Using next-generation sequencing for the diagnosis of paediatric-onset genetic diseases

Abstract

Next Generation Sequencing (NGS) is a versatile technology that revolutionizes the practice of clinical genetics. The sequencing advancement has been adopted in the genetic diagnosis over the last decade. A proper genetic diagnosis can help the patients by providing better clinical management.

The primary objective of this project is to investigate the application of NGS in diagnosing genetic diseases using two NGS approaches. Multi-gene sequencing panel was used for early-onset bronchiectasis and RASopathies, while whole-exome sequencing (WES) was utilized for prenatally detected structural congenital anomalies (SCAs), and rapid WES for patients with suspected genetic disorders. Functional characterization was integrated into the diagnostic pipeline of NGS gene panel, and the feasibility of performing WES in diverse patients with genetic disorders was discussed.

Gene-panel sequencing was used in the diagnosis of childhood bronchiectasis and identification of causative mutations in patients with RASopathies. Molecular diagnosis was made in six out of 21 (28.6%) children with bronchiectasis, and a Chinese-specific CFTR mutation was identified. Functional characterization of the CFTR protein with the founder mutation was performed. Results suggested that the protein exhibit reduced glycosylation level with normal gating function. This showed that the mutant protein has an intracellular trafficking defect and causes cystic fibrosis.

On the other hand, an NGS gene panel was used to identify the causative mutations in patients with RASopathies with a primary diagnostic yield of 31.7%. In vitro and in vivo functional assays have been implemented for the interpretation of variants of unclear clinical significance (VUSs). With the integration of functional analysis, the diagnostic rate of the pipeline was improved to 36.5%.

To test the feasibility of using WES in prenatal diagnosis, a total of 33 fetuses with SCAs were tested using a stringent set of interpretation criteria. Overall, diseasecausing mutations were identified in three out of 33 (9.1%), and VUSs were found in six out of 33 (18.2%) fetuses. These findings suggested that it is feasible to adopt WES in a local prenatal setting. And a systematic review of published clinical WES studies showed that the diagnostic yield in this study was comparable with the previous prenatal studies.

To evaluate the clinical utility of rapid WES in patients with an urgent need of genetic diagnosis, 35 patients with suspected genetic conditions were recruited from intensive care units or out-patient clinic. The sequencing pipeline was assessed in three areas: diagnostic yield, turnaround time and clinical implication. The results suggested that disease-causing mutations were found in nine out of 35 (25.7%) patients, with an average turnaround time of eight days. It also showed that all the nine patients who received a genetic diagnosis could also benefit their clinical management. In summary, the application of both gene-panel sequencing and WES are feasible in the molecular diagnosis of genetic conditions. Furthermore, both sequencing strategies can facilitate clinical diagnosis, and provide necessary medical management for patients. Integration of functional analysis into the sequencing pipeline can also improve the diagnostic performance of an NGS pipeline and clinical values of genetic diagnosis.