Discussion
This study identified that increasing sw[Cl-] values and ever having a respiratory culture positive for PSA are associated with risk of reclassification from CRMS/CFSPID to CF in a population with high IRT and two CFTR variants. These findings emphasize that continuous follow up, allowing for observation of changes in phenotype over time, will help define the prognosis for children with CRMS/CFSPID. Multiple factors that could influence CF reclassification were studied, and we found that children who reclassified to CF had more frequent reporting of crackles, respiratory symptoms, and growth concerns compared to those who retained the CRMS/CFSPID designation. CF-reclassified children also had higher rates of pneumonia, hospitalizations for respiratory symptoms, use of antibiotics, and a steeper slope of rising sw[Cl-] values. Measures of ppFEV1, LCI2.5, IRT, and history of asthma/reactive airways disease were not significantly associated with the risk of CRMS/CFSPID-to-CF reclassification; this is notable considering the clinical relevance of these variables within the CF population.
A rising sw[Cl-] predicts CF reclassification, confirming previous reports. The precise predictive threshold is unclear and may vary by cohort. In prior studies, CRMS/CFSPID cohorts that have reclassified to CF had sw[Cl-] increases from 5.3-7.3 mmol/L/year and an increase in mean sw[Cl-] from 40 (±11) to 82 (±26) mmol/L.21 Our group and others have also reported that healthy controls and those who retained the CRMS/CFSPID designation through childhood did not have significant increase in sw[Cl-] over time.2021 Here, we present that a rate of sw[Cl-] increase of 4.71 mmol/L/year (95%CI 2.45 – 6.97) is an independent risk factor for CF reclassification. These combined findings may guide providers to be more attentive to children with CRMS/CFSPID showing increase sw[Cl-] ≥4 mmol/L/year, and more reasured by others showing increase at ~1 mmol/L/year. This also suggests that there is more value in making clinical decision based on multiple data points collected over time, rather than based on one data point from an intial assessment. Notably, 50% of those who reclassified had an intial sw[Cl-] <30 mmol/L, which is considered “CF unlikely” by current guidelines.16 Although an initial intermediate sw[Cl-] (30-59 mmol/L) naturally raises concern for likelihood of CFTR dysfunction,23 recent longitudinal data from the CF-reclassified group suggests that providers should also focus on the rate at which sw[Cl-] rises as an indication for change in diagnosis, even before it reaches the diagnostic threshold of ≥60 mmol/L.21
CFTR genotypes from children who reclassified to CF included at least one F508del (58%) or a nonsense variant (G542X and R1162X;Table 2 ). Genotypes containing a CF-causing variant and 5T;TG12 or 5T;TG13 were seen in 7 (58%) of the 12 who reclassified. Notably, as the CF NBS algorithm in California includes CFTRsequencing, it is unlikely that other variants influenced the phenotypes in these individuals. As we previously reported, 38% of children with a CF-causing variant and 5T;TG13 reclassified to CF in their first 8 years of life.20 This is aligned with previous reports that determination of TG repeat number allows for more accurate prediction of benign versus pathogenic 5T alleles.24 Other variants observed in the second allele of the CF-reclassified group were missense variants of varying clinical consequence per CFTR2, and which are approved for CFTR modulators: c.1853T>C (I618T), c.3454G>C (D1152H) , and c.3154T>G (F1052V). (https://cftr2.org) The non CF-causing variant c.3705T>G (S1235R) was observed as part of a complex allele. Variants c.94C>A (L32M) and c.1393-42G>A (1525-42G>A) are frequent in California and are of unknown consequence.25
LCI2.5 was non-discriminatory between those with CRMS/CFSPID who reclassified to CF and those who did not (Figure 1, Supplemental Figure 2, Table 3) and did not influence reclassification when tested in the multivariate logistic regression model. As children with CRMS/CFSPID may have abnormal LCI2.5 despite being asymptomatic,11longer follow-up will reveal if those with abnormal measurements at a young age will progress to have symptoms and abnormal ppFEV1 later in life.26 Munck and colleagues reported that asymptomatic children with CRMS/CFSPID may have bronchiectasis;27 therefore, MBW should continue to be explored as a sensitive and safe method to detect early lung disease in this population.11,28
Assessment of CFTR protein function was performed in a subgroup of participants. This method was chosen because it directly estimates protein function, is not expected to change over time, and is anex-vivo test that can be tolerated by children. Our results suggest greater overlap in HNE assay results between children who reclassified to CF and those who retained the CRMS/CFSPID designation compared to sw[Cl-] values in these two groups (Figure 2 ). On the other hand, HNE assays show greater distinction between the CRMS/CFSPID group and healthy controls than sw[Cl-], as healthy controls and carriers may have sw[Cl-] from <30 to >60 mmol/L.29 Additionally, HNE assay can be performed at any time, whereas sw[Cl-] slope requires repeated measurements over time. We recruited a selected genotype group to evaluate using HNE assays based on higher risk of reclassification: a CF-causing variant on one allele and R117H;7T, 5T;TG12, or 5T;TG13 on the other.20,30 A larger sample is needed to draw conclusions based on genotype groups. Additionally, a non-linear association between HNE and sw[Cl-] has been reported in a cohort of individuals with CF 18 that was not found in this CRMS/CFSPID cohort. However, this association was difficult to interpret when sw[Cl-] was <60 mmol/L. Finally, the HNE assay reported here was completed in 10 subjects but a total of 17 underwent sampling (including some individuals who were sampled twice; data not shown). The ~50% test yield due to contamination and poor cell growth may be a reflection of the discomfort caused by sampling, which could be a barrier to utilizing this assay for young pediatric patients. However, considering the potential benefit of having the accurate estimation of CFTR function early in life, HNE assays should be explored further in a larger sample of the CRMS/CFSPID population.
Microbiology characteristics included at least one positive respiratory culture for PSA in 12% of the CRMS/CFSPID group, similar to other US reports and lower than in European cohorts (~10% and 24% respectively)3,27 (Table 1 ). One child with two positive cultures in the previous 12 months was placed in the chronic category; in this case, the second positive culture was immediately after 28 days of inhaled tobramycin (Tobi®), which triggered a second course of treatment.
CFQ-R questionnaires were used as a patient reporting outcome for patients ≥6 years of age.14 Among the different domains, social showed significant higher results for children with CRMS/CFSPID, likely because these children are asymptomatic and their social lives are less impacted. Respiratory and emotional domains were statistically similar, which may be a reflection of early interventions related to early diagnosis of CF through NBS and to the young age group.
IRT results were statistically similar between the CF-reclassified subjects and those who retained the CRMS/CFSPID designation (Table 3 ). This contrasts our previous findings, where an IRT>80 ng/mL increased the likelihood of CF reclassification.20 Others have reported no difference in baseline IRT when reclassified and non-reclassified children with CRMS/CFSPID were compared.21 Thus, it remains unclear whether the baseline IRT measured at birth can be used as a predictor of CF reclassification.31
Other findings included no identification of CFTR-related disorder (CFTR-RD) and no association of asthma or reactive airways disease with reclassification to CF. CFTR-RD was defined in 2011 as a diagnosis related to CFTR dysfunction that does not fulfill the diagnosis criteria for CF and typically manifests in one affected organ system (congenital bilateral absence of the vas deferens [CBAVD], pancreatitis, or bronchiectasis).32 As CFTR variants associated with CFTR-RD are also found in CRMS/CFSPID cohorts, it is possible that some children with CRMS/CFSPID will eventually fulfill diagnostic criteria for CFTR-RD but not CF. The CFTR-RD definition is under needed revision, as it likely that a child with CRMS/CFSPID, two CFTRvariants, and evidence of CFTR dysfunction (i.e., intermediate sw[Cl-]) who develops chronic respiratory symptoms and bronchiectasis will be diagnosed as CF.4, 6Chronic cough and wheezing can be the initial manifestations of CF in childhood;33 however, they may lead to the diagnosis of asthma/reactive airway disease instead of CF, considering the increasing prevalence of asthma in childhood.34Interestingly, CFTR dysfunction is associated with an increased likelihood of asthma, as seen among CFTRcarriers.35 In this cohort, the asthma prevalence was statistically similar between the CF-reclassified group and those who kept the CRMS/CFSPID designation, and asthma did not increase the risk of reclassification. It is unclear if the high prevalence seen in both groups (42% in CF-reclassified and 36% in CRMS/CFSPID; Table 3 ) indicates misdiagnosis of CF, CFTR dysfunction, or a public health trend. Nevertheless, primary care providers should avoid misdiagnosis and refer a child with CRMS/CFSPID and who has chronic respiratory signs and symptoms to a CF center for re-evaluation.36
This study is not without limitations. The recruitment was limited to two sites, making the sample size relatively small and limited to a specific geographic area. Both sites also had a high lost-to-follow-up rate among CRMS/CFSPID children (51% at CHLA and 34% at KP), potentially introducing bias to the population continuing to receive care. We did attempt to address this by specifically recruiting from this population – 13 of the 59 (22%) subjects in the CRMS/CFSPID group were recruited from a lost-to-follow-up pool. Tobacco smoke exposure, a known risk factor for CFTR dysfunction,37 could not be studied because of the low prevalence of exposure to secondhand smoking.
A final thought is that although currently published data showed that most children with CRMS/CFSPID designation have a favorable outcome, data is limited to a small number entered in national registries and short follow up time.27,31,38 The short follow up may be attributed to young age of this population, early discharge per physician’s decision, and high rates of lost-to-follow up.39 The second challenge for defining prognosis in this population is high variability in duration of follow up, care practices, and CF reclassification criteria despite improved guidelines.40
CF providers and investigators are still in the beginning of understanding the intricacies of the CRMS/CFSPID population, and longer longitudinal observational studies are needed to better define the risk of reclassification to CF. Although recent literature suggests that most persons with CRMS/CFSPID will not develop any symptoms, more current findings indicate that some will develop CF, others CFTR-RD, and a larger group may be at risk for a wide range of cystic fibrosis-related conditions such as those seen in carriers. Key strengths of our work are the size of the cohort, the diversity of fully-sequenced CFTRgenotypes, and a wide age range with longitudinal data up to 13 years. We showed that the two independent risk factors for manifesting signs and symptoms to fulfil CF diagnosis criteria are rapid rising sw[Cl-] values and the detection of PSA from respiratory cultures. Yearly visits at the CF center and greater awareness from the primary care providers will promote monitoring of chronic respiratory symptoms and other signs and symptoms seen in early CF disease, so treatment can start early fulfilling the preventive purpose of NBS. The CF scientific community should continue to invest in alternative CFTR function assays that can complement sw[Cl-] testing in defining risk of disease for each unique combination of CFTR variants. With all this put in place we will better define each child’s needs from infancy, which will avoid unnecessary medical care for those at low risk and close follow up for those at a higher risk of developing CF disease.