A disorder clinically resembling cystic fibrosis caused by biallelic variants in the AGR2 gene

Abstract

Purpose: We sought to describe a disorder clinically mimicking cystic fibrosis (CF) and to elucidate its genetic cause.

Methods: Exome/genome sequencing and human phenotype ontology data of nearly 40 000 patients from our Bio/Databank were analysed. RNA sequencing of samples from the nasal mucosa from patients, carriers and controls followed by transcriptome analysis was performed.

Results: We identified 13 patients from 9 families with a CF-like phenotype consisting of recurrent lower respiratory infections (13/13), failure to thrive (13/13) and chronic diarrhoea (8/13), with high morbidity and mortality. All patients had biallelic variants in AGR2, (1) two splice-site variants, (2) gene deletion and (3) three missense variants. We confirmed aberrant AGR2 transcripts caused by an intronic variant and complete absence of AGR2 transcripts caused by the large gene deletion, resulting in loss of function (LoF). Furthermore, transcriptome analysis identified significant downregulation of components of the mucociliary machinery (intraciliary transport, cilium organisation), as well as upregulation of immune processes.

Conclusion: We describe a previously unrecognised autosomal recessive disorder caused by AGR2 variants. AGR2-related disease should be considered as a differential diagnosis in patients presenting a CF-like phenotype. This has implications for the molecular diagnosis and management of these patients. AGR2 LoF is likely the disease mechanism, with consequent impairment of the mucociliary defence machinery. Future studies should aim to establish a better understanding of the disease pathophysiology and to identify potential drug targets.

Keywords: RNA-Seq; genetic research; genetics.

Figure 1

Summarised family trees of the nine families and the identified AGR2 variants. Variants are colour-coded, the founder missense variant is shown in red font, with the corresponding haplotypes (families 2, 3, 4 and 7). Genotypes are shown below available individuals. AGR2 genotypes show full co-segregation with the phenotype.

Figure 2

AGR2 variants identified in the patients and abnormal splicing caused by an intronic variant. (A) The deletion region of AGR2/3 is shown in the Integrative Genome Viewer (IGV). Reads for the exonic regions of the AGR2/3 genes can be seen in the control (lower panel), whereas no reads are seen in the index sample III-1 (deleted region is boxed, chr7:16834456–16918247). This deletion was confirmed by qPCR. (B) Schematic representation of AGR2 gene, with the detected variants shown (font colours match the respective family). (C) Sashimi plots from IGV, illustrating AGR2 splicing junctions. Arcs represents splice junctions and connect the exons, the number of reads split is displayed across the junction. The variant c.330+1del causes aberrant splicing, note the junctions skipping exon 5 (arrow). See also online supplemental figure 2A–C.

Figure 3

Structural protein analysis of the missense variants. (A) Dimer of the AGR2 residues 36–175. Monomers are individually coloured in grey or blue. Side chains of the residues are not shown, except for the mutated P71, H117 and G143 in red. (B) Overview of AGR2 as seen from the side; one monomer is shown as grey surface only. This view shows the distance between the mutated residues (red side chains) and the putative active site of the protein CPHS-motif (orange). (C) Variant P71T: The proline side chain is shown in magenta; note the attachment of the side chain to its own backbone; the threonine side chain is shown in yellow. Side chains of the protein are coloured by atom type (carbon=cyan, oxygen=red, nitrogen=blue, sulfur=green). The proline side chain is slightly larger than threonine, but the main differences between these two residues lie in the shape of the side chain and proline tendency to make rigid turns that stabilise the protein structure. (D) Variant H117Y: The histidine side chain is shown in magenta, whereas the tyrosine side chain is yellow. Other atoms are coloured as described. The change from histidine to tyrosine indicates a small difference in size, and a different potential for interactions since histidine’s side chain can be used for electron storage. (E) Variant G143E: The side chain of the mutant residue glutamic acid will not fit in the same space (note that wild-type glycine does not have a side chain). The change in charge and side chain size will affect the local structure and may affect interactions with other proteins.