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Cystic fibrosis of the pancreas was described in a study of clinical reports with autopsy analysis of the pancreas that aimed to find more reliable criteria for differentiating causes of steatorrhea. 1 49 cases of cystic fibrosis of the pancreas were described Autosomal recessive single-gene defect was postulated as leading to cystic fibrosis of the pancreas based on an analysis of the inheritance characteristics of 105 cases with documented fibrosis of the pancreas out of 365 children (well and sick) from 118 families 2 Defects in both bicarbonate and chloride were shown in a study of 56 patients with CF with a wide range of pancreatic function and 56 non-cf subjects who where healthy (6 subjects), had no evidence of pancreatic disease but had function testing to rule out Schwachmans syndrome (6 subjects) or CF (3 subjects), had Schwachman s syndrome (10 subjects), or had other diagnoses with variable pancreatic dysfunction (31). 3 Pancreatic output samples were obtained by a marker perfusion pancreatic function test, using a double-lumen tube positioned to perfuse a nonabsorbable marker across the ampulla of Vater and aspirate the marker and pancreatic secretions distally. Samples were analyzed for chloride, bicarbonate, and other secretions. Three pivotal papers were published simultaneously in Science in 1989 describing the discovery and description of the CF gene A detailed analysis of 280 kb of contiguous DNA isolated by chromosome jumping and walking permitted the cloning of the locus responsible for cystic fibrosis without prior knowledge of the basic defect. 4 Analysis of cdna clones identified a region of 6.1 kb that was capable of encoding a 1480 amino acid protein. Hybridization experiments localized the protein to pancreas, primary cultures of nasal polyps, lung, colon, and cultured epithelial cells from sweat glands. 5 When CF sequences were compared with normal sequences, a 3-base-pair deletion difference was found that produced F508del. 5 Predicted structure was a protein with resemblance to membrane transporter proteins. 5 Analysis of CF carriers using restriction fragment length polymorphisms were used to study determine the relation of each of the DNA segments isolated from the chromosome walking and jumping experiments to CF. These studies were able to show a relationship between pancreatic sufficiency/insufficiency and severity of mutations in the gene product. 6 Two studies identified the single channel properties of the CFTR protein expressed in cells that do not normally have a camp-regulated chloride conductance, 7 and synthetic phospholipid vesicles, 8 demonstrating unequivocally that CFTR is a membrane ion channel with a chloride conductance that is activated by camp-dependent protein kinase A. A chloride-independent bicarbonate flux was demonstrated using pancreatic duct epithelia from guinea pigs and wild type mice that was absent in CF mice. 9 References 1. Andersen DH. Am J Dis Child 1938; 56:344-399. 2. Lowe CU et al. Am J Dis Child. 1949;78:349-74. 3. Kopelman H et al. Gastroenterol. 1988;95:349-355. 4. Rommens JM et al. Science. 1989;245:1059-1065. 5. Riordan JR et al. Science. 1989;245:1066-1073. 6. Kerem B et al. Science. 1989;245:1073-1080. 7. Berger HA et al. J Clin Invest. 1991;88:1422-1431. 8. Bear CE et al. Cell. 1992;68:809-818. 9. Ishiguro H et al. J Gen Physiol. 2009;133:315-326. 4

Cystic fibrosis is a rare life-long genetic disease that affects approximately 29,000 people in the United States and about 70,000 worldwide regardless of race or ethnicity but is more common in Caucasians 1,2 CF generally manifests in infancy or early childhood and leads to a shortened lifespan. Availability of multiple treatments and an improved understanding of the course of the disease has led to increased life expectancy; however, the median predicted age of survival was reported to be 39.3 years in the US in 2014 1 Clinical manifestations of CF occur throughout the body and affect many key organs and systems, including lungs, sinuses, pancreas, digestive and reproductive tracts, sweat glands and others 3 References 1. Cystic Fibrosis Foundation (CFF) Patient Registry. 2014 Annual Data Report. Bethesda, MD: CFF; 2015. 2. Sosnay PR et al. Nat Genet. 2013;45(10):1160-1167. 3. O Sullivan BP, Freedman SD. Lancet. 2009;373:1891-1904. 5

CFTR protein channels transport ions, such as chloride and bicarbonate, to regulate fluid and electrolyte balance in epithelial tissues throughout the body Reference MacDonald KD et al. Pediatr Drugs. 2007;9:1-10. 6

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The expression of the CF clinical phenotype in a specific patient is affected by total CFTR activity (determined by by CFTR genotype) as well as by modifier genes and environmental factors 1 Generally, 2 mutations with little or no CFTR activity are associated with a more classic phenotype. The presence of a complex allele may also contribute to reduction in CFTR activity 2-3 Ongoing active research identified many modifier genes that have been implicated in modifying function of various organs affected in cystic fibrosis, including those that :4 Are directly involved in CFTR function (β-adrenergic receptor) Encode alternate ion transport pathways (voltage-gated chloride channel 2) Affect immune responses (mannose binding lectin-2, tissue necrosis factor (TNF)-α) Impact lung function/disease course (transforming growth factor-β1, α-1 antitrypsin, TNF-α) Assessing environmental contribution to the CF pulmonary phenotype is challenging due to various components involved and the lack of objective measurements for many components. Despite these limitations, passive cigarette smoking and ambient air pollution have been linked with reduced pulmonary function. Pulmonary colonization with Pseudomonas aeruginosa and Burkholderia cepacia is an environmentally mediated event that is associated with reduced longevity 4 References 1. Cutting GR et al. Nat Rev Genet. 2015;16:45-56. 2. DeGracia J et al. Thorax. 2005;60:558-563. 3. Castellani C et al. J Cyst Fibros. 2008;7:179-196. 4. Cutting GR. Annu Rev Genomics Hum Genet. 2005;6:237-260. 8

Studies have shown that the increased chloride level in sweat in patients with cystic fibrosis (CF) is linked to dysfunction or absence of CFTR protein. 1 Generally, the sweat chloride concentration in normal subjects is below 40 mmol/l and in individuals with CF, sweat chloride concentrations are 60 mmol/l 2 CF patients with partial CFTR function and exocrine pancreatic sufficiency tend to have lower sweat chloride levels than patients who have pancreatic insufficiency (PI) 1 Individuals with CF and PI have the lowest level of CFTR protein activity and the highest sweat chloride content 1 Therefore, sweat chloride level is a biomarker of pancreatic status in CF 1 Note for the graph: 3 important assumptions were made: 1) sweat chloride levels are versus predicted CFTR activity; 2) normal individuals are assumed to have 100% CFTR activity; 3) carriers are assumed to have 50% CFTR activity 1 References 1. Rowe SM et al. Proc Am Thorac Soc. 2007;4(4):387-398. 2. Farrell PM et al. J Pediatr. 2008;153:S4-S14. 9

A single center study in Toronto evaluated 742 patients with a confirmed clinical diagnosis of CF who had been followed between 1990 and 1997; at diagnosis, 610 (82.2%) were exocrine pancreas insufficient (PI), 110 (14.8%) remained exocrine pancreas sufficient (PS), and 22 patients, who were PS at diagnosis, became PI during follow up (PS PI) 1 Among the 569 patients carrying two severe mutations, 546 (96.0%) were PI, while 61/62 (98%) with at least one mild mutation were PS A subset of patients with severe mutations on both alleles were PS at diagnosis but progressed to PI. Only four individuals (0.7%) with two severe mutations remained PS The correlation between CFTR mutation and pancreatic status is, however, incomplete: pancreatic insufficiency has been observed in some patients with CFTR mutations usually associated with pancreatic sufficiency. This may suggest that factors other than CFTR contribute to the maintenance of the PS state, like modifier genes 2 Genetic variation in SLC26A9 modifier gene, which encodes a bicarbonate and chloride transport protein that interacts with CFTR, is associated with cystic fibrosis-related diabetes (CFRD), and 3 additional type 2 diabetes loci also affect CFRD risk. 3 SLC26A9 was also recently shown to be associated with prenatal exocrine pancreatic damage as measured by newborn screened (NBS) immunoreactive trypsinogen (IRT) levels 4 References 1. Ahmed N et al. Gut. 2003;52:1159-1164. 2. Cutting GR. Annu Rev Genomics Hum Genet. 2005;6:237-260. 3. Blackman SM et al. Diabetes. 2013;62:3627-3635. 4. Miller MR et al. J Pediatr. 2015;166:1152-1157. 10

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The exocrine pancreas secretes digestive enzymes and sodium bicarbonate which are ultimately carried through the pancreatic duct into the duodenum 1 Digestive enzymes include α-amylase, lipase, DNase, RNase and the proenzymes trypsinogen, chymotrypsinogen, procarboxypeptidase A, procarboxypeptidase B, prophospholipase, proelastase, and mesotrypsin 1 The endocrine pancreas secretes hormones into the bloodstream, including insulin, somatostatin, and glucagon 2 References 1. Pandol SJ. The Exocrine Pancreas. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. 2. Begg DP, Woods SC. Adv Physiol Educ. 2013;37:53-60. 13

Pancreatic gross anatomy is depicted on this slide References Pandol SJ. The Exocrine Pancreas. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. Avisse C, Flament JB, Delattre JF. Surg Clin North Am. 2000;80:201-212. 14

Acinar cells store digestive enzymes in forms that prevent destruction of intracellular structures and the acinar cells themselves. Some are stored as inactive proenzymes, others are packaged into acidic zymogen granules that are secreted 1 The lumen of the acini is connected to intercalated ducts, which drain into intralobular collecting ducts, which in turn drain into the main pancreatic duct 1 CFTR protein is located in the duct cell membrane and helps regulate ion and fluid secretion 2 References 1. Pandol SJ. The Exocrine Pancreas. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. 2. Macdonald KD et al. Pediatr Drugs. 2007;9:1-10. 15

Digestive enzymes include α-amylase, lipase, DNase, RNase and the proenzymes trypsinogen, chymotrypsinogen, procarboxypeptidase A, procarboxypeptidase B, prophospholipase, proelastase, and mesotrypsin Water and ions (sodium, potassium, chloride and bicarbonate) are the main inorganic constituents of pancreatic secretion; these constituents and their flow during a meal are necessary for transporting pancreatic enzymes secreted from the acinar cell to the intestinal lumen Another key function of pancreatic water and ion secretion is neutralization of gastric acid emptied into the duodenum Pancreatic enzymes have optimal activity at neutral ph, and are ineffective at the acid ph of gastric secretions Ductal secretion during the intestinal phase of the meal is much greater in volume than acinar secretion. The flow rate increases from an average rate of 0.2 or 0.3 ml/min in the resting state to 4.0 ml/min during stimulation of a meal. The total daily volume of secretion is about 2.5 L The concentration of bicarbonate increases dramatically during a meal with a reciprocal decrease in chloride; The exocrine pancreatic duct is unique in its ability to secrete a fluid with bicarbonate concentrations as great as 140 mm Reference Pandol SJ. The Exocrine Pancreas. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. 16

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Direct harvesting of pancreatic secretions is possible, but invasive and requires duodenal intubation 1 The 72-hour quantitative fecal fat balance study measures pancreatic function by determining coefficient of fat absorption 2 Patients must not be taking pancreatic enzyme supplements (PERT) during the test period. Fat intake must be carefully recorded during the test period 3 Two proxies are often used for pancreatic function: Serum immunoreactive trypsinogen is a useful tool to define PI in patients older than 7 years of age, 4 but the mean age of diagnosis of patients is 3.7 years (median, 4 months) 5 A human monoclonal or polyclonal ELISA test for Fecal Elastase 1 References 1. Daftary A et al. J Cyst Fibros. 2006;5:71-76. 2. Borowitz D et al. J Pediatr Gastro Nutr. 2007;44: 219-223. 3. O Sullivan BP et al. J Pediatr. 2013;162:808-812. 4. Durie PR et al. Pediatr Res. 1986;20:209-213. 5. CFF Patient Registry 2013 Annual Data Report to the Center Directors. Bethesda, MD: 2014 CFF. 18

For research purposes, the 72-hour quantitative fecal fat balance test involves 1 : 3 days of a high-fat diet (100mg fat/d or 60 g fat/m 2 ) controlled and measured by a research dietitian Stool collection between the appearance of 2 orally ingested dye markers to mark the beginning and end of the controlled high-fat diet Fecal fat analysis performed in a central laboratory The formula used to calculate the coefficient of fat absorption is: (grams of fat consumed - grams of fat excreted) X100 grams of fat consumed In a head-to-head comparison, the 72-hour fecal fat balance test was more sensitive and more specific than the fecal elastase and immunoreactive trypsinogen tests for distinguishing between pancreatic insufficiency and pancreatic sufficiency 2 References 1. Borowitz D et al. J Pediatr Gastro Nutr. 2007;44:219-223. 2. Weintraub A et al. J Pediatr Gastroenterol Nutr. 2009;48:306-310. 19

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Fecal elastase is detected using ELISA using either monoclonal or polyclonal antibodies 1 and levels of fecal elastase below 100 µg/g of stool indicate pancreatic insufficiency 1 Monoclonal antibody test can be performed while patients are taking exogenous PERT because the human monoclonal elastase antibody does not cross-react with porcine elastase 2 Limitations of the fecal elastase test 1 : Polyclonal antibodies react with some as yet unknown antigen associated with elastase. Falsely elevated E1 levels may be detected when using the polyclonal antibody test even in the presence of clinical steatorrhea Care should be taken in interpreting values from diarrheal stools and in the presence of intestinal inflammation or enteropathy as underestimation of true E1 levels may result Intestinal inflammation can cause low enzyme levels, which may take several months to recover It is unable to distinguish primary exocrine pancreatic insufficiency from exocrine insufficiency secondary to intestinal villous damage References 1. Daftary A et al. J Cyst Fibros. 2006;5:71-76. 2. Borowitz D et al. J Pediatr Gastro Nutr. 2007;44:219-223. 21

To produce active trypsin, the cells that line the duodenum secrete an enzyme, enteropeptidase, that hydrolyzes a unique lysineisoleucine peptide bond in trypsinogen as the zymogen (inactive precursor or proenzyme) enters the duodenum from the pancreas. The small amount of trypsin produced in this way activates more trypsinogen and the other zymogens The digestion of proteins in the duodenum requires the concurrent action of several proteolytic enzymes, because each is specific for a limited number of side chains. Thus, the zymogens must be switched on at the same time Coordinated control is achieved by the action of trypsin as the common activator of all the pancreatic zymogens trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase, and prolipase, a lipid degrading enzyme Thus, the formation of trypsin by enteropeptidase is the master activation step Reference Berg JM, Tymoczko JL, Stryer L (eds). Chapter 10.5 In: Biochemistry. 5th Edition. New York (NY): W H Freeman; 2002. 22

The mechanism by which trypsinogen enters the systemic circulation remains unknown Inspissated protein plugging of small pancreatic ductules is thought to be a consequence of diminished fluid output resulting from protein hyperconcentration, ductal stasis, and protein precipitation. Regurgitant release of trypsinogen into the circulation from obstructed acinar-ductular units may be an end result of this process Alternatively, increased serum levels of cationic trypsinogen may reflect increased ductular concentrations of pancreatic enzymes, with back diffusion into the systemic circulation The high serum trypsinogen levels in infants with PI and CF probably reflect the presence of functional acini proximal to obstructed ductules Reference Couper RT et al. J Pediatr. 1995;127:408-413. 23

The IRT test can be done with random blood samples 1 An enzyme immunoassay (EIA) with monoclonal antibodies against human trypsinogen in neonatal bloodspots has been evaluated for screening for neonatal CF, and found to be quicker and less labor intensive than conventional assays for the detection of immunoreactive trypsin 2 References 1. Durie PR et al. Pediatr Res. 1986;20:209-213. 2. Bowling FG et al. Lancet. 1987;1:826-827. 24

These data came from a study of 381 children with CF and 99 healthy controls conducted at the Hospital for Sick Children, Toronto, between 1981 and 1983 CF patients had known pancreatic status based on fecal fat balance studies or direct pancreatic stimulation Figures show the least squares mean and 95% confidence intervals that were modeled from the data derived from the 314 patients with pancreatic insufficiency. On the left, the data from those patients is plotted. On the right, the data from the pancreatic sufficient patients is plotted The non-cf range (16.6-46.2 pg/liter) is shaded The results show that after age 7, the IRT curves distinguished PS from PI patients Reference Durie PR et al. Pediatr Res. 1986;20:209-213. 225

Wireless motility capsule (WMC) System Measures ph and transit time Use of ph capsule devise is limited in young children and infants due to its size Reference Bodewes FA et al. J Cyst Fibros. 2015;14:169-177. 26

This pilot study was conducted in 10 subjects with CF and pancreatic insufficiency, and 10 controls matched for sex, age, and BMI Subjects swallowed the wireless motility capsule and carried a receiver to track duodenal ph Following an overnight fast, eligible subjects ate a standardized low fat meal bar and swallowed the WMC with water. CF subjects took half their usual enzyme dose with the meal bar Analysis of CF subjects matched to the controls yielded statistically significant results with time required to reach and sustain ph of 5.5 in CF vs. control subjects (17.7 ± 6.8 vs. 6.3 ± 6.4 min, respectively; p<0.001) and time required to reach and sustain ph of 6.0 in CF vs. control subjects (42.2 ± 23.1 vs. 12.6 ± 17.1 min, respectively; p<0.001) Reference Gelfond D et al. Dig Dis Sci. 2013;58:2275-2281. 27

PAP is proposed as a less costly and invasive alternative to the DNA testing or IRT retesting after the initial elevated IRT test results in newborns 1,2 Blood from neonatal samples (pinpricks/bloodspots) are analyzed using ELISA method 2 According to one proposed screening strategy (France), all newborns are tested for IRT. Newborns with IRT > 50 ng/ml are tested for PAP. Those with PAP > 1.8 ng/ml and those with PAP >1.0 ng/ml and IRT > 100 ng/ml are recalled for sweat testing 2 References 1. Sarles J et al. Arch Dis Child Fetal Neonatal Ed. 1999;80:F118-F122. 2. Sarles J. et al. J Pediatr. 2005;147:302-305. 28

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Under normal conditions in the pancreas, CFTR channels secrete Cl into the lumen of pancreatic ducts to maintain the appropriate fluid volume of pancreatic secretions. These channels also secrete HCO 3 (bicarbonate) to maintain a more alkaline ph inside the lumen of the ducts 1 Pancreatic secretions contain digestive enzymes that flow from the pancreatic duct into the small intestine to properly break down nutrients 1 In CF disease, dysfunction of CFTR channels leads to defective HCO 3 and Cl secretion into the ducts of the pancreas. This impairs the fluid and ph balance of secretions containing digestive enzymes 1-3 As a result, pancreatic secretions become reduced in volume and more acidic, obstructing the pancreatic ducts. This causes progressive damage to the pancreas and may lead to nutrient malabsorption, malnutrition, failure to thrive, and reliance on enzyme replacement therapy 1,2 Pancreatic damage is thought to occur, in part, because inadequate HCO 3 secretion creates an acid environment inside the pancreatic ducts. This acidic environment, normally reached when the digestive enzymes are secreted into the small intestine, begins to prematurely activate digestive enzymes 3 This causes proteolysis (digestion of pancreatic proteins) and inflammation that destroys the exocrine pancreas 3 References 1. Wilschanski M et al. Gut. 2007;56:1153-1163. 2. Walkowiak J et al. Eur J Gastroenterol Hepatol. 2008;20:157-160. 3. Quinton PM. Acta Physiologica Sinica. 2007;59:397-415. 30

In the pancreas, loss of CFTR gene can lead to progressive deterioration of structure and function 1 The physiological effects of defective CFTR in pancreas are reduced volume of secretions (fluid and bicarbonate), and associated increased protein concentration in secretions 1 As a consequence of secretion precipitation, early pathology of duct plugging and dilatation and inflammation occur. 1 Later, fibrosis and fatty replacement of tissue and atrophy occur The clinical picture includes pancreatic insufficiency if the damage is severe, pancreatitis, and with sufficient loss of islets, cysticfibrosis related diabetes (CFRD) may develop 2 References 1. Wilschanski M, Novak I. Cold Spring Harb Perspect Med. 2013;3:a009746. 2. Moran A et al. Pediatr Diabetes. 2014;15(Suppl 20):65-76. 31

Data on timing of pancreatic insufficiency are from a longitudinal evaluation of pancreatic function in patients with CF based on fecal elastase values measured during the first year of life. 1 The evaluable cohort of 61 infants provided their first stool sample for testing before age 3.5 months and their final sample at age 9 months to ensure that assessment of changes in fecal elastase occurring over the entire first year of life 48 of these infants had an initial fecal elastase value <200 µg/g indicating pancreatic insufficiency, and an additional 3 had a starting value above 200 and ending value <200 µg/g, indicating that about 84% of infants in this study were PI within the first year of life A cutoff of 2% of functioning pancreas for pancreatic sufficiency was shown in a study of 24 patients with steatorrhea (fecal fat excretion > 7% of fat intake) and 54 patients with normal fat absorption. 2 The 24 patients with steatorrhea included 3 patients aged 5-12 yr with Shwachman syndrome (exocrine pancreatic hypoplasia and neutropenia), 1 patient aged 1.5 yr with pancreatic hypoplasia of unknown etiology, and 20 patients aged 0.2-15 yr with cystic fibrosis All patients were assessed with direct pancreas stimulation; any pancreatic supplementation was stopped 48 hours before the study Development issues with exocrine pancreas were evaluated in a retrospective study in 60 pancreatic autopsy specimens of infants who died within 4 months of birth, collected over a period of 25 years. 3 Subjects were included in 1 of 3 groups: Group 1: 29 control subjects with no evidence of CF or of any pancreatic involvement in the cause of death Group 2: 24 subjects with a history of meconium ileus and exocrine changes compatible with cystic fibrosis, based on the examination of at least three organ systems Group 3: 7 subjects with pathologic findings compatible with CF and with no history of meconium ileus Control subjects: Ratio of acinar to connective tissue increased 4-fold from birth to 4 months Lumenal volume =0.2% of pancreas volume, not age-dependent CF subjects Ratio of acinar to connective tissue diminished progressively with age 83% of cases had lumenal volume >0.5% of pancreas volume Similar failure of acinar development in CF subjects with meconium ileus and pulmonary failure as cause of death References 1. O Sullivan BP et al. J Pediatr. 2013;162:808-812. 2. Gaskin KJ et al. Gastroenterol. 1984;86:1-7. 3. Imrie J et al. Am J Path. 1979;95:697-708. 32

The study by Augarten et al was an Israeli cohort study of 505 CF patients, 139 of whom were pancreatic sufficient, 366 were pancreatic insufficient, and 4 were PS who converted to PI during the period in their charts 1 Pancreatitis was diagnosed based on episodes of abdominal pain associated with elevation of serum amylase or serum pancreatic lipase levels The study by Ooi et al looked at CF patients from the Canadian Consortium for CF Genetic Studies and Verona CF Centre databases and identified 277 who were PS and met inclusion criteria 2 Pancreatitis was diagnosed by abdominal pain (with no other attributable cause) in association with (1) elevated serum amylase and/or lipase level (>2 x ULN) and/or (2) imaging evidence of acute (such as pancreatic edema, hemorrhage, or necrosis) or chronic (such as calcification or characteristic ductal changes) The study by Durno et al was a cohort study involving 1075 patients with CF in a regional referral center database with acute recurrent or chronic pancreatitis over a 30-year period (1966 1996) 3 Pancreatitis was diagnosed if a patient experienced an acute episode of abdominal pain in association with elevated serum amylase level ( 1.5 x ULN) provided the pain could not be attributed to another cause References 1. Augarten A et al. Eur J Gastroenterol Hepatol. 2008;20:164-168. 2. Ooi CY et al. Gastroenterol. 2011;140:153-161. 3. Durno G et al. Gastroenterol. 2002;123:1857-1864. 33

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Anatomic studies demonstrate that the blood flow from the endocrine pancreas enters the capillaries of the exocrine tissue surrounding each of the islets before entering the general circulation This "portal" system provides for the delivery of very high concentrations of hormones from the islets of Langerhans to the exocrine tissue surrounding the Islets The hormones from the islets of Langerhans include insulin, and although the full significance of the effects of these hormones on the exocrine pancreas is not known, the acinar cells of the pancreas have insulin receptors that are involved in regulation of digestive enzyme synthesis of the exocrine pancreas Reference Pandol SJ. The Exocrine Pancreas. San Rafael (CA): Morgan & Claypool Life Sciences; 2010. 36

Islet cells include β-cells that secrete insulin and amylin, α-cells that secrete glucagon, δ-cells that secrete somatostatin, ε-cells that secrete ghrelin, and F-cells that secrete pancreatic polypeptide 1 Insulin s best known action is to stimulate glucose transporters in cell membranes of most peripheral tissues to activate glucose uptake into the cells for use as fuel or storage as glycogen 1 Glucagon functions as a counter-regulatory hormone to insulin, increasing blood glucose by stimulating glycogenolysis and gluconeogenesis 2 Amylin produces a reduction in eating, gastric acid and glucagon secretion, and limits the rate of gastric emptying 3 Somatostatin inhibits a wide range of endocrine secretions, from the pituitary (growth hormone, prolactin and thyrotropin), gastrointestinal tract (cholecystokinin, gastric inhibitory peptide, gastrin, motilin, neurotensin and secretin) and pancreas (glucagon, insulin and pancreatic polypeptide). It is also a transmitter and neuromodulator in the nervous system 4 Ghrelin increases growth hormone release and stimulates appetite, and also causes a slight increase in glucose levels and reduction in circulating insulin 5 Pancreatic polypeptide inhibits gastric emptying, which may account for its ability to reduce appetite; it can also inhibit intestinal electrolyte and water secretion, and has an inhibitory action on intestinal motor activity and peristalsis 6 References 1. Begg DP, Woods SC. Adv Physiol Educ. 2013;37:53-60. 2. Charron MJ, Vuguin PM. J Endocrinol. 2015;224:R123-R130. 3. Lutz TA. Am J Physiol Regul Integr Comp Physiol. 2010;298:R1475-R1484. 4. Krantic S et al. Eur J Endocrinol. 2004;151:643-655. 5. Lengyel AM. Braz J Med Biol Res. 2006;39:1003-1011. 6. Holzer P et al. Neuropeptides. 2012;46:261-274. 37

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The oral glucose tolerance test is preferred for CFRD based on poor performance of other tests in this population, data linking OGTT with clinical outcomes, and the importance of detecting CFRD early, when fasting glucose levels are still normal Hemoglobin A1c is a measure of glycated hemoglobin in the blood, and therefore gives a measure of the average blood glucose level over the past 3 months, based on the turnover time of erythrocytes 2 Hemoglobin A1c is not sensitive enough to use as a screening test for CFRD 3 Low or normal HbA1c levels do not exclude the diagnosis of CFRD because HbA1c is often spuriously low in CF. This has been postulated to be due to increased red blood cell turnover related to inflammation 1 References 1. Moran A et al. Pediatr Diabetes. 2014;15(Suppl 20):65-76. 2. National Clearinghouse of Diabetes Information. The A1C Test and Diabetes. Available at http://diabetes.niddk.nih.gov/dm/pubs/a1ctest/a1c_test_dm_508.pdf. Accessed March 17, 2015. 3. Moran A et al. Diabetes Care. 2010;33:2697-2708. 39

Testing should be done on 2 separate days to rule out laboratory error unless there are unequivocal symptoms of hyperglycemia (polyuria and polydipsia) A positive FPG or A1c can be used as a confirmatory test, but if it is normal the OGTT should be performed or repeated Reference Moran A et al. Diabetes Care. 2010;33:2697-2708. 40

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Several hypotheses have been proposed for the mechanism of CFRD pathogenesis: Pancreatic destruction leads to loss if islet cells, fibrosis and amyloid deposits in islets 1 Defective CFTR activity led to insufficient depolarization and Ca 2+ mobilization in response to glucose in cultured beta cells 2 β-cells synthesize large amounts of protein. Chronic accumulation of misfolded CFTR may overwhelm the β-cell proteolytic degradation pathways leading to sustained endoplasmic reticulum stress responses (unfolded protein response, endoplasmic reticulum-associated protein degradation) and β-cell apoptosis 3 A chronic inflammatory state produced by oxidative stress, ROS production, 4 elevated pro-inflammatory cytokines, 1 defective fat-soluble antioxidant absorption, and possibly vitamin D deficiency contribute to β-cell destruction and dysfunction and insulin resistance 1,4 Insulin sensitivity is normal in patients with CF in most studies, 1,5 and in studies showing reduced insulin sensitivity, issues of study populations, methods, and interpretation may be factors 5 Insulin resistance in CF may develop because of abnormal localization of the GLUT-4 glucose transporter and/or elevated TNF-α levels 6 References 1. Barrio R. Eur J Endocrinol. 2015;172:R131-R141. 2. Guo JH et al. Nat Commun. 2014;5:4420. 3. Ali BR. Med Hypotheses. 2009;72:55-57. 4. Galli F et al. Biochimica et Biophysica Acta. 2012;1822:690-713. 5. Mohan K et al. Diabet Med. 2009;26:582-588. 6. Hardin DS et al. Am J Physiol Endocrinol Metab. 2001;281:E1022-E1028. 42

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A number of clinical consequences of pancreatic insufficiency have been documented, including steatorrhea, fat-soluble vitamin deficiency, nutrient malabsorption leading to malnutrition, weight loss and failure to thrive, lung function decline, and increased mortality 1-3 Development of CFRD may also occur, and is associated with protein catabolism, lung function decline and increased mortality such that these conditions will be worse in patients with PI and CFRD 4 References 1. O Sullivan BP, Freedman SD. Lancet. 2009;373:1891-1904. 2. Cystic Fibrosis Foundation. Patient Registry 2013 Annual Data Report to the Center Directors. Bethesda, MD. 2014. 3. Schaedel C et al. Pediatr Pulmonol. 2002;33:483-491. 4. Davis PB et al. Pediatr Pulmonol. 2004;38:204-209. 5. Moran A et al. Diabetes Care. 2010;33:2697-2708. 45

Key Points In cystic fibrosis, nutritional deficiency is common due to pancreatic insufficiency and malabsorption. 1 Evidence shows that growth and nutritional status is strongly associated with pulmonary function in children with cystic fibrosis 2,3 Patient Registry data show a strong association between a higher BMI percentile and better lung function in children with CF 2 The graph above on the left shows the association between higher BMI percentile and better lung function in children ages 6 through 19 years. The bar in the middle is the BMI 50th percentile goal for children with CF 2 Shown in the figure on the right, relative weight gain from age 3 to 6 was associated with improved pulmonary function at age 6 (when FEV 1 becomes a reliable measure) 3 Data suggest that early nutritional intervention may improve pulmonary health 2,3 Additional Information A large cohort of patients were prospectively followed and evaluated for growth indexes and pulmonary health measures between age 3 and 6 2 Data revealed that patients with lower growth indexes at age 3 had lower pulmonary function at age 6. This was strongest for measures of weight for age 2 As shown in the figure, relative weight gain from age 3 to 6 was also associated with better pulmonary function at age 6 2 Children whose weight for age was greater than the 10th percentile at age 3 and remained greater than the 10th percentile at age 6 had the highest FEV 1 scores Conversely, patients whose weight for age was less than the 10th percentile at age 3 and remained less than the 10th percentile at age 6 had the lowest FEV 1 scores References 1. O Sullivan BP, Freedman SD. Lancet. 2009;373:1891-1904. 2. Konstan et al. J Pediatr. 2003;142:624-630. 3. Cystic Fibrosis Foundation. Patient Registry 2013 Annual Data Report to the Center Directors. Bethesda, MD. 2014. 46

Occurrence of abnormal glucose and CFRD was assessed in patients recruited during a longitudinal, prospective, multicenter study on Early Diagnosis of Diabetes Mellitus in Patients with Cystic Fibrosis (Trial No. NCT00662714) carried out between 2001 and 2010 in 43 specialized centers (40 German, 3 Austrian) 1 Standardized OGTTs performed to screen serially for CFRD starting at age 10. In case of a pathological OGTT, testing was repeated: according to study protocol, a diabetic OGTT was to be repeated after 4 to 6 weeks and an OGTT revealing impaired glucose tolerance (IGT) or impaired fasting glucose (IFG) was to be repeated after 6 months The final study population comprised 5,179 oral glucose tolerance tests performed in 1,658 cystic fibrosis patients Prevalence of CFRD was evaluated based on a total of 527 patients followed at the University of Minnesota Cystic Fibrosis Center on 15 September 2008 2 Routine annual OGTT screening has been recommended at the University of Minnesota since the early 1990s for patients aged 6 years (1.75 g/kg glucose [maximum 75g]). OGTTs are performed when patients are in their usual baseline state of health References 1. Scheuing N et al. PLoS ONE. 2013;8:e81545. 2. Moran A et al. Diabetes Care. 2009;32:1626-1631. 47

Key Point Patients with CF and mutations associated with pancreatic sufficiency (PS) have a better survival rate than those homozygous for the F508del mutation, 98% of whom are PI 1,2 Notes The clinical impact of CFTR protein activity can be illustrated by the impact of pancreatic manifestations of CFTR impairment on patient survival. Pancreatic sufficiency is associated with lesser impairment of CFTR protein function, whereas PI is associated with greater impairment. 3 In a study of patients with CF (N=264), those with PS had a significantly better survival rate compared with those who were homozygous for F508del-CFTR (P=0.0083) 2 References 1. CFTR2.org F508del mutation details. http://www.cftr2.org/mutation.php?view=general&mutation_id=1. Accessed 5/15/15. 2. Davis PB et al. Pediatr Pulmonol. 2004;38:204-209. 3. Ahmed N et al. Gut. 2003;52:1159-1164. 48

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