MR is a complex disorder, and according to WHO, it consists of three following main criteria: below-average intelligence quotient (IQ < 70), noticeable adaptive functioning (such as communication and social and interpersonal skills), and the manifestation before the age of 18 (WHO 1980). In this study, proband 1 and 2 present fundamental characteristics of moderate and severe MR, respectively. Exome sequencing detected hemizygote DLG3 mutation and based on the clinical features of both affected males, and the MR diagnosis matches the previously reported cases (Isrie et.al 2012; Kumar et.al 2016; Philips et.al 2014; Tzschach et.al 2015; Zanni et.al 2010; Gieldon et.al 2017). DLG3 gene, also known as Synapse-associated protein 102 (SAP102), belongs to the SAP subfamily of neuronal membrane-associated guanylate kinase (MAGUK) proteins, including SAP90/postsynaptic density (PSD)95, SAP97/hDLG, and Chapsyn110/PSD93 (Fujita & Kurachi 2000; Laumonnier et.al 2007). SAP proteins are extensively expressed in the brain: SAP102 is found in dendrites and axons and is abundant in the postsynaptic density as well as in the cytoplasm. Furthermore, SAP102 is highly expressed in early postnatal brain development, unlike PSD95 and PSD97 predominant at later stages (Sans et.al 2000). DLG3 is a known XLMR gene that encodes SAP102, a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. MAGUKs are a group of ionotropic scaffolding proteins located at the postsynaptic density (PSD), including PSD-95, PSD-93, PSD-97 and SAP102, and their function is in the formation and plasticity of excitatory synaptic terminals of neurons in the brain. DLG3 mutation is proved to be one of the causative factors in MR. A study on mouse showed a significant role of DLG3 in neural induction and development (Van Campenhout et.al 2011). In human, DLG mutation showed to have resulted in synaptic dysfunction and intellectual disability (Zanni et.al 2010; Cuthbert et.al 2007; Gardoni 2008). Cellular study of DLG3 in human neural cells supports the direct relation between DLG3 mutation and neural induction failure during early embryo development and may give rise to abnormal cortical development and eventually lead to defective forebrain in embryos (Lickert & Van Campenhout 2012). This is due to the role of dlg3 protein in the central nervous system (CNS). Dlg3 interacts with N-methyl-D-aspartate (NMDA) receptors and maintain their functions during synaptogenesis (Sans et.al 2003). In this study, both cases manifested delayed psychomotor development and behavioral problems associated with the cause of mutation in the DLG3 gene. Further molecular analysis of the impaired gene showed the mutation in the guanylate kinase (GK) domain of the protein. The SAP102 has 3 PDZ domains (PDZ1, PDZ2, and PDZ3), Src homology 3 (SH3) domain, and GK domain (Cuthbert et.al 2007). The protein is abundant in nonproliferating cells that express their function in cell growth maintenance by interacting with the carboxy terminus of Adenomatous Polyposis Coli (APC) tumor suppressor protein (Makino et.al 1997; Masuko et.al 1999). SAP10 protein is abundant in dendritic cells and synaptic junctions and links NMDA receptors to susbmembraneous cytomatrix, and the linkage regulates plasticity, behaviour and signal transduction (Cuthbert et.al 2007; Lau et.al 1996). Some studies demonstrated the possibility of SAP10 protein in autism as they bind directly to neuroligin, a gene recognized as susceptible in autism (Cuthbert et.al 2007; Yan et.al 2005, 48). However, the relationship between autism and MR has not been elucidated. To date, over 90 genes underlying XLMR have been identified, each of them contributing to the disease group with a small number of individual mutations (Ge´cz et.al 2009). The prevalence of autism is four times higher in males compared to females, and about 80% of cases express intellectual disability (Smalley 1997). This is intriguing as a higher prevalence in males and intellectual disability are also characteristic for XLMR (Ropers & Hamel 2005). In our study, the second case was diagnosed with atypical autism previously. The relationship between ASD and XLMR is still unclear. However, we identified DLG3 deletion, which appears to be the cause of mental impairment in this individual who was previously diagnosed with atypical autism.
Protein structure analysis of the variants by Swissmodel (swissmodel.expasy.org) revealed remarkable alteration in the protein structure and bonding pockets of the GK domain. Previous studies suggested the importance of intramolecular interaction between the SH3 domain and GK domain for cytoplasmic localization of human dlg (hDLG) (Nix et.al 2000; McGee et.al 2001; Tavares et.al 2001; Kohu et.al 2002), and mutation in either of the domains can lead to nuclear translocation (Kohu et.al 2002).
In proband 1 (Fig. 4B), Arginine was substituted with glutamine (non-essential amino acid). Arginine is considered as one of the essential amino acids and frequently found in protein structures due to its amine-containing side chain and was shown to have enhanced protein folding and expression (Tsumoto et.al 2004). Substitution of arginine by glutamine in the TNNI3 gene was shown to have been associated with hypertrophic obstructive cardiomyopathy (Rani et.al 2012). It is believed that the substitution of arginine by glutamine might have affected the intra- and intermolecular interaction of the GK domain that could affect the efficacy of the protein in binding with NMDA receptors and eventually cause synaptic disorder leading to behavioral dysfunction and abnormality in movements.
In proband 2 (Fig. 5B), glycine was substituted with serine. Glycine is a non-essential non-polar amino acid with a significant function as a neurotransmitter by facilitating an excitatory potential at NMDA receptors. Although serine has an important role in muscle development and immune system stability, the maintenance of NMDA receptor by glycine controls neural development and muscular function. Therefore, it is assumed that the substitution might have caused withdrawn behaviors and neurological disorders.