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

X.F Colin C Wong,Simon L.I.J Denil,Jia Nee Foo,Huijia Chen,Angeline Su Ling Tay,Rebecca L Haines,Mark B.Y Tang,W.H Irwin Mclean,Aileen Sandilands,Frances J.D Smith,E Birgitte Lane,Jianjun Liu,John E.A Common

doi:10.1016/j.jaci.2017.10.001

Abstract

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation with links to skin diseases, however it has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using polymerase chain reaction (PCR) and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques such as oligoprobe-based exome or customized targeted-capture can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection across different sequencing methodologies. We applied this methodology to analyze cohorts from ethnicities previously not studied for FLG variants and demonstrate usefulness for discovery projects. This comprehensive coverage sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.

Highlights

We developed a robust and cost-effective high-throughput PCR-based method for analyzing the entire coding region of filaggrin gene (FLG) using Fluidigm microfluidics technology and next-generation sequencing (NGS)
We identified all FLG LoF variants originally identified by Sanger in the 14 samples as well as additional variants in 2 samples and documented LoF variants in the remaining 78 samples that passed quality control testing
In 279 Chinese Singaporean samples, we identified a further 11 additional LoF variants, raising the total number identified in this population to 33 with 5 not previously reported in the literature; 85 of these samples had been previously Sanger sequenced[7] and the concordance profile was near identical

Summary

Introduction

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. We designed and optimized overlapping FLG-specific primer assays (containing NGS adapters) to span the entire FLG coding region including known intragenic copy number variation (CNV) (see Fig 1, A; see Table E1 in this article’s Online Repository at www.jacionline.org).

Results

Conclusion