The CC2D1A gene located on chromosome 19 (19p13.12) has 29 exons and a length of about 24,679 bp. The protein encoded by this gene contains two isoforms [long (951aa) and short (388aa)] (Basel-Vanagaite et al., 2006). The long isoform consists of two domains: the C2 domain located at the C-terminal and DM14 domains located at the N-terminals. The short isoform only has a C2 domain. It seems that the long isoform has the main function because the loss-of-function mutations of CC2D1A occur in this isoform (Basel-Vanagaite et al., 2006; Chiara Manzini et al., 2014).
The C2 motif located in the C-terminal region of the gene at positions 656-770, is a protein structural domain involved in targeting proteins in cell membranes (Bateman et al., 2021) and calcium-dependent phospholipid binding (Davletov & Sudhof, 1993). Zhao M et al. in 2010 demonstrated that CC2D1A activates NF-ƙB through conserved C2 and DM14 domains. They observed that the lack of N-terminal 138 amino acids and the absence of first and second DM14 motifs caused NF-ƙB activity reduction by 50 %. Further deletion, including third and fourth DM14 and C2 domains, reduces the activity to less than 20 %. Therefore, the C2 domain and DM14 domains, primarily the C2 domain, are essential for NF-ƙB activation (Zhao et al., 2010).
The DM14 motifs located at N-terminal at position [1:(138-195), 2:(257-315), 3:(349-407), 4:(494-552)] (https://smart.embl.de/smart/show_motifs.pl?ID=Q6P1N0), and their role are unknown (figure 2c). The number of DM14 repeats is four times in the human CC2D1A gene and three times in the Caenorhabditis elegans orthologue sequence. The DM14 motifs are present only in the long isoform of CC2D1A (Basel-Vanagaite et al., 2006). The research by Tawashi et al. has shown that the CC2D1A gene is a novel regulator of phosphodiesterase 4D (PDE4D). The CC2D1A bound to PDE4D could fine-tune cyclic adenosine 3', 5'-monophosphate (cAMP), a key regulator of various cellular signaling, especially in neuronal systems. Although the DM14 motifs, particularly the first motif, are essential for CC2D1A-PDE4D5 binding, they cannot prevent increased PDE4D5 activity (PKA-dependent phosphorylation) alone. The C2 domain too has no role in this connection. The absence of second and third DM14 domains causes PDE4D hyperphosphorylation at the serine 126 positions through the cAMP-dependent protein kinase A (PKA). This hyperphosphorylation causes continuous PDE4D activity and eventually disrupts the homeostasis of the cAMP and the downstream signaling pathway, especially phosphorylation at S133 in CREB. This finding highlights the importance of the role of the second and third DM14 motifs. When the first three motifs from four DM14 motifs are absent in the CC2D1A gene, it causes intellectual disability without physical defects. Still, when only the first motif is present, it leads to death in mice at eight to twelve hours after birth, suggesting that the regulatory role of the CC2D1A gene is critical for neuronal function (Al-Tawashi & Gehring, 2013).
So far, three pathogenic mutations have been reported in this gene, which have been recorded in ClinVar: one splicing mutation and two deletion variants that lead to a frameshift mutation (Landrum et al., 2020). One deletion mutation was also reported by McSherry et al. in 2018 that has not been recorded in ClinVar (McSherry et al., 2018). In 2006, Basel-Vanagaite et al. first identified the CC2D1A gene as the cause of non-syndromic intellectual disability (NSID) in nine consanguineous families with severe ID. Using homozygosity mapping and sequencing, they were able to detect deletion in this gene (IVS13-16DEL). The resulting protein lacks one of the four DM14 and C2 domains. By identifying two isoforms of this protein (951aa & 388aa) in normal individuals and the presence of the C2 domain in both, the researchers concluded that the absence of the C2 domain in the truncated protein in patients does not lead to ID, and the role of DM14 has probably been more fundamental in this regard.
Patients in this study had a psychomotor developmental delay during early childhood and could not speak except for a few words. All of them had severe ID and normal physical conditions (Basel-Vanagaite et al., 2006). The next researchers who studied the association of the CC2D1A gene with ID were Manzini et al. in 2014. They detected "c.748+1G>T mutation" in the CC2D1A gene in three families and "c.346delA mutation" in one family with ID, ASD, and seizure. This change (c.748+1G>T) leads to complete removal of exon 6, disrupts the reading frame at position 172 in the protein, and creates an early stop codon at position 223 (p. Thr172Valfs*51). Truncated protein contains N-terminal fragments and lacks DM14 and C2 domains. It can be concluded that the loss of function of CC2D1A is the result of this mutation. The next mutation (c.346delA) is a 1bp deletion in exon 3, resulting in an early frameshift (p.Lys116Argfr*81). This mutation creates N-terminal fragments without any known domains and results in CC2D1A loss of function (Chiara Manzini et al., 2014). Patients (16 individuals affected in four families) in that study had a spectrum of cognitive disorders and social impairments including ASD, ID, aggressive behavior, and seizure, suggesting shared developmental mechanisms (Chiara Manzini et al., 2014). McSherry et al. in 2018 also used whole-exome sequencing on 21 Turkish families with non-syndromic ID, and found causative variants in known genes. The CC2D1A (c.811delG, p.A271Pfs*30) was one of those genes. This variant causes frameshift mutation by deletion of one nucleotide in exon seven and creates an early stop codon in exon eight at position 301. The resulting truncated protein lacks the third and fourth DM14 and C2 domains and is predicted to cause a loss of function of the CC2D1A gene. The family with this causative variant has three females affected with NSID. All of them suffer from ID, ASD, and seizures, and have normal physical conditions without facial dysmorphism (McSherry et al., 2018). In the current study, the variant found in patients is (c.1641+1G>A), a novel splicing variant in exon 14 of the CC2D1A gene. It is assumed that this variant disrupts normal splicing, could eliminate the fourth DM14 and C2 domains, and probably results in the loss of function of the CC2D1A gene. Performing a functional study would shed more light on the functional consequences of this genome and its variants. The patients in this study had ID, ASD, speech delay, and seizures. They were normal in the physical examination and did not show any dysmorphic features. The clinical characteristics of all the patients are summarized in table 1.
Sener EF et al. in 2020 evaluated the association of the CC2D1A gene and ASD. Based on the involvement of the CC2D1A gene in NSID and the presence of ID in one-third of autistic patients, these researchers considered it a new candidate gene in autism. They performed a genetic analysis on the CC2D1A gene in 44 patients with ASD and 27 normal individuals. All the variants identified in the patients were missense and were reported as variants of uncertain significance (VUS). Only one variant was splicing and was reported as pathogenic (c. 2520-1G>T). The patient carrying this pathogenic variant had autism without ID or birth defect. These missense variants are located in exons 12, 13, 15, and 19 (figure 2d). (Sener et al., 2020). As shown in figure 2, none of them affected DM14 domains.
Although the mutations found in CC2D1A were connected with NSID, for the first time, Ma ACH in 2020 established a novel association between CC2D1A and ciliary dysfunction. Using whole-exome sequencing on 26 individuals with heterotaxy, they were able to identify novel rare damaging mutations in the CC2D1A gene. A functional study was performed, and heterotaxy
phenotypes of the gastrointestinal and cardiovascular systems were observed in zebrafish knockout models. All of these mutations were missense and were considered benign, likely benign, and VUS. These variants and their locations are demonstrated in figure 2e. Investigation of the positions revealed that only two variants were located in the fourth DM14 domain (exon 14), and the rest were found in exons 6 and 23, which do not interfere with other DM14 domains. On the other hand, exon 14 variants that were likely benign and VUS do not probably have pathogenic effects on the fourth DM14 domain (Ma et al., 2020). Tuncel G. also reported a patient with Joubert syndrome that is classified as a ciliopathy disease caused by two homozygous variants, c.1739C>T (rs202057391) in AHI1 and c.1739C>T (p.Thr580Ile) in CC2D1A. This variant was also located in exon 15, which is not located in DM14 domains (Tuncel et al., 2021).
Based on previous studies, the variants of CC2D1A have a different phenotypic spectrum. Splicing and frameshift mutations cause the loss of function of the CC2D1A gene and disrupt DM14 domains leading to ID, ASD, and seizure. These mutations are pathogenic according to the ACMG guidelines. On the other hand, variants found in ASD patients were missense with uncertain significance (VUS) according to the ACMG guideline, and the variants detected in ciliopathy were all missense and were reported as benign, likely benign, and VUS. Complete loss of function of the CC2D1A gene appears to create a severe phenotype such as ID plus ASD and, in some cases, seizures and developmental delay. Whereas in missense mutations, the loss of function does not occur, and the resulting phenotype is autism, which in some cases, is accompanied by ID or causes ciliopathy. In these cases, it can be speculated that the effects of this gene, along with the cumulative effects of the other genes, may contribute to the final phenotype characterization.
Comparison of the clinical features of the patients with loss-of-function mutations in CC2D1A revealed no phenotype-genotype correlation. Almost all the patients had ID, and a high percentage of them had ASD and seizures. Language impairment is another common symptom in these patients. Some of the patients showed developmental delay. Dysmorphic features were not seen in patients, and all of them were considered normal after physical examination.