The zebrafish provides insight into the genes implicated in IS
Late onset idiopathic scoliosis (IS) is a fixed lateral rotatory curvature of the spine that affects approximately 2 percent of prepubertal and adolescent individuals who are otherwise normal. The incidence, potential relentless progression, and morbidity surrounding current treatment modalities have frustrated both physicians and families.
Recent advances in the areas of genetics and genomics have afforded significant new insights into the understanding of the etiologic mechanisms of IS and the genetic underpinnings of this complex disease. This article reviews the genetic infrastructure of IS and the progress in the understanding of disease pathogenesis as related to gene discovery.
Population and family studies
Using a scoliosis curvature equal to or greater than 10⁰ as a threshold, the prevalence of IS across most population studies is 2 percent to 3 percent. Clinical observations, family studies, and twin studies have well established the familial nature of IS and have yielded sibling risk and heritability estimates. Calculated familial risk values, the lack of concordance in monozygotic twins, and the variability in heritability—both within and among families—are indicative of the complex genetic architecture of IS. A spectrum of inheritance patterns exists and unknown environmental and genetic modifications influence disease susceptibility and severity of disease progression.
Identification of IS genes
Candidate gene approaches—Prior to the availability of genome-wide techniques, the candidate gene approach was a classic hypothesis-driven approach for the identification of disease genes. Genes with known protein products that appeared to be relevant to the physiologic basis of the disease were tested in individuals, families, or identified sample populations.
The association of scoliosis with multiple connective tissue disorders led to the study of extracellular matrix genes, including collagens, elastin, fibrillin, and aggrecan. Most early studies, however, did not identify distinctive positive findings at the gene level. For those studies that did report positive findings, the results could not be replicated by independent laboratories.
Mapping of genetic susceptibility loci—Advances in the mapping of the human genome and in genetic technology led to hypothesis-free, genome-wide approaches that allow for the screening of the entire genome with known genetic markers (for example, tandem repeats) located at specified intervals across the genome or high-density single nucleotide polymorphisms (SNPs).
Within families, the physical proximity of a genetic marker to a functional area of the genome in affected individuals identifies potential regions that may harbor disease-specific mutations.
Family-based linkage studies have identified multiple genetic loci related to familial IS. Five loci have been listed in the Online Mendelian Inheritance in Man, including chromosomes 19p13.1, 17p11, 17q25, 8q12, and 9q31–q34. To date, the area on chromosome 9 remains the only locus independently identified in unrelated familial cohorts.
Each study, however, has been constructed around a different set of variables and different genetic statistical programs have been applied. Despite their differing results, these studies should not be construed as conflicting in nature, but as examples of the genetic heterogeneity of IS that exists both between and among families.
Population-based studies directed to the identification of disease loci utilize high-density panels of common SNPs that span the entire genome in a large population of affected individuals (cases) versus a control population. Statistical association analyses then compare the frequencies of the SNP alleles between cases and controls (known as genome-wide association studies or GWAS). This results in an array of SNPs that may be associated to the observed phenotype, in this case IS.
Due to the high cost and investment of GWAS, the National Human Genome Research Institute (NHGRI) and the European Bioinformatics Institute (EMBL-EBI) have collaborated to produce a GWAS catalog available to the public. The catalog is a collection of all published GWAS that have adequate quality controls, SNP density, and statistical analyses (www.ebi.ac.uk/gwas/).
Eight publications on IS have met the catalog criteria and collectively report the association of 22 common SNP variants with IS. Of note is the omission of the GWAS studies related to the DNA-based prognostic test known as the ScoliScore. The studies included within the catalog have involved multiple ethnic groups, varying study designs, and differing study parameters of affectation status of cases versus controls. The identified variants reported are located on multiple chromosomes and in a variety of functional areas of the genome, including both exon and intron areas of specific genes, and in gene regulatory regions. Associated genes include LBX1, CHL1, BNC2, GPR126, MEIS1, BCL-2, MAGI1, TNIK, SOX9, TNIK, AJAP1, FOXA2, and PAX1.
Genes vary in functionality from spinal cord nerve development, axon guidance, expression of cartilage-associated genes, cilia-directed symmetry of right and left, extracellular matrix structure, and intercellular communication. Associations that have been replicated across populations are variants of LBX1, a strong influencer of dorsal spinal cord neural development through Wnt/B-catenin signaling, and GPR126, a gene associated with height, body mass, and pulmonary function. Conceptually, these genes and their associated molecular pathways can be related to the pathophysiology of IS, however, these genes remain candidates of IS because causality has not yet been demonstrated.
Recent research avenues
Genomic studies are continuing with larger populations to identify common genetic variants associated with IS. Other studies have used exome sequencing approaches, with or without the aid of previous linkage results, in defined populations and families to identify rare variants that may contribute to disease. Novel variants in extracellular matrix genes include the collagens (most significantly COL11A2) and HSPG2 and have been identified in specific families and populations. This indicates that a polygenic burden of rare variants across extracellular matrix genes potentially contributes strongly to the risk of IS.
Disease modeling in animals has been an important step in the transition from genetic discovery to translational medicine. Recently, the zebrafish has proven to be a potentially powerful animal model system for IS due to its ease of genetic manipulation, genetic resources surrounding its genome, and short breeding cycles (Fig. 1). As an identified IS model, the zebrafish provides insight into the genes implicated in IS and has become a vehicle for functional validation of human IS-associated genes and variants. Studies of the ptk7 mutant zebrafish, which exhibit an IS-type curvature, have determined that motile cilia function and cerebrospinal fluid flow are essential to spinal cord development. Additional studies report the potential functional validation of genes such as GPR126 and MAPK7 within the zebrafish animal model.
Efforts to understand the etiology of IS may lead to more effective, less invasive treatment methods. Because mild and moderate curvatures do not lead to significant morbidity, the more substantial goal is the ability to define those scoliosis curvatures that are relentlessly progressive and lead to significant complications during adulthood.
Research has confirmed the complexity of the genetic architecture of IS and has provided great insight into candidate genes, genetic variants, and molecular pathways that are potentially related to disease pathogenesis.
To date, many results indicate elements of the neurologic and developmental mechanisms, lending some support to long-held theories of IS etiology. These studies, in conjunction with new genomic methodologies, allow for hypothesis-driven testing in animal models. The creative application of these methodologies will add to the understanding of the functional pathogenesis of IS.
Although this research is still in its infancy in relation to clinical applicability, ultimately it will provide the basis for new therapeutic interventions to minimize the severity and/or penetrance of IS in the lives of individuals and families.
Nancy Hadley Miller, MS, MD, is a pediatric orthopaedic clinician scientist. She instigated her study of idiopathic scoliosis under the mentorship of Ignacio Ponseti, MD, and Stuart Weinstein, MD, during her time at the University of Iowa.
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