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HuGENet Review
“The findings and conclusions in this review are those of the author(s) and do not
necessarily represent the views of the funding agency.”
This HuGE Review was published in the Am J of Epidem 2001; 154(1):1-13

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δ-Aminolevulinic Acid Dehydratase (ALAD) Genotype and Lead Toxicity
Samir N. Kelada1,4, Erin Shelton2, Rachel B. Kaufmann3, and Muin J. Khoury1

1 Office of Genomics and Disease Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia 30341; e-mail: muk1@cdc.gov (MJK)
2 Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109; e-mail: erin.shelton@chmcc.org
3 Lead Poisoning Prevention Branch, Division of Environmental Hazards and Health Effects, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia; email: RKaufmann@cdc.gov
4 Address Correspondence to: Samir N. Kelada, Department of Environmental Health, School of Public Health and Community Medicine, University of Washington, Box 357234, Seattle, WA 98195; email: skelada@u.washington.edu

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 ABSTRACT

The ALAD gene (chromosome 9q34) codes for d-aminolevulinic acid dehydratase (ALAD) (E.C. 4.2.1.24). ALAD catalyzes the second step of heme synthesis and is polymorphic. The ALAD G177C polymorphism yields two codominant alleles, ALAD-1 and ALAD-2, and has been implicated in susceptibility to lead toxicity. Genotype frequencies vary by geography and race. The rarer ALAD-2 allele has been associated with high blood lead levels and was thought to increase the risk of lead toxicity by generating a protein that binds lead more tightly than the ALAD-1 protein. Other evidence suggests that ALAD-2 may confer resistance to the harmful effects of lead by sequestering lead, making it unavailable to participate pathophysiologically. Recent studies have shown that individuals homozygous for the ALAD-1 allele have higher cortical bone lead levels, implying that they may have a greater body burden and may be at higher risk of the long-term effects of lead. Individuals exposed to lead in occupational settings have been the most frequent subjects of study. Genotype selection bias may limit inferences from these studies. No firm evidence exists for an association between ALAD genotype and lead toxicity susceptibility at background exposure levels; population testing for the ALAD polymorphism is therefore not justified.


Keywords: lead, d-Aminolevulinic Acid Dehydratase (ALAD), genetic susceptibility, epidemiology
Abbreviations: BLL = Blood lead level (mg/dl), ALAD = Aminolevulinic acid dehydratase, activity, ALA = Aminolevulinic acid.

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 GENE

The ALAD gene is located on chromosome 9q34 and is approximately 16 kilobases long (1). This gene codes for the d-aminolevulinic acid dehydratase (ALAD) enzyme (E.C. 4.2.1.24), also known as porphobilinogen synthase, a 280 kilodalton protein that is composed of eight identical subunits and requires eight zinc ions as cofactors for full activity (2). The ALAD enzyme catalyzes the second step in heme synthesis, the asymmetric addition of two molecules of aminolevulinic acid (ALA) to form the monopyrrole porphobilinogen (PBG) (Figure 1), which is the precursor of heme, as well as cytochromes and cobalamins. ALAD is expressed in all tissues, but the highest levels of expression are found in erythrocytes and the liver (3).

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 GENE VARIANTS

We searched Medline for relevant publications using the MeSH headings “ALAD” and “d-aminolevulinic acid dehydratase.” Eight ALAD gene variants have been described in the literature. This review will focus on one polymorphism that yields two alleles, designated ALAD-1 and ALAD-2, which exhibit a codominant pattern of inheritance (4). The ALAD-2 allele contains a G ® C transversion at position 177 of the coding region, resulting in the substitution of asparagine for lysine at amino acid 59 (5). These two alleles determine three isozymes, designated 1-1, 1-2, and 2-2, all of which display similar activities but have different charges (4). Asparagine is a neutral amino acid, whereas lysine is positively charged. Therefore, ALAD 1-2 heterozygotes produce an enzyme that is more electronegative than that of ALAD-1 homozygotes, and ALAD-2 homozygotes produce an enzyme that is more electronegative than 1-2 heterozygotes. This forms the basis of the electrophoretic technique originally used to identify the polymorphism and phenotype individuals (4).

The prevalence of the ALAD-2 allele ranges from 0 to 20 percent depending on the population. Generally, Caucasians have the highest frequency of ALAD-2 allele, with approximately 18 percent of the population being ALAD 1-2 heterozygotes and 1 percent being 2-2 homozygotes. In comparison, African and Asian populations have low ALAD-2 allele frequencies, with few or no ALAD-2 homozygotes found in such populations. Table 1 lists genotype frequencies from around the world (6-26). All of these frequencies are in Hardy-Weinberg equilibrium. The listed genotype frequencies were determined in the early 1980s by phenotyping. In 1991, Wetmur et al. (5) devised a PCR-based genotyping technique, which correctly identified all 93 ALAD-2 heterozygotes and homozygotes tested, i.e., there was a 100% genotype-phenotype correspondence. Most investigators have subsequently used this technique.

 Table 1: ALAD genotype frequencies

 

Most of the studies presented in Table 1 that document genotype or phenotype frequencies gave little detail about the study population (e.g., age or source of donors), making it hard to rule out any potential biases due to subject selection. These populations are referred to as “general” in the table. Alternatively, some studies used hospital-based study samples. Other studies (e.g., 6, 7, 26) used samples comprised of individuals with relatively high levels of lead exposure from occupational studies. This may also promote bias in the results, as persons with “at risk” genotypes may have been selected against during the course of employment and therefore may not have been represented in the study sample. Background and Epidemiology of Lead Poisoning.

Lead has been a known toxin for thousands of years and remains a persistent environmental health threat. Exposure to lead can result in significant adverse health effects to multiple organ systems. Toxic effects to the nervous, hematological, renal, and reproductive systems have been studied extensively and are well documented (27, 28). Since lead was phased out as a gasoline additive (tetra-ethyl lead) in the 1970s and its use in paint and food containers (e.g., ceramicware and tin cans) was curtailed, blood lead concentrations have decreased significantly; however, other sources of lead and its unknown threshold of subclinical toxicity continue to make lead an issue of public health concern.

There are many risk factors for lead poisoning. Generally, living in a home built before 1950 is considered a risk factor because of the presence of multiple avenues of exposure to lead. Old pipes with lead solder can contaminate the water supply, and lead-based paint is still a notorious source of lead in these houses (29). Additionally, living in close proximity to lead-emitting industrial facilities can present a significant source of cumulative exposure to lead via air, water, and soil. Occupational exposure to lead is most often encountered at lead smelters and battery manufacturing facilities, as well as in housing renovation projects in which workers inhale and ingest lead-contaminated fumes and dust from lead-based paint.

Children’s hand-to-mouth activity, increased respiratory rates, and increased intestinal absorption of lead make them more susceptible than adults to lead exposure (30, 31). Lead-based paint remains the predominant source of high-dose lead poisoning in children. Poor nutrition, particularly inadequate calcium and iron intake, is likely an important risk factor for children as well (32).

Blood lead level (BLL, mg/dl) is the biological index most often used by health care providers as an indicator of recent lead exposure (33). Two analytical techniques, anodic stripping voltammetry (ASV) and atomic absorption spectroscopy (AAS), are used to measure BLL and have detection limits < 1 mg/dl (34). In addition to BLL, other lead exposure indices include free erythrocyte protoporphyrin (FEP) and zinc protoporphyrin (ZPP); both are precursors of heme whose levels elevate upon moderate to high exposure to lead. However, FEP and ZPP are neither sensitive enough nor specific enough to be used as primary indicators of lead exposure (27, 35). Lead levels in plasma, urine, bone, and teeth (dentin lead) are less commonly used measures of exposure and body burden.

At steady state, 90 percent of lead is in the skeleton (27). The association of lead and bone is due to lead’s similar valence to calcium. Measurements of lead in trabecular or spongy bone (e.g., patella), in which lead has a relatively short half-life, and lead in cortical bone (e.g. tibia), which represents a site of long-term lead storage, have been used to estimate the distribution of lead in bone and total body burden (24, 27). Reliable, non-invasive techniques such as X-ray fluorescence (XRF) have been developed to measure bone lead levels. Lead in bone can leach out and constitutes a significant long-term source of lead to the blood (27). Chelating agents such as dimercaptosuccinic acid (DMSA) have been used therapeutically to extract lead from tissues (28). It has been shown that chelatable lead correlates well with lead in trabecular bone (36). Administration of chelators has also been used in research studies to estimate body burden.

Subclinical lead toxicity remains a problem for both adults and children (37-39). Blood lead concentrations of 10 mg/dl in children have been associated with cognitive deficits, aggressive behavior, and hearing dysfunction (40-45). Alarmingly, evidence is indicating that no detectable threshold exists for the adverse effects of lead exposure on neurodevelopment (45, 46). Using National Health and Nutrition Examination Survey (NHANES) III survey data, the Centers for Disease Control and Prevention (CDC) estimated that 890,000, or 4.4 percent, of U.S. children aged 1 through 5, have blood lead concentrations of 10 mg/dl, the current level of concern, or higher (47). The current mean blood lead concentration for children 1-5 years old is 2.7 mg/dl (48). In the adult population, BLLs measured in NHANES II and Phase I of NHANES III showed a decrease from 13.1 mg/dl to 3.0 mg/dl, and currently more than 90 percent of adults have BLLs < 10 mg/dl (49). With respect to the occupational arena, the current goal of the U.S. Department of Health and Human Services (DHHS) is to eliminate all occupational exposures resulting in BLLs > 25 mg/dl (33). The National Institute for Occupational Safety and Health (NIOSH) used to maintain the Adult Blood Lead Epidemiology and Surveillance (ABLES) program, which reported the prevalence of elevated BLLs among adults in 28 US states. At last report, in the third quarter of 1998, 3322 (16%) of the 20,511 adults for whom BLLs were reported had BLLs ³ 25 mg/dl; of these, 182 (6%) had BLLs ³ 50 mg/dl (50). Both of these prevalence statistics represent declines from previous quarters of ABLES reporting.

Lead and ALAD
One of lead’s primary effects is hematotoxicity, specifically heme synthesis inhibition. Lead inhibits three enzymes in the heme biosynthesis pathway (Figure 1) -- ALAD, coporphyrinogen oxidase, and ferrochelatase -- but its effects on ALAD are most profound (51). Lead inhibits ALAD stoichiometrically (52-54), and the degree of erythrocyte ALAD inhibition has been used clinically to gauge the degree of lead poisoning. At the molecular level, lead displaces a zinc ion at the metal binding site, not the active site (55), producing inhibition through a change in the enzyme’s quaternary structure. ALAD inhibition results in the build up of ALA, detectable in the plasma and urine at BLLs < 10 mg/dl. ALA resembles g-aminobutyric acid (GABA) and can stimulate GABA receptors in the nervous system; this is thought to be one of primary mechanisms of lead-induced neurotoxicity (55-57).

 Figure 1: The heme biosynthesis pathway. A = – CH2COOH; M = – CH2; P = – CH2CH2COOH; V = – CH═CH2; ALA = Aminolevulinic acid; PBG = Porphobilinogen. Apostrophe denotes ‘porphyrino’ abbreviation. (Taken with permission from Scriver C. et al. The Metabolic and Molecular Basis of Inherited Disease, 7th ed., McGraw Hill publishing, 1994).

The ALAD-1/2 Polymorphism as a Modifier of Lead’s Effects Initial Studies
Early studies conducted on the ALAD polymorphism and lead poisoning focused on differences in BLLs by genotype in populations with relatively high levels of lead exposure, either from home or occupation (Table 2). Ziemsen et al. (58) were the first to describe differences in BLLs by genotype. They found that lead-exposed workers (n = 202) with the ALAD 1-2 genotype* had higher BLLs than ALAD 1-1 homozygotes (44 vs. 38 mg/dl) and that ALAD 2-2 homozygotes had higher BLLs at 56 mg/dl. Astrin et al. (25) subsequently found a higher than expected proportion of individuals with the ALAD 1-2 or 2-2 genotype among individuals with lead poisoning screened by BLLs > 50 mg/dl or FEP > 30 mg/dl (n = 1074). The ascertainment bias in the sampling technique was noted in the paper. Astrin et al. also reported that the ALAD-2 allele was associated with a four-fold increase in the ability to retain BLLs above 30 mg/dl. Further, Wetmur et al. (26) found significant differences in BLLs in a group of lead-exposed workers (n = 202) and New York City children (n = 1278) screened by elevated FEP. They found median BLLs 9 mg/dl and 11 mg/dl higher among ALAD-2 carriers in these two populations, respectively. All three of these studies examined populations with exposure levels higher than normal whose BLLs were often > 30 mg/dl, a previously designated cutoff used as evidence of lead poisoning.

Hypotheses generated to support these results were based on the charge of the ALAD-2 isozyme (3, 25, 26). Since the ALAD-2 allele codes for a more electronegative enzyme, the ALAD-2 protein is thought to be able to bind positively charged lead ion more tightly than the ALAD-1 protein. Carriers of the ALAD-2 allele who are exposed to lead might then retain it in their blood and tissues longer, increasing the chance of an adverse effect due to inhibition of ALAD and consequent build up of ALA and perhaps due to lead itself, which can initiate oxidative damage and change the structure of cellular components (27). From these initial studies, it is safe to conclude that the kinetics of lead in blood are modified by ALAD genotype, although perhaps only at relatively high levels of exposure. These studies also imply that the ALAD 1-2 and 2-2 genotypes are the “at risk genotypes” at high exposure levels.

Further Studies
Subsequent studies (Table 2) were again primarily occupational epidemiologic studies but often used new sets of measures for lead exposure and body burden. Bone lead measurements, in particular, began to be used as measures of outcome. In 1995, Schwartz et al. (59) used an occupational cohort of employees from three lead storage battery factories (n = 307) and found that the ALAD-2 allele was not clearly associated with higher blood levels (i.e., no difference in BLL by genotype) but that individuals with the 1-2 genotype (there were no 2-2 subjects) were 2.3 times more likely to have BLL3 40 mg/dl, although the 95 percent confidence interval (CI) contained 1.0. No relationship was found between genotype and ZPP. They did find, however, that the 1-2 genotype was associated with occupational exposures of more than 6 years (Odds Ratio (OR) = 2.6, 95 percent CI: 1.2, 5.8), suggesting that the ALAD-2 allele conferred a protective effect. In support, ALAD 1-2 heterozygote workers with high exposure histories had lower ZPP levels than ALAD-1 homozygotes with equivalent exposure histories. The authors cited this as a possible genotype-selection factor and proposed that the ALAD-2 subunit of the protein keeps lead in a non-bioavailable form so that these individuals (ni = 4) were protected from lead’s effects and could tolerate longer exposures to lead than ALAD 1-1 subjects.

 Table 2: Summary of studies on ALAD genotype and lead exposure

Using a group of 122 carpenters with relatively low blood lead levels for study (average BLL = 7.8 mg/dl), Smith et al. (24) avoided the bias of previous studies that used individuals with high blood lead levels. They found no association between ALAD genotype and BLL, implying that ALAD genotype may be a modifier of BLL only at high blood lead concentrations. They also found no association between genotype and tibial or patellar bone lead levels, which was measured using XRF methods. However, using the difference of lead levels in patellar versus tibial bone as an indicator of effect of the genotype on the distribution of lead in bone, a difference of borderline significance was found between 1-1 and 1-2/2-2 genotypes (p = 0.06). ALAD-1 homozygotes had a smaller difference in patella – tibia bone lead levels than 1-2/2-2 individuals (3.4 ± 12.0 vs. 8.6 ± 9.5 mg Pb/g bone mineral). This indicates that 1-1 individuals have increased uptake of lead into cortical bone, the long-term storage depot, relative to 1-2/2-2 individuals. It was hypothesized that 1-2/2-2 individuals partition less lead into cortical bone because of the increased affinity of the ALAD-2 subunit for lead. Hence, ALAD-1 homozygotes would be at increased long-term risk as they build up higher cortical bone lead levels that can leech out at times of bone lead redistribution (e.g., during pregnancy). These investigators also observed a relationship between ALAD-2 and subclinical renal toxicity, as evidenced by elevated blood, urea, and nitrogen (BUN), uric acid, and creatinine levels in ALAD-2 subjects.

In contrast, Bergdahl et al. (60) found lower levels of urinary creatinine and calcium in 1-2/2-2 genotype subjects in a study of 89 lead-exposed and 34 unexposed workers in Sweden. No association between genotype and lead in blood, bone, or urine among the exposed group was observed in this study. The frequency of the ALAD-2 allele was less than expected among lead workers (p value from c2 test = 0.0025), and the authors cited this finding as potential evidence of a genetic-healthy worker effect, in which ALAD-2 individuals who reach high blood lead levels would be removed from the workplace (by Swedish occupational health standards) and therefore would not be represented in the study sample.

Several studies have yielded supporting evidence for the hypothesis that ALAD genotype also modifies the kinetics of lead in bone. In a study of 381 lead smelter workers, Fleming et al. (6) observed increased uptake of lead from blood into bone in ALAD-1 homozygotes, which was seen by the increased slope of the line relating bone lead levels to a cumulative blood level index (CBLI, mg/dl) in 1-1s compared to 1-2/2-2s. This effect was most pronounced in trabecular bone (the calcaneus) of workers hired after the implementation of a lead safety initiative at the plant in 1977 (p < 0.001, vs. p < 0.04 in cortical bone). This study also provided more evidence for the influence of ALAD genotype on the kinetics of lead in blood at moderate to high exposure levels (mean BLL = 23.3 mg/dl), as 1-2/2-2 genotype individuals had 10 percent greater blood lead levels (p < 0.04).

Schwartz et al. (61) used the chelator DMSA to test for differences in bioavailable lead by genotype in a group of 57 lead battery manufacturing workers. The data showed that 1-1 individuals yielded more lead in 4-hour urine samples in response to chelation therapy (5 mg/kg orally) versus 1-2/2-2 individuals with the same exposure history (92.9 ± 45.1 vs. 70.3 ± 42.1 mg, respectively, p = 0.07). Another study by Schwartz et al. (62) corroborated these findings (Table 2). Given that DMSA chelatable lead is used as a measure of bioavailable lead, these data indicate that ALAD 1-2 subjects have lower levels of bioavailable lead and therefore may be at decreased risk compared to ALAD-1 homozygotes. Schwartz and colleagues (61) also saw higher levels of ALA in plasma (ALAP) of 1-1 individuals (17.3 vs. 11.8 ng/ml, p = 0.03), a finding that was replicated by Sithisarankul et al. (p = 0.012) (63). Similar differences in ALAP were seen in a recent study of 192 Japanese male lead workers by Sakai et al (17). These findings suggest that ALAD-1 homozygotes may be at greater neurological risk due to the build up of ALA in the plasma. Finally, Schwartz et al. (62) noted an elevation in hemoglobin A1 (HbA1) levels in 1-1 subjects (n = 38) versus 1-2 subjects (n = 19) (6.3 ± 1.0 vs. 5.9 ± 1.0 %, p = 0.08), which led them to conclude that both ALAD and HbA1 are important lead binding sites that influence the excretion of chelated lead.

It should be noted that the studies by Schwartz et al. (59, 61, 62) and Sithisarankul et al. (63) contained overlapping study samples. The degree of overlap is, however, unknown. The studies are presented separately for the purposes of this review, but the results should be interpreted with caution since they are based on study samples that may have contained substantial redundancy.

Most recently, Schwartz et al. (64) reported on a study of 798 Korean lead workers and 135 unexposed controls. ALAD 1-1 workers yielded substantially more chelated lead after 10 mg/kg DMSA (see Table 2). Logistic regression modeling of chelated lead showed that creatinine clearance was an important predictor (b = 0.006, p < 0.001) and ALAD genotype modified this relationship (p value for ALAD-creatinine interaction = 0.04). ALAD-2 subjects had larger increases in chelated lead with increasing creatinine clearance. (The effect of a polymorphism in the Vitamin D Receptor gene was also investigated and is discussed below under gene-gene interactions.)

In a study by Bergdahl et al. (65), the ALAD enzyme was found to be the principal lead- binding site in erythrocytes. The investigators found a higher percentage of lead bound to erythrocyte ALAD in lead-exposed ALAD 1-2 subjects versus 1-1 subjects (84 percent vs. 81 percent, p = 0.03).

One study of a group of 134 lead smelter workers in British Columbia by Alexander et al. (7) examined differences in sperm count and sperm concentration by genotype, in addition to BLL, ZPP, heme, and coporphyrin (CPU). In this group, blood lead levels were higher in ALAD 1-2 subjects, 28.4 versus 23.1 mg/dl for 1-1s (p = 0.08); ALAD 1-2 subjects had higher sperm counts but the difference between the two groups was not significant. Focusing on the relationships of ALAD genotype, ZPP, and CPU at blood lead levels ³ 40 mg/dl, Alexander et al. observed that ALAD-1 homozygotes had significantly higher ZPP levels (86.0 ± 41.1 vs. 50.0 ± 18.4 mg/dl, p = 0.03) and higher CPU levels (46.5 ± 31.6 vs. 13.6 ± 7.8 mg/l, p = 0.01). Markers other than BLL, in other words, indicated that ALAD-1 homozygotes in this study exhibited more inhibition of heme synthesis after exposure to lead. The authors noted that the non-random method of study subject ascertainment (solicitation by postal questionnaire) and the lack of women in the sample were limitations of the study.

The sole population-based study was conducted by Hsieh et al. (23) in a Taiwanese population (n = 660). They measured BLL and found 20 percent higher levels in the 1-2/2-2 group (7.83 ± 5.95 vs. 6.51 ± 5.03 mg/dl), but this result was not statistically significant (p = 0.17). The authors suggested that the difference in BLL by genotype was not significant possibly because of the small number of individuals in the 1-2/2-2 group (n = 30). They also postulated that blood lead levels in Taiwan may be relatively low due to the rarity of the ALAD-2 allele in that population.

ALAD Genotype and Neurological Outcomes
In 1994, Bellinger et al. (66) published a report in which adolescents (n = 72) with high (> 24 mg/g) and low (< 8.7 mg/g) dentin lead levels were studied, and the results suggested that the body burden and the effects of lead were worse in ALAD-1 homozygotes. Although the study had only five subjects with the 1-2 genotype, these five had lower dentin lead levels than subjects with the 1-1 genotype and consistently scored better on neuropsychological tests (no p values given because of small n).> This is the only study on the ALAD polymorphism to date that has used a neurological outcome measure. The 1-2 genotype subjects were also less likely to have tibial lead concentrations > 6 mg/g and more likely to have patellar lead concentrations > 6 mg/g.

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 INTERACTIONS

In addition to ALAD genotype, other factors to consider in determining overall susceptibility to lead toxicity include substances that inhibit the ALAD enzyme and nutritional status, primarily calcium and iron intake. The interaction between these factors and ALAD may be important when considering the health effects of lead. Additionally, the genes encoding the vitamin D receptor (VDR) and the hemochromotosis-MHC Class I protein (HFE), are both polymorphic and have been recently implicated in lead poisoning susceptibility. Thus, gene-environment and gene-gene interactions may produce enhanced effects and deserve further exploration

Gene-Environment Interactions
The ALAD enzyme is inhibited by alcohol and smoking (67). Three of the studies examined in this review measured smoking (24, 61, 66), and one of them controlled for smoking in regression models of outcome (61). Three studies measured alcohol use (24, 61, 66), and one controlled for alcohol use in a model (24). No studies explicitly examined ALAD-alcohol or ALAD-smoking interactions.

Calcium status has been shown to influence the intake and effects of lead. Lead binds to calcium binding proteins and may also compete directly for absorption in the intestine. Mahaffey et al. (68) showed that blood lead levels are lower in children with higher calcium intakes. In addition, several studies in experimental animals have clearly demonstrated that prior intake of calcium reduces the absorption of lead and that absorption of lead is higher in calcium-deficient animals compared to normal animals. Studies also show that persons subjected to fasting absorb more lead than when not fasting (69); dietary intake in general is therefore an important factor as well. No studies have explored interactions between ALAD and any of these factors.

Gene-Gene Interactions
The effects of calcium on lead intake and absorption are mediated through calcium binding proteins that are, in turn, mediated through the bloodborne form of vitamin D, calcitriol. Calcitriol binds to the Vitamin D receptor, and thus genetic variations in the vitamin D receptor are also important in this pathway. A common polymorphism in the VDR gene, a restriction fragment length polymorphism detected by digestion with BsmI that results in the B and b alleles, has already been shown to affect bone mineral density (BMD). The BB genotype, which signals no BsmI restriction site, exists in about 7-32 percent of the population (70) and has been shown by meta-analysis to be associated with lower BMD (70). Two recent studies by Schwartz et al. (65, 71) explored the role of this polymorphism in tibial bone lead levels in lead workers. Schwartz et al. (71) first reported small differences in bone lead levels by VDR genotype among a group of former lead workers (13.9 ± 7.9, 14.3 ± 9.5, and 15.5 ± 11.1 mg Pb/g bone mineral in the bb, Bb, and BB genotypes, respectively; adjusted p value for linear trend = 0.16). The relationship between years since last exposure and tibial bone lead concentration was also modified by VDR genotype. In their second report (65), Schwartz and colleagues noted larger differences in BLL by VDR genotype than by ALAD genotype. On average, the VDR B allele gave a 4.2 mg/dl increase in BLL, while the ALAD-2 allele yielded an increase of 3.6 mg/dl in BLL. They also explored the role of a possible gene-gene interaction between VDR and ALAD and found no evidence of an interaction. Interestingly, they did find an association between ALAD and VDR genotypes which varied by exposure status. Lead workers with ALAD 1-1 genotype were less likely to have the VDR bb genotype (OR = 0.29, 95 percent CI: 0.06 – 0.91) while unexposed controls with the ALAD 1-1 genotype were more likely to have the VDR bb genotype (OR=2.5, 95 percent CI: 0.23-14.84). This may be indicative of genotype selection in the occupational environment.

Like calcium status, iron deficiency also increases the absorption and toxic effects of lead (72). Ferritin, the iron transport protein, binds lead and is increased in iron-deficient anemia. Counter-intuitively, persons homozygous for the HFE mutation that induces hemochromotosis, a disease of iron overload, have been shown to have higher blood lead levels than wild-type HFE persons (5.6 ± 0.6 vs. 3.6 ± 0.5 mg/dl, respectively, [73]), and heterozygotes had intermediate levels (4.1 ± 0.5 mg/dl), suggesting that carriers of the mutant gene absorb more lead. This finding was not, however, replicated in a recent study by Åkesson et al. (74). To date, no studies have evaluated an HFE-ALAD interaction.

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 LABORATORY TESTING

Early studies used the phenotyping technique developed by Battistuzzi et al. (4) to classify individuals as ALAD 1-1, 1-2, or 2-2. In this procedure, whole blood samples are taken and the red blood cells are isolated and lysed. Isolation and electrophoresis of the ALAD protein enables distinction of the phenotypes due the charge differences of the isozymes. Wetmur et al. (5) developed the PCR-based genotyping technique that has been used by most investigators. A 916 base-pair sequence containing the ALAD-1/2 polymorphic site is amplified and then cleaved with MspI. The cleavage products are then analyzed on an agarose gel. Studies using this technique should include positive (e.g., a gene encoding an essential enzyme) and negative controls.

 POPULATION TESTING

There is inadequate evidence to support population-based testing at this time.

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 CONCLUSIONS AND RECOMMENDATIONS

The evidence surrounding the ALAD G177C polymorphism and lead poisoning can be summarized as follows: at high levels of exposure and in comparison to ALAD 1-1 genotype individuals, ALAD 1-2/2-2 genotype individuals have increased blood lead levels, lower concentrations of ALAP, lower ZPP levels, lower cortical bone lead concentrations, higher concentrations of trabecular (spongy) bone lead, and lower amounts of DMSA chelatable lead. Thus, ALAD genotype modifies the kinetics of lead in both blood and bone. Although ALAD-2 subjects may achieve higher BLLs when exposed to lead, they may experience less heme synthesis inhibition compared to ALAD-1 homozygotes. When lead binds and inhibits the ALAD enzyme, ALAD expression is increased in response (75, 76). Therefore individuals with ALAD-2 may be better able to compensate than ALAD-1 homozygotes as more lead is bound to ALAD-2 enzyme (65). This hypothesis might help explain the genotype selection observed by Schwartz et al. (59) in which ALAD-2 subjects seemed to tolerate longer exposures to lead in the occupational setting. The data also suggest that ALAD-1 homozygotes may be at greater risk of neurotoxicity than ALAD 1-2 individuals, as ALAD-1 homozygotes have higher levels of ALAP (61, 63). Finally, a study by Bellinger et al. (66) gave preliminary evidence that ALAD 1-2 individuals may have better neuropsychological performance than ALAD-1 homozygotes of similar lead exposure history.

It is difficult to make a decision as to which genotype is in fact “at risk” because different measures of outcome indicate that each genotype is more susceptible to one or more adverse effects compared to the other. This problem is complicated by the use of different measures in studies. Indeed, the question of which measures are most appropriate for estimating body burden and health risk is one that remains to be answered and merits discussion. The lack of available data on the effect of this polymorphism on endpoints such as cognitive deficits and/or neuropsychological performance, in particular, is troubling. In addition, most studies used occupationally exposed individuals with relatively high levels of lead exposure. Bias in study subject selection is often encountered in these studies. Very few studies used samples from the general population, for whom exposure levels are generally much less than in occupational settings; and in both these and the occupational studies the percentage of samples including women is unknown. Thus, it is difficult to make inferences for the general population. Results from current research projects investigating these issues using community samples may help resolve these issues.

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 TABLES AND FIGURES

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 REFERENCES

List of References

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 INTERNET SITES

Page last reviewed: June 8, 2007 (archived document)
Page last updated: November 2, 2007
Content Source: National Office of Public Health Genomics