Array CGH - College of American Pathologists

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Courtesy: Bassem Bejjani, MD Profile for deletion of 15q11-13. Each clone on the plot is arranged along the x-axis according to its location on chromosome 15. The most proximal (centromeric) long-arm clones are on the left and the most distal/telomeric long-arm clones are on the right. The dark blue line represents the control:patient fluorescence intensity ratios for each clone, whereas the pink line represents the fluorescence intensity ratios obtained from a second hybridization in which the dyes have been reversed (patient:control). Normal chromosome 15 Above: Normal chromosome 15 plot: The normal chromosome 15 plot shows a ratio of 0 on a log2 scale for all clones. Below: Deleted chro- mosome 15 in an individual with Prader-Willi syndrome: The abnormal chromosome 15 plot shows a plot for individual with a typical del(15)(q11q13). The plot of the deletion of 15q11q13 shows a signifi- cant deviation from 0 for those five clones within the deletion region that have a single-copy loss in the individual (arrow). Note that for a deletion, the blue line deviates up and the pink line deviates down. Prader-Willi/Angelman deletion 1.8 1.5 1.2 0.9 0.6 0.3 0 -0.3 -0.6 -0.9 -1.2 -1.5 -1.8 1.8 1.5 1.2 0.9 0.6 0.3 0 -0.3 -0.6 -0.9 -1.2 -1.5 -1.8

Transcript of Array CGH - College of American Pathologists

jani BA, et al. Am J Med Genet A. 2005;1 3 4 :259–267). Dr. Bejjani emphasizes that this is at a rgeted array—one in which every BAC corre-sponds to a genetic locus implicated in a con-genital disord e r. Arrays can be made that coverthe entire genome, but with that type of arraymany results will not be interpretable in clinicalterms.

At Signature, each array CGH analysis in-cludes a male-female comparison—normal DNAof the opposite gender from the test sample—asan internal control. Between this pair of sam-ples, there will always be a gene dosage diff e r-ence for the X chromosome. If a run shows no dif-f e rence between the test and re f e rence samples,but does have the expected diff e rence betweenthe male and female samples, that shows that thelack of diff e rence is not due to a technical defi-ciency in the run.

Each patient dataset is actually made up of twoexperiments on the Signature platform—one inwhich the test and re f e rence DNA samples eachhave a diff e rent dye color and a second in whichthe dyes are switched. Both experiments areplotted on a single graph and should show theexact inverse of each other (see box at right).For technical reasons, data are plotted as log 2,rather than arithmetically.

S i g n a t u re’s first array con-tained 831 BACs covering 126loci. Dr. Bejjani’s laboratory isdeveloping a new array withg reater coverage of the sub-telomeric and pericentro m e r i cregions. Signature first off e re darray CGH as a clinical service inM a rch 2004. “In the first coupleof months we had only a hand-ful of cases,” Dr. Bejjani says. “Now we are do-ing 65 to 70 cases a week. We started with thre eemployees, now we have 20.” To accommodatethe increasing demand, Signature is moving to-w a rd automation. “This technology allows youto automate more easily than conventional cy-

togenetics,” Dr. Bejjani says. “It is not as lim-ited by human skills and training.”

Dr. Beaudet at Baylor also uses BAC ar-rays, but

says, “There is plenty of literature to indicatethat oligo arrays have enormous attraction.Maybe they will be the final format for thist e c h n o l o g y.” They can print BAC arrays nowc o s t - e ff i c i e n t l y, he says: “We can print thearrays and do a complete analysis for a littleover $1,000.” If they switched to oligo ar-rays, simply purchasing the arrays them-selves would cost $1,000 or more, Dr. Beaudetsays. A ffymetrix, which holds the right toprinting oligo arrays, “is unwilling to nego-tiate a license,” according to Dr. Beaudet.Nonetheless, he believes that oligo technol-ogy will eventually predominate. “Signal-to-noise problems can be overcome,” he says.

Just as Dr. Bejjani did, Dr. Beaudet had tovalidate his array in-house (Cheung SW, et al.Genet Med. 2005;7: 422–432). Regarding poly-morphisms, or benign variants, Dr. Beaudetsays, “We have tried to weed out those clones.If we do use one, we check the [healthy] par-ents to see if the same gain or loss is pre s e n tin their genome,” which would indicate thatit is probably not the cause of the child’sp roblem.

“ We are just beginning to offer array CGHon a prenatal basis,” Dr. Beaudet says. “Ithas tremendous attraction. It can diagnose anumber of disorders that would not otherwise bediagnosed.” Examples include diGeorge syn-d rome, Angelman syndrome, and Prader- Wi l l is y n d rome. “For someone having an amniocen-tesis, array CGH becomes a possibility,” Dr.Beaudet says. “Should we be offering all womenan invasive test such as amniocentesis?” he asks.“Some people are arguing that the answer isyes” (Caughey AB, et al. Obstet Gynecol. 2004;103:539–545).

Noninvasive alternatives include finding fe-tal cells in maternal circulation, isolating fetal cellswith a cervical cell collection, and isolating fetalD N A f rom maternal plasma. It is unlikely that

p rolonged presence of cells or DNA f rom pre v i-ous pregnancies would be a problem with the lat-ter two methods, according to Dr. Beaudet. “Infive to 10 years we may be able to do array CGHon noninvasive samples,” he says. “That would

be a huge revolution in geneticdiagnosis.” When they counselp a rents-to-be now they say thatt h e re is “some merit” to doingamniocentesis in all pre g n a n twomen and, if parents elect anamniocentesis, they offer arrayCGH as an additional test.

Array CGH will definitely re-main a sendout for a while, says

D r. Amos Wilson. Laboratories de-siring array CGH analysis will besending samples to either the Sig-n a t u re or Baylor laboratories. Theywould have a choice of how to pro-ceed, Dr. Amos Wilson explains. “Ifthe clinician suspects an unbalancedc h romosome rearrangement, thechoices are not doing routine cyto-genetics at all, or maybe doing ro u-tine cytogenetics to look for larg ealterations, then sending a sample toone of these laboratories.” For now,array CGH will be secondary to ro u-tine cytogenetics but will allow med-ical geneticists to skip FISH, in Dr.Amos Wilson’s view. “CGH couldbe considered superFISH,” she says.“ You do FISH because you suspecta specific chromosome alteration.With array CGH you can look atthe whole genome when you don’thave a suspected target.” Referringlaboratories will also have to makea financial decision, she says. “Lab-oratories make no money if theysend out.”

A case studied by Quest’s Dr. Mo-hammed and Shelley Gunn, MD,PhD, illustrates the power of array

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Courtesy: Bassem Bejjani, MD

Profile for deletion of 15q11-13. Each clone on the plot is arrangedalong the x-axis according to its location on chromosome 15. The mostproximal (centromeric) long-arm clones are on the left and the mostdistal/telomeric long-arm clones are on the right. The dark blue linerepresents the control:patient fluorescence intensity ratios for eachclone, whereas the pink line represents the fluorescence intensity ratiosobtained from a second hybridization in which the dyes have beenreversed (patient:control).

Normal chromosome 15

Above: Normal chromosome 15 plot: The normal chromosome 15 plotshows a ratio of 0 on a log2 scale for all clones. Below: Deleted chro-mosome 15 in an individual with Prader-Willi syndrome: The abnormalchromosome 15 plot shows a plot for individual with a typicaldel(15)(q11q13). The plot of the deletion of 15q11q13 shows a signifi-cant deviation from 0 for those five clones within the deletion regionthat have a single-copy loss in the individual (arrow). Note that for adeletion, the blue line deviates up and the pink line deviates down.

Prader-Willi/Angelman deletion

Dr. Mohammed

Dr. Beaudet

1 . 81 . 51 . 20 . 90 . 60 . 3

0- 0 . 3- 0 . 6- 0 . 9- 1 . 2- 1 . 5- 1 . 8

1 . 81 . 51 . 20 . 90 . 60 . 3

0- 0 . 3- 0 . 6- 0 . 9- 1 . 2- 1 . 5- 1 . 8

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