Using 2 complementary techniques, transmission disequilibrium and SNP linkage, we show that SNP variations adjacent to and within the human gene C21orf91 yield the best fine-mapping linkage and TDT association results for susceptibility or resistance to labial herpes outbreaks. None of the 39 SNPs in the other 5 genes investigated appeared to be related to the cold-sore phenotype by both methods.
These results are somewhat surprising because the coxsackie and adenovirus receptor gene (CXADR), a well-known viral receptor, also lies within the chromosome 21 region studied. SNPs within the other genes in the chromosome 21 region, USP25, C21orf34, CXADR, and BTG3, were not linked with the cold-sore frequency phenotype nor were they significantly associated by ParenTDT. Although 2 SNPs within CHODL had LOD scores >1, the scores were not confirmed by ParenTDT.
For all SNPs with identified genotypes in the region, the total LOD score was lower in the full set of 43 families compared with that of the original families (data not shown). An examination of the LOD scores at each SNP by individual family (data not shown) revealed that this decrease was because 28% of the added families showed positive linkage to the chromosome 21 region, 44% were linkage neutral, and 28% were negative or unlinked. The additional families provided some support for the original locus but also demonstrated that cold-sore frequency in some of the families was not attributable to SNPs in susceptibility genes within the chromosome 21q region. It is possible, however, that genes outside the 21q region regulate the function or expression of 21q genes in these families.
Within the nonrecombinant region of chromosome 21 under investigation, a maximum LOD of 2.26 for short tandem repeat (STR) marker abmc65 (D21S409) was found under the dominant model within the original families [2
]. This marker maps the region between C21orf91 and CHODL
. Therefore, in this study the positive SNP LOD scores in and near these genes correlate well with the previous findings. The D21S409 marker is a 10-allele system, whereas all the SNPs genotyped here are 2-allele systems. This fact likely accounts for the higher STR marker LOD scores compared with the SNP LOD scores observed in this region.
The highest 2-point LOD scores were consistently found for SNP rs1047987 under the dominant model tested with the original families, the expanded 43-family data set, and the 25-family ParenTDT data set. This SNP encodes a nonsynonymous change of residue 136 from aspartic acid to glutamic acid (D136E). Positive LOD scores were also observed with the SNP rs243588 in all 3 data sets. This SNP lies 3′ (downstream) from the end of the predicted transcript and shows some linkage disequilibrium (r2 = 0.71) with the D136E SNP. SNP rs243588 is found in both haplotypes that may protect against frequent cold sores, H2 and H3 (see ). An additional C21orf91 SNP, rs1062202, was identified by ParenTDT analysis. This 3′-UTR SNP was found in the haplotypes that may increase susceptibility to frequent cold sores, H4 and H5. SNPs in 3′ UTR of other genes have been shown to alter messenger RNA (mRNA) stability or protein translation. The specific effects of these C21orf91 genotype changes on mRNA and protein expression in different tissues are unknown at this time.
Because of the limited number of SNPs available for genotyping, it is likely that additional SNPs are present within the main candidate gene, C21orf91. Deletions, frame-shifts, and insertions may also be present, genetic features not detectable in this study. The possibility that the SNPs from C21orf91 might be in linkage disequilibrium (LD) with neighbouring genes was addressed and no significant LD was detected. Because the LD data does not include rare variants (SNP frequency <1%), it is unlikely but still possible that there are rare variants of larger effect in the neighbouring genes that the C21orf91 SNPs could be tagging. Another limitation is the size and familial structure of the population being studied. The conclusions stated here are based on ParenTDT testing from 75 individuals plus linkage on 355 individuals. Despite the limited number of SNPs and individuals analyzed, positive linkage signals and a significant association (P < .005) by ParenTDT analysis were seen with SNPs mapping in or near C21orf91. Although these results do not definitively rule out a role for other genes in this region, they support a potential role for C21orf91 in modulation of the cold-sore phenotype.
The results of this study indicate that the C21orf91 gene may play a role in the frequency of labial herpes outbreaks. This gene has an open reading frame of 293 amino acids, a 3′ UTR of about 4400 nucleotides, and encodes a protein of unknown function. The C21orf91 protein is the sole member of the “Early Undifferentiated Retina and Lens” proteins [30
]. It is expressed in chick retinal precursor cells as well as in the anterior epithelial cells of the lens during early development. We demonstrated that the protein was localized to the cytoplasm. Additional studies are needed to determine how C21orf91 affects HSV-1 pathogenesis.
In summary, this study reports a new association between a previously obscure human gene, C21orf91, and frequent cold sores. The authors propose that C21orf91 be designated the Cold Sore Susceptibility Gene 1 (CSSG1). Although these findings await confirmation in a larger, unrelated population, they could have important implications for the development of new drugs that affect determinants of the cold-sore phenotype.