Diane Cairns (Liverpool )

1. Describe the variable clinical phenotypes that arise due to mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). What are the molecular determinants of phenotypic variability in the so-called CFTR-Related Disorders? Diane Cairns (Liverpool)

2. Key Words • Severe • Mild • Variable • Classical • Non-classical • Variant – Atypical • CFTR-related

3. Cystic Fibrosis • Hereditary disorder affecting mainly lungs & digestive system. • Autosomal recessive. • Disease frequency - ~1 in 2500. (European Caucasians) • Carrier frequency - ~1 in 25. • Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene mutations. • Chloride channel for secretory endothelial cells.

4. Gene • Cystic fibrosis transmembrane conductance regulator (CFTR) 7q31.2, 27 exons. • spans ~190kb of genomic DNA. • mature transcript of 6.5kb that is translated into a 1480aa membrane bound glycoprotein of approximately 168kDa. • member of the ATP binding cassette (ABC) superfamily of proteins. • CFTR functions as a cyclic adenosine monophosphate (cAMP) dependent chloride channel, important in chloride transport across epithelial surfaces.

5. CFTR protein • Comprised of two, 6 span membrane bound regions each connected to a nuclear binding factor which binds ATP. • Between these two units is an R-domain which is comprised of many charged amino acids. • R-domain is a unique feature of CFTR within the ABC superfamily.

6. Mutations • Over 1600 mutations described • CF mutation database (http://www.genet.sickkids.on.ca/cftr) • 1 very common mutation – p.Phe508del. • 4 other mutations, p.Gly542X, p.Asn1303Lys, p.Gly551Asp + p.Trp1282X, frequencies >1%. • other mutations rare or private. • mutations throughout gene BUT hotspots nucleotide binding domains + regulatory domain.

7. CF Mutation Frequencies LocalNational F508del 80.6% F508del 75.3% G551D 3.0% G551D 3.1% 621+1 G>T 1.9% G542X 1.7% G542X 1.5% 621+1 G>T 0.9% R117H 1.5% 1717-1 G>A 0.6% 1898+1 G>A 1.3% R117H 0.5% R560T 1.3% R553X 0.5% R553X 0.5% 1898+1 G>A 0.5% 1717-1 G>A 0.3% N1303K 0.5% I507del 0.3% R560T 0.4% E60X 0.3% N1303K 0.3%

8. Phenotypes multisystem disease, can include: • exocrine pancreas (pancreatic insufficiency - ~85% cases). • intestine (meconium ileus = distal intestinal obstruction). • respiratory tract (pulmonary infection + obstructive lung disease). • male genital tract (infertility). • hepatobilliary system (liver disease). • exocrine sweat glands (high sweat electrolyte causing salt depletion).

9. CF(Classical) • affects the lungs, pancreas, sweat glands, testis, ovaries + intestines (mild to severe). • >95% males CF infertile due to azoospermia. • pulmonary disease major cause morbidity + mortality. Variant forms of CF (non-classical, CFTR-related) • Male infertility eg. CBAVD. • Idiopathic pancreatitis. • Pulmonary CF, eg bronchiectasis, allergic bronchopulmonary aspergillosis, chronic rhinosinusitus.

10. Protein classification • Severity of CF mutations can be classified according to the dysfunction of the protein. • Severe mutations that abolish protein function include nonsense, frame shift or amino acid deletions. • Mild mutations that only partially decrease protein function consist of missense or splice site mutations.

12. Molecular determinants of phenotypic variability • Carriers usually no symptoms. • Threshold activity. • Milder mutation acts as dominant – variable phenotypes. • Higher proportion missense mutations found in CBAVD - ? milder mutations. • Patients homozygous for severe mutations can show a milder phenotype, this may be due to the presence of modifier genes. • Pancreatic sufficiency/insufficiency correlates with genotype. • Genotype-phenotype correlations in pulmonary disease poor – environmental influences – modifier genes.

13. Examples of phenotypic variability in CFTR related disorders • Poly thymidine tract in intron 8. • 3 alleles 5T, 7T or 9T. • 5T allele results in incorrect splicing (majority exon 9 absent). • Frequency of 5T allele = ~5%. • Increased frequency in some CFTR-related disorders. • Modifies other mutations - e.g. p.Arg117His. • polymorphic TG tract adjacent to T tract - 11TG, 12TG and 13TG variants. • found that 12TG/13TG in cis with 5T increased likelihood symptomatic -> ? effect TG on splicing. • Higher TG and lower T number on allele increased chance exon 9 skipping.

14. p.Leu206Trp • Processing class II mutation as the CFTR biosynthetic pathway is severely impaired – however single-channel ion measurements indicate ion conductance similar to wild-type protein. • Question of phenotypic variability in class II mutations including p.Phe508del. • Since multiple potential partners could modify processing of CFTR during its course to the cell surface, environmental and other genetic factors may contribute to variability. • For 30/36 patients p.Leu206Trp was combined with severe mutation – patient conditions varied from isolated CBAVD to severe CF, even within the same genotype. • Results indicate milder CF disease in patients carrying p.Leu206Trp + p.Phe508del, based on the pancreatic sufficient phenotype (>90%) of patients.

15. Complex alleles • c.-102T>A + p.Ser549Arg is associated with a milder phenotype than p.Ser549Arg alone. • p.Ser549Arg + severe mutation. • Promoter mutation in cis with p.Ser549Arg attenuates phenotype. Variants • Missense change p.Met470Val. p.Met470 CFTR proteins have 1.7 fold increased intrinsic chloride channel activity compared to p.Val470 CFTR proteins.

16. Alternative splicing • c.3849+10kb – the ‘mild’ mutation c.3849+10kbC>T results in the insertion of a cryptic exon containing a stop codon in a proportion of the transcripts from that allele. • Significant correlation between level of cryptic exon inclusion and pulmonary function. Modifier genes • Modifiers may include non-CFTR ion channels or genes involved in host defence, inflammation, epithelial repair, mucin production and airway responsiveness. • Several candidate modifier genes identified. • Modifier locus for meconium ileus on chromosome 19q13.

17. Conclusion Changes at the molecular level in the CFTR gene can result in variable disease presentation, affecting many or just one organ. These effects can range from mild to severe. Many factors influence this such as genotype, alternative splicing, modifier genes and environment.

18. References • Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype. Clain et al, Human Mutation 24:360-371 (2005). • Complex alleles -102T>A+S549R(T>G) is associated with milder forms of cystic fibrosis than allele S549R(T>G) alone, Romey et al., 1999 Human Genetics 105 145-150. • Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes., Cuppens et al., 1998 J. Clin Invest. 101 487-496. • Modultation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Rozmahel et al, 1996 Nature Genetics 12 280-287. • Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13. Zielenski et al, 1999 Nature Genetics 22 128-129. • The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10kb C>T mutation. Chiba-Falek et al, 1998 Genomics 53 276-283. • The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. Rave-Harel et al, 1997 Am. J. Hum. Genet. 60 87-94. • Cystic fibrosis modifier genes, Davies et al, Journal of the Royal Society of Medicine, Supplement No:45, Volume 98 2005. • Gene Modifiers in cystic fibrosis, Accurso et al, The Journal of Clinical Investigation, Volume 118, Number 3, March 2008. • Identification of IFRD1 as a modifier for cystic fibrosis lung disease, Gu et al, Nature 458, 1039-1042, 2009. • CMGS Best Practice Guidlines – www.CMGS.org • Gene Reviews – http://www.ncbi.nlm.nih.gov/sites/GeneTests • CF mutation database - http://www.genet.sickkids.on.ca/cftr