Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Tests for developmental diseases provide useful information for both clinicians and families.

Detection and analysis of structural variability within chromosomes have become an integral part of genomic medicine. Because genomic instability and chromosomal abnormalities characterize cancer as well as many developmental diseases, understanding these structural aberrations can provide insight into disease etiology, treatment options, and prognosis.

According to physicians, current cytogenetic tests for developmental diseases tests, including G-banded chromosome analysis and fluorescence in situ hybridization (FISH), provide useful information for both clinicians and families, allowing identification of potential medical interventions for the patients. This information, they say, also enables accurate recurrence-risk counseling and helps families plan for the expected natural history of the disease.

G-banded chromosome analysis, for example, enables examination of recognized banding patterns on Giemsa-stained metaphase chromosomes, allowing microscopic visualization of all 23 pairs of chromosomes in a single assay. The technique has led to the characterization of syndromes associated with aneuploidy (gains or losses of whole chromosomes) including Down, Cri-du-chat, and Smith-Magenis syndromes.

But, cytogeneticists say, while G-banding detects aneuploidy and large structural rearrangements including deletions, duplications, inversions, and translocations, it doesn’t consistently detect gains or losses of less than approximately 5–10 million base pairs.

FISH has complemented chromosome studies, detecting chromosomal rearrangements as small as 100,000 base pairs. Due to its comparatively high resolution, FISH has been used as the definitive diagnostic test for microdeletion/duplication disorders such as Williams syndrome and 22q11.2 deletion (DiGeorge/velocardiofacial) syndrome, as well as submicroscopic chromosomal rearrangements involving the subtelomeric regions that cause congenital abnormalities and developmental disabilities. FISH, however, can only detect deletions or duplications of regions specifically targeted by the probe used and which are larger than the probe. The technique, therefore, may miss small deletions.

Copy Number Variation

As copy number variation (CNV) has been increasingly recognized as playing a role in in such human diseases as obesity, heart disease, cancer, autism, and schizophrenia, and as a major cause of structural variation in the genome, the need for higher-resolution technologies for chromosomal analysis has emerged. CNVs involve both duplications and deletions of sequences that typically range in length from 1,000–5,000,000 base pairs, in many cases below the cytogenetic level of resolution.

Based on the length of the affected sequence, scientists assign CNVs to one of two main categories. The first includes copy number polymorphisms (CNPs), found commonly in the general population, at an overall frequency of greater than 1%. CNPs are typically small (most are less than 10 kilobases in length), and they are often enriched for genes that encode proteins important in drug detoxification and immunity. CNPs associated with immune response genes have recently been associated with susceptibility to complex genetic diseases, including psoriasis, Crohn's disease, and glomerulonephritis.

The second class of CNVs includes relatively rare variants that are much longer than CNPs, ranging in size from hundreds of thousands of base pairs to over 1 million base pairs in length. Also known as microdeletions and microduplications, these large and rare structural variants occur disproportionately in patients with mental retardation, developmental delay, schizophrenia, and autism, suggesting to some scientists that their appearance in such patients has led to speculation that large and rare CNVs may be more important in neurocognitive diseases than other forms of inherited mutations, including single nucleotide substitutions.

CNVs, which result from larger-scale genomic events such as deletions, duplications, inversions, and translocations, occur more commonly than previously thought, according to Steve Scherer, Ph.D., director of the Centre for Applied Genomics at the Hospital for Sick Children in Toronto.

“One of the surprising findings is that CNVs and indels [insertions or deletions] encompass over 10 times the total number of nucleotides of genetic variation when compared to SNPs,” he points out.

Emerging Diagnostic Tool

Cytogenomic microarray analysis (CMA), which can involve comparative genomic hybridization (aCGH) and cytogenomic constitutional microarray analysis, has emerged as a novel diagnostic tool for individuals with unexplained developmental delay, autism spectrum disease, and mental retardation. aCGH is used in addition to clinical evaluation and conventional genetic testing.

For chromosomal microarray testing, highly specific oligonucleotide probes are designed and distributed throughout the genome, allowing for a whole genome survey in a single assay with very high resolution analysis. CMA, reports the Mayo Clinic, has quickly moved from research to the clinical setting and has emerged as the recommended first-tier postnatal test for individuals with multiple anomalies not specific to a well-delineated genetic syndrome, apparently nonsyndromic developmental delay/intellectual disability, and autism spectrum disorder.

Chromosomal microarray testing detects a genetic cause for these clinical features in 15–20% of cases, a substantial increase in the diagnostic yield in this patient population. For individuals with multiple miscarriages, infertility, or who are suspected of having sex chromosome abnormalities (such as Turner or Klinefelter syndromes), a conventional chromosome study remains the most appropriate test.

In a communique, “Clinical Utility of Chromosomal Microarray Testing,” the Mayo Clinic warns, however, that the interpretation of chromosomal microarray test results is a “complex and evolving process that is aided by collaboration between the clinician and clinical laboratory, particularly regarding the submission of detailed clinical information at the time of test referral. A genetic consultation is often of benefit to ensure the appropriate clinical interpretation of chromosomal microarray test results.”

And, it goes on to say, the rapid proliferation of array-based technology into clinical laboratories has led to a lack of uniform guidelines for the clinical interpretation of observed copy number variations, at times causing confusing results or conflicting reports between laboratories.

To address these issues, the International Standards for Cytogenomic Arrays (ISCA) Consortium, now consisting of about 160 international laboratories, including the Mayo Clinic, was formed with the goals of developing evidence-based standards for chromosomal microarray design, to build a public database of clinical array data as a resource for the clinical and research communities, and to utilize the database to develop standards and guidelines for the interpretation of copy number changes in the clinical setting.

The ISCA reports that its rapidly growing group of clinical cytogenetics and molecular genetics laboratories remains committed to improving the quality of patient care related to clinical using new molecular cytogenetic technologies, including aCGH and quantitative single nucleotide polymorphism analysis using microarrays or bead chip technology.

In an issue brief from the Leonard Davis Institute of Health Economics, Marian Reiff, Ph.D., et al., write, “New technologies have given us the ability to detect genomic variation at resolutions 50–100 times greater than earlier tests. The good news is that we can now detect variations that help explain developmental delays, autism, or multiple congenital anomalies in up to 20% of children.

“The bad news is that we can also detect small missing or extra pieces of chromosomes that remain unexplained: that is, we don’t know whether they have any clinical significance at all. The rapid pace of technological change may have outpaced the lab’s ability to interpret.”

Hopefully, organizations such as the ISCA consortium will continue to work with scientists and clinicians in facilitating interpretation of a whole new world of data and enable the clinical utility of advanced cytogenomic tools.

Patricia Fitzpatrick Dimond, Ph.D. ([email protected]), is technical editor at Genetic Engineering & Biotechnology News.

This article was originally published in the August 13 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this new digital publication, go to

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