Amyotrophic lateral sclerosis, a lethal progressive neurologic disease, is characterized by selective degeneration of the lower and upper motor neurons. Approximately 5–10% of patients with ALS have a positive family history, suggesting that genetic factors substantially contribute to its pathogenesis. Frontotemporal dementia (FTD) is a spectrum of syndromes characterized by a progressive deterioration in behavior, personality, language, and cognition, associated pathologically with frontotemporal lobar degeneration (FTLD). ALS is closely related to FTD. Up to ~50% of patients with ALS show behavioral dysfunction and/or subtle cognitive impairment, while about 15% meet the psychiatry diagnostic criteria of FTD (termed as ALS–FTD) (1–3). A similar scenario is observed in FTD. Approximately 30% of patients with FTD have motor impairments, and 12.5% meet the diagnostic criteria for ALS (4, 5).

In the past few years, owing to the rapid development of next-generation sequencing, ALS–FTD-associated genes have been progressively identified. For example, mutations of C9orf72, TARDBP, and TBK1 have been identified as major genetic causes of ALS–FTD. The aggregation of TAR DNA-binding protein 43 (TDP-43) in the affected brain regions and motor neurons is a common pathological characteristic of each of these variants (6–10) in up to 97% of ALS and 50% of FTD cases. Beyond that, mutations in CCNF, CHCHD10, FUS, SQSTM1, UBQLN2, and VCP are also associated with ALS–FTD (11). However, the genetic etiology of ALS–FTD in some patients remains unclear. In the current study, mutation in the Annexin A11(AXAN11) gene was proved to be linked to ALS–FTD in a Chinese clinical cohort. We also included a review of previously reported mutations with ALS or ALS–FTD in the AXAN11 gene.

Located on the human chromosome 10q22.3, the ANXA11 gene encodes the 505 amino acid annexin A11 protein, which is a member of a calcium-dependent phospholipid-binding annexin protein family. The primary function of the annexin protein family is to bind Ca2+, RNA, other proteins, and lipid membranes. Unlike other family members, ANXA11 shows a uniquely long N-terminal domain that contains the calcyclin binding site (residues 50–62). Calcyclin can mediate ubiquitination and proteasome degradation of many target proteins (27). In total, four conserved annexin domains, including annexin1-4, constitute the conserved C terminus (28).

ANXA11-related ALS was initially identified in 2017 by whole-exome sequencing in 180 sporadic-ALS (SALS) cases and 751 European familial-ALS (FALS) (17). Smith et al. identified six ANXA11 mutations (G38R, D40G, G175R, G189E, R235Q, and R346C) in 9 patients from 6 families, and 3 SALS cases without FTD. In the aforementioned study, the D40G mutation was found to be the most common mutation. Patients carrying the D40G mutation presented a delayed-onset of classical ALS symptoms, with 5/6 cases having the bulbar-onset disease. Subsequently, a study in a non-Caucasian population supported the pathogenicity of D40G in the ANXA11 mutation associated with ALS. Of note, a sporadic ALS case was found once in a Chinese mainland cohort of 383 patients with ALS or ALS–FTD (15). There was also another reported study of 500 Korean patients with SALS (16). Liu et al. failed to discover D40G; instead, they found two rare heterozygous missense variants, namely, c.878C>T (p.A293V) and c.921C>G (p.I307M), in another Chinese cohort with 434 patients with SALS and 50 patients who had the index FALS (24). If the results of the two Chinese cohorts are combined, the D40G mutation rate is rarely low (0.12%, 1/867) in the Chinese patients with ALS or ALS–FTD. The aforementioned results suggest that p.D40G mutation is not the primary cause of ALS in the Chinese population (24).

According to the functional analysis, p.D40G being located near the calcyclin-binding region could cause abnormal binding of calcyclin. Analyses from a postmortem p.D40G ALS case showed profuse annexin A11-positive aggregates in neurons and neuropil of the neocortex and hippocampus, and motor neurons of the spinal cord (17).

In the current study, patients with the same D40G mutation have different clinical symptoms: (1) five of six European patients and one Korean patient who carried the mutation initially showed difficulty in swallowing and speaking (bulbar-onset ALS) (17); (2) a Chinese patient initially displayed left arm weakness at the age of 59 years (15); (3) in the present study, proband 1 with the ANXA11 p.D40G mutation initially presented abnormal behaviors, executive deficits, and anomia, and later progressed to classic upper motor nervous system damage in the bulbar and limbs. MRI showed significant bilateral temporal lobe atrophy and bilateral signal hyperintensities along the corticospinal tracts. The patient was diagnosed with ALS with bv-FTD. To our knowledge, this study is the first to associate the D40G mutation with ALS–FTD. Our results provided more genetic support for ALS and FTD.

Reviewing the literature, the spectrum of genotypes and phenotypes associated with ANXA11-related diseases has expanded as follows: (14–26) (i) late-onset or early-onset ALS (black mutations in Figure 2); (ii) ALS with FTD (P36R, G38R, D40Y, D40G, I278V, and G491R); (iii) inclusion body myopathy (hIBM), isolated or in combination with ALS/FTD (D40Y). In addition, the ordinary single nucleotide polymorphism (rs1049550, C>T, p.R230C, and MAF 0.44) in ANXA11 may enhance the risk of sarcoidosis (29). Furthermore, the rs1049550T in the ANXA11 allele plays a protective role for sarcoidosis in the Chinese Han nationality (30). Like other multisystem proteinopathies (MSP), ANXA11-related disorders possess a high clinical heterogeneity (Table 2), suggesting that diverse phenotypes driven by the ANXA11 mutations require long-term patient follow-ups. Of the six mutations, four mutations that were related to the ALS–FTD phenotype were clustered in ANXA11 within the long N terminus. The P36R, G38R, D40Y, and D40G mutations are near the calcyclin-binding domain in annexin 11, indicating the functional importance of this region. We know that calcyclin forms a regulatory complex with the calcyclin-binding protein (CACYBP) and RING-type E3 ubiquitin ligase SIAH-1, thereby regulating the ubiquitination and degradation of many proteins, including β-catenin (27). Therefore, calcyclin plays a critical role in proteostasis. However, the pathogenetic mechanism of ANXA11 mutations leading to ALS–FTD is unclear. Teyssou et al. performed the neuropathological analysis for the G38R case and revealed that FTLD–TDP type A allocations were elicited by the deposition of a mass of TDP-43 lesions in the cortex (31). In patients with ALS, TDP-43 lesion allocations are common because it is associated with a pure FTD phenotype or behavior, related to non-fluent aphasia, or linked to the GRN or C9orf72 mutation (32). Currently, in vivo and in vitro experiments are warranted to further this area of research.

In conclusion, this study confirmed the essential role of ANXA11 mutations in ALS and ALS–FTD. Our results enhanced the understanding of the clinical spectrum and the underlying mechanisms of ANXA11-related diseases, including typical ALS, hIBM, FTD, and their combinations.

The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found at: National Center for Biotechnology Information (NCBI) BioProject, https://www.ncbi.nlm.nih.gov/bioproject/, PRJNA832024.

The studies involving human participants were reviewed and approved by the Ethics Committee of China-Japan Friendship Hospital (2021-1-Y0). The patients/participants provided their written informed consent to participate in this study.

YW, XD, and DP designed the study. YW, XD, XZho, RW, and DP contributed patient material and clinical data. XW, ZC, XZho, ZZ, XZha, and YS carried out the experiments. YW, XD, DP, and RW analyzed and interpreted the data. YW and XD wrote the manuscript. All authors have made significant contributions and have approved the final version of this manuscript.

This study received funding from Deutsche Herzstiftung e.V. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. All authors declare no other competing interests. Constanze Pfitzer is participant in the BIH Charité Clinician Scientist Program funded by the Charité—Universitätsmedizin Berlin and the Berlin Institute of Health. This work was supported by the Competence Network for Congenital Heart Defects (Federal Ministry of Education and Research/grant number 01GI0601) and the National Register for Congenital Heart Defects (Federal Ministry of Education and Research/grant number 01KX2140). We acknowledge financial support by Land Schleswig- Holstein within the funding program Open Access Publikationsfonds.

Authors ZC and XW are employed by Running Gene Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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