Linzy Hill

DNA by Mail: An Inexpensive and Noninvasive Method for Collecting DNA Samples from Widely Dispersed Populations

As specific genes are identified that are associated with behavior, it becomes increasingly important for behavioral geneticists to be able to incorporate these genes in their research. Rather than using blood, DNA can be extracted from cheek swabs, which makes it possible to obtain DNA inexpensively by mail from large, widely dispersed individuals. The purpose of this paper is to recommend this technique to the behavioral genetics community and to present results of our use of this technique to obtain DNA by mail for 114 2-year-olds and 116 adults.

The serotonin transporter gene and peer-rated neuroticism

POLYMORPHISMS in the serotonin transporter gene (5HTT) have been reported to be associated with neuroticism (emotionality) and with depression. A recent report of an association between 5HTT and neuroticism involved unselected samples and self-report questionnaires.1 We attempted to extend these findings using a selected extremes design and peer ratings. From a sample of 2085 individuals, each assessed on neuroticism by two independent peers, we selected 52 individuals from the top 5% and 54 individuals from the bottom 5%. No association was found for either a functional 44 bp insertion/deletion polymorphism in 5HTT regulatory sequence (5HTTLPR) or for a non-functional variable number tandem repeat 5HTT polymorphism.

A Genome-Wide Scan of 1842 DNA Markers for Allelic Associations with General Cognitive Ability: A Five-Stage Design Using DNA Pooling and Extreme Selected Groups

All measures of cognitive processes correlate moderately at the phenotypic level and correlate substantially at the genetic level. General cognitive ability (g) refers to what diverse cognitive processes have in common. Our goal is to identify quantitative trait loci (QTLs) associated with high g compared with average g. In order to detect QTLs of small effect size, we used extreme selected samples and a five-stage design with nominal alpha levels that permit false positive results in early stages but remove false positives in later stages. As a first step toward a systematic genome scan for allelic association, we used DNA pooling to screen 1842 simple sequence repeat (SSR) markers approximately evenly spaced at 2 cM throughout the genome in a five-stage design: (1) case-control DNA pooling (101 cases with mean IQ of 136 and 101 controls with mean IQ of 100), (2) case-control DNA pooling (96 cases with IQ >160 and 100 controls with mean IQ of 102), (3) individual genotyping of Stage 1 sample, (4) individual genotyping of Stage 2 sample, (5) transmission disequilibrium test (TDT; 196 parent-child trios for offspring with IQ >160). The overall Type I error rate is 0.000125, which robustly protects against false positive results. The numbers of markers surviving each stage using a conservative allele-specific directional test were 108, 6, 4, 2, and 0, respectively, for the five stages. A genomic control test using DNA pooling suggested that the failure to replicate the positive case-control results in the TDT analysis was not due to ethnic stratification. Several markers that were close to significance at all stages are being investigated further. Relying on indirect association based on linkage disequilibrium between markers and QTLs means that 100,000 markers may be needed to exclude QTL associations. Because power drops off precipitously for indirect association approaches when a marker is not close to the QTL, we are not planning to genotype additional SSR markers. Instead we are using the same design to screen markers such as cSNPs and SNPs in regulatory regions that are likely to include functional polymorphisms in which the marker can be presumed to be the QTL.

A functional polymorphism in the succinate-semialdehyde dehydrogenase (aldehyde dehydrogenase 5 family, member A1) gene is associated with cognitive ability

Succinate-semialdehyde dehydrogenase (SSADH) deficiency is a rare cause of learning disability. We have investigated SSADH to assess its contribution to cognitive ability in the general population in both case–control- and family-based analyses. Sequence analysis of SSADH revealed four changes affecting the encoded protein, only one of which had a minor allele whose frequency is even moderately common. We genotyped this functional polymorphism in 197 high-IQ cases, 201 average-IQ controls and 196 parent high-IQ offspring trios. The minor allele was significantly less frequent in high-IQ cases and was significantly less frequently transmitted by parents to high-IQ subjects than chance expectation. A previous study has shown that the minor allele encodes a lower activity enzyme than the major allele. These data suggest that higher SSADH activity is associated with higher intelligence across the general population. The effect is small, with each allele having an effect size translating to about 1.5 IQ points.

DNA pooling and dense marker maps: a systematic search for genes for cognitive ability

Pooling DNA from subjects within a group and comparing the pooled DNA across groups for a dense map of DNA markers offers a solution to the conundrum that linkage is systematic but not powerful whereas allelic association is powerful but not systematic. We used DNA pooling to screen 66 markers on chromosome 22 in original and replication samples of children of high general cognitive ability (g) and controls of average g. Although none of these markers survived our three-stage screening design (original pooling, replication pooling, individual genotyping), the results of DNA pooling were largely confirmed by individual genotyping. We can therefore exclude associations of major effect size on chromosome 22 for g, a key variable for cognitive neuroscience research on learning and memory.

Dopamine markers and general cognitive ability

BECAUSE general cognitive ability (g) is among the most heritable behavioural traits, it is a reasonable target for a search for quantitative trait loci (QTLs). We used a selected-extremes design to test candidate genes for allelic association with g. Polymorphisms in four genes in the dopamine system (DRD2, DRD3, DRD4, DAT1) were genotyped for 51 high g children with IQ scores > 130 and for 51 average g control children. No significant allelic or genotypic differences were found between the high g and average g groups for these markers of the dopamine system, even though the selected-extremes design provides power to detect QTL associations that involve a relative risk of about 1.5.

A Quantitative Trait Locus Not Associated with Cognitive Ability in Children: A Failure to Replicate

In 1998 in this journal, we reported results suggesting that a gene (insulin-like growth factor-2 receptor, IGF2R) on chromosome 6 was associated with general cognitive ability ( g ) in two independent casecontrol samples of children selected for very high g (cases) or for average g (controls; Chorney et al., 1998). The suggested association with the IGF2R gene was especially interesting because IGF2R had recently been shown to be active in brain regions most involved in learning and memory (Wickelgren, 1998). The IGF2R association emerged in the first step of our attempts to move in the direction of a systematic genome scan for association in which we genotyped 47 simple-sequence repeat (SSR) DNA markers on the long arm of chromosome 6 (Chorney et al., 1998). In two independent samples, we found significant associations for an SSR marker that happened to be in the IGF2R gene. The SSR marker in the IGF2R gene involved two nucleotide base pairs of DNA (TG in this case) that repeat several times; the number of repeats varies and is stably inherited. One allele (allele 5) for this TG repeat marker yielded frequencies of about 30% in the two highg groups and about 15% in the two control groups of average g , with a p value of .02 in each of the two samples and a combined p value of .001. We also found higher than average allele 5 frequencies for two other groups of highg individuals who were either especially high in mathematics ability or especially high in verbal ability, although these two additional comparisons were only marginally significant ( p s .06 and .08). We concluded that “our results suggest that the IGF2R gene is associated with high g ” (Chorney et al., 1998, p. 164). Because of the likelihood of false positive results in the quest for quantitative trait loci (QTLs) of small effect size using many DNA markers, replication is crucial (Cardon & Bell, 2001). We had hoped that other laboratories would attempt to replicate the IGF2R association with g, but 4 years after the original publication in this journal, we are not aware of such efforts. For this reason, we conducted our own replication analysis. The purpose of the present letter is to report results for the IGF2R gene for a new sample that is as large as the two previously reported samples combined. A more detailed description of this replication study and its implications is available on the Web at www.sgdp.org.uk. The new sample was obtained in the same manner as in the original study (Chorney et al., 1998). The samples were restricted to non-Hispanic, Caucasian participants to reduce the likelihood that differences in marker allele frequencies between the groups would be due to ethnic stratification. For the new sample, DNA was extracted from cheek swabs (Freeman et al., 1997), yielding an average of 50 g of DNA for each individual. Genotyping was conducted using procedures similar to those described in our earlier publication (Chorney et al., 1998). The results we reported for the combined original and replication samples yielded an allelic frequency for allele 5 of 32% in the highg group and 16% in the control group, 2 (1, N 186) 12.41, p .0004. In the present sample, the frequency of allele 5 was 19% in the highg group and 24% in the control group, 2 (1, N 188) 1.54, p .22. Tests of other alleles and genotypic comparisons also failed to replicate our previous results. After publishing our article using the TG repeat marker in the IGF2R gene, we discovered that there is another polymorphism within the sequence of 100 base pairs that we had amplified (Hol, Geurds, Hamel, & Mariman, 1992). This polymorphism, which involves the insertion or deletion of a sequence of 4 base pairs (ACAA), is within the forward primer itself and would complicate analysis of our original marker because DNA strands with the ACAA deletion would not be amplified (Hill, Craig, Chorney, Chorney, & Plomin, 1999). To address this problem, we genotyped the samples again using a different set of primers (Hol et al., 1992) with a forward primer upstream from the original primer. The new primer set thus encompassed both the ACAA insertion/deletion polymorphism and the TG repeat marker. Analyses taking into account both the TG repeat marker and the ACAA insertion/deletion polymorphism yielded similar results in that the significant results found in our previous study were not replicated in the present sample. (For details, see Hill, 2002.) The present sample was as large as our original and replication samples combined and provided 98% power to detect a QTL association with an effect size as small as 1%. Thus, we conclude that the TG repeat polymorphism in IGF2R is not associated with high g . This finding joins a long list of reported associations that have failed to be replicated (Cardon & Bell, 2001), and those associations that have shown some replication, such as dopamine gene associations with hyperactivity (Thapar, 2003), have not done so consistently. The most likely reason is that the ubiquitous heritability of complex traits such as cognitive abilities and disabilities is due to many QTLs, each of very small effect size, which means that substantially greater power is needed to identify and especially to replicate QTLs (Cardon & Bell, 2001). It has been recommended that QTL association studies be designed to break the 1% QTL barrier, which no behavioral study has yet done (Plomin, DeFries, Craig, & McGuffin, 2003).

Failure To Replicate a QTL Association between a DNA Marker Identified by EST00083 and IQ.

Abstract In a paper published in this journal, a possible QTL association was reported between general cognitive ability and a marker, identified by an expressed sequence tag, EST00083 (Skuder et al., 1995). In two small samples, the frequency of the common allele of this DNA marker, which was shown to be in the threonine transfer RNA gene in mitochondrial DNA, was significantly greater in a high-IQ group than in a low-IQ group. As part of the ongoing IQ QTL Project (Plomin et al., 1995), we have attempted to replicate this QTL association. First, we found that the QTL association remained significant when we compared 51 high- and 51 -average IQ subjects, drawn in part from the samples used in the previous report. However, when we examined the association in new samples of 40 extremely high-IQ subjects and 50 average-IQ subjects, the association did not replicate. This underlies the need for replication in case-control studies of allelic association.

Single-nucleotide polymorphism genotyping in DNA pools.

To undertake partial, or complete, genome screens by association-based methodology for quantitative trait loci, multiple individuals have to be screened for large numbers of genetic markers. Consequently, much recent interest has focused on methods enabling accurate allele quantification in pooled deoxyribonucleic acid (DNA) samples. Microsatellites were the favored markers in initial studies, but the extraordinary wealth of data concerning single-nucleotide polymorphisms (SNPs) has turned attention to the quantification of SNP alleles in pools. All such approaches require accurate estimation of DNA concentrations, followed by the preparation of replicate pools, their validation, and application of procedures for determining allele frequencies. This chapter describes the important steps in preparing pools and surveys a variety of techniques that have been proposed for SNP detection. Finally, we describe the application of a generic approach using pooled DNA for detection of allele frequency differences between case and control populations based on primer extension protocols and outline a strategy for estimating SNP allele frequencies employing microarrays.

Validation of single nucleotide polymorphism quantification in pooled DNA samples with SNaPIT. A glycosylase-mediated methods for polymorphism detection method.

Association studies using genome scans to identify quantitative trait loci for multifactorial disorders, with anything approaching reasonable power, have been compromised by the need for a very dense array of genetic markers and large numbers of affected individuals. These requirements impose enormous burdens on the genotyping capacity for most laboratories. DNA pooling has been proposed as a possible approach to reduce genotyping costs and effort. We report on the application of the SNaPIT™ technology to evaluate allele frequencies in pooled DNA samples and conclude that it offers a cost effective, efficient and accurate estimator and provides several advantages over competing technologies in this regard.