L13 Genetics Molecular diagnosis and prognosis in inherited disorders

Overview

This lecture uses cystic fibrosis (CF) as a worked example of precision/personalised medicine in a monogenic disorder. It moves from variant nomenclature and CF epidemiology, through CFTR gene/protein structure and the molecular classification of CFTR mutations, to newborn screening and diagnostic testing, then to the development, mechanism and New Zealand funding history of CFTR modulator drugs, closing with a Hardy-Weinberg revision link to carrier frequency.

Variant nomenclature and classification

• The genetics community (led by the American College of Medical Genetics and Genomics, ACMG) recommends replacing the terms “mutation” and “polymorphism” with the neutral term variant, because those older terms wrongly imply pathogenic or benign effects respectively.
• Five-tier variant classification: pathogenic, likely pathogenic, uncertain significance, likely benign, benign.
• Any assertion of pathogenicity should be reported together with the condition and inheritance pattern, e.g. c.1521_1523delCTT (p.Phe508del), pathogenic, cystic fibrosis, autosomal recessive.
• Source: 2015 ACMG/Association for Molecular Pathology guidelines.

Cystic fibrosis: the disorder

• Severe, early-onset, autosomal recessive disorder with high penetrance; incidence about 1/3000 newborns in NZ; roughly 400 patients in NZ.
• Carrier frequency: about 4% of Caucasians (1/25) carry a CFTR variant.
• Caused by defects in the single CFTR gene, which encodes the Cystic Fibrosis Transmembrane Conductance Regulator, a chloride channel protein.
• Core pathophysiology: defective CFTR leads to thick, sticky mucus that blocks ducts throughout the body.
• Organs affected and mechanism:
  • Airways: mucus clogs bronchial passages and promotes chronic infection; progressive lung destruction is the leading cause of CF death.
  • Pancreas: duct occlusion stops digestive enzymes reaching the bowel in about 65% of patients; can also cause diabetes.
  • Small intestine: obstruction by thick stool requires surgery in about 10% of newborns.
  • Liver: plugging of small bile ducts disrupts liver function in about 5% of patients.
  • Reproductive tract: absence of the vas deferens renders about 95% of males infertile; occasionally a mucus plug causes female infertility.
  • Skin/sweat glands: malfunctioning sweat glands cause excess NaCl in sweat, so sweat chloride measurement is a mainstay of diagnosis.
• Historical life expectancy rose from under 1 year (1959) to over 40 years (2016), reflecting better respiratory physiotherapy, infection control, nutrition and, more recently, mutation-specific drugs.

Framework: diseases with a genetic component

• Monogenic disorders (“rare”) are the focus of the individual specific-disorder study.
• Polygenic disorders include diabetes, autism and obesity.
• Cancer is covered separately in the cancer module and tutorial.
• General workflow: identify the genetic defect(s) and pathophysiology, then apply this in two directions:
  • Diagnostics (e.g. haemochromatosis, CF affected/carrier screening, inherited cancer screening) leading to preventative medicine.
  • Pharmacogenomics (e.g. mutation-specific CF treatment, targeted cancer therapies) leading to genetics-informed personalised/precision medicine.

CFTR gene and protein structure

• CFTR gene: DNA 189 kb, mRNA 6 kb, protein 1480 amino acids, spanning 24 exons and their introns.
• Pathway: gene (exons/introns) -> transcription -> primary transcript -> RNA processing -> mRNA with poly-A tail -> translation -> CFTR protein -> folding and insertion into the membrane -> functional folded cell-surface protein.
• Protein domains: NH2 terminus, two ATP-binding sites/domains, a central R (regulatory) domain, hydrophobic transmembrane regions, COOH terminus, and carbohydrate side chains; together these form a chloride ion channel spanning the lipid bilayer.
• The gene was cloned and characterised in 1989 (Riordan, Rommens, Kerem and colleagues, including Francis Collins and Lap-Chee Tsui).

Allelic heterogeneity and mutation testing

• CFTR shows allelic heterogeneity: many different disease-causing variants exist. Clinical testing panels: a 50-variant panel ($402)orfullCFTRgenesequencing($710).
• F508del (HGVS c.1521_1523del, legacy name DeltaF508/DF508) is by far the most common CF variant, accounting for 72.42% of alleles in the tested non-Hispanic Caucasian US reference population.
• Other variants and their frequencies in that population: G542X 2.28%, G551D 2.25%, 621+1G>T 1.57%, DI507 0.88%, R117H 0.70%, 1717-1G>A 0.48%, R347P 0.45%, A455E 0.34%, G85E 0.29%, R334W 0.14%; 394delTT, 1078delT and R347H are listed without a stated frequency.
• Molecular basis of F508del: an in-frame three-nucleotide deletion removes the phenylalanine at codon 508. Normal sequence ATC ATC TTT GGT GTT (Ile-Ile-Phe508-Gly-Val510) becomes ATC ATT GGT GTT (Ile-Ile-Gly-Val506) in DeltaF508.

Molecular pathophysiology: CFTR mutation classes

CFTR variants are grouped into six functional classes by the step of protein synthesis, processing or function each disrupts:

• Class I, no synthesis: nonsense (e.g. G542X), frameshift (e.g. 394delTT), or splice-junction (e.g. 1717-1G>A) variants that stop CFTR protein being made at all.
• Class II, block in processing: misfolded protein (e.g. F508del, N1303K) is retained in the endoplasmic reticulum and degraded rather than trafficked to the Golgi and cell surface.
• Class III, block in regulation: protein reaches the cell surface (e.g. G551D) but the channel gate fails to open normally.
• Class IV, altered conductance: protein reaches the surface and opens (e.g. R117H, R347P) but conducts chloride poorly.
• Class V, reduced synthesis: normal protein is made but in reduced amount (e.g. A455E missense; 3849+10kbC>T alternative splicing).
• Class VI, reduced surface stability: mutations that shorten the time functional CFTR spends at the cell surface.
• Trafficking pathway these classes map onto: CFTR gene in the nucleus -> transcription/translation and folding in the endoplasmic reticulum (Class I blocks synthesis here; Class II blocks exit/processing) -> Golgi complex -> onward trafficking to the cell surface, where Class III and IV act (gating and conductance); misfolded protein can be degraded via the proteosome.

Reducing the burden of genetic disorders: levels of intervention

A framework for approaches to CF and similar disorders, moving from the molecular to the societal level:

• DNA level: gene therapy, e.g. CRISPR-Cas delivered to the lung.
• RNA level: nucleic-acid drugs.
• Protein level: small-molecule drugs.
• Functional-protein level: small-molecule replacement drugs.
• Cell level: cell transplant.
• System/organ level: lung transplant.
• Person/family level: genetic counselling and lifestyle measures.
• Society level: support and funding.

CFTR modulator therapies

Two broad classes of modulator drug, described in the lecture with a “door” metaphor:

• Correctors (“makers”: elexacaftor, tezacaftor, lumacaftor) help misfolded CFTR protein fold correctly and traffic to, and remain at, the cell surface.
• Potentiators (“openers”: ivacaftor) bind CFTR protein already at the cell surface and hold the channel gate open, increasing chloride flow.

Ivacaftor (Kalydeco)

• A potentiator, approved for people over 6 months old.
• First CF drug approved (US, February 2012); subsequently funded in the UK (2012), Australia (2014) and by Pharmac in NZ (2020).
• Targets gating mutations, chiefly p.G551D (about 2% of NZ CF mutations, roughly 18 people) plus other gating and residual-function mutations (about 6% of all CF patients).
• List price roughly US$294,000to$350,000 per year (about NZ$200,000/yearcitedelsewhere);2014worldwidesaleswereUS$464 million.
• Clinical effect illustrated with before/after patient photos and chest X-rays showing clearer lung fields after treatment.
• Pharmac confirmed funding of ivacaftor for G551D and other class III gating mutations on 26 February 2020, via agreement with Vertex Pharmaceuticals.

Lumacaftor/ivacaftor (Orkambi)

• Combination of a corrector (lumacaftor) and a potentiator (ivacaftor).
• For patients with two copies of F508del, or one of 26 specified residual-function mutations; approved from age 2 years.
• With lumacaftor alone only about one-third of CFTR protein reaches the cell surface and those channels do not open enough; adding ivacaftor increases chloride flow.
• Clinical benefit is only modest: chloride transport rose from about 3% of normal with no drug, to about 14% with the corrector alone, to about 26% with corrector plus potentiator (trial p-values 0.0033 and 0.0119/0.0288). Lung function improved by only about 3% in trials, yet patient testimony (e.g. to the FDA, 2015) described major quality-of-life benefit.

Tezacaftor/ivacaftor (Symdeko)

• Tezacaftor is another corrector, combined with the potentiator ivacaftor.
• Approved for a single copy of one of 26 specified mutations, from age 6 years; also an option for F508del homozygotes unable to tolerate lumacaftor/ivacaftor.
• FDA approved 2018; list price about US$292,000/year.

Trikafta (Elexacaftor/tezacaftor/ivacaftor)

• Elexacaftor and tezacaftor act as correctors, binding different sites on F508del-CFTR protein to help more of it reach the cell surface; ivacaftor is the potentiator that helps it stay open there.
• Suitable for about 90% of CF patients: those aged 12 years and over with at least one F508del allele.
• Trial results versus placebo: 10% increase in lung function with two F508del copies, 14% increase with one copy.
• List price about NZ$430,000 per patient per year.
• Pharmac confirmed funding on 7 March 2023 for patients aged 6 years and over meeting eligibility criteria, following an application from Vertex received in July 2021; wider access to ivacaftor and a possible funding application for Symdeko were flagged as still under consideration.

Funding, access and public advocacy in New Zealand

• High list prices created long gaps between overseas approval and NZ Pharmac funding, e.g. ivacaftor was approved in the US in 2012 but not Pharmac-funded until 2020.
• Media coverage highlighted the human cost of funding delays through named patient cases, including Mia James, Bella Powell, Kase Williams and Hamish Mountfort, whose families publicly campaigned for Kalydeco or Trikafta funding.
• The estimated cost to fund Trikafta for all NZ CF patients was cited as about $236 million per year.
• These cases illustrate the recurring tension in precision medicine between very high per-patient drug cost and substantial clinical/quality-of-life benefit, and the ethical and public-health dimension of funding decisions.

Screening and diagnosis

• NZ newborn metabolic screening: a heel-prick blood sample is taken from about 60,000 babies per year onto a “Guthrie card” and screened for more than 20 metabolic diseases.
• CF newborn screening is mainly biochemical, using tandem mass spectrometry to measure immunoreactive trypsinogen in blood.
• A positive screen is confirmed by direct genetic (DNA) analysis or a sweat chloride test.
• Carrier testing is possible by DNA analysis (genotyping) and underpins genetic counselling; carriers are shown on pedigree diagrams with a diamond-within-circle/square symbol.
• Key distinction: screening tests (population-level, e.g. the Guthrie card) versus diagnostic tests (confirmatory, e.g. sweat test or direct DNA analysis).

Hardy-Weinberg equilibrium (revision)

• Used to estimate carrier and affected frequencies for an autosomal recessive condition such as CF, with genotypes AA (unaffected, non-carrier), Aa (carrier) and aa (affected).
• Genotype frequencies from a Punnett-style cross of A(p)/a(q) gametes: frequency of AA = $p^2$, frequency of Aa = $2pq$, frequency of aa = $q^2$.
• Hardy-Weinberg equation: $p^2+2pq+q^2=1$.

Summary

• CF is a well-studied disorder with a clear genetic basis.
• Understanding of CFTR variants and their pathophysiology has driven: newborn/molecular screening, improved general treatments, mutation-specific drugs, and carrier testing and counselling.
• The lecture frames CF as a model for personalised medicine generally: matching a diagnostic biomarker (the patient’s specific CFTR variant/class) to the right targeted therapy for that patient.

Self-test

1. Explain why the ACMG recommends using the term “variant” with a modifier instead of “mutation” or “polymorphism”, and list the five classification tiers.
2. Describe the inheritance pattern, incidence and carrier frequency of cystic fibrosis, and state which single gene is defective.
3. List three organs or systems affected by CF and, for each, describe the mechanism by which thick mucus causes disease.
4. Describe the pathway from CFTR gene to functional cell-surface protein, and give the approximate sizes of the CFTR gene, mRNA and protein.
5. What proportion of CF alleles in the tested reference population carry the F508del variant, and what is the molecular basis of this variant at the nucleotide and amino acid level?
6. Describe the six functional classes of CFTR mutation (I to VI) and, for each, state the step of CFTR synthesis, trafficking or function it disrupts, with an example variant.
7. A patient has a nonsense CFTR variant that prevents any CFTR protein being synthesised. Which mutation class is this, and would a potentiator drug such as ivacaftor be expected to help? Explain why or why not.
8. Distinguish a corrector from a potentiator CFTR modulator drug, in terms of mechanism and which mutation class each targets.
9. Describe how ivacaftor works at the molecular level, which patients it is approved for, and its NZ Pharmac funding history.
10. Explain why lumacaftor/ivacaftor (Orkambi) produced only a modest improvement in lung function in trials despite substantial reported improvements in patient quality of life.
11. Describe the mechanism of Trikafta (elexacaftor/tezacaftor/ivacaftor) and state which patients are eligible for it.
12. Outline the steps of newborn CF screening in New Zealand, from heel-prick sample to diagnostic confirmation, and distinguish a screening test from a diagnostic test.
13. Describe the “levels of intervention” framework (DNA to society) for reducing the burden of a genetic disorder like CF, with one example approach at each level.
14. Using Hardy-Weinberg equilibrium and a CF incidence of 1/3000 newborns, estimate the carrier frequency and compare it with the value given in the lecture.
15. Why did high-profile NZ patient advocacy cases (e.g. Bella Powell, Kase Williams) arise around drugs like Kalydeco and Trikafta, and what tension in precision medicine funding do they illustrate?
16. Distinguish monogenic, polygenic and cancer-related genetic disease, giving an example of each from the lecture.

Answers

Reveal answers

1. Older terms wrongly imply an effect on function (“mutation” implying pathogenic, “polymorphism” implying benign), so ACMG recommends the neutral term “variant” plus a modifier. The five tiers are pathogenic, likely pathogenic, uncertain significance, likely benign, and benign.
2. CF is a severe, early-onset, autosomal recessive disorder with high penetrance, incidence about 1/3000 newborns in NZ, and a carrier frequency of about 4% of Caucasians (1/25). The defective gene is CFTR (Cystic Fibrosis Transmembrane Conductance Regulator).
3. Airways: mucus clogging and chronic infection progressively destroy the lungs (leading cause of death). Pancreas: duct occlusion stops digestive enzymes reaching the bowel (about 65% of patients) and can cause diabetes. Small intestine: thick stool obstructs the gut, requiring surgery in about 10% of newborns. (Liver duct plugging in about 5%, and vas deferens absence causing infertility in about 95% of males, are also acceptable answers.)
4. Gene (exons/introns) is transcribed to a primary transcript, RNA-processed to mRNA with a poly-A tail, translated to CFTR protein, then folded and inserted into the membrane as a functional cell-surface channel. DNA is 189 kb, mRNA is 6 kb, protein is 1480 amino acids.
5. F508del accounts for 72.42% of alleles. It is an in-frame deletion of three nucleotides removing phenylalanine at codon 508: normal ATC ATC TTT GGT GTT (Ile-Ile-Phe508-Gly-Val510) becomes ATC ATT GGT GTT (Ile-Ile-Gly-Val506) in DeltaF508.
6. Class I, no synthesis (e.g. G542X nonsense, 394delTT frameshift, 1717-1G>A splice). Class II, block in processing/ER retention and degradation (e.g. F508del, N1303K). Class III, block in regulation, protein reaches the surface but the gate will not open (e.g. G551D). Class IV, altered conductance, channel opens but conducts poorly (e.g. R117H, R347P). Class V, reduced synthesis of otherwise normal protein (e.g. A455E, 3849+10kbC>T). Class VI, reduced stability of CFTR once at the surface.
7. This is a Class I mutation. A potentiator would not be expected to help, because potentiators act on CFTR protein already present at the cell surface, and Class I mutations mean no CFTR protein is made at all, so there is nothing for the drug to act on.
8. Correctors (e.g. lumacaftor, tezacaftor, elexacaftor) help misfolded CFTR fold correctly and traffic to and remain at the cell surface, addressing Class II-type defects. Potentiators (ivacaftor) bind CFTR already at the surface and hold the channel gate open, addressing Class III (gating) defects.
9. Ivacaftor binds the defective CFTR protein at the cell surface and holds the chloride channel gate open, increasing chloride flow. It is approved from 6 months of age, mainly for the G551D gating mutation and other gating/residual-function mutations (about 6% of CF patients). It was first approved in the US in 2012, funded earlier overseas (UK 2012, Australia 2014), and only Pharmac-funded in NZ from 2020.
10. With lumacaftor alone only about one-third of CFTR protein reaches the surface, and even with ivacaftor added chloride transport only rises to about 26% of normal (versus about 3% with no drug), giving only about a 3% lung-function improvement in trials. Despite this modest measured effect, patients reported it substantially improved daily quality of life (e.g. the Jeff Masters FDA testimony).
11. Elexacaftor and tezacaftor act as correctors, binding different sites on F508del-CFTR to help more protein reach the surface, while ivacaftor potentiates the channel once there. It is approved for patients 12 years and older with at least one F508del allele, covering about 90% of CF patients.
12. A heel-prick blood sample is taken from newborns onto a Guthrie card and screened biochemically (tandem mass spectrometry for immunoreactive trypsinogen). A positive screen is confirmed diagnostically by direct genetic analysis or a sweat chloride test. Screening tests identify at-risk individuals in a population; diagnostic tests confirm the condition in an individual.
13. DNA: gene therapy, e.g. CRISPR-Cas in the lung. RNA: nucleic-acid drugs. Protein: small-molecule drugs. Functional protein: small-molecule replacement. Cell: cell transplant. System/organ: lung transplant. Person/family: genetic counselling and lifestyle. Society: support and funding.
14. q^2 = 1/3000, so q is about 0.018 and p is about 0.982. Carrier frequency = 2pq is about 2 x 0.982 x 0.018, about 3.6%, close to the approximately 4% (1/25) carrier frequency given in the lecture.
15. Drugs like Kalydeco and Trikafta had very high list prices (hundreds of thousands of dollars per patient per year) and were funded overseas well before NZ, so families campaigned publicly (e.g. Bella Powell, Kase Williams, Hamish Mountfort) to pressure Pharmac to fund them. This illustrates the tension between very high per-patient drug cost and substantial clinical/quality-of-life benefit that underlies precision-medicine funding decisions.
16. Monogenic disease is caused by a single gene defect and is individually rare, e.g. cystic fibrosis. Polygenic disease results from many genes (plus environment), e.g. diabetes, autism, obesity. Cancer has both somatic and inherited genetic contributions and is covered separately in the cancer module.