Damji K F, Gallione C J, Allingham R R, Slotterbeck B, Guttmacher A E, Pasyk K A, Vance J M, Pericak-Vance M A, Speer M C, Marchuk D A
University of Ottawa Eye Institute, Ontario, Canada.
Hum Genet. 1998 Feb;102(2):207-12. doi: 10.1007/s004390050679.
DNA pooling is an efficient method to rapidly perform genome-wide linkage scans in autosomal recessive diseases in inbred populations where affected individuals are likely to be homozygous for alleles near the disease gene locus. We wanted to examine whether this approach would detect linkage in autosomal dominant (AD) disorders where affected individuals may share one allele identical by descent at loci tightly linked to the disease. Two large outbred pedigrees in which the AD diseases familial venous malformation (FVM) and hereditary hemorrhagic telangiectasia (HHT1), linked to 9p and 9q, respectively, were investigated. Separate pools of DNA from affected (n = 21 for FVM and 17 for HHT1) and unaffected family members (n = 9 FVM and HHT1), and 25 unrelated population controls were established. Polymorphic markers spanning chromosome 9 at approximately 13.5-cM intervals were amplified using standard PCR. Allele quantitation was performed with a fluorimager. Visual inspection of allele intensities and frequency distributions suggested a shift in frequency of the most common allele in the affecteds lane when compared to control lanes for markers within 30 cM of the FVM and HHT1 loci. These subjective assessments were confirmed statistically by testing for the difference between two proportions (one-sided; P < or = 0.05). When using population controls, the true-positive rates for FVM and HHT1 were 5/5 and 2/5 markers, respectively. False-positive rates for FVM and HHT1 were 3/9 and 2/9, respectively. In both AD diseases investigated, quantitative DNA pooling detected shifts in allele frequency, thus identifying areas of known linkage in most cases. The utility of this technique depends on the size of the pedigree, frequency of the disease-associated allele in the population, and the choice of appropriate controls. Although the false-positive rate appears to be high, this approach still serves to reduce the amount of overall genotyping by about 60%. DNA pooling merits further investigation as a potential strategy in increasing the efficiency of genomic linkage scans.
DNA池化是一种有效的方法，可在近亲繁殖群体中的常染色体隐性疾病中快速进行全基因组连锁扫描，在这些群体中，受影响个体很可能在疾病基因位点附近的等位基因上是纯合的。我们想研究这种方法是否能在常染色体显性（AD）疾病中检测到连锁，在这类疾病中，受影响个体可能在与疾病紧密连锁的位点上共享一个通过遗传相同的等位基因。我们研究了两个大型远交系家系，其中分别与9p和9q连锁的AD疾病家族性静脉畸形（FVM）和遗传性出血性毛细血管扩张症（HHT1）。从受影响（FVM为21例，HHT1为17例）和未受影响的家庭成员（FVM和HHT1均为9例）中分别建立DNA池，并设置了25个无关的群体对照。使用标准PCR扩增以大约13.5厘摩间隔跨越9号染色体的多态性标记。用荧光成像仪进行等位基因定量。通过目视检查等位基因强度和频率分布发现，与FVM和HHT1位点30厘摩范围内标记的对照泳道相比，受影响个体泳道中最常见等位基因的频率发生了偏移。通过检验两个比例之间的差异（单侧；P≤0.05）对这些主观评估进行了统计学确认。使用群体对照时，FVM和HHT1的真阳性率分别为5/5和2/5个标记。FVM和HHT1的假阳性率分别为3/9和2/9。在所研究的两种AD疾病中，定量DNA池化均检测到等位基因频率的偏移，因此在大多数情况下识别出了已知的连锁区域。该技术的实用性取决于家系大小、群体中疾病相关等位基因的频率以及合适对照的选择。尽管假阳性率似乎较高，但这种方法仍可将总体基因分型量减少约60%。DNA池化作为提高基因组连锁扫描效率的潜在策略值得进一步研究。
