Acta Pædiatrica ISSN 0803–5253

REGULAR ARTICLE

Lack of association of δ-aminolevulinic acid dehydratase genotypewith blood lead levels in environmentally exposed children of Uygurand Han populationsYan Chen (ychen88@hotmail.com)1, Jiang-Xia Zhao2, Ji-Wen Liu1, Jun Cui3, Ling Li3, Wei Tian1

1.Department of Toxicology, School of Public Health, Xinjiang Medical University, Urumqi, Xinjiang, China2.School of Public Health, FuDan University, Shanghai, China3.Insitute of Health Care for Children in Urumqi, Urumqi, Xinjiang, China

Keywordsδ-aminolevulinic acid dehydratase, Geneticpolymorphism, Han, Lead poisoning, Uygur

CorrespondenceYan Chen, Department of Public Health in XinjiangMedical University, 8 XinYi Road, Urumqi, XinjiangProvince 830054, China.Tel: 86-991-4362427 |Fax: 86-991-4365505 |Email: ychen88@hotmail.com

Received29 May 2008; revised 18 July 2008;accepted 29 July 2008.

DOI:10.1111/j.1651-2227.2008.01003.x

AbstractAim: A cross-section study was conducted to explore the association between polymorphism of

δ-aminolevulinic acid dehydratase (ALAD) and lead poisoning in Uygur and Han children in China.

Methods: The ALAD genotyping was determined by PCR-RFLP in 443 Uygur and 469 Han children

aged 6–10 years from Urumqi in Xinjiang province.

Results: The blood lead levels of 912 environmentally exposed children ranged from 0.5 to

48.2 μg/dL, with a mean of 5.45 μg/dL and a standard deviation of 0.22 μg/dL, and 23. Thirty-one

percent individuals were with blood lead level ≥10 μg/dL. The mean and standard deviation of

blood lead levels were 5.57 ± 0.223 μg/dL and 5.30 ± 0.224 μg/dL in Uygur and Han children,

respectively. The frequencies of the allele ALAD1 and ALAD2 in Uygur subjects were 90.52% and

9.48%, and in Han subjects were 95.73% and 4.27%, respectively (chi-square = 19.55, p < 0.05).

No statistic correlation between the distribution of ALAD alleles and the blood lead level was found in

both populations.

Conclusion: A significant difference was seen in the frequency distribution of ALAD genotype between the

different races. The genetic susceptibility of ALAD polymorphism to lead toxicity may exhibit in a lead

dose-dependent manner.

INTRODUCTIONLead has been a known toxicant for thousands of years, andit remains a persistent environmental health threat. Expo-sure to lead can result in significant adverse health effects tomultiple organ systems. Toxic effect to the nervous, hema-tologic and reproductive systems have been studied exten-sively and are well documented (1,2). Regarding the leadpoisoning for children, the U.S. Centers for Disease Con-trol and Prevention (CDC) amended the definition of thethreshold blood lead level considered to the value 10 μg/dL(CDC, 1991). Blood lead concentrations of 10 μg/dL in chil-dren have been associated with cognitive deficits, aggressivebehaviour and hearing dysfunction (3,4). Children’s hand-to-mouth activity, increased respiratory rates and increasedintestinal absorption of lead make them more susceptiblethan adults to lead exposure (5,6).

Delta-aminolevulinic acid dehydratase (ALAD) has beenwidely acknowledged to play an important role in the patho-genesis of lead poisoning (7). The effects of lead on ALADactivity have become a subject of much recent interest be-cause ALAD is involved in the synthesis of heme, and thisenzyme is strongly inhibited by lead, thus resulting in ac-cumulated aminolevulinic acid (ALA) in urine and plasma(8). The ALAD gene is located on chromosome 9q34 andis approximately 16 kilobases long (9). This gene codes forthe ALAD enzyme E. 4.2.1.24. Human ALAD is a poly-

morphic enzyme (10) that lies in a single G-to-C transver-sion of nucleotide 177 that results in the substitution of as-parginine for lysine at residue 59 (dbSNP ID: rs1800435)in the coding region of ALAD gene, thus leading to two al-leles (ALAD1 and ALAD2) and three isozyme phenotypes,designated ALAD1-1, ALAD1-2 and ALAD2-2 (11). Sev-eral studies have suggested that carries of the ALAD2 allelewould have higher blood lead concentrations than noncar-riers, thereby increasing their susceptibility to lead toxicity(12–14). Other researchers have found no association be-tween the ALAD genotype and blood levels (15,16).

The prevalence of the ALAD2 allele ranges from 0%to 20% depending on the population. Generally, Cau-casians have the highest frequency of the ALAD2 allele,with approximately 18% of the Caucasian population beingALAD1-2 heterozygotes and 1% being 2–2 homozygotes. Incomparison, African and Asian populations have low fre-quencies of the ALAD2 allele, with few or no ALAD2 ho-mozygotes being found in such populations (8).

The cross-section study was conducted to identify the as-sociation between the ALAD genotype and the blood leadlevels of environmentally exposed children in Uygur andHan populations. It was designed to determine the frequencyof ALAD genotype in Uygur and Han population and toidentify the contribution of ALAD genotypes to body bur-den, as indexed by blood lead values.

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ALAD genotype and blood lead levels Chen et al.

MATERIALS AND METHODSStudy populationA total of 443 Uygur and 469 Han children aged 6–10 years were enrolled in the study after informed consentwas obtained from April to June 2005 at Urumqi city inChina. Each subject completed an interviewer-administeredquestionnaire that contained information on age, gender,living conditions, lifestyle, health status, etc. Blood sam-ples were collected for blood lead measurement and ALADgenotyping.

Determination of lead in bloodVenous blood was taken from study subjects and drawn intoheparin-containing tubes. Blood lead level was measuredby graphite furnace atomic absorption spectrophotometry(GFAAS).

ALAD genotypingGenomic DNA was extracted from blood samples by a rou-tine phenol–chloroform method. An assay based on poly-merase chain reaction-restriction fragment length polymor-phim (PCR-RFLP) was used to determine the genotype ofthe ALAD gene. Primer sequence was designed by Oligo6.0 software (F: 5′-ATCCTGGGCTCAAG TGATCCAC-3′,R: 5′-AATTCCGGAGCTCTGCTACTCA-3′). PCR was doneusing 50–100 ng of genomic DNA, 0.2 μmol/L of eachprimer, 1×PCR buffer, 0.2 μmol/L of each deoxynucleotidetriphosphate, 2.0 mmol/L MgCl2 and 0.75 units of Taq in a25-μL reaction volume. The PCR programme was a 5-mindenaturation step at 95◦C followed by 35 cycles of 94◦C for30 sec, 62◦C for 30 sec, 72◦C for 45 sec and a final extensionstep at 72◦C for 5 min. The amplified products were digestedwith MspI at 37◦C for 3 h. The digested PCR products wereobserved under a UV image system. All genotypes were eval-uated and agreed on by at least two persons independently.Ten percent of DNA samples were selected randomly forrepeat analyses and the concordance was 100%. Subjectswith ALAD1-1 genotype showed 144 bp and 255 bp frag-ments; subjects with ALAD1-2 genotype showed fragmentsof 144 bp, 255 bp, 184 bp and 71 bp; subjects with ALAD2-2genotype showed 144 bp, 184 bp and 71 bp fragments. PCRproducts showed 399 bp.

Statistical analysisThe distribution of genotypes was assessed for deviationfrom the Hardy–Weinberg equilibrium by using chi-squaredtest. Because of the relatively low frequency of the ALAD2-2 genotypes, we combined both ALAD1-2 and ALAD2-2genotypes together (ALAD1-2/2-2 group) and compared

Table 1 ALAD polymorphism in Ugyur and Han population: genotype and allele frequencies

Genotype frequency Allele frequency

Sample No. ALAD1-1 (%) ALAD1-2 (%) ALAD2-2 (%) ALAD1 (%) ALAD2 (%)

Uygur population 443 363(81.94) 76(17.16) 4(0.90) 802(90.52) 84(9.48)Han population 469 430(91.68) 38(8.10) 1(0.22) 898(95.73) 40(4.27)

with the ALAD2-2 genotype group. Multiple regressionmodel with adjustment for potential confounding factorswas used for estimating the risk factors to blood levels ofchildren. All tests were done by using SPSS 10.0 software(SPSS, Inc., Chicago, IL, USA).

RESULTSThe blood lead levels of 912 environmentally exposed chil-dren ranged from 0.5 to 48.2 μg/dL, with a mean of5.45 μg/dL and a standard deviation of 0.22 μg/dL, and 23.Thirty-one percent individuals were with blood lead level ≥10 μg/dL. The mean and standard deviation of blood leadlevels were 5.57 ± 0.223 μg/dL and 5.30 ± 0.224 μg/dL inUygur and Han children, respectively. The blood lead lev-els were not significantly different between Uygur and Hanchildren.

The percentage of ALAD1-1 homozygote was 81.94%,ALAD1-2/ALAD2-2 genotype were 18.06% in Urgur sub-jects and the percentage of ALAD1-1 homozygote was91.68%, ALAD1-2/ALAD2-2 genotype were 8.10% in Hansubjects, respectively (chi-square = 20.55, p < 0. 05). The fre-quencies of the allele ALAD1and ALAD2 in Uygur subjectswere 90.52% and 9.48%, and in Han subjects were 95.73%and 4.27%, respectively (chi-square = 19.55, p < 0.05)(Table 1).

To determine whether the individuals with the ALAD1-2/2-2 genotype had higher blood lead levels, the subjects, bothUgyur and Han children, were divided into two groups:one consisting of ALAD1-1 homozygote genotype and otherconsisting of ALAD1-2 hoterozygote and ALAD2-2 ho-mozygote genotypes. As shown in Table 2, the mean bloodlevels of the environmentally exposed Uygur and Han chil-dren who were ALAD1-2/2-2 genotype were little higherthan those who were ALAD1-1 genotype, but no significantdifference was found.

We used multiple liner regression analysis to examinethe effect of sex, age, parents’ smoking, parents’ occupa-tion, parents’ education, habits, housing condition, livingenvironment and ALAD genotype on blood lead levels(Tables 3 and 4). The results showed that parents’ educa-tion and their occupation and the housing condition andliving environment significantly affected blood lead levelsfor children. But the risk factors were a little different be-tween Uygur and Han population.

DISCUSSIONALAD polymorphism has been demonstrated to vary withracial group. The frequency of ALAD2 allele is found in

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Chen et al. ALAD genotype and blood lead levels

Table 2 ALAD genotype and blood lead levels of Uygur and Han children

Blood lead levelSample No. Genotype (mean ± SD, μg/dL) t-test p-value

Uygur population 363 ALAD1-1 5.52 ± 0.22 0.22 0.6480 ALAD1-2/2-2 5.79 ± 0.24

Han population 430 ALAD1-1 5.34 ± 0.22 0.01 0.9339 ALAD1-2/2-2 5.40 ± 0.25

Table 3 Multiple regression model for risk factors for blood lead levels in Uygurchildren

RegressionVariable coefficient SE p-value

Mother’s education −0.3780 0.1710 0.0271Mother’s occupational Pb exposure 0.1249 0.0648 0.0538Wall skin/paint coat falling off in room −1.8023 0.6296 0.0042Toy washing −0.4101 0.2981 0.1689Hand washing before eating 0.6268 0.3662 0.0870

Table 4 Multiple regression model for risk factors for blood lead levels in Hanchildren

RegressionVariable coefficient SE p-value

Father’s education −0.7344 0.2770 0.0080Mother’s education −0.3376 0.2341 0.1492Father’s occupational Pb exposure 0.0872 0.0385 0.0236Proximity to traffic way −0.2182 0.0957 0.0226Hand washing before eating 0.4535 0.2659 0.0881

about 10% of the Caucasian population (8,17,18), 3–4% ofChinese Han population (19,20), but is was not detected inan African population (21). This study found that the pro-portion among Uygur population with ALAD1-1/1-2 geno-type and ALAD2 allele was higher than among the Hanpopulation. The frequency of ALAD2 allele among Uygurpopulation is similar to Caucasian population’s, is typicallydifferent from Han population’s.

In this study, we found that the ALAD G177 C poly-morphism did not affect blood lead levels for Uygur andHan children. While this result confirms previous findingsshowing no effects of ALAD polymorphim on blood leadlevels concentrations (15,16,22,23), other studies have sug-gested that carriers of the ALAD2 allele would have higherPb-B concentrations than noncarriers, thereby increasingtheir susceptibility to lead toxicity (12–14,19,24). And in re-cent study, newer ALAD ploymorphisms(Rsa and Rsa 39488ALAD1-1/1-2) were found that they might influence humansusceptibility to effects of inorganic lead on the neurobe-havioural functions (25). In our study, blood lead levels var-ied from 0.5 to 48.2μg/dL, and the mean blood lead levelwas 5.45 ± 0.22 μg/dL, which is barely below the normalreference level currently set at 10 μg/dL, thus reflecting arelatively low exposure to lead. Therefore, our results sup-

port the notion that ALAD variants would significantly af-fect blood lead level only at high exposure levels (26). Wethink the genetic susceptibility of ALAD polymorphism tolead toxicity may exhibit in a lead dose-dependent manner.

There are many risk factors for children lead poisoning.We found that the high-risk factors of parents’ low educa-tion levels, parents’ occupational exposure to lead, hous-ing condition and living environment were associated withlead poisoning for children. But the risk factors affectedby the culture background, economic level and customsmore or less had little difference between Uygur and Hanpopulation.

In conclusion, different ALAD genotypes were not shownto be associated with blood lead levels in environmentallyexposed Uygur and Han children. A significant ALAD2 al-lele difference existed in Uygur and Han populations, andit is important for researchers to provide the basic informa-tion of ALAD genotype for Chinese populations, especiallyChinese Ugyur populations.

ACKNOWLEDGEMENTSThis study was supported by Xinjiang Education Depart-ment grant (XJEDU2004S16).

References

1. Mahaffey K, Mckinney J, Reigart JR. Lead and compounds. In:Loppmann M, editor. Environmental toxicants, humanexposures and their health effects. 2nd ed. New York, NY:John Wiley and Sons, 2000: 481–521.

2. Goyer RA. Toxic effects of metals. In: Klaassen CD, editor.Casarett and Doull’s toxicology: the basic science of poisons.5th ed. New York, NY: McGraw-Hill Book Company, 1996:691–736.

3. Otto DA, Fox DA. Auditory and visual dysfunction followinglead exposure. Neurotoxicology 1993; 14: 191–207.

4. Banks EC, Ferretti LE, Shucard DW. Effects of low level leadexposure on cognitive function in children: a review ofbehavioral, neuropsychological and behavioral evidence.Neurotoxicology 1997; 18: 237–81.

5. Lin-Fu JS. Vulnerability of children to lead exposure andtoxicity: part one. N Engl J Med 1973; 289: 1229–33.

6. Ziegler EE, Edwards BB, Jensen RL. Absorption and retentionof lead by infants. Pediatr Res 1978; 12: 29–34.

7. Bergdahl IA, Grubb A, Schutz A, Desnick RJ, Wetmur JG,Sassa S, et al. Lead binding to delta-aminolevulinic aciddehydratase (ALAD) in human erythrocytes. PharmacolToxicol 1997; 81: 153–8.

8. Kelada SN, Shelton E, Kaufmann RB, Khoury MJ.Deltaaminolevulinic acid dehydratase genotype and leadtoxicity: a HuGE review. Am J Epidemiol 2001; 154: 1–3.

9. Wetmur JG, Bishop DF, Cantelmo C, Desnick RJ. Humandelta-aminolevulinate dehydratase: nucleotide sequence of afull-length cDNA clone. Proc Natl Acad Sci U S A 1986; 83:7703–7.

10. Battistuzzi G, Petrucci R, Silvagni L, Urbani FR, Caiola S.Delta-Aminolevulinate dehydrase: a new geneticpolymorphism in man. Ann Hum Genet 1981; 45: 223–9.

11. Wetmur JG, Kaya AH, Plewinska M, Desnick RJ. Molecularcharacterization of the human delta-aminolevulinatedehydratase 2 (ALAD2) allele: implications for molecularscreening of individuals for genetic susceptibility to leadpoisoning. Am J Hum Genet 1991a; 49: 757–63.

C©2008 The Author(s)/Journal Compilation C©2008 Foundation Acta Pædiatrica/Acta Pædiatrica 2008 97, pp. 1717–1720 1719

ALAD genotype and blood lead levels Chen et al.

12. Astrin KH, Bishop DF, Wetmur JG, Kaul B, Davidow B,Desnick RJ. Delta-Aminolevulinic acid dehydrataseisozymes and lead toxicity. Ann NY Acad Sci 1987; 514:23–9.

13. Wetmur JG, Lehnert G, Desnick RJ. The delta-aminolevulinatedehydratase polymorphism: higher blood lead levels in leadworkers and environmentally exposed children with the 1-2and 2-2 isozymes. Environ Res 1991b; 56: 109–19.

14. Ziemsen B, Angerer J, Lehnert G, Benkmann HG. GoeddeHWPolymorphism of delta-aminolevulinic acid dehydratase inlead-exposed workers. Int Arch Occup Environ Health 1986;58: 245–7.

15. Marcelo FM, Fernando B, Raquel F. A polymorphism in thedelta-aminolevulinic acid dehydratase gene modifies plasma/whole blood lead. Arch Toxicol 2006; 80: 394–8.

16. Wu FY, Chang PW, Wu CC, Lai JS, Kuo HW. Lack ofassociation of δ-aminolevulinic acid dehydratase genotype withcytogenetic damage in lead workers. Int Arch Occup EnvironHealth 2004; 77: 395–400.

17. Raczek E. Polymorphismof delta-aminolevulinate dehydratasein the upper Silesian population. Hum Hered 1994; 44: 172–4.

18. Fleming DEB, Chettle DR, Wetmur JG, Desnick RJ, Robin JP,Boulay D, et al. Effects of the delta- aminolevulinatedehydratase polymorphism on the accumulation of lead inbone and blood in lead smelter workers. Environ Res 1998; 77:49–61.

19. Shen XM, Wu SH, Yan CH, Zhao W, Ao LM, Zhang YW,et al. Delta- aminolevulinate dehydratase polymorphism andblood lead levels in Chinese children. Environ Res 2001; 85:185–90.

20. Zheng YX, Song WJ, Wang YW, He DS, Liu YY, Wu YQ. Thegene polymorphism of δ-aminolevulinate dehydratase (ALAD)in 530 cases of Chinese Han population. Chin J of Prev Med2001; 1: 16–8.

21. Sousa MG, Silva MC, Duarte AF, Azevedo ES. Delta-aminolevulinate dehy-dratase (ALAD) polymorphism in mixedBrazilians from the State of Bahia. Gene Geogr 1991; 5: 33–7.

22. Hu H, Wu MT, Cheng Y, Sparrow D, Weiss S, Kelsey K. Thedelta-aminolevulinic acid dehydratase (ALAD) polymorphismand bone and blood lead levels in community-exposed men:the Normative Aging Study. Environ Health Perspect 2001;109: 827–32.

23. Schwartz BS, Lee BK, Stewart W, Ahn KD, Springer K, KelseyK. Associations of delta-aminolevulinic acid dehydratasegenotype with plant, exposure duration, and blood lead andzinc protoporphyrin levels in Korean lead workers. Am JEpidemiol 1995; 142: 738–45.

24. Perez-Bravo F, Ruz M, Moran-Jimenez MJ, Olivares M,Rebolledo A, Codoceo J, et al. Association betweenaminolevulinate dehydrase genotypes and blood lead levels inchildren from a lead-contaminated area in Antofagasta, Chile.Arch Environ Contam Toxicol 2004; 47: 276–80.

25. Chia SE, Huijun Z, Theng TM, Yap E. Possibilities of newerALAD polymorphism influencing human susceptibility toeffects of inorganic lead on the neurobehavioral functions.Neurotoxicity 2007; 28: 312–7.

26. Scinicariello F, Murry HE, Mofferett DB, Abadin HG, SextonMJ. Lead and δ-aminolevulinate dehydratase polymorphism:where dose it lead? A meta-analysis. Environ Health Perspect2007; 115: 35–40.

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