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Genetics

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Prof Graeme Suthers
Clinical Articles iconClinical Articles

Pharmacogenomics in general practice

Despite potential savings of more than $1 billion annually, awareness of pharmacogenomic tests among Australian prescribers is low and national guidelines for their use have not been developed. This void contributes directly to the continued prescribing of ineffective medications, unacceptably high rates of adverse drug reactions and associated personal and economic costs.Pharmacogenomics (PGx) is the study of how the genome of an individual patient influences their response to a medication.

Expert/s: Prof Graeme Suthers, Dr Kym Mina, Dr Tom Polasek
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Dr Kym Mina
Clinical Articles iconClinical Articles

Lactose intolerance and genetic testing

A genetic test is now available to assist in the diagnosis of lactose intolerance in both children and adults.

Key points

  • Lactose intolerance affects approximately 75% of the population.
  • Genetic testing can confirm lactose tolerance (also referred to as lactase persistence).
  • The test differentiates between primary lactose intolerance, due to lactase deficiency, and secondary causes of lactose intolerance, due to other more serious conditions that affect the small bowel.
  • The test is not affected by intercurrent illness and can be performed non-invasively on patients of all ages.
The test only needs to be performed once during a person’s lifetime.

How does the test work?

Testing is now available to detect the genetic variant (LCT-13910C>T) that accounts for close to 100% of lactase persistence in Europeans. Three other genetic variants that have a similar effect and are more common in non-European populations are also detected.These variants are thought to act as enhancers of the lactase gene that in turn stimulates lactase production.When one of these variants is found, a diagnosis of primary lactose intolerance can be excluded.Lactose intolerance can be secondary to other conditions that affect the small bowel, such as gastroenteritis, inflammatory bowel disease and coeliac disease. Genotyping can help to distinguish these causes of intolerance.

What causes lactose intolerance?

Lactose is the major carbohydrate in mammalian milk. Lactose intolerance is caused by deficiency of lactase, the enzyme required for digestion of lactose.Symptoms include abdominal pain, diarrhoea, nausea, flatulence and/or bloating, following the consumption of lactose-containing foods.

Who is affected by lactose intolerance?

After infancy, approximately 75% of the population lose the ability to digest lactose, due to a deficiency in lactase, referred to as primary lactose intolerance. The remainder of people maintain their tolerance for lactose-containing foods because of genetic variants that enable continued production of lactase, referred to as lactase persistence.The prevalence of primary lactose intolerance varies significantly with ethnic background. Lactose intolerance is uncommon in populations that consume large amounts of dairy, for example, northern Europeans (as low as 10%), but is frequent in other populations (as high as 100% in Asiatic countries). It is hypothesised that this is the result of selective genetic advantage; populations that have historically been dependent on dairy food sources for nutrition have survived by having genetic variants that allow tolerance for lactose.

Other testing alternatives

Currently, testing for lactose intolerance can also be performed by a hydrogen breath test with lactose load, or by measurement of intestinal lactase enzyme activity in a biopsy obtained during endoscopy.These tests may give a false-positive result when lactase levels have been affected by a recent viral illness or coeliac disease.These procedures are also not suitable for testing children younger than seven years old. Genotyping is not affected by intercurrent illness and can be performed non-invasively on patients of all ages.

Genetic testing limitations

Please note that genotyping will not identify very rare genetic variants associated with persisting lactase activity, and therefore the absence of a variant can only be used to support a diagnosis of lactose intolerance along with other clinical and laboratory findings.

Arranging a test

  1. Complete a standard pathology request form to refer the patient for ‘lactase persistence’ or ‘lactose intolerance genetic testing’.
  2. Collect or send the patient to a pathology collection centre for a blood test or buccal swab. No special preparation or booking is necessary.
  3. The result is reported back to the doctor, usually within five business days of the laboratory receiving the patient’s sample.

Cost

Medicare does not cover the cost of this test and the patient will receive an invoice for $75.*

References

  • Bayless T, Brown E, Paige DM. Lactase non-persistence and lactose intolerance. Curr Gastroenterol Rep. 2017 May; 19(5): 23. DOI: 10.1007/s11894-017-0558-9
  • Mattar R, de Campos Mazo DF, Carrilho FJ. Lactose intolerance: diagnosis, genetic, and clinical factors. Clin Exp Gastroenterol. 2012; 5: 113-21. DOI: 10.2147/CEG.S32368
  • Tishkoff SA, Reed FA, Ranciaro A, Voight BF, Babbitt CC, Silverman JS, et al. Convergent adaptation of human lactase persistence in Africa and Europe. Nat Genet. 2007 Jan; 39(1): 31-40. DOI: 10.1038/ng1946
  • Heyman MB, Committee on Nutrition. Lactose intolerance in infants, children, and adolescents. Pediatrics. 2006 Sep; 118(3): 1279-86. DOI: 10.1542/peds.2006-1721
*Correct at time of printing. Please to refer to the Sonic Genetics website, www.sonicgenetics.com.au, for current pricing.General Practice Pathology is a regular 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.
Expert/s: Dr Kym Mina
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Dr Linda Calabresi
Clinical Articles iconClinical Articles

Gene-directed prescribing coming to general practice

It’s been around for some time now. The idea of checking a person’s genes to guide appropriate prescribing is not new. It is pretty much standard practice when treating many if not most cancers.But pharmacogenomics in general practice? Looking at an individual’s genetic variants to work out the best treatment for their depression, high cholesterol or gout? Yes – it’s coming.As authors of a recently published review in the Australian Journal of General Practice say, all practising clinicians will have had the experience of patients responding differently to medications despite every indication the medication should be effective, based on all the evidence from randomised controlled trials.It is known that certain genes that regulate the absorption, distribution, metabolism and excretion (ADME) of medications are commonly responsible for this difference in response, as these genes can vary between individuals. Researchers have also identified another group of genes that can influence medication responses directly, which, while less common can have important implications for prescribing.“For example, carbamazepine should not be prescribed to patients with certain human leucocyte antigen (HLA) genotypes because of an increased risk of Stevens-Johnson syndrome and toxic epidermal necrolysis,” the Australian review authors said.Overall, there have been at least 15 genes identified for which testing can be useful and clinically beneficial in guiding prescribing of 30 different medications.How important will this be in general practice?According to this review at least, very. Firstly the likelihood of having a genetic variation that will influence how a person responds to a common medication is incredibly high.“A recent study of 5400 Australians who underwent testing of just four ADME genes showed that 96% had at least one clinically actionable pharmacogenomic variant,” the review authors said.And then the likelihood that a person will be prescribed one of the drugs that we can now predict the response based on genetic testing is also incredibly high. On analysis of PBS data from 2017, the researchers determined that in that year approximately 1.7 million Australians had filled a prescription for at least one the drugs that has the highest level of evidence of clinically relevant gene-medication association.In their review the authors present a number of case studies which demonstrate the usefulness of genetic testing in clinical practice. These include a patient with anxiety and depression who fails to respond to standard treatment leading to a significant deterioration of her condition. Genetic testing of CYP enzymes found the patient had a genetic variation that meant they rapidly metabolised certain antidepressants, but could be prescribed an alternative medication that wasn’t as dependent on the affected enzyme.Another example involved an older Han Chinese man who needed to be prescribed allopurinol for gout. Because this particular ethnicity has a 20% chance of carrying a gene that puts them at risk of developing severe cutaneous adverse reaction to allopurinol, gene testing was done to help ensure this risk was minimised.Similarly, a genetic variation that gives a higher than normal risk of muscle toxicity when taking simvastatin or atorvastatin can be tested for prior to patients being prescribed these medications, and a safer alternative statin offered.Fundamentally this type of testing will lead to more effective, safer prescribing. The problem is of course, pharmacogenomic testing does not attract a Medicare rebate in Australia and the cost is prohibitive for most patients, even though, according to the review authors the costs are decreasing all the time. A panel of common CYP enzymes now costs between $150 and $200.But the authors suggest this situation will have to change. Even on the basis of economics alone, the government needs to consider allowing rebates for at least some pharmacogenomic tests.“A report in 2008 estimated that the widespread implementation of such testing in Australia could yield savings in excess of $1 billion annually by the avoidance of adverse medication reactions alone,” they said.And imagine how much time, money and angst could be saved if doctors could ensure the first antidepressant a patient is prescribed had a high likelihood of having an effect, rather than the current trial and error approach.However while we wait for better reimbursement for this testing, the authors suggest the doctors consider using the tests where appropriate in patients who are prepared to pay.“Responsible doctors can use the tests and evidence that are already available to improve prescribing decisions for their patients,” they conclude.

Reference

Polasek TM, Mina K, Suthers G. Pharmacogenomics in general practice: The time has come. AJGP. 2019 March; 48(3): 100-5. Available from: https://www1.racgp.org.au/ajgp/2019/march/pharmacogenomics-in-general-practice
Expert/s: Dr Linda Calabresi
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Dr Linda Calabresi
Clinical Articles iconClinical Articles

Reproductive carrier screening

Have you seen this? This little print-out could save you a good 30 minutes in valuable consulting time.It’s the information from Sonic for couples who are planning a family about the potential value for testing their carrier status for conditions such as cystic fibrosis and fragile X.Even though the information is coming from an organisation with a vested interest in promoting the testing, there is not even a suggestion of bias. It is all straight down the line – here are the risks – this is what is available for testing should you choose to pursue it.There’s no denying it is worth considering. RANZCOG recommends that information about reproductive carrier screening be offered to every woman either prior to conception (preferred) or in early pregnancy.Having this site bookmarked and ready to print off ensures your advice when advising women pre-conception is in keeping with best practice.>> Click here for resource

Expert/s: Dr Linda Calabresi
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Sullivan Nicolaides Pathology
Clinical Articles iconClinical Articles

Prenatal Screening for Chromosome Disorders

Prenatal screening for chromosome disorders by maternal serum screening, ultrasound and non-invasive prenatal tests, such as Harmony®, is an established part of reproductive care in Australia.The overall risk of chromosome disorders rises markedly with maternal age, as shown in Figure 1. (There are two exceptions: Monosomy X, also known as Turner syndrome, and microdeletions, such as 22q11.2, occur independently of maternal age). This does not mean that chromosome screening should be restricted to older mothers.Younger mothers have more babies than older mothers, and the overall outcome is that the majority of pregnancies with a serious chromosome disorder occur in mothers under 35 years of age. For this reason, screening for chromosome disorders in pregnancy should be offered to mothers of all ages.The great majority of these chromosome disorders are new abnormalities that have happened for the first time in this pregnancy. They are not inherited disorders, and genetic testing of the parents provides no information about the risk of such an abnormality. This provides another reason for offering screening for chromosome disorders to all mothers, irrespective of family history. 

The frequency of single-gene disorders at birth

Chromosome disorders are not the only type of genetic condition which can affect the developing foetus. Many serious childhood disorders are due to recessive mutations that have been inherited from parents, with the parents being unaffected by these mutations. A parent who is a carrier of a recessive mutation, that is, having one normal and one abnormal copy of a gene, will not be affected by the abnormal gene. Everyone is a carrier for one or more disorders; this is of no immediate consequence and there usually is no family history of the disorder.The situation changes if both parents are carriers of mutations in the same gene located on one of the autosomes (chromosomes 1-22). The chance of their child inheriting the abnormal gene from each parent, and so developing an autosomal recessive disorder, is 25%. The situation is a little different for a woman with a recessive mutation on an X-chromosome: each of her sons is at 50% risk of inheriting the abnormal gene and being affected, and half of her daughters will be carriers. Overall, the risk of a woman who is an X-linked carrier having an affected child is approximately 25%.There are hundreds of inherited autosomal and X-linked recessive disorders that present in infancy and early childhood. These disorders are individually rare but, together, they are more common than the chromosome disorders for which prenatal screening is widely available and accepted. Further, the risk of these recessive disorders does not vary with maternal age (Figure 1). For mothers under 35 years of age, the risk of having a child with a serious childhood-onset recessive disorder is greater than the risk of having a child with a chromosome disorder. 

Screening potential parents for recessive disorders

These disorders are inherited but there is usually no family history to provide a clue.Until recently, the only way of identifying a carrier of a rare recessive disorder was to diagnose the disorder in their affected child.This has now changed.It is possible to screen a couple for mutations in autosomal genes, and a woman for mutations in X-linked genes, to determine whether they are at 25% risk of having an affected child. This screening test is called ’reproductive carrier screening’.From both a technical and clinical perspective, the challenge lies in choosing which genes to analyse. A number of providers, including Sonic Genetics, offer reproductive carrier screening for mutations responsible for three common disorders: cystic fibrosis and spinal muscular atrophy (both autosomal recessive) and Fragile X syndrome (X-linked recessive). Approximately 6% of people are carriers of one or more of these conditions, and 0.6% (one in 160) couples are at 25% risk of having an affected child.Those couples who are identified as carriers can consider a variety of options, including IVF with a donor gamete, pre-implantation genetic diagnosis, prenatal diagnosis by CVS, or they may make an informed decision to accept the risk.RANZCOG recommends that couples be offered such screening.The cost of this three-gene panel is approximately $400* per person. There is no Medicare rebate for carrier screening; there are exceptions (and restrictions) for people with a documented family history of cystic fibrosis or Fragile X syndrome. 

Expanded reproductive screening

If we were to screen more genes, we would identify more carriers.Sonic Genetics offers a screen of over 300 genes (autosomal and X-linked) which cause serious recessive childhood disorders. We estimate that approximately 70% of Australians are carriers for one or more conditions included in this screen and 3% (one in 30) couples are at 25% risk of having an affected child. This amounts to five times more information than is provided by the three-gene panel.This screen, the Beacon Expanded Carrier Screen, currently costs $995* per person or $1,750* for couples tested together.It is tempting to think that ‘more genes tested = more information for a couple’. This is not the case because the information provided by a carrier screen is also determined by the carrier frequency, mode of inheritance and detection rate of the assay for each gene.Some currently available screens of more than 100 genes provide less information than the three-gene screen described earlier. 

Implementing reproductive screening

Before offering reproductive carrier screening to your patients, it is important to consider some of the nuances, particularly in relation to the Fragile X syndrome (some carriers will develop premature ovarian failure or a tremor/ataxia syndrome in later life) and when there is a family history of a recessive disorder (seek expert advice; do not rely on screening).It is also important to recognise that some couples will not want this carrier information – and others will demand it. Each person needs to be free to make their own decision about what information they wish to have.We provide information about the three-gene and Beacon screens for both requestors and patients on our website.Sonic Genetics also offers genetic counselling free-of-charge for couples who are identified by either of these reproductive carrier screens as being at high risk of having an affected child (see www.sonicgenetics.com.au/rcs/gc). 

Conclusion

It is accepted practice that every woman is offered screening for chromosome disorders in pregnancy, irrespective of age and family history.In a similar vein, every couple should be offered reproductive carrier screening for recessive disorders, irrespective of age and family history. For women under 35 years, the risk of their child having a recessive disorder is greater than the risk of a chromosome disorder. Offering reproductive carrier screening simply represents good medical practice. 

References

RANZCOG. Prenatal screening and diagnostic testing for fetal chromosomal and genetic conditions. 2018 Aug. 35 p. Available from: https://www.ranzcog.edu.au/RANZCOG_SITE/media/RANZCOG-MEDIA/Women%27s%20Health/Statement%20and%20guidelines/Clinical-Obstetrics/Prenatal-screening.pdf?ext=.pdfArchibald AD, Smith MJ, Burgess T, Scarff KL, Elliott J, Hunt CE, et al. Reproductive genetic carrier screening for cystic fibrosis, fragile X syndrome, and spinal muscular atrophy in Australia: outcomes of 12,000 tests. Genet Med. 2018; 20(5): 513-523 Available from https://www.ncbi.nlm.nih.gov/pubmed/29261177 doi:10.1038/gim.2017.134.Sonic Genetics [Internet]. c2015. Reproductive Carrier Screening; 2018. Available from: www.sonicgenetics.com.au/rcs General Practice Pathology is a new regular 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.
Expert/s: Sullivan Nicolaides Pathology
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Dr Linda Calabresi
Clinical Articles iconClinical Articles

New Medicare-Funded BRCA Testing – Use With Caution

GPs may have to correct some patients’ misunderstanding following reports in the general media suggesting that testing for high risk cancer genes was now available to everyone free of charge.Writing in the latest issue of the MJA, Australian genetics experts say that testing for specific high- risk genetic mutations, especially BRCA1 and BRCA2 has been available to appropriate patients free of charge (but not Medicare-rebated) by genetic specialists in public clinics for over 20 years.What’s new is that these tests now attract a Medicare rebate and you don’t have to be a genetic specialist to order them, but they are still only available to selected patients.“Testing is appropriate when there is at least a 10% chance of identifying a gene mutation responsible for the personal or family history of cancer,” the authors of the article wrote.There are a number of algorithms available to help clinicians calculate whether the likelihood of having one of these cancer-causing genetic mutations is at least 10%.Usually testing is initially considered for patients who have been diagnosed with either breast or ovarian cancer, and because of their young age and/or their strong family history are considered at high possibility of having a genetic mutation that explains their condition.The new item numbers (73295,73296 and 73297) cover testing for heritable germline mutations in seven genes including BRCA1 and BRCA2. If such a mutation is found, then at risk adult relatives will be justified in also accessing testing.However, as the article authors point out there are limitations with this type of genetic testing. Firstly most breast and ovarian cancers occur in people without an identifiable underlying genetic variant.“Only 5% of female breast cancers, 15% of invasive epithelial ovarian cancers and up to 14% of male breast cancers are related to BRCA1 or BRCA2 mutations, thus, most patients with breast cancer do not need, nor will they benefit from, a genetic test,” they said.That’s not to say the absence of BRCA1 or BRCA2, or one of the other rarer high-risk mutations currently tested for, excludes the possibility that the patient has inherited a predisposition to the cancer. Families that appear to have a high prevalence of these types of cancer may indeed have an inherited genetic mutation, it is just that because of limitations of technology and knowledge it is yet to be isolated.What’s more, the sensitivity of the current testing methods, means that a number of incidental genetic mutations may be noted, but the significance of these is as yet unknown. It is critical that when testing is requested for a relative of an affected patient, the laboratory is informed of the exact genetic variation found in the original affected patient, to ensure pathologists distinguish between the disease-causing mutation and variants of undetermined significance. The authors also suggest confining testing to only the most likely variant/s rather than requesting testing for mutations in multiple genes.“[T]he testing of multiple genes may uncover unclassified variants, variants outside the usual clinical context, variants unrelated to the current cancer, or unexpected important variants for which the patient has not been well prepared,” they said.They also suggest education and counselling be given to patients considering this genetic testing, and written consent obtained.The new Medicare item numbers represent a major step forward in terms of genetic and genomic testing becoming mainstream, but, as the current incorrect media headlines demonstrate, this transition is going to require information and education. Clinicians who order these tests are likely to benefit from establishing close ties with genetic services and specialists to ensure best and appropriate practice in this ever-expanding area of medicine.Ref:Med J Aust 2018; 209 (5): 193-196. || doi: 10.5694/mja17.01124

Expert/s: Dr Linda Calabresi
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Dr Vivienne Miller
Clinical Articles iconClinical Articles

Can My Genes Be Making Me Overweight?

Based on an interview with endocrinologist and obesity expert, Professor Joseph Proietto at the Annual Women and Children’s Health Update, Melbourne, March 2018There are many reasons proposed for our society becoming more overweight than ever before.The commonest explanation is that people are overeating because they have more refined, energy dense foods easily available and requiring little physical effort to access.The other consideration is that people are not moving and exercising as much, due to increased sedentary employment and entertainments that are clearly less effort. Once people become overweight, they feel less like exercising and so the situation worsens.Unfortunately, in our society, food (including alcohol), socialising and entertainment are all strongly associated. Food is easily obtained and is abundant in variety and quantity.Previous generations ate less because of cost, availability and the fact that food generally plainer and perhaps less tasty. This was especially true for the poorer in society, who also tended to have more physically demanding jobs, with less time and money to spend on eating during the day.On a scientific level, genetics and epigenetics are now known to play an important role in the development of obesity.In particular, there are many genes currently being researched in relation to appetite and obesity including leptin (a hormone made mostly by adipose cells that inhibits hunger) and its receptor, and the melanocortin 4 receptor."For obvious evolutionary reasons, there are no genes (yet) identified that reduce metabolic rate," said Professor Joseph Proietto.So far, all genes that have been found to be associated with obesity have been linked to increased hunger. There are no genes known that reduce metabolism.It is interesting that force-feeding increases energy expenditure while weight loss reduces energy expenditure and, in both cases, it is spontaneous activity that changes, with only minor alteration in basal metabolic rate. This has been demonstrated in overfeeding experiments.Some causes of obesity may be epigenetic. For example, some women who gain excess weight during pregnancy find it more difficult to lose after the pregnancy. This is likely to be due to epigenetic change in the expression of genes connected with obesity. Unfortunately, the offspring of mothers who become overweight before or during pregnancy are likely to inherit these genes, and hence themselves have trouble with weight gain.Certain medical conditions (hypothyroidism, Cushing's syndrome) may induce modest weight gain, but the extreme numbers of people in our society with serious weight problems mean that endocrinological causes are very much in the minority. Hence, we need to look for other causes for obesity in the modern age.One of the biggest problems with healthy lifestyle programmes and extensive community information about diet, weight and exercise in our society is that genetics trump willpower in many cases, especially over the long-term.Following weight loss there are hormonal changes that lead to increased hunger (leptin levels fall and ghrelin levels increase) and in 2011 these changes were shown to be long lasting, so the weight-reduced individual has to fight increased hunger.Given the prolific amount of available food, temptation adds to the problem. In effect, one is then fighting nature.

Expert/s: Dr Vivienne Miller
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Prof Sally Ferguson
Clinical Articles iconClinical Articles

Nobel prize winner: What they discovered and why it matters

Today, the “beautiful mechanism” of the body clock, and the group of cells in our brain where it all happens, have shot to prominence. The 2017 Nobel Prize in Physiology or Medicine has been awarded to Jeffrey C. Hall, Michael Rosbash and Michael W. Young for their work on describing the molecular cogs and wheels inside our biological clock.In the 18th century an astronomer by the name of Jean Jacques d'Ortuous de Marian noted his plants opening and closing their leaves with the cycle of light and dark, with the leaves opening towards the sun. Being an inquisitive chap, he placed the plants in constant darkness and observed that the daily opening and closing of the leaves continued even in the absence of sunlight – indicative of an internal clock.Subsequent work by others also showed innate daily rhythms in other animals and plants, but the location and inner workings of the biological timing system remained a mystery.
Read more - Keeping time: how our circadian rhythms drive us
The discovery of a misfiring gene that resulted in disrupted daily rhythms in fruit flies (the unsung heroes of the story) gave the first hint. Over several years, Hall, Rosbash and Young uncovered the machinery of the biological clock.It’s in your genes.From the latin circa “about” and diem “a day”, circadian rhythms are internally driven cycles in all living things - including humans - that continue in the absence of external time cues. The sleep/wake cycle is one daily rhythm; core body temperature is another. While we have known since de Marian that physiological systems are controlled internally, the way in which the clock works was a mystery.The biological clock’s cycle is generated by a feedback loop. Genes are activated which trigger the production of proteins. When protein levels build up to a critical threshold in the cells, the genes are switched off. The proteins then degrade over time to a point that allows the genes to switch back on, starting the cycle again. This takes about 24 hours.But it isn’t just one gene doing all the work. Hall, Rosbach and Young found that many genes, proteins and regulators are involved in the complex machinery that keeps us ticking. Some molecules control the activation of genes, some are involved in the translation of light information from the eyes, and some govern the clock’s stability and precision, ensuring that it keeps ticking and remains in sync with the external environment.While we already knew that the internally generated cycle existed, Hall, Rosbach and Young described the mechanisms by which the cycle is created and maintained at the molecular level. As a result of this work we now understand how internal rhythms remain synchronised with each other and with the external environment.We are starting to understand the range of health challenges experienced by those who have to work against their internal clocks, such as shift workers. We can predict times of the day and night where alertness and performance are likely to be impaired and thus control the health and safety risks.
Read more: Power naps and meals don’t always help shift workers make it through the night
The ConversationAnd we can explain why, on the first morning after the start of daylight savings, waking up is so much harder. But don’t worry, the beautiful mechanism in your biological clock is designed to make adjustments based on the information it gets from the external environment, and those molecules will have you resynchronised in just a couple of days.Sally Ferguson, Research professor, CQUniversity AustraliaThis article was originally published on The Conversation. Read the original article.

Expert/s: Prof Sally Ferguson
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Prof Graeme Suthers
Clinical Articles iconClinical Articles

Studying Chromosomes In 2017

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.
Expert/s: Prof Graeme Suthers

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