A, Multiple collagenomas on the lower back; B, a small shagreen patch on the mid back; C, a shagreen patch composed of 2 medium-sized lesions on the lower back; and D, a large shagreen patch on the left posterior thigh.
A, Composite of 100 lesions that were mapped from photographs. Grey represents lesions on the posterior surface; blue represents lesions on anterior surface. B, Number and corresponding percentages of tuberous sclerosis complex (TSC)-related connective tissue nevi found on various regions of the posterior trunk based on photographs or clinical records. The total lesion count was 120, including 2 lesions on the anterior trunk and 4 lesions on the thighs for which percentages are not shown.
A, Hematoxylin-eosin stain shows thickened, disorganized collagen bundles in the reticular dermis (original magnification ×40). B, Elastin stain shows little elastin in connective tissue nevi (original magnification ×40).
Patients are indicated as P numbers. Immunoblotting is shown for TSC2, TSC1, pS6, S6, and β-actin. AF indicates angiofibroma cells; C, control fibroblasts; FCP, fibrous cephalic plaque cells; PF, periungual fibroma cells; pS6, phospho-S6 (Ser-235/236); S6, ribosomal protein S6; SP, shagreen patch cells.
eTable. Size distribution of connective tissue nevi in tuberous sclerosis complex.
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Bongiorno MA, Nathan N, Oyerinde O, et al. Clinical Characteristics of Connective Tissue Nevi in Tuberous Sclerosis Complex With Special Emphasis on Shagreen Patches. JAMA Dermatol. 2017;153(7):660–665. doi:10.1001/jamadermatol.2017.0298
What are the clinical characteristics of connective tissue nevi in patients with tuberous sclerosis complex (TSC)?
In this retrospective analysis of patient medical records and clinical photography of 104 adult patients with TSC, more than half of patients with TSC had at least 1 connective tissue nevus, and 51% of all nevi appeared on the lower back, while the other 49% were distributed throughout the upper and middle back, buttocks, and thighs.
Tuberous sclerosis complex–related connective tissue nevi have various presentations, and their recognition is important for timely diagnosis.
Patients with tuberous sclerosis complex (TSC) frequently develop collagenous connective tissue nevi. The prototypical lesion is a large shagreen patch located on the lower back, but some patients only manifest small collagenomas or have lesions elsewhere on the body. The ability to recognize these variable presentations can be important for the diagnosis of TSC.
To describe the clinical characteristics of connective tissue nevi on the trunk and extremities of patients with tuberous sclerosis complex.
Design, Setting, and Participants
A retrospective analysis of patient medical records and skin photography was performed; 104 adult patients with TSC were enrolled in an observational cohort study that was enriched for those with pulmonary lymphangioleiomyomatosis, and was therefore composed mostly of women (99 women, 5 men). All patients included were examined at the National Institutes of Health (NIH) in Bethesda, Maryland, from 1998 to 2013. Connective tissue nevi were categorized per anatomic location and size. Lesions less than 1 cm in diameter were termed collagenomas. Shagreen patches were characterized as small (1 to <4 cm), medium (4 to <8 cm), and large (≥8 cm).
Main Outcome and Measures
Frequency, anatomic location, size, and histological appearance of connective tissue nevi in patients with TSC.
Overall, 58 of 104 patients (median [range] age, 42 [19-70] years) with TSC (56%) had at least 1 connective tissue nevus on the trunk or thighs; of these, 28 of 58 patients (48%) had a solitary lesion, and 30 of 58 patients (52%) had 2 or more lesions. Overall, 120 lesions from 55 patients were classified by size; 46 lesions (38%) were collagenomas; 39 lesions (32%) were small shagreen patches; 21 lesions (18%), medium shagreen patches; and 14 lesions (12%), large shagreen patches. The distribution of lesions was 9% (n = 11), upper back; 29% (n = 35), middle back; 51% (n = 61), lower back; and 11% (n = 13), other locations. All 26 shagreen patches that were analyzed histopathologically had coarse collagen fibers and 24 of 26 stained with Miller elastic stain had decreased elastic fibers. On immunoblot analysis, fibroblasts grown from shagreen patches expressed higher levels of phosphorylated ribosomal protein S6 than paired fibroblasts from normal-appearing skin.
Conclusions and Relevance
Tuberous sclerosis complex–related connective tissue nevi are not limited to the lower back, and occasionally present on the central or upper back, buttocks, or thighs. Elastic fibers are typically decreased. Recognition of these variable presentations can be important for TSC diagnosis.
Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome that is characterized by benign tumor formation in the brain, lung, heart, kidneys and skin.1 Most patients with TSC will have dermatologic manifestations such as multiple facial angiofibromas, fibrous cephalic plaques (forehead plaques), hypomelanotic macules, ungual fibromas, or shagreen patches.2 Accordingly, these lesions constitute several of the major diagnostic criteria from the 2012 International Tuberous Sclerosis Complex Consensus Conference,3,4 making physician recognition imperative for diagnosis.
The shagreen patch is a type of connective tissue nevus that is considered highly diagnostic of TSC when it has the classic appearance of a large, irregular, firm plaque located on the lower back. The texture of shagreen patches has been described as “pigskin” or peau d’orange with a pink or skin-colored hue, or prominent, dilated follicular openings. Previous studies5-9 have documented their presence in approximately 21% to 83% of patients with TSC.
Tuberous sclerosis complex is caused by germline mutation in 1 of 2 tumor suppressor genes, TSC1 or TSC2.10 The formation of tumors in TSC is driven by a somatic second-hit mutation of the wild-type allele.11,12 These inactivating mutations in either of the tumor suppressor genes lead to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1), causing downstream kinase activation and phosphorylation of ribosomal protein S6 (phospho-S6).13 While angiofibromas, ungual fibromas, and fibrous cephalic plaques have been shown to develop secondary to a second-hit mechanism in tumor fibroblast–like cells,14-16 molecular alterations in the shagreen patch have not yet been studied.
Not all connective tissue nevi in patients with TSC fit the shagreen patch description. Presence outside of the lower back may hinder recognition as a TSC manifestation. Other connective tissue nevi in TSC may present as smaller papules that can mimic collagenomas observed in other conditions. To help improve diagnostic accuracy, the current study was undertaken to describe the spectra of locations and sizes of connective tissue nevi in patients with TSC.
A total of 104 patients with TSC with or without pulmonary lymphangioleiomyomatosis were examined at the National Institutes of Health (NIH) in Bethesda, Maryland, from 1998 to 2013. Each patient provided written informed consent, and this study was approved by the National Heart, Lung, and Blood Institute institutional review board. All patients met the revised diagnostic criteria of the 2012 International Tuberous Sclerosis Complex Consensus.3 Information on sex, race, and ethnicity was self-reported during patient registration at the NIH and assessed in accordance with the NIH policy on inclusion of women and minorities. Patients were evaluated for the presence or absence of connective tissue nevi and queried regarding the age of onset of lesion(s). Connective tissue nevi involving the head or neck (fibrous cephalic plaques) were not assessed for this study. Paired samples of shagreen patch and normal skin were obtained from 26 patients, and the sections were stained with routine hematoxylin-eosin and Miller elastin stain. In tissue sections stained for elastic fibers, a semiquantitative scale ranging from 0 to 4 was used, with 0 showing no elastin and 4 showing the most.
Photographs were taken, and lesions were classified according to size based on the length of the major axis: smaller than 1 cm (collagenomas), 1 cm or larger but less than 4 cm (small shagreen patches), 4 cm or larger but less than 8 cm (medium shagreen patches), and 8 cm or larger (large shagreen patches). Anatomic location was mapped if the connective tissue nevus could be localized based on the presence of at least 2 position-defining landmarks (ie, gluteal crease, axillary crease, midline). Lesions were outlined on a representative torso using Adobe Illustrator version CS5 (Adobe Systems Incorporated) and the outlines overlaid to form a composite. The trunk was divided into 6 regions (upper, middle, and lower thirds bisected into right and left), and location was assigned by its majority in a region.
For Western blot analysis, cells grown from samples of shagreen patch, angiofibroma, ungual fibroma, fibrous cephalic plaque, and normal-appearing skin were seeded overnight into 60-mm dishes at 5 × 105 cells in DMEM (Dulbecco Modified Eagle Medium) with 10% FBS (fetal bovine serum) and switched to serum-free DMEM for 24 hours. Cells were lysed in protein extraction buffer and immunoblotting performed as previously described16 with anti-Tuberin/TSC2 (D93F12), anti-Hamartin/TSC1, anti-phospho-S6 ribosomal protein (Ser-235/236), anti-S6 ribosomal protein antibodies (Cell Signaling), β-actin (Sigma-Aldrich), and horseradish peroxidase-conjugated secondary antibodies (GE Healthcare).
A paired t test was used to analyze elastin stain score for paired samples of shagreen patch and normal skin.
The 104 adult patients with TSC were comprised of 99 women and 5 men (median [range] age, 42 [19-70] years) (Table). Fifty-eight patients (56%) had a connective tissue nevus. Most reported onset during early childhood. A total of 120 lesions were classified as 46 collagenomas, 39 small shagreen patches, 21 medium shagreen patches and 14 large shagreen patches. The median (range) number of lesions per patient was 2 (1-14) (eTable in the Supplement). Forty-six patients (79%) had either 1 lesion (28 patients) or 2 lesions (18 patients).
Collagenomas appeared as firm, skin-colored to slightly hypopigmented, well-circumscribed, dome-shaped papules (Figure 1A). Shagreen patches were typically skin-colored to pink plaques, sometimes hypopigmented or hyperpigmented. Small shagreen patches tended to be oval, whereas medium (Figure 1B) and large shagreen patches (Figure 1C) were irregular ovals. Shagreen patches on the back were mostly firm whereas those on the thighs (Figure 1D) were soft and had less distinct margins. The elevation of large shagreen patches often appeared as irregular bumps. The surfaces of some were smooth, while others had either dilated follicular openings (peau d’orange) or small follicular papules. The long axis of shagreen patches tended to fall along Langer lines.
One-hundred seven lesions (89%) were observed on the back, with 11 (9%) in the upper third, 35 (29%) in the middle third, and 61 (51%) in the lower third (Figure 2). Thirteen (11%) connective tissue nevi were present at sites other than the back, predominantly buttocks or thighs. Lesions were equally distributed on the right and left except for large shagreen patches that predominated on the left (9, left; 5, right).
Routine hematoxylin-eosin staining revealed thickened, disorganized collagen bundles in the reticular dermis consistent with the diagnosis of connective tissue nevus in all 26 samples (Figure 3A). Miller elastin stain showed decreased elastin fibers in 24 of 26 samples within the connective tissue nevus (Figure 3B). On average, the elastin score for connective tissue nevi was 2.24 points lower than normal controls (P < .001).
Fibroblasts were grown from shagreen patches and normal-appearing skin from 8 patients. Western blot analysis of cells under serum-starved conditions showed that fibroblasts grown from shagreen patches had greater phosphorylation of ribosomal protein S6 (Ser235/236) than fibroblasts from normal-appearing skin (Figure 4), consistent with increased signaling through mTORC1. The levels of expression of TSC2 (tuberin) and TSC1 (hamartin) in shagreen patch cells were similar to those observed in cells grown from normal-appearing skin. Patient P50, known to have a germline mutation in TSC1 rather than TSC2, appeared to show slightly lower levels of TSC1 expression in both shagreen patch cells and normal skin fibroblasts. Representative results using cells grown from an angiofibroma, ungual fibroma, and fibrous cephalic plaque are also shown. In contrast to the shagreen patches, these tumors, obtained from patients with documented mutations in TSC2, show loss of TSC2 expression and decreased TSC1 expression.
The most common location for a connective tissue nevus in individuals with TSC is the lower back; however, nearly one-third of lesions may occur on the central back, with fewer lesions located on the upper back, buttocks, or upper thighs. They are rare on the chest or abdomen. About one-half of patients with a connective tissue nevus have a solitary lesion. When multiple lesions are present, they may be either scattered or grouped. Histologically, collagen fibers are thickened and increased in amount whereas elastic fibers are decreased. Most appear in the first decade of life. They are typically asymptomatic and generally do not require treatment except for cosmetic reasons. Their main medical significance is as a major feature for the diagnosis of TSC.
Clinical confidence that a connective tissue nevus represents a manifestation of TSC is greatest for a large shagreen patch on the lower back, which tend to resemble a peau d’orange. Some small and medium shagreen patches have features similar to connective tissue nevi observed in Buschke Ollendorff Syndrome (BOS).17,18 However, skin lesions in patients with BOS tend to be more yellow, occur in greater numbers, and more frequently manifest on the anterior trunk or extremities than in TSC. Some connective tissue nevi in BOS show findings of collagenomas but most show increased elastic fibers histologically.18,19 Elastomas of other etiologies (sporadic or familial) also have increased elastic fibers, distinguishing them from the collagenous TSC connective tissue nevi.17
Connective tissue nevi in TSC that are smaller than 1 cm were termed collagenomas to distinguish them from shagreen patches that serve as a major feature for clinical diagnosis. In most cases, collagenomas were accompanied by a shagreen patch; however, we found that nearly one-fifth of patients with a connective tissue nevus only had 1 or more collagenomas, which could cause a diagnostic challenge. Collagenomas should raise suspicion for TSC, particularly when located on the lower back, but they are not specific. Without additional manifestations of TSC, the observation of collagenomas should also prompt consideration of multiple endocrine neoplasia type 1 (MEN1), Birt-Hogg-Dubé syndrome, familial cutaneous collagenomas, eruptive collagenoma, and storiform collagenomas of Cowden syndrome.20 Patients with MEN1 may manifest both collagenomas and angiofibromas, but MEN1 collagenomas usually occur on the anterior chest, abdomen, as well as proximal arms in addition to the back, and the angiofibromas and collagenomas have later onset.21
In most cases, patients with TSC will not require biopsy of a shagreen patch because they will typically manifest other skin features such as facial angiofibromas, hypomelanotic macules, or ungual fibromas. In those rare patients without accompanying features, we recommend performing a biopsy of the lesion.22 Pathological interpretation may benefit from staining for elastic fibers and concurrent evaluation of a biopsy of adjacent normal-appearing skin.
Shagreen patches were located dorsally and rarely ventrally. It is not clear why skin at one body region is more susceptible to formation of a TSC connective tissue nevus than others, but it may reflect embryonic origins. Dermal fibroblasts in dorsal skin are derived from paraxial mesoderm whereas those in ventral skin and extremities arise from lateral plate mesoderm.23 These fibroblast populations may respond differently to impaired TSC1-TSC2 function and mTORC1 activation. Our data also show that the long axis of shagreen patches tended to align with skin tension lines. It is known that the distribution and orientation of collagen differs when analyzed either in parallel or perpendicular to the Langer lines.24 The pattern of shagreen patches suggest that fibroblasts with impaired TSC1-TSC2 function exhibit abnormalities in collagen deposition and/or remodeling. Abnormalities in collagen remodeling are expected because fibrous plaques and angiofibromas of patients with TSC show increased expression of collagenases (MMP-1 and MMP-13) and reduced expression of tissue inhibitor of metalloproteinase 1 (TIMP-1 and TIMP-2).25
Immunoblot analysis was performed to identify molecular alterations in the connective tissue nevi compared with alterations in other TSC-related skin lesions. Our prior studies of angiofibromas, ungual fibromas, and forehead plaques have shown that TSC skin tumors arise by a 2-hit mechanism, with tumor fibroblasts showing biallelic mutations in TSC2 and loss of TSC2 expression.15,16,26 In contrast, none of the samples of shagreen patch cells showed loss of TSC1 or TSC2 expression, despite showing increased phospho-S6 consistent with mTORC1 activation. It is possible that shagreen patches, unlike other types of TSC skin tumors, form by haploinsufficiency. However, biallelic mutations in TSC2 were identified in shagreen patch cells from 1 patient, albeit with very low allele frequencies.16 This result suggests that the cultures of shagreen patches include 2-hit tumor cells and contaminating 1-hit fibroblasts. In either case, the activation of mTORC1 appears to be important for tumor formation, as they regress in response to treatment with an mTOR inhibitor, sirolimus.27
Limitations of this study include the retrospective design, use of patient-reported onset of nevi, and the small sample size for immunblot analysis. Our cohort was predominantly adult women due to enrichment for those patients with TSC with pulmonary lymphangioleiomyomatosis, which occurs at much higher frequencies in women than men. Others have not observed significant differences in frequencies of shagreen patches in men vs women,28 but it is possible that the patterns of lesion size and distribution in women may not be generalizable to men.
In addition to the classic large shagreen patch on the lumbosacral region, connective tissue nevi in TSC include smaller collagenomas and lesions occurring on the middle to upper back, buttocks, and thighs. The histological appearance comprises coarse collagen fibers and usually decreased elastic fibers compared to normal skin. Fibroblasts grown from the lesions exhibit increased signaling through mTORC1 as seen in other TSC tumors. Awareness of the clinical appearance, location, and pathological composition of these and other TSC skin lesions is necessary to enable prompt disease identification and initiation of management.
Corresponding Author: Thomas Darling, MD, PhD, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD 20814 (email@example.com).
Accepted for Publication: January 26, 2017.
Published Online: April 26, 2017. doi:10.1001/jamadermatol.2017.0298
Author Contributions: Drs Bongiorno and Darling had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Bongiorno, Moss, Darling.
Acquisition, analysis, or interpretation of data: Bongiorno, Nathan, Oyerinde, Wang, Lee, Brown, Darling.
Drafting of the manuscript: Bongiorno, Nathan, Oyerinde, Moss, Darling.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Bongiorno, Darling.
Obtained funding: Nathan, Moss, Darling.
Administrative, technical, or material support: Bongiorno, Wang, Lee, Moss.
Study supervision: Moss, Darling.
Conflict of Interest Disclosures: None reported.
Funding/Support: This study was supported in part by the National Institutes of Health (NIH) (grant No. NIH R01AR062080); the Sulzberger Dermatological Research and Education Endowment; the Intramural Research Program of the NIH; the National Heart, Lung, and Blood Institute; and the NIH Medical Research Scholars Program, a public-private partnership supported jointly by the NIH; generous contributions to the Foundation for the NIH from the Doris Duke Charitable Foundation; the American Association for Dental Research; the Colgate-Palmolive Company; Genentech; and other private donors. For a complete list, visit http://www.fnih.org.
Role of the Funder/Sponsor: The funders/sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: We thank Claire Hong, BS, who assisted with figure creation and review of manuscript as part of her role as a student intern at the Uniformed Services University of the Health Sciences.