Biology and Biomedical Sciences: Molecular Genetics and Genomics
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Christina A Gurnett
Scoliosis is characterized by a lateral curvature of the spine that requires long-term bracing and invasive spinal surgery in cases with progressive deformity. Some individuals develop scoliosis secondary to congenital malformations or syndromic disorders, but most scoliosis is considered idiopathic and has no known cause. Adolescent idiopathic scoliosis (AIS) onsets in late childhood and causes spinal deformity in approximately 3% of the pediatric population. Despite a strong genetic basis, genetic risk factors for AIS are unknown and the pathogenesis remains poorly understood, which has been further hindered by the lack of a relevant animal model. Therefore, we used multiple approaches to better understand the genetic and molecular etiology of AIS using human and animal-based studies.
First, we performed copy number variation analysis on 143 patients with isolated scoliosis using the Affymetrix Genome-wide Human SNP Array 6.0. We identified a duplication of chromosome 1q21.1 in 2.1% (N=3/143) of AIS patients, which was enriched compared to 0.09% (N=1/1079) of controls (P=0.0057) and 0.07% (N=6/8329) of published controls (P=0.0004). Other notable findings include trisomy X, which was identified in 1.8% (N=2/114) of female AIS patients, and rearrangements of chromosome 15q11.2 and 16p11.2 that may be relevant to scoliosis susceptibility. We also report rare CNVs that will be of use to future studies investigating candidate genes for AIS.
Second, we performed a genome-wide rare variant burden analysis using exome sequence data and identified FBN1 (fibrillin-1) as the most significantly associated gene with AIS. Mutations in FBN1 are most frequently association with Marfan syndrome, a syndromic condition that causes scoliosis in 60% of patients. Based on these results, FBN1 and a related gene, FBN2 (fibrillin-2), 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 to 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 have utility 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 TGF-β pathway.
Finally, we characterized a recessive zebrafish mutant, called skolios, which develops a spinal deformity phenotype that parallels many features of human AIS, including an isolated lateral curvature of the spine that arises independent of congenital vertebral malformations. Skolios was identified in a mutagenesis screen and previously mapped to a 2.7 Mb region on chromosome 17. Because skolios may be an informative model of scoliosis, we sought to identify its genetic basis. We performed low coverage whole genome sequencing on a skolios mutant and identified a single nonsense mutation in the mapped region, which caused a premature stop in kinesin family member 6 (kif6), a poorly characterized kinesin of unknown function. To determine if the loss of kif6 is responsible for the skolios phenotype, we used TALENs to create additional mutant alleles. We isolated three new TALEN-induced mutations that caused frameshift mutations in kif6. All zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed body axis curvature that was indistinguishable from skolios mutants, verifying kif6 as the causative gene. Preliminary investigation into the mechanism of spinal deformity revealed no association with vertebral malformations or cilia defects in skolios mutants.
Overall, these results identify important genetic risk factors for AIS, including clinically relevant copy number variants and rare genetic variation in FBN1 and FBN2. Moreover, we have identified an animal model with scoliosis and demonstrated a novel role for kif6 in the developing spine. These findings have significant impact on our understanding of the genetic basis of AIS and reveal new strategies to identify and treat AIS.
Buchan, Jillian Gwen, "Genomic Analysis of Human Spinal Deformity and Characterization of a Zebrafish Disease Model" (2014). All Theses and Dissertations (ETDs). 1223.