Association of HK1 with Glycated Hemoglobin Table 2. Multiple Linear Regression Statistics of SNPs Retained by the Forward Model Selection Algorithm. Locus 2q24.3 7p13 8q24.11 10q21.3 10q21.3 SNP rs1402837 rs730497 rs13266634 rs7072268 rs2305198 Nearest Gene Function G6PC2 GCK SLC30A8 HK1 HK1 - intron NSd intron intron MAFa 0.23 0.17 0.30 0.50 0.38 HWb 0.87 0.82 0.50 0.10 0.05 Betac 0.0236 0.0291 20.0189 0.0465 0.0409 P-Valuec 4.7E-10 5.7E-12 5.4E-08 1.8E-25 4.3E-19 aMAF: Minor Allele Frequency. bHW: Deviation from Hardy-Weinberg equilibrium P-value. cAll analyses were performed using adjusted glycated hemoglobin values (see text for details). Beta coefficients and P-values were derived form a multiple linear model that included all 5 SNPs. dNS: Non-Synonymous Coding SNP. doi:10.1371/journal.pgen.1000312.t002 Figure 2. Quantile-Quantile plot of Glycated Hemoglobin Association P-Values. The quantile-quantile plot of glycated hemoglobin association P-values is shown on the left. On the right, the same quantile-quantile plot is shown, but after adjusting glycated hemoglobin values for the additive genetic effect of the 5 SNPs retained by the model selection algorithm (rs1402837, rs730497, rs13266634, rs7072268 and rs2305198; see text for details). doi:10.1371/journal.pgen.1000312.g002 Discussion Four loci – GCK, SLC30A8, HK1 and G6PC2 – have been identified in this report as being significantly associated with glycated hemoglobin levels in non-diabetic individuals. While genetic variants of GCK [2–4] and G6PC2 [5,6] are known to influence fasting glucose concentrations in non-diabetics, the association with the diabetes gene SLC30A8 [7] has not been previously identified. In contrast, genetic associations with glycated hemoglobin have previously been described with the candidate genes calpain-10 (CAPN10) [35], resistin (RETN) and adiponectin (ADIPOQ) loci [36], although none of these associations were replicated in our data. The most striking association observed in the current report involves HK1, which is a novel finding. Four different hexokinase isozymes, named type I, II, III and glucokinase (GCK), are present in mammalian tissues. Hexokinase type I (HK1) is the predom- inant isoform found in erythrocytes, although it is also expressed in other human tissues that depend strongly on glucose utilization for their physiologic functioning, such as brain and muscle [37,38]. Despite this wide tissue distribution, the main clinical manifesta- tion of rare HK1 mutations is severe nonspherocytic hemolytic anemia [38]. To our knowledge, no common genetic variation has previously been correlated with any phenotype. The reason why erythrocytes are particularly susceptible to these rare mutations is not clear, but the prevailing assumption is that tissues strongly dependent on HK1, such as erythrocytes, might be preferably affected. One hypothesis to explain the strong association of rs7072268 and rs2305198 with glycated hemoglobin levels is that these SNPs affect hexokinase concentration or activity in Table 3. Haplotype Analysis of rs7072268 and rs2305198 (HK1 locus) in Relation to Adjusted Glycated Hemoglobin. Haplotype rs7072268 A G A G rs2305198 G G A A Frequency 0.02 0.36 0.48 0.14 Beta (%HbA1c) 0.033 0.006 0.017 20.049 P-Value 0.012 0.095 2.2E-07 1.5E-26 The omnibus (3 df) p-value from the joint haplotype analysis is 2.1E-25. doi:10.1371/journal.pgen.1000312.t003 PLoS Genetics | www.plosgenetics.org 5 December 2008 | Volume 4 | Issue 12 | e1000312