The Genom of Homo sapiens.pdf

328 LI ET AL.Figure 5. Comparison of CGH and aCGH profiles for chromosome 5. A and B are aCGH profiles of normal vs. normal hybridizationand patient vs. normal control hybridization, respectively. The X axis represents linear positions of clones in 100 kb along the chromosomefrom p to q arm. Y axis is a normalized log 2 fluorescence ratio calculated from a pair of dye-reversal experiments. (C)Metaphase CGH profile of chromosome 5 of the same patient. Both the aCGH and chromosomal CGH show gain of the terminal endof 5q. In addition, several clones along the terminal end of 5p show a gain.genomic rearrangements exist in idiopathic MR, we initiatedthe genetic analysis of patients with idiopathic MRusing human WGAs. All the recruited patients had mildMR as determined by formal testing (IQ 50–70). Followinga thorough genetic evaluation at Baylor College ofMedicine, none of the patients had detectable genetic etiologysuch as fragile X, microdeletion syndromes, subtelomererearrangements, serious neurologic impairments(such as structural brain anomaly on imagingstudies), or severe dysmorphic features.We used the whole-genome BAC arrays for primaryscreening of potential regions of subtle chromosomal gainor loss in eight patients. For each patient sample we performedtwo hybridizations using the principle of dye reversal(Cai et al. 2002). The false-positive predictions dueto random fluorescence ratio drifts or ratio normalizationartifacts are greatly reduced by dye-reversal experiments.However, because the number of hybridization targets ishigh (over 20,000 spots), the number of gains and losseswas also high in each of the eight patients, suggesting thata number of them are false-positive gains or losses. Sincethe verification of every clone showing a copy numberchange using conventional techniques such as FISHwould be very time-consuming, we created subarrays containingclones from areas of loss or gain. We selectedclones that were balanced (control clones) as well asclones showing a gain or loss. These small subarrays hadfive replicate spots for each of the selected clones. Dye-reversalhybridizations were repeated for subarrays for eachsample, resulting in five pairs of data points for each positiveand control clone. Arbitrarily, we defined a verystringent cutoff in which a clone must have at least fourpairs of ratio data points outside the 2.5 S.D. units from thenormalized average ratio of control clones to qualify for atrue gain or loss. Among the 959 control BAC clones thatdid not show gain or loss in the HWG arrays, no clone wasfound to pass this cutoff and was therefore truly balanced.On the other hand, and as expected, only a fraction of thecandidate positive BAC clones were verified in the secondaryscreen using these verification subarrays (Table 1).It is obvious that a verification step is necessary in order toeliminate false positives due to experimental variations onthe HWG arrays. The results of our preliminary studiesshowed that gains of clones are much more frequent thanlosses. As previously discussed, consistent gains can befalse positives caused by cross-hybridization from a regioncontaining low copy repeats. Thus, further study isrequired to verify whether all regions showing gains aretrue positives.CONCLUSIONWe demonstrate that high-resolution array-based comparativegenomic hybridization can serve as a powerfulapproach for discovering small chromosomal rearrangementsin a genome as complex as the human genome.Preliminary analysis of patients with known chromosomalabnormalities, as well as patients with normal karyotypesand idiopathic MR, were encouraging and identifiedareas of gain and loss. The biggest challenge in thefuture will be to distinguish between the changes due tonormal variability in the genome versus gains and lossesas the cause of the disorders. Population studies involvinglarger numbers of normal individuals are therefore requiredto establish the baseline variability and to identifyregions most prone to polymorphisms. Large-scale studiesof patients with mild MR are also required to resolvethe issue of whether a combination of multiple polymorphicgenomic imbalances may contribute to mild MR. Inaddition, the development of high-throughput molecularTable 1. Losses and Gains in Idiopathic MR Patient in PrimaryHWG Arrays Screening and Secondary Subarray VerificationPrimary HWG array screen Subarray verificationSample loss BAC gain BAC loss BAC gain BAC26–03 20 80 0 1527–03 25 155 0 3235–03 9 87 0 437–03 119 156 3 214–03 193 218 5 1618–03 112 341 2 1619–03 25 97 1 017–03 21 200 0 81