CMT 1A new gene research - Determination of genomic copy number with quantitativ

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Hum Mutat. 2006 Mar 15;27(4):376-386

Determination of genomic copy number with quantitative microsphere

hybridization.

Newkirk HL, Rogan PK, Miralles M, Knoll JH.

Laboratory of Genomic Disorders, Children's Mercy Hospitals and

Clinics, University of Missouri-Kansas City, Kansas City, Missouri.

We developed a novel quantitative microsphere suspension

hybridization (QMH) assay for determination of genomic copy number by

flow cytometry. Single copy (sc) products ranging in length from 62

to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from

ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1

(17q21) were conjugated to spectrally distinct polystyrene

microspheres. These conjugated probes were used in multiplex

hybridization to detect homologous target sequences in biotinylated

genomic DNA extracted from fixed cell pellets obtained for

cytogenetic studies. Hybridized targets were bound to phycoerythrin-

labeled streptavidin; then the spectral emissions of both target and

conjugated microsphere were codetected by flow cytometry. Prior

amplification of locus-specific target DNA was not required because

sc probes provide adequate specificity and sensitivity for accurate

copy number determination. Copy number differences were

distinguishable by comparing the mean fluorescence intensities (MFI)

of test probes with a biallelic reference probe in genomic DNA of

patient samples and abnormal cell lines. Concerted 5' ABL1 deletions

in patient samples with a chromosome 9;22 translocation and chronic

myelogenous leukemia were confirmed by comparison of the mean

fluorescence intensities of ABL1 test probes with a HOXB1 reference

probe. The relative intensities of the ABL1 probes were reduced to

0.59+/-0.02 & !ndash;fold in three different deletion patients and

increased 1.42+/-0.01 & !ndash;fold in three trisomic 9 cell lines.

TEKT3 and PMP22 probes detected proportionate copy number increases

in five patients with Charcot-Marie-Tooth Type 1a disease and

chromosome 17p12 duplications. Thus, the assay is capable of

distinguishing one allele and three alleles from a biallelic

reference sequence, regardless of chromosomal context.