Title: Inappropriate interpretation of non‐pathogenic <scp><i>HTRA1</i></scp> variant as pathogenic
Abstract: Recently, novel high-temperature requirement A serine peptidase 1 (HTRA1) mutations have been identified as a cause of cerebral small vessel disease (CSVD) in increasing numbers of patients. Zhang et al. reported eight HTRA1 variants, including A20V (rs369149111), as causative mutations for CSVD in a sample of 181 Chinese patients.1 While they demonstrated a loss-of-function property for A20V using in vitro assay, we have several concerns about this study. First, A20V is a common variant, particularly among East Asians, with an allele frequency of 0.1480 according to the gnomAD browser (https://gnomad.broadinstitute.org/) and 0.2112 among Japanese individuals according to dbSNP (https://www.ncbi.nlm.nih.gov/snp/). To determine the pathogenicity of such common variants, it is necessary to investigate whether the clinical features or frequency of A20V carriers differ significantly between CSVD patients and the general population. Second, previous studies have shown that the location of the variant can strongly influence the protease activity of HTRA1.2 Variants located outside the linker region or protease domain are unlikely to affect protease activity. Therefore, we retrospectively investigated the frequency of A20V carriers in our database of CSVD patients. Clinical information and blood samples were collected from neurological centers in Japan. Patients with severe white matter hyperintensity corresponding to Fazekas grade 3/III and an age of onset of neurological symptoms/signs ≤70 years were included. After purifying genomic DNA, genetic testing for NOTCH3 and HTRA1 was performed, and whole exome sequencing was additionally performed if hereditary CSVD was strongly suspected. After excluding patients with causative mutations for CSVD, we identified 13 heterozygous A20V carriers among 62 CSVD patients (21.0%). No homozygous mutations were detected in this study. Next, we investigated the protease activity of A20V. Expression plasmids for wild-type (WT), A20V, and S328A complementary DNA were generated. Because A20V is located in the signal peptide,3 we also examined whether the A20V variant influenced the secretion of the HTRA1 protein. Culture supernatants containing overexpressed HTRA1 proteins after transfection into HEK293T cells were used to measure protease activity (Supplementary methods). The protease activity of A20V was not significantly different from that of WT HTRA1 (Fig. 1) and insufficient secretion of the A20V variant was not detected. Collectively, these results suggested that the A20V variant is not pathogenic for CSVD. Inappropriate interpretation of HTRA1 variants may unnecessarily cause anxiety in patients. A thorough assessment including epidemiological and biological factors is necessary for the identified HTRA1 variants. Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Writing – original draft; Writing – review & editing: Masahiro Uemura. Investigation: Sho Kitahara. Methodology; Investigation: Taisuke Kato. Genetic testing; Obtaining patient information; Writing – review & editing: Hiroaki Nozaki. Data curation; Writing – review & editing: Shoichiro Ando. Writing – review & editing: Tomohiko Ishihara. Conceptualization; Data curation; Formal analysis; Funding acquisition; Resources; Supervision; Writing – review & editing: Osamu Onodera. The study was funded by a grant-in-aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control; 26117006) from MEXT; a grant-in-aid for Practical Research Project for Rare/Intractable Diseases (19ek0109236h0003) from AMED; a grant-in-aid for Scientific Research (A) (19H01043); a grant-in-aid for Medical Research from the Takeda Science Foundation; and a grant-in-aid for Research on Intractable Disease (21FC0201) from the Japanese Ministry of Health, Labor and Welfare, Japan. Osamu Onodera is a speaker honorarium for Kyowa Hakko Kirin, Bristol-Myers Squibb, Ono Pharmaceutical, Mitsubishi Tanabe Pharm, Takeda, Daiichi-Sankyo, FUJIFILM, SANOFI, FP-pharm. The other authors have no conflicts of interests to declare. Data S1 Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.