Are copy number variants associated with adolescent idiopathic scoliosis?

BACKGROUND: Adolescent idiopathic scoliosis (AIS) is a complex genetic disorder that causes spinal deformity in approximately 3% of the population. Candidate gene, linkage, and genome-wide association studies have sought to identify genetic variation that predisposes individuals to AIS, but the genetic basis remains unclear. Copy number variants are associated with several isolated skeletal phenotypes, but their role in AIS, to our knowledge, has not been assessed. QUESTIONS/PURPOSES: We determined the frequency of recurrent copy number rearrangements, chromosome aneuploidy, and rare copy number variants in patients with AIS. METHODS: Between January 2010 and August 2014, we evaluated 150 patients with isolated AIS and spinal curvatures measuring 10° or greater, and 148 agreed to participate. Genomic copy number analysis was performed on patients and 1079 control subjects using the Affymetrix(®) Genome-wide Human SNP Array 6.0. After removing poor quality samples, 143 (97%) patients with AIS were evaluated for copy number variation. RESULTS: We identified a duplication of chromosome 1q21.1 in 2.1% (N = 3/143) of patients with AIS, which was enriched compared with 0.09% (N = 1/1079) of control subjects (p = 0.0057) and 0.07% (N = 6/8329) of a large published control cohort (p = 0.0004). Other notable findings include trisomy X, which was identified in 1.8% (N = 2/114) of female patients with AIS, and rearrangements of chromosome 15q11.2 and 16p11.2 that previously have been associated with spinal phenotypes. Finally, we report rare copy number variants that will be useful in future studies investigating candidate genes for AIS. CONCLUSIONS: Copy number variation and chromosomal aneuploidy may contribute to the pathogenesis of adolescent idiopathic scoliosis. CLINICAL RELEVANCE: Chromosomal microarray may reveal clinically useful abnormalities in some patients with AIS.

Pitx1 haploinsufficiency causes clubfoot in humans and a clubfoot-like phenotype in mice.

Clubfoot affects 1 in 1000 live births, although little is known about its genetic or developmental basis. We recently identified a missense mutation in the PITX1 bicoid homeodomain transcription factor in a family with a spectrum of lower extremity abnormalities, including clubfoot. Because the E130K mutation reduced PITX1 activity, we hypothesized that PITX1 haploinsufficiency could also cause clubfoot. Using copy number analysis, we identified a 241 kb chromosome 5q31 microdeletion involving PITX1 in a patient with isolated familial clubfoot. The PITX1 deletion segregated with autosomal dominant clubfoot over three generations. To study the role of PITX1 haploinsufficiency in clubfoot pathogenesis, we began to breed Pitx1 knockout mice. Although Pitx1(+/-) mice were previously reported to be normal, clubfoot was observed in 20 of 225 Pitx1(+/-) mice, resulting in an 8.9% penetrance. Clubfoot was unilateral in 16 of the 20 affected Pitx1(+/-) mice, with the right and left limbs equally affected, in contrast to right-sided predominant hindlimb abnormalities previously noted with complete loss of Pitx1. Peroneal artery hypoplasia occurred in the clubfoot limb and corresponded spatially with small lateral muscle compartments. Tibial and fibular bone volumes were also reduced. Skeletal muscle gene expression was significantly reduced in Pitx1(-/-) E12.5 hindlimb buds compared with the wild-type, suggesting that muscle hypoplasia was due to abnormal early muscle development and not disuse atrophy. Our morphological data suggest that PITX1 haploinsufficiency may cause a developmental field defect preferentially affecting the lateral lower leg, a theory that accounts for similar findings in human clubfoot.

Familial isolated clubfoot is associated with recurrent chromosome 17q23.1q23.2 microduplications containing TBX4.

Clubfoot is a common musculoskeletal birth defect for which few causative genes have been identified. To identify the genes responsible for isolated clubfoot, we screened for genomic copy-number variants with the Affymetrix Genome-wide Human SNP Array 6.0. A recurrent chromosome 17q23.1q23.2 microduplication was identified in 3 of 66 probands with familial isolated clubfoot. The chromosome 17q23.1q23.2 microduplication segregated with autosomal-dominant clubfoot in all three families but with reduced penetrance. Mild short stature was common and one female had developmental hip dysplasia. Subtle skeletal abnormalities consisted of broad and shortened metatarsals and calcanei, small distal tibial epiphyses, and thickened ischia. Several skeletal features were opposite to those described in the reciprocal chromosome 17q23.1q23.2 microdeletion syndrome associated with developmental delay and cardiac and limb abnormalities. Of note, during our study, we also identified a microdeletion at the locus in a sibling pair with isolated clubfoot. The chromosome 17q23.1q23.2 region contains the T-box transcription factor TBX4, a likely target of the bicoid-related transcription factor PITX1 previously implicated in clubfoot etiology. Our result suggests that this chromosome 17q23.1q23.2 microduplication is a relatively common cause of familial isolated clubfoot and provides strong evidence linking clubfoot etiology to abnormal early limb development.

Soft-Tissue Abnormalities Associated with Treatment-Resistant and Treatment-Responsive Clubfoot: Findings of MRI Analysis.

BACKGROUND: Clubfoot treatment commonly fails and often results in impaired quality of life. An understanding of the soft-tissue abnormalities associated with both treatment-responsive and treatment-resistant clubfoot is important to improving the diagnosis of clubfoot, the prognosis for patients, and treatment. METHODS: Twenty patients with clubfoot treated with the Ponseti method were recruited for magnetic resonance imaging (MRI) of their lower extremities. Among these were seven patients (six unilateral cases) with treatment-responsive clubfoot and thirteen patients (five unilateral cases) with treatment-resistant clubfoot. Demographic information and physical examination findings were recorded. A descriptive analysis of the soft-tissue abnormalities was performed for both patient cohorts. For the patients with unilateral clubfoot, we calculated the percentage difference in cross-sectional area between the affected limb and the unaffected limb in terms of muscle, subcutaneous fat, intracompartment fat, and total area. With use of the Wilcoxon signed-rank test, we compared inter-leg differences in cross-sectional areas and the intracompartment adiposity index (IAI) between treatment-responsive and treatment-resistant groups. The IAI characterizes the cross-sectional area of fat within a muscle compartment. RESULTS: Extensive soft-tissue abnormalities were more present in patients with treatment-resistant clubfoot than in patients with treatment-responsive clubfoot. Treatment-resistant clubfoot abnormalities included excess epimysial fat and intramuscular fat replacement as well as unique patterns of hypoplasia in specific muscle groups that were present within a subset of patients. Among the unilateral cases, treatment-resistant clubfoot was associated with a significantly greater difference in muscle area between the affected and unaffected limb (-47.8%) compared with treatment-responsive clubfoot (-26.6%) (p = 0.02), a significantly greater difference in intracompartment fat area between the affected and unaffected limb (402.6%) compared with treatment-responsive clubfoot (9%) (p = 0.01), and a corresponding higher inter-leg IAI ratio (8.7) compared with treatment-responsive clubfoot (1.5) (p = 0.01). CONCLUSIONS: MRI demonstrated a range of soft-tissue abnormalities in patients, including unique patterns of specific muscle-compartment aplasia/hypoplasia that were present in patients with treatment-resistant clubfoot and not present in patients with treatment-responsive clubfoot. Correlations between MRI, physical examination, and treatment responsiveness may aid in the development of a prognostic classification system for clubfoot. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Optical mapping of the isolated coronary-perfused human sinus node.

OBJECTIVES: We sought to confirm our hypothesis that the human sinoatrial node (SAN) is functionally insulated from the surrounding atrial myocardium except for several exit pathways that electrically bridge the nodal tissue and atrial myocardium. BACKGROUND: The site of origin and pattern of excitation within the human SAN has not been directly mapped. METHODS: The SAN was optically mapped in coronary-perfused preparations from nonfailing human hearts (n = 4, age 54 ± 15 years) using the dye Di-4-ANBDQBS and blebbistatin. The SAN 3-dimensional structure was reconstructed using histology. RESULTS: Optical recordings from the SAN had diastolic depolarization and multiple upstroke components, which corresponded to the separate excitations of the SAN and atrial layers. Excitation originated in the middle of the SAN (66 ± 17 beats/min), and then spread slowly (1 to 18 cm/s) and anisotropically. After a 82 ± 17 ms conduction delay within the SAN, the atrial myocardium was excited via superior, middle, and/or inferior sinoatrial conduction pathways. Atrial excitation was initiated 9.4 ± 4.2 mm from the leading pacemaker site. The oval 14.3 ± 1.5 mm × 6.7 ± 1.6 mm × 1.0 ± 0.2 mm SAN structure was functionally insulated from the atrium by connective tissue, fat, and coronary arteries, except for these pathways. CONCLUSIONS: These data demonstrated for the first time, to our knowledge, the location of the leading SAN pacemaker site, the pattern of excitation within the human SAN, and the conduction pathways into the right atrium. The existence of these pathways explains why, even during normal sinus rhythm, atrial breakthroughs could arise from a region parallel to the crista terminalis that is significantly larger (26.1 ± 7.9 mm) than the area of the anatomically defined SAN.