Utility of array-based technology for detection of chromosomal abnormalities

Chromosome analysis by karyotyping has proven to be a robust technique in detecting the majority of chromosomal abnormalities and is a ‘gold standard’ method for prenatal clinical genetic testing for several decades. This method enables diagnosis of aneuploidy, structural rearrangements, deletions, and duplications of 3-10 Mb. There is a need for additional diagnostic tests with higher resolution since the main disadvantage with this technique is the inability to detect abnormalities smaller than 2 to 3 Mb. Microarray-based genomic copy-number analysis also known as “chromosomal microarray” (CMA) and “molecular karyotyping” circumvents the limitations of conventional cytogenetic techniques. CMA simultaneously evaluates regions across the entire genome with a higher resolution and offers high throughput to analyze patient samples with suspected genome imbalances. Array technology has rapidly taken over cytogenetics laboratories and has become a commonly ordered clinical genetic test for patient populations.

Deletions and duplications of chromosomal segments (copy-number variations or CNVs) constitute a major source of variation between individual humans, underlying human evolution and many diseases from mental illness and developmental disorders to cancer. CNVs are determined by the differences in hybridization pattern intensities between patient DNA and control DNA. For these analyses, the patient DNA is labeled and hybridized to the microarray, and the patient results are compared with a well-studied reference DNA. Population studies suggest that >99% of all benign CNVs are inherited, and the vast majority of inherited CNVs are much smaller than 500 kb. Most pathogenic copy-number alterations are larger than 1 Mb, and most occur de novo. Not all CNVs are pathologic, as it has been demonstrated that the mean number of benign CNVs per person could be as high as 800 or more and determining the clinical significance of variants identified by CMA can be challenging. CNVs are determined by computer analysis of the array patterns and intensities of the hybridization signals.

The resolution and yield of an array is limited by the genomic coverage, i.e. the length of and spacing between probes on the microarray and by the specific statistical algorithms used to set the criteria for gains and losses. Array resolution is also dependent on the number and types of probes used and how they are distributed across the genomes. CMA is currently performed in many different laboratories with different technology platforms and different array design and content. For example, BAC arrays oligo arrays targeted whole genome, and single nucleotide polymorphisms (SNPs). Therefore, uniform interpretation of results presents a big challenge in addition to the need for standardization. A whole genome oligo array can detect clinically significant copy number changes missed on a targeted BAC array. A SNP array can detect long contiguous stretches of homozygosity that can be associated with increased risk for autosomal recessive conditions.

The proliferation of in-house developed and commercially available platforms prompted the American College of Medical Genetics (ACMG) to publish guidelines for the design and performance expectations for clinical microarrays and associated software in the postnatal setting. Targeted CMA provides high-resolution coverage of the genome primarily in areas containing known, clinically significant CNVs. The ACMG guideline for designing microarrays recommends probe enrichment in clinically significant areas of the genome to maximize detection of known abnormalities. Ahn et. al. at NHS Foundation Trust in the United Kingdom developed CMA testing strategy to detect CNVs > 3 Mb at a specific deletion region with prenatal samples labeled with CGH labeling kit from Enzo Life Sciences. The choice of 3 Mb as a cut-off for “calling” CNV was based on prior data. This prenatal CMA strategy detected all CNVs of clinical significance and avoided problems associated with interpreting CNVs of uncertain prognosis. In 2008, the International Standards for Cytogenomic Arrays (ISCA) Consortium was organized to improve the quality of research and clinical care in genetics. The ISCA Consortium has initiated a new database for CNV and phenotype data generated from clinical CMA laboratories. This database is set to receive and manage raw data and normalized data files from all current CMA platforms, including both copy-number and SNP arrays. Implementation of CMA for prenatal testing continues to be debated and no international consensus has been reached. It is hoped that through sharing of information across laboratories would provide the most benefit to laboratories, clinicians, and—ultimately and most importantly—patients. Enzo Life Sciences offers geneticists a comprehensive portfolio for epigenetics and molecular biology including CGH labeling kits.

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