Multilineage somatic activating mutations in HRAS and NRAS cause mosaic cutaneous and skeletal lesions, elevated FGF23 and hypophosphatemia

Abstract

Pathologically elevated serum levels of fibroblast growth factor-23 (FGF23), a bone-derived hormone that regulates phosphorus homeostasis, result in renal phosphate wasting and lead to rickets or osteomalacia. Rarely, elevated serum FGF23 levels are found in association with mosaic cutaneous disorders that affect large proportions of the skin and appear in patterns corresponding to the migration of ectodermal progenitors. The cause and source of elevated serum FGF23 is unknown. In those conditions, such as epidermal and large congenital melanocytic nevi, skin lesions are variably associated with other abnormalities in the eye, brain and vasculature. The wide distribution of involved tissues and the appearance of multiple segmental skin and bone lesions suggest that these conditions result from early embryonic somatic mutations. We report five such cases with elevated serum FGF23 and bone lesions, four with large epidermal nevi and one with a giant congenital melanocytic nevus. Exome sequencing of blood and affected skin tissue identified somatic activating mutations of HRAS or NRAS in each case without recurrent secondary mutation, and we further found that the same mutation is present in dysplastic bone. Our finding of somatic activating RAS mutation in bone, the endogenous source of FGF23, provides the first evidence that elevated serum FGF23 levels, hypophosphatemia and osteomalacia are associated with pathologic Ras activation and may provide insight in the heretofore limited understanding of the regulation of FGF23.

Figure 1.

Clinical and histological features of CSHS. All photomicrographs are at ×10 magnification. (A and B) Affected individual CSHS101 is a 7-year-old Caucasian female who presented at birth with linear epidermal nevi restricted to the left side of her body distributed from the neck to the calf. Histopathology shows thickening of the epidermis (acanthosis) and papillomatosis. (C and D) CSHS102 is a 12-year-old Caucasian female with nevus sebaceus on the left side of her head and neck. Histology of lesional skin from the cheek shows sebaceous hyperplasia, thickening of the stratum corneum (hyperkeratosis) and papillomatosis. (E and F) CSHS103 is a 15-year-old black female with widespread keratinocytic epidermal nevi on the torso and sebaceous nevi on the scalp and cheek, with brown verrucous papules and plaques covering the scalp, face, torso and extremities, as well as linear white plaques on the scalp and torso. Histological examination shows marked sebaceous hyperplasia, hyperkeratosis and papillomatosis. (G and H) CSHS104 is a black female who presented with a giant melanocytic nevus covering the entire posterior torso at birth, extending across the flanks to the anterior chest. Round, raised, hairy and pigmented plaques determined to be satellitosis of the congenital nevi were located on her extremities. There was a 0.5 × 12 cm linear tan epidermal nevus on the left forearm (not shown). Histology of tissue from her back tissue shows melanocytes infiltrating the full thickness of the dermis, melanin deposition and hyperkeratosis. She died at 4 years of age from a large pericardial effusion that occurred during sedation for an MRI. (I and J) CSHS105 is a 16-year-old Caucasian male born with whorls of raised pink to tan plaques across the central right chest and nipple, as well as lesions extending around the flank towards the right back, all consistent with keratinocytic epidermal nevi. Histology of lesions shows hyperkeratosis, acanthosis and papillomatosis.

Figure 2.

Radiographic features of CSHS. (A) A CT scan of the jaw of patient CSHS103 shows large areas of dysplastic bone (asterisks) that appear as lytic lesions. The skeletal disease in the mandible displays an aggressive expansion that displaces and resorbs the tooth roots. (B) A radiograph of the femurs of patient CSHS104 demonstrates healing fractures due to osteomalacic bone (solid arrows), evidence of rickets (ill-defined, frayed and widened growth plates, dotted arrows), and dysplastic bone with mixed lytic and sclerotic changes (asterisk). (C) A radiograph of the femur of patient CSHS101 at age 7 reveals a stretch of dysplastic bone with a primarily sclerotic appearance at this age (solid arrows). At a younger age, the same lesion was more lytic in nature (not shown). As a result of medical therapy, the growth plate is normal, with no evidence of rickets (dotted arrows). (D) A radiograph of the arm of patient CSHS102 shows multiple unhealed fractures (arrows). Dysplastic lesions with lytic changes are seen in some of the phalanges (asterisks).

Figure 3.

Dysplastic skeletal lesions from the rib of CSHS104. (A) Low power (×2) view from the pleural of the side of the rib shows a thin layer of cells that underlie the periostium along the length of the section of rib depicted (arrows). Asterisk denotes focus of dysplastic cells. (B) Higher power (×4) H&E of the area of dysplastic tissue marked by the asterisk in section (A) is shown. The lesion consists of a collection of fibroblast-like spindle-shaped cells in a relatively dense collagen matrix that appears to arise from the layer of dysplastic cells demonstrated in (A). (C) A Masson's trichrome stain of the same region seen in (B) displays both blue-staining collagen in the matrix surrounding the dysplastic fibroblast-like cells and an extensive area of osteoid surrounding the collection of dysplastic cells (asterisk). (D) A higher power (×10) view of the smaller area dysplastic cells seen in (B and C) show a collection of cells similarly arising from the layer of dysplastic cells seen in (A) as well as active osteoclast resorption of adjacent lamellar bone (arrows), suggesting that the dysplastic cells of CSHS can induce osteoclastogenesis. (E) A high power (×40) view of the region seen in (C) demonstrates collagen bundles connecting the osteoid with the pericellular matrix. The collagen bundles are parallel to the mineralizing surface (Sharpey's fibers) and interdigitate between the atypical but osteoblast-like cells that appear to have produced the adjacent osteoid.

Figure 4.

NRAS Q61R is present in dysplastic, but not normal, bone in CSHS102 and 104. (A) The region within the dotted line on a ×2 view of an affected rib from patient CSHS104 shows a representative region of dysplasia. DNA was extracted from cores of tissue excised from this region. (B) A ×20 view of the same region shown in (A) demonstrates an area of fibrous tissue composed of spindle-shaped cells set in a dense collagen matrix and in intimate association with the adjacent lamellar bone. (C) Sanger sequencing of DNA from affected bone tissue from patient CSHS104 shows the same heterozygous NRAS Q61R mutation that was found in the patient's cutaneous lesion (left panel). Sequence of DNA extracted from the patient CSHS104′s unaffected rib is wild type (right panel). (D) Sanger sequencing of DNA from a bone sample appearing grossly sclerotic from patient CSHS102 shows the heterozygous HRAS G13R mutation that was found in the patient's nevus (left panel). DNA from a region near the cortical bone is wild type (right panel).

Figure 5.

Affected skin, but not affected bone, shows staining for MelanA. Skin (A) and bone (B) specimens from patient CSHS104 confirmed to carry the NRAS Q61R mutation were sectioned and immunostained with melanocyte marker MelanA, to confirm the affected bone to be osteogenic and independent. Nuclei of abundant dermal melanocytes within a congenital nevus stain strongly brown for MelanA (A: ×10, ×20 insert), while a cellular region of dysplastic bone shows no staining (B: ×10, ×20 insert). A hematoxylin counterstain highlights nuclei in both panels.