Allison Scott

Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes.

The EXT family of putative tumor suppressor genes affect endochondral bone growth, and mutations in EXT1 and EXT2 genes cause the autosomal dominant disorder Hereditary Multiple Exostoses (HME). Loss of heterozygosity (LOH) of these genes plays a role in the development of exostoses and chondrosarcomas. In this study, we characterized EXT genes in 11 exostosis chondrocyte strains using LOH and mutational analyses. We also determined subcellular localization and quantitation of EXT1 and EXT2 proteins by immunocytochemistry using antibodies raised against unique peptide epitopes. In an isolated non-HME exostosis, we detected three genetic hits: deletion of one EXT1 gene, a net 21-bp deletion within the other EXT1 gene and a deletion in intron 1 causing loss of gene product. Diminished levels of EXT1 and EXT2 protein were found in 9 (82%) and 5 (45%) exostosis chondrocyte strains, respectively, and 4 (36%) were deficient in levels of both proteins. Although we found mutations in exostosis chondrocytes, mutational analysis alone did not predict all the observed decreases in EXT gene products in exostosis chondrocytes, suggesting additional genetic mutations. Moreover, exostosis chondrocytes exhibit an unusual cellular phenotype characterized by abnormal actin bundles in the cytoplasm. These results suggest that multiple mutational steps are involved in exostosis development and that EXT genes play a role in cell signaling related to chondrocyte cytoskeleton regulation.

Hereditary multiple exostosis and pain.

This study was undertaken to characterize pain in individuals with hereditary multiple exostosis (HME). Two hundred ninety-three patients with HME completed a questionnaire designed to assess pain as well as its impact on their life. Eighty-four percent of participants reported having pain, indicating that pain is a real problem in HME. Of those with pain, 55.1% had generalized pain. Two factors were found to be associated with pain outcome: HME-related complications and surgery. Individuals who had HME-related complications were five times more likely to have pain, while those who had surgery were 3.8 more likely to have pain. No differences were found between males and females with respect to pain, surgery, or HME-related complications. The results of this study indicate that the number of individuals with HME who have pain has been underestimated and that pain is a problem that must be addressed when caring for individuals with HME.

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME‐associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage‐capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non‐HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5‐μm repeat cross‐bridges of α‐actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle‐specific α‐actin, whereas β‐actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

Altered transmission of HOX and apoptotic SNPs identify a potential common pathway for clubfoot.

Clubfoot is a common birth defect that affects 135,000 newborns each year worldwide. It is characterized by equinus deformity of one or both feet and hypoplastic calf muscles. Despite numerous study approaches, the cause(s) remains poorly understood although a multifactorial etiology is generally accepted. We considered the HOXA and HOXD gene clusters and insulin‐like growth factor binding protein 3 (IGFBP3) as candidate genes because of their important roles in limb and muscle morphogenesis. Twenty SNPs from the HOXA and HOXD gene clusters and 12 SNPs in IGFBP3 were genotyped in a sample composed of non‐Hispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of non‐Hispanic white simplex trios (validation sample). Four SNPs (rs6668, rs2428431, rs3801776, and rs3779456) in the HOXA cluster demonstrated altered transmission in the discovery sample, but only rs3801776, located in the HOXA basal promoter region, showed altered transmission in both the discovery and validation samples (P = 0.004 and 0.028). Interestingly, HOXA9 is expressed in muscle during development. An SNP in IGFBP3, rs13223993, also showed altered transmission (P = 0.003) in the discovery sample. Gene–gene interactions were identified between variants in HOXA, HOXD, and IGFBP3 and with previously associated SNPs in mitochondrial‐mediated apoptotic genes. The most significant interactions were found between CASP3 SNPS and variants in HOXA, HOXD, and IGFBP3. These results suggest a biologic model for clubfoot in which perturbation of HOX and apoptotic genes together affect muscle and limb development, which may cause the downstream failure of limb rotation into a plantar grade position.

NAT2 variation and idiopathic talipes equinovarus (clubfoot)

Idiopathic talipes equinovarus (ITEV), or isolated clubfoot, is a common developmental anomaly that is characterized by a rigid foot, adducted forefoot, cavus midfoot, equinovarus of the hindfoot, and hypoplastic calf musculature. The etiology of this common birth defect is largely unknown, but genetic factors have been implicated in population and family studies and maternal smoking during pregnancy has been identified as an environmental risk factor. The biotransformation of exogenous substances, such as tobacco smoke, is modulated by numerous genes including N‐acetylation genes, NAT1 and NAT2. Functional variants of these genes exist and can be distinguished by genotyping. We hypothesized that variation in NAT1 and NAT2 genes might be associated with ITEV. To test this hypothesis, NAT1 and NAT2 were genotyped in a sample of 56 multiplex ITEV families, 57 trios with a positive family history and 160 simplex trios with ITEV. The results detected a slight decrease in the expected number of homozygotes for the NAT2 normal allele in the Hispanic simplex trios. In addition, in a pilot case–control study of ITEV, there were significantly more slow NAT2 acetylators among the cases. This suggests that slow acetylation may be a risk factor for ITEV. This study is the first to find evidence suggesting a role for a biotransformation candidate gene in the etiology of ITEV and provides a scientific foundation to further explore the contributions of other tobacco metabolism genes in the etiology of clubfoot.

Evidence for locus heterogeneity in the Camurati–Engelmann (DPD1) Syndrome

To the Editor: Camurati–Engelmann Syndrome (DPD1) (MIM 131300) is a rare, autosomal dominant sclerosing bone disorder belonging to the osteopetroses group of bone conditions (1–5). Waddling gait, muscle weakness and wasting, pain and generalized fatigue are characteristic clinical features. Progressive cortical sclerosis involving the diaphyses of the long bones and the base of the skull are the major radiographic findings. The locus for DPD1 has recently been mapped to chromosome 19q13.1-13.3 (6, 7). Subsequently, mutations have been identified in individuals with DPD1 in the human transforming growth factor b1 gene (TGFb1), a candidate gene because of its location on chromosome 19 (8). We report a family with typical DPD1 in which no TGFb1 mutation can be identified, and in which linkage analysis excludes the disease from the region surrounding TGFb1. The multigenerational family with DPD1 is shown in Fig. 1. The DPD1 in affected individual II-1 appears to be the result of a new mutation as he is the first affected individual in his family. He has three affected children with two different women. Individual III-2 (proband) was diagnosed with Camurati–Engelmann Syndrome at 14 years after a 3-year history of lower extremity bone pain. Her musculoskeletal symptoms consist of joint aching, particularly in the lower extremities, as well as pain in the hips predominantly with walking or lying on her side. X-rays documented increased bone density in the petrous and frontal bone of the skull. Femurs and tibias show symmetric expansion of the mid-shaft with undulating cortical thickening, also present in the radius and humeri, bilaterally. Corticosteroids have not significantly improved her clinical symptomatology. All of the affected individuals had onset of symptoms in late childhood or early adolescence and demonstrate the characteristic sclerosing diaphyseal dysplasia (Fig. 2). Linkage analysis was performed using the following PCR STR markers: D19S587, D19S8881, D19S223, D19S400, D19S718, D19S211, D19S913, D19S408, D19S178 (7). Two-point linkage analysis was conducted using MLINK of the package LINKAGE version 5.1 (9). Multipoint analysis was conducted using VITESSE (10). SimWalk2 (11) was used to reconstruct haplotypes from data converted using Mega2 (12). Two point lod scores for all informative markers (markers D19S211 and D19S408 were not informative) were –3.1 at u= 0.0 and –2.1 at u=0.001. Multipoint lod scores over the entire region ranged from –3.2 to –6.2. Haplotypes of the informative markers, as reconstructed with SimWalk2, are depicted in Fig. 1. It has previously been shown that the DPD1 locus lies in the 3.2 cM region flanked by D19S881 and D19S211 (7). As D19S211 was uninformative in this pedigree, D19S913 becomes the next closest flanking marker. The pedigree clearly demonstrates that individual III-3 inherited different chromosome 19 marker alleles from her affected parent II-2 than were inherited by her other two affected siblings, III-1 and III-2. The inter-marker distances are small making a double recombination in the region between two markers unlikely. However, to rule this possibility out, we sequenced TGFb1 to

Adductor transfers in cerebral palsy: long-term results studied by gait analysis.

Thirty-three ambulatory patients with spastic cerebral palsy underwent evaluation and gait analysis an average of 9.6 years after bilateral posterior adductor transfers to the ischium. All patients showed functional improvement postoperatively, which was maintained at long-term follow-up in 94%. Gait analysis, however, documented pelvic obliquity in 85% of this group of patients. Associated with pelvic obliquity was a 36% incidence of unilateral hip subluxation. These complications of posterior adductor transfers have been difficult to treat and have resulted in the abandonment of this procedure at our institution.

Correction and Recurrence of Ankle Valgus in Skeletally Immature Patients With Multiple Hereditary Exostoses

Background: Ankle valgus is encountered in children with a variety of congenital musculoskeletal disorders, including multiple hereditary exostoses (MHE). Guided growth with temporary distal tibial medial hemiepiphysiodesis (DTMH) may correct the deformity; however, exostoses about the ankle commonly observed in MHE patients may hinder correction and increase the risk of recurrence. Thus, the purpose of this study was to review the outcomes of DTMH in treatment of ankle valgus in MHE versus other diagnosis (non-MHE). Methods: Medical records and radiographs of patients undergoing DTMH for ankle valgus between January 1, 2005, and November 1, 2010, at a single pediatric orthopedic hospital were retrospectively analyzed. Radiographs obtained preoperatively and at 6-month intervals postoperatively were reviewed and the tibiotalar angle was measured. Results: Fifty-eight ankles in 41 patients met inclusion criteria, with minimum follow-up of 12 months (mean, 34 months). Mean age was 10 years (range, 4-14 years). MHE was the most common underlying diagnosis (19 ankles, 33%). The rate of tibiotalar angle correction (mean ± standard deviation) with hardware in place was 0.37 ± 0.28 deg/mo in MHE ankles and 0.51 ± 0.42 deg/mo in non-MHE ankles (P = .161). Following hardware removal, the rate of recurrence was faster in MHE (0.29 ± 0.25 deg/mo) compared with non-MHE ankles (0.12 ± 0.19 deg/mo) (P = .059), and more total recurrent valgus deformity was observed in MHE (7.8 ± 8.2 degrees) than non-MHE ankles (3.4 ± 4.6 degrees) (P = .08) over a similar follow-up period (mean 23.4 vs 23.6 months, respectively), with differences approaching statistical significance. Conclusion: MHE is a common cause of ankle valgus in children. Guided growth interventions in this population can be successful but require special consideration given the potential for relatively gradual deformity correction and rapid recurrence following hardware removal in the skeletally immature. Level of Evidence: Level III, retrospective comparative study.

Medial malleolar screw versus tension-band plate hemiepiphysiodesis for ankle valgus in the skeletally immature

Background: Ankle valgus is frequently encountered in skeletally immature patients in association with a variety of musculoskeletal disorders. Guided growth with temporary medial malleolar transphyseal screw (MMS) hemiepiphysiodesis is an established surgical treatment capable of correcting the angular deformity, but is often complicated by symptomatic screw head prominence and difficult hardware removal. Tension-band plate (TBP) hemiepiphysiodesis has recently been advocated as an alternative; however, the relative efficacy of these 2 techniques has not been directly investigated. Thus, the purpose of this study was to compare MMS and TBP in treatment of pediatric ankle valgus deformity. Methods: Medical records and radiographs of all patients undergoing distal tibial medial hemiepiphysiodesis for ankle valgus between January 1, 2005 and November 1, 2010 at a pediatric orthopaedic specialty hospital were retrospectively reviewed. Radiographs obtained preoperatively and at 6-month intervals postoperatively were reviewed and the tibiotalar angle was measured. Patient age, sex, underlying diagnosis, concurrent surgical procedures, surgical and postoperative complications, and the presence or absence of symptomatic hardware complaints were documented. Results: Sixty ankles in 42 patients met the inclusion criteria, with adequate radiographs and minimum postoperative follow-up of 12 months (mean: 34 mo). Thirty-five ankles were treated with MMS, and 25 with TBP. Good mean correction of the tibiotalar angle was achieved in both groups (MMS: pre—77.1 degrees to post—87.8 degrees over 25.2 mo; TBP: pre—81.3 to post—87.6 over 20.0 mo). The mean rate of correction was faster in ankles treated with MMS than TBP, but differences did not reach statistical significance (0.55 vs. 0.36 degrees/mo, respectively; P=0.057). Complications included 6 hardware-related surgical complications in MMS ankles (17.1%) and 1 in TBP ankles (4.0%). The incidence of symptomatic hardware complaints was low in both groups (MMS, 5.7%; TBP, 0%). Conclusions: Both MMS and TBP techniques can result in successful correction of ankle valgus in the growing child. Although the rate of deformity correction may be faster with MMS, TBP seems to be associated with fewer hardware-related complications. This information may aid the clinician in selecting the surgical option most appropriate for each individual patient. Level of Evidence: Level II—retrospective study.