Chromogranins from Cell Biology to Physiology and Biomedicine

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clusters, which define a deep division in the human lineage. Resequencing also
identified four different naturally occurring SNPs in the CHGA 3 ′-UTR (untrans-
lated region): C+87T, (C11825T; rs7610); C+96T (C11834T; rs9658672),
C+274T (C12012T; rs9658673), and G+864C (G12602C) (Fig.  2 ).

3 Function of SNPs in the Regulatory Region of CHGA

3.1 Regulation of CHGA Expression by Promoter SNPs

The functional significance of CHGA promoter variants was verified at UCSD by
examining the influence of individual SNPs on the expression of CHGA gene
products in vivo. Testing associations of SNPs across CHGA with four plasma
CgA peptide fragments revealed significant association with plasma CgA for only
three SNPs (T-1014C, T-988G, and G-462A), which showed absolute linkage
disequilibrium (LD) with each other in the promoter region (Fig. 3b). The pro-
moter haplotype- specific reporter constructs were made by placing eight inferred
promoter haplotypes upstream of a luciferase reporter and evaluated CHGA
expression in PC12 cells (Wen et  al. 2004 ). The lowest CHGA expression was
detected in two common haplotypes containing minor alleles at the four com-
plete-LD sites (Fig. 3c). In addition, CHGA expression was different in haplo-
types 3 (GACGATAC) and 6 (GACGATCC), which differ in sequence only at
−89 bp position (bold letter). Based on these findings, it was suspected that SNP
C-89A and at least one of the three SNPs in absolute LD would play a prominent
role in expression of CHGA. To validate this, each of the three SNPs in absolute-
LD was mutated in the promoter reporter constructs for high-expressing haplo-
type 1 (GATTGTCC) and low-expressing haplotype 6 (GACGATCC). While
mutation of G-462 to A-462  in haplotype 1 caused significant decrease in pro-
moter activity, mutation of A-462 to G-462 resulted in significant increase in
CHGA expression (Wen et al. 2004 ), implicating a prominent role for this SNP in
regulation of CHGA expression (Fig. 3d).

Cap

site

C-57T

C-89T

T-415C

G-462A

T-988G Start

T-1014C

A-1018T

G-1106A

C+87T

Stop

C+274T

G+864C

E-1 E-2 E-3 E-4 E-5 E-6 E-7 E-8

// // // // // // //

100 bp

C+96T

Promoter

5’-UTR

3’-UTR

3’-UTR and its downstream region

Exon

Intron

Fig. 2 Schematic diagram showing promoter and 3’UTR SNPs in CHGA gene

Chromogranin A SNPs and Disease Association