A Quick Guide to the

Mutation2789+5G–›A

C F T R S C I E N C E

© 2016 Vertex Pharmaceuticals Incorporated | VXR-HQ-02-00045a(1) | 03/2016

1

Loss of CFTR activity is the underlying cause of cystic fibrosis (CF)1

• People with 2 CFTR mutations resulting in loss of CFTR activity generally have a CF phenotype, which may include1-3,6

– Elevated sweat chloride (>60 mmol/L)

– Pancreatic insufficiency

– CBAVD a

– Lung function decline over time

– Pseudomonas aeruginosa colonization

aCBAVD, congenital bilateral absence of the vas deferens.

References: 1. Davis PB et al. Am J Respir Crit Care Med. 1996;154(5):1229-1256. 2. Rowe SM et al. Proc Am Thorac Soc. 2007;4(4):387-398. 3. Zielenski J. Respiration. 2000;67(2):117-133. 4. Sheppard DN et al. Nature. 1993;362(6416):160-164. 5. Welsh MJ, Smith AE. Cell. 1993;73(7): 1251-1254. 6. Castellani C et al. J Cyst Fibros. 2008;7(3):179-196.

Some CFTR mutations result in little to no CFTR activity3-5

0% 100%

Total CFTR Activity

0% 100%

Total CFTR Activity

Some CFTR mutations result in residual or partial CFTR activity3-5

Spectrum of Phenotypes Associated With Total CFTR Activity1,2

Total CFTR Activity

% of Normal

No CF Disease

CFTR-relatedDisorders

Cystic Fibrosis

Individuals with 2 normal alleles and CFTR mutation carriers

Clinical entities associated with CFTR dysfunctionthat do not fulfill diagnostic criteria for CF, e.g., CBAVD, acute recurrent or chronic pancreatitis and bronchiectasis

Typical phenotype: sinopulmonary disease, sweatchloride >60 mmol/L, pancreatic insufficiency, appearing early in life. In some patients, progressionof disease is delayed or not all features are present

100%

0%

Clinical Phenotype

2

1

Levels of CFTR activity affect survival in CF1

References: 1. McKone EF et al. Chest. 2006;130(5):1441-1447. 2. The World Bank. http://data.worldbank.org/indicator/SP.DYN.LE00.IN?page=2. Accessed November 12, 2015. 3. MacKenzie T et al. Ann Int Med. 2014;161:233-241.

1.00

0.75

0.50

0.25

0.00

0 10 20

Risk Time (Age)

Surv

ival

Pro

babi

lity

30 40 50

Residual CFTR activity (Class IV, V): R117H, R334W, R347P, 3849+10KbC–›T, 2789+5G–›A, A455E

Severely reduced CFTR activity (Class I, II, III): G542X, R553X, W1282X, R1162X, 621-IG–›T, 1717-1G–›A, 1078delT, 3659delC, I507del, N1303K, S549N, G85E, F508del, G551D, R560T

n=1126

n=14,525

• Life expectancy in Western countries (general population born in 2000) is ~79 years2

• Between 1993 and 2002, median survival for US patients with genotypes associated with little to no CFTR activity was 36.3 years (95% CI, 35.5 to 37.6 years), while median survival for those having genotypes associated with residual CFTR activity was 50 years (95% CI, 47.1 to 55.9 years)1

– In this study, patients with a 2789+5G–›A mutation (Class V) were part of the residual CFTR activity group

• More recent US data (2000-2010) suggest median survival across genotypes continues to improve3

Survival Curves by CFTR Activity During a 10-Year Follow-Up (1993-2002) of Patients From the US CFF Registrya

a Data are from a retrospective study of patients enrolled in the Cystic Fibrosis Foundation patient registry measuring risk of death over a 10-year observation period from 1993 to 2002. Patients were grouped as having a high-risk or low-risk genotype based on the functional effects of their class of CFTR mutation on phenotype and mortality. Patients having a Class I, II, or III mutation on both alleles were considered high-risk, while patients having at least 1 Class IV or V mutation were categorized as low-risk. A total of 15,651 patients had a CFTR genotype of a known functional class; 14,525 (93%) had a high-risk CFTR genotype and 1126 (7%) had a low-risk CFTR genotype.1

This survival curve represents population-based outcomes.1 Individual outcomes in cystic fibrosis are variable.

Adapted with permission from McKone EF et al. Chest. 2006;130(5):1441-1447.

3

1

Country registries listing the 2789+5G–›A mutation report 0.3% to 2% prevalence among patients with CF1-5

References: 1. Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry 2013 Annual Data Report. Bethesda, MD.© 2014; Cystic Fibrosis Foundation. 2. Belgisch Mucoviscidose Register – Registre Belge de la Mucoviscidose. The Belgian Cystic Fibrosis Registry. Summary Report 2011.© 2014; Institute of Public Health (WIV-ISP), Brussels, Belgium. 3. French Cystic Fibrosis Registry. Annual Report 2013. © Vaincre la Mucoviscidose and Ined, 2015; Paris, France. 4. Cystic Fibrosis Trust. UK Cystic Fibrosis Registry Annual Data Report 2014. © 2015; Cystic Fibrosis Trust; London, UK. 5. Cystic Fibrosis Australia. Australian Cystic Fibrosis Data Registry 2013–16th Annual Report. © 2015; Cystic Fibrosis Australia; Baulkham Hills NSW, Australia. 6. US CF Foundation, Johns Hopkins University, The Hospital for Sick Children. The Clinical and Functional TRanslation of CFTR (CFTR2). http://www.cftr2.org. Accessed November 12, 2015. 7. European Cystic Fibrosis Society. ECFS Patient Registry 2010 Annual Data Report. 2014. 8. Bobadilla JL et al. Hum Mutat. 2002;19(27):575-606. 9. World Health Organization. The molecular genetic epidemiology of cystic fibrosis: report of a joint meeting of WHO/ECFTN/ICF(M)A/ECFS; June 19, 2002; Genoa, Italy. © World Health Organization; 2004.

Europe: Belgium2: 2%France3: 2%UK4: 0.4%

• In the CFTR2 global database, ~1% of patients with CF have at least 1 copy of the 2789+5G–›A mutation6

Aus5: 0.3%

US1: 1%

Prevalence of the 2789+5G–›A Mutation in Patients With Cystic Fibrosis (% of Patients With at Least 1 Allele)

5%

4%

4%

3%

2%

2%

0.2%

Republic of Moldova7

Slovenia8

Turkey8

Lebanon8

Austria (Tyrol)8

Greece8

Canada9

Country % of Alleles

Additional sources report frequency of the 2789+5G–›A mutation on CF alleles

4

1

References: 1. Zielenski J. Respiration. 2000;67(2):117-133. 2. Highsmith WE Jr et al. Hum Mutat. 1997;9(4):332-338. 3. Wilschanski M et al. J Pediatr. 1995;127(5):705-710.

2789+5G–›A is a splicing mutation that results in some functional CFTR at the apical cell surface1-3

• 2789+5G–›A is associated with alternative splicing and reduced synthesis of CFTR proteins, typical of a Class V mutation1-3

• This results in some functional CFTR proteins at the apical cell surface1-3

Illustrative Example of Class V Defect

CFTR Processing

CFTR Synthesis

CFTR Trafficking

Endoplasmicreticulum

Proteosome

Golgicomplex

DNA

mRNA

Transcription

mRNA

EndoplasmicReticulum

DNA

CFTR gene

mRNA Pre-mRNA

RNA processing

Splicingdefect

Chromosome 7

CFTRTrafficking

CFTR Turnover

CFTR Function

5

1

The 2789+5G–›A allele results in residual total CFTR activity1-3

2789+5G–›A allele results in reduced quantity of CFTR channels at apical

surface

NormalChannel-open

Probability

NormalConductance

Residual2789+5G–›A-CFTR Activity

2789+5G–›A reduces CFTR protein quantity…

…but does not appear to reduce CFTR function…

…and results in residual total CFTR activity

References: 1. Zielenski J. Respiration. 2000;67(2):117-133. 2. Welsh MJ, Smith AE. Cell. 1993;73(7):1251-1254. 3. Sheppard DN et al. Nature. 1993;362(6416):160-164.

1

1

2

2

3

3

Total CFTR activity can be defined as total ion transport mediated by CFTR protein channels at the cell surface, depending on CFTR protein quantity and function.3

=x x

Channel-openProbability

Conductance

CFTR FunctionTotal

CFTR ActivityxCFTR Quantity x =

DefectiveSplicing(Class V)

6

1

Both CFTR alleles play a role in determining phenotype or disease severity1-7

• A 2789+5G–›A allele results in residual CFTR activity. The phenotype of a particular patient is also influenced by the mutation on the other allele4,5

• 2789+5G–›A typically results in the indicated phenotypes

No CF DiseaseNormal individuals and CF carriers

Cystic Fibrosis

CFTR-related DisorderClinical entities associated with CFTR dysfunction that do not fulfill diagnostic criteria for CF

Normal Normal Normal

Normal Residual

Residual Residual

Residual

Littleto None

Littleto None

Littleto None

Littleto None

Allele 1Total CFTR Activity

Allele 2Total CFTR Activity

TotalCFTR

Activity

0%

100%

References: 1. US CF Foundation, Johns Hopkins University, The Hospital for Sick Children. The Clinical and Functional TRanslation of CFTR (CFTR2). http://www.cftr2.org. Accessed November 12, 2015. 2. deGracia J et al. Thorax. 2005;60(7):558-563. 3. Davis PB et al. Am J Respir Crit Care Med. 1996;154(5): 1229-1256. 4. Duguépéroux I, De Braekeleer M. Eur Respir J. 2005;25:468-473. 5. Highsmith WE Jr et al. Hum Mutat.1997;9(4):332-338. 6. Castellani C et al. J Cyst Fibros. 2008;7(3):179-196. 7. Zielenski J. Respiration. 2000;67(2):117-133.

Adapted from Zielenski J. Respiration. 2000;67(2):117-133.

7

1

2789+5G–›A in combination with another allele that produces little to no CFTR activity can result in CF symptoms that may emerge later in life1-5

References: 1. US CF Foundation, Johns Hopkins University, The Hospital for Sick Children. The Clinical and Functional TRanslation of CFTR (CFTR2). http://www.cftr2.org. Accessed November 12, 2015. 2. Duguépéroux I, De Braekeleer M. Eur Respir J. 2005;25:468-473. 3. deGracia J et al. Thorax. 2005;60(7):558-563. 4. Castellani C et al. J Cyst Fibros. 2008;7(3):179-196. 5. Hubert D et al. Eur Respir J. 1996(9):2207-2215.

CF Phenotype In patients registered in the CFTR2 database with a 2789+5G–›A mutation on 1 allele and a pancreatic insufficient mutation on the second allele1:

CFTR Genotype

Residual Total CFTR Activity

EnvironmentalFactors

Modifier Genes

Residual CFTR Protein Activity

Allele #1: 2789+5G–›A

Little to NoCFTR Protein

Activity

Allele #2

• Elevated sweat chloride (average): 97 mmol/L

• Later onset of CF lung disease compared to homozygous F508del patients2

• Pseudomonas colonization: 39% of patients

• Pancreatic insufficiency: 42% of patients

8

1

Summary• Loss of CFTR activity is the underlying cause of CF

• Levels of CFTR activity affect survival in CF

• Country registries listing the 2789+5G–›A mutation report 0.3% to 2% prevalence among patients with CF

• 2789+5G–›A is a splicing mutation that results in some functional CFTR at the apical cell surface

• The 2789+5G–›A allele results in residual total CFTR activity

• Both CFTR alleles play a role in determining phenotype or disease severity

• 2789+5G–›A in combination with another allele that produces little to no CFTR activity can result in CF symptoms that may emerge later in life

9