Mutation-specific reporter for optimization and enrichment of prime editing.

Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

Cirrhosis associated with decreased survival and a 10-year lower median age at death of cystic fibrosis patients in the Netherlands.

BACKGROUND: Up to 10% of patients with Cystic Fibrosis develop cirrhotic CF-related liver disease with portal hypertension: CF cirrhosis (CFC). In a nationwide study, we aimed to determine the role of CFC on survival in the Netherlands between 1 and 1-2009 and1-1-2015. METHODS: We identified all CFC patients in the Netherlands, based on ultrasonographic liver nodularity and portal hypertension. A non-cirrhotic control group was obtained from the national Dutch CF patient registry. We compared groups with regards to baseline lung function and nutritional status and survival and age at death over a 6-year period. In case of death of CFC patients, the clinical reported cause was recorded. RESULTS: At baseline, we found no significant difference in lung function and nutritional status between the CFC patients (N = 95) and controls (N = 980). Both the 6-year survival rate (77 vs. 93%; P < .01) and the median age at death (27 vs. 37 years; P = .02) was significantly lower in CFC compared to controls. In the deceased CFC patients, the reported primary cause of death was pulmonary in 68% of cases, and liver failure related in 18% of cases. CONCLUSIONS: In the Netherlands, the presence of CFC is associated with a higher risk for early mortality and an approximately 10-year lower median age at death. This substantial poorer outcome of CFC patients was not reflected in a lower baseline lung function or a diminished nutritional status. However, in the case of mortality, the reported primary cause of death in CFC patients is predominantly pulmonary failure and not end-stage liver disease.

Progress towards non-invasive diagnosis and follow-up of celiac disease in children; a prospective multicentre study to the usefulness of plasma I-FABP.

This prospective study investigates whether measurement of plasma intestinal-fatty acid binding protein (I-FABP), a sensitive marker for small intestinal epithelial damage, improves non-invasive diagnosing of celiac disease (CD), and whether I-FABP levels are useful to evaluate mucosal healing in patients on a gluten-free diet (GFD). Ninety children with elevated tTG-IgA titres and HLA-DQ2/DQ8 positivity were included (study group). Duodenal biopsies were taken, except in those fulfilling the ESPGHAN criteria. Plasma I-FABP levels and tTG-IgA titres were assessed sequentially during six months of follow-up. Eighty children with normal tTG-IgA titres served as control group. In 61/90 (67.8%) of the children in the study group an increased I-FABP level was found; in all these children CD diagnosis was confirmed. Interestingly, in 14/30 (46.7%) children with slightly elevated tTG-IgA titres (<10x upper limit of normal), an increased I-FABP level was found. In all these children the diagnosis of CD was confirmed histologically. After gluten elimination for six weeks I-FABP levels had decreased towards levels in the control group. Measurement of plasma I-FABP, in addition to tTG-IgA, EMA-IgA and HLAtyping, enables non-invasive diagnosing of CD in a substantial number of children, and might therefore be of value in the diagnostic approach of CD.

Effects and efficacy of laparoscopic fundoplication in children with GERD: a prospective, multicenter study.

INTRODUCTION: Laparoscopic antireflux surgery (LARS) in children primarily aims to decrease reflux events and reduce reflux symptoms in children with therapy-resistant gastroesophageal reflux disease (GERD). The aim was to objectively assess the effect and efficacy of LARS in pediatric GERD patients and to identify parameters associated with failure of LARS. METHODS: Twenty-five children with GERD [12 males, median age 6 (2-18) years] were included prospectively. Reflux-specific questionnaires, stationary manometry, 24-h multichannel intraluminal impedance pH monitoring (MII-pH monitoring) and a 13C-labeled Na-octanoate breath test were used for clinical assessment before and 3 months after LARS. RESULTS: After LARS, three of 25 patients had persisting/recurrent reflux symptoms (one also had persistent pathological acid exposure on MII-pH monitoring). New-onset dysphagia was present in three patients after LARS. Total acid exposure time (AET) (8.5-0.8 %; p < 0.0001) and total number of reflux episodes (p < 0.001) significantly decreased and lower esophageal sphincter (LES) resting pressure significantly increased (10-24 mmHg, p < 0.0001) after LARS. LES relaxation, peristaltic contractions and gastric emptying time did not change. The total number of reflux episodes on MII-pH monitoring before LARS was a significant predictor for the effect of the procedure on reflux reduction (p < 0.0001). CONCLUSIONS: In children with therapy-resistant GERD, LARS significantly reduces reflux symptoms, total acid exposure time (AET) and number of acidic as well as weakly acidic reflux episodes. LES resting pressure increases after LARS, but esophageal function and gastric emptying are not affected. LARS showed better reflux reduction in children with a higher number of reflux episodes on preoperative MII-pH monitoring.

Impact of laparoscopic antireflux surgery on belching in pediatric GERD patients.

BACKGROUND: Laparoscopic antireflux surgery (LARS) is a well-established treatment option for children with proton pomp inhibitor (PPI)-resistant gastroesophageal reflux disease (GERD). Besides preventing reflux of gastric fluid and solid content, LARS may also impair the ability of the stomach to vent intragastric air (i.e. gastric belching) and induce gas-related complications, such as bloating and/or hyperflatulence. Furthermore, it was previously hypothesized that LARS induces a behavioral type of belching, not originating from the stomach, called supragastric belching. The aim of this study was to objectively evaluate the impact of LARS on gastric (GB) and supragastric belching (SGB) in children with GERD. METHODS: We performed a prospective, Dutch multicenter cohort study including 25 patients (12 males, median age 6 (range 2-18) years) with PPI-resistant GERD who were scheduled for LARS. Twenty-four-hour multichannel intraluminal impedance pH monitoring (MII-pH monitoring) was performed before and 3 months after fundoplication. Impedance pH tracings were analyzed for reflux episodes and GBs and SGBs. KEY RESULTS: LARS reduced acid exposure time from 8.5% (6.0-16.2%) to 0.8% (0.2-2.8%), p < 0.001. The number of GBs also significantly decreased after LARS (59 [43-77] VS 5 [2-12], p < 0.001). The number of air swallows remained unchanged after LARS. SGBs were infrequent before LARS with no change in the number of SGB observed after the procedure. Postoperative belching symptoms were associated with GBs, not with SGBs. CONCLUSION & INFERENCES: LARS significantly reduces the number of GBs in children with GERD, whereas the number of air swallows remains unchanged. Postoperative symptomatic belching is associated with GBs, but not with SGBs. These findings suggest that LARS does not induce the occurrence of SGBs in children, but longer follow-up is required.

Serum retinol levels and pulmonary function in children and adolescents with cystic fibrosis.

BACKGROUND: It has been suggested that higher serum retinol levels could have protective effects on pulmonary function (PF) in patients with cystic fibrosis (CF). However, serum retinol levels will be transiently decreased during pulmonary exacerbation. Therefore, the extent of chronic pulmonary inflammation should be included when describing the association between PF and serum retinol. We assessed the longitudinal relation between serum retinol, immunoglobulin G (IgG) and PF in paediatric CF patients. METHODS: We studied the serum retinol, IgG and forced expiratory volumes in one second (FEV(1)% pred.) of 228 CF patients during a seven-year follow up period. The cross-sectional and longitudinal relations between these variables were assessed. RESULTS: Serum retinol, with medians levels between 1.2 and 1.4 µmol/l, were relatively stable, while median serum IgG gradually increased during the age years. The FEV(1)% pred. was longitudinally inversely associated with serum IgG and age, but not with serum retinol. Each g/l increase in serum IgG level was associated with an accelerated yearly decline in FEV(1)% pred. of 0.5% (95% CI -0.8 to -0.1, p=0.008), and each year increase in age was associated with a 1.7% (95% CI -2.1 to -1.3, p=0.000) decline in FEV(1)% pred. This effect was not observed with respect to serum retinol levels (95% CI -1.9 to 2.2, p=0.570). CONCLUSIONS: In this large sample of children and adolescents with CF, we found no evidence that higher serum retinol levels had protective effects on PF.

Dietary intake in children and adolescents with cystic fibrosis.

BACKGROUND & AIMS: The recommendation for caloric intake in CF patients is to obtain intakes between 110 and 200% of the estimated average requirement (EAR) for age groups and gender, of which 35-40 energy% should be from fat. It is questionable whether the advice is met. METHODS: 1726 Completed 3-day dietary food records of 234 CF patients (111 girls) and 2860 completed two non-consecutive 24-h dietary assessments of healthy controls (1411 girls) were studied. The dietary intake in CF patients was compared with that of healthy controls by using independent sample t tests. RESULTS: Caloric intake in children with CF varied highly with age (88-127% EAR), which is below or in the lower range of the recommended 110-200% EAR. However the absolute caloric intake in CF children was significantly higher compared to controls at all ages. In addition, apart from boys aged 1-3 years, all CF children had a fat intake of 35 energy% or more. This fat intake was significantly higher than in controls, as was the consumption of saturated fat, the latter being well above 10% of the total energy intake. CONCLUSION: Although CF patients generally do not meet the EAR recommendations, they had a significantly higher caloric intake than controls. Moreover fat intake in CF patients does generally meet recommendations, but this resulted in a considerable consumption of saturated fat; a reduction of the latter seems appropriate.

The long-term outcome of the Kasai operation in patients with biliary atresia: a systematic review.

BACKGROUND: Biliary atresia (BA) is a progressive inflammatory destructive process of the bile ducts occurring in about one of every 20,000 live births. If left untreated, biliary atresia can lead to liver failure. The only effective treatments for BA at the moment are the Kasai operation and liver transplantation. Kasai portoenterostomy increases the survival of children with BA and postpones subsequent liver transplantation. Because long-term survival is rare, there is not much known about the long-term efficacy of the Kasai operation. METHODS: The aim of this review was to study the outcome of patients with BA who survived more than 20 years on their native liver. We performed a systematic search on PubMed using MeSH terms for articles describing the long-term outcomes of patients with biliary atresia. We searched for patients who have lived at least 20 years with their native liver and we registered the number of complications. The endpoints identified in these articles were: death, cholangitis, portal hypertension and gastrointestinal bleeding. RESULTS: From 53 articles we included 14 articles for analysis. In total 184 patients were above the age of 20 years. Of these 162 patients, 88% (162/184) were still alive with their native liver and 60.5% (98/162) were suffering from liver-related complications. CONCLUSIONS: It is possible for patients with biliary atresia to survive more than 20 years on their native liver after undergoing the Kasai operation during early infancy. However, 60.5% of the long-term survivors alive on their native liver end up suffering from progressive liver-related complications.

Nutritional intervention in patients with Cystic Fibrosis: a systematic review.

BACKGROUND: To systematically assess the literature published after 1997 describing the effectiveness of nutritional interventions in Cystic Fibrosis patients. METHODS: An online search in PUBMED, EMBASE and COCHRANE databases was conducted. Original studies with 4 patients or more, describing a nutritional intervention and giving at least weight as an outcome parameter were included. RESULTS: The inclusion criteria were met by 17 articles, focusing on respectively behavioural interventions (n=6), oral supplementation (n=4) or enteral tube feeding (n=7). This latter intervention was universally successful to induce weight gain. One behavioural study and 2 oral supplementation studies also reported significant weight gain. CONCLUSION: Enteral tube feeding is effective to improve nutritional status, while the described effects of behavioural intervention and oral supplementation are not consistent at present.

Course of life into adulthood of patients with biliary atresia: the achievement of developmental milestones in a nationwide cohort.

PURPOSE: To investigate the course of life of young adults diagnosed with biliary atresia (BA) in infancy by comparing patients who did and did not underwent transplantation with an age-matched Dutch reference group. METHODS: All patients from the Dutch BA registry, aged >18 years, were invited to complete the course of life questionnaire. RESULTS: Forty patients participated (response = 74%). Twenty-five had not undergone transplantation; 15 had undergone orthotopic liver transplantation. One significant between-group difference was found, namely in substance use and gambling. BA patients who underwent transplantation reported less use than the reference group (p = .01, moderate effect size). Additional moderate effect sizes were found for differences in psychosexual and social development and antisocial behavior. Patients who underwent transplantation had lower scores than one or both other groups. CONCLUSIONS: Development of BA survivors who did not undergo transplantation seems not delayed, whereas that of transplanted patients does seem somewhat delayed. However, patients who underwent transplantation display less risk behavior. Larger samples are necessary to confirm these findings.

Celiac disease: an overview from pathophysiology to treatment.

Celiac disease (CD) is one of the most common immune-mediated diseases with a worldwide prevalence of around 1%, although a couple of decades ago the disease was thought to be very rare. CD is characterized by an inadequate inflammatory response to gluten in genetically susceptible individuals. In this inflammatory response both the adaptive and innate immunity are involved. The clinical picture of CD is variable ranging from severe malabsorption syndrome to silent cases. Disease specific antibodies can aid in selecting patients for a small intestinal biopsy, which is thought to be the gold standard investigation to diagnose CD. However, in selected patients, serology can be sufficient to confirm the diagnosis and a biopsy is not needed. Hitherto, the only treatment for CD is adherence to a lifelong strict gluten-free diet. The purpose of this review was to summarize current literature on the epidemiology and pathophysiology of CD and to discuss diagnostic and therapeutic approaches.

Immunoglobulin G antibodies against deamidated-gliadin-peptides outperform anti-endomysium and tissue transglutaminase antibodies in children <2 years age.

To investigate the usefulness of deamidated-gliadin-peptides-antibodies in the diagnosis of celiac disease, serology was tested in 212 children suspected with celiac disease who had undergone a small-intestinal-biopsy. For deamidated-gliadin-peptides-antibodies, two kits were tested. Positive and negative predictive values for IgA deamidated-gliadin-peptides-antibodies using the Bindazyme-kit were 89% and 74%, while the Quanta-Lite-kit had values of 89% and 85%, respectively. For the IgG subtype using the Bindazyme-kit, these values were 85% and 89%, while they were 85% and 91% for the Quanta-Lite-kit. The positive predictive values for endomysium and tissue-transglutaminase antibodies were disappointing (77% and 87%), although the negative predictive values were better (97% and 96%). When the analysis was restricted to the 41 children aged <2 years, no misclassifications occurred with IgG deamidated-gliadin-peptides-antibodies giving 100% accuracy in both kits. The positive predictive value reached 100% for tissue-transglutaminase antibodies and both kits for IgA deamidated-gliadin-peptides-antibodies, while the negative predictive value was 94% in these assays. Positive and negative predictive values for endomysium antibodies were 96% and 93%, respectively. In conclusion, although deamidated-gliadin-peptides-antibodies do not outperform anti-endomysium antibodies in the total study population, the IgG subtype seems to be the best test in children aged <2 years, reaching 100% accuracy.

Hydrolysed formula is a risk factor for vitamin K deficiency in infants with unrecognised cholestasis.

OBJECTIVES: Vitamin K deficiency (VKD) may cause life-threatening haemorrhages, especially in breast-fed infants with unrecognised cholestasis. Interestingly, hypoallergenic formulas appear overrepresented in reported cases of VKD bleeding (VKDB) in formula-fed infants. We therefore assessed whether the risk of VKD in formula-fed infants with cholestasis is associated with hypoallergenic formulas. PATIENTS AND METHODS: Infants born in the Netherlands between January 1991 and December 2006 with cholestatic jaundice due to biliary atresia (BA) or to α-1-antitrypsin deficiency (A1ATD) were identified in the Netherlands Study Group for Biliary Atresia Registry and the A1ATD registry, respectively. The relative risk (RR) of VKDB in patients with BA or A1ATD was calculated for different formula types. The influence of prior or ongoing breast-feeding on the RR of VKDB was also assessed. RESULTS: A total of 179 infants with either BA (139) or A1ATD (40) were included. One hundred eighteen infants were formula fed; 8 presented with VKD. Six of these 8 infants (75%) received hypoallergenic formula (whey-based hydrolysate in 4). One infant on whey-based hydrolysed formula presented with VKDB. Risk factor analysis revealed that infants receiving hydrolysed, especially whey-based, formula, had a strongly increased risk of VKD (RR 25.0 [6.4-97.2], P < 0.001)) compared with infants receiving regular formula. Prior or ongoing breast-feeding was not significantly associated with VKD. CONCLUSIONS: Infants with cholestasis receiving (whey-based) hydrolysed formula are at increased risk of developing VKD, compared with infants receiving regular formula. Because VKD may lead to serious haemorrhages, infants receiving whey-based hydrolysed formulas may need additional vitamin K supplementation.

Association of the CLCA1 p.S357N variant with meconium ileus in European patients with cystic fibrosis.

In Cftr-/- mice that mostly die because of intestinal obstruction, intestinal expression of Clca3 is decreased, whereas upregulation of Clca3 results in amelioration of intestinal disease. The aim of the study was to investigate whether the p.S357N variant in CLCA1, the human orthologue of Clca3, acts as a modifier gene in a cohort of 682 European patients with cystic fibrosis (CF)-99 patients with meconium ileus. The 357SS genotype was significantly overrepresented in both patients with meconium ileus and also with a severe CFTR genotype (P = 0.009) and in p.F508del homozygotes (P = 0.002). This suggests that CLCA1 has similar important functions in CF-related intestinal obstruction in humans as in Cftr-/- mice.

Constipation in pediatric cystic fibrosis patients: an underestimated medical condition.

BACKGROUND: The aims of this study were to determine prevalence, risk factors and treatment of constipation in patients with Cystic Fibrosis (CF), as well as the diagnostic value of abdominal radiography. METHODS: A cohort of 214 pediatric CF patients was investigated. Furthermore, 106 abdominal radiographs of CF patients with or without constipation were independently assessed by three observers on two separate occasions using the Barr and Leech scores. RESULTS: The prevalence of constipation was 47%. Low total fat absorption and meconium ileus were independent risk factors for constipation in CF, while fiber and fluid intake were not associated. In CF patients the inter and intraobserver variabilities of the Barr and Leech scores were poor to moderate. CONCLUSION: Constipation is a significant medical issue in CF and was associated with low total fat absorption and a history of meconium ileus. Finally, abdominal radiography seems of little value in the regular follow-up of CF patients.

Vitamin K deficiency bleeding in cholestatic infants with alpha-1-antitrypsin deficiency.

OBJECTIVE: Exclusively breastfed infants with unrecognised cholestatic jaundice are at high risk of a vitamin K deficiency (VKD) bleeding. It is presently unknown whether (the size of) this risk depends on the degree of cholestasis. Since alpha-1-antitrypsin deficiency (A1AD) induces a variable degree of cholestasis, we assessed the risk of VKD bleeding in infants with cholestatic jaundice due to A1AD. PATIENTS AND METHODS: Infants with a ZZ or SZ phenotype born in The Netherlands between January 1991 and December 2006 were identified from the databases of the five Dutch diagnostic centres for alpha-1-antitrypsin phenotyping and/or genotyping. We determined the risk of VKD bleeding upon diagnosis of A1AD in breastfed and formula fed infants and searched for correlations between serum levels of conjugated bilirubin and the risk of bleeding. RESULTS: A total of 40 infants with A1AD were studied. VKD bleeding was noted in 15/20 (75%) of breastfed infants, compared with 0/20 of formula fed infants with A1AD. The relative risk for VKD bleeding in breastfed versus formula fed infants was at least 15.8 (95% CI 2.3 to 108). Conjugated bilirubin levels at diagnosis did not correlate with the risk of VKD bleeding. CONCLUSIONS: The risk of VKD bleeding in breastfed infants with A1AD was high and did not correlate with serum level of conjugated bilirubin at diagnosis. A similar absolute risk was previously reported in breastfed infants with biliary atresia under the same prophylactic regimen. This confirms that-without adequate prophylaxis-the risk of VKD bleeding is uniformly high in exclusively breastfed infants with cholestatic jaundice, irrespective of underlying aetiology.