Examining the structure of chromosomes The first studies in human genetics were done in the early 1900s, well before we had any idea of the structure of DNA or chromosomes. It was not until the late 1950s that the double helix was deciphered, that we realised that chromosomes were large bundles of DNA, and that we were able to visualise the number and shape of chromosomes under the microscope. In just a few years, numerous clinical disorders were identified as being due to abnormalities in the number or shape of chromosomes, and the field of “cytogenetics” was born. Over the next five decades, techniques improved. With the right sample and a good microscope, the laboratory could detect an abnormal gain or loss that was as small as 5-10 million base pairs of DNA on a specific chromosome. The light microscope reigned supreme as the ultimate tool for genetic analysis!

Examining the mass of chromosomes

In the last 10-15 years, a different technology called “microarrays” has challenged the supremacy of the microscope in genetic analysis. There are many different implementations of microarrays, but in essence they are all based on breaking the chromosomes from a tissue sample into millions of tiny DNA fragments, thereby destroying the structural cues used in microscopy. Each fragment then binds to a particular location on a prepared surface, and the amount of bound fragment is measured. The prepared surface, a “microarray”, is only a centimetre across and can have defined locations for millions of specific DNA fragments. The relative amounts of specific fragments can indicate tiny chromosomal regions in which there is a relative deficiency or excess of material. For example, in a person with Down syndrome (trisomy 21), the locations on the microarray that bind fragments derived from chromosome 21 will have 1 ½ times the number of fragments as locations which correspond to other chromosomes (three copies from chromosome 21 versus two copies from other chromosomes). The microarray could be regarded as examining the relative mass, rather than the shape, of specific chromosomal regions. Current microarrays can identify loss or gain of chromosomal material that is 10-100 times smaller than would be visible with the microscope. This has markedly improved the diagnostic yield in many situations but, as described below, conventional cytogenetics by light microscopy still has a role to play.

Microarrays in paediatrics

Conventional cytogenetics will identify a chromosome abnormality in 3-5% of children with intellectual disability or multiple malformations. A microarray will identify the same abnormality in those children, plus abnormalities in a further 10-15% i.e. the yield from microarray studies is approximately 15-20% (1). For this reason, microarray studies are the recommended type of cytogenetic analysis in the investigation of children or adults with intellectual disability or multiple malformations. There is a specific Medicare item for “diagnostic studies of a person with developmental delay, intellectual disability, autism, or at least two congenital abnormalities” by microarray. Requestors should request microarray analysis (item 73292) rather than use the less specific request for chromosome studies (item 73289). There are three cautions about microarray studies in this setting. First, a microarray will not detect every familial disorder. Intellectual disability due to a single gene disorder e.g. fragile X syndrome, will not be detected by a microarray. Second, experience with microarrays has demonstrated that some gains and losses of genetic material are benign and familial. It may be necessary to test the parents as well as the child to clarify the clinical significance of an uncommon change identified by microarray; the laboratory would provide guidance in such instances. And third, a microarray may identify an unexpected abnormality that has clinical consequences other than those which triggered the investigation.

Microarrays in antenatal care

The use of microarrays to investigate children with multiple malformations has now been extended to the investigation of fetuses with malformations. By using microarrays rather than conventional microscopy, the diagnostic yield from antenatal cytogenetics has increased by 6%(2). The cautions noted above still apply i.e. a microarray cannot detect every genetic cause of malformations, and determining the clinical significance of an uncommon finding may require additional studies. Microarrays can also be useful in the investigation of miscarriage and stillbirth. Most miscarriages are due to chromosome abnormalities which occur during the formation of the sperm or egg, or during early embryogenesis(3). These abnormalities are not inherited from either parent and hence do not constitute a hazard in subsequent pregnancies. Many clinicians and couples wish to confirm that a miscarriage was due to a sporadic chromosome abnormality that carries little risk for a subsequent pregnancy. This analysis can be done by either microarray or microscopic analysis of the products of conception. Microscopic analysis requires viable tissue, and up to 30% of studies may fail. Microarray analysis is preferred because it has better resolution and does not require living cells; as a result, the yield from microarray analysis is much higher(2). Requesters should specifically request microarray analysis, utilising the non-specific MBS item (73287).

Situations in which microarrays should not be used

There are two important antenatal situations in which microarrays should not be used: preconception screening, and investigation after a high risk non-invasive prenatal testing (NIPT) result. As noted above, a microarray measures the relative amount of genetic material from a specific location on a chromosome; it does not evaluate the shape of that chromosome. Approximately 1:1,000 healthy people has a balanced translocation i.e. part of one chromosome is attached to a different chromosome. The overall amount of genetic material is normal and there is usually no clinical consequence of this rearrangement. A balanced translocation would not be detected by microarray because there is not net gain or loss of chromosomal material. Microscopic analysis is likely to detect the translocation because of the change in shape of the two chromosomes involved. A person with a translocation can produce eggs or sperm that are unbalanced, having an abnormal gain or loss of chromosome material. This can cause infertility, recurrent miscarriages, or the birth of a child with intellectual disability or malformations. The unbalanced abnormality in the child would be detected by microarray, but the balanced precursor in the parent would not. For this reason, cytogenetic investigation of infertility and recurrent miscarriages requires microscopic cytogenetic studies of both partners (MBS item 73289). Approximately 4% of couples with recurrent miscarriages are found to have a balanced translocation in one or both partners. For similar reasons, microarray testing is not recommended for follow-up studies of CVS or amniotic fluid after a high risk result from NIPT. A microarray would identify the trisomy, but may not detect the rare instance of trisomy due to a familial translocation. Prenatal testing for autosomal trisomy requires microscopic cytogenetic studies (MBS item 73287).

The future of microarrays

Rapid developments in DNA sequencing have raised the possibility that microarrays will themselves be displaced as the preferred method of cytogenetic analysis(4). It is already possible to replicate many of the functions of a microarray by advanced sequencing methods. However, the microarray currently has the advantages of precision, reproducibility, and affordability that will ensure its continuing use for at least the next few years. And, as already demonstrated above, there may still be clinical questions that require the older methods. Cytogenetics is changing, but it is not dead. Sonic Genetics offers cytogenetic studies by both microscopic and microarray methods. General Practice Pathology is a new fortnightly column each authored by an Australian expert pathologist on a topic of particular relevance and interest to practising GPs. The authors provide this editorial, free of charge as part of an educational initiative developed and coordinated by Sonic Pathology. References

1. Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet. 2010 May 14;86(5):749–64.
2. Dugoff L, Norton ME, Kuller JA. The use of chromosomal microarray for prenatal diagnosis. Am J Obstet Gynecol. 2016;215(4):B2–9.
3. van den Berg MMJ, van Maarle MC, van Wely M, Goddijn M. Genetics of early miscarriage. Biochim Biophys Acta - Mol Basis Dis. 2012;1822(12):1951–9.
4. Downie L, Donoghue S, Stutterd C. Advances in genomic testing. Aust Fam Physician. 2017;46(4):200–4.