Genetic variation in severe cystic fibrosis liver disease is associated with novel mechanisms for disease pathogenesis.

BACKGROUND AND AIMS: It is not known why severe cystic fibrosis (CF) liver disease (CFLD) with portal hypertension occurs in only ~7% of people with CF. We aimed to identify genetic modifiers for severe CFLD to improve understanding of disease mechanisms. APPROACH AND RESULTS: Whole-genome sequencing was available in 4082 people with CF with pancreatic insufficiency (n = 516 with severe CFLD; n = 3566 without CFLD). We tested ~15.9 million single nucleotide polymorphisms (SNPs) for association with severe CFLD versus no-CFLD, using pre-modulator clinical phenotypes including (1) genetic variant ( SERPINA1 ; Z allele) previously associated with severe CFLD; (2) candidate SNPs (n = 205) associated with non-CF liver diseases; (3) genome-wide association study of common/rare SNPs; (4) transcriptome-wide association; and (5) gene-level and pathway analyses. The Z allele was significantly associated with severe CFLD ( p = 1.1 × 10 -4 ). No significant candidate SNPs were identified. A genome-wide association study identified genome-wide significant SNPs in 2 loci and 2 suggestive loci. These 4 loci contained genes [significant, PKD1 ( p = 8.05 × 10 -10 ) and FNBP1 ( p = 4.74 × 10 -9 ); suggestive, DUSP6 ( p = 1.51 × 10 -7 ) and ANKUB1 ( p = 4.69 × 10 -7 )] relevant to severe CFLD pathophysiology. The transcriptome-wide association identified 3 genes [ CXCR1 ( p = 1.01 × 10 -6 ) , AAMP ( p = 1.07 × 10 -6 ), and TRBV24 ( p = 1.23 × 10 -5 )] involved in hepatic inflammation and innate immunity. Gene-ranked analyses identified pathways enriched in genes linked to multiple liver pathologies. CONCLUSION: These results identify loci/genes associated with severe CFLD that point to disease mechanisms involving hepatic fibrosis, inflammation, innate immune function, vascular pathology, intracellular signaling, actin cytoskeleton and tight junction integrity and mechanisms of hepatic steatosis and insulin resistance. These discoveries will facilitate mechanistic studies and the development of therapeutics for severe CFLD.

Airway Mucosal Host Defense Is Key to Genomic Regulation of Cystic Fibrosis Lung Disease Severity.

RATIONALE: The severity of cystic fibrosis (CF) lung disease varies widely, even for Phe508del homozygotes. Heritability studies show that more than 50% of the variability reflects non-cystic fibrosis transmembrane conductance regulator (CFTR) genetic variation; however, the full extent of the pertinent genetic variation is not known. OBJECTIVES: We sought to identify novel CF disease-modifying mechanisms using an integrated approach based on analyzing "in vivo" CF airway epithelial gene expression complemented with genome-wide association study (GWAS) data. METHODS: Nasal mucosal RNA from 134 patients with CF was used for RNA sequencing. We tested for associations of transcriptomic (gene expression) data with a quantitative phenotype of CF lung disease severity. Pathway analysis of CF GWAS data (n = 5,659 patients) was performed to identify novel pathways and assess the concordance of genomic and transcriptomic data. Association of gene expression with previously identified CF GWAS risk alleles was also tested. MEASUREMENTS AND MAIN RESULTS: Significant evidence of heritable gene expression was identified. Gene expression pathways relevant to airway mucosal host defense were significantly associated with CF lung disease severity, including viral infection, inflammation/inflammatory signaling, lipid metabolism, apoptosis, ion transport, Phe508del CFTR processing, and innate immune responses, including HLA (human leukocyte antigen) genes. Ion transport and CFTR processing pathways, as well as HLA genes, were identified across differential gene expression and GWAS signals. CONCLUSIONS: Transcriptomic analyses of CF airway epithelia, coupled to genomic (GWAS) analyses, highlight the role of heritable host defense variation in determining the pathophysiology of CF lung disease. The identification of these pathways provides opportunities to pursue targeted interventions to improve CF lung health.

Immunoreactive trypsinogen levels in newborn screened infants with an inconclusive diagnosis of cystic fibrosis.

BACKGROUND: Newborn screening (NBS) for cystic fibrosis (CF) not only identifies infants with a diagnosis of CF, but also those with an uncertain diagnosis of cystic fibrosis (CF), i.e. CF transmembrane conductance regulator (CFTR)-related metabolic syndrome (CRMS) or CF screen positive inconclusive diagnosis (CFSPID). These infants have an uncertain long-term outcome and it is currently unclear around time of diagnosis, which infants are at higher risk of later fulfilling a CF diagnosis. In this study, we hypothesised that immunoreactive trypsinogen (IRT) levels, used in NBS as a marker of pancreatic disease and function, may reflect the degree of CFTR dysfunction in each individual and therefore would help to identify those with CRMS/CSPID who are later at risk for meeting the criteria of CF. METHODS: In this longitudinal, prospective study, infants with CRMS/CFSPID and CF were recruited and followed in 9 CF clinics (Canada and Italy). We compared NBS IRT levels between CF and CRMS/CFSPID, and between children with CRMS/CFSPID→CF and CRMS/CFSPID→CRMS/CFSPID during the period of June 2007 to April 2016. RESULTS: Ninety eight CRMS/CFSPID and 120 CF subjects were enrolled. During the study period, 14 (14.3%) CRMS/CFSPID subjects fulfilled the diagnostic criteria for CF (CRMS/CFSPID→CF), while the diagnosis remained uncertain (CRMS/CFSPID→ CRMS/CFSPID) in 84 (85.7%) subjects. Significantly higher NBS IRT concentrations (ng/ml) were present in CF than CRMS/CFPSID (median (interquartile range): 143.8 (99.8-206.2) vs. 75.0 (61.0-105.9); P < 0.0001). Infants with CRMS/CFSPID→CF (n = 14) had significantly higher NBS IRT concentrations (ng/ml) than CRMS/CFSPID→ CRMS/CFSPID (n = 83) (median (interquartile range): 108.9 (72.3-126.8) vs. 73.7(60.0-96.0); P = 0.02). CONCLUSIONS: Amongst infants who tested positive on NBS for CF, there is a gradation of elevated NBS IRT concentrations. Infants with CF have higher NBS IRT levels than CRMS/CFPSID, and higher NBS IRT concentrations were present in infants with CRMS/CFSPID→CF than CRMS/CFSPID→ CRMS/CFSPID. NBS IRT concentrations, in concert with other factors, may have the potential to predict the likelihood of CF amongst infants with CRMS/CFSPID.

A Joint Location-Scale Test Improves Power to Detect Associated SNPs, Gene Sets, and Pathways.

Gene-based, pathway, and other multivariate association methods are motivated by the possibility of GxG and GxE interactions; however, accounting for such interactions is limited by the challenges associated with adequate modeling information. Here we propose an easy-to-implement joint location-scale (JLS) association testing framework for single-variant and multivariate analysis that accounts for interactions without explicitly modeling them. We apply the JLS method to a gene-set analysis of cystic fibrosis (CF) lung disease, which is influenced by multiple environmental and genetic factors. We identify and replicate an association between the constituents of the apical plasma membrane and CF lung disease (p = 0.0099 and p = 0.0180, respectively) and highlight a role for the SLC9A3-SLC9A3R1/2-EZR complex in contributing to CF lung disease. Many association studies could benefit from re-analysis with the JLS method that leverages complex genetic architecture for SNP, gene, and pathway identification. Analytical verification, simulation, and additional proof-of-principle applications support our approach.

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfß, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfß molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.

Features of Severe Liver Disease With Portal Hypertension in Patients With Cystic Fibrosis.

BACKGROUND & AIMS: Liver disease is the third leading cause of death in patients with cystic fibrosis (CF), but features of patients with CF, severe liver disease, and portal hypertension have not been characterized fully. METHODS: We performed a retrospective analysis of data from 561 patients with CF (63% male, 99% with pancreatic insufficiency), liver disease (hepatic parenchymal abnormalities consistent with cirrhosis, confirmed by imaging), and portal hypertension (esophageal varices, portosystemic collaterals, or splenomegaly), with no alternate causes of liver disease. All patients were enrolled in the Genetic Modifier Study of Severe CF Liver Disease at 76 international centers, from January 1999 through July 2013. RESULTS: Male patients were diagnosed with liver disease at a younger age than female patients (10 vs 11 y; P = .01). Splenomegaly was observed in 99% of patients and varices in 71%. Levels of liver enzymes were near normal in most patients. Thrombocytopenia affected 70% of patients and was more severe in patients with varices (88 × 10(9)/L vs 145 × 10(9)/L; P < .0001). Ninety-one patients received liver transplants (16%), at a median age of 13.9 years. Compared with patients who did not receive liver transplants, patients who received liver transplants had lower platelet counts (78 × 10(9)/L vs 113 × 10(9)/L; P < .0001), higher international normalized ratios (P < .0001), and lower levels of albumin (P = .0002). The aminotransferase to platelet ratio index (APRi) and fibrosis index based on 4 factor (FIB-4) values were higher than the diagnostic thresholds for CF liver disease in 96% and in 90% of patients, respectively. Patients who received liver transplants or who had varices had higher APRi and FIB-4 values than patients who did not. CONCLUSIONS: In patients with CF, severe liver disease develops early in childhood (approximately 10 years of age), and is more common in boys than in girls. Patients with varices and those who receive liver transplants have more abnormal platelet counts and APRi and FIB-4 scores.

Direct Costs of Acute Recurrent and Chronic Pancreatitis in Children in the INSPPIRE Registry.

OBJECTIVE: To estimate selected direct medical care costs of children with chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP). METHODS: We performed a cross-sectional study of data from International Study Group of Pediatric Pancreatitis: In Search for a Cure (INSPPIRE), a multinational registry of children with ARP or CP. We determined health care utilization and estimated costs of hospitalizations, surgical and endoscopic procedures, and medications in our study population. Health care utilization data were obtained from all subjects enrolled in the study, and costs were calculated using national United States costs. RESULTS: We included 224 subjects (median age 12.7 years), 42% of whom had CP. Mean number of hospitalizations, including for surgery and endoscopic retrograde cholangiopancreatography, was 2.3 per person per year, costing an estimated average $38,755 per person per year. Including outpatient medications, estimated total mean cost was $40,589 per person per year. Subjects using surgical procedures or endoscopic retrograde cholangiopancreatography incurred mean annual costs of $42,951 per person and $12,035 per person, respectively. Estimated annual costs of pancreatic enzyme replacement therapy, diabetic medications, and pain medications were $4114, $1761, and $614 per person, respectively. In an exploratory analysis, patients with the following characteristics appear to accrue higher costs than those without them: more frequent ARP attacks per year, reported constant or episodic pain, family history of pancreatic cancer, and use of pain medication. CONCLUSIONS: ARP and CP are uncommon childhood conditions. The severe burden of disease associated with these conditions and their chronicity results in high health care utilization and costs. Interventions that reduce the need for hospitalization could lower costs for these children and their families.

Variants in Solute Carrier SLC26A9 Modify Prenatal Exocrine Pancreatic Damage in Cystic Fibrosis.

OBJECTIVES: To test the hypothesis that multiple constituents of the apical plasma membrane residing alongside the causal cystic fibrosis (CF) transmembrane conductance regulator protein, including known CF modifiers SLC26A9, SLC6A14, and SLC9A3, would be associated with prenatal exocrine pancreatic damage as measured by newborn screened (NBS) immunoreactive trypsinogen (IRT) levels. STUDY DESIGN: NBS IRT measures and genome-wide genotype data were available on 111 subjects from Colorado, 37 subjects from Wisconsin, and 80 subjects from France. Multiple linear regression was used to determine whether any of 8 single nucleotide polymorphisms (SNPs) in SLC26A9, SLC6A14, and SLC9A3 were associated with IRT and whether other constituents of the apical plasma membrane contributed to IRT. RESULTS: In the Colorado sample, 3 SLC26A9 SNPs were associated with NBS IRT (min P=1.16×10(-3); rs7512462), but no SLC6A14 or SLC9A3 SNPs were associated (P>.05). The rs7512462 association replicated in the Wisconsin sample (P=.03) but not in the French sample (P=.76). Furthermore, rs7512462 was the top-ranked apical membrane constituent in the combined Colorado and Wisconsin sample. CONCLUSIONS: NBS IRT is a biomarker of prenatal exocrine pancreatic disease in patients with CF, and a SNP in SLC26A9 accounts for significant IRT variability. This work suggests SLC26A9 as a potential therapeutic target to ameliorate exocrine pancreatic disease.

Pediatric chronic pancreatitis is associated with genetic risk factors and substantial disease burden.

OBJECTIVE: To determine the clinical presentation, diagnostic variables, risk factors, and disease burden in children with chronic pancreatitis. STUDY DESIGN: We performed a cross-sectional study of data from the International Study Group of Pediatric Pancreatitis: In Search for a Cure, a registry of children with acute recurrent pancreatitis and chronic pancreatitis. Between-group differences were compared using Wilcoxon rank-sum test. RESULTS: Among 170 subjects in the registry, 76 (45%) had chronic pancreatitis; 57% were female, 80% were white; median age at diagnosis was 9.9 years. Pancreatitis-predisposing genetic mutations were identified in 51 (67%) and obstructive risk factors in 25 (33%). Toxic/metabolic and autoimmune factors were uncommon. Imaging demonstrated ductal abnormalities and pancreatic atrophy more commonly than calcifications. Fifty-nine (77%) reported abdominal pain within the past year; pain was reported as constant and receiving narcotics in 28%. Children with chronic pancreatitis reported a median of 3 emergency department visits and 2 hospitalizations in the last year. Forty-seven subjects (70%) missed 1 day of school in the past month as the result of chronic pancreatitis; 26 (34%) missed 3 or more days. Children reporting constant pain were more likely to miss school (P = .002), visit the emergency department (P = .01), and experience hospitalizations (P = .03) compared with children with episodic pain. Thirty-three children (43%) underwent therapeutic endoscopic retrograde pancreatography; one or more pancreatic surgeries were performed in 30 (39%). CONCLUSIONS: Chronic pancreatitis occurs at a young age with distinct clinical features. Genetic and obstructive risk factors are common, and disease burden is substantial.

Variation in MSRA modifies risk of neonatal intestinal obstruction in cystic fibrosis.

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5' to and within the MSRA gene were associated with MI (P = 1.99 × 10(-5) to 1.08 × 10(-6); Bonferroni P = 0.057 to 3.1 × 10(-3)). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53-0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2 × 10(-4)). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr(-/-) and Cftr(-/-)Msra(-/-) mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function.

Unraveling the complex genetic model for cystic fibrosis: pleiotropic effects of modifier genes on early cystic fibrosis-related morbidities.

The existence of pleiotropy in disorders with multi-organ involvement can suggest therapeutic targets that could ameliorate overall disease severity. Here we assessed pleiotropy of modifier genes in cystic fibrosis (CF). CF, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, affects the lungs, liver, pancreas and intestines. However, modifier genes contribute to variable disease severity across affected organs, even in individuals with the same CFTR genotype. We sought to determine whether SLC26A9, SLC9A3 and SLC6A14, that contribute to meconium ileus in CF, are pleiotropic for other early-affecting CF co-morbidities. In the Canadian CF population, we assessed evidence for pleiotropic effects on (1) pediatric lung disease severity (n = 815), (2) age at first acquisition of Pseudomonas aeruginosa (P. aeruginosa) (n = 730), and (3) prenatal pancreatic damage measured by immunoreactive trypsinogen (n = 126). A multiple-phenotype analytic strategy assessed evidence for pleiotropy in the presence of phenotypic correlation. We required the same alleles to be associated with detrimental effects. SLC26A9 was pleiotropic for meconium ileus and pancreatic damage (p = 0.002 at rs7512462), SLC9A3 for meconium ileus and lung disease (p = 1.5 × 10(-6) at rs17563161), and SLC6A14 for meconium ileus and both lung disease and age at first P. aeruginosa infection (p = 0.0002 and p = 0.006 at rs3788766, respectively). The meconium ileus risk alleles in SLC26A9, SLC9A3 and SLC6A14 are pleiotropic, increasing risk for other early CF co-morbidities. Furthermore, co-morbidities affecting the same organ tended to associate with the same genes. The existence of pleiotropy within this single disorder suggests that complementary therapeutic strategies to augment solute transport will benefit multiple CF-associated tissues.

Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?

BACKGROUND: The phenotypic spectrum of cystic fibrosis (CF) has expanded to include patients affected by single-organ diseases. Extensive genotyping and nasal potential difference (NPD) testing have been proposed to assist in the diagnosis of CF when sweat testing is inconclusive. However, the diagnostic yield of extensive genotyping and NPD and the concordance between NPD and the sweat test have not been carefully evaluated. METHODS: We evaluated the diagnostic outcomes of genotyping (with 122 mutations included as disease causing), sweat testing and NPD in a prospectively ascertained cohort of undiagnosed patients who presented with chronic sino-pulmonary disease (RESP), chronic/recurrent pancreatitis (PANC) or obstructive azoospermia (AZOOSP). RESULTS: 202 patients (68 RESP, 42 PANC and 92 AZOOSP) were evaluated; 17.3%, 22.8% and 59.9% had abnormal, borderline and normal sweat chloride results, respectively. Only 17 (8.4%) patients were diagnosable as having CF by genotyping. Compared to sweat testing, NPD identified more patients as having CF (33.2%) with fewer borderline results (18.8%). The level of agreement according to kappa statistics (and the observed percentage of agreement) between sweat chloride and NPD in RESP, PANC and AZOOSP subjects was 'moderate' (65% observed agreement), 'poor' (33% observed agreement) and 'fair' (28% observed agreement), respectively. The degree of agreement only improved marginally when subjects with borderline sweat chloride results were excluded from the analysis. CONCLUSIONS: The diagnosis of CF or its exclusion is not always straightforward and may remain elusive even with comprehensive evaluation, particularly among individuals who present at an older age with single-organ manifestations suggestive of CF.

Design and implementation of INSPPIRE.

OBJECTIVES: Acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP) are rare and poorly understood diseases in children. Better understanding of these disorders can only be accomplished via a multicenter, structured, data collection approach. METHODS: The International Study Group of Pediatric Pancreatitis: In Search for a Cure (INSPPIRE) consortium was created to investigate the epidemiology, etiologies, pathogenesis, natural history, and outcomes of pediatric ARP and CP. Patient and physician questionnaires were developed to capture information on demographics, medical history, family and social history, medications, hospitalizations, risk factors, diagnostic evaluation, treatments, and outcome information. Information collected in paper questionnaires was then transferred into Research Electronic Data Capture (REDCap), tabulated, and analyzed. RESULTS: The administrative structure of the INSPPIRE consortium was established, and National Institutes of Health funding was obtained. A total of 14 sites (10 in the United States, 2 in Canada, and 2 overseas) participated. Questionnaires were amended and updated as necessary, followed by changes made into the REDCap database. Between September 1, 2012 and August 31, 2013, a total of 194 children were enrolled into the study: 54% were girls, 82% were non-Hispanic, and 72% were whites. CONCLUSIONS: The INSPPIRE consortium demonstrates the feasibility of building a multicenter patient registry to study the rare pediatric diseases, ARP and CP. Analyses of collected data will provide a greater understanding of pediatric pancreatitis and create opportunities for therapeutic interventional studies that would not otherwise be possible without a multicenter approach.

Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome).

Johanson-Blizzard syndrome (OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, multiple malformations such as nasal wing aplasia, and frequent mental retardation. We mapped the disease-associated locus to chromosome 15q14-21.1 and identified mutations, mostly truncating ones, in the gene UBR1 in 12 unrelated families with Johanson-Blizzard syndrome. UBR1 encodes one of at least four functionally overlapping E3 ubiquitin ligases of the N-end rule pathway, a conserved proteolytic system whose substrates include proteins with destabilizing N-terminal residues. Pancreas of individuals with Johanson-Blizzard syndrome did not express UBR1 and had intrauterine-onset destructive pancreatitis. In addition, we found that Ubr1(-/-) mice, whose previously reported phenotypes include reduced weight and behavioral abnormalities, had an exocrine pancreatic insufficiency, with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury. Our findings indicate that deficiency of UBR1 perturbs the pancreas' acinar cells and other organs, presumably owing to metabolic stabilization of specific substrates of the N-end rule pathway.

Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.

BACKGROUND: A new approach in the treatment of cystic fibrosis involves improving the function of mutant cystic fibrosis transmembrane conductance regulator (CFTR). VX-770, a CFTR potentiator, has been shown to increase the activity of wild-type and defective cell-surface CFTR in vitro. METHODS: We randomly assigned 39 adults with cystic fibrosis and at least one G551D-CFTR allele to receive oral VX-770 every 12 hours at a dose of 25, 75, or 150 mg or placebo for 14 days (in part 1 of the study) or VX-770 every 12 hours at a dose of 150 or 250 mg or placebo for 28 days (in part 2 of the study). RESULTS: At day 28, in the group of subjects who received 150 mg of VX-770, the median change in the nasal potential difference (in response to the administration of a chloride-free isoproterenol solution) from baseline was -3.5 mV (range, -8.3 to 0.5; P=0.02 for the within-subject comparison, P=0.13 vs. placebo), and the median change in the level of sweat chloride was -59.5 mmol per liter (range, -66.0 to -19.0; P=0.008 within-subject, P=0.02 vs. placebo). The median change from baseline in the percent of predicted forced expiratory volume in 1 second was 8.7% (range, 2.3 to 31.3; P=0.008 for the within-subject comparison, P=0.56 vs. placebo). None of the subjects withdrew from the study. Six severe adverse events occurred in two subjects (diffuse macular rash in one subject and five incidents of elevated blood and urine glucose levels in one subject with diabetes). All severe adverse events resolved without the discontinuation of VX-770. CONCLUSIONS: This study to evaluate the safety and adverse-event profile of VX-770 showed that VX-770 was associated with within-subject improvements in CFTR and lung function. These findings provide support for further studies of pharmacologic potentiation of CFTR as a means to treat cystic fibrosis. (Funded by Vertex Pharmaceuticals and others; ClinicalTrials.gov number, NCT00457821.).

Deficiency of Sbds in the mouse pancreas leads to features of Shwachman-Diamond syndrome, with loss of zymogen granules.

BACKGROUND & AIMS: Shwachman-Diamond syndrome (SDS) is the second leading cause of hereditary exocrine pancreatic dysfunction. More than 90% of patients with SDS have biallelic loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene, which encodes a factor involved in ribosome function. We investigated whether mutations in Sbds lead to similar pancreatic defects in mice. METHODS: Pancreas-specific knock-out mice were generated using a floxed Sbds allele and bred with mice carrying a null or disease-associated missense Sbds allele. Cre recombinase, regulated by the pancreatic transcription factor 1a promoter, was used to disrupt Sbds specifically in the pancreas. Models were assessed for pancreatic dysfunction and growth impairment. RESULTS: Disruption of Sbds in the mouse pancreas was sufficient to recapitulate SDS phenotypes. Pancreata of mice with Sbds mutations had decreased mass, fat infiltration, but general preservation of ductal and endocrine compartments. Pancreatic extracts from mutant mice had defects in formation of the 80S ribosomal complex. The exocrine compartment of mutant mice was hypoplastic and individual acini produced few zymogen granules. The null Sbds allele resulted in an earlier onset of phenotypes as well as endocrine impairment. Mutant mice had reduced serum levels of digestive enzymes and overall growth impairment. CONCLUSIONS: We developed a mouse model of SDS with pancreatic phenotypes similar to those of the human disease. This model could be used to investigate organ-specific consequences of Sbds-associated ribosomopathy. Sbds genotypes correlated with phenotypes. Defects developed specifically in the pancreata of mice, reducing growth of mice and production of digestive enzymes. SBDS therefore appears to be required for normal pancreatic development and function.

ß-adrenergic sweat secretion as a diagnostic test for cystic fibrosis.

RATIONALE: ß-Adrenergically induced sweat secretion offers an expedient method to assess native cystic fibrosis transmembrane conductance regulator (CFTR) secretory function in vivo. OBJECTIVES: To evaluate the sensitivity, specificity, and reliability of a test based on the activity and secretory function of CFTR in the sweat gland. METHODS: Primary and validation trials with prospectively ascertained healthy control subjects, obligate heterozygotes, and patients with a CFTR-related disorder and CF (pancreatic sufficient and insufficient). MEASUREMENTS AND MAIN RESULTS: Diagnostic accuracy and reliability of ß-adrenergic sweat secretory rates using an evaporimeter was assessed and compared with sweat chloride concentrations. The cholinergically stimulated mean sweat rate did not differ among groups. The mean maximal ß-adrenergically stimulated sweat rate in heterozygotes was about half the rate of healthy control subjects, and completely absent in pancreatic-insufficient patients with CF and pancreatic-sufficient patients with CF (P < 0.0001). Subjects with a CFTR-related disorder showed reduced or absent ß-adrenergic sweat secretion. The ß-adrenergic secretory response demonstrated high diagnostic accuracy (area under a characteristic receiver-operator curve = 0.99; 95% confidence interval, 0.97-1.00) and reliability (intraclass correlation, 0.90; 95% confidence interval, 0.81-0.95). The diagnostic cutoff level for CF, derived from the primary trial, correctly identified all control subjects, heterozygotes, and patients with CF in the validation cohort, whereas concurrent sweat chloride measurements misclassified one heterozygote and five subjects with CF. The cholinergic and ß-adrenergic sweat secretion rates were lower in women compared with men (P < 0.001). CONCLUSIONS: ß-Adrenergic sweat secretion rate determined by evaporimetry is an accurate and reliable technique to assess different levels of CFTR function and to identify patients with CF.

Mutations in SBDS are associated with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS; OMIM 260400) is an autosomal recessive disorder with clinical features that include pancreatic exocrine insufficiency, hematological dysfunction and skeletal abnormalities. Here, we report identification of disease-associated mutations in an uncharacterized gene, SBDS, in the interval of 1.9 cM at 7q11 previously shown to be associated with the disease. We report that SBDS has a 1.6-kb transcript and encodes a predicted protein of 250 amino acids. A pseudogene copy (SBDSP) with 97% nucleotide sequence identity resides in a locally duplicated genomic segment of 305 kb. We found recurring mutations resulting from gene conversion in 89% of unrelated individuals with SDS (141 of 158), with 60% (95 of 158) carrying two converted alleles. Converted segments consistently included at least one of two pseudogene-like sequence changes that result in protein truncation. SDBS is a member of a highly conserved protein family of unknown function with putative orthologs in diverse species including archaea and eukaryotes. Archaeal orthologs are located within highly conserved operons that include homologs of RNA-processing genes, suggesting that SDS may be caused by a deficiency in an aspect of RNA metabolism that is essential for development of the exocrine pancreas, hematopoiesis and chrondrogenesis.

Comparing the American and European diagnostic guidelines for cystic fibrosis: same disease, different language?

BACKGROUND: The American and European cystic fibrosis (CF) guidelines recommend different diagnostic criteria. This study assessed diagnostic concordance between these recommendations. METHODS: Subjects with single organ manifestations suggestive of CF (chronic sinopulmonary disease (RESP), chronic/recurrent pancreatitis (PANC) or obstructive azoospermia (AZOOSP)) were prospectively evaluated by sweat test, nasal potential difference and genotyping. Concordance in diagnostic outcomes between the two algorithms was measured using observed agreement and κ statistics. RESULTS: A total of 208 subjects were evaluated. Observed agreement was 84.8% and level of agreement was excellent (κ=0.87) between the American and European recommendations. The RESP phenotype was associated with the highest degree of concordance (observed agreement ≥90%, κ=0.92) compared with the PANC (observed agreement 86%, κ=0.65) and AZOOSP (observed agreement 80%, κ=0.87) phenotypes. Incorporation of nasal potential difference into the American algorithm failed to improve the overall degree of concordance (good agreement level; κ=0.75); the level of agreement was unchanged in RESP and PANC subjects, but reduced in AZOOSP subjects (from excellent to good). Extensive genotyping had limited clinical utility in the diagnosis of CF in both algorithms. CONCLUSIONS: Despite inconsistencies between the American and European diagnostic recommendations, concordance in diagnostic outcomes among subjects presenting with single organ manifestations of CF was good to excellent. These diagnostic guidelines provide guidance and promote rigorous evaluation for the diagnosis of CF but neither guideline should be regarded as dogma.