Neil W. Bulstrode

Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS

Congenital melanocytic nevi (CMN) can be associated with neurological abnormalities and an increased risk of melanoma. Mutations in NRAS, BRAF, and Tp53 have been described in individual CMN samples; however, their role in the pathogenesis of multiple CMN within the same subject and development of associated features has not been clear. We hypothesized that a single postzygotic mutation in NRAS could be responsible for multiple CMN in the same individual, as well as for melanocytic and nonmelanocytic central nervous system (CNS) lesions. From 15 patients, 55 samples with multiple CMN were sequenced after site-directed mutagenesis and enzymatic digestion of the wild-type allele. Oncogenic missense mutations in codon 61 of NRAS were found in affected neurological and cutaneous tissues of 12 out of 15 patients, but were absent from unaffected tissues and blood, consistent with NRAS mutation mosaicism. In 10 patients, the mutation was consistently c.181C>A, p.Q61K, and in 2 patients c.182A>G, p.Q61R. All 11 non-melanocytic and melanocytic CNS samples from 5 patients were mutation positive, despite NRAS rarely being reported as mutated in CNS tumors. Loss of heterozygosity was associated with the onset of melanoma in two cases, implying a multistep progression to malignancy. These results suggest that single postzygotic NRAS mutations are responsible for multiple CMN and associated neurological lesions in the majority of cases.

The role of surgery in the management of congenital melanocytic naevi in children: a perspective from Great Ormond Street Hospital

Recent advances in research have prompted this review of the role of surgery in the management of congenital melanocytic naevi (CMNs). Good data on the incidence of neurological and malignant complications of CMNs have re-fuelled the debates on whether surgery decreases the risk of malignant melanoma and whether early surgery is advantageous. We conclude the following: 1) untreated CMNs can lighten spontaneously, sometimes dramatically, 2) routine surgery has not been demonstrated to reduce the risk of malignancy and is, therefore, for cosmetic reasons only, 3) early surgery has not been shown to be advantageous and carries increased anaesthetic risk and 4) there is some evidence that surgical intervention may adversely affect the behaviour of the CMN cells. Our current practice is based on the following guidelines: 1) patients are treated in a multidisciplinary-team setting which includes the specialties of paediatric dermatology, plastic surgery and neuroradiology with access to neurology, neurosurgery and oncology, 2) serial photographs are taken at yearly intervals to assess spontaneous lightening. 3) all routine surgery is delayed for at least the first year. 4) patients with facial CMNs (either the principal lesion or large satellites) are offered surgery for cosmetic reasons, 5) patients with a single, easily excisable CMN are offered surgery for cosmetic reasons and 6) all families are made aware of the possibility of spontaneous lightening and the possibility that surgery may have effects on the behaviour of naevus cells.

Germline Melanocortin-1-Receptor Genotype Is Associated with Severity of Cutaneous Phenotype in Congenital Melanocytic Nevi: A Role for MC1R in Human Fetal Development

Congenital melanocytic nevi (CMN) are pigmented birthmarks that affect up to 80% of the skin surface area. The increased frequency of CMN in families of severely affected individuals is suggestive of a predisposing germline genotype. We noted a high prevalence of red hair in affected families, and considered a role for MC1R in this condition. A cohort of 166 CMN subjects underwent pigmentary phenotyping, with MC1R genotyping in 113. Results were compared with a local control group of 60 unrelated children and with 300 UK children without CMN. CMN subjects had higher prevalences of red hair and a red-haired parent than local controls and had a higher rate of compound heterozygosity and homozygosity for MC1R variants. The presence of a V92M or R allele (D84E, R151C, R160W, D294H) was associated with increasing size of the CMN, implying a growth-promoting effect of these alleles. Unexpectedly, the V92M and R151C alleles were also strongly associated with birth weight in the CMN cohort, a finding confirmed in the control group. The effect of germline MC1R genotype on development and severity of CMN led us to investigate potential broader effects on growth, revealing a role for MC1R in normal fetal development.

Chondrogenic differentiation of adipose tissue-derived stem cells within nanocaged POSS-PCU scaffolds: A new tool for nanomedicine

UNLABELLED Scaffold cellularization for cartilage engineering can aid implant properties, their retention and minimize repeated intervention, particularly in paediatric reconstructive craniofacial surgery. We developed novel bionanoscaffolds using paediatric adipose tissue-derived stem cells (hADSCs), an accessible autologous cell source, and POSS-PCU. Little is known about cellular responses to this nanomaterial, though it was used in human. We assessed: 1) POSS-PCU cellularization and bioaffinity to hADSCs; 2) hADSC chondrogenic differentiation ability in POSS-PCU; 3) whether bionanoscaffolds became encased within a vascular network and/or vascularised. POSS-PCU supported ADSC survival and proliferation and their migration and differentiation into cartilage within the nanoscaffold. Furthermore, after CAM-grafting, bionanoscaffolds were rapidly surrounded by blood vessels without any apparent negative reaction and erythrocytes of host origin were detected inside the scaffold, suggesting invasion from some capillaries. Altogether, this study demonstrates that POSS-PCU displays excellent bioactivity and hADSC/POSS-PCU bionanoscaffolds offer much promise for autologous cell-based tissue engineering for clinical applications. FROM THE CLINICAL EDITOR In this study, human adipose tissue derived stem cells were used in combination with POSS-PCU nanoscaffolds to generate cartilage tissue demonstrating excellent bioactivity for autologous cell-based tissue engineering for clinical applications.

Health-related quality-of-life assessment and surgical outcomes for auricular reconstruction using autologous costal cartilage.

Background: This study aims to assess the health-related quality-of-life benefit following auricular reconstruction using autologous costal cartilage in children. In addition, key aspects of the surgical reconstruction are assessed. Methods: After auricular reconstruction, patients completed two questionnaires. The first was a postinterventional health-related quality-of-life assessment tool, the Glasgow Benefit Inventory. A score of 0 signifies no change in health-related quality-of-life, +100 indicates maximal improvement, and –100 indicates maximal negative impact. The second questionnaire assessed surgical outcomes in auricular reconstruction across three areas: facial integration, aesthetic auricular units, and costal reconstruction. These were recorded on a five-point ordinal scale and are presented as mean scores of a total of 5. Results: The mean total Glasgow Benefit Inventory score was 48.1; significant improvements were seen in all three Glasgow Benefit Inventory subscales (p < 0.0001). A mean integration score of 3.8 and a mean aesthetic auricular unit reconstruction score of 3.4 were recorded. Skin color matching (4.3) of the ear was most successfully reconstructed and auricular cartilage reconstruction scored lowest (3.5). Of the aesthetic units, the helix scored highest (3.6) and the tragus/antitragus scored lowest (3.3). Donor-site reconstruction scored 3.9. Correlation analysis revealed that higher reconstruction scores are associated with a greater health-related quality-of-life gain (r = 0.5). Ninety-six percent of patients would recommend the procedure to a friend. Conclusions: Auricular reconstruction with autologous cartilage results in significant improvements in health-related quality-of-life. In addition, better surgical outcomes lead to a greater improvement in health-related quality-of-life. Comparatively poorer reconstructed areas of the ear were identified so that surgical techniques may be improved. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

High Plasticity of Pediatric Adipose Tissue-Derived Stem Cells: Too Much for Selective Skeletogenic Differentiation?

Stem cells derived from adipose tissue are a potentially important source for autologous cell therapy and disease modeling, given fat tissue accessibility and abundance. Critical to developing standard protocols for therapeutic use is a thorough understanding of their potential, and whether this is consistent among individuals, hence, could be generally inferred. Such information is still lacking, particularly in children. To address these issues, we have used different methods to establish stem cells from adipose tissue (adipose‐derived stem cells [ADSCs], adipose explant dedifferentiated stem cells [AEDSCs]) from several pediatric patients and investigated their phenotype and differentiation potential using monolayer and micromass cultures. We have also addressed the overlooked issue of selective induction of cartilage differentiation. ADSCs/AEDSCs from different patients showed a remarkably similar behavior. Pluripotency markers were detected in these cells, consistent with ease of reprogramming to induced pluripotent stem cells. Significantly, most ADSCs expressed markers of tissue‐specific commitment/differentiation, including skeletogenic and neural markers, while maintaining a proliferative, undifferentiated morphology. Exposure to chondrogenic, osteogenic, adipogenic, or neurogenic conditions resulted in morphological differentiation and tissue‐specific marker upregulation. These findings suggest that the ADSC “lineage‐mixed” phenotype underlies their significant plasticity, which is much higher than that of chondroblasts we studied in parallel. Finally, whereas selective ADSC osteogenic differentiation was observed, chondrogenic induction always resulted in both cartilage and bone formation when a commercial chondrogenic medium was used; however, chondrogenic induction with a transforming growth factor β1‐containing medium selectively resulted in cartilage formation. This clearly indicates that careful simultaneous assessment of bone and cartilage differentiation is essential when bioengineering stem cell‐derived cartilage for clinical intervention.

Elevated FGF21 Leads to Attenuated Postnatal Linear Growth in Preterm Infants Through GH Resistance in Chondrocytes

CONTEXT The hormone fibroblast growth factor 21 (FGF21) is a key metabolic regulator in the adaptation to fasting. In food-restricted mice, inhibition of skeletal growth is mediated by the antagonistic effect of FGF21 on GH action in the liver and growth plate. OBJECTIVE The objective of the study was to assess the role of FGF21 in growth regulation in humans using postnatal growth failure of very preterm infants as a model. DESIGN FGF21 levels were measured serially in very preterm infants, and their linear growth evaluated from birth to term-equivalent age. Primary chondrocytes obtained from pediatric donors were used to test whether FGF21 can directly interfere with GH signaling. RESULTS A negative association (β -.415, P < .005, linear regression model) of FGF21 levels with the change in SD score for length was found. In primary chondrocytes, FGF21 upregulated basal and GH-induced SOCS2 expression and inhibited GH-induced signal transducer and activator of transcription 5 (STAT5) phosphorylation as well as GH-induced COLII and ALP expression. Finally, FGF21 inhibited GH-induced IGF-1 expression and cell proliferation, indicating GH resistance. However, FGF21 did not affect IGF-1-induced cell proliferation. CONCLUSIONS Elevated FGF21 serum levels during the first weeks of life are independently associated with postnatal growth failure in preterm infants. Furthermore, our data provide mechanistic insights into GH resistance secondary to prematurity and may offer an explanation for the growth failure commonly seen in chronic conditions of childhood.

Squamous cell carcinoma in a child with Clericuzio-type poikiloderma with neutropenia.

MADAM, Poikiloderma with neutropenia (PN) is a rare disorder attributed to mutations in the USB1 gene, with many similarities to other hereditary poikilodermas (HP). The risks ascribed to this condition are yet to be fully elucidated. We report the case of a patient with PN who developed a squamous cell carcinoma (SCC) of the toe at an early age, and we include studies demonstrating abnormal neutrophil function. Our patient developed pigmented, reticulate skin lesions with atrophic change and telangiectasia on the arms, legs, face and trunk from the age of 3 months. Additional features include pachyonychia of the toenails, occasional mouth ulcers and plantar keratoderma. Distinctive facies were noted: a saddle nose, flat nasal bridge, flared nostrils and prominent forehead with some mid-face hypoplasia (Fig. S1; see Supporting Information). She was initially on the 25th centile for height and weight; however, this fell after her third year to her current stature below the 2nd centile for height and weight. Ophthalmology reviews have shown no abnormality. There is no history of photosensitivity, noting that the family lives in Cyprus. However, the patient has taken great care to avoid sun exposure, using high factor sun protection cream and covered shoes whenever outside. The initial diagnosis was considered to be Rothmund– Thomson syndrome; however, she had a normal karyotype and no evidence of a RECQL4 mutation. A persistent neutropenia became apparent from the age of 3 years. This was associated with frequent admissions for infections requiring intravenous antibiotics, and necessitating the initiation of granulocyte colony stimulating factor (GCSF) at 5 years. A diagnosis of PN was made and genetically proven. Direct sequencing shows the patient to be homozygous for a USB1 gene mutation. Mutation analysis revealed a point mutation 541C>T, protein Gln181X. The patient’s asymptomatic parents are heterozygous for this mutation. Her haemoglobin and platelet counts have remained low ⁄normal to low since first recorded at 2 years. Neutrophils have remained very low, in the absence of GCSF support. Recently she has relied on antibiotic prophylaxis, with GCSF for breakthrough infections only. She presented at 13 years with a nonhealing skin lesion on the right fourth toe (Fig S1; see Supporting Information). This was treated at an outside hospital as an infection, by debridement and repeat dressings, but with no improvement. She was subsequently evaluated at our institution 16 months after onset. Examination revealed the toe to be swollen and purple with no adjacent spread. Biopsy revealed it to be a SCC (Fig. 1). The fourth toe was amputated and healed well; 3 months postsurgery she has recovered completely. This patient had been referred to in the literature prior to the development of the SCC. Histology showed an acanthotic epidermis with hypergranulosis and compact hyperkeratosis (Fig. 1). A longitudinal section through the junction of the ulcerated area demonstrated an invasive well-differentiated SCC, with adjacent basal atypia that was completely excised by at least 4 mm. Neutrophil function studies have been performed twice. Phagocytosis of Escherichia coli and shedding of L-selectin in response to lipopolysaccharide or phorbol 12-myristate 13acetate (PMA) were normal. On both occasions patient neutrophils produced a significantly lowered respiratory burst when stimulated with PMA (8% of the median response in

The surgical management of Treacher Collins syndrome.

Treacher Collins syndrome (TCS), mandibulofacial dysostosis, or Franceschetti-Zwahlen-Klein syndrome, is a rare genetic disorder characterised by dysgenesis of the hard and soft tissues of the first and second branchial arches. Early operations focus on maintaining the airway, protecting the eyes, and supporting auditory neurological development. Later operations include staged reconstruction of the mouth, face, and external ear. Bimaxillary surgery can improve the maxillomandibular facial projection, but correction of malar, orbital rim, and temporal defects may be more difficult. We present a clinical review of the syndrome with a chronological approach to the operations.

Donor site reconstitution for ear reconstruction

BACKGROUND Current techniques of autologous ear reconstruction involve the soft tissue coverage of a carved costal cartilage framework. However, assessment of the morbidity associated with this donor site has been little documented. This study describes a method to reconstruct the defect and analyses the outcomes with or without donor site reconstitution. METHODS The donor site was reconstituted by wrapping morcelised cartilage in a vicryl mesh. Twenty-one patients with reconstitution and nine without were recruited to the study. Scar quality and length, dimensions of donor defect and visible deformity were recorded according to a modified Vancouver scar scale. Patients were also assessed by the SF36 questionnaire, a well-validated health survey. In a subset of our study group, we assessed the fate of the donor site reconstitution by direct visualisation in situ and histological analysis. RESULTS Fifteen donor sites of patients without donor site reconstitution were compared to 23 reconstructed donor sites. In those without, all had a palpable defect with nearly half exhibiting visible chest deformity. In contrast, those that had rib reconstitution did not demonstrate significant chest wall deformity. Intraoperative examination demonstrated formation of a neo-rib, histologically proven to comprise hyaline cartilage admixed with fibrous tissue. Analysis of SF36 results showed a higher satisfaction in the reconstituted group, but in both groups, the donor site was of little overall morbidity. CONCLUSIONS Although there is little difference between the groups in terms of subjectively perceived benefit, rib reconstitution is objectively associated with better costal margin contour and less chest wall deformity.