Background: Aspergillus fumigatus is the most common mould causing pulmonary disease. The
global burden of aspergillosis is higher than 10 million excluding sensitization. Invasive aspergillosis
which develops in immunocompromised patients is the most severe form of the disease resulting in
mortality >90% if untreated. However, there are other clinical forms as chronic pulmonary aspergillosis
(CPA) and allergic bronchopulmonary aspergillosis (ABPA), which account for more than 80% of
aspergillosis cases but affect patients without obvious immune compromise or those with atopy. Our
inability to interrogate the multifactorial nature of disease within the context of gene function has
motivated this study.
Material/methods: We have explored the utility of whole-exome sequencing (WES) to discover
genetic variants associated with susceptibility to fungal disease in a cohort of 113 ABPA, 156 CPA and
119 atopic asthmatic patients. Four genetic variants in three regulatory elements, two transcription
factors and a long non coding RNA (LncRNA), strongly associated with ABPA and/or CPA (AF>90% in
disease) were selected for fungal disease modelling. Bronchial epithelial cells (16HBE) were genomeedited
by using CRISPR-Cas9 system and used in Aspergillus challenge experiments. Moreover,
alterations in the epithelial monolayer in the re-created cell lines were determined.
Results: WES of aspergillosis patients has identified a strong association of mutations in regulatory
elements, such as transcription factors and LncRNA, with ABPA and CPA. We have successfully
optimized a CRISPR-Cas9 genome-editing system to re-introduce the precise genetic variants
discovered in these regulatory elements into “healthy” human bronchial epithelial cells. Epithelial cells
carrying the mutations did not form confluent layers and were hypersusceptible to damage caused by
A.fumigatus exposure, which could be detected 4h after co-culture. Therefore, the cell lines carrying
the mutations in all of these regulatory elements showed alterations in the structure of the monolayer,
a reduction in the transepithelial resistance and changes in the expression of tight junction genes.
Conclusions: CRISPR-Cas9 is a suitable system for modelling genetic susceptibility to aspergillosis
in bronchial epithelial cells. Successful precise reintroduction of disease associated variants into
healthy cell lines resulted in phenotypes consistent with hypersusceptibility to A. fumigatus damage
This system opens the door to potential research in fungal diseases. Studies to confirm the importance
of these variants in the host immune response to Aspergillus exposure are needed.
Full conference title:
- ECCMID 26th (2016)