A mutation in *CCDC91*, Homo sapiens coiled-coil domain containing 91 protein, cause autosomal-dominant acrokeratoelastoidosis

Acrokeratoelastoidosis (AKE; OMIM: 101850) is a rare, inherited skin disorder characterized by bilateral, hyperkeratotic papules primarily on the palms and soles. First described in 1953 by Oswaldo Costa, AKE typically manifests in childhood or early adulthood, with lesions progressively increasing in size and number. While both familial (autosomal dominant inheritance) and sporadic cases exist, the underlying etiology remains largely unknown. Previous studies suggested a potential linkage to chromosome 2, but the causative gene remained unidentified. This study aimed to identify the causative gene for AKE in a large Chinese family exhibiting classic symptoms and explore the underlying biological mechanisms involved in the pathogenesis of the disease.

Previous research on AKE has been limited, with the primary focus on clinical description and histological findings. While a potential linkage to chromosome 2 was suggested in 1983 by Greiner et al., this lead was not pursued further. The classification of AKE as palmoplantar keratoderma type III (PPKP3) in 1996 did not provide definitive insights into its genetic basis, as investigations into genes linked to other palmoplantar keratoderma types, such as *AAGAB* in PPKP1, proved fruitless. The lack of a known pathogenic gene for AKE highlighted the need for further genetic investigation. Studies have proposed potential roles of light exposure and trauma in sporadic cases but lacked the conclusive evidence provided by genetic identification.

A three-generation Chinese family (22 individuals) with AKE was recruited. Genome-wide linkage analysis using Illumina Human 660W-Quad_v1 BeadChips identified a susceptibility region on chromosome 12. Whole-exome sequencing (WES) on four family members identified a heterozygous splice mutation (c.1101+1G>A) in the *CCDC91* gene. Sanger sequencing validated this mutation. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed that this mutation caused exon 11 skipping, resulting in a 59-amino-acid deletion (L309-Q367del) in the CCDC91 protein. Functional studies were performed using shRNA knockdown in human skin fibroblasts (HSF) and CRISPR/Cas9 knockout of exon 11 in HEK293T cells. Immunofluorescence analysis assessed Golgi apparatus structure (using GM130 as a marker), tropoelastin, elastin, and fibrillin-1 expression and localization. Transmission electron microscopy (TEM) examined cellular ultrastructure. Enzyme-linked immunosorbent assay (ELISA) quantified insoluble elastin. Bioinformatics analysis predicted the three-dimensional structure of the wild-type and mutant CCDC91 proteins.

Linkage analysis revealed a susceptibility locus on chromosome 12 (between rs7296765 and rs10784618) with a maximum LOD score of 3.55. WES identified a heterozygous splice site mutation (c.1101+1G>A) in the *CCDC91* gene, confirmed by Sanger sequencing. This mutation resulted in the skipping of exon 11 and a 59-amino-acid deletion (L309-Q367del) in the CCDC91 protein. Functional studies in CCDC91 knockdown HSF and exon 11 knockout HEK293T cells revealed distended Golgi cisternae, cytoplasmic vesicle accumulation, and increased lysosomes. Immunofluorescence staining showed tropoelastin accumulation in the Golgi and abnormal extracellular aggregates. ELISA revealed a significant decrease in insoluble elastin production in CCDC91 knockdown fibroblasts. Bioinformatics analysis predicted structural changes in the mutant CCDC91 protein that could impair its function. No significant changes in fibrillin-1 microfibril assembly or lysyl oxidase activity were observed.

This study identifies *CCDC91* as a novel causative gene for AKE. The identified splice site mutation and subsequent protein truncation likely disrupt the function of CCDC91 in elastin transport. The observed Golgi distension, vesicle accumulation, and lysosome increase suggest defects in protein trafficking and secretion, consistent with the accumulation of tropoelastin within the Golgi apparatus. The reduced insoluble elastin production further supports this hypothesis. The findings are in line with previous observations of abnormal elastic fiber structure in AKE. While the precise mechanism by which CCDC91 facilitates elastin transport remains to be fully elucidated, this study provides a significant advance in our understanding of AKE pathogenesis. The findings suggest that CCDC91 may play a role in the intracellular trafficking and secretion of elastin. The study also highlights the importance of considering both familial and sporadic cases when investigating genetic causes of AKE.

This study definitively identifies a mutation in the *CCDC91* gene as the cause of autosomal-dominant acrokeratoelastoidosis in a Chinese family. Functional studies support a role for CCDC91 in elastin transport within the Golgi apparatus. Future research should focus on clarifying the precise mechanisms of CCDC91 action in elastin trafficking and investigate the potential role of CCDC91 in other conditions involving elastic fiber abnormalities.

The study is based on a single family, limiting the generalizability of the findings. Further studies in larger cohorts and other populations are needed to confirm the association between *CCDC91* mutations and AKE. The functional studies were performed in vitro, and further in vivo studies are needed to validate the findings. The mechanism by which CCDC91 affects elastin transport requires more detailed investigation. The study did not identify causative genes in sporadic cases of AKE, suggesting potential differences in pathogenesis between familial and sporadic forms of the disease.