SPAG1

Inheritance Pattern: AR

Disease ID:MONDO:0014216

Submitter: Labcorp Genetics (formerly Invitae)

Inheritance Pattern: AD

Disease ID:MONDO:0016575

Submitter: Orphanet

Inheritance Pattern: AR

Disease ID:MONDO:0014216

Submitter: G2P

Inheritance Pattern: AR

Disease ID:MONDO:0014216

Submitter: PanelApp Australia

Inheritance Pattern: AR

Disease ID:MONDO:0014216

Submitter: ClinGen

Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous group of disorders, usually beginning in early childhood, characterized by chronic cough, recurrent infections of the upper and lower respiratory tract, randomization of left/right body asymmetry, and subfertility (PMID: 32943623). The PCD phenotype results from structural and/or functional abnormalities of motile cilia and flagella. Ciliary motility is powered by a set of axoneme-specific dynein motor complexes. These large multiprotein complexes are assembled within the cytoplasm by dynein axonemal assembly factors (DNAAFs) before their transport into cilia. Mutations in genes encoding DNAAFs result in the improper cytoplasmic preassembly of dynein chains, causing defects in both inner (IDA) and outer dynein arms (ODA) that can result in cilia immotility (PMID: 31624012). SPAG1 (Sperm Associated Antigen 1) was initially identified and characterized as a human testis-specific protein related to female infertility and spermatogenesis (PMIDs: 1299558, 11517287) and was later also identified as a marker of pancreatic cancer progression and proposed to promote motility of cancer cells (PMID: 16983343). The *SPAG1* (*DNAAF13*) gene was first reported in relation to PCD by Knowles et al. in 2013 when whole exome sequencing identified *SPAG1* biallelic variants in individuals with PCD and defective ODAs and IDAs (PMID: 24055112). This study also showed that *SPAG1* was expressed in human airway epithelial cells and it was proposed that SPAG1 plays a role in the cytoplasmic preassembly of IDAs and ODAs. The specific disease entity, Primary Ciliary Dyskinesia 28 is an autosomal recessive disorder caused by homozygous or compound heterozygous mutation in the *SPAG1* gene. Per criteria outlined by the ClinGen Lumping and Splitting Working Group, we found the molecular mechanism and autosomal recessive mode of inheritance to be consistent among unrelated patients, while the phenotypic variability among them appeared to represent a spectrum of disease rather than separate disease entities. Therefore, cases caused by inherited *SPAG1* variants have been lumped into a single disease entity, Primary Ciliary Dyskinesia 28 (MONDO: 0014216, OMIM # 615505). Evidence from 16 probands in 5 publications have been included in this curation representing 13 unique variants (1 missense, 3 nonsense, 7 frameshift, 1 splice site, and 1 large deletion) (PMIDs: 24055112, 32502479, 33577779, 26139845, 35178554). Of note, three of these variants have been identified as founder mutations by haplotype analysis: NM_003114.5:c.902_906del (p.Lys301fs), Clin Var ID:209000; NM_003114.5:c.2014C>T (p.Gln672Ter), ClinVar ID: 88683; and NC_000008.11:g.100151617_100163589del, Clin Var ID:88681 (PMID: 24055112). These founder mutations are seen in multiple studies (PMIDs: 24055112, 35178554, 26228299) and are seen repeatedly in this curation. Five of the probands in this curation carry NC_000008.11:g.100151617_100163589del; two carry NM_003114.5:c.902_906del; and 6 carry NM_003114.5:c.2014C>T. The mechanism of pathogenicity in *SPAG1* mutations appears to be biallelic loss of function. There is additional case-level evidence available in the literature, but its inclusion in this curation was not necessary to reach the maximum score for genetic evidence (12 points). The gene-disease relationship between *SPAG1* and PCD 28 is supported by protein expression data. Immunoblot analysis shows expression of full length SPAG1 in healthy differentiated respiratory epithelial whole cell lysates but not in ciliary axonemes or undifferentiated respiratory epithelial cell lysates (PMID: 24055112). This pattern is consistent with a role of full length SPAG1 in the cytoplasmic preassembly of dynein arm complexes in the respiratory epithelial cell. More recent studies show that full length SPAG1 is absent in respiratory epithelial cells of three PCD patients with biallelic *SPAG1* mutations, but present in control samples (PMID: 35178554). The absence of full length SPAG1 is consistent with the absence of ODAs and IDAs and ciliary immotility in most PCD patients with *SPAG1* mutations, strengthening the *SPAG1*-PCD 28 gene-disease association. Of note, at least three major isoforms of SPAG1 have been described in human respiratory epithelial cells: a full length 926 amino acid (~106-kDa) protein, a 416 amino acid (~94-kDa) protein product of alternative splicing (containing first 10 exons), and a 545 amino acid (~60-kDa) protein product of an alternative in-frame start codon (starts at pMet382 and contains exons 11-19) (PMIDs: 16983343, 24055112, 35178554). The role these alternative isoforms might play in the preassembly of dynein arms is still not well understood. Transmission electron microscopy of cilia cross sections from respiratory epithelial cells of two of the three patients presented by Smith et al 2022 (PMID: 35178554) have an atypical *SPAG1* PCD ultrastructural phenotype with no IDAs but some normal ODAs and HSVM shows residual ciliary motility. Immunolocalization studies to further characterize cilia structure in the cells of these patients showed an absence of DNAH5 and DNAI1 (ODA proteins) and an absence of the DNALI1 (IDA protein) in the patient with typical PCD ultrastructure, while the two atypical phenotype patients had no DNALI1 staining of the IDAs but still had DNAI1 and DNAH5 present along the full length of the ciliary axoneme (albeit at a reduced level compared to control cilia) confirming the absence of IDAs and the presence of ODAs. These two patients retain expression of the 60-kDa isoform while the other patient (lacking ODAs and IDAs, no ciliary motility) does not. These authors propose that the SPAG1 60-kDA isomer may be able to compensate for a lack of full length SPAG1 by assembling some normal ODAs. While the 106-kDa and 94-kDa isoforms are expressed specifically during ciliogenesis, the 60-kDa isoform is constitutively expressed, suggesting that the 60-kDA isoform may play a motile cilia-independent function in normal epithelium. The cytoplasmic folding and preassembly of dynein arm proteins into larger complexes takes place in a stepwise manner involving interactions with various DNAAFs, chaperones, and co-chaperones (PMID: 31817850). SPAG1 has been hypothesized to be one of many DNAAFs that interact to form dynein arm complexes in ciliated cells (PMID: 30561330). Immunoprecipitation of SPAG1 using SPAG1 antibody shows that SPAG1 interacts with dynein chains DNAH6 and DNAH5. SPAG1 also co-precipitates with co-chaperone complex proteins RPAP3, RUVBL1, and RUVBL2 as well as other DNAAFs: DNAAF1, DNAAF2, DNAAF3, DNAAF10 (WDR92), and DNAAF15 (PIH1D2). DNAAF1, DNAAF2, and DNAAF3 are DNAAFs that have been previously associated with PCD (PMID: 32943623). DNAAF10, RUVBL1 and RUVBL2 are components of the R2TP/Prefoldin-like complex, an HSP90 co-chaperone complex involved in the assembly process of many molecular machines. These protein interactions identified by immunoprecipitation are consistent with SPAG1's involvement in axonemal dynein arm assembly. The interaction of SPAG1 with a large co-chaperone complex has also been confirmed by Chagot et al 2019 (PMID: 31118266) who used NMR and molecular dynamics simulations to characterize the molecular level structure and dynamics of SPAG1 in complex (via its TPR domains) with the tail of HSP70 and HSP90. SPAG1 was shown to be part of the R2SP co-chaperone complex, which combines SPAG1, PIH1D2, RUVBL1 and RUVBL2 proteins to work with protein chaperones HSP70 and HSP90 in the quaternary preassembly of dynein arm protein complexes. Two animal models have been used to support the gene-disease relationship between *SPAG1* and PCD 28. Morpholino knockdown of *spag1* in zebrafish embryos produced zebrafish exhibiting dorsal body axis curvature and hydrocephalus (PMID: 24055112). Zebrafish have motile cilia in the left-right organizer (Kupffer's vesicle), the spinal canal, pronephric ducts, brain ventricles, and olfactory placode. The defects seen in zebrafish when *spag1* is knocked down using morpholinos are consistent with the phenotype one might see in zebrafish with defective motile cilia (hydrocephalus, curved axis), however there were non-significant laterality defects and cilia function was not tested. RNAi knockdown of *Drosophila CG18472/Spag1* produces flies with loss of both ODAs and IDAs. Male infertility, immotile sperm, the loss of auditory/vibration mechanotransduction in Ch neurons (impaired larval hearing), and defective adult proprioception in SPAG1 depleted *Drosophila* mutants are phenotypes consistent with ciliary/flagellar immotility and in line with human *SPAG1* mutation ciliary phenotypes. In summary, there is definitive evidence supporting a gene-disease relationship between variants in *SPAG1* and Primary Ciliary Dyskinesia 28. This has been repeatedly demonstrated in both research and clinical diagnostic settings, and has been upheld over time. This classification was approved by the ClinGen Motile Ciliopathy GCEP on June, 15, 2022 (SOP Version 8).