Somatic gain-of-function mutations in PIK3CA in patients with macrodactyly

Abstract

Macrodactyly is a discrete congenital anomaly consisting of enlargement of all tissues localized to the terminal portions of a limb, typically within a 'nerve territory'. The classic terminology for this condition is 'lipofibromatous hamartoma of nerve' or Type I macrodactyly. The peripheral nerve, itself, is enlarged both in circumference and in length. It is not related to neurofibromatosis (NF1), nor is it associated with vascular malformations, such as in the recently reported CLOVES syndrome. The specific nerve pathophysiology in this form of macrodactyly has not been well described and a genetic etiology for this specific form of enlargement is unknown. To identify the genetic cause of macrodactyly, we used whole-exome sequencing to identify somatic mutations present in the affected nerve of a single patient. We confirmed a novel mutation in PIK3CA (R115P) present in the patient's affected nerve tissue but not in blood DNA. Sequencing PIK3CA exons identified gain-of-function mutations (E542K, H1047L or H1047R) in the affected tissue of five additional unrelated patients; mutations were absent in blood DNA available from three patients. Immunocytochemistry confirmed AKT activation in cultured cells from the nerve of a macrodactyly patient. Additionally, we found that the most abnormal structure within the involved nerve in a macrodactylous digit is the perineurium, with additional secondary effects on the axon number and size. Thus, isolated congenital macrodactyly is caused by somatic activation of the PI3K/AKT cell-signaling pathway and is genetically and biochemically related to other overgrowth syndromes.

Figure 1.

Macrodactyly causes disproportionate overgrowth of affected digits. (A) Patient with type I macrodactyly affecting the index and middle fingers shown at 5 months of age. (B) Macrodactyly affects a ‘nerve territory’, invariably affecting the median nerve of the affected hand.

Figure 2.

Overgrowth is apparent in disorders distinct from type I macrodactyly. (A) The left hand of an infant with Klippel–Trenaunay Syndrome (KT). KT is caused by increased vascularity causing severe overgrowth. Vascular markings are evident with soft tissue overgrowth characteristic of KT. (B) Overgrowth of the left arm of a patient with Neurofibromatosis type I (NF1). Overgrowth of the hand or macrodactyly is rarely seen in NF1 patients. (C) Infant with a keloid-forming overgrowth. This keloid-forming overgrowth is different from the overgrowth present in type I macrodactyly, which does not form keloid scars.

Figure 3.

Immunocytochemistry reveals increased AKT activation in cultured cells from the macrodactylous nerve of Patient 6. (A and B) Negative (no antibody) immunocytochemistry of cells cultured from the nerves of patients with polydactyly (A) and macrodactyly (B). (C and D) Representative immunocytochemistry showing no significant increase of Ser₄₇₃-phosphorylated AKT in cells from the nerve of a polydactylic digit (C) compared with a clear increase in cells from the macrodactylous nerve (D). Scale bars, 200 μm.

Figure 4.

Perineurial overgrowth of the peripheral nerve from the macrodactylous digit of Patient 6. (A and B) Hematoxylin and eosin (A) and Gomori trichrome (B) stains of the paraffin section of peripheral nerve. Nerve fascicles (F) are separated by endoneurial connective tissue and have thickened, concentrically arrayed perineurium (arrowhead). Scale bars, 250 μm. (C and D) Toluidine blue-stained resin sections from the macrodactylous digit of Patient 6 (C) and a polydactylous control digit (D). Macrodactylous nerve has fewer myelinated axons (arrows) and thicker perineurial sheath (arrowhead). Scale bar, 250 μm. (E and F) Uranyl acetate-lead citrate-stained sections from the macrodactylous digit (E) and a polydactyly control (F). Macrodactylous nerve has fewer myelinated axons with disproportionately thinner myelin sheaths (arrow). The macrodactylous nerve also has an increased number of swollen Schwann cell processes (arrowhead). Scale bar, 20 μm.