With an ever-increasing resource of validated single-nucleotide polymorphisms (SNPs), the limiting factors in genome-wide association analysis have become genotyping capacity and the availability of DNA. We provide a proof of concept of the use of pooled DNA as a means of efficiently screening SNPs and prioritizing them for further study. This approach reduces the final number of SNPs that undergo full, sample-by-sample genotyping as well as the quantity of DNA used overall. We have examined 15 SNPs in the cholesteryl ester transfer protein (CETP) gene, a gene previously demonstrated to be associated with serum high-density lipoprotein cholesterol levels. The SNPs were amplified in two pools of DNA derived from groups of individuals with extremely high and extremely low serum highdensity lipoprotein cholesterol levels, respectively. P values <0.05 were obtained for 14 SNPs, supporting the described association. Genotyping of the individual samples showed that the average margin of error in frequency estimate was Ϸ4% when pools were used. These findings clearly demonstrate the potential of pooling techniques and their associated technologies as an initial screen in the search for genetic associations.A ssociation testing may be regarded as a comparison of allele frequency between cases and controls, in which a statistically significant difference is used to implicate a locus in a disease etiology. The methodology relies on the investigator testing either the causal variant itself or a marker in linkage disequilibrium (LD) with the causal variant. It is now well known that the distance over which LD extends is highly variable, not only between populations (1-3) but also across genomic regions (4, 5). A comprehensive genome scan by association analysis is therefore likely to require tens or hundreds of thousands of markers (6, 7).To date, we have performed validation experiments on Ϸ200,000 putative single-nucleotide polymorphisms (SNPs) from the public domain, and the confirmation rate is Ϸ60% in a Caucasian sample (unpublished results). The theoretical advantages of analyzing these SNPs by an association rather than by a linkage approach are clear (8). The reality, however, is hindered by the expense of generating the necessarily large number of genotypes. The use of pooled DNA has been offered as an alternative in the case of qualitative traits (9-11), but practical extensions to quantitative traits have not been presented thus far.To evaluate the applicability of pooled samples, SNPs in the cholesteryl ester transfer protein (CETP) gene were tested for association by using a pair of pools containing DNA from individuals with low and high serum high-density lipoprotein cholesterol (HDL-C) levels, respectively. CETP is known to play an essential role in reverse cholesterol metabolism. It is believed to mediate the exchange of cholesteryl ester in HDL-C for triglyceride in low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL) (12)(13)(14).

The imprinted H19 gene produces a fully processed transcript that does not exhibit any conserved open reading frame between mouse and man. Although transcriptional control elements associated with the mouse H19 locus have been shown to control the neighboring Igf2 gene in cis, the prevailing view is that the cytoplasmic H19 transcript does not display any function. In contrast to earlier reports, we show here that the H19 transcript is associated with polysomes in a variety of cell types, in both mouse and man. A possible transfunction of the H19 gene is suggested by a reciprocal correlation in trans between cytoplasmic H19 and IGF2 mRNA levels, as well as IGF2 mRNA translatability. We discuss these results in terms of their challenge to the prevailing dogma on the function of the enigmatic H19 gene, as well as with respect to the ontogeny of the Beckwith-Wiedemann syndrome, and propose that the human H19 gene is an antagonist of IGF2 expressivity in trans.

Potential cis‐acting regulatory elements of the human platelet derived growth factor‐B (PDGF‐B) gene were identified by DNase I hypersensitive site mapping. The transcription unit was examined for the presence of hypersensitive sites in chromatin DNA isolated from human term placental cytotrophoblasts, human placental fibroblasts, the JEG‐3 choriocarcinoma cell line and the U2‐OS osteosarcoma cell line. A number of cell type‐specific hypersensitive sites were identified, all within the 1st intron. Transient transfection of JEG‐3 cells with CAT constructs containing regions of the c‐sis 1st intron linked to the basal c‐sis promoter identified a cell type‐specific positive regulatory activity within the intron, composed of at least two distinct elements. One element appeared to be specific for JEG‐3 cells, while the other was also active in U2‐OS cells. The overall positive regulatory activity of the 1st intron region was specific for JEG‐3 cells, but did not function as a classically defined enhancer, as it was orientation‐dependent (unless stably integrated into chromatin DNA). In addition, the activator appears to require interaction with the c‐sis promoter, as little or no activation was seen when either the SV40 or human beta‐globin promoters were substituted for the c‐sis promoter. The 1st intron also contained a negative regulatory element, which was specific for U2‐OS cells and silenced an abnormally high basal c‐sis promoter activity in these cells. The complexity of the transcriptional control of the PDGF‐B gene is discussed.

Obesity is a multifactorial disorder with a complex phenotype. It is a significant risk factor for diabetes and hypertension. We assessed obesity-related traits in a large cohort of twins and performed a genome-wide linkage scan and positional candidate analysis to identify genes that play a role in regulating fat mass and distribution in women. Dizygous female twin pairs from 1094 pedigrees were studied (mean age 47.0711.5 years (range 18 -79 years)). Nonparametric multipoint linkage analyses showed linkage for central fat mass to 12q24 (141 cM) with LOD 2.2 and body mass index to 8q11 (67 cM) with LOD 1.3, supporting previously established linkage data. Novel areas of suggestive linkage were for total fat percentage at 6q12 (LOD 2.4) and for total lean mass at 2q37 (LOD 2.4). Data from follow-up fine mapping in an expanded cohort of 1243 twin pairs reinforced the linkage for central fat mass to 12q24 (LOD 2.6; 143 cM) and narrowed the -1 LOD support interval to 22 cM. In all, 45 single-nucleotide polymorphisms (SNPs) from 26 positional candidate genes within the 12q24 interval were then tested for association in a cohort of 1102 twins. Single-point Monks -Kaplan analysis provided evidence of association between central fat mass and SNPs in two genes -PLA2G1B (P ¼ 0.0067) and P2RX4 (P ¼ 0.017). These data provide replication and refinement of the 12q24 obesity locus and suggest that genes involved in phospholipase and purinoreceptor pathways may regulate fat accumulation and distribution.

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