A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.

Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

Rate and predictors of insufficient sweat volume in very young infants after pilocarpine gel iontophoresis: prospective, population-based study.

OBJECTIVE: To verify the rate and predictors of 'quantity not sufficient' (QNS) among Brazilian infants younger than 3 months with positive newborn screening (NBS) for cystic fibrosis (CF). DESIGN: Prospective, population-based study. SETTING: Public Statewide Newborn Screening Programme where the incidence rate of CF is ≈1:11 000. PATIENTS: Subjects with positive two-tiered immunoreactive trypsinogen. INTERVENTIONS: Sweat induction and collection were performed in the same facility; one sweat sample was obtained per individual. MAIN OUTCOME MEASURES: The QNS rate and its predictors; analysis corresponded to the day of sweat collection. RESULTS: Among the 975 participants, QNS rates for 10 and 15 µL were 3.6% (95% CI 2.5% to 4.9%) and 8.3% (95% CI 6.6% to 10.2%). Infants weighing >3056 and >3845 g and with gestational age higher than 37 weeks had a greater likelihood (5.5 and 6.7, and 2.7 and 5.8 times more, respectively) of avoiding QNS than their peers. CONCLUSION: QNS rates fulfilled the requirements, but predictors differed from those recommended by the Cystic Fibrosis Foundations guidelines.

Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens.

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

Severe lung disease in children with cystic fibrosis missed in newborn screening.

BACKGROUND: Cystic fibrosis (CF) is now routinely diagnosed through newborn screening (NBS), but the tests employed in the USA have been evolving for two decades as missed cases become recognized and lab methods improve in association with more knowledge about CF genetics. New Jersey was among the first states to implement CF NBS in 2001 when it introduced the original two-tiered method that combined measurements of immunoreactive trypsinogen (IRT) with detection of the principal pathogenic variant (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. OBJECTIVE: With continuation of the IRT/DNA (F508del) algorithm for two decades and identification of screening false negative children, we decided to examine the condition of some missed cases with special attention to their respiratory status. METHODS: To strengthen the arguments for quality improvement in New Jersey's CF NBS program, we reviewed and evaluated false negative cases to determine the potential extent of preventable patient suffering as a consequence of delayed diagnoses. RESULTS: Five children with CF who had false negative screening results were studied in detail. In each case there was a different cause of the negative screening results. They all had clinically significant/severe lung disease, ranging from chronic cough with CF pathogens on respiratory culture at a young age to respiratory failure. CONCLUSION: This case series highlights the consequences of false negative screening results, which served as the impetus to upgrade New Jersey's CF NBS algorithm. Implemented changes include lowering the IRT cutoff to 70 ng/mL and expanding to a 139 variant CFTR panel. In 2023, a floating IRT cutoff is anticipated to be implemented.

Why cystic fibrosis newborn screening programs have failed to meet original expectations thus far.

This Commentary summarizes what the author has learned in 46 years of research on newborn screening (NBS) for cystic fibrosis (CF) combined with healthcare and public health practice. The original expectation was that screening for this relatively common, life-threatening genetic disorder would lead to consistently timely diagnoses in the neonatal period and be equitable. Unfortunately, this ambitious goal has not been achieved in the USA despite the availability of an excellent, although imperfect, 2-tiered screening test employing immunoreactive trypsinogen (IRT) and DNA analysis for pathogenic variants in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR). In fact, variations in the quality of NBS programs, inconsistencies in their operations, and disparities in outcomes have been prominent features. The causes include leadership challenges and deficiencies among both CF centers and NBS labs; failures to form effective partnerships among CF centers and with NBS programs; relatively rapid implementation after 2005 with variable quality planning; misunderstandings and erroneous dogma about CF; data limitations regarding IRT, especially cutoff values, and CFTR genetics; tolerance of suboptimal protocols and false negative results; problems in dried blood spot collections plus a lack of transparency and national oversight; partial lack of readiness, qualifications, funding and/or willingness to innovate with floating IRT cutoffs and DNA/CFTR analyses; follow up challenges/deficiencies impairing timeliness, including sweat testing limitations; and published guidelines that are more descriptive than sufficiently critical and directive. But the lessons learned through uniquely intensive CF NBS research have been enlightening and guided the U.S. Cystic Fibrosis Foundation to nationwide quality improvement initiatives.

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Classification of PRSS1 variants responsible for chronic pancreatitis: An expert perspective from the Franco-Chinese GREPAN study group.

BACKGROUND: PRSS1 was the first reported chronic pancreatitis (CP) gene. The existence of both gain-of-function (GoF) and gain-of-proteotoxicity (GoP) pathological PRSS1 variants, together with the fact that PRSS1 variants have been identified in CP subtypes spanning the range from monogenic to multifactorial, has made the classification of PRSS1 variants very challenging. METHODS: All currently reported PRSS1 variants (derived primarily from two databases) were manually reviewed with respect to their clinical genetics, functional analysis and population allele frequency. They were classified by variant type and pathological mechanism within the framework of our recently proposed ACMG/AMP guidelines-based seven-category system. RESULTS: The total number of distinct germline PRSS1 variants included for analysis was 100, comprising 3 copy number variants (CNVs), 12 5' and 3' variants, 19 intronic variants, 5 nonsense variants, 1 frameshift deletion variant, 6 synonymous variants, 1 in-frame duplication, 3 gene conversions and 50 missense variants. Based upon a combination of clinical genetic and functional analysis, population data and in silico analysis, we classified 26 variants (all 3 CNVs, the in-frame duplication, all 3 gene conversions and 19 missense) as "pathogenic", 3 variants (missense) as "likely pathogenic", 5 variants (four missense and one promoter) as "predisposing", 13 variants (all missense) as "unknown significance", 2 variants (missense) as "likely benign", and all remaining 51 variants as "benign". CONCLUSIONS: We describe an expert classification of the 100 PRSS1 variants reported to date. The results have immediate implications for reclassifying many ClinVar-registered PRSS1 variants as well as providing optimal guidelines/standards for reporting PRSS1 variants.

Neutrophil lymphocyte ratio, mean platelet volume, and immunoreactive trypsinogen as early inflammatory biomarkers for cystic fibrosis in infancy: A retrospective cohort study.

BACKGROUND: Airway inflammation starts in early life in cystic fibrosis (CF) and limited, objective markers are available to help identify infants with increased inflammation. We aimed to investigate neutrophil, lymphocyte ratio (NLR), mean platelet volume (MPV) and immunoreactive trypsinogen (IRT) to be a possible inflammatory biomarker for CF in infancy. METHODS: This was a retrospective cohort study in three centers. Between January 2015 and December 2022, children with CF newborn screening (NBS) positivity and diagnosed as CF were included in the study. Correlation analysis were performed with NLR, MPV, IRT and follow-up parameters such as z-scores, modified Shwachman-Kulczycki score (mSKS) at the first, second, third and sixth ages and pulmonary function test (PFT) at the sixth age. RESULTS: A total of 92 children with CF included in the study and 47.8% of them were female. There were no correlations between NLR, MPV and weight and height z-scores for all ages (p > 0.05), a negative correlation was found between MPV and body mass indexes (BMI) z-score at the age of 6 (r = -0.443, p = 0.038). No correlation was found between NLR, MPV and PFT parameters and mSKS at all ages (p > 0.05). There was a negative correlation between first IRT and BMI z-score at 6 years of age (r = -0.381, p = 0.046) and negative correlations between second IRT and weight and BMI z-score at the age of 6 (r = -0.462, p = 0.010; r = -0.437, p = 0.016, respectively). CONCLUSION: Higher MPV and IRT levels during NBS period are associated with worse nutritional outcome which may reflect chronic inflammation. Children with higher MPV and IRT should be followed up closely in terms of chronic inflammation and nutritional status.

PRSS2 regulates EMT and metastasis via MMP-9 in gastric cancer.

Serine protease 2 (PRSS2) is upregulated in gastric cancer tissues, correlates with poor prognosis and promotes migration and invasion of gastric cancer cells. However, the exact mechanism by which PRSS2 promotes metastasis in gastric cancer is unclear. We examined serum PRSS2 levels in healthy controls and gastric cancer patients by enzyme linked immunosorbent assay (ELISA) and analyzed the correlation between PRSS2 serum level with the clinicopathological characteristics of gastric cancer patients and matrix metalloproteinase-9 (MMP-9) expression. A lentiviral MMP-9 overexpression vector was constructed and used to transfect gastric cancer cells with stable silencing of PRSS2, and migration, invasion and epithelial-mesenchymal transition (EMT) of gastric cancer cells were examined. High serum PRSS2 levels were detected in gastric cancer patients and associated with lymphatic metastasis and TNM stage. Serum PRSS2 was positively correlated with serum MMP-9 level. PRSS2 silencing inhibited EMT, and knock-down of PRSS2 partially abrogated cell metastasis and EMT caused by overexpression of MMP-9. These results suggest that PRSS2 promotes the migration and invasion of gastric cancer cells through EMT induction by MMP-9. Our findings suggest that PRSS2 may be a potential early diagnostic marker and therapeutic target of gastric cancer.

Spectrum of PRSS1, SPINK1, CTRC, CFTR, and CPA1 Gene Variants in Chronic Pancreatitis Patients in Russia.

The aim of the study was to define the spectrum of genetic risk factors of chronic pancreatitis (CP) development in patients living in the European part of the Russian Federation. Materials and Methods: The study group included 105 patients with CP, with the age of the disease onset under 40 years old (the average age of onset was 26.9 years). The control group consisted of 76 persons without clinical signs of pancreatitis. The diagnosis of chronic pancreatitis in patients was made on the basis of clinical manifestations and the results of laboratory and instrumental investigations. Genetic examination of patients was conducted using the next-generation sequencing (NGS) technology and included targeted sequencing of all exons and exon-intron boundaries of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes. The genotyping of the rs61734659 locus of the PRSS2 gene was also conducted. Results: Genetic risk factors of the CP development were found in 61% of patients. Pathogenic and likely-pathogenic variants associated with the risk of CP development were identified in the following genes: CTRC (37.1% of patients), CFTR (18.1%), SPINK1 (8.6%), PRSS1 (8.6%), and CPA1 (6.7%). The frequent gene variants in Russian patients with CP were as follows: CTRC gene - c.180C>T (rs497078), c.760C>T (rs121909293), c.738_761del24 (rs746224507); cumulative odds ratio (OR) for all risk alleles was 1.848 (95% CI: 1.054-3.243); CFTR gene - c.3485G>T (rs1800120), c.1521_1523delCTT (p.Phe508del, rs113993960), and c.650A>G (rs121909046); OR=2.432 (95% CI: 1.066-5.553). In the SPINK1, PRSS1, and CPA1 genes, pathogenic variants were found only in the group of patients with CP. The frequent variants of the SPINK1 gene include c.101A>G (p.Asn34Ser, rs17107315) and c.194+2T>C (rs148954387); of the PRSS1 gene - c.86A>T (p.Asn29Ile, rs111033566); of the CPA1 gene - c.586-30C>T (rs782335525) and c.696+23_696+24delGG. The OR for the CP development for the c.180TT genotype (rs497078) CTRC according to the recessive model (TT vs. CT+CC) was 7.05 (95% CI: 0.86-263, p=0.011). In the CTRC gene, the variant c.493+49G>C (rs6679763) appeared to be benign, the c.493+51C>A (rs10803384) variant was frequently detected among both the diseased and healthy persons and did not demonstrate a protective effect. The protective factor c.571G>A (p.Gly191Arg, rs61734659) of the PRSS2 gene was detected only in the group of healthy individuals and confirmed its protective role. 12.4% of the patients with CP had risk factors in 2 or 3 genes. Conclusion: Sequencing of the coding regions of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes allowed to identify genetic risk factors of the CP development in 61% of cases. Determining the genetic cause of CP helps to predict the disease course, perform preventive measures in the proband's relatives, and facilitate a personalized treatment of the patient in future.

High expression of serine protease 2 (PRSS2) associated with invasion, metastasis, and proliferation in gastric cancer.

BACKGROUND: Accumulating evidence indicates that the occurrence and development of tumors are related to the activation of oncogenes and the inactivation of tumor suppressor genes caused by epigenetic mechanisms. However, the function of serine protease 2 (PRSS2) in gastric cancer (GC) is still unknown. Our study aimed to find a regulation network involved in GC. METHODS: The mRNA data (GSE158662 and GSE194261) of GC and normal tissues were downloaded from the Gene Expression Omnibus (GEO) dataset. Differential expression analysis was performed using R software, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted by using Xiantao software. Besides, we used Quantitative Real-time PCR (qPCR) to verify our conclusions. After gene knockdown, cell migration and CCK-8 experiment were carried out to detect the effect of gene on cell proliferation and invasion. RESULTS: Totally, 412 differentially expressed genes (DEGs) were identified from GSE158662 and 94 DEGs were identified from GSE196261. Km-plot database results indicated that PRSS2 exhibited high diagnosis worth for GC. Gene functional annotation enrichment analysis revealed that these hub mRNAs were mainly taken part in the process of tumorigenesis and development. Besides, vitro experiments showed that down-regulation of PRSS2 gene reduced the proliferation and invasion ability of GC cells. CONCLUSIONS: Our results indicated that PRSS2 may play vital roles in the carcinogenesis and progression of GC and can be potential biomarkers for patients with GC.

Sweat conductivity for diagnosing cystic fibrosis after positive newborn screening: prospective, diagnostic test accuracy study.

OBJECTIVE: To assess the accuracy of sweat conductivity among newborns and very young infants. DESIGN: Prospective, population-based, diagnostic test accuracy study. SETTING: Public Statewide Newborn Screening Programme where the incidence rate of cystic fibrosis (CF) is ≈1:11 000. PATIENTS: Newborns and very young infants with positive two-tiered immunoreactive trypsinogen. INTERVENTIONS: Sweat conductivity and sweat chloride were performed simultaneously, on the same day and facility by independent technicians, with the cut-off values of 80 mmol/L and 60 mmol/L, respectively. MAIN OUTCOME MEASURES: Sensitivity, specificity, positive and negative predictive values (PPV and NPV), overall accuracy, positive and negative likelihood ratios (+LR, -LR) and post (sweat conductivity (SC)) test probability were calculated to assess SC performance. RESULTS: 1193 participants were included, 68 with and 1108 without CF, and 17 with intermediate values. The mean (SD) age was 48 (19.2) days, ranging from 15 to 90 days. SC yielded sensitivity of 98.5% (95% CI 95.7 to 100), specificity of 99.9% (95% CI 99.7 to 100), PPV of 98.5% (95% CI 95.7 to 100) and NPV of 99.9% (95% CI 99.7 to 100), overall accuracy of 99.8% (95% CI 99.6 to 100), +LR of 1091.7 (95% CI 153.8 to 7744.9) and -LR of 0.01 (95% CI 0.00 to 0.10). After a positive and negative sweat conductivity result, the patient's probability of CF increases around 350 times and drops to virtually zero, respectively. CONCLUSION: Sweat conductivity had excellent accuracy in ruling in or ruling out CF after positive two-tiered immunoreactive trypsinogen among newborns and very young infants.

Genetic determinants of pancreatitis: relevance in severe hypertriglyceridemia.

PURPOSE OF REVIEW: Not all patients with severe hypertriglyceridemia develop acute pancreatitis. We surveyed recent literature on inter-individual genetic variation in susceptibility to pancreatitis. RECENT FINDINGS: Genetic determinants of pancreatitis include: rare Mendelian disorders caused by highly penetrant pathogenic variants in genes involved in trypsinogen activation; uncommon susceptibility variants in genes involved in trypsinogen activation, protein misfolding as well as calcium metabolism and cystic fibrosis, that have variable penetrance and show a range of odds ratios for pancreatitis; and common polymorphisms in many of the same genes that have only a small effect on risk. The role of these genetic variants in modulating pancreatitis risk in hypertriglyceridemia is unclear. However, among genetic determinants of plasma triglycerides, those predisposing to more severe hypertriglyceridemia associated with chylomicronemia appear to have higher pancreatitis risk. SUMMARY: Currently, among patients with severe hypertriglyceridemia, the most consistent predictor of pancreatitis risk is the triglyceride level. Furthermore, pancreatitis risk appears to be modulated by a higher genetic burden of factors associated with greater magnitude of triglyceride elevation. The role of common and rare genetic determinants of pancreatitis itself in this metabolic context is unclear.

Proteome-Wide Mendelian Randomization Identifies Causal Links Between Blood Proteins and Acute Pancreatitis.

BACKGROUND & AIMS: Acute pancreatitis (AP) is a complex disease and the leading cause of gastrointestinal disease-related hospital admissions. Few therapeutic options exist for AP prevention. Blood proteins with causal evidence may represent promising drug targets, but few have been causally linked with AP. Our objective was to identify blood proteins linked with AP by combining genome-wide association meta-analysis and proteome-wide Mendelian randomization (MR) studies. METHODS: We performed a genome-wide association meta-analysis totalling 10,630 patients with AP and 844,679 controls and a series of inverse-variance weighted MR analyses using cis-acting variants on 4719 blood proteins from the deCODE study (N = 35,559) and 4979 blood proteins from the Fenland study (N = 10,708). RESULTS: The meta-analysis identified genome-wide significant variants (P <5 × 10-8) at 5 loci (ABCG5/8, TWIST2, SPINK1, PRSS2 and MORC4). The proteome-wide MR analyses identified 68 unique blood proteins that may causally be associated with AP, including 29 proteins validated in both data sets. Functional annotation of these proteins confirmed expression of many proteins in metabolic tissues responsible for digestion and energy metabolism, such as the esophagus, adipose tissue, and liver as well as acinar cells of the pancreas. Genetic colocalization and investigations into the druggable genome also identified potential drug targets for AP. CONCLUSIONS: This large genome-wide association study meta-analysis for AP identified new variants linked with AP as well as several blood proteins that may be causally associated with AP. This study provides new information on the genetic architecture of this disease and identified pathways related to AP, which may be further explored as possible therapeutic targets for AP.

Mouse model of PRSS1 p.R122H-related hereditary pancreatitis highlights context-dependent effect of autolysis-site mutation.

Mutation p.R122H in human cationic trypsinogen (PRSS1) is the most frequently identified cause of hereditary pancreatitis. The mutation blocks protective degradation of trypsinogen by chymotrypsin C (CTRC), which involves an obligatory trypsin-mediated cleavage at Arg122. Previously, we found that C57BL/6N mice are naturally deficient in CTRC, and trypsinogen degradation is catalyzed by chymotrypsin B1 (CTRB1). Here, we used biochemical experiments to demonstrate that the cognate p.R123H mutation in mouse cationic trypsinogen (isoform T7) only partially prevented CTRB1-mediated degradation. We generated a novel C57BL/6N mouse strain harboring the p.R123H mutation in the native T7 trypsinogen locus. T7R123H mice developed no spontaneous pancreatitis, and severity parameters of cerulein-induced pancreatitis trended only slightly higher than those of C57BL/6N mice. However, when treated with cerulein for 2 days, more edema and higher trypsin activity was seen in the pancreas of T7R123H mice compared to C57BL/6N controls. Furthermore, about 40% of T7R123H mice progressed to atrophic pancreatitis in 3 days, whereas C57BL/6N animals showed full histological recovery. Taken together, the observations indicate that mutation p.R123H inefficiently blocks chymotrypsin-mediated degradation of mouse cationic trypsinogen, and modestly increases cerulein-induced intrapancreatic trypsin activity and pancreatitis severity. The findings support the notion that the pathogenic effect of the PRSS1 p.R122H mutation in hereditary pancreatitis is dependent on its ability to defuse chymotrypsin-dependent defenses.

Five-year performance analysis of a cystic fibrosis newborn screening program in northeastern Brazil.

OBJECTIVE: To analyze the performance of the cystic fibrosis (CF) newborn screening (NBS) program over its first five years in a Brazilian northeastern state. METHOD: A population-based study using a screening algorithm based on immunoreactive trypsinogen (IRT)/IRT. Data were retrieved from the state referral screening center registry. The program performance was evaluated using descriptive indicators such as the results of an active search, coverage, newborn's age at the time of blood sampling, the time between sample collection and its arrival at the laboratory, and the child's age at diagnosis of disease. RESULTS: The public CF screening program covered 82.6% of the 1,017,576 births that occurred, with an accumulated five-year incidence of 1:20,767 live births. The median (25th-75th) age at diagnosis was 3.5 (2.3-7.3) months. The sampling before 7 days of life for the first IRT (IRT1) increased between 2013 and 2017 from 42.2 to 48.3%. Around 5% of IRT1 samples and 30% of the second samples were collected after 30 days of life. In the first and second stages of screening, 23.6% and 19.9% of the infants, respectively, were lost to follow-up. In both stages of screening, the samples were retained at the health units for a median (25th-75th) of 9.0 (7.0-13.0) days. CONCLUSIONS: The coverage by the CF-NBS program was satisfactory as compared to other Brazilian state rates and the percentage of IRT1 samples collected within the first week of life increased progressively. However, time of samples retention at the health units, inappropriate sampling, inherent methodological problems, and loss of follow-up need to improve.