Blinded Ultrasound Examination of the Subscapularis Following Anatomic Shoulder Arthroplasty.

BACKGROUND: The subscapularis tendon is commonly released during shoulder arthroplasty, and its integrity and repair postoperatively have been shown important to help maximize patient function. However, diagnosing subscapular tendon failure can be difficult with magnetic resonance imaging secondary to metal artifact as well as very costly. PURPOSE: The purpose of this study was to assess the utility of ultrasound imaging in evaluating subscapularis integrity at specific time points following shoulder arthroplasty, in a blinded fashion. Secondarily, we report on the correlation between the condition of the subscapularis and quality-of-life outcome measures. STUDY DESIGN: Prospective case series. METHODS: Ultrasounds were completed preoperatively and postoperatively at 1 week as well as at 1, 3, and 6 months. Each was read by a single musculoskeletal radiologist and categorized as "intact," "torn," or "unclear." Clinical outcome was evaluated using the Western Ontario Osteoarthritis Shoulder (WOOS) index at these same time points. RESULTS: The final study group consisted of 35 procedures in 33 patients (19 females and 14 males, mean age 66 ± 9 years). Three patients had postoperative subscapularis failures that were confirmed in the operating room at the time of repair. Of 24 sonographs categorized as "unclear" in the postoperative period, the majority (n = 12, 50%) were taken at 1 week. Compared to preoperative scores, patients had lower WOOS scores at 1, 3, and 6 months postoperatively (P < .001). Correlation analysis did not reveal an association between the ultrasound readings and the WOOS scores postoperatively. CONCLUSION: The utility of ultrasound examination of the subscapularis tendon following shoulder arthroplasty is limited by timing and may be most useful when used by the physician within clinical context. Significant improvement was noted in disease-specific quality-of-life scores regardless of the status of the subscapularis tendon as read on ultrasound.

Lateral clavicle fracture with coracoclavicular ligament injury: a biomechanical study of 4 different repair techniques.

PURPOSE: To investigate the biomechanical performance of four different methods used for coracoclavicular (CC) ligament reconstruction in a lateral clavicle fracture repair. METHODS: Native displacement, translation, and rotation at the acromioclavicular joint of 24 fresh-frozen cadaveric shoulders were tested. A reproducible fracture in the lateral third of the clavicle was created by dissecting both CC ligaments. Each specimen was then repaired with plate fixation of the fracture and the following CC repair technique: (1) Cortical button. (2) Suture anchor and plate button. (3) Suture anchor no plate button, and (4) Suture around coracoid. All reconstructed specimens were then re-tested for displacement, translation, and load to failure, and compared to their native results. Groups 1 and 3 were investigated for rotational load. RESULTS: There was no difference in load to failure between the repaired groups (p: ns). Group 1 showed less superior and anterior translations (p < 0.05). Group 2 showed significantly less superior translation (p = 0.003), but no significance with anterior and posterior translations to the native joint. Group 3 showed less superior and posterior translations (p = 0.005 and p = 0.039). Anterior and posterior translations were increased in group 4 (p < 0.05). CONCLUSION: The biomechanical analyses did not show any significance in load to failure or displacement after cyclic loading among the study groups. All repairs were effective in preventing superior translation. Groups 1 and 2 demonstrated increased horizontal stability compared to the native state. All 4 methods are clinically viable options for CC ligament repair.

Regeneration of Articular Cartilage by Human ESC-Derived Mesenchymal Progenitors Treated Sequentially with BMP-2 and Wnt5a.

The success of cell-based therapies to restore joint cartilage requires an optimal source of reparative progenitor cells and tight control of their differentiation into a permanent cartilage phenotype. Bone morphogenetic protein 2 (BMP-2) has been extensively shown to promote mesenchymal cell differentiation into chondrocytes in vitro and in vivo. Conversely, developmental studies have demonstrated decreased chondrocyte maturation by Wingless-Type MMTV Integration Site Family, Member 5A (Wnt5a). Thus, we hypothesized that treatment of human embryonic stem cell (hESC)-derived chondroprogenitors with BMP-2 followed by Wnt5a may control the maturational progression of these cells into a hyaline-like chondrocyte phenotype. We examined the effects of sustained exposure of hESC-derived mesenchymal-like progenitors to recombinant Wnt5a or BMP-2 in vitro. Our data indicate that BMP-2 promoted a strong chondrogenic response leading to terminal maturation, whereas recombinant Wnt5a induced a mild chondrogenic response without promoting hypertrophy. Moreover, Wnt5a suppressed BMP-2-mediated chondrocyte maturation, preventing the formation of fibrocartilaginous tissue in high-density cultures treated sequentially with BMP-2 and Wnt5a. Implantation of scaffoldless pellets of hESC-derived chondroprogenitors pretreated with BMP-2 followed by Wnt5a into rat chondral defects induced an articular-like phenotype in vivo. Together, the data establish a novel role for Wnt5a in controlling the progression from multipotency into an articular-like cartilage phenotype in vitro and in vivo. Stem Cells Translational Medicine 2017;6:40-50.

Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing.

Purpose of this study: To elucidate the origin of cell populations that contribute to rotator cuff healing, we developed a mouse surgical model where a full-thickness, central detachment is created in the supraspinatus. MATERIALS AND METHODS: Three different inducible Cre transgenic mice with Ai9-tdTomato reporter expression (PRG4-9, αSMA-9, and AGC-9) were used to label different cell populations in the shoulder. The defect was created surgically in the supraspinatus. The mice were injected with tamoxifen at surgery to label the cells and sacrificed at 1, 2, and 5 weeks postoperatively. Frozen sections were fluorescently imaged then stained with Toluidine Blue and re-imaged. RESULTS: Three notable changes were apparent postoperatively. (1) A long thin layer of tissue formed on the bursal side overlying the supraspinatus tendon. (2) The tendon proximal to the defect initially became hypercellular and disorganized. (3) The distal stump at the insertion underwent minimal remodeling. In the uninjured shoulder, tdTomato expression was seen in the tendon midsubstance and paratenon cell on the bursal side in PRG4-9, in paratenon, blood vessels, and periosteum of acromion in SMA-9, and in articular cartilage, unmineralized fibrocartilage of supraspinatus enthesis, and acromioclavicular joint in AGC-9 mice. In the injured PRG4-9 and SMA-9 mice, the healing tissues contained an abundant number of tdTomato+ cells, while minimal contribution of tdTomato+ cells was seen in AGC-9 mice. CONCLUSIONS: The study supports the importance of the bursal side of the tendon to rotator cuff healing and PRG4 and αSMA may be markers for these progenitor cells.

A Biomechanical Analysis of Different Clavicular Tunnel Diameters in Anatomic Acromioclavicular Ligament Reconstruction.

PURPOSE: To evaluate the biomechanical stability of a tendon-to-clavicle bone interface fixation of a graft in revision acromioclavicular reconstruction. METHODS: Fifteen fresh-frozen cadaveric shoulders were used. All specimens underwent bone density evaluation. For the primary reconstruction, a 5-mm semitendinosus allograft was inserted into a 5-mm bone tunnel at 25 and 45 mm from the lateral end of the clavicle using a 5.5 × 8-mm PEEK (polyether ether ketone) tenodesis screw. Each single graft was fixed in a cryo-clamp and cyclically loaded from 5 to 70 N for 3,000 cycles, followed by load-to-failure testing at a rate of 120 mm/min to simulate the revision case. To simulate tunnel widening, the tunnels of the revision series were over-drilled with an 8-mm drill, and a 5-mm semitendinosus graft with an 8 × 12-mm PEEK tenodesis screw was inserted. Biomechanical testing was then repeated. RESULTS: The bone mineral density analysis showed a significantly higher density at the 45-mm hole compared with the 25-mm hole (P = .001). The ultimate load to failure increased from the 5.5-mm screw to the 8-mm screw at the 45-mm hole position (P = .001). There was no statistically significant difference at the 25-mm hole position (P = .934). No statistical significance for graft elongation comparing the 5.5-mm screw and the 8-mm screw at the 25-mm (P = .156) and 45-mm (P = .334) positions could be found. CONCLUSIONS: Comparable biomechanical stability for the tendon-to-bone interface fixation in different clavicular tunnel diameters simulating primary and revision reconstruction was achieved. CLINICAL RELEVANCE: There is a lack of literature regarding revision acromioclavicular joint reconstruction, but our biomechanical results show comparable stability to primary reconstruction. These data provide support for the use of anatomic acromioclavicular ligament reconstruction in revision cases.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity.

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.

Biodistribution of LV-TSTA transduced rat bone marrow cells used for "ex-vivo" regional gene therapy for bone repair.

"Ex vivo" regional gene therapy using lentiviral (LV) vectors to over-express bone morphogenetic protein 2 (BMP-2) is an effective way to enhance bone healing in animal models. Here, we evaluated two different "ex vivo" approaches using either "same day" rat bone marrow cells (SDRBMCs) or cultured rat bone marrow cells (C-RBMCs), both transduced with a LV based two-step transcriptional activation system overexpressing GFP (LV-TSTA-EGFP), to assess the fate of the transduced cells and the safety of this approach. The transduced cells were implanted in femoral defects of syngeneic rats. Animals were sacrificed at 4, 14, 28 and 56 days after surgery (n=5 per group). Viral copies were detectable in the defect site of SD-RBMC group and gradually declined at 8w (5 log decrease compared to 4d). In the C-RBMC animals, there was a 2-4 log decline in the viral copy numbers at 2w and 4w, but at 8w there was a relative rise (about 100 fold) in the number of the viral vectors in the defect site of 4 (out of 5) animals compared to the previous time points. For both gene transfer approaches, the pattern of tissue distribution was non-specific and no histological abnormalities were noted in either group. In summary, we demonstrated that the LV-TSTA transduced cells remain in the defect site for at least 56 days, though the numbers decreased over time. There were no consistent findings of viral copies in internal organs which is encouraging with respect to the development of this strategy for use in humans.

The enthesis: a review of the tendon-to-bone insertion.

The integration of tendon into bone occurs at a specialized interface known as the enthesis. The fibrous tendon to bone enthesis is established through a structurally continuous gradient from uncalcified tendon to calcified bone. The enthesis exhibits gradients in tissue organization classified into four distinct zones with varying cellular compositions, mechanical properties, and functions in order to facilitate joint movement. Damage to tendinous insertions is common in the field of orthopaedic medicine and often involves surgical intervention that requires the attempted recreation of the natural organization of tendon into bone. The difficulty associated with recreating the distinct organization may account for the surgical challenges associated with reconstruction of damaged insertion sites. These procedures are often associated with high failure rates and consequently require revision procedures. Management of tendinous injuries and reconstruction of the insertion site is becoming a popular topic in the field of orthopaedic medicine.

Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice.

We evaluated the osteoprogenitor response to rhBMP-2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre-osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and α-smooth muscle actin (α-SMA-Cherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP-2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre-osteoblasts demonstrated a maximal response (% GFP + cells/TV) in the Col3.6GFPtopaz mice at day 7 (7.2% ± 6.0, p < 0.05 compared to days 14, 21, 28, and 56). The maximal response of the Col2.3GFP cells was seen at days 14 (8.04% ± 5.0) and 21 (8.31% ± 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes.

Evaluation of the effects of systemic treatment with a sclerostin neutralizing antibody on bone repair in a rat femoral defect model.

Systemic administration of a sclerostin neutralizing antibody (Scl-Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl-Ab to improve healing in a bone defect model,we evaluated Scl-Ab in a 3mm femoral defect in young male outbred rats. Scl-Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, µ-CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl-Ab treated 5/30, vehicle treated 1/15). µ-CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p<0.05), but a non-significant increase in the continuous group (35%, p = 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un-operated femurs. We demonstrate that treatment with Scl-Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity.

Constructing the toolbox: Patient-specific genetic factors of altered fracture healing.

The multifaceted sequence of events that follow fracture repair can be further complicated when considering risk factors for impaired union, present in a large and growing percentage of the population. Risk factors such as diabetes, substance abuse, and poor nutrition affect both the young and old alike, and have been shown to dramatically impair the body's natural healing processes. To this end, biotherapeudic interventions such as ultrasound, electrical simulation, growth factor treatment (BMP-2, BMP-7, PDGF-BB, FGF-2) have been evaluated in preclinical models and in some cases are used widely for patients with established non-union or risk/indication or impaired healing (ie. ultrasound, BMP-2, etc.). Despite the promise of these interventions, they have been shown to be reliant on patient compliance and can produce adverse side-effects such as heterotopic ossification. Gene and cell therapy approaches have attempted to apply controlled regimens of these factors and have produced promising results. However, there are safety and efficacy concerns that may limit the translation of these approaches. In addition, none of the above mentioned approaches consider genetic variation between individual patients. Several clinical and preclinical studies have demonstrated a genetic component to fracture repair and that SNPs and genetic background variation play major roles in the determination of healing outcomes. Despite this, there is a need for preclinical data to dissect the mechanism underlying the influence of specific gene loci on the processes of fracture healing, which will be paramount in the future of patient-centered interventions for fracture repair.

Systemic administration of sclerostin antibody enhances bone repair in a critical-sized femoral defect in a rat model.

BACKGROUND: Systemic administration of sclerostin neutralizing antibody has led to increased bone formation in animal models of osteoporosis. The purpose of this study was to determine if systemic administration of sclerostin neutralizing antibody could increase the healing response in a critical-sized femoral defect in rats. METHODS: Critical-sized femoral defects were created in Lewis rats, and the rats were randomized into four groups. The sclerostin antibody (Scl-Ab) treatment groups included the continuous Scl-Ab group (twenty-one animals), the early Scl-Ab group (fifteen animals), and the delayed Scl-Ab group (fifteen animals), which received sclerostin antibody (25 mg/kg) twice weekly for weeks 0 through 12; weeks 0 through 2; and weeks 2 through 4; respectively. Twenty-one animals in the control group received vehicle from weeks 0 through 12. In a subsequent study, bone turnover markers were measured at zero, two, six, and twelve weeks after surgery in rats receiving vehicle or sclerostin neutralizing antibody for twelve weeks (fifteen rats per group). The quality of bone formed was evaluated with radiographs, microcomputed tomography, biomechanical testing, and histologic and histomorphometric analysis. RESULTS: In the primary study, four of fifteen defects in the continuous (zero to twelve-week) Scl-Ab group, three of fifteen defects in the early (zero to two-week) Scl-Ab group, and four of fifteen defects in the delayed (two to four-week) Scl-Ab group healed at twelve weeks, while none of the defects healed in the control group. In both studies, treatment with sclerostin antibody for twelve weeks demonstrated a significant increase in new bone formation (p < 0.05) compared with the control group. The three treatment groups did not differ significantly with respect to the healing rates and the quality of new bone formed in the defect. The serum markers of bone formation were significantly elevated in the animals in the continuous Scl-Ab group (p < 0.05) compared with the controls. CONCLUSIONS: Administration of sclerostin neutralizing antibody led to increased bone formation, resulting in complete healing of femoral defects in a small subset of rats, with a majority of the animals not healing the defect by twelve weeks.

In vitro evaluation of a double-stranded self-complementary adeno-associated virus type2 vector in bone marrow stromal cells for bone healing.

BACKGROUND: Both adenoviral and lentiviral vectors have been successfully used to induce bone repair by over-expression of human bone morphogenetic protein 2 (BMP-2) in primary rat bone marrow stromal cells in pre-clinical models of ex vivo regional gene therapy. Despite being a very efficient means of gene delivery, there are potential safety concerns that may limit the adaptation of these viral vectors for clinical use in humans. Recombinant adeno-associated viral (rAAV) vector is a promising viral vector without known pathogenicity in humans and has the potential to be an effective gene delivery vehicle to enhance bone repair. In this study, we investigated gene transfer in rat and human bone marrow stromal cells in order to evaluate the effectiveness of the self-complementary AAV vector (scAAV) system, which has higher efficiency than the single-stranded AAV vector (ssAAV) due to its unique viral genome that bypasses the rate-limiting conversion step necessary in ssAAV. METHODS: Self-complementaryAAV2 encoding GFP and BMP-2 (scAAV2-GFP and scAAV2-BMP-2) were used to transduce human and rat bone marrow stromal cells in vitro, and subsequently the levels of GFP and BMP-2 expression were assessed 48 hours after treatment. In parallel experiments, adenoviral and lentiviral vector mediated over-expression of GFP and BMP-2 were used for comparison. RESULTS: Our results demonstrate that the scAAV2 is not capable of inducing significant transgene expression in human and rat bone marrow stromal cells, which may be associated with its unique tropism. CONCLUSIONS: In developing ex vivo gene therapy regimens, the ability of a vector to induce the appropriate level of transgene expression needs to be evaluated for each cell type and vector used.

Combined inhibition of the BMP pathway and the RANK-RANKL axis in a mixed lytic/blastic prostate cancer lesion.

The purpose of this study was to investigate the influence of combined inhibition of receptor activator of nuclear factor kappa-B ligand (RANKL) and bone morphogenetic protein (BMP) activity in a mixed lytic/blastic prostate cancer lesion in bone. Human prostate cancer cells (C4 2b) were injected into immunocompromised mice using an intratibial injection model to create mixed lytic/blastic lesions. RANK-Fc, a recombinant RANKL antagonist, was injected subcutaneously three times a week (10mg/kg) to inhibit RANKL and subsequent formation, function and survival of osteoclasts. Inhibition of BMP activity was achieved by transducing prostate cancer cells ex vivo with a retroviral vector expressing noggin (retronoggin; RN). There were three treatment groups (RANK-Fc treatment, RN treatment and combined RN and RANK-Fc treatment) and two control groups (untreated control and empty vector control for the RN treatment group). The progression of bone lesion and tumor growth was evaluated using plain radiographs, hindlimb tumor size, (18)F-Fluorodeoxyglucose and (18)F-fluoride micro PET-CT, histology and histomorphometry. Treatment with RANK-Fc alone inhibited osteolysis and transformed a mixed lytic/blastic lesion into an osteoblastic phenotype. Treatment with RN alone inhibited the osteoblastic component in a mixed lytic/blastic lesion and resulted in formation of smaller osteolytic bone lesion with smaller soft tissue size. The animals treated with both RN and RANK-Fc demonstrated delayed development of bone lesions, inhibition of osteolysis, small soft tissue tumors and preservation of bone architecture with less tumor induced new bone formation. This study suggests that combined inhibition of the RANKL and the BMP pathway may be an effective biologic therapy to inhibit the progression of established mixed lytic/blastic prostate cancer lesions in bone.

Early results of the Ponseti method for the treatment of clubfoot associated with myelomeningocele.

BACKGROUND: Myelomeningocele is a common birth defect that is often accompanied by clubfoot deformity. Treatment of clubfoot associated with myelomeningocele traditionally has consisted of extensive soft-tissue release operations, which are associated with many complications. The purpose of the present study was to evaluate the early results of the Ponseti method for the treatment of clubfoot associated with myelomeningocele. METHODS: Sixteen consecutive patients with myelomeningocele (twenty-eight clubfeet) and twenty consecutive patients with idiopathic clubfeet (thirty-five clubfeet) were followed prospectively while being managed with the Ponseti method. The average duration of follow-up was thirty-four months for the myelomeningocele group and thirty-seven months for the idiopathic group. Clubfoot severity was graded at the time of presentation with use of the Diméglio system. The initial correction that was achieved, casting and/or bracing difficulties, recurrences, and subsequent treatments were evaluated and compared between the two cohorts by means of appropriate statistical analysis. RESULTS: Eleven (39%) of the twenty-eight clubfeet in the myelomeningocele group were graded as Diméglio grade IV, compared with only four (11%) of the thirty-five clubfeet in the idiopathic group (p = 0.014). Initial correction was achieved in thirty-five clubfeet (100%) in the idiopathic group and in twenty-seven clubfeet (96.4%) in the myelomeningocele group (p = 0.16). Relapse of deformity was detected in 68% of the feet in the myelomeningocele group, compared with 26% of the feet in the idiopathic group (p = 0.001). Relapses were treated successfully without the need for extensive soft-tissue release surgery for all but four of the clubfeet in the myelomeningocele group and for all but one of the clubfeet in the idiopathic group (p = 0.16). CONCLUSIONS: Our data support the use of the Ponseti method for the initial treatment of clubfoot deformity associated with myelomeningocele, although attention to detail is crucial in order to avoid complications. Longer follow-up will be necessary to assess the risk of late recurrence and the potential need for more extensive clubfoot corrective surgery in this patient population.

Skeletal muscle contractile gene (TNNT3, MYH3, TPM2) mutations not found in vertical talus or clubfoot.

UNLABELLED: Arthrogryposis presents with lower limb contractures that resemble clubfoot and/or vertical talus. Recently, mutations in skeletal muscle contractile genes MYH3 (myosin heavy chain 3), TNNT3 (troponin T3), and TPM2 (tropomyosin 2) were identified in patients with distal arthrogryposis DA2A (Freeman-Sheldon syndrome) or DA2B (Sheldon-Hall syndrome). We asked whether the contractile genes responsible for distal arthrogryposis are also responsible for cases of familial clubfoot or vertical talus. We determined the frequency of MYH3, TNNT3, and TPM2 mutations in patients with idiopathic clubfoot, vertical talus, and distal arthrogryposis type 1 (DA1). We resequenced the coding exons of the MYH3, TNNT3, and TPM2 genes in 31 patients (five with familial vertical talus, 20 with familial clubfoot, and six with DA1). Variants were evaluated for segregation with disease in additional family members, and the frequency of identified variants was determined in a control population. In one individual with DA1, we identified a de novo TNNT3 mutation (R63H) previously identified in an individual with DA2B. No other causative mutations were identified, though we found several previously undescribed single-nucleotide polymorphisms of unknown importance. Although mutations in MYH3, TNNT3, and TPM2 are frequently associated with distal arthrogryposis syndromes, they were not present in patients with familial vertical talus or clubfoot. The TNNT3 R63H recurrent mutation identified in two unrelated individuals may be associated with either DA1 or DA2B. LEVEL OF EVIDENCE: Level II, prospective study. See the Guidelines for Authors for a complete description of levels of evidence.

Genetic linkage localizes an adolescent idiopathic scoliosis and pectus excavatum gene to chromosome 18 q.

STUDY DESIGN: A single large family, in which adolescent idiopathic scoliosis (AIS) and pectus excavatum (PE) segregate as an autosomal dominant condition, was evaluated. Genome-wide linkage analysis and candidate gene sequencing were performed. OBJECTIVE: To map the disease-causing locus in a large white family in which AIS and PE cosegregate. SUMMARY OF BACKGROUND DATA: AIS and PE are common musculoskeletal conditions known to have a genetic component, though few genes have been identified for either. Genetic studies have been confounded by a lack of large families in which the disorders segregate. METHODS: Clinical examinations were performed on the proband, who underwent posterior spinal fusion, and 12 additional affected family members. To map a gene causing AIS and PE, a genome-wide linkage analysis was performed with the Affymetrix Mapping 10 K XbaI array on 13 affected and 10 unaffected family members. Candidate genes were sequenced. RESULTS: AIS was present in 13 female family members and PE was present in 3 males and 1 female. Genome-wide linkage analysis resulted in a linkage peak on chromosome 18 q with a maximum parametric multipoint logarithm of the odds score of 3.86. Recombinants delineated the critical genetic region to an interval of 6.4 cM between SNP_A-1519369 and SNP_A-1507702, corresponding to a 7.06-Mb region (hg18: chr18:26342508-34395660). The chromosome 18 q linkage region contains more than 30 genes. Resequencing of the coding regions of 21 candidate genes in the region did not reveal any causative mutation. CONCLUSION: Linkage analysis in this large family demonstrated a novel locus for AIS and PE on chromosome 18 q. Because of the increased frequency of PE in family members of AIS patients, consideration of family members with PE as affected may increase the power of AIS genetic linkage studies.

Asymmetric lower-limb malformations in individuals with homeobox PITX1 gene mutation.

Clubfoot is one of the most common severe musculoskeletal birth defects, with a worldwide incidence of 1 in 1000 live births. In the present study, we describe a five-generation family with asymmetric right-sided predominant idiopathic clubfoot segregating as an autosomal-dominant condition with incomplete penetrance. Other lower-limb malformations, including patellar hypoplasia, oblique talus, tibial hemimelia, developmental hip dysplasia, and preaxial polydactyly, were also present in some family members. Genome-wide linkage analysis with Affymetrix GeneChip Mapping 10K mapping data from 13 members of this family revealed a multipoint LOD(max) of 3.31 on chromosome 5q31. A single missense mutation (c.388G-->A) was identified in PITX1, a bicoid-related homeodomain transcription factor critical for hindlimb development, and segregated with disease in this family. The PITX1 E130K mutation is located in the highly conserved homeodomain and reduces the ability of PITX1 to transactivate a luciferase reporter. The PITX1 E130K mutation also suppresses wild-type PITX1 activity in a dose-dependent manner, suggesting dominant-negative effects on transcription. The propensity for right-sided involvement in tibial hemimelia and clubfoot suggests that PITX1, or pathways involving PITX1, may be involved in their etiology. Implication of a gene involved in early limb development in clubfoot pathogenesis also suggests additional pathways for future investigations of idiopathic clubfoot etiology in humans.

Early results of the Ponseti method for the treatment of clubfoot in distal arthrogryposis.

BACKGROUND: Clubfoot occurs in approximately one in 1000 live births and is one of the most common congenital birth defects. Although there have been several reports of successful treatment of idiopathic clubfoot with the Ponseti method, the use of this method for the treatment of other forms of clubfoot has not been reported. The purpose of the present study was to evaluate the early results of the Ponseti method when used for the treatment of clubfoot associated with distal arthrogryposis. METHODS: Twelve consecutive infants (twenty-four feet) with clubfoot deformity associated with distal arthrogryposis were managed with the Ponseti method and were retrospectively reviewed at a minimum of two years. The severity of the foot deformity was classified according to the grading system of Diméglio et al. The number of casts required to achieve correction was compared with published data for the treatment of idiopathic clubfoot. Recurrent clubfoot deformities or complications during treatment were recorded. RESULTS: Twenty-two clubfeet in eleven patients were classified as Diméglio grade IV, and two clubfeet in one patient were classified as Diméglio grade II. Initial correction was achieved in all clubfeet with a mean of 6.9 +/- 2.1 casts (95% confidence interval, 5.6 to 8.3 casts), which was significantly greater than the mean of 4.5 +/- 1.2 casts (95% confidence interval, 4.3 to 4.7 casts) needed in a cohort of 219 idiopathic clubfeet that were treated during the same time period by the senior author with use of the Ponseti method (p = 0.002). Six feet in three patients had a relapse after initial successful treatment. All relapses were related to noncompliance with prescribed brace wear. Four relapsed clubfeet in two patients were successfully treated with repeat casting and/or tenotomy; the remaining two relapsed clubfeet in one patient were treated with extensive soft-tissue-release operations. CONCLUSIONS: Our early-term results support the use of the Ponseti method for the initial treatment of distal arthrogrypotic clubfoot deformity. Longer follow-up will be necessary to assess the risk of recurrence and the potential need for corrective clubfoot surgery in this patient population, which historically has been difficult to treat nonoperatively.