Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

• Main Authors: Wong, Colin C. X. F., Denil, Simon L. I. J., McLean, W. H. Irwin, Smith, Frances J. D., Common, John E. A., Tang, Mark B. Y., Foo, Jia Nee, Chen, Huijia, Tay, Angeline Su Ling, Haines, Rebecca L., Sandilands, Aileen, Lane, E. Birgitte, Liu, Jianjun
• Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
• Format: Article
• Language: English
• Published: 2019
• Subjects: Filaggrin Gene; Disease-associated Variants; Science::Medicine
• Online Access: https://hdl.handle.net/10356/84165; http://hdl.handle.net/10220/49132
• Institution: Nanyang Technological University

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation. FLG loss-of-function (LoF) variants are associated with ichthyosis vulgaris and the major genetic risk factor for developing atopic dermatitis (AD).1, 2, 3 Genetic stratification of patients with AD according to FLG LoF risk is a common practice for both research and clinical studies; however, few studies comprehensively sequence the entire FLG coding region. Most studies that include FLG genotyping have screened for common predominant LoF variants to report allele frequencies after full Sanger sequencing of a smaller batch of test patient samples or previously published data. This strategy potentially results in underreporting of the genetic contribution especially in ethnicities where FLG LoF variants are highly diverse.4 Distinct LoF variants have been reported for most ethnicities studied to date. For example, 2 predominant sequence variants (p.R501X and c.2282del4) make up approximately 80% of the mutation burden in northern Europeans,5 whereas in East Asian ethnicities, a larger FLG LoF mutation spectrum is found with fewer predominating variants.6, 7 However, routinely Sanger sequencing the entire FLG coding region for large cohorts is not always feasible, although desirable as it is essential to correctly stratify patients. To address this, we developed a robust and cost-effective high-throughput PCR-based method for analyzing the entire coding region of FLG using Fluidigm microfluidics technology and next-generation sequencing (NGS). We have applied this method to fully resequence cohorts of Chinese, Malay, and Indian patients with AD from the Singaporean population.