The sensitivity of NBS for PCD has been unsatisfactory since a small proportion of PCD patients are missed by current MS/MS-based screening approach. Our findings revealed that an overall proportion of missed cases of 24% (6/25) at conventional NBS, physicians should note thata normal C0 level during the recall stage does not necessarily rule out PCD. Incorporating second-tier method into the current NBS program may introduce an opportunity for missed PCD cases. The high throughput iPLEX genotyping assay we used has proven to be robust and reliable in the validation. Six PCD patients who would have been missed were successfully identified when we applied the iPLEX assay as a second-tier screening method. Therefore, we have clearly demonstrated that incorporating the second-tier molecular genetic testing into current NBS program could increase PCD detection.
The iPLEX assay has several advantages, our previous work have demonstrated that it is a powerful tool for population-based genetic screening [18, 19]. In this study, the iPLEX assay was applied in NBS for second-tier mutation screening of suspected PCD patients, this provides rapid diagnose of most PCD patients and helps shorten the time of disease diagnosis. The application of second-tier testing lead to early identification of all six PCD patients, which enables them to receive timely treatment and prevent the occurrence of adverse symptoms in these patients. These six PCD patients would be excluded from conventional NBS based on the normal second screen results, which means that all patients would have escaped detection if the new screening algorithm not been implemented, indicating the important value of second-tier genetic testing for discovering latent PCD patients in the NBS program. However, it is noteworthy that the improvement of sensitivity comes at the expense of increased carrier identification. A previous study in Taiwan used second-tier molecular test to screen c.760C > T in 206 newborns with low C0 levels and found 10 carriers, these carriers were directly excluded due to the normal C0 levels on second screen . In contrast, our study found that 4% (4/99) of carriers had a second SLC22A5mutation/variant were true patients rather than carriers, especially two of them have normal C0 levels on second screen. The challenge is how best to use this assay to increase the detection and minimize the detection of unaffected carriers that require further genetic analysis. Further large-scale studies are needed to optimize the workflow of second-tier genetic screening. Notably, despite the designed panel with 17 hotspots represents the majority of SLC22A5 mutations in China, one patient with extremely low C0 levels was missed by our second-tier genetic testing. Nevertheless, all predefined panels are faced with this deficiency because the known mutations targeted for a specific population have been studied far less. Due to the extremely low C0 levels on second screen, NGS was performed and two novel SLC22A5 pathogenic variants were identified in this patient. Therefore, further genetic analysis is also required when newborns with persistently low C0 levels but no mutation was found in the second-tier genetic screening.
The incidence of PCD in selected population was estimated at 1:8191 by incorporating second-tier genetic screening into NBS programs. The incidence is higher than that reported in most region of China [9, 27, 28]. Of course there may be regional differences, but our findings suggest that the true incidence of PCD in China might be much underestimated. Consistent with most previous studies [22, 29], c.760C > T (p.R254*) was the most frequently occurring mutation in this cohort. The three mutationsc.760C > T, c.1400C > G and c.51C > G together had a relative frequency of 60%, confirming that these are hotspot mutations among Chinese populations [22, 23].
One limitation of this study is our selection of subjects based solely on the population with abnormal NBS results. It is well known that the C0 levels in newborns can be affected by the mother because C0 is transported to the fetus via the placenta [6, 12, 30]. If PCD patients with falsely normal C0 levels during NBS, the second-tier SLC22A5 mutation analysis would not have been done. It is therefore possible that some PCD cases missed during NBS may not come to our attention. Utilizing a higher C0 cut-off value could reduce the number of missed cases during NBS, but was expected to produce an unacceptable percent of false positives, balancing these two metrics remains challenging.
To summarize, the incidence of PCD is relatively high in Quanzhou, China. The high throughput iPLEX assay is a powerful tool for PCD genotyping. The incorporation of second-tier genetic screening into current NBS program could increase PCD detection.PCD NBS continues to be a challenge, newborns with persistently low C0 levels would require combined genetic analyses, and further studies are needed to optimize the workflow of new screening algorithm.