DLG3 Impairment Caused by Missense Variants in Non-Syndromic X-Linked Mental Retardation

X-lined intellectual disability (XLID), formerly known as X-lined mental retardation, is dened as genetically heterogeneous disorders with remarkable cognitive impairment and abnormal adaptive behaviour skills. This study demonstrates the Disc-large homolog 3 (DLG3) gene impairment in 2 different unrelated male probands. The results detected two missense mutations in the DLG3 gene, c.2267 G > A (p.Arg756Gln) and c.2359G > A p. (Gly787Ser) using by NGS. Both mutations were run in the PolyPhen2 program for mutation sensitivity check and showed to have 0.709 and 1, respectively. The familial transmission pattern of MR detected both mothers to be heterozygote. The mutations were shown to have caused non-syndromic XLMR (NS-XLMR) as both males did not show any abnormal facial or physiological features. Based on the IQ measurement, proband 1 and 2’ IQs were measured 40 and 33, and they were diagnosed with moderate and severe XLMR, respectively. Both affected males showed signicant deterioration in neural development and behaviour abilities, which indicates the signicant impact of the mutation on neurotransmitters and maintenance of NMDA receptors in neural functions. However, further molecular and functional studies are necessary to provide more conclusive evidence of the detailed abnormalities caused by the reported mutations.

Although many factors including pre-, peri-, and post-natal problems (Strømme & Hagberg 2007;Piecuch et.al 1977;Kolevzon et.al 2007;Zoghbi 2003), metabolic disorders such as sphingolipidoses (Scriver 1995;McDermott 2007), iodine de ciency (Gaitan & Dunn 1992), and malnutrition (Wines 2006) showed to have caused MR, X-linked gene defects and have been reported to be the signi cant cause as MR incidence rate in males is higher than females (Penrose 1938; Lehrke R.G 1972; Lehrke R.A 1974). The clinical analysis of collected data and linkage studies describes X-linked mental retardation (XLMR) as a highly heterogenous condition with more than 23 identi ed XLMR genes and has an estimated prevalence of 1 in 1,000 males, with fragile X syndrome accounting for 10 to 15% of cases, the prevalence of most other cloned X-linked genes being very low (0.5-1.0%) with the exception of Aristaless X (Mandel & Chelly 2004). It is classi ed as syndromic XLMR (S-XLMR), which accounts for less than 30% of the cases, and non-syndromic XLMR (NS-XLMR) cognitive dysfunction is the only distinctive feature and no other clinical, radiological, or biochemical alterations are present and accounts for the rest (Fishburn et.al 1983). The main features of S-XLMR can vary based on the affected gene. For instance, mutation on the FMR1 gene causes Fragile X syndrome and is presented with facial anomalies and macroorchidism (Verkerk et.al 1981;Jin & Warren 2003;Huber et.al 2002). Investigation of X-linked chromosome MR forms has led to discovering pathogenic gene variants and molecular pathways in MR.
This study reports 2 MR male cases with novel DLG3 mutations and X-linked recessive inheritance with cognitive anomalies correlated with NS-XLMR.

Patients
The male proband 1 was examined and diagnosed at the age of 5. He was the rst child of healthy nonconsanguineous Turkish parents (Fig. 1). He was born with average birth measurements after a normal pregnancy in the 38th gestational week. Delay in psychomotor development was observed. The child started sitting at the age of 13 months and walking at almost 17 months. He spoke sentences with 3-5 words with poor articulation and slurry speech and required special educational aid at three years old. Physical examination showed normal body measurements with no dysmorphic features. He exhibited behavioral problems with epilepsy and severe conclusions at the age of 10 controlled with pharmacological therapy and antiepileptic medications, respectively. The electroencephalography detected a severe epileptic pattern. Wechsler Intelligence Scale for Children (WISC) full-scale IQ test was performed and measured 40, which indicated moderate MR A brain computed performed tomography scan, and it was normal.
The male Proband 2 was examined and diagnosed at the age of 6. He was born to healthy nonconsanguineous Turkish parents with no affected member in the family (Fig. 2). The proband manifested delayed psychomotor development and movement disorder at the age of 4. The patient was diagnosed with atypical autism at the age of 6 and showed limited communication skills with slow and poor speech. WISC full-scale IQ test was performed and measured 33, which was categorized as severe MR. the male proband showed withdrawn behaviour with no abnormal physical or neurological characteristics. Further examinations showed no seizure, hearing and visual disability, and dysmorphic features. A brain computed tomography scan was performed, and it was normal.

WES
Whole Exome Sequencing (WES) was carried out to analyse the coding exons and the exon-intron boundaries of protein-coding genes. The preparation of genomic DNA, exome capture, and Illumina® sequencing (NextSeq500 platform) of the probands and the parents were carried out according to the manufacturer's manual. Genomic DNA was isolated from the whole blood sample (collected prior to analysis) by Invitrogen® NextSeq500 iPrep PureLink gDNA blood kit (Thermo Fisher Scienti c, Inc.). Preparation of the gene library was performed by Agilent SureSelect Target Enrichment System (Agilent Technologies, Inc.) and the coding exons and anking intronic regions were enriched by the Agilent SureSelect Human All Exon V6 reagent (Agilent Technologies, Inc.) according to the manufacturer's protocol ( Bonnefond et.al 2012). Sequencing was accomplished by the Illumina® NextSeq500 system (Illumina, Inc.), and the sequences were mapped to the human genome (GRCh37/hg19) using the Burrows-Wheeler Aligner (version 0.6.1; algorithm 'BWA-SW'; default parameters) to acquire targeted sequencing data. To obtain a more e cient analysis, variants that showed less than 1% frequency in the gene pool were eliminated from the nal data. Following the data collected, the variants were annotated by Alamut® Visual (a decision-support software dedicated to variant diagnostics used by clinical and research molecular laboratories worldwide; see https://www. interactive-biosoftware.com/alamut-visual/). Finally, the frequency of allele was determined by the following databases: The National Center for Biotechnology Information database for nucleotide variations dbSNP (https://www.ncbi.nlm. nih.gov/snp), the exome aggregation consortium (ExAC) and the 1000 Genomes Project (https://www.internation [1]algenome.org/). Disease causality was assessed using ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and exome sequencing project (ESP) variants and ExAC variants.

Sanger Sequencing Of The Detected Mutations
DNA from the patients and the parents was isolated from whole blood using the QIAamp DNA Blood Mini kit (Qiagen, Inc.). Exon 18 of the DLG3 geneusing primer sets (forwards, 5'CAAGAAATTCACTATGCAAG3'; and reverse, 5'CTTGCATAGTGAATTTCTTG − 3') for proband 1 and exon 19 (forwards, 5' TACTACCCGGCCTCGACGTG3'; and reverse, 5' CACGTCGAGGCCGGGTAGTA − 3') for proband 2 with and MyTaq™ DNA Polymerases Mix (Bioline Reagents Ltd.) were ampli ed. PCR was carried out with 40 ng of genomic DNA in 30 µl, with an initial denaturation at 94˚C for 3 min, 30 cycles of 94˚C for 30 sec, 55˚C for 45 sec and 72˚C for 2 min, followed by a nal extension at 72˚C for 10 min. The SERAC1 gene was sequenced directly from puri ed PCR products using a BigDye Terminator Cycle Sequencing kit (version 3.1; Applied Biosystems; Thermo Fisher Scienti c, Inc.) prior to analysis on an ABI 3130 automated DNA sequencer (Applied Biosystems; Thermo Fisher Scienti c, Inc.).

Genetic Analysis
DLG3 gene validation analysis for the rst case was revealed DNA material mutation in the DLG3 gene of c.2267 G > A (p.Arg756Gln) sequence. In the validation study performed by Sanger sequence analysis, c.2267 G > A (p.Arg756Gln) variant was hemizygote at scans in the DLG3 gene ( Fig. 1A1 and A2). The Fathers' DLG3 gene c.2267 G > A (p.Arg756Gln) mutation was screened in situ. No mutation was detected at the scanned point in the validation study performed by Sanger sequence analysis (Fig. 1C). The Mothers' DLG3 gene c.2267 G > A (p.Arg756Gln) mutation was screened in situ. In Sanger sequence analysis, c.2267 G > A (p.Arg756Gln) mutation was screened as heterozygous (Fig. 1B). PolyPhen2 analysis (genetics.bwh.harvard.edu/pph2) of the mutation indicated the mutation to have been possibly damaging with a score of 0.709 DLG3 gene validation analysis for the second case revealed DNA material mutation in the DLG3 gene of c.2359G > A p. (Gly787Ser) sequence. In the validation study performed by Sanger sequence analysis, C.2359G > A, p.Gly787Ser variant was hemizygote at scans in the DLG3 gene (Fig. 2C). The fathers' DLG3 gene was screened for c.2359G > A p. (Gly787Ser) mutation.
In the validation study performed by Sanger sequence analysis, no mutation was detected in the scanned region (Fig. 2D). The mothers' DLG3 gene for c.2359G > A p. (Gly787Ser) mutation was screened. In the validation study performed by Sanger sequence analysis, at scanned points c.2359G > A, p.Gly787Ser variant was a heterozygous (Fig. 2A). Using sequencing data and information collected from family members, the hereditary pattern of the NS-XLMR syndrome in the family was elaborated in a pedigree charts for Proband 1 (Fig. 3A) and 2 ( Fig. 3B) that was con rmed using the analysis on the blood sample of the probands and their parents. The parents were both found to be unaffected while the both male cases of the families manifested mild and severe NS-XLMR in order.

Protein Analysis
PolyPhen2 analysis (genetics.bwh.harvard.edu/pph2) of the proband 1 mutation indicated the mutation to have been possibly damaging with a score of 0.709. PolyPhen2 analysis (genetics.bwh.harvard.edu/pph2) of the proband 2 mutation indicated the mutation to have been probably damaging with the score of 1. The protein structure of the wild and the impaired proteins for both probands were designed by Swissmodel (swissmodel.expasy.org). And the remarkable changes in binding pockets were observed with the arginine to glutamine substitution (Fig. 4B) in proband 1 in comparison with the wild type (Fig. 4A). Similarly, substitution of glycine by serine in proband 2 (Fig. 5B) determine noticeable alteration in the protein's binding sites compared to the wild type (Fig. 5A). The observation on the changes could altogether suggest the impact of the mutation on protein function that may cause further neural complications.

Discussion
MR is a complex disorder, and according to WHO, it consists of three following main criteria: belowaverage 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 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). . 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 identi ed, 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 identi ed 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 e cacy 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 signi cant 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.

Conclusion
As a result of the whole Exome Sequence Analysis, the mutation detected as hemizigote in the DLG3 gene is thought to cause the XLMR since both patients are male. In our second case, who previously diagnosed with atypical autism, we have identi ed DLG3 deletion seem to be the cause of XLMR. This data is important to emphasize the genetic relationship between ASD and XLMR. New studies in the future will strengthen this thesis and enlighten the genetic etiology of mental retardation and ASD.

Declarations
Ethics approval and consent to participate have been taken from Bruni University Medicine School Ethical Committie Consent for publication have been taken from the patients' parents. Patient's parents gave informed written consent for their personal or clinical details along with any identifying images to be published in this study.

Availability of data and material statement Not applicable
Competing interests None declerated Funding None declareted Sanger Sequencing analysis of proband 1 and his family. The analysis shows proband 1 to be hemizygote with c.2267 G> A (A1 and A2). The result showed overlapping area of back (Guanine) and green (adenine) in c.2267 in mother with adenine and Guanine that indicates mother to be heterozygote (B) and father shows no mutation at the speci ed position (C).

Figure 2
Sanger Sequencing analysis of proband 1 and his family. The analysis shows proband 1 to be hemizygote with c.2359G> A (C). The result showed overlapping area of back (Guanine) and green (Adenine) in c.2267 in mother with adenine and Guanine that indicates mother to be heterozygote (C) and father shows no mutation at the speci ed position (C).

Figure 3
The pedigree chart was drawn to determine the inheritance pattern of the mutation in proband 1's family (A).The pedigree chart was drawn to determine the inheritance pattern of the mutation in the proband 2's family (B).

Figure 4
Protein con guration of DLG3 gene in wild type (A) and proband 1 mutations (B). Figure 1B represents protein con guration change and its binding ligand caused by substitution of Arginine with Glutamine.