Dominant Splice Site Mutations in PIK3R1 Cause Hyper IgM Syndrome, Lymphadenopathy and Short Stature

Abstract

The purpose of this research was to use next generation sequencing to identify mutations in patients with primary immunodeficiency diseases whose pathogenic gene mutations had not been identified. Remarkably, four unrelated patients were found by next generation sequencing to have the same heterozygous mutation in an essential donor splice site of PIK3R1 (NM_181523.2:c.1425 + 1G > A) found in three prior reports. All four had the Hyper IgM syndrome, lymphadenopathy and short stature, and one also had SHORT syndrome. They were investigated with in vitro immune studies, RT-PCR, and immunoblotting studies of the mutation's effect on mTOR pathway signaling. All patients had very low percentages of memory B cells and class-switched memory B cells and reduced numbers of naïve CD4+ and CD8+ T cells. RT-PCR confirmed the presence of both an abnormal 273 base-pair (bp) size and a normal 399 bp size band in the patient and only the normal band was present in the parents. Following anti-CD40 stimulation, patient's EBV-B cells displayed higher levels of S6 phosphorylation (mTOR complex 1 dependent event), Akt phosphorylation at serine 473 (mTOR complex 2 dependent event), and Akt phosphorylation at threonine 308 (PI3K/PDK1 dependent event) than controls, suggesting elevated mTOR signaling downstream of CD40. These observations suggest that amino acids 435-474 in PIK3R1 are important for its stability and also its ability to restrain PI3K activity. Deletion of Exon 11 leads to constitutive activation of PI3K signaling. This is the first report of this mutation and immunologic abnormalities in SHORT syndrome.

Keywords: Hyper IgM syndrome; PIK3R1 splice site mutations; SHORT syndrome; lymphadenopathy; mTOR pathway; next generation sequencing; short stature.

Fig. 1

Histopathology of cervical lymph node biopsy of Patient 1. a A low magnification demonstrates reactive follicular hyperplasia with expanded germinal centers and attenuated mantle zones, as indicated by the arrows. Note the polarity of well-defined germinal centers. H&E stain, ×40. b Immunohistochemical stain for CD20 highlights well defined germinal centers with diminished mantle zones or absence of mantle zones, as indicated by the arrows. Note an apparently naked germinal center without mantle zone in upper left, as indicated by the arrowhead, and increased B-cells in the interfollicular area. Anti-CD20 stain, ×40

Fig. 2

a–c Aberrant splicing in PIK3R1 mRNA in patient 1. a Detection of mutant form of PI3K1R mRNA by RT-PCR. b Sequence of normal and mutant allele cDNA. c Chromatographs showing aberrant exon 10 to 12 splicing in patient 1′s mutant PIK3R1 allele. d Detection of p85α (PIK3RI) protein in lysates from EBV-immortalized B cells from patient 1 and his parents. In the patient sample, the intensity of the band corresponding to p85α is decreased and there is a weak band (blue arrow) that could be vaguely seen right beneath the p85α band, which is likely the exon 11 deletion mutant of p85α. The band indicated with a red arrow is only seen in patient, which is likely a degraded product of the mutant p85α. The identities of the other two bands indicated by *, whether they are non-specific proteins detected by the anti-p85α antibody or degradation products of p85α, are unknown at present. e Assessment of S6 and Akt phosphorylation in EBV-B cells following anti-CD40 stimulation. EBV-B cells from patient 1 and her parents were rested in PBS at 37 °C for 30′ and then stimulated with an anti-CD40 antibody (10 μg/ml) for 10 or 30 min. Cell lysates were subjected to immunoblotting analysis with the indicated antibodies

Fig. 3

a Assessment of S6 phosphorylation in EBV-B cells following anti-CD40 stimulation. EBV-B cells from patients 1 and 4 and their mothers were rested in PBS at 37 °C for 30′ and then either unstimulated or stimulated with insulin (1 μM) for the indicated minutes. Cell lysates were subjected to immunoblotting analysis with the indicated antibodies. b Assessment of glucose uptake before and after insulin stimulation: EBV-transformed B cells from Patients1, 2 and 4 their parents (controls) were rested in PBS for 30 min followed by culture in the presence or absence of 1–2 μM insulin for 30–60 min. Glucose uptake was determined