High-quality read-based phasing of cystic fibrosis cohort informs genetic understanding of disease modification.

Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. We sequenced 477 individuals with cystic fibrosis (CF) using linked-read sequencing, which display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, demonstrating its utility for understanding complex CF alleles. These are visualized in a Web app, CFTbaRcodes, that enables interactive exploration of CFTR haplotypes present in this cohort. We perform fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus, an intestinal obstruction at birth associated with more severe CF outcomes and pancreatic disease. A 20-kb deletion polymorphism and a PRSS2 missense variant p.Thr8Ile (rs62473563) are shown to independently contribute to meconium ileus risk (p = 0.0028, p = 0.011, respectively) and are PRSS2 pancreas eQTLs (p = 9.5 × 10-7 and p = 1.4 × 10-4, respectively), suggesting the mechanism by which these polymorphisms contribute to CF. The phase information from linked reads provides a putative causal explanation for variation at a CF-relevant locus, which also has implications for the genetic basis of non-CF pancreatitis, to which this locus has been reported to contribute.

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease.

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.

Outcomes of Cystic Fibrosis Screening-Positive Infants With Inconclusive Diagnosis at School Age.

BACKGROUND AND OBJECTIVES: Cystic fibrosis (CF) screen-positive infants with an inconclusive diagnosis (CFSPID) are infants in whom sweat testing and genetic analysis does not resolve a CF diagnosis. Lack of knowledge about the health outcome of these children who require clinical follow-up challenges effective consultation. Early predictive biomarkers to delineate the CF risk would allow a more targeted approach to these children. METHODS: Prospective, longitudinal, multicenter, Canada-wide cohort study of CF positive-screened newborns with 1 to 2 cystic fibrosis transmembrane conductance regulator gene variants, of which at least 1 is not known to be CF-causing and/or a sweat chloride between 30 and 59 mmol/L. These were monitored for conversion to a CF diagnosis, pulmonary, and nutritional outcomes. RESULTS: The mean observation period was 7.7 (95% confidence interval 7.1 to 8.4) years. A CF diagnosis was established for 24 of the 115 children with CFSPID (21%) either because of reinterpretation of the cystic fibrosis transmembrane conductance regulator genotype or because of increase in sweat chloride concentration ≥60 mmol/L. An initial sweat chloride of ≥40 mmol/l predicted conversion to CF on the basis of sweat testing. The 91 remaining children with CFSPID were pancreatic sufficient and showed normal growth until school age. Pulmonary function as well as lung clearance index in a subgroup of children with CFSPID were similar to that of healthy controls. CONCLUSIONS: Children with CFSPID have good nutritional and pulmonary outcomes at school age, but rates of reclassifying the diagnosis are high. The initial sweat chloride test can be used as a biomarker to predict the risk for CF in CFSPID.

Shrinking lung syndrome treated with rituximab in pediatric systemic lupus erythematosus: a case report and review of the literature.

BACKGROUND: Shrinking lung syndrome (SLS), a rare complication of systemic lupus erythematosus (SLE) characterized by dyspnea, low lung volumes, and a restrictive pattern on pulmonary function tests (PFTs), has only been reported in a few children. Given the rarity of SLS there is a paucity of literature regarding its optimal treatment. Outcomes are variable, with case reports documenting some improvement in most patients treated with corticosteroids, with or without additional immunosuppressive agents. However, most reported patients did not recover normal lung function. We report full recovery of a child with SLE and SLS following treatment with rituximab and review the current literature. CASE PRESENTATION: An 11-year-old boy presented with a malar rash, myositis, arthritis, oral ulcers, leukopenia, anemia, positive lupus autoantibodies and Class II nephritis. He was diagnosed with SLE and treated with corticosteroids, hydroxychloroquine, azathioprine, and subsequently mycophenolate with symptom resolution. At age 14, his SLE flared coincident with a viral chest infection. He presented with a malar rash, polyarthritis, increased proteinuria and pleuritis which all improved with corticosteroids and ongoing treatment with mycophenolate. Six weeks later he presented with severe dyspnea, markedly decreased lung volumes, but otherwise normal chest X-ray (CXR) and high-resolution chest computed tomography (HRCT). He was found to have severely restricted PFTs (FEV1 27%, FVC 29%; TLC 43%). After additional investigations including echocardiography, pulmonary CT angiography, and diaphragmatic fluoroscopy, he was diagnosed with SLS and treated with rituximab and methylprednisolone. At 1 month his symptoms had improved, but he still had dyspnea with exertion and severely restricted PFTs. At 6 months his FVC and TLC had improved to 51 and 57% respectively, and were 83 and 94% respectively at 4 years. He had returned to all baseline activities, including competitive hockey. CONCLUSIONS: Although extremely rare, it is important to recognize SLS as a possible cause of dyspnea and chest pain in a child with SLE. Optimal treatment strategies are unknown. This is the second reported case of a child treated with rituximab for SLS who recovered normal lung function. International lupus registries should carefully document the occurrence, treatment and outcome of patients with SLS to help determine the optimal treatment for this rare complication.

Cystic fibrosis-related diabetes onset can be predicted using biomarkers measured at birth.

PURPOSE: Cystic fibrosis (CF), caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR), affects multiple organs including the exocrine pancreas, which is a causal contributor to cystic fibrosis-related diabetes (CFRD). Untreated CFRD causes increased CF-related mortality whereas early detection can improve outcomes. METHODS: Using genetic and easily accessible clinical measures available at birth, we constructed a CFRD prediction model using the Canadian CF Gene Modifier Study (CGS; n = 1,958) and validated it in the French CF Gene Modifier Study (FGMS; n = 1,003). We investigated genetic variants shown to associate with CF disease severity across multiple organs in genome-wide association studies. RESULTS: The strongest predictors included sex, CFTR severity score, and several genetic variants including one annotated to PRSS1, which encodes cationic trypsinogen. The final model defined in the CGS shows excellent agreement when validated on the FGMS, and the risk classifier shows slightly better performance at predicting CFRD risk later in life in both studies. CONCLUSION: We demonstrated clinical utility by comparing CFRD prevalence rates between the top 10% of individuals with the highest risk and the bottom 10% with the lowest risk. A web-based application was developed to provide practitioners with patient-specific CFRD risk to guide CFRD monitoring and treatment.

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.

Predictors for surgical intervention and surgical outcomes in neonates with cystic fibrosis.

BACKGROUND/PURPOSE: To identify prenatal and perinatal predictors of surgery and describe surgical findings/outcomes of neonates with Meconium Ileus (MI) secondary to Cystic Fibrosis (CF). METHODS: Potential risk factors (prenatal bowel echogenicity, CF genotype, birthweight, prematurity and sex) for MI and surgery were examined in a retrospective cohort of neonates with CF presenting to a tertiary center between 1997 and 2015. Following univariable analysis, predictors of MI and surgery were determined using multivariable logistic regression. For surgical patients, detailed operative findings and outcomes were examined. RESULTS: MI was diagnosed in 26/120 (21.7%) neonates with CF and 19/26 (73.0%) required surgery. Prematurity was significantly associated with increased risk of MI and operative intervention (p-value 0.022 and p-value 0.016 respectively); lower birthweight was associated with operative intervention (p-value 0.039); genotype and echogenic bowel were associated with neither. Surgical data were available for 17/19 patients; median age at surgery was 2 days (IQR1-3), 4/17 had an atresia and 6/17 received an ostomy. Median NICU and hospital stays were 34.5 and 70 days while median time on TPN and time to ostomy reversal were 28.5 and 97 days, respectively. CONCLUSIONS: In patients with CF, prematurity and lower birthweight were identified as risk factors for meconium ileus and need for surgery. Specific genotypes and echogenic bowel were not predictors of either. LEVEL OF EVIDENCE: Level III.

Lower airway nitric oxide is increased in children with sickle cell disease.

OBJECTIVES: To determine alveolar and airways nitric oxide (NO) levels in children with sickle cell disease (SCD). STUDY DESIGN: Multiple flows fractional exhaled NO (FE(NO)), bronchial NO flux (J'aw(NO)), and alveolar NO concentration (Ca(NO)) were determined prospectively in 16 non-atopic children with SCD in a tertiary ambulatory clinic and compared with those in 10 children with primary ciliary dyskinesia and 22 healthy control subjects. Differences in FE(NO), J'aw(NO), and Ca(NO) were compared with mixed model analysis and Mann-Whitney tests. RESULTS: Children with SCD had reference range FE(NO) at 50 mL/sec, but FE(NO) was elevated across all flows compared with healthy control subjects (mean difference=2.10±0.91 parts per billion, P=.03). Subjects with SCD had increased J'aw(NO) (1177±533 picoliters per second versus 833±343 picolitres per second, P=.03), and Ca(NO) was no different from control subjects. In contrast, children with primary ciliary dyskinesia had decreased FE(NO) (mean difference=3.36±1.24 parts per billion, P<.01) and J'aw(NO) (507±259 picoliters per second versus 833±343 picoliters per second, P<.01). CONCLUSIONS: Lower airways NO is increased in children with SCD. Elevation of J'aw(NO) may represent dysregulation of NO metabolism or subclinical airways inflammation.

Diagnostic value of nasal nitric oxide measured with non-velum closure techniques for children with primary ciliary dyskinesia.

OBJECTIVES: Nasal nitric oxide (nNO) is a reliable non-invasive screening test for primary ciliary dyskinesia (PCD), but the recommended technique, exhalation against resistance (ER), requires cooperation limiting its use in young children. Our objectives were to determine whether easier non-velum closure techniques have the ability to discriminate PCD and longitudinal reproducibility. STUDY DESIGN: We conducted a case-control study evaluating 5 breathing techniques (ER, breath hold, tidal breathing mouth open, tidal breathing mouth closed, and humming) for measuring nNO in patients with PCD compared with control subjects (cystic fibrosis [CF], non-PCD non-CF bronchiectasis, and healthy). A subgroup repeated measurements 1 month later. Sensitivity, specificity, and intraclass correlation coefficient of each nNO technique were determined. RESULTS: We tested 85 children (20 PCD, 32 CF, 14 broncheoctasis, and 19 healthy), aged 5 to 18 years (mean age, 11.5 years); 52% of children were male. All breathing techniques discriminated patients with PCD from control subjects with high specificity (>90%), 100% sensitivity, and intraclass correlation coefficient >0.8. nNO output cutoff values for diagnosing PCD varied with techniques (ER, 59 nL/min; breath hold, 61 nL/min; tidal breathing mouth open, 37 nL/min; tidal breathing mouth closed, 30 nL/min; humming, 41 nL/min). CONCLUSION: Non-velum closure techniques are reproducible and valid to discriminate PCD; however, they generally yield lower values than ER.

Plastic bronchitis as an unusual cause of mucus plugging in cystic fibrosis.

Cystic fibrosis patients are known to produce abundant, purulent sputum consisting mainly of DNA and cellular debris. We present a case of a CF patient with recurrent airway obstruction caused by a rare condition known as plastic bronchitis (PB). PB is characterized by the formation of casts of the airways that cause obstruction. Multiple etiologies have been proposed, but to our knowledge, no CF patient has been reported in any PB classification. Histological analysis and in-vitro testing of the cast were important factors in choosing the adequate therapy in this patient.

Two reports of acute neonatal acalculous cholecystitis (necrotizing cholecystitis) in a 2-week-old premature infant and a term neonate.

We present 2 cases of acute acalculous cholecystitis, an extremely rare entity in newborns. A number of risk factors have been identified, such as inspissated bile, prematurity, sepsis, dehydration, total parenteral nutrition, medications, and Escherichia coli lipopolysaccharide endotoxin. In our cases, gallbladder bile had positive bacterial cultures for E coli. We present the cases of a premature infant and a term neonate who developed acute acalculous cholecystitis: one with several risk factors and the second with a very atypical presentation.

Double anomalous coronary origin from the pulmonary artery: successful surgical correction in an infant.

We report an extremely rare entity in which all coronary arteries originate from the pulmonary artery. Only a few cases have been reported and corrected, and fewer still have had a favorable outcome. The survival range reported for these patients is from 9 hours to 1 year. This case was diagnosed and surgically corrected in a patient at the age of 2 months, and the patient is doing well 27 months after the procedure.