnosis among NHB veterans; however, no evidence for the hypothesized 5-HTTLPR x combat interaction was found.

Gene-gene interactions are likely involved in many complex genetic disorders and new statistical approaches for detecting such interactions are needed. We propose a multi-analytic paradigm, relying on convergence of evidence across multiple analysis tools. Our paradigm tests for main and interactive effects, through allele, genotype and haplotype association. We applied our paradigm to genotype data from three GABAA receptor subunit genes (GABRB3, GABRA5, and GABRG3) on chromosome 15 in 470 Caucasian autism families. Previously implicated in autism, we hypothesized these genes interact to contribute to risk. We detected no evidence of main effects by allelic (PDT, FBAT) or genotypic (genotype-PDT) association at individual markers. However, three two-marker haplotypes in GABRG3 were significant (HBAT). We detected no significant multi-locus associations using genotype-PDT analysis or the EMDR data reduction program. However, consistent with the haplotype findings, the best single locus EMDR model selected a GABRG3 marker. Further, the best pairwise genotype-PDT result involved GABRB3 and GABRG3, and all multi-locus EMDR models also selected GABRB3 and GABRG3 markers. GABA receptor subunit genes do not significantly interact to contribute to autism risk in our overall data set. However, the consistency of results across analyses suggests that we have defined a useful framework for evaluating gene-gene interactions.

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. For the novel SPG31 locus on chromosome 2p12, we identified six different mutations in the receptor expression–enhancing protein 1 gene (REEP1). REEP1 mutations occurred in 6.5% of the patients with HSP in our sample, making it the third-most common HSP gene. We show that REEP1 is widely expressed and localizes to mitochondria, which underlines the importance of mitochondrial function in neurodegenerative disease.

The multifactor dimensionality reduction (MDR) is a model-free approach that can identify gene × gene or gene × environment effects in a case-control study. Here we explore several modifications of the MDR method. We extended MDR to provide model selection without crossvalidation, and use a chi-square statistic as an alternative to prediction error (PE). We also modified the permutation test to provide different levels of stringency. The extended MDR (EMDR) includes three permutation tests (fixed, non-fixed, and omnibus) to obtain p-values of multilocus models. The goal of this study was to compare the different approaches implemented in the EMDR method and evaluate the ability to identify genetic effects in the Genetic Analysis Workshop 14 simulated data. We used three replicates from the simulated family data, generating matched pairs from family triads. The results showed: 1) chi-square and PE statistics give nearly consistent results; 2) results of EMDR without cross-validation matched that of EMDR with 10-fold cross-validation; 3) the fixed permutation test reports false-positive results in data from loci unrelated to the disease, but the non-fixed and omnibus permutation tests perform well in preventing false positives, with the omnibus test being the most conservative. We conclude that the non-cross-validation test can provide accurate results with the advantage of high efficiency compared to 10-cross-validation, and the non-fixed permutation test provides a good compromise between power and false-positive rate.

Several genome-wide screens have indicated the presence of an autism susceptibility locus within the distal long arm of chromosome 7 (7q). Mapping at 7q22 within this region is the candidate gene reelin (RELN). RELN encodes a signaling protein that plays a pivotal role in the migration of several neuronal cell types and in the development of neural connections. Given these neurodevelopmental functions, recent reports that RELN influences genetic risk for autism are of significant interest. The total data set consists of 218 Caucasian families collected by our group, 85 Caucasian families collected by AGRE, and 68 Caucasian families collected at Tufts University were tested for genetic association of RELN variants to autism. Markers included five single-nucleotide polymorphisms (SNPs) and a repeat in the 5'-untranslated region (5'-UTR). Tests for association in Duke and AGRE families were also performed on four additional SNPs in the genes PSMC2 and ORC5L, which flank RELN. Family-based association analyses (PDT, Geno-PDT, and FBAT) were used to test for association of single-locus markers and multilocus haplotypes with autism. The most significant association identified from this combined data set was for the 5'-UTR repeat (PDT P-value=0.002). These analyses show the potential of RELN as an important contributor to genetic risk in autism.

Apolipoprotein E (APOE) is the only universally confirmed susceptibility gene for late-onset Alzheimer disease (LOAD), although many loci are believed to modulate LOAD risk. The genetic homogeneity of isolated populations, such as the Amish, potentially provide increased power to identify LOAD susceptibility genes. Population homogeneity in these special populations may reduce the total number of susceptibility genes contributing to the complex disorder, thereby increasing the ability to identify any one susceptibility gene. Dementia in the Amish is clinically indistinguishable from LOAD in the general population. Previous studies in the Amish demonstrated a significantly decreased frequency of the APOE-4 susceptibility allele, but significant familial clustering of dementia [M.A. Pericak-Vance, C.C. Johnson, J.B. Rimmler, A.M. Saunders, L.C. Robinson, E.G. D'Hondt, C.E. Jackson, J.L. Haines, Alzheimer's disease and apolipoprotein E-4 allele in an Amish population, Ann. Neurol. 39 (1996) 700-704]. These data suggested that a genetic etiology independent of APOE may underlie the dementia observed in this population. In the present analysis, we focused on a large, multiplex, inbred Amish family (24 sampled individuals; 10 of whom are affected). We completed a genomic screen to identify novel LOAD loci (n=316 genetic markers), using both model-dependent "affecteds-only" analysis (dominant and recessive) and model-independent affected relative pair analysis. Interesting results (lod>1.5 or p

Autism has a strong and complex genetic component, involving several genes. Genomic screens, including our own, have shown suggestive evidence for linkage over a 20-30 cM region on chromosome 2q31-q33. Two subsequent reports showed that the linkage evidence increased in the subset of families with phrase speech delay (PSD), defined as onset of phrase speech later than 3 years of age. To further investigate the linkage in the presumptive candidate region, microsatellite markers in a 2 cM grid covering the interval from 164 to 203 cM were analyzed in 110 multiplex (2 or more sampled autism patients) families. A maximum heterogeneity LOD (HLOD) score of 1.54 was detected at D2S1776 (173 cM) in the overall dataset (dominant model), increasing to 1.71 in the PSD subset. While not conclusive, these data continue to provide suggestive evidence for linkage, particularly considering replication by multiple independent groups. Positive LOD scores extended over the entire region, continuing to define a broad candidate interval. Association studies were performed on several functional candidates mapping within the region. These included GAD1, encoding GAD67, whose levels are reduced in autopsy brain material from autistic subjects, and STK17B, ABI2, CTLA4, CD28, NEUROD1, PDE1A, HOXD1 and DLX2. We found no evidence for significant allelic association between autism and any of these candidates, suggesting that they do not play a major role in the genetics of autism or that substantial allelic heterogeneity at any one of these loci dilutes potential disease-allele association.

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disease characterized by wide variability in phenotypic expression, both within and among families. The most-common cause of autosomal dominant HSP is mutation of the gene encoding spastin, a protein of uncertain function. We report the existence of intragenic polymorphisms of spastin that modify the HSP phenotype. One (S44L) is a previously described recessively acting allele and the second is a novel allele affecting the adjacent amino acid residue (P45Q). In 4 HSP families in which either L44 or Q45 segregates independently of a missense or splicing mutation in the AAA domain of spastin, L44 and Q45 are each associated with a striking decrease in age at onset in the presence of the AAA domain mutations. Using a bioinformatics approach, we found that the highly conserved S44 is predicted to be phosphorylated by a number of family members of the proline-directed serine/threonine cyclin-dependent kinases (Cdks). Cdk1 and Cdk5 showed no kinase activity toward synthetic spastin peptide in an in vitro kinase assay, suggesting that this serine residue may be phosphorylated by a different Cdk. Our identification of S44L and P45Q as modifiers of the HSP phenotype suggests a role for spastin phosphorylation by Cdks in the neurodegeneration of the most-common form of HSP.

Rett disorder and autistic disorder are both pervasive developmental disorders. Recent studies indicate that at least 80% of Rett Disorder cases are caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. Since there is some phenotypic overlap between autistic disorder and Rett disorder, we analyzed 69 females clinically diagnosed with autistic disorder for the presence of mutations in the MeCP2 gene. Two autistic disorder females were found to have de novo mutations in the MeCP2 gene. These data provide additional evidence of variable expression in the Rett disorder phenotype and suggest MeCP2 testing may be warranted for females presenting with autistic disorder.

To the editor: Fetal hemoglobin (HbF) protects against many but not all of the hematologic and clinical complications of sickle cell anemia.[1][1],[2][2] This protection is dependent on the ability of HbF to hinder deoxyHbS polymerization. HbF level is variable and highly heritable. Previous

Item does not contain fulltext Buitelaar J, Bulik CM, Cichon S, Collier D, Corvin A, Craddock N, DeLisi LE, Ebstein R, Edenberg HJ, Estivill X, Faraone SV, Farmer AE, Fernandez-Aranda F, Flint J, Foroud T, Franke B, Gelernter J, Gill M, Grabe HJ, Hamilton SP, Hickie IB, Hoogendijk W, Jablensky A, Kalaydjieva L, Kaprio J, Kas M, Keller M, Kelsoe J, Kendler KS, Kennedy M, Kent L, Klump K, Knowles JA, Levinson DF, Lewis CM, Lichtenstein P, Lucae S, Martin NG, McCarroll S, McGough J, McGrath JJ, McGuffin P, Mitchell P, Mowry B, Muglia P, Müller-Myhsok B, Neale B, Nöthen M, Nurnberger JI, Oades RD, O'Donovan M, Ophoff R, Owen M, Pato CN, Pato MT, Penninx BW, Perlis RH, Porjesz B, Posthuma D, Potash JB, Preisig M, Reichborn-Kjennerud T, Rice JP, Rietschel M, Riley B, Ripke S, Rothenberger A, Rujescu D, Santangelo SL, Schachar R, Schellenberg GD, Scherer SW, Schofield P, Schulze TG, Sklar P, Smoller JW, Stefansson H, Stefansson K, Sullivan P, Todorov A, Tozzi F, Tzeng JY, van den Oord EJ, Vorstman JA, Wang K, Weissman MM, Werge T, Wray NR, Zeggini E 01 januari 2012

Since acquired von Willebrand syndrome (AVWS) was first described in 1968, approximately 270 cases have been reported in the literature. In 12% of these cases AVWS was secondary to cardiovascular disorders.1 Only two of these reports dealt with children.2,3 Gill and colleagues reported on 12 patients with non-cyanotic heart defects (atrial and ventricular septal defects, aortic stenosis) and abnormal von Willebrand factor (VWF) subunits.2 Four out of five patients with ventricular septal defect who underwent repeat examination following surgical repair showed normalisation of the multimeric pattern. We recently described the first case of AVWS in an infant with persistently patent ductus arteriosus (PDA).3 To study the frequency of this association and its aetiology, we examined 12 children (eight girls, four boys, median age 3.4 years) …

Autistic disorder (AutD) is a neurodevelopmental disorder characterized by significant disturbances in social, communicative, and behavioral functioning. A two-stage genomic screen analysis of 99 families with AutD revealed suggestive evidence for linkage to chromosome 2q (D2S116 nonparametric sib-pair LOD score [MLS] 1.12 at 198 cM). In addition, analysis of linkage disequilibrium for D2S116 showed an allele-specific P value of 36 mo) of phrase speech (PSD). We similarly classified our data set of 82 sib pairs with AutD, identifying 45 families with AutD and PSD. Analysis of this PSD subset increased our support for linkage to 2q (MLS 2.86 and HLOD 2.12 for marker D2S116). These data support evidence for a gene on chromosome 2 contributing to risk of AutD, and they suggest that phenotypic homogeneity increases the power to find susceptibility genes for AutD.

Autistic disorder (AutD) is a neurodevelopmental disorder characterized by significant impairment in social, communicative, and behavioral functioning. A genetic basis for AutD is well established with as many as 10 genes postulated to contribute to its underlying etiology. We have completed a genomic screen and follow-up analysis to identify potential AutD susceptibility loci. In stage one of the genome screen, 52 multiplex families (two or more AutD affected individuals/family) were genotyped with 352 genetic markers to yield an approximately 10 centimorgan (cM) grid, inclusive of the X chromosome. The selection criterion for follow-up of interesting regions was a maximum heterogeneity lod score (MLOD) or a maximum nonparametric sib pair lod score (MLS) of at least 1.0. Eight promising regions were identified on chromosomes 2, 3, 7, 15, 18, 19, and X. In the stage two follow-up study we analyzed an additional 47 multiplex families (total=99 families). Regions on chromosomes 2, 3, 7, 15, 19, and X remained interesting (MLOD> or =1.0) in stage two analysis. The peak lod score regions on chromosomes 2, 7, 15, 19, and X overlap previously reported peak linkage areas. The region on chromosome 3 is unique.

BACKGROUND: Alloimmunization to minor blood group antigens is more common in sickle cell disease (SCD) than in the general population. Causes include the high frequency of transfusion as well as phenotypic differences between the largely African-American SCD population and mostly Caucasian blood donors. Alloimmunization, as well as the risk of alloimmunization, results in higher costs as well as reduced availability of and delays in obtaining antigen-matched blood to meet clinical needs. We used a candidate gene approach to determine if specific genetic signatures were associated with alloimmunization and thus could identify patients at high risk for alloimmunization and therefore requiring prospective antigen matching for transfusion. METHODS: Patient data were previously collected through a multicenter study to identify genetic factors associated with SCD complications (Duke and UNC; DU) and through the Cooperative Study of SCD (CSSCD). All subjects were adults with HbSS genotype (DU mean yrs 34.9 ± 12.1, CSSCD mean yrs 29.1 ± 9.5, p < .0001). The DU data set was 43.7% male while the CSSCD data set was 37.1% male (p = .18). A genome-wide association study (GWAS) was conducted with the Illumina 610 BeadChips for ­­­390 individuals (215 DU, 175 CSSCD). Quality control processing of these data was published previously (Solovieff et al, 2010). Additionally, imputation of the DU data set was conducted using IMPUTE2 (Howie et al, 2009) and a global reference panel from 1000 Genomes in order to have consistent genotypes across both studies. Based on previous literature, we studied 255 SNPs in the HLA locus as well as 684 SNPs in 53 genes previously associated with immune responses, totaling 939 SNPs. Additive logistic regression was performed separately for the DU and CSSCD data sets with PLINK (Purcell et al, 2008), controlling for age at enrollment. Results from the two data sets were combined by meta-analysis, using the invariance weight method in METAL (Willer et al, 2010). False discovery rate (FDR) q-values were generated using PROC MULTTEST in SAS v9.4 (SAS Systems). Contingency tables were produced using SAS and association between candidate SNPs and specific antibodies was tested using PLINK. RESULTS: Alloimmunization rates were different in the DU and CSSCD cohorts (30.7% vs. 18.9%, respectively; p = .0075) but were not associated with age or number of transfusions > 0 in either cohort. The most significant genetic associations with alloimmunization (FDR q = .28) occurred for 46 SNPs in the HLA locus and 10 other genes (Table 1). Two of these SNPs were missense mutations in GZMB (granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1)), one of which (rs8192917) has previously been associated with acute kidney transplant rejection and acute GVHD after stem cell transplant. A missense mutation in TRIM31 (E3 ubiquitin-protein ligase) was also associated with alloimmunization (p = .01). TRIM31 regulates Src-induced anchorage-independent cell growth and has been implicated in antiretroviral defense, cancer and ADHD. Within the DU cohort, we conducted a sub-analysis among only antigen-negative individuals, in whom immunization rates were 15.8%, 14%, 4.5%, 10.2%, 11%, and 8.3% to E, C, K, S, Jkb and Fya, respectively. Regression analysis of all 939 SNPs identified nominally significant associations predicting specific antibody alloimmunization. A missense mutation in PRKCQ (rs2236379) was associated with both anti-E and –C alloimmunization. Additional missense mutations were identified as follows: in TGFBR3 and GZMB with anti-C; in CFH, andTLR6 with anti-K, and in TLR10 with anti-Jkb. CONCLUSIONS: We have identified several immune response genes that are also associated with increased risk of alloimmunization in SCD (nominal p ≤ .006, FDR q = .28). Some associations may arise from the prevalence of alloimmunization to specific antigens. For example, PRKCQ was associated with development of alloantibodies to both E and C antigens, which reside on the same nonglycosylated RH protein. Expanded studies may identify those patients who would benefit from intensive or specific antigen-matching transfusion protocols. TableGenes Associated with AlloimmunizationGene or Locus (ordered by descending p value)SNPs with FDR q = 0.28 SNPs with p ≤ .006GZMB3 (2 missense)3TGFBR242TLR311HLA locus262TGFBR332PRKCQ3CD801VAV21TRIM312 (1 missense)RNF391ZNRD11 Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Mean life expectancy among adults with sickle cell disease (SCD) remains in the sixth decade of life (Elmariah et al. Am J Hem. 2014). We examined the relationship of genetic differences among SCD patients to survival, in order to gain greater understanding of contributors to early mortality. Genetic signatures associated with survival could identify high risk patients who would be optimal candidates for advanced therapies. METHODS: Patient data were previously collected through a multicenter study to identify genetic factors associated with SCD complications. All enrolled patients were 18 years or older, with a diagnosis of SCD (SS, Sβ0, Sβ+, SC). Of the patients enrolled, 542 were followed for a mean of 9.3 years for incidence of death. Genome-wide association study (GWAS) data from the Illumina 610 BeadChips were available for 503 of these individuals. We used the software program IMPUTE2 with a global reference panel from the 1000Genomes Project to impute missing genotypes up to ~3 million single nucleotide polymorphisms (SNPs). Time to death was correlated with genotype using a Cox Proportional Hazards model implemented in an R plugin to PLINK (Purcell et al. AJHG. 2007), controlling for age at enrollment, gender, and population substructure. Candidate genes were prioritized for follow-up using the following criteria: at least one SNP with p RESULTS: Median survival was 61 years for all subjects. Median survival for patients with hemoglobin (Hb) SS and Sβ0 was 58 years and for Hb Sβ+ and SC was 66 years. GWAS results are shown in FIGURE 1. No genome-wide associations with survival met adjustment for multiple testing. The hazard ratios for the most significant SNPs increased risk for mortality 2-3 fold with each minor allele present. Five candidate genes were selected for follow-up: G-Protein Coupled Receptor 158 (GPR158), G protein alpha-12 (GNA12), Neurobeachin (NBEA), RAR-related orphan receptor alpha (RORA), and Sortilin-related VPS10 Domain Controlling Receptor (SORCS2). Follow-up genotyping of fifteen selected imputed SNPs within the candidate genes confirmed the imputed genotypes and associations from GWAS (p's CONCLUSIONS: We have identified genes that were nominally associated with survival in our SCD cohort and had clinical relevance. GNA12 has been associated with cellular adhesion, proteinuria, and renal failure (Boucher et al. Lab Invest. 2012). We previously linked kidney disease to early mortality in SCD (Elmariah et al. Am J Hem. 2014)and confirm here that GNA12 is associated with GFR and survival. In our cohort, GPR158 is associated with seizures, and other candidate genes, including NBEA, RORA, and SORC2, have previously been associated with neurologic development and function. These findings corroborate our prior results that seizures and strokes are strongly associated with decreased survival in SCD patients. The association between RORA and survival might be attributable to its role in nitric oxide biosynthesis. Several nominally significant SNPs lie in known regulatory regions, which may explain mechanisms by which those SNPs influence mortality. Our study does not prove causality or confirm mechanism of action. Further, we had limited statistical power due to the relatively small cohort, evidenced by the lack of significance on false discovery rate testing. Despite these limitations, our results demonstrate that genetic signatures may identify high-risk SCD patients who might benefit from more aggressive novel therapies and provide interesting mechanistic hypotheses. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Sickle cell disease (SCD) patients have a heterogeneous clinical course, and as patient survival has improved, end-organ damage has become an emergent clinical priority. End stage renal disease (ESRD), occurring in 5-18% of SCD patients, is a particular concern, because it is a major risk factor for early mortality (Platt et al. 1994). Detection of SCD nephropathy (SCDN) relies on relatively late-stage markers, namely proteinuria and reduced glomerular filtration rate (GFR). Consequently, at-risk SCD patients are not identified prior to end-organ damage. In non-SCD nephropathy, ESRD risk among African American individuals has been attributed to coding variants (termed G1 and G2) in apolipoprotein L1 (APOL1). The G1 allele consists of two nonsynonymous variants in perfect LD, rs73885319 and rs60910145 (encoding S384G and I384M), while the G2 variant consists of a six base pair deletion removing amino acids N388 and Y389 (~21% and ~13% allelic frequency in African Americans for G1 and G2, respectively). We demonstrated that these variants in APOL1 are strong predictors of risk for proteinuria in SCD (Ashley-Koch et al. 2011). Here, we use zebrafish as an in vivo model to both examine the role of apol1 in glomerular development and pronephric filtration and also to test the effects of APOL1 G1 and G2 expression in the developing kidney. METHODS: A morpholino (MO) was designed by Gene Tools, LLC (Philomath, OR) to target the translation initiation site of zebrafish apol1. APOL1 G1 and G2 allelic constructs were synthesized from a wild-type (WT) APOL1 human open reading frame clone (GenBank: BC112943) using site-directed mutagenesis (Stratagene, QuikChange II), subsequently transcribed (mMESSAGE mMACHINE®, Life Technologies) into capped mRNA and co-injected with apol1-MO into zebrafish embryos. To assay glomerular filtration, 70 kDa FITC-conjugated dextran was injected into the cardiac venous sinus of 48 hour post-fertilization embryos. The eye vasculature of individual fish was imaged at two, 12, and 36 hours after dextran injection. The average fluorescence intensity was measured across the eye, and changes in intensity relative to the 2-hour post-injection measurements were calculated for comparison. Electron microscopy sections of five days post-fertilization embryos were cut on a Leica-Reichert Ultracut E ultramicrotome, and semi-thin sections (1.0μm) were stained and examined on a Phillips CM12 electron microscope. RESULTS: As we showed previously (Anderson et al., ASH 2013), MO-induced suppression of apol1 in zebrafish embryos results in pericardial edema, glomerular filtration defects, and extensive podocyte loss. Importantly, complementation of apol1 morphants with WT human APOL1 mRNA rescues the observed kidney defects. However, we now show that neither APOL1 G1 nor G2 risk alleles ameliorate defects caused by apol1 suppression. Notably, injection of APOL1 G2 alone results in renal defects, as indicated by increased dextran clearance and the presence of microvillus protrusions in the urinary space. Injection of APOL1 G1 alone, however, does not induce noticeable kidney dysfunction. Furthermore, when APOL1 G2 injected embryos were titrated with increasing concentrations of human APOL1 WT mRNA, we observed a significant reduction of edema formation in developing embryos, suggesting a possible dominant-negative effect of the altered protein. CONCLUSIONS: Unlike the WT APOL1 mRNA, neither the APOL1 G1 or G2 risk alleles could rescue kidney dysfunction due to knockdown of apol1 in zebrafish embryos, suggesting that these SCDN risk alleles impact the normal function of APOL1 in the kidney. Furthermore, development of edema with concomitant defects in glomerular ultrastructure in zebrafish embryos injected with APOL1 G2 mRNA alone suggest that this allele may act in a dominant-negative manner to induce kidney defects. Interestingly, it has been shown that APOL1 may cause toxic renal effects through programmed cell death pathways leading to glomerulosclerosis (Wan et al. 2008). Thus, apol1 suppression could result in the dysregulation of autophagic pathways, causing podocyte malformation and thereby affecting the susceptibility of the pronephros to glomerular injury. In summary, these data provide essential insight into the biological mechanisms by which APOL1 variants confer disease risk in human SCDN and other nondiabetic nephropathies. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Nephropathy is a common and devastating complication of sickle cell disease (SCD) associated with mortality (Elmariah et al, 2014). However, early detection of SCD nephropathy (SCDN) has proven difficult. Discovery of genetic markers associated with SCDN could greatly improve our ability to identify patients at risk for renal decline. To that end, we have performed a genome-wide association study (GWAS) of glomerular filtration rate (GFR) in our adult SCD cohort. METHODS: Medical history, laboratory values and DNA for genotyping were collected as part of a multicenter study of outcome-modifying genes in SCD. Participating institutions included sickle cell centers at Duke University, University of North Carolina at Chapel Hill, Emory University, and East Carolina University. GFR was estimated using the ‘Modification of Diet in Renal Disease’ (MDRD) study definition (Levey et al, 1999) and dichotomized at the clinically relevant threshold of 90 ml/min/1.73m2. 463 patients with complete data were included in the analysis (54.6% female, mean age 34.2 ± 12 years, 17.5% GFR < 90 ml/min/1.73m2). Genotyping was performed using the Illumina Human610-Quad BeadChip (Illumina, San Diego, CA), and a global reference panel from the 1000 Genomes project was used to impute genotypes not covered on the GWAS chip. Samples were pre-phased with SHAPEIT (Delaneau et al, 2012) and genotypes inferred using IMPUTE2 (Howie et al, 2009). After data cleaning and removal of single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) < 5%, 3,021,990 SNPs were available for analysis. Principal component (PC) analysis was performed to obtain measures of population stratification in our data set using EIGENSOFT (Patterson et al, 2006). Logistic regression was utilized to test for association between each SNP and dichotomized GFR, controlling for genome-wide PCs using PLINK (Purcell et al, 2008). False discovery rate (FDR) p-values were generated using PROC MULTTEST in SAS (SAS Systems, Cary, NC). RESULTS: Because GWAS chips capture common variation, several of the most highly associated SNPs were intergenic. The SNP with the most evidence for association that also lies within a gene was rs72777730 in XYLT1 (p=6.8E-6). For each additional C allele, the odds of having GFR < 90 ml/min/1.73m2 increased by 2.7. Other top hits included rs1990628 in ZNF423 (p=2.7E-5) and rs6449202 in SLC2A9 (p=3.8E-5). None of the findings met FDR significance. To further investigate our findings, we assessed the role of rare variants on renal function in our data set. We observed SNPs in XYLT1 from the Illumina HumanExome BeadChips associated with proteinuria (p=0.008) using the gene-based SKAT test (Wu et al, 2011). Further, we performed whole-exome sequencing of 15 patients with disparate GFR levels using VCRome 2.1 capture and 100bp paired-end reads generated from an Illumina HiSeq. One novel non-synonymous SNP (chr16:17353261, C>T) was identified in XYLT1 in 6 of 7 patients with low GFR (≤ 70 ml/min/1.73m2) but was absent in patients with normal GFR (> 90 ml/min/1.73m2). Additionally, four known non-synonymous SNPs were observed in low GFR patients but were absent in those with normal GFR levels. DISCUSSION: Xylosyltransferase 1 (XYLT1, also known as XT-1) encodes the enzyme responsible for biosynthesis of heparin sulfate proteoglycans, which affect permeability of the glomerular basement membrane. The c.343G>T polymorphism in XYLT1 has been implicated in diabetic nephropathy (Schön et al, 2006), and XT-1 is up-regulated in rats with adriamycin nephropathy (Sichuan et al, 2007). Zinc finger protein 423 (ZNF423) is involved in DNA damage response signaling, and mutations in ZNF423 have been identified in patients with nephronophthisis and Joubert syndrome (Chaki et al, 2012). Finally, solute carrier family 2 member 9 (SLC2A9, also known as GLUT9) is a transporter involved in urate reabsorption in the renal tubules. Mutations in this gene have been identified in patients with renal hypouricemia-2 (Matsuo et al, 2008). Thus, while none of the SNPs reached genome-wide significance, likely due to the modest size of our data set, we have identified several promising candidate genes nominally associated with renal function in our cohort of SCD patients and demonstrate that both common and rare variation in these genes likely contributes to risk for renal decline. Disclosures No relevant conflicts of interest to declare.