Haplotype-Aware Detection of SERPINA1 Variants by Nanopore Sequencing

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Alpha-1 antitrypsin (AAT) deficiency (AATD) is linked to pulmonary and hepatic diseases, often due to SERPINA1 gene variants.
• While common alleles like PI*Z and PI*S are known, the impact of SERPINA1 haplotypes on AAT function and disease remains unclear.
• Current diagnostic methods may not fully capture the genetic diversity contributing to AATD.

Purpose of the Study:

• To develop and validate a nanopore sequencing workflow for comprehensive SERPINA1 haplotype analysis.
• To identify novel single-nucleotide variants (SNVs) and insertion/deletions within the SERPINA1 gene.
• To investigate the population-specific allele frequencies and haplotype diversity in a Spanish cohort.

Main Methods:

• PCR amplification of the SERPINA1 gene from 94 asthma patients using indexed primers.
• High-throughput sequencing on a MinION-Mk1C device with subsequent basecalling and demultiplexing.
• Bioinformatic analysis including filtering, mapping, variant calling, and haplotype phasing using advanced algorithms.

Main Results:

• Achieved 100% gene coverage with a minimum sequencing depth of 500× for all samples.
• Detected 75 SNVs and 4 indels, with 45 SNVs and 2 indels being highly polymorphic.
• Identified nine SNVs with differing allele frequencies compared to the Spanish population, and phased over 90% of heterozygous SNVs into 91 and 58 distinct haplotypes.

Conclusions:

• The developed nanopore sequencing workflow enables accurate, haplotype-aware genotyping of the SERPINA1 gene.
• This approach can reveal novel genetic variations and haplotype structures influencing AATD.
• The findings pave the way for improved diagnostic strategies for AATD, considering a broader spectrum of genetic factors.