Genetic Disorders of the Pancreas and Pancreatic Disorders in Childhood

Models of Pancreatic Biology and Disease

Fig. 57.1 shows a progressive model of CP that organizes genetic and environmental risks and modifying factors, clinical features, biomarkers, and complications in response to dysfunction of the injury → inflammation → resolution → regeneration sequence. Some patients develop evidence of CP without going through all of the steps, but the model still allows for the complex risk profile and probabilities of progression to be calculated in most patients. The model is agnostic to the mechanism of injury or progression through the various stages as long as it includes significant injury and/or stress, and inflammation. The later complications of fibrosis, pancreatic exocrine insufficiency, diabetes mellitus, various chronic pain syndromes, and cancer risk are not surrogates of each other, but represent disease features of different cell types or systems linked to the pancreas.

There are many independent and combined risk factors for CP, which become pathogenic once the inflammatory process has been initiated (see Fig. 57.1 , Stage A). TIGAR-O is an acronym for classes of etiological factors including Toxic-metabolic, Idiopathic, Genetic, Autoimmune, Recurrent acute or severe AP, and Obstructive causes. An updated TIGAR-O list ( Box 57.1 ) organizes the major etiological factors found in an individual recognizing that most patients have multiple risk factors. The TIGAR-O approach is also integrated into the M-ANNHEIM classification system using a modified acronym.

The Acinus: An Exocrine Pancreas Functional Unit Model

Although physicians typically view the exocrine pancreas as a unit, the etiology and mechanism of pancreatitis generally begins within either the acinar cell or duct. The distinction is important for targeting treatments and developing management plans.

The acinar and duct cells are organized in functional units called acini (the plural of acinus) (see Chapter 55 ). A simplified model of the acinus and duct is given in Fig. 57.2 . An acinus (top) is a local organization of acinar cells with the apical membrane facing the lumen at the most upstream part of the pancreatic duct. The duct (bottom) is an organization of duct cells to form a tube that extends from the center of each acini to the lumen of the duodenum. The nerves, blood vessels, islets, immune cells, and supportive tissue, which are important for various aspects of complex pancreatic disease, are not shown here because the initiation of AP originates within the context of Fig. 57.2 . Many of these factors affecting the acinar cell or duct interact with each other as a complex, disease susceptibility mechanism with different combinations of risk factors in different subjects.

Acinar Cell Dysfunction and Disease

The acinar cells make up the bulk of the parenchymal mass and are responsible for most of the inflammatory diseases of the pancreas, either directly or indirectly, as their products are the primary contributor to duct content. The principle function of the pancreatic acinar cell is to synthesize and secrete pancreatic digestive enzymes (see Chapter 56 ). The process includes the synthesis of a range of pancreatic pro-enzyme proteins (zymogens) in the rough endoplasmic reticulum (RER), transportation of the properly folded proteins to the Golgi apparatus for sorting and packaging in zymogen vesicles, vesicular trafficking of the zymogens to the apex, and apical secretion of the zymogens into the pancreatic ducts ( Fig. 57.3 ). The process requires large amounts of energy for protein synthesis and transport of ions, such as calcium, from one compartment to another.

The maintenance of low calcium concentrations within the acinar cells is critical to protecting them from premature trypsinogen activation. Acinar cell calcium can rise through neurohormonal hyperstimulation ^, ; high extracellular calcium concentrations ; bile acid reflux, which opens apical membrane calcium pathways ^, ; prolonged, high-dose alcohol consumption, which lowers the threshold for stimulation-induced AP ; mitochondrial damage ; and other factors that regulate intracellular calcium. Any process that increases acinar cell calcium will predispose to AP through a calcium-dependent trypsinogen activation and stabilization mechanism.

Trypsin-Dependent Pancreatitis Pathway

Acute and CP can be triggered and driven from within the acinar cell by several mechanisms. Trypsin is a key to triggering AP. Trypsin is the master digestive enzyme because it activates itself and all the other zymogens to their active form (see Chapter 56 ). Activation of digestive enzymes within the pancreas leads to autodigestion, the release of immune activating molecules, and direct cross-activation of components of the immune system further amplifying the immune response to injury. ^, The importance of trypsin in AP is illustrated by genetically engineered trypsin “knockout” mice that are resistant to experimental AP. Thus, minimizing generation of uncontrolled trypsin activity within the pancreas is critical to protecting the patient from AP.

Acinar cells synthesize trypsin as the pro-enzyme trypsinogen, a zymogen that normally remains inactive until it reaches the duodenum and is activated by the duodenal enzyme enterokinase. Cationic trypsinogen ( PRSS1 ) is the major form of trypsinogen (≈65%) followed by anionic trypsinogen ( PRSS2, ≈30%) and mesotrypsin ( PRSS3, ≈5%).

The trypsinogen molecule has 2 globular domains linked by a single connecting chain ( Fig. 57.4 ). Activation of trypsinogen normally occurs when enterokinase or trypsin cleave an 8 amino acid peptide, the trypsinogen activation peptide (not shown), from trypsinogen to form trypsin.

Trypsinogen also has 2 sites that allow it to be digested by proteolytic enzymes. Trypsin can be digested by cleavage at the Arg122-Val123 peptide bond by another trypsin molecule or at the Leu81-Glu82 peptide bond by another digestive enzyme, chymotrypsin C (CTRC). ^,

The trypsinogen molecule also has 2 calcium binding pockets that determine if the trypsinogen molecule will be activated by trypsin (in high calcium concentrations) or destroyed by trypsin (in low calcium concentrations). Thus, local calcium concentrations serve as a critical switch between trypsin activation and inactivation.

Trypsinogen can be prematurely activated to trypsin by autoactivation (facilitated by elevated calcium and lower cell pH), by another trypsin, by other enzymes in other cellular compartments (e.g., lysosomes), and/or other mechanisms. Furthermore, multiple mechanisms of controlling trypsin activity in the wrong place at the wrong time have been deduced based on studies of humans with idiopathic RAP, cell biology studies, animal models, and in vitro experiments.

Acinar Cell Dysfunction/Failure Without Pancreatitis

Shwachman-Diamond syndrome gene mutations (SBDS). SDS is an autosomal recessive, multisystem Mendelian disorder characterized by EPI rather than pancreatitis, cyclic neutropenia, bone malformation, and other features. ^, A previously uncharacterized causative gene was discovered and named the Shwachman-Bodian-Diamond syndrome gene ( SBDS ). The gene product participates in ribosome maturation, a process critical to many cell types. In addition, mutations in other genes such as DNAJC21 , SRP54, EFL1 , and others appear to cause a similar syndrome. The molecular mechanism of disease and clinical syndrome will be discussed later.

Johanson-Blizzard syndrome (UBR1). Johanson-Blizzard syndrome is an autosomal recessive, multisystem Mendelian characterized by pancreatic exocrine destruction beginning in utero, although milder cases may present later in life. ^, Johanson-Blizzard syndrome is caused by mutations in UBR1 , which encodes one of the E3 ubiquitin ligases that is normally involved in removing and degrading various cytosolic digestive enzymes from the cell through the proteasome, a system that is critical to intracellular amino acid generation. The clinical spectrum of Johanson-Blizzard syndrome is further described later.

Duct Cell–Related Pancreatitis Mechanisms

The pancreatic duct system serves 2 important functions. First, it connects every acinus to the duodenum for delivery of pancreatic digestive enzymes. Secondly, it generates large volumes of sodium bicarbonate to neutralize gastric hydrochloric acid. The alkaline pancreatic juice also protects the pancreas from premature activation of pancreatic digestive enzymes, and likely other protective functions.

There are 2 general pathologic conditions that link the duct to pancreatitis (see Fig. 57.2 ). The first is failure of the duct cells to generate sufficient bicarbonate-rich fluid on demand. The second is duct obstruction (see Fig. 57.2 and Box 57.1 ). Reduction in flow can therefore be caused by low head pressure, high distal resistance, or a combination of both.

Overview of Duct Cell Physiology and Duct-Associated Pancreatitis

The principal function of the proximal (upstream) duct cells is to secrete a bicarbonate-rich fluid to flush the zymogens out of the pancreas and into the duodenum. The bicarbonate-rich fluid serves 2 critical functions. First, pancreatic sodium bicarbonate secretion buffers the acid secretion from the stomach as it enters the duodenum. A feedback mechanism involving release of the duodenal hormone secretin perfectly matches bicarbonate secretion with the hydrochloric acid secretion of the stomach (see Chapters 4 and 56 ). Secondly, the sodium bicarbonate in the ducts maintains an alkaline pH which helps keep trypsinogen in an inactive confirmation. Duct cell bicarbonate secretion appears to be further regulated by protease activated receptors on the duct cell as well as calcium sensors and other receptors linked to injury and inflammation.

The process of bicarbonate secretion includes the energy-dependent transport of anions (chloride and/or bicarbonate) across the basolateral membrane of the duct cell to the apical membrane and then secreted into the duct ( Fig. 57.5 ). Secretion occurs because the addition of anions to the duct lumen increases the negative electrical potential in the duct to attract positively charged sodium ions into the lumen between the duct cells. The addition of sodium and chloride/bicarbonate increases solute concentration and osmotic pressure and draws water into the duct as well. The duct is a cul-de-sac , and with increasing hydrostatic pressure the solution is forced out of the acinus, ducts, and pancreas and into the duodenum.

AP and CP can be triggered when the duct flushing mechanism fails, trypsin is generated, other zymogens are activated, and ensuing injury begins. The best-known example of this failure is obstruction of the pancreatic duct with a gallstone causing biliary AP. In addition to obstruction, the pancreas becomes susceptible to AP with diminished secretion that occurs with mutations in CFTR or failure to activate CFTR and flush the duct when conditions favoring trypsin activation are present. Environmental factors such as cigarette smoking also diminish duct cell function and reduce fluid secretion, in part by causing internalization of CFTR. ^, Cigarette smoking and other environmental factors may also cause mucin accumulation or protein plugging. ^, Taken together, disorders of the duct produce a “plumbing” problem, and require different management strategies from acinar cell-associated mechanisms.

CF Transmembrane Conductance Regulator Gene (CFTR) Variants

The CFTR gene is the most important molecule for regulating pancreatic duct cell function. Loss of functional CFTR molecules from biallelic severe pathogenic genetic mutations results in CF, which is the only known major, Mendelian (autosomal recessive) genetic disorder of the duct cells. The human proximal (upstream) pancreatic duct cells use CFTR to secret bicarbonate, and there is no alternative—so loss of CFTR results in damage to the pancreas that correlates directly with genotype. The pancreas is also the first organ to fail in CF patients, and the early CP with pseudocyst and fibrosis resulted in the name “cystic fibrosis of the pancreas,” now simply referred to as CF. Milder mutations, and complex genotypes that include mutations in CFTR cause CF-like diseases limited to one or more organs and are called CFTR-related disorders, or CFTR-RD.

The CFTR molecule forms a regulated ion channel expressed on epithelial cells in the respiratory system, sweat glands, digestive tract mucosa, biliary epithelium, pancreatic duct cells, and other locations. The primary anions conducted through CFTR under physiologic conditions are chloride and, under some conditions, bicarbonate. The CFTR gene contains 24 exons and 3 splice variants that code for a single protein of 1480 amino acids. The CFTR molecule has 12 membrane-spanning domains, 2 nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain (R domain) with multiple phosphorylation sites ( Fig. 57.6 ). Genetic variants causing amino acid substitutions within the various domains determine different types of dysfunction, which are further divided into different classes (later).

CFTR-mediated pancreatic fluid secretion is stimulated when the duct cell is activated by secretin or vasoactive intestinal peptide acting on basolateral receptors that increase intracellular cyclic adenosine monophosphate (see Chapter 56 ). The cyclic adenosine monophosphate activates protein kinase A–mediated phosphorylation of various sites in the R domain, followed by increased anion conductance (e.g., chloride, bicarbonate) through the CFTR channel. Duct cell stimulation by cholinergic agents or other agonists that increase intracellular calcium also potentiate anion secretion.

CFTR genetic variant classes. CFTR genetics plays a major role in disorders of CFTR function, with clinical disease features reflecting the effect of other factors such as sex, modifier genes, environmental factors, metabolic states, lifestyles, and comorbidities. Although about 2000 CFTR variants are known ( www.cftr2.org ) less than 100 comprise the vast majority of clinical cases of CF or CFTR-RD. In populations with a Northern European ancestry only about 20 variants have a MAF above 0.1% and with only p.F508del, p.G551D, p.W1282X, and p.N1303K having a MAF greater than 1%. Other populations have much lower allele prevalences for the top variants seen in CF patients including the USA where MAF in the ExAC and TOPMED databases have the variant associated with p.F508del (rs113993960) at 0.004 to 0.007, pG542X (rs113993959) at 0.0003 to 0.0004, pG551D (rs75527207) at 0.0001 to 0.0003, p.W1282X (rs77010898) at 0.0003 to 0.0004, p.N1303K (rs80034486) at 0.001 to 0.002, p.R5553X (rs74597325) at 0.0001, and p.R117H (rs78655421) at 0.0015 to 0.0016 (with variable severity depending on linkage with the 5T allele). CFTR variants are classified according to impact on the patient phenotype as severe, mild-variable, borderline, or benign. CFTR variants are also organized into 1 of 5 mechanistic classes according to their effect on protein production, stability, and channel function ( Table 57.1 ). Class I variants include premature stop codons that result in a truncated, nonfunctional protein. Class II variants cause defective processing, including p.F508del, due to protein misfolding or other features. Class III variants alter CFTR channel regulation and are considered gating mutations, as the channel fails to open or stay open. Class IV variants affect CFTR channel conductance so that the ion flux through the channel occurs, but at a significantly reduced level. Class V variants alter the amount of functional CFTR on the cell surface due to exon skipping (e.g., the IVS8-T5 allele causing a high rate of exon skipping) or causing decreased stability. Class I-III are functionally severe, and no or minimal functional CFTR protein reaches the cell surface. Class IV and V are functionally mild-variable and borderline because CFTR protein reaches the plasma membrane but does not function adequately. A Class VI category has been proposed that represents rapid molecule turnover at the cell membrane and Class VII for variants that impact generation of mRNA. The organization of the functional classes along the protein synthesis and processing pathway is highlighted in Fig. 57.3 (bottom row) .

CFTR genotypes. The genotype is a combination of the 2 alleles at the CFTR locus that define the overall function of CFTR in cells, with one allele being inherited from the father and the other from the mother. CFTR variants can be on one or both alleles, and functionally severe variants must be on both alleles to cause CF. If a severe variant is on one allele, then the person is typically asymptomatic and considered a carrier—even though cellular CFTR function is reduced approximately 50%. If a person has 3 or more CFTR gene variants, then at least 2 of them must be on the same allele (known as complex alleles). Variants that are on the same allele are said to be in cis, whereas those on opposite alleles are in trans. If there are multiple pathogenic CFTR variants that are all in cis, then the person is a CFTR variant carrier and will be asymptomatic unless there are unidentified variants on the trans allele (e.g., Class VII) or if they have a complex disorder involving other genes and environmental factors.

CFTR functional phenotypes. Many of the organs that are affected in patients with CF have alternative pathways and protective mechanisms that minimize the impact of complete loss of CFTR function. The pancreas and sweat gland are 2 exceptions, where there is good genotype-phenotype correlation. Because complete loss of one CFTR copy has no phenotype, the relative severity of bi-allelic pathogenic CFTR variants is defined by the least severe variant. Thus, a person with 2 severe CFTR genotypes will likely have classic, early-onset CF with pancreatic insufficiency (PI), whereas a person with one severe and one mild-variable CFTR genotype may have a milder form of CF, later age on onset, and pancreatic sufficiency (PS). However, the pancreas is not easily studied, and therefore testing CFTR function in an individual is typically done by sweat chloride testing.

Measures of variant CFTR function. Of the approximately 2000 CFTR variants, a clear majority are tentatively classified as severe, mild-variable, borderline, or benign based on the patient phenotype when the unclassified variant is in trans with a known, severe variant. The most accurate measure of CFTR protein function is to perform site-directed mutagenesis and test the permeability and conductance characteristics of the mutant CFTR in an optimized cell system under a variety of experimental conditions. ^, This information provides direct functional insight into the effect of protein sequence-altering variants. The sum of the most damaging variants on each allele (in trans) should hypothetically predict the severity of disease in the patient. Although this approach is a useful approximation, many other factors contribute to function including other pathogenic variants in complex haplotypes, the mechanistic role of CFTR and other molecules within various organs, modifying factors, epigenetics, environmental factors, and so on. Thus, even within patients with similar or identical genotypes, such as identical twins, there may be a wide spectrum of phenotypic features or severity. Furthermore, because the clinical spectrum of symptoms in patients with CF overlaps other non-CF diseases, a diagnosis of CF or CFTR-RD requires not only the clinical setting, the family history, and/or the CFTR genotype, but also testing of CFTR function in the patient.

Bicarbonate defective CFTR variants. Phenotyping diseases by focusing on one organ may lead to classifying variants as benign, whereas they are strongly associated with disease in other organs. Such is the case of a class of CFTR variants that are associated with pancreatitis but classified as benign by investigators seeing patients referred for lung disease. Epithelial cells have an internal chloride concentration monitoring receptor called WNK1 that regulates the activity of multiple ion channels, transporters, and pumps. WNK1 directly regulates CFTR, dynamically changing the permeability and conductance characteristics from a chloride-type channel to a bicarbonate-type channel. LaRusch and colleagues demonstrated the critical role of CFTR-dependent bicarbonate secretion in the human pancreas based on this paradigm and previous mathematical modeling. They screened 984 well-phenotyped pancreatitis cases from the NAPS2 study for candidate mutations in CFTR with bicarbonate-defective conductance (CFTR-BD) from among 81 previously described CFTR variants in pancreatitis patients. Nine variants (CFTR p.R74Q, p.R75Q, p.R117H, p.R170H, p.L967S, p.L997F, p.D1152H, p.S1235R, and p.D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, P = 0.002). The variants were cloned and tested for chloride and bicarbonate conductance in EK 293T cells, and although chloride was normal, bicarbonate permeability and conductance were significantly diminished in the presence of WNK1. A 3-dimensional model suggests that defective bicarbonate conductance may be caused by at least 4 mechanisms ( Fig. 57.7 ). Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Because several other organs use CFTR to secrete bicarbonate, the NAPS2 cohort was further evaluated for chronic sinusitis (because bicarbonate is needed for mucus hydration), and male infertility because bicarbonate is necessary for vas deference development (avoiding congenital bilateral absence of the vas deferens; CBAVD) and sperm survival. CFTR-BD variants significantly increased risk for rhinosinusitis (OR 2.3, P < 0.005) and male infertility (OR 395, P < 0.0001). Furthermore, heterozygous CFTR-BD variants plus SPINK1 p.N34S variant genotypes were strongly associated with pancreatitis without the sinus or infertility effects. ^,

Sweat chloride testing. Sweat chloride testing remains the most reliable, standardized, and widely available functional test of CFTR function, because normal sweat gland function is directly dependent on CFTR function and because most of the other glands and organs linked to the morbidity of CF disease are relatively inaccessible. The sweat gland is composed of a secretory coil (eccrine gland) that generates an isotonic salt solution and a sweat duct connecting the gland to the skin surface. CFTR and epithelial sodium channels (ENaC) are expressed in both the eccrine gland and especially the duct, where they absorb chloride and sodium resulting in a hypotonic (low sodium and chloride concentrations) solution (sweat) that evaporates to provide body cooling, without the loss of electrolytes. Normally, the concentration of chloride in sweat is less than 20 mmol/L, but levels increase with increasing rates of secretion and can reach nearly 60 mmol/L in some people.

In CF, the concentrations of chloride are 3 to 5 times higher than normal, with resting concentrations above 60 mmol/L and stimulated chloride levels approaching 120 mmol/L. Subjects who are heterozygous for severe, CF-causing mutations typically have nearly normal sweat chloride testing. Of note, extensive genetic testing of some patients with clinical CF and with a very abnormal sweat chloride test have only one identifiable pathogenic CFTR variant (i.e., they appear to be heterozygous), suggesting that other important factors that strongly affect CFTR function are yet to be identified. Current clinical guidelines for the diagnosis of CF include a sweat chloride of ≥60 mmol/L as well as clinical features consistent with CF (including positive newborn screening, NBS) and/or a positive family history. Patients with features of CF and an intermediate sweat chloride test results of 30 to 59 mmol/L may still have CF. Patients with CFTR variants that only affect bicarbonate conductance may be missed by sweat chloride testing.

Sweat chloride testing may also be important in the evaluation of symptomatic patients who may have undiagnosed CF or CFTR-RD. CFTR-RD includes RAP and CP, CBAVD, disseminated bronchiectasis, and sclerosing cholangitis, alone or in combination with other features. When a patient with unexplained RAP and/or early CP are found to have likely pathogenic CFTR variants, the clinically validated functional test to determine if they have CF or CFTR-RD is the sweat chloride test. The importance of diagnosing CF or CFTR-RD is highlighted by the availably of new therapies that specifically target CFTR function (later). Although secretin-stimulated pancreatic function testing also measures CFTR function, the interpretation of the results is confounded by the diminished bicarbonate concentrations in pancreatic juice in progressive pancreatic disease and by environmental factors such as smoking.

The Cystic Fibrosis Foundation Clinical Care Guidelines suggests further evaluation of intermediate sweat chloride levels as outlined in Fig. 57.8 . The algorithm begins with “clinical presentation of CF,” which may include patients with elevated immunoreactive trypsinogen levels during NBS in the USA, but who have intermediate sweat chloride test results. This approach may also be useful with CFTR-RD affecting only the pancreas, but the utility of this approach in an adult RAP or CP population has not yet been reported.

Newborn CF screening—CRMS/CFSPID. The vast majority of infants with NBS and an intermediate sweat chloride test result remained disease free for an indeterminate time. These infants have therefore been classified as CF transmembrane conductance regulator-related metabolic syndrome in the USA (the “metabolic syndrome” reflected a billing code issue rather than any metabolic feature) and CF screen positive, inconclusive diagnosis (CFSPID) in other countries. A new unified definition by a US/European consensus group defines CRMS/CFSPID as a feature in an infant who has a positive NBS test for CF and either (a) a sweat chloride less than 30 mmol/L and 2 CFTR mutations, at least 1 of which has unclear phenotypic consequences, or (b) an intermediate sweat chloride value (30 to 59 mmol/L) and 1 or 0 CF-causing mutations. The CF-causing mutations are generally defined in the CFTR2 database ( www.cftr2.org ) with others variants classified as mutations of varying clinical consequence (MVCC, e.g., Class IV or Class V), non CF-causing mutation when the mutation in trans with another CF-causing mutation will not result in CF (which does not exclude the possibility that the mutation may contribute to CF-like clinical characteristics resembling mild CF or CFTR-RD), variants of unknown significance, or benign. Genetic counseling and follow-up clinical evaluations by qualified physicians and further testing is recommended.

CFTR disease mechanism. The medical approach to CF is presented later whereas the mechanism of CFTR in disease is discussed here. Severe mutations in both CFTR alleles leading to total or near-total loss of CFTR function results in CF. The molecular consequences of CF include inability to adequately hydrate mucus and other macromolecules, leading to accumulation of viscid material and inspissated glands. This condition results in progressive organ destruction of the pancreas and respiratory system, and dysfunction of the liver, intestine, sweat glands, and other sites where epithelial cell secretion plays an important physiologic role. As noted earlier, the pancreas incurs a double risk because most of its proteins are zymogens and trypsin activation will lead to recurrent injury and eventually destruction of the pancreas through progressive fibrosis. Trypsin-mediated injury and destruction of the pancreas in children with CF is consistent with this model because the pancreatic pathology in CF is pseudocyst formation and fibrosis rather than atrophy alone (as expected with duct obstruction). It appears that pancreatic gland injury in CF children roughly parallels the expression of trypsinogen in the developing acinar cells, which begins at 16 weeks gestation and gradually increases in concentration until birth and through the first 6 months of life when levels markedly rise. ^, The resulting histology has many of the features of end-stage CP that develops in children and adults, but also has striking expanded ducts that appear as multiple protein-filled cysts ( Fig. 57.9 ).

The overall clinical picture in an individual with pathogenic CFTR variants depends on the nature of the combined CFTR mutations, the genetic background in which the defective genes operates (e.g., modifier genes), and environmental factors. ^{, ,} About 70 to 90% of non-Hispanic white patients with CF have p.F508del. Distinct mutations are common to other ethnic and ancestral groups, including 3120 + 1G greater than A which is the second most frequent CF allele in African Americans (9.5 to 12.3%), the p.R334W mutation which is common in Hispanics, and the p.W1282X in Ashkenazi Jews (∼45%). ^{, ,}

Patients with 2 CFTR ^severe mutations in trans typically develop classic features of CF, with elevated chloride levels in sweat glands, PI, recurrent and chronic pulmonary infections, and CBAVD in males. Complicating manifestations of severe CF can also include meconium ileus, distal intestinal obstruction syndrome (DIOS), gallbladder dysfunction, liver cirrhosis, and other GI problems.

Patients with one severe CFTR mutation (Class I-III, e.g., p.F508del) and one mild-variable CFTR mutation (Class IV or V, e.g., p. R117H or p. R334W ) typically have CF with PS CF due to incomplete loss of CFTR function, partially reduced chloride and/or bicarbonate conductance, and subsequent residual duct cell function, resulting in acinar cell survival. ^, This residual pancreatic parenchyma with abnormal CFTR function leaves a PS-CF patient at high risk for AP and RAP, with an incidence of 22%. These patients are more likely to have only a subset of organs expressing CFTR affected and presenting symptoms may occur later in life (teens or 20s).

Environment and modifier gene variants. Many of the features of CF cannot be explained by variations in CFTR sequence. Instead, these features are caused by specific environmental factors or modifier genes. ^, Environmental factors, such as bacterial colonization of the respiratory system, tobacco smoke, poor nutritional status, and environmental allergens contribute to the severity of lung disease. The other major factors are modifier genes that strongly contribute to the wide range of clinical features in patients with apparently identical CFTR genotypes. ^{, ,}

In 1998 2 groups ^, demonstrated that pathogenic CFTR variants were also very common in idiopathic and alcoholic CP, suggesting that CFTR mutations may be part of a more complex trait. ^, Because heterozygous pathogenic CFTR variants are common in populations with European ancestry, and because the parents of CF children (obligate CFTR mutation carriers without CF) do not appear to have an increased incidence of AP or CP compared with the normal population, it is likely that a second factor that specifically targets the pancreas is required. ^, In early-onset idiopathic pancreatitis, this second factor may be a genetic variant in SPINK1 , CTRC , or CASR , an anatomical factor like pancreatic divisum, an environmental factor, or other mechanism. ^{, , ,} Although high-quality treatment trials for these patients are still needed, the problem is one of “plumbing” and methods to restore lost CFTR function and/or reduce resistance to pancreatic juice flow should be considered.

Calcium-Sensing Receptor Gene (CASR) Variants

Calcium plays multiple roles in pancreatic physiology and pathophysiology. On one hand the regulation of intra-acinar cell calcium is critical for the prevention of pancreatic injury, ^, whereas increasing concentrations of calcium in the pancreatic duct increases the risk of sustained trypsin activation and precipitation as calcium-containing stones. The calcium-sensing receptor gene (CASR) is a membrane-bound member of the G-protein–coupled receptor superfamily. CaSR plays an important role in calcium homeostasis, as is reflected in its expression by cells of the parathyroid gland and renal tubules that are involved in the calcium homeostasis. CaSR has been identified in human pancreatic acinar and ductal cells, as well as in various non-exocrine tissues, although its functional significance in the pancreas has not yet been determined. A possible role of the CaSR in the pancreatic duct is plausible by extension of duodenal physiology, noting that CaSR is coexpressed with CFTR in bicarbonate-secreting epithelial cells. CaSR activation dose-dependently raises intracellular calcium levels which causes calcium-dependent CFTR bicarbonate secretion as well as modulating other molecules involved in the process. More than 170 functional mutations (activating and inactivating) have been described in the CASR related to familial hypocalcuric hypercalcemia, neonatal severe primary hyperparathyroidism, autosomal dominant hypocalcemia, and related hypercalcemic or hypocalcemic disorders. The common CASR variants p.R990G, p.A986S, and p.Q1011E are strongly associated with urolithiasis and hypercalcuria in various populations.

In 2003 Felderbauer investigated a kindred with familial pancreatitis and the SPINK1 p.N34S variant. However, only 2 of these family members had CP, and both were found to have a novel CASR c.518T>C mutation that was linked to hypercalcemia. An association between additional CASR variants, with or without SPINK1 mutations, was subsequently identified in patients in India with TP, as well as in the USA in sporadic and alcoholic CP in which the CASR p.R990G (rs1042636) variant doubles and triples the relative risk, respectively (the rs number is given because the amino acid number may change based on the CASR transcript used). Multiple rare CASR variants were also identified in a French cohort and the p.A986S variant (rs1801725) in multiple Chinese pancreatitis patients. CASR p.Q1011E (rs1801726) is also widely studied, but a clear role in pancreatic disease has not been established. The finding of different CASR polymorphisms in different populations is intriguing, but it appears that the presumed mild hypercalcemia is a cofactor for pancreatitis rather than an independent risk factor, as seen in animal models of hypercalcemia, ^, or as part of complex functional genotypes with SPINK1 or CFTR or other factors. CASR variants that reduce CaSR function may also contribute to pancreatic disease because CaSR also serves as an amino acid receptor in the duodenum, linking luminal nutrients with release of cholecystokinin that subsequently stimulates pancreatic enzyme secretion.