Volume 21, Issue 2, June 2005

Table of Contents 21-2

ROMA – A New Addition to Cytogenetic Analysis

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The resolution of cytogenetic analysis has steadily improved with the implementation of new techniques such as high-resolution banding, FISH analysis for microdeletions and subtelomeric rearrangments, multichromosome painting and, most recently, chromosomal comparative genomic hybridization (CGH) to assess copy number variation. CGH on genomic microarrays allows for high resolution scanning. This is usually accomplished on bacterial artificial chromosome (BAC) arrays, which, when ordered to cover the genome, have a theoretical resolution of 100 Kb to 200 Kb, although clinical application has largely been limited to ≥1 Mb resolution. A group headed by Dorothy Warburton has now introduced a new very high resolution genomic scanning technique referred to as Representational Oligonucleotide Microarray Analysis (ROMA). ROMA involves digesting a subject’s DNA followed by PCR-based amplification of the resulting small DNA fragments to generate a representation of the genome, which is hybridized against a large number of probes on chips—85 000 in this study—to give a resolution of approximately 35 kb. The results are subjected to extensive computational analysis to plot segmental deletions and duplications against current human gene map and sequence databases. To validate the technique, ROMA was carried out in a blinded fashion on 3 patients in whom the authors had documented a cytogenetic abnormality by conventional cytogenetic analysis and FISH. Patient 1 had a complex rearrangement which included a balanced translocation between the long arms of chromosomes 3 and 10 and an interstitial deletion of chromosome 13; the karyotype was 46,XY,t(3:10)(q23;q11.2),del(13)(q14.3q21.2). Patient 2 had a large deletion of chromosome 4q; karyotype was 46,XY,del(4)(q12q21.2). Patient 3 had an unbalanced terminal rearrangement with an additional copy of 16q subtelomeric probe on the end of a small acrocentric chromosome and a deleted copy of the 22q subtelomeric probe. The karyotype was 46,XX,der(22)t(16:22)(q24.3;q13.3)pat. The father carried a balanced translocation. ROMA confirmed all 3 previously defined abnormalities and provided additional information. Patient 1 best illustrates the latter as depicted in the figure. ROMA showed no evidence of lost or gained DNA sequence associated with the balanced translocation. However, it revealed that the cytogenetically visible interstitial deletion of 13q consisted of 2 distinct noncontiguous deletions. The first deletion detected by 221 probes extended from 53 075 360 to 61 396 479 bp on the chromosome 13 bp map. The second deletion detected by 68 probes extended from 72 882 686 to 74 827 260 bp. Plotting these refined deletions on the chromosome 13 gene map allowed identification of which specific genes were deleted and prompted speculation on how the deletion of these genes could contribute to the clinical phenotype. Interestingly, patient 1 had few clinical abnormalities, which was surprising given the 10 Mb size of the deletion. However, ROMA revealed that the deletion mapped to a gene-poor region of chromosome 13, presumably explaining the discrepancy. The authors caution that ROMA detects smaller-sized copy number differences referred to as copy number polymorphisms, or CNPs, that reflect structural polymorphisms present in the normal population. However, they point out that these can be distinguished from deletions and duplications reported here and that ROMA has several advantages over BAC microarray systems, including higher resolution, higher signal-to-noise ratio, flexible chip design, and powerful computational analysis to yield meaningful information.

Jobanputra V, Sebat J, Troge J, Chung W, Anyane-Yeboa K, Wigler M, Warburton D. Application of ROMA (representational oligonucleotide microarray analysis) to patients with cytogenetic rearrangements. Genet Med. 2005;7(2):111–118.

Editor’s Comment: ROMA provides a valuable link between cytogenetics and molecular genetics and a means to begin to understand how clinical features associated with cytogenetic abnormalities can be coupled to an abnormal copy number of specific genes. The technique is not widely available and is expensive, but as the authors note the cost should come down, potentially making it a highly useful tool in the assessment of infants and children with unexplained clinical features suggestive of a chromosomal abnormality.

William A. Horton. MD

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