Purinergic receptors P2RX4 and P2RX7 in familial multiple sclerosis

Abstract

Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study, we set out to evaluate whether rare coding variants of major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T [p.T205M], P2RX7 rs201921967:A>G [p.N361S], and P2RX4 rs765866317:G>A [p.G135S]) segregating with disease in a multi-incident family with six family members diagnosed with MS (logarithm of odds = 3.07). Functional analysis of this haplotype in HEK293 cells revealed impaired P2X7 surface expression (P < 0.01), resulting in over 95% inhibition of adenosine triphosphate (ATP)-induced pore function (P < 0.001) and a marked reduction in phagocytic ability (P < 0.05). In addition, transfected cells showed 40% increased peak ATP-induced inward current (P < 0.01), and a greater Ca²⁺ response to the P2X4 135S variant compared with wild type (P < 0.0001). Our study nominates rare genetic variants in P2RX4 and P2RX7 as major genetic contributors to disease, further supporting a role for these purinergic receptors in MS and the disruption of transmembrane cation channels leading to impairment of phagocytosis as the pathological mechanisms of disease.

Keywords: P2RX4, P2RX7, P2X4, P2X7, variant; familial, Mendelian, multiple sclerosis; mutation.

Figure 1. Pedigree presenting the P2X7 p.T205M, P2X7 p.N361S and P2X4 p.G135S haplotype and protein conservation

A) Males are represented by squares and females by circles with the proband indicated win an arrow head. Patients diagnosed with MS have black filled symbols with corresponding age at onset of disease when available. Heterozygote carriers for all three mutations (M) and wild type (wt) haplotypes are indicated. B) Conservation of P2X7 and P2X4 variants in orthologs and human paralogs are highlighted in black. Protein homologs were aligned via ClustalO, and RefSeq accession numbers are provided.

Figure 2. P2X7 functional assay

A) ATP induced ethidium uptake was used to measure P2X7 pore formation in HEK293 cells transfected with various P2X7 and/or P2X4 constructs. The mean fluorescent intensity of ethidium uptake into cells was measured at 5 sec intervals by two-color flow-cytometry. B) P2X7 expression on the surface of HEK 293 cells transfected with AcGFP-tagged P2X7 and/or P2X4 constructs. Bars represent mean values and SEM of three replicate experiments. WT = wild type. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. P2X4 functional assay using HEK293 cells transfected with AcGFP, wild type P2X4-AcGFP or P2X4-G135S-AcGFP

A) Whole-cell averaged current traces from mock vector control alone (n = 6 cells), P2X4-WT (n = 6 cells) and P2X4-G135S (n = 10 cells) in response to ATP (100 μM). B) Typical Ca²⁺ influx traces. Ca²⁺ (3 mM) was added 40 sec prior to the addition of 100 μM ATPγS. Normalized Ca²⁺ influx from four separated experiments. *p<0.01; **p<0.0001.