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Identification of CFTR variants in Clinically Diagnosed Cystic Fibrosis Patients from Pakistan using Next Generation Sequencing

Clinical Genetics and Therapeutics
  • Primary Categories:
    • Clinical- Pediatric
  • Secondary Categories:
    • Clinical- Pediatric
Introduction:
Cystic Fibrosis (CF) is a multisystem, life-limiting autosomal recessive disease. There is paucity of CF-related data from Pakistan due to limited resources and lack of diagnostic tools. We used Next Generation Sequencing (NGS) based CFTR sequencing for patients with high clinical suspicion of CF. We aimed to differentiate between ‘True CF’ and ‘Labeled Suspected CF’ patients and report variants unique to the Pakistani population, and determine candidates for future CFTR (Cystic Fibrosis Transmembrane Receptor) modulator therapies.

 

Methods:
92 patients were prospectively recruited after IRB approval based on clinical and laboratory findings, raising suspicion for CF. Inclusion criteria were high sweat chloride levels (>40 mmol/L) and low levels of fecal elastase (<200 μg/ml). NGS-based CFTR analysis was done using xGenTM Custom Amplicon Panel for CFTR by Integrated DNA Technologies (IDT). Variants were prioritized based on ACMG classification which was followed by genotype-phenotype correlation. Further analysis was carried out using various in-silico tools.

Results:
Thirty patients (33%) received a molecular diagnosis of CF, based on having biallelic P/LP variants, with reported consanguinity in 58 (63%) families. Of a total of 82 variants detected, 22 were classified as pathogenic/ likely pathogenic (P/LP), while seven variants were classified as variants of uncertain significance (VUS) and the remaining 53 were classified as benign/ likely benign (B/LB). 40 patients harbored 22 P/LP variants, of which 23 patients were homozygous (including one patient with two homozygous, and one patient with homozygous and compound heterozygous variants, respectively), seven were compound heterozygous, while 10 patients were heterozygous only for P/LP variants. Amongst these 40 patients, the most frequently identified variants included Phe508del identified in 15 (38%) patients (3 homozygous, six compound heterozygous, and six heterozygous), c.2T>C, p.Met1? identified in four (10%) patients. Two patients born to non-consanguineous parents were homozygous and four patients, born to consanguineous parents harbored compound heterozygous variants.

Conclusion:
The diagnostic yield of our study was 33%, and some patients had variants amenable to CFTR modulator therapy. The low diagnostic yield may indicate that in our low-resource setting, clinically labelled CF patients may have an alternate diagnosis such as Primary Ciliary Dyskinesia or Primary Immunodeficiency, or that they have deep intronic variants which remain unidentified. Our study highlights the need for improved screening and molecular diagnosis for patients who are clinically labeled and chronically managed as CF.

 

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