Genetic and metabolic test selection — formative SAQs

SAQ 1 (10 marks)

A 4-year-old boy is referred to your general paediatric clinic with global developmental delay and mild dysmorphism. He walked at 22 months and speaks in two-word phrases. His chromosomal microarray, requested by the general practitioner a year ago, is normal. Both parents are available and willing to provide blood. [1] [2]

1. State the next genetic test of choice, the platform, and the sample strategy, and defend the choice against the alternatives of repeating the microarray and requesting a karyotype. (4) [1]
2. Explain why a trio sample (child and both parents) is preferred over a proband-only sample, and quantify the expected effect on the diagnostic yield. (3) [2]
3. The exome returns a pathogenic variant in BRCA1, reported as an incidental actionable secondary finding. Outline the consent that should have been taken before the test, the post-test counselling, and the rules around testing this child's siblings. (3) [11]

Model answer — SAQ 1

(1) Next test of choice and defence (4). The next test is whole-exome sequencing as a trio (the child and both parents). The chromosomal microarray is the first-tier test for unexplained developmental delay and detects copy-number variants with a yield of around 10 to 20 percent, but a normal microarray does not exclude a genetic diagnosis — it directs the next step. Repeating the microarray is not indicated because it will not find a new class of variant. A karyotype is the wrong test for developmental delay because its resolution (5 to 10 megabases) is too low; it is reserved for suspected aneuploidy or a known familial balanced rearrangement. The exome is the platform that answers the question a normal microarray raises — a single-nucleotide variant or small indel in a coding gene — and Yang and colleagues' series gives a diagnostic yield of around 25 percent in ambulant children with a suspected monogenic disorder. [1] [2]

(2) Trio over proband-only (3). The single most powerful signal in exome interpretation is the de novo variant — a variant present in the affected child and absent from both unaffected parents, which arose in the germline of one parent or in early embryogenesis. A trio sample lets the bioinformatician filter the roughly 20,000 coding variants found in a typical child down to the small handful that are de novo and plausibly disease-causing. A proband-only sample leaves a much larger variant list that the laboratory must prioritise by phenotype alone. The effect on yield is large — trio sampling roughly doubles the diagnostic yield of exome over proband-only testing. Both parents should have their blood drawn at the same time as the child's, and the family structure documented on the request form. [2]

(3) Incidental actionable secondary finding (3). Before the test, the consent must have recorded the family's opt-in or opt-out for the ACMG secondary-findings list, which in the v3.2 update stands at 81 reportable genes covering hereditary cancer and cardiovascular risk syndromes among others. The family should have been told that an exome can return a diagnostic finding, a variant of uncertain significance, an incidental actionable secondary finding, or an unexpected finding such as non-paternity, and that the secondary finding is a family finding, not the child's finding. Post-test, the counselling frames the BRCA1 variant as an actionable risk rather than a diagnosis: predictive testing is offered to at-risk adult relatives; a minor is not tested for an adult-onset condition without a childhood intervention, so the siblings would not be tested now; and the child is referred, when an adult, to a familial cancer service for their own predictive testing and surveillance. The discussion is documented. [11]

SAQ 2 (10 marks)

A term infant is referred from the maternity unit on day 7 because the newborn bloodspot screen taken at 72 hours has returned a markedly elevated octanoylcarnitine (C8). The infant is feeding normally and is clinically well. [12] [14]

1. State what this result suggests, why the heel-prick timing matters, and the immediate confirmatory diagnostic pathway. (4) [12]
2. Explain the principle that newborn bloodspot screening is a screen rather than a diagnosis, the role of tandem mass spectrometry, and the evidence for expanded screening. (3) [14]
3. Suppose instead that the screen was normal but the infant now presents at 6 months with an acute decompensation and a metabolic panel is being considered. Explain why the panel must be sent during the decompensation, and list the acute-episode samples to draw. (3) [12]

Model answer — SAQ 2

(1) Suggested diagnosis and pathway (4). A markedly elevated octanoylcarnitine (C8) on newborn bloodspot screening is the classic flag for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, a fatty-acid oxidation disorder in which the child cannot mobilise fat during fasting and is at risk of hypoketotic hypoglycaemia. The heel-prick timing matters because the analytes the screen measures only become informative once the infant has been feeding and metabolising for a day or two; a spot taken before 48 hours can be falsely normal, and a spot after a transfusion or from a squeezed, layered card can be falsely reassuring. The immediate pathway is to recall and examine the infant, send the confirmatory diagnostic test — a quantitative plasma acylcarnitine profile and plasma amino acids, and an organic acid analysis on a urine sample — and refer urgently to the metabolic service. The family is told that the screen is a flag, not a diagnosis, until the confirmatory test is back, and the ACMG ACT sheet for elevated C8 specifies the timeframe and the immediate actions, including counselling on fasting avoidance and an emergency regimen. [12]

(2) Screen, not diagnosis; MS/MS; expanded screening evidence (3). Newborn bloodspot screening is a population-level screen, not a diagnostic test — its purpose is to flag the infant who needs a confirmatory diagnostic test, and every abnormal result is confirmed quantitatively before a label is attached to a child. The engine of the expanded panel is tandem mass spectrometry, which ionises each analyte, separates it by mass-to-charge ratio in a first mass analyser, fragments it, and separates the fragments in a second mass analyser — yielding a quantitative fingerprint of dozens of acylcarnitines and amino acids from a single punch of a dried blood spot. The evidence base for expanded screening includes the Waisbren 2003 study showing improved outcomes for the disorders it detected without a measurable increase in parental stress, while recognising that the screen produces false positives and mild variants that require confirmatory testing. The Wilson and Jungner principles govern the design: an important condition, an accepted treatment, available facilities, a recognisable latent stage, and a suitable test. [14]

(3) Metabolic window and acute samples (3). The metabolic panel must be sent during the decompensation because most analytes — amino acids, organic acids, acylcarnitines, lactate, ammonia — normalise between episodes, once the child is treated with dextrose, protein restriction and detoxification. The well-interval panel is often normal and may falsely close the diagnostic door, which is why the acute-episode sample is the diagnostic one. The samples to draw during the decompensation, before treatment blunts the abnormality, are: venous blood gas; glucose with beta-hydroxybutyrate (a hypoketotic hypoglycaemia is the flag for a fatty-acid oxidation disorder); ammonia on ice, to the laboratory within 15 minutes; lactate; plasma amino acids (heparin or EDTA plasma on ice); a urine sample for organic acids (any urine is better than none); a free-carnitine and acylcarnitine profile; and saved aliquots of plasma and urine at minus 80 degrees for later testing. Treatment begins in parallel — resuscitation precedes and runs with the diagnosis. [12]