Association of Rare Loss-Of-Function Alleles in HAL, Serum HistidineCLINICAL PERSPECTIVE
Levels and Incident Coronary Heart Disease
Background—Histidine is a semiessential amino acid with antioxidant and anti-inflammatory properties. Few data are available on the associations between genetic variants, histidine levels, and incident coronary heart disease (CHD) in a population-based sample.
Methods and Results—By conducting whole exome sequencing on 1152 African Americans in the Atherosclerosis Risk in Communities (ARIC) study and focusing on loss-of-function (LoF) variants, we identified 3 novel rare LoF variants in HAL, a gene that encodes histidine ammonia-lyase in the first step of histidine catabolism. These LoF variants had large effects on blood histidine levels (β=0.26; P=1.2×10−13). The positive association with histidine levels was replicated by genotyping an independent sample of 718 ARIC African Americans (minor allele frequency=1%; P=1.2×10−4). In addition, high blood histidine levels were associated with reduced risk of developing incident CHD with an average of 21.5 years of follow-up among African Americans (hazard ratio=0.18; P=1.9×10−4). This finding was validated in an independent sample of European Americans from the Framingham Heart Study (FHS) Offspring Cohort. However, LoF variants in HAL were not directly significantly associated with incident CHD after meta-analyzing results from the CHARGE Consortium.
Conclusions—Three LoF mutations in HAL were associated with increased histidine levels, which in turn were shown to be inversely related to the risk of CHD among both African Americans and European Americans. Future investigations on the association between HAL gene variation and CHD are warranted.
The ability to identify novel genetic variants influencing health and disease is a function of sample size, effect size, and frequency. Effect size can be maximized by focusing on variants that obliterate gene/protein function and by analyzing intermediate analytes that are closer to the gene level. Loss-of-function (LoF) variants have been reported to have a strong effect on a spectrum of phenotypes from rare Mendelian disease1 to associations with common complex traits. For example, LoF variants in PCSK9 have a significant effect on low-density lipoprotein-cholesterol levels and incident coronary heart disease (CHD).2–4
Histidine, an essential amino acid in humans,5 is a powerful antioxidant and anti-inflammatory factor.6 Blood levels of histidine are tightly regulated and are controlled by dietary intake, enzymatic catabolism, and urinary excretion. Histidinemia, characterized by elevated blood levels of histidine, is an autosomal recessive disorder caused by missense variation in the histidine ammonia-lyase (HAL) gene (MIM: 609457). In population studies, low levels of histidine have been associated with inflammation and oxidative stress in obese women and in patients with chronic kidney disease.7,8 Although histidine has a potential effect on human health, it is unknown whether the genetic variants are associated with histidine levels in the general population. In addition, no study has been conducted to examine the association between histidine levels and the onset of cardiovascular disease (CVD). The goal of this study was to identify LoF variants influencing histidine levels using whole exome sequencing, and then, subsequently testing the associations among the identified LoF variants, histidine levels, and incident CHD.
Clinical Perspective on p 355
Study Population and Phenotype Measurements
The Atherosclerosis Risk in Communities (ARIC) study is a longitudinal cohort study designed to ascertain the cause and predictors of CVD. The ARIC study enrolled 15 792 middle-aged adults from 4 US communities (Forsyth County, NC; Jackson, MS; suburbs of Minneapolis, MN; and Washington County, MD) between 1987 and 1989 (baseline) and followed by 4 completed visits in 1990 to 1992, 1993 to 1995, 1996 to 1998, and 2011 to 2013. A detailed description of the ARIC study design and methods was published elsewhere.9 The study was approved by the institutional review committee, and the participants gave the informed consents. Histidine levels were measured by Metabolon Inc. (Durham) using liquid chromatography-mass spectrometry in baseline serum from African Americans selected from the Jackson, MS field center. Incident CHD was defined as a definite or probable myocardial infarction, definite coronary death, or coronary revascularization procedure. Individuals were followed up for CHD events from enrollment (baseline) until death or December 31, 2010, and those who were lost to follow-up were censored at the date of last contact.
The Framingham Heart Study (FHS) Offspring Cohort was initiated in 1971, and a sample of 5124 men and women, consisting of the offspring of the Original Cohort and their spouses, were recruited. A detailed description of the study design was published elsewhere.10 Histidine levels were measured at Examination 5, and the genotypes were measured by Illumina Omni 5M platform. The FHS data were downloaded from dbGaP Framingham Cohort (study accession: phs000007.v21.p8) and then analyzed locally.
The Cardiovascular Health Study (CHS) is a prospective observational cohort study designed to investigate CVD in older adults, which enrolled 5201 individuals in the original cohort at 1989 to 1990, with further enrollment of a minority sample of African Americans in 1992 to 1993. The Rotterdam Study (RS) is a prospective cohort study started in 1990 at Ommoord, a suburb of Rotterdam, to investigate the prevalence, incidence, and risk factors for chronic diseases in the elderly among 10 994, participants aged ≥55 years. The Jackson Heart Study (JHS) participants were recruited in 2000 to 2004, and the study was designed to monitor the cause and progression of CVD among noninstitutionalized African Americans aged 35 to 84 years living in the 3 counties (Hinds, Madison, and Rankin) that comprise the Jackson, MS metropolitan area. A detailed description of study designs for CHS, RS, and JHS has been published elsewhere.11–15 The definition of incident CHD in CHS was incident fatal and nonfatal myocardial infarction, fatal CHD (possible and definite), or sudden death within 1 hour of onset of symptoms, while RS and JHS used the same definition as ARIC.
Whole Exome Sequencing and ExomeChip Genotyping
The whole exome was captured using Nimblegen’s VChrome2.1 (Roche NimbleGen, Madison, WI), and the captured exons were sequenced on Illumina HiSeq 2000 (Illumina, San Diego, CA). Sequence reads were aligned to the hg19 reference genome using Burrows–Wheeler Aligner.16 Single nucleotide variants were called using AtlasSNP,17 and variants were annotated using Cassandra.18 Variants were excluded if they had posterior probability <0.95, had variant read ratio <0.1, or had total coverage <6. An average of 92% of the targeted bases was covered to a depth of ≥20× in each individual.
ARIC, CHS, RS, and JHS participants were genotyped by the Illumina Human Exome BeadChip (exome chip) v.1.0 and were jointly called as described previously.19 Individuals were excluded if they had missing genotypes >5%, population structure outliers, sex mismatch, high inbreeding coefficients, and evidence of relatedness.
Whole exome sequence analyses were performed in ARIC study participants focusing on LoF variants (defined as stop-gain or stop-gain/splice by University of California Santa Cruz genes annotation) with a minor allele frequency ≤5%. A single summary T5 test20 and a Sequence Kernel Association Test (SKAT)21 were conducted to evaluate the joint effects of mutated alleles in a gene on natural log-transformed histidine levels by comparing these mutation carriers and noncarriers after adjusting for age, sex, and population structure. Statistical significance was defined as a P<3.4×10−5 with Bonferroni correction of 1470 tests (735 genes×2 tests). The genotype–phenotype association was first validated in an independent sample of ARIC African Americans with exome chip genotypes and then replicated in FHS Offspring Cohort. Linear regression was applied for the replication analyses, and a kinship matrix was used in FHS Offspring Cohort to account for the family structure.
The association between histidine levels with incident CHD was evaluated in ARIC and FHS Offspring Cohort using Cox proportional hazards models adjusting for age, sex, body mass index, current smoking, systolic blood pressure, antihypertensive medication use, diabetes mellitus, high-density lipoprotein, total cholesterol, and triglycerides. Histidine levels were natural log-transformed before the analyses. In FHS Offspring Cohort, the family structure was taken into account using Cox proportional hazards model with a frailty term clustering on family.22
Cox proportional hazards models were applied in ARIC, CHS, RS, and JHS, respectively, to evaluate whether the mutated LoF alleles in HAL, genotyped by the exome chip, were associated with the incidence rate of CHD adjusting for age and sex. An indicator variable indicating the presence of ≥1 LoF allele in HAL was generated in each sample for the analysis. Inverse variance fixed-effect meta-analyses were used to combine the results within each race strata and also to meta-analyze the race-specific results to obtain an overall disease risk estimate, standard error, and P value. Among the sample of African Americans, ≈80% power would be reached at α=0.05 if the hazard ratio (HR) lies outside the range of (0.50, 2.00), with a 1.1% risk-changing allele frequency. All the analyses were performed by R (www.r-project.org).
A total of 1152 ARIC African Americans (demographics in Table I in the Data Supplement) with both histidine measurements and whole exome sequence data were included, and a set of 735 genes with ≥5 minor allele copies of rare or low frequency LoF variants presented in the study sample were tested. On average, each individual possessed 47 LoF nucleotide variants with a standard deviation at 6.8 (Figure I in the Data Supplement).
Two gene-based analyses, T5 and SKAT, were conducted across the exome, and the results were well calibrated (Figure II in the Data Supplement). The gene, HAL, with 3 LoF variants, was strongly associated with histidine levels in both tests (both P=1.2×10−13, β=0.26 for T5 test; Table), where each mutated allele was associated with a 29.7% increase in histidine’s geometric mean and explained 4.8% of its variance. All 3 LoF variants had an elevating effect on histidine levels (Figure 1); 2 of them were singleton splice variants (rs141634423 and rs76945459; Table), and the other was a stop-gain variant (rs34457757, R322X; Table). There were 24 mutation carriers (all heterozygotes), and 22 of them carried the stop-gain mutation (R322X). R322X was present on the exome chip, and the concordance of this variant between exome sequence and exome chip genotypes was 100%. The association between R322X and histidine levels was replicated in an independent sample of 718 ARIC study participants with both exome chip data and blood histidine levels (P=1.2×10−4, demographics in Table I in the Data Supplement).
We next examined the association between histidine levels and incident CHD among 1812 ARIC African Americans (demographics in Table II in the Data Supplement, and histidine distribution in Figure III in the Data Supplement). A total of 175 incident CHD events occurred during a median follow-up of 21.5 years with a range from 0.04 to 24.1 years. High histidine levels were related to reduced risk of incident CHD after adjusting for traditional risk factors (HR=0.18; 95% confidence interval=0.07–0.44; P=1.9×10−4; Figure 2).
These primary findings were replicated in a sample of European Americans from FHS Offspring Cohort: R322X variant was associated with increased histidine levels (β=0.34; P=0.05; minor allele frequency=0.09%). Likewise, elevated histidine levels were associated with decreased risk of incident CHD (HR=0.25; 95% confidence interval=0.08–0.74; P=0.01; Figure 2) after accounting for established risk factors and family structure among 753 European Americans, with an average of 15.5 years follow-up (number of events=118; demographics in Table II in the Data Supplement, and histidine distribution in Figure III in the Data Supplement).
Given that histidine is a potential predictor of CHD, we next tested the direct association between LoF variants in HAL with incident CHD in 6039 African American individuals (demographics in Table III in the Data Supplement) and 16 647 European American individuals (demographics in Table IV in the Data Supplement) from 4 large cohorts, including ARIC, CHS, RS, and JHS. For incident CHD cases, the average follow-up time was from 3.5 to 11.7 years among African Americans and from 8.7 to 12.0 years among European Americans. The cumulative minor allele frequency for LoF variants in HAL was higher in African Americans compared with that in European Americans (1.10% versus 0.06%, detailed variants information in Table V in the Data Supplement). Carriers of LoF mutations in HAL showed lower risk of CHD in each race group compared with that in noncarriers (except European Americans from CHS), although not statistically significant (HR=0.79; P=0.35; Figure 3).
We report the occurrence of LoF variants in HAL resulting in elevated histidine levels, especially among African Americans with the R322X LoF allele. We show that elevated histidine levels were associated with reduced risk for incident CHD among African Americans and European Americans. Although the direction of the effect was consistent, the presence of LoF variants in HAL was not statistically significantly associated with incident CHD in 22 686 individuals of African and European descent.
Histidine is an essential amino acid in humans and other mammals. It is a precursor for histamine and carnosine biosynthesis. HAL is an enzyme, encoded by the HAL gene, catalyzing the first reaction in histidine catabolism. A rare autosomal recessive metabolic disorder, histidinemia, is caused by missense mutations in HAL.23 Histidinemia is generally believed to lead to mental impairment and speech difficulties, and its incidence is ≈1:20 000.24 Neonatal screening suggested infants demonstrating a blood histidine level of 6 mg/dL or more as suspected histidinemia cases, and the excretion of histidine by affected children was ≈15-fold greater than the excretion by normal children.25 We report that 3 novel LoF variants in HAL, each with 1 copy of mutated allele, are associated with elevated histidine in the general population with an effect of 29.7% increase in the geometric mean. The mutated LoF allele is expected to have an additive effect on histidine levels, but no homozygotes were observed in our study sample. Therefore, lack of homozygotes in this cohort study from the general population may be because of its low minor allele count or an association with serious disabilities precluding study participation.
Histidine is considered an antioxidant and anti-inflammatory factor.6 Studies have shown that people under oxidative stress or having inflammatory diseases, such as chronic kidney disease or rheumatoid arthritis, tend to have low histidine levels.7,26 Inflammation plays a critical role in atherosclerosis and CVD,27 and proinflammatory cytokines are related to the early stages of atherosclerosis, a primary cause of CHD.28 In the present study, we show that high histidine levels were associated with reduced risk of developing CHD in 2 independent populations. Histidine suppresses proinflammatory cytokine expression, possibly via the nuclear factor-κB pathway, in adipocytes.29 In addition, the efficacy of histidine in protecting inflamed tissue is attributed to the capacity of scavenging reactive oxygen species generated by cells during acute inflammatory response.6 These studies suggest that histidine may have a protective effect on incident CHD via reducing oxidative stress or suppression of inflammation.
Although LoF variants in HAL showed a large effect on histidine levels and histidine levels were strongly related to the occurrence of CHD, we were not able to establish a direct path between HAL gene variation and incident CHD, which may be because of insufficient statistical power. We estimate that ≈80% power would be achieved if the HR was <0.5 or >2.0. The HR after meta-analysis in this sample was 0.79, which was inside this range. Given the fact that a causal association was not established, the immediate clinical implication of HAL gene variation on CHD is limited. Nevertheless, we show that low histidine levels were associated with high CHD risk in 2 independent populations, implying that interventions that raise blood histidine levels may prevent CHD.
In summary, we used whole exome sequence data to identify 3 LoF mutations in HAL that were associated with blood histidine levels, which in turn were shown to be inversely related to the risk of CHD among both African Americans and European Americans. These data provide new insight into the cause of CHD and possible prevention.
We acknowledge the essential role of the CHARGE Consortium in developing and support for this article. The authors also thank the staff and participants of the ARIC, CHS, RS, and JHS studies for their important contributions.
Sources of Funding
The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C), R01HL087641, R01HL59367 and R01HL086694; National Human Genome Research Institute contract U01HG004402; and National Institutes of Health contract HHSN268200625226C. The histidine measurement work obtained through support from the National Genome Research Institute (HG004402). The DNA sequence data work obtained through support from the National Heart Lung and Blood Institute (HL102419) and National Human Genome Research Institute (HG003273 and HG006542) of the National Institute of Health. The Framingham Heart Study is conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with Boston University (Contract No. N01-HC-25195). This manuscript was not prepared in collaboration with investigators of the Framingham Heart Study and does not necessarily reflect the opinions or views of the Framingham Heart Study, Boston University, or NHLBI. SHARe Illumina genotyping was provided under an agreement between Illumina and Boston University. Funding support for the Framingham Metabolomics (HILIC-Installment 1) data set was provided by National Institutes of Health grant R01DK081572. The Cardiovascular Health Study (CHS) research was supported by NHLBI contracts HHSN268201200036C, HHSN268200800007C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086; and NHLBI grants HL080295, HL087652, HL105756, HL103612, HL068986, HL120393 with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided through AG023629 from the National Institute on Aging (NIA). A full list of principal CHS investigators and institutions can be found at CHS-NHLBI.org/. The provision of genotyping data was supported, in part, by the National Center for Advancing Translational Sciences, CTSI grant UL1TR000124, and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. The generation and management of the Illumina exome chip v1.0 array data for the Rotterdam Study (RS-I) was executed by the Human Genotyping Facility of the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands. The Exome chip array data set was funded by the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, from the Netherlands Genomics Initiative (NGI)/Netherlands Organisation for Scientific Research (NWO)-sponsored Netherlands Consortium for Healthy Aging (NCHA; project nr. 050-060-810); the Netherlands Organization for Scientific Research (NWO; project number 184021007) and by the Rainbow Project (RP10; Netherlands Exome Chip Project) of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL; www.bbmri.nl). We thank Ms. Mila Jhamai, Ms. Sarah Higgins, and Mr. Marijn Verkerk for their help in creating the exome chip database, and Carolina Medina-Gomez, Lennard Karsten, and Dr. Linda Broer for QC and variant calling. The Jackson Heart Study is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. Dr Wei was partially supported by National Institutes of Health grant R01HL116720. Dr Dehghan is supported by NWO grant (veni, 916.12.154) and the EUR Fellowship.
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.114.000697/-/DC1.
- Received May 13, 2014.
- Accepted December 2, 2014.
- © 2015 American Heart Association, Inc.
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Histidine is a semiessential amino acid with antioxidant and anti-inflammatory properties, and thus, it may influence coronary heart disease risk. By conducting whole exome sequencing in a large sample of African Americans, 3 novel rare loss-of-function variants in HAL, a gene which encodes histidine ammonia-lyase that catalyzes the first step in histidine catabolism, were related to increased level of serum histidine. Elevated histidine levels were associated with reduced risk of developing incident coronary heart disease after 21.5 years of follow-up. These findings were replicated in an independent sample of European Americans. Loss-of-function variants in HAL were not significantly associated with incident coronary heart disease. These results contribute to the knowledge base of histidine regulation and suggest that histidine has a generalized protective effect on incident coronary heart disease.