Gabe Haller

Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence

Genome-wide association studies have identified common variation in the CHRNA5-CHRNA3-CHRNB4 and CHRNA6-CHRNB3 gene clusters that contribute to nicotine dependence. However, the role of rare variation in risk for nicotine dependence in these nicotinic receptor genes has not been studied. We undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes in African American and European American nicotine-dependent smokers and smokers without symptoms of dependence. Carrier status of individuals harboring rare missense variants at conserved sites in each of these genes was then compared in cases and controls to test for an association with nicotine dependence. Missense variants at conserved residues in CHRNB4 are associated with lower risk for nicotine dependence in African Americans and European Americans (AA P = 0.0025, odds-ratio (OR) = 0.31, 95% confidence-interval (CI) = 0.31-0.72; EA P = 0.023, OR = 0.69, 95% CI = 0.50-0.95). Furthermore, these individuals were found to smoke fewer cigarettes per day than non-carriers (AA P = 6.6 × 10(-5), EA P = 0.021). Given the possibility of stochastic differences in rare allele frequencies between groups replication of this association is necessary to confirm these findings. The functional effects of the two CHRNB4 variants contributing most to this association (T375I and T91I) and a missense variant in CHRNA3 (R37H) in strong linkage disequilibrium with T91I were examined in vitro. The minor allele of each polymorphism increased cellular response to nicotine (T375I P = 0.01, T91I P = 0.02, R37H P = 0.003), but the largest effect on in vitro receptor activity was seen in the presence of both CHRNB4 T91I and CHRNA3 R37H (P = 2 × 10(-6)).

Rare variants in FBN1 and FBN2 are associated with severe adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) causes spinal deformity in 3% of children. Despite a strong genetic basis, few genes have been associated with AIS and the pathogenesis remains poorly understood. In a genome-wide rare variant burden analysis using exome sequence data, we identified fibrillin-1 (FBN1) as the most significantly associated gene with AIS. Based on these results, FBN1 and a related gene, fibrillin-2 (FBN2), were sequenced in a total of 852 AIS cases and 669 controls. In individuals of European ancestry, rare variants in FBN1 and FBN2 were enriched in severely affected AIS cases (7.6%) compared with in-house controls (2.4%) (OR = 3.5, P = 5.46 × 10(-4)) and Exome Sequencing Project controls (2.3%) (OR = 3.5, P = 1.48 × 10(-6)). Scoliosis severity in AIS cases was associated with FBN1 and FBN2 rare variants (P = 0.0012) and replicated in an independent Han Chinese cohort (P = 0.0376), suggesting that rare variants may be useful as predictors of curve progression. Clinical evaluations revealed that the majority of AIS cases with rare FBN1 variants do not meet diagnostic criteria for Marfan syndrome, though variants are associated with tall stature (P = 0.0035) and upregulation of the transforming growth factor beta pathway. Overall, these results expand our definition of fibrillin-related disorders to include AIS and open up new strategies for diagnosing and treating severe AIS.

A polygenic burden of rare variants across extracellular matrix genes among individuals with adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) is a complex inherited spinal deformity whose etiology has been elusive. While common genetic variants are associated with AIS, they explain only a small portion of disease risk. To explore the role of rare variants in AIS susceptibility, exome sequence data of 391 severe AIS cases and 843 controls of European ancestry were analyzed using a pathway burden analysis in which variants are first collapsed at the gene level then by Gene Ontology terms. Novel non-synonymous/splice-site variants in extracellular matrix genes were significantly enriched in AIS cases compared with controls (P = 6 × 10(-9), OR = 1.7, CI = 1.4-2.0). Specifically, novel variants in musculoskeletal collagen genes were present in 32% (126/391) of AIS cases compared with 17% (146/843) of in-house controls and 18% (780/4300) of EVS controls (P = 1 × 10(-9), OR = 1.9, CI = 1.6-2.4). Targeted resequencing of six collagen genes replicated this association in combined 919 AIS cases (P = 3 × 10(-12), OR = 2.2, CI = 1.8-2.7) and revealed a highly significant single-gene association with COL11A2 (P = 6 × 10(-9), OR = 3.8, CI = 2.6-7.2). Importantly, AIS cases harbor mainly non-glycine missense mutations and lack the clinical features of monogenic musculoskeletal collagenopathies. Overall, our study reveals a complex genetic architecture of AIS in which a polygenic burden of rare variants across extracellular matrix genes contributes strongly to risk.

Exome Sequencing Identifies a Rare HSPG2 Variant Associated with Familial Idiopathic Scoliosis

Idiopathic scoliosis occurs in 3% of individuals and has an unknown etiology. The objective of this study was to identify rare variants that contribute to the etiology of idiopathic scoliosis by using exome sequencing in a multigenerational family with idiopathic scoliosis. Exome sequencing was completed for three members of this multigenerational family with idiopathic scoliosis, resulting in the identification of a variant in the HSPG2 gene as a potential contributor to the phenotype. The HSPG2 gene was sequenced in a separate cohort of 100 unrelated individuals affected with idiopathic scoliosis and also was examined in an independent idiopathic scoliosis population. The exome sequencing and subsequent bioinformatics filtering resulted in 16 potentially damaging and rare coding variants. One of these variants, p.Asn786Ser, is located in the HSPG2 gene. The variant p.Asn786Ser also is overrepresented in a larger cohort of idiopathic scoliosis cases compared with a control population (P = 0.024). Furthermore, we identified additional rare HSPG2 variants that are predicted to be damaging in two independent cohorts of individuals with idiopathic scoliosis. The HSPG2 gene encodes for a ubiquitous multifunctional protein within the extracellular matrix in which loss of function mutation are known to result in a musculoskeletal phenotype in both mouse and humans. Based on these results, we conclude that rare variants in the HSPG2 gene potentially contribute to the idiopathic scoliosis phenotype in a subset of patients with idiopathic scoliosis. Further studies must be completed to confirm the effect of the HSPG2 gene on the idiopathic scoliosis phenotype.

Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration–response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC50 (β = −0.67, r2 = 0.017, P = 2×10−4) or by response to low nicotine (β = −0.29, r2 = 0.02, P = 6×10−5) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = −0.04, r2 = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.

Genome-wide association study identifies new disease loci for isolated clubfoot

Background Clubfoot is a common congenital birth defect with complex inheritance patterns. Currently, the genetic and morphological basis of clubfoot is poorly understood. To identify genetic risk factors associated with clubfoot, we performed a genome-wide association study of common genetic variants. Methods The DNA of 396 isolated clubfoot patients and 1000 controls of European descent was genotyped for >600 000 single nucleotide polymorphisms (SNP) using the Affymetrix 6.0 array. Replication was performed with an independent cohort of 370 isolated clubfoot cases and 363 controls of European descent. Results Strongest evidence for an association of clubfoot was found with an intergenic SNP on chromosome 12q24.31 between NCOR2 and ZNF664 (rs7969148, OR=0.58, p=1.25×10−5) that was significant on replication (combined OR=0.63, p=1.90×10−7). Additional suggestive SNPs were identified near FOXN3, SORCS1 and MMP7/TMEM123 that also confirmed on replication. Conclusions Our study suggests a potential role for common genetic variation in several genes that have not previously been implicated in clubfoot pathogenesis.

Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine

Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.

Positive selection on loci associated with drug and alcohol dependence

Much of the evolution of human behavior remains a mystery, including how certain disadvantageous behaviors are so prevalent. Nicotine addiction is one such phenotype. Several loci have been implicated in nicotine related phenotypes including the nicotinic receptor gene clusters (CHRNs) on chromosomes 8 and 15. Here we use 1000 Genomes sequence data from 3 populations (Africans, Asians and Europeans) to examine whether natural selection has occurred at these loci. We used Tajima’s D and the integrated haplotype score (iHS) to test for evidence of natural selection. Our results provide evidence for strong selection in the nicotinic receptor gene cluster on chromosome 8, previously found to be significantly associated with both nicotine and cocaine dependence, as well as evidence selection acting on the region containing the CHRNA5 nicotinic receptor gene on chromosome 15, that is genome wide significant for risk for nicotine dependence. To examine the possibility that this selection is related to memory and learning, we utilized genetic data from the Collaborative Studies on the Genetics of Alcoholism (COGA) to test variants within these regions with three tests of memory and learning, the Wechsler Adult Intelligence Scale (WAIS) Block Design, WAIS Digit Symbol and WAIS Information tests. Of the 17 SNPs genotyped in COGA in this region, we find one significantly associated with WAIS digit symbol test results. This test captures aspects of reaction time and memory, suggesting that a phenotype relating to memory and learning may have been the driving force behind selection at these loci. This study could begin to explain why these seemingly deleterious SNPs are present at their current frequencies.

Genetic Risk for Aortic Aneurysm in Adolescent Idiopathic Scoliosis

BACKGROUND Scoliosis is a feature of several genetic disorders that are also associated with aortic aneurysm, including Marfan syndrome, Loeys-Dietz syndrome, and type-IV Ehlers-Danlos syndrome. Life-threatening complications of aortic aneurysm can be decreased through early diagnosis. Genetic screening for mutations in populations at risk, such as patients with adolescent idiopathic scoliosis, may improve recognition of these disorders. METHODS The coding regions of five clinically actionable genes associated with scoliosis (COL3A1, FBN1, TGFBR1, TGFBR2, and SMAD3) and aortic aneurysm were sequenced in 343 adolescent idiopathic scoliosis cases. Gene variants that had minor allele frequencies of <0.0001 or were present in human disease mutation databases were identified. Variants were classified as pathogenic, likely pathogenic, or variants of unknown significance. RESULTS Pathogenic or likely pathogenic mutations were identified in 0.9% (three) of 343 adolescent idiopathic scoliosis cases. Two patients had pathogenic SMAD3 nonsense mutations consistent with type-III Loeys-Dietz syndrome and one patient had a pathogenic FBN1 mutation with subsequent confirmation of Marfan syndrome. Variants of unknown significance in COL3A1 and FBN1 were identified in 5.0% (seventeen) of 343 adolescent idiopathic scoliosis cases. Six FBN1 variants were previously reported in patients with Marfan syndrome, yet were considered variants of unknown significance based on the level of evidence. Variants of unknown significance occurred most frequently in FBN1 and were associated with greater curve severity, systemic features of Marfan syndrome, and joint hypermobility. CONCLUSIONS Clinically actionable pathogenic mutations in genes associated with adolescent idiopathic scoliosis and aortic aneurysm are rare in patients with adolescent idiopathic scoliosis who are not suspected of having these disorders, although variants of unknown significance are relatively common. CLINICAL RELEVANCE Routine genetic screening of all patients with adolescent idiopathic scoliosis for mutations in clinically actionable aortic aneurysm disease genes is not recommended on the basis of the high frequency of variants of unknown significance. Clinical evaluation and family history should heighten indications for genetic referral and testing.

ATRAID, a genetic factor that regulates the clinical action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFFs) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase (FDPS), resulting in defects in protein prenylation. Yet it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID. Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Lastly, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare non-synonymous coding variants in patients with ONJ or AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people. One Sentence Summary ATRAID is essential for responses to the commonly prescribed osteoporosis drugs nitrogen-containing bisphosphonates. Overline BONE