Chromogranin B P413L variant as risk factor and modifier of disease onset for amyotrophic lateral sclerosis

Abstract

Recently, chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). This interaction led us to analyze the frequencies of sequence variants of the CHGB gene in ALS patients and matched controls from three different countries. Of particular interest was the finding of the P413L CHGB variant present in 10% of ALS patients (n = 705) as compared to 4.5% in controls (n = 751), conferring a 2.2-fold greater relative risk to develop the disease (P < 0.0001). This effect was mainly contributed by the samples of French origin that yielded a frequency of the P413L variation at 17% in ALS (n = 289) and 5% in controls (n = 448), conferring a 3.3-fold greater risk to develop ALS. Furthermore, the P413L CHGB variant is associated with an earlier age of onset by almost a decade in both sporadic ALS and familial ALS cases. Genetic variation influencing age of onset in ALS had not previously been reported. Expression of fusion CHGB-EGFP constructs in SHSY-5Y cells revealed that the P413L variation can cause defective sorting of CHGB into secretory granules. The finding that CHGB may act as a susceptibility gene and modifier of onset in ALS is consistent with the emerging view that dysfunction of the secretory pathway may contribute to increased vulnerability of motor neurons.

Fig. 1.

Onset probability of ALS patients among carriers and noncarriers of the CHGB P413L variant. (A) The presence of the CHGB P413L variant among SALS patients (Median onset, 55-years) significantly reduced the age of onset (P = 0.0001) as compared to non-P413L SALS carriers (Median onset, 62-years). (B) The presence of the CHGB P413L variant among FALS carriers (Median onset, 43 years) significantly reduced the age of onset (P = 0.01) as compared to non-P413L FALS carriers (Median onset, 54.5 years). (C) Mean age of onset also significantly differ amongst P413L carriers vs. noncarriers among ALS patients. SALS patients carrying the P413L CHGB variant showed a mean age of onset of 53.3 ± 1.8 years as compared to 59.7 ± 0.7 years for noncarrier SALS patients (P = 0.008). Furthermore, FALS patients carrying the P413L CHGB variant also demonstrated a lower age of onset (49.8 ± 2.8 years) as compared noncarrier FALS patients (57.1 ± 1.9 years) (P = 0.05). These results strongly suggest that CHGB may be a modifier of the disease. The “n” number between brackets represents the number of patients for which we had clinical information.

Fig. 2.

Defective sorting and maturation of CHGB variants. (A) Confocal laser microscopy of human neuroblastoma SHSY-5Y cells that were cotransfected with EGFP-tagged human WT CHGB, H230R, or P413L CHGB variants (green) and DsRed-fused Golgi marker (red). After 48 h, the cells were fixed with 4% PFA and counterstained with DAPI (blue). (Scale bar, 10 μm.) (B) Quantification data of CHGB vesicles colocalizing with the DsRed-Golgi marker. The merged and unmerged CHGB vesicles with DsRed-Golgi marker were counted, and the percentage of CHGB in the Golgi apparatus calculated (P = 0.009). Note that six different cells have been counted for each conditions.