Case-parent Triads Research Articles

Genetic polymorphisms in arginase I and II and childhood asthma and atopy

A recent microarray study implicated arginase I (ARG1) and arginase II (ARG2) in mouse allergic asthma models and human asthma. To examine the association between genetic variation in ARG1 and ARG2 and childhood asthma and atopy risk. We enrolled 433 case-parent triads, consisting of patients with asthma 4 to 17 years old and their biologic parents, from the allergy clinic of a public hospital in Mexico City between 1998 and 2003. Atopy to 24 aeroallergens was determined by skin prick tests. We genotyped 4 single nucleotide polymorphisms (SNPs) of ARG1 and 4 SNPs of ARG2 with minor allele frequencies higher than 10% by using the TaqMan assay (Roche Molecular Systems, Pleasanton, Calif). ARG1 SNPs and haplotypes were not associated with asthma, but all 4 ARG1 SNPs were associated with the number of positive skin tests (P = .007-.018). Carrying 2 copies of minor alleles for either of 2 highly associated ARG2 SNPs was associated with a statistically significant increased relative risk (RR) of asthma (1.5, 95% CI = 1.1-2.1 for arg2s1; RR = 1.6, 95% CI = 1.1-2.3 for arg2s2). The association was slightly stronger among children with a smoking parent (arg2s1 RR = 2.1, 95% CI = 1.2 - 3.9 with a smoking parent; RR = 1.2, 95% CI = 0.8-1.9 without; interaction P = .025). Haplotype analyses reduced the sample size but supported the single SNP results. One ARG2 SNP was related to the number of positive skin tests (P = .027). Variation in arginase genes may contribute to asthma and atopy in children.

Stochastic Search Gene Suggestion: A Bayesian Hierarchical Model for Gene Mapping

Mapping the genes for a complex disease, such as diabetes or rheumatoid arthritis (RA), involves finding multiple genetic loci that may contribute to the onset of the disease. Pairwise testing of the loci leads to the problem of multiple testing. Looking at haplotypes, or linear sets of loci, avoids multiple tests but results in a contingency table with sparse counts, especially when using marker loci with multiple alleles. We propose a hierarchical Bayesian model for case-parent triad data that uses a conditional logistic regression likelihood to model the probability of transmission to a diseased child. We define hierarchical prior distributions on the allele main effects to model the genetic dependencies present in the human leukocyte antigen (HLA) region of chromosome 6. First, we add a hierarchical level for model selection that accounts for both locus and allele selection. This allows us to cast the problem of identifying genetic loci relevant to the disease into a problem of Bayesian variable selection. Second, we attempt to include linkage disequilibrium as a covariance structure in the prior for model coefficients. We evaluate the performance of the procedure with some simulated examples and then apply our procedure to identifying genetic markers in the HLA region that influence risk for RA. Our software is available on the website http://www.epigenetic.org/Linkage/ssgs-public/.

Risk of Testicular Germ Cell Cancer in Relation to Variation in Maternal and Offspring CytochromeP450 Genes Involved in Catechol Estrogen Metabolism

The incidence of testicular germ cell carcinoma (TGCC) is highest among men ages 20 to 44 years. Exposure to relatively high circulating maternal estrogen levels during pregnancy has long been suspected as being a risk factor for TGCC. Catechol (hydroxylated) estrogens have carcinogenic potential, thought to arise from reactive catechol intermediates with enhanced capability of forming mutation-inducing DNA adducts. Polymorphisms in maternal or offspring genes encoding estrogen-metabolizing enzymes may influence prenatal catechol estrogen levels and could therefore be biomarkers of TGCC risk. We conducted a population-based, case-parent triad study to evaluate TGCC risk in relation to maternal and/or offspring polymorphisms in CYP1A2, CYP1B1, CYP3A4, and CYP3A5. We identified 18- to 44-year-old men diagnosed with invasive TGCC from 1999 to 2004 through a population-based cancer registry in Washington State and recruited cases and their parents (110 case-parent triads, 50 case-parent dyads). Maternal or offspring carriage of CYP1A2 -163A was associated with reduced risk of TGCC [maternal heterozygote relative risk (RR), 0.6; 95% confidence interval (95% CI), 0.2-1.7; offspring heterozygote RR, 0.7; 95% CI, 0.3-1.5)]. Maternal CYP1B1 (48)Gly homozygosity was associated with a 2.7-fold increased risk of TGCC (95% CI, 0.9-7.9), with little evidence that Leu(432)Val or Asn(453)Ser genotypes were related to risk. Men were also at increased risk of TGCC if they carried the CYP3A4 -392G (RR, 7.0; 95% CI, 1.6-31) or CYP3A5 6986G (RR, 2.4; 95% CI, 1.1-5.6) alleles. These results support the hypothesis that maternal and/or offspring catechol estrogen activity may influence sons' risk of TGCC.

Assessing Maternal Genetic Associations

In utero exposures, including maternal phenotypes, are potential risk factors for both early-onset and adult-onset diseases. Two alternative study designs use maternal genotypes at polymorphic loci as biomarkers of an offspring's in utero exposure: (1) a traditional case-control study with logistic regression analysis, in which cases, controls, and mothers of both types of subjects are genotyped; and (2) a case-parent triad study with log-linear analysis, in which cases and both parents are genotyped. We used computer simulations to compare the operating characteristics of the log-linear approach to case-parent triad data and the case-control approach for assessing relative risks (RRs) associated with maternal genotypes. For high-risk allele frequencies (chromosomal prevalence; f) between 0.20 and 0.75, both methods allowed for valid, unbiased estimates of maternal RRs. The case-parent triad approach, however, had 43% greater power, on average, than the case-control approach with an equal number of genotypes, and 13% greater power with an equal number of cases. For example, under dominant inheritance, to detect 2-fold maternal RRs with 200 (or 150) cases when allele prevalence is between 0.15 and 0.40, the case-parent triad and equal-genotype case-control designs had, on average, 87% and 62% power, respectively. As f approached 0 or 1, the power of both methods decreased sharply. The greater efficiency of case-parent triads may be due to the inclusion of paternal genotype information, which allows for independent tests of disease association with maternal or offspring genotypes. These results highlight one potential advantage of case-parent triad data in assessing maternal genetics as risk factors for offspring disease. We discuss these findings and other considerations between the 2 methodological approaches.

Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.

Performance of the Log-Linear Approach to Case-Parent Triad Data for Assessing Maternal Genetic Associations with Offspring Disease: Type I Error, Power, and Bias

Maternal genetic variation may serve as a biomarker in studies aimed at clarifying fetal determinants of infant or adult disease. The log-linear approach to case-parent triad data (LCPT) can be used to investigate maternal genetic polymorphisms in relation to offspring disease risk, but LCPT operating characteristics have been reported for only a limited range of situations. The authors performed a simulation study to investigate the performance of the LCPT for assessing maternal associations with offspring disease risk over a wide range of scenarios with varying sample sizes (n), high-risk allele frequencies (f ), and modes of inheritance, all of which greatly affect the expected number of triads in informative categories. For most f values less than 0.5, the LCPT approach with 200 triads allowed for approximately 80% power to detect valid, unbiased maternal relative risks of 2 when inheritance was log-additive or dominant. When inheritance was recessive, this was true for most f 's greater than 0.35. Outside of this range, however, power and bias depended greatly on the mode of inheritance, f, and n. On the basis of these findings, epidemiologists may consider the LCPT a useful approach for assessing maternal relative risks unless one expects a very rare or fairly common maternal allele to increase offspring disease risk.

Continuous Covariates in Genetic Association Studies of Case-Parent Triads: Gene and Gene-Environment Interaction Effects, Population Stratification, and Power Analysis

We propose a multinomial logistic regression method which permits estimation and likelihood ratio tests for allele effects, their interactions with continuous covariates, and assessment of the degree of population stratification in genetic association studies of case-parent triads. Our approach overcomes the constraint imposed by the categorical nature of explanatory variables in the log-linear model. We also demonstrate that the multinomial logistic method can yield efficient inference in the presence of missing parental genotype data via the use of the Expectation-Maximization (EM) algorithm. We performed simulations to compare the multinomial logistic model with the case-pseudosibling conditional logistic model approach, both of which permit the incorporation of continuous covariates. Simulation results indicate that the multinomial logistic model and the conditional logistic model lead to similar estimates in large samples. A simulation-based method of sample size estimation is also used to show that the two models are approximately equivalent in sample size requirements. When parental genotype data are missing, either completely at random or dependent on covariates, the use of the EM algorithm gives multinomial logistic model greater power. Since the multinomial logistic model offers the possibility of assessing the degree of population stratification in the sample and can also provide efficient inference in the presence of missing parental genotypes, the proposed model has an important application in epidemiological family-based association studies.

On the combination of transmission/disequilibrium test and mean test for linkage detection using affected sib pairs

The transmission/disequilibrium test (TDT) and the mean test are the two major nonparametric methods used in linkage analysis. Using case-parent triad data the TDT method tests the equality of the probabilities of a marker allele transmitted or not transmitted with the disease gene of a heterozygous parent. On the other hand, the mean test tests the goodness-of-fit of the observed and expected distributions of the number of alleles shared identical by descent using affected sib-pair data. It has been clarified in the literature that the mean test has higher power than the TDT when linkage disequilibrium (LD) is weak but the TDT has higher power than the mean test when LD is strong. These results indicate that both tests are sensitive to linkage disequilibrium, though their responses are different. The development of a robust method against the interference of linkage disequilibrium is considered. The combination idea is employed to combine the TDT and the mean test to search for such a robust statistic using case-parent tetrad data (two parents and two affected children). Several available combining methods are taken into account in this study. Numerical comparisons of these methods with the published ones are presented.

Bias in parental transmission of the HLA-DR3 allele in Sardinian multiple sclerosis.

The authors analyzed the female: male (F:M) ratio according to the HLA-DRB1-DQB1 genotype in a cohort of multiple sclerosis (MS) patients from Sardinia, where the disease is associated with DR3 and DR4. In the whole cohort of 1,097 patients, F:M ratio was 2.24; however, it was 2.88 in DR3/DR3 and 2.52 in DR3/DRX (X#DR3 and DR4) individuals. Parental transmission of DR3 and DR4, assessed in a set of 565 case-parent triads, showed evidence of paternal inheritance of DR3 in affected women, thus explaining the excess of females in the DR3 category.

Maternal and fetal variants of genetic thrombophilias and the risk of preeclampsia.

A woman's thrombophilic genes may increase her risk of preeclampsia in pregnancy. Vascular conditions of the placenta related to thrombophilic genes of the fetus could also be relevant for preeclampsia. The case-parent triad study design provides separate estimation of maternal and fetal genes. We recruited 92 mother-father-child triads of preeclamptic pregnancies from a birth clinic in Stavanger, Norway. All parents were of Norwegian origin. Maternal, paternal, and fetal DNA were genotyped for the methylenetetrahydrofolate reductase (MTHFR) C677T and Factor V Leiden (FVL) G1691A SNPs. Estimation of the relative risk (RR) associated with fetal and maternal genetic variants was performed by log-linear models. There was no indication of an effect of the child's FVL alleles on preeclampsia risk. For case babies with 2 copies of the variant allele, the association with the MTHFR variant was inconclusive (RR = 1.6; 95% confidence interval [CI] = 0.6-4.3). Case mothers who were homozygous for the MTHFR variant had a relative risk of 2.0 (CI = 1.0-4.1) assuming a recessive gene effect. A 2.5-fold risk (CI = 1.1-5.7) of preeclampsia was estimated when the mother carried one copy of the FVL. Among mothers homozygous for the MTHFR variant, the relative risk of the FVL variant was 4.6-fold (CI = 1.0-21). We found little evidence of an effect of the child's MTHFR or FVL alleles on the risk of preeclampsia. Our estimates of effects of maternal MTHFR and FVL alleles were consistent with estimates from case-control studies. The case-parent triad design may be a useful tool for studies of pregnancy complications such as preeclampsia.

Tests of Gene‐Environment Interaction for Case‐Parent Triads with General Environmental Exposures

As knowledge of the human genome continues to grow, more progress is being made towards not only identifying the genes involved in disease susceptibility but also in defining the synergistic role genes play with environmental exposures. The detection of gene-environment interactions is important as it can offer clinicians a potential means of intervention. The discovery of interactions relies heavily on powerful statistical methods. We present a test, FBAT-I, that can be used to investigate gene-environment interaction. The test uses the case-parent triad design and protects the statistical inference from potential spurious results due to population admixture.

Cleft palate, transforming growth factor alpha gene variants, and maternal exposures: assessing gene-environment interactions in case-parent triads.

We have previously reported a threefold risk of cleft palate only (CPO) among children homozygous for the less common allele A2 at the TaqI marker site of the transforming growth factor alpha gene (TGFA) (Jugessur et al. [2003a] Genet. Epidemiol. 24:230-239). Here we assess possible interaction between the child's TGFA TaqI A2A2 genotype and maternal cigarette smoking, alcohol consumption, use of multivitamins and folic acid. This was done by comparing the strength of genetic associations between strata of exposed and unexposed case-parent triads. We also looked for possible gene-gene interaction with the polymorphic variant C677T of the folic acid-metabolizing gene MTHFR. We analyzed a total of 88 complete CPO triads selected from a population-based study of orofacial clefts in Norway (May 1996-1998). No evidence of interaction was observed with either smoking or alcohol use. The risk associated with two copies of the A2 allele at TGFA TaqI was strong among children whose mothers did not use folic acid (relative risk=4.5, 95% confidence interval=1.3-15.7), and was only marginal among children whose mothers reported using folic acid (RR=1.4, 95% CI=0.2-12.7). Although the interaction between the child's genotypes at TGFA TaqI and MTHFR-C677T was not statistically significant, the effect of the TGFA TaqI A2A2 genotype appeared to be stronger among children with either one or two copies of the T-allele at C677T (RR=4.0, 95% CI=1.1-13.9) compared to children homozygous for the C-allele (RR=1.7, 95% CI=0.2-15.7). In conclusion, we find little evidence of interaction between the child's genotypes at TGFA TaqI and various exposures for cleft palate, with the possible exception of folic acid intake.

Nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase and glutathione S-transferase M1 polymorphisms and childhood asthma.

Nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) M1 are phase II enzymes important in response to oxidative stress, such as occurs during exposure to ozone. We examined the relationship between functionally significant polymorphisms in NQO1 (Pro187Ser) and GSTM1 (homozygous deletion) and asthma risk in children with high lifetime exposure to ozone. We enrolled children with asthma from the allergy referral clinic at a public pediatric hospital in Mexico City, together with their parents. We assayed for the Pro187Ser polymorphism in NQO1 using a polymerase chain reaction-restriction fragment length polymorphism assay and for the presence of GSTM1 by polymerase chain reaction among 218 case-parent triads. We did not find strong evidence of an association between NQO1 genotype alone and asthma risk. However, among subjects with homozygous deletion of GSTM1, carriers of a serine allele were at significantly reduced risk of asthma compared with Pro/Pro homozygotes (relative risk = 0.4; 95% confidence interval, 0.2-0.8). The p value for difference in relative risk for NQO1 by GSTM1 genotype = 0.013. These data are consistent with a protective effect of the NQO1 Ser allele in this population of GSTM1-null children with high ozone exposure.

Exploring the effects of methylenetetrahydrofolate reductase gene variants C677T and A1298C on the risk of orofacial clefts in 261 Norwegian case-parent triads.

Folic acid and the methylenetetrahydrofolate reductase (MTHFR) gene have both been implicated in the etiology of orofacial clefts. The authors selected 261 case-parent triads (173 cases with cleft lip with or without cleft palate (CL/P) and 88 cases with cleft palate only (CPO)) from a Norwegian population-based study of orofacial clefts (May 1996-1998). A case-parent triad design was used to examine whether MTHFR variants C677T and A1298C, and their haplotypes, are risk factors for orofacial clefts. Among CL/P cases, the child's genotype at C677T or A1298C did not influence the risk. However, children of mothers carrying the C677T variant allele had a lower risk of CL/P. For CPO, children carrying the C677T variant allele had about a twofold increased risk, whereas the mother's genotypes did not contribute to the risk. The haplotype-based transmission/disequilibrium test showed that except for 677T/1298A (p = 0.06), none of the other haplotypes showed evidence of excess transmission to the offspring. The authors also explored interaction of C677T with maternal use of folic acid among children with CPO. Surprisingly, the risk associated with the child's carrying either CT or TT was higher (fourfold) when the mother used folic acid. These findings suggest a possible role of MTHFR and folic acid in the causation of orofacial clefts, but the strength and direction of these effects remain to be clarified.

Variants of developmental genes (TGFA, TGFB3, and MSX1) and their associations with orofacial clefts: a case-parent triad analysis.

We selected 262 case-parent triads from a population-based study of orofacial clefts in Norway, and examined variants of developmental genes TGFA, TGFB3, and MSX1 in the etiology of orofacial clefts. One hundred seventy-four triads of cleft lip cases (CL+/-P) and 88 triads of cleft palate only cases (CPO) were analyzed. There was little evidence for an association of any of these genes with CL+/-P. The strongest association was a 1.7-fold risk with two copies of the TGFB3-CA variant (95% CI=0.9-3.0). Among CPO cases, there was a 3-fold risk with two copies of the TGFA TaqI A2 allele, and no increase with one copy. Assuming this to be a recessive effect, we estimated a 3.2-fold risk among babies homozygous for the variant (95% CI=1.1-9.2). Furthermore, there was strong evidence of gene-gene interaction. While there was only a weak association of the MSX1-CA variant with CPO, the risk was 9.7-fold (95% CI=2.9-32) among children homozygous for both the MSX1-CA A4 allele and the TGFA A2 allele. No association of CPO with the TGFA variant was seen among the other MSX1-CA genotypes. In conclusion, no strong associations were found between CL+/-P and variants at these three genes. There was a possible recessive effect of the TGFA TaqI variant on the risk of CPO, with a 3-fold risk among children homozygous for the variant. The effect of this TGFA genotype was even stronger among children homozygous for the MSX1-CA A4 allele, raising the possibility of interaction between these two genes.

Detecting genotype combinations that increase risk for disease: maternal-fetal genotype incompatibility test.

Biological mechanisms that involve gene-by-environment interactions have been hypothesized to explain susceptibility to complex familial disorders. Current research provides compelling evidence that one environmental factor, which acts prenatally to increase susceptibility, arises from a maternal-fetal genotype incompatibility. Because it is genetic in origin, a maternal-fetal incompatibility is one possible source of an adverse environment that can be detected in genetic analyses and precisely studied, even years after the adverse environment was present. Existing statistical models and tests for gene detection are not optimal or even appropriate for identifying maternal-fetal genotype incompatibility loci that may increase the risk for complex disorders. We describe a new test, the maternal-fetal genotype incompatibility (MFG) test, that can be used with case-parent triad data (affected individuals and their parents) to identify loci for which a maternal-fetal genotype incompatibility increases the risk for disease. The MFG test adapts a log-linear approach for case-parent triads in order to detect maternal-fetal genotype incompatibility at a candidate locus, and allows the incompatibility effects to be estimated separately from direct effects of either the maternal or the child's genotype. Through simulations of two biologically plausible maternal-fetal genotype incompatibility scenarios, we show that the type-I error rate of the MFG test is appropriate, that the estimated parameters are accurate, and that the test is powerful enough to detect a maternal-fetal genotype incompatibility of moderate effect size.

Relative risk for genetic associations: the case-parent triad as a variant of case-cohort design.

The contribution of this paper is to conceptualize the case-parent triad within an epidemiological framework. We propose that the case-parent triad design is a variant of the case-cohort design. The affected offspring of case-parent triads come from a source cohort of all offspring of parents in a population. We first demonstrate that if the source cohort is restricted to offspring of a certain parental mating type then the relative risk in relation to genetic exposure can be estimated simply from the ratio of the number of exposed to the number of unexposed affected offspring. We then extend the logic to studies including offspring of all parental mating types; provided that the allele frequencies and possible parental mating types are specified, a valid relative risk can still be estimated. Compared to prior descriptions of the case-parent triad design, the proposed approach is readily understandable, epidemiologically meaningful and provides a relatively simple perspective for estimating valid measure of effect. Also, by allowing the potential sources of selection bias to be revealed more easily the design is made more accessible both conceptually and practically to epidemiologists.

Transmission/disequilibrium test when neither parent is available in some families: a non-iterative approach.

The transmission/disequilibrium test (TDT) is a simple method of detecting linkage between a marker locus and a disease-susceptibility locus. The test requires genotype data from the affected individuals (the probands) and their parents, the 'case-parents triads'. However, missing data from parents often complicate the situations. Several non-iterative methods, such as the sibling-based TDT, the reconstruction-combined TDT, and the 1-TDT have been proposed to deal with the problem. But when the father, the mother, and other non-proband siblings in a family are all unavailable for study, that family has to be excluded from analysis by any of the above methods. The author proposes a new non-iterative method to deal with the situation when the data contain some 'orphaned' probands. The method utilises the complete data from case-parents triads to infer probabilistically the missing parental genotypes, under the assumption that the missingness is independent of genotype and ethnic origin. The methods can be tailored to specific genetic models, such as the gene-dose, dominance, and recessive alternatives. Simulation analysis shows that, under the null hypothesis of no linkage, the proposed method is not prone to produce an excess of false positive results, even in complex situations such as population stratification or assortive mating. Under the linkage alternative, the new method recaptures much of the information that would be lost by discarding the orphaned probands. The method provides a way for dealing with the situation where neither parents nor siblings are available in a TDT study.

Power Calculations for a General Class of Tests of Linkage and Association That Use Nuclear Families with Affected and Unaffected Sibs

Family-based tests of association are now often used when trying to fine-map a disease susceptibility locus. Recently, several tests of linkage and association have been proposed that use nuclear families with multiple affected and unaffected sibs rather than just case–parent triads. In this paper we propose a test that generalizes these previous tests. Formulae are derived to calculate the power of the test for a randomly mating population. These power calculations are used to determine conditions under which it is advantageous to include unaffected sibs in the analysis.

Association between historically high frequencies of neural tube defects and the humanT homologue of mouseT (Brachyury)

The human T developmental gene has been implicated in the etiology of neural tube defects (NTDs) on the basis both of mouse studies of its homologue, T (Brachyury), and of allelic association in a Caucasian population. We have investigated the frequency of the T allelic variant TIVS7-2 in 218 Irish NTD case-parent triads. This population showed the same trend as previously reported, with an excess of the TIVS7-2 allele among cases. Log-linear modeling of case and maternal genotypic effects within families indicated that TIVS7-2 was elevated in cases (relative risk, RR = 1.36) but not in mothers (RR = 0.91). The TIVS7-2 allele is markedly associated with cases born before 1980 (RR = 2.09; CI = 1.23-3.55; corrected p = 0.030), but not with more recent cases (RR = 0.92). Cases carrying a TIVS7-2 allele did not show any increased tendency to be homozygous for the thermolabile variant of the folate-dependent enzyme 5,10-methylene tetrahydrofolate reductase, which is an established genetic risk factor for NTDs. Since the incidence of NTDs has declined markedly in Ireland over the last few decades, we suggest that the T-associated risk is potentiated by nutritional or environmental risk factor(s), the impact of which have been diminishing over time.