Genomic Testing 101

A question from the paediatric intensivist reaches me via the registrar. ‘

Does this normal array result exclude 22q11.2 deletion?’

“Yes!” I say emphatically, delighted to finally have a definitive answer to a genetic question.

Genetic investigations are so full of caveats and uncertainty, and very rarely able to exclude anything, that I relish standing on a small island of certainty.

That our esteemed colleagues are wrapping their heads around chromosome microarray results a decade after arrays became mainstream makes a lesson on genomic testing all the more pressing. Why? Despite the devastating global pandemic caused by a maleficent 29,844-base-pair RNA, the genomic revolution has arrived, and our patients stand to benefit.

 

Who Can Order Genomic Sequencing?

For the Australians in the audience, a Medicare item for genomic sequencing was released on 1st May 2020. Whole exome sequencing, known as WES (think Wes Anderson), and whole-genome sequencing (WGS), are now government-funded for children ten and under with:

• non-familial facial features and a major congenital anomaly OR
• moderate global developmental delay
• AND a non-informative chromosome microarray result.

A paediatrician can now order genomic sequencing in conjunction with a clinical geneticist. What ‘in conjunction’ will look like varies by service, but the goal is to usher in the safe adoption of this powerful diagnostic technology.

Not to stoke inter-hemispheric rivalry, but the Australian approach lags behind the UK, which for years has had a coordinated and mainstreamed approach to genomic testing, first through the Deciphering Developmental Disorders study and now the 100,000 Genomes Project. The NHS has implemented an excellent genomics education program for clinicians and patients alike.

 

The Burden of Genetic Disorders

Whilst individually rare, the combined incidence of rare diseases is common, affecting approximately 8% of the Australian and UK populations, with the vast majority of cases being genetic in origin. These numbers are similar to the proportion of people living with diabetes or asthma. Children with rare diseases have disproportionate representation in the emergency department, NICU, PICU, general and developmental paediatrics clinics, often with the input of many paediatric subspecialists. In years past, many of these individuals were thoroughly reviewed by clinical geneticists, occasionally received a diagnosis, often after many years, but the majority remained undiagnosed, occasionally adopting the moniker of a syndrome without a name (SWAN).

 

Enter the genomic revolution

Our genome, the instructions that make us who we are, is in almost every cell in our body (enucleated red blood cells are a notable exception). These instructions, coded via 3 billion DNA base pairs, are tightly packaged into chromosomes. Typically, a person has 46 chromosomes, 1 to 22 (longest to shortest, in theory, but not quite) and the sex chromosomes, XX or XY. Genes, the individual recipes for proteins and enzymes, are spaced out irregularly along the chromosomes, akin to legible sentences in a sea of lorum ipsum.

 

Remarkably, the approximately 20,000 genes that humans have make up only 2% of the whole genome. WES sequences only the 2% slice of the apple, whereas WGS sequences the whole darned apple. Whole-genome sequencing of the apple achieves better coverage of 2%, detects structural rearrangements, and has a 5-10% higher diagnostic yield than WES. Irrespective of what is sequenced, most of the analysis remains focused on the 2%. Whilst tempting to order WGS as a first-line test, in Australia at least, there are very few places with access to WGS through public laboratories.

Only 1 out of 4 of all human genes is currently known to be associated with health conditions. The remaining 3 out of 4 genes may have pathophysiological associations yet to be discovered, or be the genetic equivalent of a gastrointestinal appendix. Genes can be disrupted in many ways, rendering them inactive, overactive, or simply a general nuisance by interfering with the normal function of proteins. These disruptions can happen at different levels.

At the chromosome level, whole genes can be deleted or duplicated. Systematically arranged photographs of chromosomes, taken under a microscope, were revolutionary when they were introduced over 50 years ago. Known as karyotyping, this was the first test to detect imbalances in chromosomal material. Generally, these imbalances contained dozens, if not hundreds, of genes.  Current-day equivalents, chromosome microarrays, offer much of what a karyotype does, but at a far higher resolution (think several orders of magnitude) and can often detect single-gene deletions or duplications. Chromosome microarrays have approximately a 10 to 15% diagnostic yield as a first-pass test for multiple congenital anomalies or developmental delay and should always be the first test ordered for these presentations. Fragile X testing should also always be ordered for children of either sex presenting with developmental delay, as it remains the most common inherited form of intellectual disability.

What microarrays do not do is read every single letter of every single gene, looking for a spelling mistake. That is what genomic sequencing does. Diagnostic yield of WES varies from paper to paper, but is typically between 35 and 60%, depending on a multitude of factors, and is slightly higher for WGS.

 

Whole exome sequencing, half exome sequencing? Genomes? What do I tell my patients?

Beyond one vial of blood in an EDTA tube, and lots of waiting, what’s involved? Consent. Consent. Consent. And then some. Educational videos on genomic consent can be found here and here. A major focus of consent is managing patient expectations about potential outcomes, including the possibility of identifying non-familial relationships (e.g., non-paternity), incidental genetic findings, and exploring implications for life and income protection insurance. Thorough consent means that most families will choose not to proceed with testing.

 

Ok. So, you either find the diagnosis, or you don’t, right? Not entirely.

Due to natural variation, each of us has 3 million base pairs that vary from the reference genome. Which of these 3 million variants is the culprit? Bioinformatics filtering removes the bulk of variants, but at the end of the residual list of variants is a very human curation effort, with scientists and genetic pathologists intently scrutinising, often for hours, variants for their pathogenicity.

Parents frequently view a genetic diagnosis as precise and unequivocal and can be perplexed by vague and uncertain interpretations of results. Imagine, for example, if all our genes were cake recipes. Our task is to compare them to a giant Martha Stewart reference compendium. WES can identify each time a recipe varies from the Martha Stewart original, but cannot bake that cake, to see what the effect of the new recipe is. Some changes are obvious. If the new recipe stops at ‘preheat the oven’, substitutes sugar with salt, or asks for vibranium, clearly there is no cake to be had. But what about subtle changes? Can sugar be substituted by honey? Can you double the chocolate (umm…yes!)? The latter constitute variants of uncertain significance (VUS) and should be considered cautiously.

Genomic testing can be either a singleton or a trio.  Singleton sequences only the child’s DNA. Trio sequencing achieves a higher diagnostic yield by analysing the child’s and parental DNA together. This ability to segregate variants in real time substantially reduces the number of red herrings and the risk of throwing the proverbial baby out with the bioinformatics bathwater.

So the potential outcomes are:

• A molecular diagnosis is made, and the family has an answer. 
• No significant variants are identified. This does not rule out a genetic origin. A variant may be outside current technical or bioinformatics capabilities for detection, or in a gene whose function has yet to be discovered. Reanalysis of previously captured genomic data can be requested through most laboratories, with a minimum recommended interval of 2 years.
• A variant of uncertain significance (VUS).
• Importantly, although rare, an incidental finding is identified in approximately 1% to 2% of cases. It is difficult to explain to a family that the cause of their child’s intractable epilepsy has not been identified, but that the child and mother carry a change in the BRCA1 gene, putting them at increased risk of breast cancer. Disclosing incidental findings is ethically fraught, with some laboratories offering an ‘opt-out’ service.

 

Is it worth it?

Information is a vital tool for empowering patients, and the value of a molecular diagnosis is hard to quantify. An early genetic diagnosis demonstrably reduces the burden of costly and often invasive investigations a child typically undergoes during their diagnostic odyssey. Some tertiary institutes may be able to offer ultra-rapid genomic testing, with turnaround times of days, for children in acute care settings, such as the NICU or PICU.

Direct changes in management are rare, but when they do occur, they can be transformative, such as novel therapies for achondroplasia or life-prolonging antisense oligonucleotides for spinal muscular atrophy (admittedly two genetic conditions which are diagnosed on clinical grounds, not genomic testing). Even without a disease-specific change in management, families perceive significantly reduced barriers to coordinated care and funding once a diagnosis is made. Disease-specific patient organisations, often hosted on social media platforms, can be a vital source of support and information for families already at risk of significant social isolation.

For couples considering further children, the phenomenon of reproductive stoppage, whereby couples elect to have no further children to avoid possible recurrence, is very real. Being able to provide an accurate estimate of the likelihood of recurrence and family planning options restores reproductive confidence.

However, a diagnosis is no panacea. With the majority of families pursuing testing in a desire to anticipate a child’s future potential complications and life expectancy, thanks to an increasing number of ultra-rare conditions, this effort at prognostication often falls short. One parent eloquently describes it as finally receiving the last piece of the puzzle, yet the picture on the puzzle remains blank. If your child is the 7^th in the world with a condition, little can be gleaned with certainty from the other n=6.

Even for well-characterised conditions, there is growing awareness of phenotypic expansion, as children at the milder end of the spectrum are diagnosed through genetic testing rather than the prototypical collection of features previously required to secure a diagnosis. Yet thanks to this space of uncertainty, there is room for genuine hope, with the very real possibility that a child proves the textbooks wrong and exceeds all expectations.

For those who remain undiagnosed, more tests are on the horizon. In the meantime, be bold, be brave, and know that there is a more powerful test to offer your patients and that clinical geneticists have your back. And that patient who didn’t have 22q11.2 deletion? His WES is in progress.

 

Further resources

Home

Genomic Testing Consent Resources for Medical Specialists: https://www.genetics.edu.au/health-professionals/genomic-videos

RACP: Clinical genomics for physicians: https://elearning.racp.edu.au/login/index.php

Explaining a VUS: https://vimeo.com/336811697

Chromosome microarray analysis: A soothing guide https://onlinelibrary.wiley.com/doi/full/10.1111/jpc.13869 

Genetic disorders UK: https://www.geneticdisordersuk.org/

Why obtaining a diagnosis is important: https://swanaus.org.au/information/diagnosis/diagnosis/#1468139158856-daf34e3b-8b25

The Voice of Rare Disease Patients in Europe: https://www.eurordis.org/

A recent publication about limitations of WES: https://pubmed.ncbi.nlm.nih.gov/32190976/