Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.     

Extensive natural variation for cellular hydrogen peroxide release is genetically controlled

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H₂O₂ release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H₂O₂ release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10−8) and 54 suggestive associations (p<1.00×10−5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H₂O₂ release was observed in Down Syndrome (DS) individuals (p<2.88×10−12). Taken together, our results show strong evidence of genetic control of H₂O₂ in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.     

YAC cloning of DNA embedded in an agarose matrix

Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200-1000 kb) and efficiency of transformation of ligation products (200-1000 cfu/micrograms) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.     

Chromosome 21: a small land of fascinating disorders with unknown pathophysiology

In the year 2000 we celebrated the sequencing of the entire long arm of human chromosome 21. This achievement now provides unprecedented opportunities to understand the molecular pathophysiology of trisomy 21, elucidate the mechanisms of all monogenic disorders of chromosome 21, and discover genes and functional sequence variations that predispose to common complex disorders. All of that requires the functional analysis of gene products in model organisms, and the determination of the sequence variation of this chromosome.