Mutations in PIK3R1 cause SHORT syndrome

Abstract

SHORT syndrome is a rare, multisystem disease characterized by short stature, anterior-chamber eye anomalies, characteristic facial features, lipodystrophy, hernias, hyperextensibility, and delayed dentition. As part of the FORGE (Finding of Rare Disease Genes) Canada Consortium, we studied individuals with clinical features of SHORT syndrome to identify the genetic etiology of this rare disease. Whole-exome sequencing in a family trio of an affected child and unaffected parents identified a de novo frameshift insertion, c.1906_1907insC (p.Asn636Thrfs*18), in exon 14 of PIK3R1. Heterozygous mutations in exon 14 of PIK3R1 were subsequently identified by Sanger sequencing in three additional affected individuals and two affected family members. One of these mutations, c.1945C>T (p.Arg649Trp), was confirmed to be a de novo mutation in one affected individual and was also identified and shown to segregate with the phenotype in an unrelated family. The other mutation, a de novo truncating mutation (c.1971T>G [p.Tyr657*]), was identified in another affected individual. PIK3R1 is involved in the phosphatidylinositol 3 kinase (PI3K) signaling cascade and, as such, plays an important role in cell growth, proliferation, and survival. Functional studies on lymphoblastoid cells with the PIK3R1 c.1906_1907insC mutation showed decreased phosphorylation of the downstream S6 target of the PI3K-AKT-mTOR pathway. Our findings show that PIK3R1 mutations are the major cause of SHORT syndrome and suggest that the molecular mechanism of disease might involve downregulation of the PI3K-AKT-mTOR pathway.

Figure 1

Facial Features of Individuals Clinically Diagnosed with SHORT Syndrome (A–F) Photographs of affected individuals 1 (A and B), 3 (C and D), and 4 (E and F) capture the core facial gestalt, including the broad triangular facies, deep-set eyes, hypoplastic alae nasae, low-hanging columella, and downturned mouth. (G) Photograph of individual 2, who has a diagnosis of SHORT syndrome. This individual has several features of SHORT syndrome; however, he appears to lack the facial features of the individuals in (A)–(F). Specifically, his face does not appear to be as triangular, and his eyes are not as deep set. His columella is not low hanging, and the ala nasi are not overly hypoplastic. His mouth is not downturned, and he does not have obvious micrognathia. This image was reproduced with permission from John Wiley & Sons (license number 3112610845148).

Figure 2

PIK3R1 Mutations in Families Affected by SHORT Syndrome (A) Family pedigrees and the heterozygous mutations identified in PIK3R1 (RefSeq NM_181523.2). (B) Electropherograms showing the mutation position in the father (top), mother (middle), and affected child (bottom). That of family 5 shows the mutation position in the mother, brother, and proband. (C) A schematic of PI3K demonstrates the position of the substitutions and truncations in the SH2 domains of the protein. The alterations in red are those from the literature and are not associated with SHORT syndrome.

Figure 3

Immunoblot Analysis from LCL Extracts Derived from Individuals with SHORT Syndrome and Hemimegalencephaly (A) The upper panel shows p85α-specific levels (top band) in extracts from the control, individual 1 (c.1906_1907insC [p.Asn636Thrfs^∗18]), and two previously described individuals with hemimegalencephaly. The latter two individuals exhibit hyperactivating mutations in PIK3R2 (c.1117G>A [p.Gly373Arg]), encoding the p85β regulatory subunit of PI3K, and PIK3CA (c.1359_1361del [p.Glu453^∗]), encoding the p110α catalytic subunit of PI3K. The middle panel shows the results of using a pan-p85 antibody capable of detecting the p85α isoform (encoded by PIK3R1), as well as p85β (encoded by PIK3R2) and p55γ (encoded by PIK3R3). A truncated product (red arrow) was detected in extracts from individual 1 only (p.Asn636Thrfs^∗18). Both p85α and p110α directly interact and costabilize. The lower panel shows a band corresponding to p110α in LCLs from individual 1, potentially suggestive of stable interaction. (B) Phosphorylation of S6 (by S6 kinase), which is directly activated by mTOR, is a terminal readout of PI3K-AKT-mTOR-pathway activity. Titration of whole-cell extract shows that compared to control extracts, those from individual 1 exhibit reduced S6 phosphorylation on serine 240 and 244. Cells from an individual with a PIK3CA mutation and hemimegalencephaly show elevated S6 phosphorylation, as previously demonstrated. These data are suggestive of diminished signaling through this pathway in the LCLs of individual 1.