Evaluation of the <i><scp>CAV</scp>1</i> gene in clinically, sonographically and histologically proven morphea patients

The inflammatory skin disorder morphea (circumscribed cutaneous scleroderma) is characterized by significant fibrosis (collagen deposition) of the dermis, evident throughout all of its pathologic phases 1. Morphea association with fibrosis may even extend to other fibrotic diseases such as lichen sclerosus, which has been reported to coexist with localized scleroderma within the genetic background of female monozygotic twins 2. Besides its typical dermal involvement, ultrasound of morphea has revealed alteration of the underlying subcutaneous tissue consisting of inflammatory signs (i.e. increased echogenicity and vascularity) during the active phase, and decrease or even total absence of the subcutaneous fatty lobules, suggestive of lipodystrophy, in the atrophic phase 3. Within the context of tissue fibrosis, recent research has identified an important pathogenic mechanism: the deficiency of caveolin 1 (CAV1) the main coating protein of caveola (plasma membrane invaginations rich in the cell surface receptors critical for the initiation of intracellular signalling cascades) 4, 5. Thus, CAV1 (determined as mRNA, by immunofluorescence, or directly using chemiluminescence) has been found decreased in affected lungs and skin of patients with systemic sclerosis (scleroderma; SSc), as well as in lungs and fibroblasts of patients with idiopathic pulmonary fibrosis. Experimentally, the CAV1 knockout mouse develops striking pulmonary and skin fibrosis 4, 6, while the CAV1 protein has antifibrotic actions from the inhibition of transforming growth factor-β (TGFβ) receptor internalization 7. In addition, the effect of CAV-1 interfering with intracellular signalling by vascular endothelial growth factor A (VEGF-A, hereafter referred to as VEGF) may also be important for the production of fibrosis in SSc. In this disease, fibrosis may result from vascular alterations via dedifferentiation of endothelial cells and pericytes towards myofibroblasts. CAV 1 deficiency would induce the profibrotic phenotype through suppression of VEGF signalling, by default stimulation (derepression) of VEGF receptor 2 degradation. Such mechanism is consistent with the reportedly low levels of CAV-1 with upregulation of VEGF signalling in bone marrow SSc mesenchymal stem cells, a pericyte surrogate 8. Is CAV1 gene positive in morphea? We therefore analysed the coding sequence of the CAV1 gene in a population of well-characterized patients with morphea, fully assessed for disease activity, both clinically and by skin ultrasound. The genetic study with DNA sequencing was performed at Columbia University (from 1 February 2014 to 30 July 2014) in patients recruited from among all cases with morphea diagnosed by dermatologic examination and histologic evaluation from 1 March 2008 to 30 July 2013 (n = 138), sonographically positive and seen at a single referral centre (Clinical Servet, Santiago, Chile). For this study, we selected the first ten patients enrolled, while excluding cases with the Parry–Romberg (PR) syndrome (facial morphea with ipsilateral hemiatrophy of the face). The test group consisted of nine females and one male, with mean age of 24.8 ± 13.1 years (SD); they had a mean of 1.9 morphea lesions. The distribution of the cutaneous lesions was as follows: 40% truncal, 30% facial and 30% extremities. On ultrasound, the lesions were predominantly inactive in one patient; mostly in the atrophic phase in three patients; and active, with subcutaneous fat involvement, in six patients. More data are available in the supplemental material. For the genetic studies, genomic DNA was extracted from whole blood with PureGene reagents (Gentra Systems Inc., Minneapolis, MN, USA). Genomic DNA was extracted from saliva using Oragene kits (Omnigene-Discover OM-501 kits; Genotek Inc., Ottawa, ON, Canada), according to the manufacturer's instructions. More data are available in the supplemental material. All participants signed an informed consent form, and the study was approved by the Institutional Review Board at Clinica Servet, Santiago, Chile. None of the patients had positive family history for morphea; eight had the normal, reference CAV1 sequence, while the remaining two subjects (# 1 and # 7) had novel variants. Patient #1 was heterozygous for two variants: c. 66 C>A (p. C22Ter) and c. 284 C>T (p. T95M). Patient #7 was heterozygous for one variant: 284 C>T (p. T95M). Segregation analysis showed that the unaffected father of patient # 1 was heterozygous for the c. 284 C>T (p. T95M) variant, and the unaffected mother was heterozygous for c. 66 C>A (p. C22Ter). The C22Ter variant is predicted to be pathogenic at the protein level and to result in haploinsufficiency. However, as it is also carried by the clinically unaffected mother, if pathogenic, the variant has incompletely penetrance. In the family of patient #7, the mother had the wild-type sequence, and an unaffected sister was heterozygous for c. 284 C>T (p. T95M) (Table 1). Therefore, T95M is, in all likelihood, an inherited normal variant. Father: c. 284 C>T (p. T95M) Mother: 66 C>A (p. C22Ter) Mother: Wild-type Sister: c. 284 C>T (p. T95M) The present work represents the first attempt at evaluating genetic contributions to morphea pathogenesis using a candidate gene approach. While the results of our genetic analysis do not support the involvement of CAV1 in morphea, there are some limitations to our study. (i) The sample size is small, although the age, distribution of location and activity of the lesions are representative of morphea. (ii) We did not quantify CAV1 protein, nor performed assessment of the gene regulatory sequences. (iii) We purposely restricted the study to patients with the most common form of the disease and excluded those with the clinically more homogeneous Parry–Romberg form. (iv) It is possible that some of the asymptomatic relatives of patients harbouring CAV1 variants (who did not undergo sonographic examination) could have cryptic lesions that were not symptomatic, because patients with morphea can have subclinical lesions evident only on ultrasound 3. While morphea is not a Mendelian disorder, excessive fibrosis is common to morphea, scleroderma, idiopathic pulmonary hypertension, lipodystrophy, and even allograft failure after renal transplantation 2-7, 9 S1–S3. TGFβ-responsive gene signature has been found in a subset of diffuse scleroderma patients (S4). Given the effects of TGFβ, CAV1 and VEGF in fibrosis and antifibrosis, it becomes increasingly important to determine the role of these proteins in these diseases to develop novel therapies. Within this context, our examination of the CAV1 gene in the commonest form of morphea may represent an important step in that direction. Ximena Wortsman, MD, worked in the design, gathering and analysis of the data, and writing of the manuscript. Lijiang Ma, PhD, worked in the gathering and analysis of the data as well as the revision of the manuscript. Wendy K. Chung, MD, PhD, worked in the design, gathering and analysis of the data, and writing of the manuscript. Jacobo Wortsman, MD, worked in the design, analysis of the data and writing of the manuscript. All participants signed an informed consent form, and the study was approved by the Institutional Review Board at Clinica Servet, Santiago, Chile. None for all authors. Data S1 Material. Supplementary References - S1-S4 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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