Elucidating the Genetic Basis of Chiari I Malformation.

Several studies have been performed to elucidate the genetic basis of Chiari I malformation (CM1). The heritability of CM1 is clear from twin studies, familial clustering, and the prevalence of CM1 among certain classes of Mendelian disorders, namely connective tissue disorders, brain overgrowth disorders, disorders of CSF homeostasis, certain tumors, disorders of skull development and vascular conditions. A comprehensive understanding of the causes of CM1 will require large cohorts of patients for genetic studies and in-depth phenotyping of cases to better understand the biological mechanisms underlying disease.

Whole-exome analysis of adolescents with low VWF and heavy menstrual bleeding identifies novel genetic associations.

Adolescents with low von Willebrand factor (VWF) levels and heavy menstrual bleeding (HMB) experience significant morbidity. There is a need to better characterize these patients genetically and improve our understanding of the pathophysiology of bleeding. We performed whole-exome sequencing on 86 postmenarchal patients diagnosed with low VWF levels (30-50 IU/dL) and HMB and compared them with 660 in-house controls. We compared the number of rare stop-gain/stop-loss and rare ClinVar "pathogenic" variants between cases and controls, as well as performed gene burden and gene-set burden analyses. We found an enrichment in cases of rare stop-gain/stop-loss variants in genes involved in bleeding disorders and an enrichment of rare ClinVar "pathogenic" variants in genes involved in anemias. The 2 most significant genes in the gene burden analysis, CFB and DNASE2, are associated with atypical hemolytic uremia and severe anemia, respectively. VWF also surpassed exome-wide significance in the gene burden analysis (P = 7.31 × 10-6). Gene-set burden analysis revealed an enrichment of rare nonsynonymous variants in cases in several hematologically relevant pathways. Further, common variants in FERMT2, a gene involved in the regulation of hemostasis and angiogenesis, surpassed genome-wide significance. We demonstrate that adolescents with HMB and low VWF have an excess of rare nonsynonymous and pathogenic variants in genes involved in bleeding disorders and anemia. Variants of variable penetrance in these genes may contribute to the spectrum of phenotypes observed in patients with HMB and could partially explain the bleeding phenotype. By identifying patients with HMB who possess these variants, we may be able to improve risk stratification and patient outcomes.

Rare and de novo duplications containing SHOX in clubfoot.

INTRODUCTION: Congenital clubfoot is a common birth defect that affects at least 0.1% of all births. Nearly 25% cases are familial and the remaining are sporadic in inheritance. Copy number variants (CNVs) involving transcriptional regulators of limb development, including PITX1 and TBX4, have previously been shown to cause familial clubfoot, but much of the heritability remains unexplained. METHODS: Exome sequence data from 816 unrelated clubfoot cases and 2645 in-house controls were analysed using coverage data to identify rare CNVs. The precise size and location of duplications were then determined using high-density Affymetrix Cytoscan chromosomal microarray (CMA). Segregation in families and de novo status were determined using qantitative PCR. RESULTS: Chromosome Xp22.33 duplications involving SHOX were identified in 1.1% of cases (9/816) compared with 0.07% of in-house controls (2/2645) (p=7.98×10-5, OR=14.57) and 0.27% (38/13592) of Atherosclerosis Risk in Communities/the Wellcome Trust Case Control Consortium 2 controls (p=0.001, OR=3.97). CMA validation confirmed an overlapping 180.28 kb duplicated region that included SHOX exons as well as downstream non-coding regions. In four of six sporadic cases where DNA was available for unaffected parents, the duplication was de novo. The probability of four de novo mutations in SHOX by chance in a cohort of 450 sporadic clubfoot cases is 5.4×10-10. CONCLUSIONS: Microduplications of the pseudoautosomal chromosome Xp22.33 region (PAR1) containing SHOX and downstream enhancer elements occur in ~1% of patients with clubfoot. SHOX and regulatory regions have previously been implicated in skeletal dysplasia as well as idiopathic short stature, but have not yet been reported in clubfoot. SHOX duplications likely contribute to clubfoot pathogenesis by altering early limb development.

Obex position is associated with syringomyelia and use of posterior fossa decompression among patients with Chiari I malformation.

OBJECTIVE: Chiari I malformation (CM-I) has traditionally been defined by measuring the position of the cerebellar tonsils relative to the foramen magnum. The relationships of tonsillar position to clinical presentation, syringomyelia, scoliosis, and the use of posterior fossa decompression (PFD) surgery have been studied extensively and yielded inconsistent results. Obex position has been proposed as a useful adjunctive descriptor for CM-I and may be associated with clinical disease severity. METHODS: A retrospective chart review was performed of 442 CM-I patients with MRI who presented for clinical evaluation between 2003 and 2018. Clinical and radiological variables were measured for all patients, including presence/location of headaches, Chiari Severity Index (CSI) grade, tonsil position, obex position, clival canal angle, pB-C2 distance, occipitalization of the atlas, basilar invagination, syringomyelia, syrinx diameter, scoliosis, and use of PFD. Radiological measurements were then used to predict clinical characteristics using regression and survival analyses, with performing PFD, the presence of a syrinx, and scoliosis as outcome variables. RESULTS: Among the radiological measurements, tonsil position, obex position, and syringomyelia were each independently associated with use of PFD. Together, obex position, tonsil position, and syringomyelia (area under the curve [AUC] 89%) or obex position and tonsil position (AUC 85.4%) were more strongly associated with use of PFD than tonsil position alone (AUC 76%) (Pdiff = 3.4 × 10-6 and 6 × 10-4, respectively) but were only slightly more associated than obex position alone (AUC 82%) (Pdiff = 0.01 and 0.18, respectively). Additionally, obex position was significantly associated with occipital headaches, CSI grade, syringomyelia, and scoliosis, independent of tonsil position. Tonsil position was associated with each of these traits when analyzed alone but did not remain significantly associated with use of PFD when included in multivariate analyses with obex position. CONCLUSIONS: Compared with tonsil position alone, obex position is more strongly associated with symptomatic CM-I, as measured by presence of a syrinx, scoliosis, or use of PFD surgery. These results support the role of obex position as a useful radiological measurement to inform the evaluation and potentially the management of CM-I.

Prevalence and Impact of Underlying Diagnosis and Comorbidities on Chiari 1 Malformation.

BACKGROUND: Chiari malformation type 1 affects approximately one in 1,000 people symptomatically, although one in 100 meet radiological criteria, making it a common neurological disorder. The diagnosis of underlying conditions has become more sophisticated, and new radiological markers of disease have been described. We sought to determine the prevalence and impact of additional comorbidities and underlying diagnoses in patients with Chiari malformation type 1 on symptomatology and surgical treatment. METHODS: A retrospective review of 612 pediatric patients with a Chiari malformation type 1 diagnosis and imaging data evaluated between 2008 and 2018 was performed. Because of extensive clinical heterogeneity, patients were separated into four categories based on their primary comorbidities (nonsyndromic, central nervous system, skeletal, and multiple congenital anomalies) to identify associations with age of onset, radiographic measurements, syringomyelia, and surgical treatment. RESULTS: The largest group had nonsyndromic Chiari malformation type 1 (70%) and the latest age at diagnosis of any group. In the syndromic group, 6% were diagnosed with a known genetic abnormality, with overgrowth syndromes being the most common. Patients with multiple congenital anomalies had the earliest Chiari malformation type 1 onset, the most severe tonsillar ectopia and obex position, and were overrepresented among surgical cases. Although there were no statistically significant differences between groups and rates of syrinx, we observed differences in individual diagnoses. CONCLUSION: The underlying diagnoses and presence of comorbidities in patients with Chiari malformation type 1 impacts rates of syringomyelia and surgery. Although most Chiari malformation type 1 cases are nonsyndromic, clinical evaluation of growth parameters, scoliosis, and joint hypermobility should be routine for all patients as they are useful to determine syringomyelia risk and may impact treatment.

Lack of joint hypermobility increases the risk of surgery in adolescent idiopathic scoliosis.

Generalized joint hypermobility (GJH) is a risk factor for developing adolescent idiopathic scoliosis (AIS); however, it is not known whether joint hypermobility influences the risk of progression to surgery. Beighton joint hypermobility scores were assessed in 570 female AIS patients. Multivariate analysis was carried out to determine whether Beighton hypermobility scores were predictors of surgical intervention. In this female AIS cohort, 24.7% (141/570) had GJH (Beighton score ≥4). Multivariate analysis showed that GJH did not influence the risk of surgery, although having no joint hypermobility (Beighton score=0) increased risk (odds ratio: 1.89; P=0.003). Females who had no hypermobility (score=0) had significantly larger curves than individuals who scored at least one point on the Beighton scale [50° (interquartile range: 26) vs. 42° (interquartile range: 24), P=0.001]. Evaluation of specific measures of joint hypermobility indicated that females who could not touch their palms to the floor were 2.1-fold more likely to have surgery than patients who could perform this task (P=0.001). None of the other features measured on the Beighton score correlated with surgical risk. The lack of joint hypermobility increases the odds of surgery in females with AIS. Specifically, inability to touch the palms to the floor is an indicator of progression to surgery.

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.

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 (ß = -0.67, r2 = 0.017, P = 2 × 10(-4)) or by response to low nicotine (ß = -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 (ß = -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.

Variants near CHRNB3-CHRNA6 are associated with DSM-5 cocaine use disorder: evidence for pleiotropy.

In the U.S.A., cocaine is the second most abused illicit drug. Variants within the CHRNB3-A6 gene cluster have been associated with cigarette consumption in several GWAS. These receptors represent intriguing candidates for the study of cocaine dependence because nicotinic receptors are thought to be involved in generalized addiction pathways. Using genotypic data from a GWAS of the Study of Addiction: Genetics and Environment (SAGE) dataset, we tested for association of CHRNB3-A6 SNPs with DSM-5 cocaine use disorder. Multiple SNPs in the region were significantly associated with increased risk of cocaine use disorder. Inclusion of the most significant SNP as a covariate in a linear regression model provided evidence for an additional independent signal within this locus for cocaine use disorder. These results suggest that the CHRNB3-A6 locus contains multiple variants affecting risk for vulnerability to cocaine and nicotine dependence as well as bipolar disorder, suggesting that they have pleiotropic effects.

Rare missense variants in CHRNB3 and CHRNA3 are associated with risk of alcohol and cocaine dependence.

Previous findings have demonstrated that variants in nicotinic receptor genes are associated with nicotine, alcohol and cocaine dependence. Because of the substantial comorbidity, it has often been unclear whether a variant is associated with multiple substances or whether the association is actually with a single substance. To investigate the possible contribution of rare variants to the development of substance dependencies other than nicotine dependence, specifically alcohol and cocaine dependence, we undertook pooled sequencing of the coding regions and flanking sequence of CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 in 287 African American and 1028 European American individuals from the Collaborative Study of the Genetics of Alcoholism (COGA). All members of families for whom any individual was sequenced (2504 African Americans and 7318 European Americans) were then genotyped for all variants identified by sequencing. For each gene, we then tested for association using FamSKAT. For European Americans, we find increased DSM-IV cocaine dependence symptoms (FamSKAT P = 2 × 10(-4)) and increased DSM-IV alcohol dependence symptoms (FamSKAT P = 5 × 10(-4)) among carriers of missense variants in CHRNB3. Additionally, one variant (rs149775276; H329Y) shows association with both cocaine dependence symptoms (P = 7.4 × 10(-5), ß = 2.04) and alcohol dependence symptoms (P = 2.6 × 10(-4), ß = 2.04). For African Americans, we find decreased cocaine dependence symptoms among carriers of missense variants in CHRNA3 (FamSKAT P = 0.005). Replication in an independent sample supports the role of rare variants in CHRNB3 and alcohol dependence (P = 0.006). These are the first results to implicate rare variants in CHRNB3 or CHRNA3 in risk for alcohol dependence or cocaine dependence.

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)).