Objectives To test the hypothesis that somatic PIK3CA mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). Study design We used next generation sequencing, droplet digital PCR (ddPCR), and single molecule molecular inversion probes (smMIPs) to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Children’s Hospital who had an isolated LM (n=17), KTS (n=21), fibro-adipose vascular anomaly (FAVA; n=8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (CLOVES; n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n=31) from Seattle Children’s Hospital. Results Most individuals from Boston Children’s Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), FAVA (4/8), and CLOVES (30/32) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ~ 80% of cases. Seventy-four percent of patients with LM from Seattle Children’s Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. Conclusions Somatic PIK3CA mutations are the most common cause of isolated lymphatic malformations and disorders in which lymphatic malformation is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism, because the abundance of mutant cells in a malformed tissue can be low.

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.

Moderate to severe slipped capital femoral epiphysis leads to premature osteoarthritis resulting from femoroacetabular impingement. We believe surgical correction at the site of deformity through capital reorientation is the best procedure to fully correct the deformity but has traditionally been associated with high rates of osteonecrosis. We describe a modified capital reorientation procedure performed through a surgical dislocation approach. We followed 40 patients for a minimum of 1 year and 3 years from two institutions. No patient developed osteonecrosis or chondrolysis. Slip angle was corrected to 4°to 8°and the mean alpha angle after correction was 40.6°. Articular cartilage damage, full-thickness loss, and delamination were observed at the time of surgery, especially in the stable slips. This technique appears to have an acceptable complication rate and appears reproducible for full correction of moderate to severe slipped capital femoral epiphyses with open physes.

Level IV-therapeutic study.

The surgical dislocation approach is useful in assessing and treating proximal femoral hip deformities commonly due to pediatric conditions. We sought to demonstrate the efficacy and problems associated with this technique. Diagnoses included slipped capital femoral epiphysis, Perthes disease, developmental dysplasia of the hip, osteonecrosis, and exostoses. Through this approach, femoral head-neck osteoplasty (22), intertrochanteric osteotomy (eight), femoral head-neck osteoplasty plus intertrochanteric osteotomy (15), femoral neck osteotomy (five), open reduction and internal fixation of an acute slipped capital femoral epiphysis with callus resection (five), open reduction and internal fixation of an acetabular fracture (one), trapdoor procedure (one), and acetabular rim osteoplasty (one) were performed. The average patient age was 16 years. The minimum followup was 12 months (average, 41.6 months; range, 12-73 months). Patients with Perthes disease and SCFE had preoperative and postoperative WOMAC scores of 9.6 and 5.1, and 7.9 and 3.5 respectively. In patients with unstable SCFEs, the average postoperative WOMAC score was 1.2. Seven patients underwent THAs and two patients underwent hip fusion. Complications in the 58 procedures included four cases of osteonecrosis: three after femoral neck osteotomy and one after intertrochanteric osteotomy. The surgical dislocation technique can be utilized to effectively treat these deformities and improve short-term symptoms. Although the technique is demanding, we believe surgical dislocation offers sufficient advantages in assessing and treating these complex deformities that it justifies judicious application.

Pistol grip deformity of the femoral head-neck junction and slipped capital femoral epiphysis can cause anterior impingement leading to pain, cartilage damage and eventual osteoarthritis. Osteoplasty of this metaphyseal prominence, with or without concomitant intertrochanteric osteotomy, using a surgical dislocation approach, can effectively treat this problem. Clinical and radiographic outcomes were assessed in 19 patients who underwent osteoplasty or osteoplasty/intertrochanteric osteotomy via Ganz-type surgical dislocation with average 12-month follow-up. Preoperative and postoperative Western Ontario and McMaster Osteoarthritis Index (WOMAC) questionnaires and radiographs were obtained and evaluated. In the osteoplasty group, 7 patients improved, 4 were unchanged, and 2 worsened. In the osteoplasty/osteotomy group, 5 of 6 patients improved. Patients with chondral flaps had less improvement. No avascular necrosis was noted postoperatively, and all trochanteric osteotomies healed. The surgical dislocation approach, combined with osteoplasty and/or bony realignment, is a safe efficacious treatment option for symptomatic pistol grip deformity. Outcomes are worse if there is preexisting cartilage damage.

Achondroplasia is the most common inherited disorder of bone growth (skeletal dysplasia). Despite this fact, consistent and evidence-based management approaches to recognized, life-threatening complications, such as foramen magnum stenosis, are lacking. This study aims to outline best practice, based on evidence and expert consensus, regarding the diagnosis, assessment, and management of foramen magnum stenosis in achondroplasia during infancy. A panel of 11 multidisciplinary international experts on skeletal dysplasia was invited to participate in a Delphi process. They were: 1) presented with a list of 26 indications and a thorough literature review, 2) given the opportunity to anonymously rate the indications and discuss in face to face discussion; 3) edit the list and rate it in a second round. Those indications with more than 80% agreement were considered as consensual. After two rounds of rating and a face-to-face meeting, consensus was reached to support 22 recommendations for the evaluation and treatment of foramen magnum stenosis in infants with achondroplasia. These recommendations include indications for surgical decompression, ventriculomegaly, and hydrocephalus, sleep-disordered breathing, physical exams and the use of polysomnography and imaging in this condition. We present a consensus-based best practice guidelines consisting of 22 recommendations. It is hoped that these guidelines will lead to more uniform and structured evaluation, standardizing care pathways, and improving mortality and morbidity outcomes for this cohort.

Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of tfec mutants demonstrates that Tfec also functions in the initial specification of all three pigment cell-types, although the melanocyte and xanthophore lineages recover later. We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.