THE ROLE OF CFTR MUTATIONS IN CAUSING CYSTIC FIBROSIS (CF)

THE ROLE OF CFTR MUTATIONS IN CAUSING CYSTIC FIBROSIS (CF) Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, MA 02210. Vertex and the Vertex triangle logo are registered trademarks of Vertex Pharmaceuticals Incorporated. 2018 Vertex Pharmaceuticals Incorporated VXR-US-20-02038(1) 11/2018 1 2018 Vertex Pharmaceuticals Incorporated

Overview CF is a complex, genetic disease CF occurs when a child inherits two copies of a CFTR gene mutation resulting in a CF genotype 1,2 CFTR protein activity is determined by CFTR protein quantity and function 1,3 In CF, there is a reduction in total CFTR protein activity leading to an imbalance of fluid and ions in organs throughout the body 1,3,4 Different CFTR mutations produce different effects on CFTR protein quantity and function 1,3,4 When patients and caregivers are educated about their genotype, they are empowered to be active partners in their own care 5 CFTR, cystic fibrosis transmembrane conductance regulator. References: 1. Boyle MP, De Boeck K. Lancet Resp Med. 2013;1(2):158-163. 2. Zielenski J. Respiration. 2000;67(2):117-133. 3. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 4. Elborn JS. Lancet. 2016;388(10059):2519-2531. 5. Cystic Fibrosis News Today. https://www.cysticfibrosisnewstoday.com/2015/02/06/cf-trust-willpay-for-patients-to-know-their-specific-genotype. February 6, 2015. Accessed October 22, 2018. 2 2018 Vertex Pharmaceuticals Incorporated

CFTR proteins in normal cells 3 2018 Vertex Pharmaceuticals Incorporated

CFTR proteins: An important regulator of fluid and ion balance in organs throughout the body CFTR proteins are found on epithelial cell surfaces in organs throughout the body 1-4 Normally, CFTR protein channels transport ions, such as chloride and bicarbonate, through the epithelial cell surface in these organs 1-4 Maintaining water and salt balance at the epithelial cell surface requires an adequate quantity and function of CFTR proteins 5 Total CFTR activity can be defined as total ion transport mediated by CFTR protein channels at the cell surface. It is dependent on CFTR protein quantity and function 4,6 CFTR CFTR function quantity X = Channel-open Conductance probability X Total CFTR activity References: 1. Zielenski J. Respiration. 2000;67(2):117-133. 2. O Sullivan BP, Freedman SD. Lancet. 2009;373(9678):1891-1904. 3. MacDonald KD et al. Pediatr Drugs. 2007;9(1):1-10. 4. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 5. Elborn JS. Lancet. 2016;388(10059):2519-2531. 6. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 4 2018 Vertex Pharmaceuticals Incorporated

CFTR protein function is determined by channel-open probability and channel conductance Channel-open probability: the fraction of time that a single CFTR protein channel is open and transporting ions. 1 Channel conductance: rate at which ions move through open CFTR protein channels. 2 Status of a single channel over time 3* Normal CFTR channel-open probability 3 Normal CFTR channel conductance 1 *Based on in vitro experimentation. Reference: 1. Wang W, Linsdell P. Biochim Biophys Acta. 2012;1818(3):851-860. 2. Hwang TC, Kirk KL. Cold Spring Harb Perspect Med. 2013;3(1):a009498. 3. Bompadre SG et al. J Gen Physiol. 2007;129(4):285-298. 5 2018 Vertex Pharmaceuticals Incorporated

CFTR proteins reach the cell membrane through a multi-step process X X X X CFTR protein function and breakdown CFTR protein trafficking (delivery to the cell surface) CFTR protein synthesis CFTR mutations may affect different steps of this process to reduce CFTR quantity and/or function and reduce total CFTR protein activity 1,2 X X CFTR gene expression References: 1. Boyle MP, De Boeck K. Lancet Resp Med. 2013;1:158-163. 2. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 6 2018 Vertex Pharmaceuticals Incorporated

The importance of understanding CFTR mutations and their nomenclature 7 2018 Vertex Pharmaceuticals Incorporated

CFTR mutations are inherited genetically If a person inherits 2 copies of a disease-causing CFTR mutation (one from each parent), CFTR protein dysfunction can occur 1 CFTR protein dysfunction impairs cellular chloride transport CF disease can be the result Reference: 1. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 8 2018 Vertex Pharmaceuticals Incorporated

CFTR mutations can be referred to by different names Names of CFTR Mutations 1,2,3 Name Definition Utilization Legacy name (F508del) cdna name (c.1521_1523delctt) Commonly used colloquial nomenclature DNA sequence Used primarily in genetic clinics and diagnostic laboratories Used to identify mutations as listed in global sequence databases such as GenBank Protein name (p.phe508del) Amino acid sequence (three-letter code) Used to identify pathogenic mechanism of mutation based on altered protein synthesis Different naming conventions are used by researchers in publications, genetic clinics, and diagnostic laboratories References: 1. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 2. Ogino S, et al. The Journal of molecular diagnostics:2007;9(1):1-6 3. NCBI GenBank. https://www.ncbi.nlm.nih.gov/genbank. Accessed October 25, 2018. 9 2018 Vertex Pharmaceuticals Incorporated

Certain CFTR mutations may affect CFTR quantity and/or function, reducing total CFTR activity Spectrum of phenotypes associated with total CFTR activity 1 People with 2 CFTR mutations resulting in loss of CFTR activity generally have a CF phenotype, which may include 1,2 Elevated sweat chloride (>60 mmol/l) Pancreatic insufficiency CBAVD Lung function decline over time Pseudomonas aeruginosa colonization Certain CFTR mutations result in some CFTR activity 1,2 Other CFTR mutations result in little to no CFTR activity 1,2 Adapted with permission from Castellani and Assael. CBAVD, congenital bilateral absence of vas deferens. References: 1. Castellani C, Assael BM. Cell Mol Life Sci. 2017;74:129-140. 2. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 10 2018 Vertex Pharmaceuticals Incorporated

The effects of CFTR mutations on total CFTR activity 11 2018 Vertex Pharmaceuticals Incorporated

Different CFTR mutations produce different effects on CFTR protein quantity and function CFTR mutations are grouped into classes according to their effects on CFTR protein synthesis, trafficking, or function 1-3 CFTR CFTR function quantity X = Channel-open Conductance probability X Total CFTR activity Defective synthesis (Class I) Defective processing and trafficking (Class II) Defective gating (Class III) Defective conductance (Class IV) Decreased splicing (Class V) Decreased protein stability (Class VI) The top 25 mutations are comprised of those in classes I-V 4 References: 1. Derichs N. Eur Respir Rev. 2013;22(127):58-65. 2. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 3. Zielenski J. Respiration. 2000;67(2):117-133. 4. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 12 2018 Vertex Pharmaceuticals Incorporated

Examples of CFTR mutations that result in defective biosynthesis of the CFTR protein CFTR quantity CFTR function Total CFTR activity Defective synthesis yields no functional X Channel-open Conductance: probability: X = Little to none 1-3 CFTR protein 1-3 Little to none Little to none An absence of CFTR protein regardless of function since few to no CFTR proteins reach the surface... results in little to no total CFTR activity Examples (Class I) Legacy Name 4,5 cdna Name 4,5 Protein Name 4,5 Type 4 US Prevalence (%) 6 G542X c.1624g>t p.gly542x Nonsense 4.7 1717-1G-->A c.1585-1g>a No Protein Name Splice defect 1.6 3659delC c.3528delc p.lys1177serfsx15 Frameshift 0.7 References: 1. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 2. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 3. Zielenski J. Respiration. 2000;67(2):117-133. 4. Ogino S et al. J Mol Diagn. 2007;9(1):1-6. 5. NCBI ClinVar https://www.ncbi.nlm.nih.gov/clinvar/rcv000029523. Accessed October 25, 2018. 6. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 13 2018 Vertex Pharmaceuticals Incorporated

Examples of CFTR mutations that result in defective processing and trafficking of the CFTR protein CFTR quantity CFTR function Total CFTR activity Defective processing and trafficking results in few CFTR proteins at cell surface 1-3 X Channel-open probability: Little to none Conductance: X = Little to none Little to none1-3 Significantly reduced CFTR protein regardless of function since few to no CFTR proteins reach the surface... results in little to no total CFTR activity Examples (Class II) Legacy Name cdna Name Protein Name Type 4 US Prevalence (%) 5 F508del c.1521_1523delctt p.phe508del Amino acid deletion 86.4 a N1303K c.3909c>g p.asn1303lys Missense 2.4 ahomozygous and heterozygous. References: 1. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 2. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 3. Zielenski J. Respiration. 2000;67(2):117 133. 4. Ogino S et al. J Mol Diagn. 2007;9(1):1-6. 5. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 14 2018 Vertex Pharmaceuticals Incorporated

Example of CFTR mutations that reduce channel-open probability in the CFTR protein CFTR quantity CFTR function Total CFTR activity Defective Normal channel Conductance: X gating at cell X = Normal Little to none 1-3 membrane 1-3 Although CFTR protein quantity may be normal function is severely reduced due to decreased channel-open probability... results in little to no total CFTR activity Examples (Class III) Legacy Name cdna Name Protein Name Type 4 US Prevalence (%) 5 G551D c.1652g>a p.gly551asp Missense 4.4 References: 1. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 2. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 3. Zielenski J. Respiration. 2000;67(2):117 133. 4. Ogino S et al. J Mol Diagn. 2007;9(1):1-6. 5. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 15 2018 Vertex Pharmaceuticals Incorporated

Example of CFTR mutations that result in a CFTR protein with defective conductance CFTR quantity CFTR function Total CFTR activity Normal X Channel-open Conductance: probability: X = Some Reduced 1-3 1-3 Normal Although CFTR protein quantity may be normal function is reduced due to decreased conductance... and results in some total CFTR activity Examples (Class IV) Legacy Name cdna Name Protein Name Type 4 US Prevalence (%) 5 R117H c.350g>a p.arg117his Missense 3.0 References: 1. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 2. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 3. Zielenski J. Respiration. 2000;67(2):117 133. 4. Ogino S et al. J Mol Diagn. 2007;9(1):1-6. 5. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 16 2018 Vertex Pharmaceuticals Incorporated

Examples of CFTR mutations that result in some functional CFTR at the cell surface CFTR quantity CFTR function Total CFTR activity Reduced 1-3 X Channel-open Conductance: probability: X = Some Little to none 1-3 Little to none CFTR protein quantity is reduced even though CFTR function is not impaired... and results in some total CFTR activity Examples (Class V) Legacy Name cdna Name Protein Name Type 4,5 US prevalence (%) 5 3849+10kbC->T c.3717+12191c>t p.phe508del Splice defect 1.5 A455E c.1364c>a p.ala455glu Missense 0.6 References: 1. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 2. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 3. Zielenski J. Respiration. 2000;67(2):117 133. 4. Ogino S et al. J Mol Diagn. 2007;9(1):1-6. 5. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 17 2018 Vertex Pharmaceuticals Incorporated

The importance of patients and caregivers knowing their CF genotype 18 2018 Vertex Pharmaceuticals Incorporated

Patients benefit from knowing their CF genotype Patients/Caregivers who are educated about their CF genotypes may be empowered to be active partners in their own care Patients/Caregivers who know about their genotype may 1 : Better understand their CF symptoms and how CF may progress Seek access to treatment plans that are most appropriate for them Actively participate in making care decisions with their doctor or healthcare team Be informed about research studies that may be an option for them Reference: 1. Cystic Fibrosis News Today. https://www.cysticfibrosisnewstoday.com/2015/02/06/cf-trust-will-pay-for-patients-to-know-their-specific-genotype. February 6, 2015. Accessed October 22, 2018. 19 2018 Vertex Pharmaceuticals Incorporated

Engaging patients and caregivers in conversations about CF Communicating with patients and/or caregivers about how their CF genotype can impact care and treatment decisions is important; but helping them understand the reasons can be challenging. Below are some points that can be useful when talking with patients/caregivers about their CF genotype: Consider discussing how: Every person with cystic fibrosis inherited two mutated CF-causing genes which together form a person s CF genotype 1 Different mutations impair CFTR function in different ways leading to variations in the symptoms, disease severity, and disease progression each person with CF experiences 2 Despite the variation in presentation, all genotypes experience disease progression, making personalized disease management important throughout a patient s lifetime 2,3 A care plan can also be personalized based on environment, physiology, patient and caregiver preferences, and lifestyle 4 The most important thing for the patient/caregiver to remember is that when the genotype is known, the care team can customize CF care and treatment plans to help optimize each patient s situation References: 1. Orenstein D et al. Cystic Fibrosis: A Guide for Patient and Family. 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2004. 2. Zielenski J. Respiration. 2000;67(2):117 133. 3. McKone EF. J Cyst Fibros. 2015;14(5):580-586. doi:10.1016/j.jcf.2015.01.005. Epub 2015 Feb 3. 4. Feng L et al. Health Aff (Millwood). 2018;37(5):773-779. 20 2018 Vertex Pharmaceuticals Incorporated

Summary THE ROLE OF CFTR IN CF 21 2018 Vertex Pharmaceuticals Incorporated

Summary CFTR protein is an important cellular regulator in organs throughout the body 1,2 CFTR mutations result in a loss of total CFTR protein activity, which is the underlying cause of CF 1,2 Different types of CFTR mutations vary in their effects on CFTR protein quantity and/or function 2,3 Both CFTR mutations play a role in determining phenotype or disease severity 4 Discussing genotype with patients helps them understand their CF disease presentation and treatment options 5 References: 1. Boyle MP, De Boeck K. Lancet Rep Med. 2013;1:158-163. 2. Derichs N. Eur Resp Rev. 2013;22(127):58-65. 3. Cystic Fibrosis Foundation. Patient Registry Annual Data Report 2016. Bethesda, MD. 2017 Cystic Fibrosis Foundation. 4. Zielenski J. Respiration. 2000;67(2):117 133. 5. Feng L et al. Health Aff (Millwood). 2018;37(5):773-779. 22 2018 Vertex Pharmaceuticals Incorporated