Early activating somatic PIK3CA mutations promote ectopic muscle development and upper limb overgrowth

Abstract PIK3CA‐related overgrowth spectrum is a group of rare genetic disorders with asymmetric overgrowth caused by somatic mosaic PIK3CA mutations. Here, we report clinical data and molecular findings from two patients with congenital muscular upper limb overgrowth and aberrant anatomy. During debulking surgery, numerous ectopic muscles were found in the upper limbs of the patients. DNA sequencing, followed by digital polymerase chain reaction, was performed on DNA extracted from biopsies from hypertrophic ectopic muscles and identified the somatic mosaic PIK3CA hotspot mutations c.3140A > G, p.(His1047Arg) and c.1624G > A, p.(Glu542Lys) in a male (patient 1) and a female (patient 2) patient, respectively. Patient 1 had four ectopic muscles and unilateral isolated muscular overgrowth while patient 2 had 13 ectopic muscles and bilateral isolated muscular overgrowth of both upper limbs, indicating that her mutation occurred at early pre‐somitic mesoderm state. The finding of PIK3CA mutations in ectopic muscles highlights the importance of PIK3CA in cell fate in early human embryonic development. Moreover, our findings provide evidence that the disease phenotype depends on the timing of PIK3CA mutagenesis during embryogenesis and confirm the diagnostic entity PIK3CA‐related muscular overgrowth with ectopic accessory muscles.


Peer Review
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| INTRODUCTION
Activating somatic mutations in the phosphatidylinositol-4,-5-bisphospate 3-kinase, catalytic subunit alpha gene (PIK3CA) occur frequently in congenital overgrowth syndromes and in human cancer. 1 PIK3CA encodes the p110 α catalytic subunit of phosphoinositide 3-kinase (PI3K), that phosphorylates phosphatidylinositol to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 plays a key role in activating signaling cascades including inhibition of apoptosis, activation of protein synthesis, and enhanced cell survival. 2 The umbrella term PIK3CA-related overgrowth spectrum (PROS) is a heterogeneous group of rare genetic disorders with overgrowth caused by somatic mosaic PIK3CA mutations. 3 The variable expression of symptoms within PROS is mainly explained by the timing and location of the initiating PIK3CA mutation, but the reason behind the high degree of interindividual phenotypic heterogeneity is unknown. 4  The terminology "aberrant muscle syndrome" or "accessory muscle syndrome" has been suggested to describe patients with "hypertrophy of the hand and arm because of aberrant muscles with or without hypertrophy of the muscles." 5 It has been proposed that an increased number of neuromuscular junctions and a change in the muscle-tendon ratio is involved in muscle hypertrophy development. 6 The etiology remains still undetermined but it has recently been reported that isolated congenital muscular upper limb overgrowth can be related to somatic mosaic PIK3CA mutations. 7,8 Somatic activating PIK3CA mutations are common in at least 12 different cancer types. 1,[9][10][11] Previous studies show that the same PIK3CA hotspot mutations found in cancer are found in PROS. [12][13][14][15][16] To date, the only malignancy that has been reported in the 419 known individuals with PROS is Wilms tumor (OMIM 194070), which has been described in 12 individuals (2.86%); six children with a confirmed molecular diagnosis 12,[17][18][19][20][21] and six children where genetic testing has not been performed. [22][23][24][25][26] It has recently been suggested to use monitoring of cell-free DNA in urine to screen for renal involvement in PROS. 27 Although it is critical for normal cell growth and survival, the role of PIK3CA in early human development is poorly characterized.
Because not all cells in overgrown tissues in PROS appear to have PIK3CA mutation, it has been suggested that PIK3CA mutationpositive cells exert growth-promoting effects on adjacent or distant cells. 4 In cells with strong activation of PI3K signaling pathway, PIK3CA mutations may lead to lineage-specific cell loss during or after differentiation because of mechanisms such as oncogene-induced senescence. 28 Previous studies have highlighted that PI3K signaling is crucial for embryonic development and plays an important role in the control of pluripotency and differentiation. 29 Recent studies have also highlighted a prominent role for PI3K signaling during cell differentiation and determination of cell fate in mouse models. 30,31 In this study, we confirm a novel PROS phenotype with isolated muscle overgrowth and ectopic muscles and show evidence that PIK3CA regulates cell fate in early human embryonic development.

| Patients and ethical approvals
We included two patients with local muscular overgrowth of the upper extremity that were seen at the Department of Clinical Genetics, Karolinska University Hospital, Stockholm. The study was per-        (Table 1).

Patient 2:
An 18-year-old female with normal physical and cognitive development was born as the second child to healthy, nonconsanguineous parents. Pregnancy and delivery were normal; her birth weight was 3300 g and her length was 49 cm. Bilateral symmetric hypertrophic hands, arms and upper part of her trunk were noted already during the neonatal period. During childhood, she was very strong in her arms and hands and could climb using only her upper limbs ( Figure 1E). She was referred for hand surgery because the muscular hypertrophy caused functional limitations with difficulties to grip small objects and problems with the pinch grip. Wide hands and abducted thumbs with difficulty to perform the thumb opposition movement were observed. Rotations osteotomy of the first metacarpal and repeated botulinum toxin injections in the hands' intrinsic muscles were performed to improve the pinch grip. The growth was proportional and no progression of the muscular hypertrophy was noted. The difficulty to move the wrists, fingers rotations and elbows deformity led to several surgical procedures. At least 13 ectopic muscles were discovered during surgery (Table 1, Figure 1D, F, G). dPCR, which showed a mutant allelic frequency of 26% in muscle biopsy I and 17% in muscle biopsy II, indicating 52%, respectively, 34% affected cells in the analyzed materials (Figure 2A-B). The mutation was not detected in blood samples from the patient. Occasional fascicles had a more normal appearance. Some adipose tissue was present in the material.

Patient 2:
The pathology was different between the two muscle biopsies I and II (Table 1). In biopsy I, widespread, prominent lesions were found with variation in fiber size and increased number of internalized nuclei. Occasional fibers appeared necrotic and some seemed to be regenerating. A few small aggregates of inflammatory cells were seen. The amount of connective tissue was increased separating the muscle fibers. Some adipose tissue was also found. In regions of more normal appearing muscle, foci of pathologically changed fibers with variation in diameter and an increase of internalized nuclei were noticed together with increased connective tissue. Some rounded eosinophilic fibers could be seen in both muscles with pronounced pathology as well as in otherwise normal tissue. In biopsy II, the pathology consisted mainly of the presence of somewhat rounded Vascular anomalies -Absent digital volar arteries Pathology Increased perimysial and endomysial fibrosis, associated with marked fiber size variability, with scattered hypertrophic fibers and many small fibers. Occasional fibers with rimmed vacuoles were found. There were also rounded eosinophilic fibers.
The pathology differed between the two muscles. In muscle I, variation in muscle fiber size and increase of connective tissue was seen while in muscle II, eosinophilic rounded fibers were present in otherwise rather well-preserved tissue. F = female, M = male. The aberrations in patient 2 seemed symmetrical but because of different operations performed on the right and left hand it was not confirmed in all locations. eosinophilic muscle fibers. In some fascicles, only one was found, in others they were frequent ( Figure 2D). Only occasionally these lesions showed variation in fiber size and an increase in connective tissue.

| DISCUSSION
In this study, we report two individuals with somatic mosaic PIK3CA Therefore, the PIK3CA mutated cells seem to have acquired an ability to become muscles when they normally should have differentiated to tendons or fascia. Studies in mouse have shown that activation of mTORC1 signaling in tendons causes impaired collagen fibrillogenesis, disorganized fibers, hypercellularity, and neovascularization 35 and it is possible that this is similar to what we describe in our two patients.
We speculate that our findings indicate that PIK3CA has a role in pluripotency and cell fate in early human development.
All vertebrate skeletal muscles, apart from superficial neck muscles, derived from the paraxial mesoderm. 36 The mesoderm is derived from the primitive streak in the middle of the epiblast plate. 37 The exact position in the primitive streak decides cell fate. 38  hemihyperplasia patients (OMIM 235000). 17,21,25 There is a possibility that the only patients who are at risk of Wilms tumor are those with a significant burden of renal cells with the activating PIK3CA mutation. 27 If so, dPCR analysis of cell-free DNA in urine seems promising to identify PROS patients at increased risk of Wilms tumor. However, more studies are needed.
In conclusion, this study adds information about timing of PIK3CA mutagenesis during embryogenesis in correlation to phenotype and confirms the diagnostic entity PIK3CA-related muscular overgrowth with ectopic muscles. However, as our study points out, our understanding of PIK3CA's role during human embryogenesis is still restricted. Further studies of induced pluripotent stem cells, animal models, and cancer cell lines on the role of somatic mosaic activating