Direct long-read visualization reveals hidden variation in GCH1 gene copy number and precise expansion steps

Area of Science:

• Genomics
• Parasitology
• Molecular Evolution

Background:

• Gene copy number amplifications are key adaptive strategies in organisms.
• Plasmodium falciparum utilizes amplifications for drug resistance and fitness.
• GTP cyclohydrolase I (GCH1) amplicon expansion was detected in parasites selected with a dihydroorotate dehydrogenase (DHODH) inhibitor.

Purpose of the Study:

• To investigate the expansion of the GCH1 locus in response to DHODH inhibitor selection.
• To characterize the structure and variation of GCH1 amplicons using long-read sequencing.
• To explore the relationship between GCH1 and DHODH amplifications in Plasmodium falciparum.

Main Methods:

• Long-read sequencing and single-read visualization.
• Direct quantification of GCH1 amplicon copy number.
• Analysis of amplicon structure and boundary sequences.
• Evaluation of historical DHODH inhibitor selection data.

Main Results:

• Selected parasite lines exhibited increased tandem GCH1 amplicons (up to 9) compared to parental lines (3).
• Long-read sequencing revealed hidden intra-parasite heterogeneity in GCH1 copy number (3, 5, or 7 amplicons).
• GCH1 amplicon expansions occurred in precise 2-unit steps, with conserved AT-rich boundary sequences.
• Parasite lines with expanded GCH1 also possessed DHODH amplicons on a separate chromosome.
• GCH1 amplification was not essential for DHODH inhibitor resistance but may facilitate DHODH locus amplification.

Conclusions:

• Long-read sequencing effectively identified previously undetected gene copy number heterogeneity.
• The tandem orientation and size of GCH1 amplicons enabled single long-read detection.
• A positive association between DHODH and GCH1 copy number was observed.
• Further research is warranted into the adaptive evolution of pyrimidine and folate biosynthesis loci in Plasmodium falciparum.