Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction

Abstract

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

Fig 1. Pedigrees and pictures of the individuals studied.

(A) Pedigree of three consanguineous families studied. Black boxes indicated affected individuals. Roman numerals indicating the generation are on the left and Arabic numerals indicating the individual are below each pedigree symbol. Genotypes are indicated below each individual with a “+” indicating a wild-type allele and a “-” indicating a mutant allele as illustrated in Fig 1G. The genotypes of individuals II-II-1, II-II-4, and II-II-5 are omitted at the family’s request. (B) Photographs of affected individuals under their corresponding pedigree symbol indicating coarse facial features including a broad nasal bridge, thick columella, and thick alae nasi. Representative images are shown of (C) an X-ray indicating square shaped vertebral bodies and a flat pelvis, subluxation of the hips, and meta- and epiphyseal dysplasia of the femoral heads in patient III-II-2, (D) the teeth in patient II-II-3 indicating broad maxilla incisors, narrow mandible teeth, and dental enamel defects, (E) the eye with a visible cataract (Cataracta polaris anterior), as indicated by a white arrow, in individual III-II-2, and (F) a brain MRI demonstrating areas of altered signal intensity as indicated by the white arrow in individual I-II-2. (G) Diagram of the intron/exon and protein domain structures of PIK3C2A indicating the location of mutations identified in the pedigrees shown in Fig 1A.

Fig 2. Protein and mRNA levels of PIK3C2A in patient-derived cells.

PIK3C2A mRNA levels were detected by qRT-PCR in patient derived fibroblasts from (A) Family I, (B) Family II, and (C) Family III. The WT sample in Fig 2A is based on n = 1 individual, the WT sample in Fig 2B is based on n = 3 individuals, and the WT sample in Fig 2C is based on n = 1 individual, totaling n = 5 different WT samples. qRT-PCR data is represented as mean ± SEM (n = 3–4 technical replicates per sample). (D, E) Whole cell lysates from fibroblasts of healthy controls (WT), heterozygous parents, and affected individuals from (D) Family III and (E) Families I and II were analyzed by Western blotting for PIK3C2A and the loading controls Actin or GAPDH. Immunogen of anti-PIK3C2A antibodies (AB1-AB4) are detailed in S5 Table. (F) Densitometry of Western blot results was performed using ImageJ. Individual samples are shown with the data combined from the four different PIK3C2A antibodies used, with the exception of the WT samples which includes fibroblasts from two individuals. * indicates p < 0.05. ** indicates p < 0.01. *** indicates p < 0.0001.

Fig 3. Impaired PI metabolism in patient-derived fibroblasts.

Quantification of PI(3,4)P2 and PI(3)P were analysed and normalized on whole cell fluorescence. (A) Immunofluorescence analysis of cellular PI(3,4)P2 levels. Results showed that PI(3,4)P2 is significantly reduced throughout the cell in -/- cells compared with +/+ cells. n = 4 independent experiments and 10 cells per genotype for each experiment (S6 Table). (B) Immunofluorescence analysis of PI(3)P localization at the base of the primary cilium. Results showed that PI(3)P is significantly reduced at the base of the primary cilium in -/- cells compared with +/+ and or +/- cells. n = 3 independent experiments and 15 cells per genotype for each experiment (S6 Table). Nuclei are stained with DAPI. ** indicates p < 0.01. *** indicates p < 0.0001.

Fig 4. Ciliary defects due to PIK3C2A deficiency in patient-derived fibroblasts.

(A) Cilia length and (B) cilia number were determined in primary fibroblasts from two affected individuals and three unrelated controls. Data is represented as mean ± SEM (n>300/sample). Raw data for cilia length is provided in S6 Table. (C) Immunofluorescence analysis of RAB11 localization at the base of the primary cilium. Results showed that RAB11 is significantly reduced at the base of the primary cilium in -/- cells compared with +/+ and or +/- cells. n = 3 independent experiments and 15 cells per genotype for each experiment. (D) Immunofluorescence analysis of IFT88 localization within the primary cilia. Results showed that IFT88 is significantly increased along the primary cilium in -/- cells compared with +/+ and or +/- cells, suggesting a defective trafficking of ciliary components. n = 3 independent experiments and 15 cells per genotype for each experiment. Quantification of IFT88 and RAB11 were normalized on whole cell fluorescence. *** indicates p < 0.0001.

Fig 5. PIK3C2A deficiency causes delayed proliferation rates in patient-derived fibroblasts.

Proliferation curve of primary fibroblasts isolated from PIK3C2A WT (+/+), heterozygous (+/-), and homozygous (-/-) individuals. Values are reported as the mean ± SEM from 3 independent experiments. * indicates p < 0.05, *** indicates p < 0.0001.

Fig 6. PIK3C2B levels are increased by PIK3C2A deficiency.

PIK3C2B mRNA levels were detected by qRT-PCR in (A) Family I, (B) Family II, and (C) Family III. qRT-PCR data is represented as mean ± SEM (n = 3 technical replicates per sample). The WT sample in Fig 6A is based on n = 1 and the WT sample in Fig 6B is based on n = 3, totaling n = 4 different WT samples. (D) PIK3C2B protein levels were detected by Western blotting in Family I from 2 independent experiments. (E) PIK3C2A and PIK3C2B protein levels were analyzed in HeLa cells by Western blotting following doxycycline inducible shRNA mediated knockdown of PIK3C2A. Data shown is representative from 3 independent experiments. * indicates p < 0.05, ** indicates p < 0.01. *** indicates p < 0.0001.