L11 Genetics Clinical genetics PODCAST

Overview

This lecture introduces Clinical Genetics as a medical discipline, worked through a single case: Amber, a healthy three-week-old who died suddenly and was found post mortem to have Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD). The case is used to trace the whole clinical-genetics pathway in order: newborn screening and biochemical diagnosis, the underlying metabolic defect, the mode of inheritance and mutation detection, the limits of DNA diagnostics and the use of linkage analysis, pedigree-based risk calculation for the wider family, and the ethical and counselling issues that diagnosis raises.

Clinical Genetics as a Discipline

• Aims of the discipline: introduce Clinical Genetics as a medical specialty; describe the process of identifying a genetic disorder; define the roles of the people involved (family, GP, Clinical Geneticist, laboratories); show its diagnostic, psychological and ethical facets; and show how molecular and clinical genetics integrate.
• Five challenge areas in genetic medicine today:
  1. Single gene disorders — their sheer number and individual rarity
  2. Chromosomal disorders — a matter of resolution (detection sensitivity)
  3. Diseases with complex aetiology — identifying multiple contributing determinants
  4. Cancer — designing treatments with sufficient specificity
  5. Drug design and response — identifying targets and clinically relevant variation
• Reference resources for an unfamiliar rare genetic disorder: Gene Reviews, Genetics Home Reference (US National Library of Medicine), NZORD (New Zealand Organisation for Rare Disorders), and disease-specific databases (e.g. SFARI Gene for autism).

Case Presentation: Amber

• Sharon and Khalid, an unrelated couple, had their first child, Amber, after an uneventful pregnancy, delivery and postnatal period.
• At three weeks of age Amber began feeding less well, with nasal congestion and a slight temperature. Her GP diagnosed a respiratory tract infection.
• The following morning Sharon found Amber dead in her cot, with vomitus on the mattress.
• Post mortem: a well-grown child with no structural anomalies; histology was unremarkable apart from fatty infiltration of the liver.
• Her newborn-screening card (dried blood spot, collected by heel-prick) was retrieved and analysed by tandem mass spectrometry.

Newborn Screening and Diagnosis by Mass Spectrometry

• Under the “old regime” (National Testing Centre, Auckland), newborn screening covered biotinidase deficiency, congenital adrenal hyperplasia, galactosaemia, phenylketonuria, maple syrup urine disease, cystic fibrosis and hypothyroidism.
  • Biotinidase deficiency, congenital adrenal hyperplasia, galactosaemia, phenylketonuria and maple syrup urine disease are grouped as “treatable genetic disorders”; the first four of these (biotinidase deficiency through phenylketonuria) are also grouped as “enzyme deficiencies”.
  • Cystic fibrosis is noted as the commonest autosomal recessive disorder; hypothyroidism is noted as predominantly non-genetic.
• Tandem Mass Spectrometry is now the newborn-screening modality used in New Zealand: it separates blood metabolites (from a day-5 heel-prick sample) by size, allowing many metabolic disorders to be detected pre-symptomatically.
• Healthy, unrelated, milk-fed infants give highly reproducible, near-identical acylcarnitine mass-spectrometry profiles.
• Amber’s mass spectrometry result showed an abnormal acylcarnitine profile, diagnostic of Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD).
• Compared with a normal profile (dominant peaks at C2 and C3 carnitine, small C16 peak), Amber’s abnormal MCAD profile showed a prominent C6 carnitine peak, a large dominant C8 carnitine peak, and a C10:1 carnitine peak — accumulation of medium-chain acylcarnitine species consistent with a block in medium-chain fatty-acid oxidation.

MCAD: Metabolic Pathway and Clinical Features

• MCAD is a “common” enzymatic defect. The enzyme normally catalyses the interconversion of fatty acids to ketones for energy utilisation; this pathway is activated during relative starvation, when simple carbohydrate is limiting.
• The pathway, in order:
  1. Carbohydrate (including glucose) feeds into citric acid cycle substrates.
  2. Citric acid cycle substrates feed into energy production.
  3. Free fatty acids would normally be converted, via the MCAD enzyme step, into ketone bodies.
  4. Ketone bodies would then feed into citric acid cycle substrates and on into energy production.
  • In MCAD deficiency, step 3 (free fatty acid → ketone bodies) is blocked, so energy production from fat during fasting/starvation is impaired, while the carbohydrate-fed route to energy remains intact.
• Epidemiology and presentation:
  • Incidence 1 in 20,000.
  • 25% of affected individuals present as sudden death below age 2 years.
  • Episodes of vomiting and hypoglycaemia.
  • Many affected individuals have no symptoms over a lifetime (non-penetrant).
  • Minimal pathology is found post mortem.
  • Clinical chemistry shows recurrent non-ketotic hypoglycaemia.
  • Unmetabolised free fatty acids are excreted as acylcarnitine conjugates in urine.
• The family’s questions to their GP, lead maternity carer and geneticist: What was it that killed my healthy child? What causes it? Will it happen again to other family members? Can we prevent it happening again?

MCAD: Inheritance and Mutation Detection

• Genetics: incidence 1 in 20,000; autosomal recessive with incomplete penetrance; gene located at 1p31, with 12 exons.
• Recurrence risk: 1 in 4, following autosomal recessive segregation.
• Diagnosis can be reached via any of: symptoms consistent with the diagnosis; acylcarnitine analysis (blood or urine); or genetic analysis (mutation detection).
• MCAD mutations are deactivating. One recurrent mutation, causing the substitution Lys304Glu (lysine replaced by glutamic acid at protein position 304), accounts for 90% of Caucasian disease alleles — a founder effect.
• The ACADM (MCAD) gene map runs from the 5’-UTR (with the ATG start codon) through 12 numbered exons to the 3’-UTR (with the TAA stop codon), with individual point mutations, deletions and insertions mapped at various exon positions (e.g. near exon 2, near exon 4, exon 5, exon 6, exon 8, exon 9, and a dense cluster near exon 11).

  The gene-map mutation labels were rendered at very small font size in the source slide; exact digits/positions for several individual variants, especially the dense cluster near exon 11, could not all be confirmed with full certainty.

Genetic Counselling and Ethics

• The genetic counsellor’s role is to establish: the family’s agenda/questions; the diagnosis, its prognosis and genetic basis; how to communicate risk; how that risk is perceived by the consultands; what the options are; and the pros and cons of genetic testing if it is contemplated.
• Ethical dimensions of genetic counselling: the re-definition of autonomy, confidentiality and privacy in this context; genetic exceptionalism; and the duty to forewarn, covering non-paternity, insurance implications, and diagnosis by proxy.

Results, DNA Diagnostics and Linkage

• DNA from Amber, obtained post mortem, showed she was heterozygous for the Lys304Glu mutation, inherited from her mother. The second mutation could not be identified.
• Usual DNA diagnostic approach: PCR amplicons corresponding to the exons; other regions of the gene are not examined. Extra-exonic DNA accounts for over 90% of the entire MCAD locus, so there is potential for mutations to go undetected by exon-only sequencing.
• A solution when no mutation is found: use genetic linkage to genotype at-risk individuals. Chromosomes can be identified and traced because they vary from one another at polymorphic marker loci.
  • Example: on the father’s chromosome 1 pair, one chromosome carries markers a, b, c, d, e with the (undetected) mutation located between markers b and c; the other chromosome carries markers A, B, C, D, E with no mutation. Even though the causal mutation itself is not directly detected, the mutation-carrying chromosome can be tracked through the family by following its flanking polymorphic markers.
• Options for this family: no further children; prenatal diagnosis; early diagnosis using biochemical screening; or early diagnosis using genetic screening (combining direct mutation detection with linkage to determine which alleles were inherited by whom).

Family Risk Calculation

• A complication arose: is a fetus in utero (in a different branch of the family) at risk of MCAD, and what is that risk?
• Using a multi-generation pedigree, carrier probabilities were assigned along the fetus’s line of descent: 0.25 and 0.5 at grandparent-generation individuals on the two sides of the family, 0.125 at the parent leading to the fetus, and 0.5 at that parent’s partner (the deceased affected relative, Amber, being a certain carrier/affected individual on the other branch).
• Risk of the fetus being affected: 0.125 × 0.5 × 0.25 = 0.015 (1 in 64).

A Confidentiality Dilemma

• Khalid does not get on with his brother (they have not spoken for years). Khalid refuses to relay his genetic risk information to his brother, and forbids the clinician from telling anyone else about his genetic status.
• This raises the questions: What do you do? Where does your duty of care lie? When does confidentiality override issues of safety?

Summary

• Clinical genetics practice combines diagnosis, management and counselling skills.
• DNA diagnosis is an imperfect instrument.
• Ancestry can guide genetic diagnosis.
• Genetic information has familial as well as individual implications.

Self-test

1. What are the aims of Clinical Genetics as a discipline, as introduced in this lecture?
2. List the five challenge areas identified in genetic medicine today.
3. What is Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD), and what metabolic step does it block?
4. Describe, in order, the metabolic pathway that MCAD disrupts, and explain what happens to energy production from fat during a period of starvation in someone with MCAD deficiency.
5. How did Amber’s acylcarnitine mass-spectrometry profile differ from a normal profile?
6. What is the incidence of MCAD deficiency, and what proportion of affected individuals present as sudden death before age 2?
7. Why do some individuals with an MCAD-deficiency genotype never develop symptoms?
8. What clinical chemistry and urine findings are characteristic of an MCAD metabolic crisis?
9. Describe the inheritance pattern of MCAD deficiency, its gene’s location, and the recurrence risk for a couple who have already had one affected child.
10. What is the most common MCAD mutation, what amino acid change does it cause, and what proportion of Caucasian disease alleles carry it?
11. List the three routes by which a diagnosis of MCAD deficiency can be reached.
12. Why can standard PCR-based exon sequencing fail to detect an MCAD-causing mutation?
13. Explain how genetic linkage analysis can be used to track a disease-associated chromosome when the causal mutation itself cannot be directly identified.
14. List the options discussed for a family who has already had one child affected by MCAD deficiency.
15. Amber was found to be heterozygous for the Lys304Glu mutation, inherited from her mother, with the second mutation undetected. Using the pedigree carrier probabilities given (0.125, 0.5, 0.25), calculate the risk that a fetus elsewhere in the family is affected.
16. Distinguish the two categories the “old regime” newborn-screening disorders were grouped into, and give one disorder found in each.
17. What must a genetic counsellor establish when working with a family facing a genetic diagnosis?
18. What ethical tension does the case of Khalid and his brother raise, and what three duty-to-forewarn issues does the lecture identify more generally?
19. (Integrative) Trace Amber’s diagnostic pathway from newborn screening through to the family’s risk calculation, naming the key test or reasoning step used at each stage.

Answers

Reveal answers

1. To introduce Clinical Genetics as a medical discipline; to discuss the processes used to identify a genetic disorder; to present the roles of the people involved (family, GP, Clinical Geneticist, laboratories); to demonstrate its diagnostic, psychological and ethical facets; and to show how molecular and clinical genetics are integrated.
2. Single gene disorders (numbers and rarity); chromosomal disorders (a matter of resolution); diseases with complex aetiology (multiple determinants); cancer (design and specificity of treatments); drug design and response (identifying targets and clinically relevant variation).
3. MCAD is an autosomal recessive enzyme deficiency; the enzyme normally converts fatty acids to ketones for energy, and this step is blocked in MCAD deficiency.
4. Carbohydrate (including glucose) feeds into citric acid cycle substrates, which feed into energy production. Free fatty acids would normally be converted, via MCAD, into ketone bodies, which would then also feed into citric acid cycle substrates and energy production. In MCAD deficiency the fatty-acid-to-ketone-body step is blocked, so energy production from fat during starvation is impaired, while the carbohydrate route to energy remains intact.
5. A normal profile shows dominant peaks at C2 and C3 carnitine and a small C16 carnitine peak. Amber’s MCAD profile instead showed a prominent C6 carnitine peak, a large dominant C8 carnitine peak, and a C10:1 carnitine peak, reflecting accumulation of medium-chain acylcarnitines from a block in medium-chain fatty-acid oxidation.
6. 1 in 20,000; 25% present as sudden death below age 2 years.
7. Because MCAD deficiency shows incomplete (non-)penetrance — many people with the genotype have no symptoms over their lifetime.
8. Recurrent non-ketotic hypoglycaemia on clinical chemistry, and unmetabolised free fatty acids excreted as acylcarnitine conjugates in urine.
9. Autosomal recessive with incomplete penetrance; the gene is at 1p31 with 12 exons; recurrence risk for a couple with one affected child is 1 in 4.
10. The Lys304Glu substitution (lysine replaced by glutamic acid at position 304), accounting for 90% of Caucasian disease alleles (a founder effect).
11. Symptoms consistent with the diagnosis; acylcarnitine analysis (blood or urine); genetic analysis (mutation detection).
12. Because the usual approach only sequences PCR amplicons corresponding to the exons; extra-exonic DNA accounts for over 90% of the entire MCAD locus and is not examined, so mutations there go undetected.
13. Chromosomes can be identified and traced because they vary from one another at polymorphic marker loci. Even if the causal mutation is not itself detected, the chromosome carrying it can be followed through a family by tracking its flanking markers (e.g. a chromosome with markers a, b, c, d, e where the undetected mutation lies between b and c can still be distinguished from the non-mutant A, B, C, D, E chromosome and traced into offspring).
14. No further children; prenatal diagnosis; early diagnosis using biochemical screening; early diagnosis using genetic screening (combining direct mutation detection with linkage).
15. Risk = 0.125 × 0.5 × 0.25 = 0.015, i.e. 1 in 64. The undetected second mutation means DNA diagnostics could confirm carrier status wherever the known Lys304Glu mutation is tracked, but could not fully resolve status via direct mutation detection alone in relatives who might carry only the unidentified second mutation — hence the added use of pedigree/carrier-probability reasoning (and linkage) alongside direct testing.
16. Biotinidase deficiency, congenital adrenal hyperplasia, galactosaemia and phenylketonuria (through maple syrup urine disease) were grouped as “treatable genetic disorders”; biotinidase deficiency through phenylketonuria were also grouped as “enzyme deficiencies” (e.g. phenylketonuria is both). Cystic fibrosis and hypothyroidism were listed separately (cystic fibrosis as the commonest autosomal recessive disorder; hypothyroidism as predominantly non-genetic).
17. The family’s agenda/questions; the diagnosis, its prognosis and genetic basis; how to communicate risk; how that risk is perceived by the consultands; what the options are; and the pros and cons of genetic testing if contemplated.
18. Khalid’s refusal to inform his brother, and to allow the clinician to tell anyone else, raises the question of where the clinician’s duty of care lies and when confidentiality should override safety concerns. More generally, the lecture identifies three duty-to-forewarn issues: non-paternity, insurance implications, and diagnosis by proxy.
19. Newborn heel-prick blood spot analysed by tandem mass spectrometry showed an abnormal acylcarnitine profile diagnostic of MCAD. The underlying enzyme defect and autosomal recessive inheritance were established, and mutation detection found Amber heterozygous for Lys304Glu (inherited from her mother) with a second, undetected mutation. Because exon-only PCR sequencing could miss extra-exonic mutations, linkage analysis was proposed as a backup to direct mutation detection. Genetic counselling then addressed the family’s options, and a pedigree-based carrier-probability calculation (0.125 × 0.5 × 0.25 = 1/64) was used to estimate the risk to a fetus elsewhere in the family.