Both rare and common polymorphisms contribute functional variation at CHGA, a regulator of catecholamine physiology

Abstract

The chromogranin/secretogranin proteins are costored and coreleased with catecholamines from secretory vesicles in chromaffin cells and noradrenergic neurons. Chromogranin A (CHGA) regulates catecholamine storage and release through intracellular (vesiculogenic) and extracellular (catecholamine release-inhibitory) mechanisms. CHGA is a candidate gene for autonomic dysfunction syndromes, including intermediate phenotypes that contribute to human hypertension. Here, we show a surprising pattern of CHGA variants that alter the expression and function of this gene, both in vivo and in vitro. Functional variants include both common alleles that quantitatively alter gene expression and rare alleles that qualitatively change the encoded product to alter the signaling potency of CHGA-derived catecholamine release-inhibitory catestatin peptides.

Figure 1

Resequencing strategy and identified variants. Sequences conserved between mouse and human CHGA were visualized with VISTA (Mayor et al. 2000). Location of common (upper) and rare (lower) SNPs relative to exons and conserved noncoding sequences is indicated by position. Red rods represent nonsynonymous SNPs, and black rods represent synonymous SNPs. Nucleotides in red in the chimpanzee haplotype indicate the minor allele in the human sequence. Computationally reconstructed haplotypes are indicated, along with their relative frequencies in ethnogeographic groups within our sample population. Nucleotide deletions in haplotype sequences are indicated by an asterisk (*).

Figure 2

Functional variation in the CHGA promoter. A, Promoter haplotypes, each represented by a circle whose area represents the overall frequency of that haplotype in the sample. Each haplotype number corresponds to haplotype numbers in table 2. Each circle is subdivided to show the proportion of the individual haplotype frequency found in each of the four populations as represented by the indicated colors. Dashed lines indicate alternative topologies of equal length. Lines connecting haplotypes represent one nucleotide substitution, except where noted in parentheses. B, Association of CHGA proximal promoter SNP genotype (G-988-T) with in vivo plasma CHGA peptide levels in 102 subjects. All CHGA peptides levels are expressed as mean±SEM. Significant differences could be observed in two peptide fragments (large fragment, CHGA_116–457; and pancreastatin, CHGA_284–301) between minor-allele homozygotes and the other two groups. N = number of subjects for each genotype group. The allele frequencies were 22.5% for G and 77.5% for T. The genotypes were in Hardy-Weinberg equilibrium (χ²=0.011; P=.91). C, In vitro haplotype-specific CHGA promoter activity assay. Two haplotypes (3 and 6) showed a marked decrease in promoter activity compared with the other four common promoter haplotypes, two rare haplotypes, and chimp haplotype. Haplotype numbers correspond to haplotype numbers in table 2. There are also significant differences in promoter activity between haplotypes 3 and 6. *P<.0001 between haplotype 6 and other haplotypes. **P<.001 between haplotype 3 and the other haplotypes except haplotype 6. D, In vitro mutated haplotype activity assay in the CHGA promoter. Each mutated promoter haplotype was derived from either haplotype 1 or haplotype 6 (see table 2).

Figure 3

Catestatin peptide variants altering cholinergic inhibition and predicted structure. A, Peptide sequence alignment from several species (and the corresponding catestatin region), showing the extent of sequence conservation at Gly364Ser and Pro370Leu (red, second and third rows). Note that 370Leu is found in all nonprimate species available. Phylogenetic relationships and bootstrap values are taken from a published multiple gene comparison (Murphy et al. 2001). B, Altered efficacy of nicotinic inhibition by variant peptides in dose response from 0.1 to 10 μM, showing functional significance to each change, but in opposite directions. C, Homology modeling, predicting altered three-dimensional structure of Gly364Ser (left) and Pro370Leu (right) variants. Point mutants were aligned to the wild-type backbone template and then subjected to energy-minimization/homology-modeling using SWISS-MODEL at the ExPASy Web site and Swiss-PDBviewer (“DeepView”) for visualization and manipulation (Peitsch 1995).

Figure 4

1-2-3 model for influence of CHGA polymorphisms on catecholamine storage and release. 1, Common promoter haplotypes affect the transcriptional level of the CHGA gene in chromaffin cells. 2, CHGA protein levels quantitatively affect pool size of catecholamine-chromogranin vesicles. 3, Variant catestatin peptides alter the release-dependent feedback inhibition of nicotinic-stimulated release. This feedback loop could explain the divergent effects of promoter polymorphisms on chromaffin cell CHGA expression and plasma levels.