Back-to-Back comparison of diagnostic efficacy between genome sequencing and exome sequencing reanalysis in suspected primary ciliary dyskinesia cohort

Abstract

Primary ciliary dyskinesia (PCD) is a heterogenous genetic disorder with more than 50 genes correlated. With the rapid improvement of next-generation sequencing, exome sequencing is frequently utilized in the clinical setting for molecular diagnosis. However, there is still an ongoing debate on whether to proceed with genome

sequencing (GS) or perform exome sequencing reanalysis (rES) if the initial results returned negative. By using a back-to-back double-blinded comparison, this study aims to evaluate the two methodologies. This study targets two main objectives of determining the genetic landscape of suspected primary ciliary dyskinesia patients in Hong Kong and a back-to-back comparison between rES and GS to inform clinicians and clinical geneticists on the optimal next action after a negative exome sequencing result.

In this study from 2015 to 2020, 61 participants (33 males and 28 females) with early-onset bronchiectasis, recurrent chest infections, and difficult to treat asthma from the Department of Paediatrics and Adolescent Medicine of HKU were recruited into this study and exome sequencing was performed. Negative or inconclusive exome sequencing results were considered for GS due to high genotype-phenotype correlation. GS was performed on 30 (13 males and 17 females) inconclusive exome sequencing results. Two teams of genome analysts and bioinformaticians were alternatively allocated to GS or rES and were blinded to the other team’s analysis. The time for bioinformatics, analysis, and diagnostic utility was recorded for evaluation.

Exome sequencing revealed 5 positive cases in the initial phase of the study. The positive case included mutations in RSPH4A, CCDC40, DNAH11, and CFTR. In the reanalysis phase of the study, five new diagnoses in genes DNAI1, DNAH11, and DNAH5 were made. Three inconclusive variants of uncertain significance results showed strong genotype-phenotype correlation with functional results in genes DNAAF3, DNAH9, and CCNO. Changes in pathogenicity classification in these cases were due to improved literature, correlation with functional investigations, and improvements in in-silico bioinformatic predictions for splicing variants. Only GS was able to detect the variant of uncertain significance DNAAF3 c.493G>C variant due to non-uniform coverage of the exons in rES. The patient’s functional investigations match previously reported DNAAF3 static ciliary movement. Beyond detection of deep intronic changes, CNVs, and SVs, GS had an additional advantage of uniform coverage over the exons.

Here we have investigated and determined the genetic landscape of suspected primary ciliary dyskinesia in Hong Kong. A back-to-back comparison in diagnostic efficacy between GS and rES could serve as a reference for which approach should be used following an exome negative result. Further investigations into other complementary approaches are required to fully evaluate what is the best choice of action.