{'fraction_non_alphanumeric': 0.03180271394362055, 'fraction_numerical': 0.028987942696298785, 'mean_word_length': 4.99748047367095, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 19, 'https://': 6, 'lorem ipsum': 0, 'www.': 4, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Exome sequencing reveled a compound heterozygous mutations in RTTN gene causing developmental delay and primary microcephaly

RTTN (Rotatin) (OMIM 614833) is a large centrosomal protein coding gene. RTTN mutations are responsible for syndromic forms of malformation of brain development, leading to polymicrogyria, microcephaly, primordial dwarfism, seizure along with many other malformations. In this study we have identified a compound heterozygous mutation in RTTN gene having NM_173630 c.5225A > G p.His1742Arg in exon 39 and NM_173630 c.6038G > T p.Cys2013Phe in exon 45 of a consanguineous Saudi family leading to brain malformation, seizure, developmental delay, dysmorphic feature and microcephaly. Whole exome sequencing (WES) techniques was used to identify the causative mutation in the affected members of the family. WES data analysis was done and obtained data were further confirmed by using Sanger sequencing analysis. Moreover, the mutation was ruled out in 100 healthy control from normal population. To the best of our knowledge the novel compound heterozygous mutation observed in this study is the first report from Saudi Arabia. The identified compound heterozygous mutation will further explain the role of RTTN gene in development of microcephaly and neurodevelopmental disorders.


Introduction
Primary Microcephaly (MCPH, OMIM 251200) is stated as the congenital genetic neurodevelopmental disorder demonstrating as small brain size along with seizure and intellectual disability. The head circumference (HC) is reduced from À4 SD (standard deviation) to À2 SD at the time of birth below the age and sex of the individual (Nicholas et al., 2010;Bilgüvar et al., 2010;Kaindl et al., 2010). In the primary microcephaly most of the patients have normal weight, height, physical appearance as well as chromoso-mal banding pattern and normal brain scan result. In some of the cases it is also observe that patients showing abnormal chromosomal analysis and short stature with mild to swear intellectual disability (Woods et al., 2005;Nicholas et al., 2010). Studies on brain growth also revealed that primary microcephaly is due to the abnormal developmental process and not because of the degeneration or regression of the neuronal tissues (Bundey, 1997;Faheem et al., 2015). The occurrences of primary microcephaly is much more in the populations ranging between 1.3 and 150 per 100,000 births where rate of consanguineous marriages is high such as Middle East and Asia and much lower in Caucasians where consanguinity is lower (Woods and Parker, 2013). In the OMIM genes database 25 genes associated with primary microcephaly has been reported so far.
RTTN gene in human with (OMIM 610436) chromosomal location 18q22.2 translates a centrosomal protein called Rotatin mainly related to ciliary functions. Kheradmand et al., for the first time in 2012 reported a homozygous mutation in RTTN gene in the three members were the product of a consanguineous family from Turkey having short stature, epilepsy, microcephaly, and polymicrogyria. Later on, Shamseldin et al. (2015) reported a homozygous https://doi.org/10.1016/j.sjbs.2021.02.014 1319-562X/Ó 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). mutation in intron 23 of the RTTN (c.2885 + 8A-G) gene resulting in a premature stop codon (S963X) in a family having primary microcephaly and severe intellectual disability along with another homozygous mutation in Saudi male where c.3190A > C and protein change p.lys1064gln with intrauterine growth retardation, short stature, primary microcephaly along intellectual disability. Further, a Canadian family was also reported having mutation where c.1732G > C p. ala578-to-pro in exon 13 of RTTN gene, and a compound heterozygous mutation c.5750A > G resulting growth retardation and primary microcephaly in the affected members of the family (Shamseldin et al., 2015). Recently, a homozygous c.2953A > G transition (chr18.6,780,1710 T-C, GRCh37) at the penultimate base of exon 23 of the RTTN gene in Marocan family, resulting in an p.Arg985Gly (R985G) substitution was reported by Grandone et al. 2016 and similarly a homozygous mutation was also reported (Cavallin et al., 2018). Moreover, a compound heterozygous mutation was also reported where c.4186delC p. E1397Kfs*7 and c.2594A > G and protein change p.H865R in RTTN gene. Later on RTTN gene mutations was linked with the brain and other organs deformities along with primary microcephaly and primordial dwarfism (Grandone et al., 2016;Shamseldin et al., 2015). The exact reason of the brain and other organs deformities along with primary microcephaly and primordial dwarfism is still unknown as recently, we have reported many novel mutations such as PGAP2 (Naseer et al., 2016a(Naseer et al., , 2016b, SATMBP (Naseer et al., 2016a(Naseer et al., , 2016b NT5C2 (Naseer et al., 2020a(Naseer et al., , 2020b, PUS7 and AASS genes related to microcephaly but the still lot of studies need to be done to identify the cause of the disease (Naseer et al., 2020a(Naseer et al., , 2020b.
Although few of RTTN gene mutations causing primary microcephaly have been reported so far. In the present study we identified a novel compound heterozygous mutation in RTTN gene that will be helpful for adding information in the disease data base and also helpful for the geneticists and clinicians to establishing a reliable diagnostic strategies for primary microcephaly families in Saudi population.

Sample collection
We recruited a Saudi family with clear phenotype of primary microcephaly. Pedigree was carefully made by interviewing the family. Peripheral blood samples of all members of the family and from 100 healthy unrelated control were collected in EDTA tubes. Genomic DNA was extracted through QIAamp genomic DNA extraction or similar kits according to the manufacturers' protocols (https://www.qiagen.com/pk/products/top-sellers/qiaampdna-minikit/#orderinginformation). Genomic DNA quantification was performed with Nanodrop spectrophotometer (https://www. thermofisher.com/order/catalog/product/ND-LITE-PR) and visualized with CYBR Safe (Thermofisher, USA) dye via running on 1% agarose horizontal gel electrophoresis apparatus. Informed consent was taken prior to the study from all participants. This study was permitted by the local ethical committee of King Abdulaziz University and followed all the guidelines according to the Helsinki's declaration 2013. The detailed family pedigree was drawn as shown in Fig. 1.

Magnetic resonance imaging (MRI)
MRI examination of the brain plain reveals, technique: T1WI axial, coronal and sagittal T2WI axial finding straight is prominent measuring in the superoanterior part. 6 mm. Sulci and extra cerebral CSF spaces are slightly prominent. Myelination pattern is nor-mal for the age of the patient. Normal ventricular system. No midline shift. No tonsillar herniation noted.

Karyotype analysis
Peripheral blood samples was used for karyotype analysis. Cells culture were subjected to multiple steps including harvesting, preparation of slide, staining and microscopic analysis, before obtaining the complete image of the chromosomes. Two lymphocyte cultures per patient were initiated in sterile T-25 flasks by adding 0.5-0.7 ml blood and 0.2 ml phytohemagglutinin solution to 10 ml of complete media consisting of RPMI 1640 50 ml, and fetal bovine serum (FBS) 10 ml along with 0.5 ml penicillin/streptomycin solution and 0.5 ml L-glutamine and the cultures were incubated at 37°C for 72 h. After incubation for 48 h, 0.1 ml 5-Fluoro-2-deoxyuridine (FUDR) solution was added in thymidine culture flask and the contents were mixed gently. Following the incubation period 0.2 ml of thymidine was also added and the contents were mixed and re-incubated at 37°C. Three hours before cell harvest, 0.1 ml of ethidium bromide solution was added in thymidine culture flask. Then, 0.65 ml of colcemid (10 lg/ml) was added to regular flask and thymidine flask 10 min and 15 min before cell harvest respectively. Following 25 min, the cultures were terminated and the contents of the two culture flasks were transferred into centrifuge tube for centrifugation for 10 min at 1500 rpm. Cells were fixed by adding 1 ml freshly made fixative. The fixed cell suspension was centrifuged at 1500 rpm for 10 min, the supernatant removed, leaving approximately 1 ml, depending on the size of pellet. The cells were resuspended, using a narrow glass pipette. One to two drops of the cell suspension were dropped onto each marked slide from a height over vapour of hot water. Slides were left to dry at room temperature overnight. The spreads quality and mitotic index of the slide was checked using an Olympus phase contrast microscope (Model No. BX51TF). For G-Banding and staining four coplin jars were prepared and each contained the following solution. Coplin jar one (48 ml of isotonic buffer + 2 ml of trypsin solution). Coplin jar two (50 ml isotonic buffer). Coplin jar three (50 ml Gurr Buffer pH 6.8) and coplin jar four (15 ml of re-filtered Leishman stain +45 ml Gurr Buffer solution pH 6.8). As for chromosome trypsin treatment, the slides were dipped in coplin jar one for 1 min. Then the slides were soaked for 3 s in coplin jars two and jar three respectively. The slides were stained with fresh Leishman stain for 2-3 min in coplin jar four. Immediately the slides were rinsed in running water and air dried and observed using the Nikon microscope for the presence of optimal chromosome bands. The position of the metaphase spreads upon finding was captured by using the Cytovision tools, the chromosomes of captured metaphases were separated, arranged in pairs, compared pairs band by band and checked for any abnormality.

Whole exome sequencing
To find out the pathogenic mutation that may be the cause of primary microcephaly WES was performed for the affected members of the family. The samples for sequencing were processed according to the guide from Agilent SureSelect Target Enrichment Kit (SureSelect_v6 Agilent USA) while whole exome sequencing was done using the Illumina HiSeq 2000/2500. Further, the libraries were sequenced using sequencer from Illumina. The variants were filtered using frequency, protein effect, quality, pathogenicity, genomic position and relations with the disease. The variants or single nucleotide polymorphisms and short indels candidates are detected at nucleotide resolution at this stage. SNPs identified are compared to 1000 genome (https://www.interna-tionalgenome.org/), SnpEff (http://snpeff.sourceforge.net/SnpEff. html) and genomAD databases (https://gnomad.broadinstitute. org/). We utilized various bioinformatics tools to find the causative variant for primary microcephaly. Laser gene Genomic Suite v. 12 (DNASTAR, Madison, WI, USA). Further, we used ArrayStar v. 12 (Rockville, MD, USA) to tag variant alleles based on dbSNP142. The sequences obtained in FASTQ were further mapped to human reference genome which is termed as ''alignment", this alignment is performed through Borrow-Wheel arrangement tool (http://biobwa.sourceforge.net/bwa.shtml) and this alignment was done with reference genome hg19 for human (http://hgdownload.cse.ucsc. edu/goldenPath). FASTQ raw data files are here converted to BAM files format. The BAM files obtained in the previous step were annotated with Toolkit for Genome Analysis (http://www.broadinstitute.org/gatk). It is an excellent and successful tool for variant discovery in next generation sequencing data.

Sanger sequencing (SS)
Selected variants were confirmed through SS so that in exome they have not been picked up as false positive. In SS first primers were selected through Primers3 plus online software, then the target DNA will be PCR amplified, purified through PCR purification kits and then Sanger sequenced through ABI genetic analyzers with help of BigDye Terminator V3.1 Cycle Sequencing kit. These technologies provide sequence reads of 400-700 nucleotides and thus the target variants are easily confirmed through electrophergrams obtained from ABI genetic analyzers.
2.6. RTTN protein docking and alignment of the protein SWISS-MODEL docking was used for the modeling of both wildtype and mutated protein structures for the position of p. His1742Arg and for the position of p.Cys2013Phe. Sequencing alignment of different species was also done and highlighted the strong conservation of the two variants at p.His1742Arg and p. Cys2013Phe by down loading the data of different proteins from Ensembl gene browser (https://m.ensembl.org/index.html) and alignment of the sequencing was done using the BioEdit software.

Clinical reports of the patient
Proband III-1 a boy was of 2 years old at time of examination. He was the first baby of the consanguineous Saudi family as shown in Fig. 1a. This was the second case in the family history as another boy of four year old first cousin from father side have the same phenotypes but blood samples was not available for analysis. His has microcephaly of -4 SD measured as occipitofrontal head cir-cumference along with mild to modest intellectual disability, dysmorphic features, developmental delay and epileptic seizure as well. Furthermore, typical feature of primary microcephaly with reduce head circumference was present. He had normal chromosomes, no metabolic as well as no neurological problem was observed. Karyotype study based on 15 metaphases analysis resulted as 46XY a normal male without any chromosomal changes. Brain MRI resulted in microcephaly with an abnormal triangular appearance of the frontal skull bones. Sulci and extra cerebral CSF spaces are slightly prominent. Myelination pattern is normal for the age of the patient. Normal ventricular system. No midline shift. No tonsillar herniation noted. The MRI report showed microcephaly with an abnormal triangular appearance of the frontal skull bones mostly related to premature closure of the metopic suture. There was no acute brain insult, mass-effect, hydrocephalus or space occupying lesion. He was intellectually disabled. He has similar feature of primary microcephaly like sloppy forehead large ears.

Whole exome sequencing
WES for the proband III-1 was done and the resulted FASTQ files were converted to BAM and then BAM files were converted to variant call format (vcf) file having 113,251 variants were obtained. These variants were utilized for the identification of mutation that may leads to the disease based on novel/rare (MAF + 0.01%) frequency, functional (predicted damaging by Polyphen/SIFT), homozygous/heterozygous state, genomic position, pathogenicity, protein effect, and earlier associations with the disease related phenotype. We applied different filters and bioinformatics tools and we did not find and any homozygous mutation than we applied more filters to find out the heterozygous mutations and we identified a compound heterozygous mutation NM_173630 c.5225A > G p.His1742Arg in exon 39 and NM_173630 c.6038G > T p.Cys2013Phe in RTTN gene. The variant was not found in the large reference population cohort of the Genome Aggregation Database (gnomeAD, n >120,000 exomes and >15,0000 genomes). To best of our knowledge these variants have been not been reported in the any medical literature or in the disease related variation database like Human Gene Mutation Database (HGMD) or ClinVar. The total number of mutations reported for RTTN gene so far collected from the literature has been mentioned in the Table 1.

Sanger sequencing
The compound heterozygous mutation identified after WES analysis were further validated by using Sanger sequencing analysis in all other available family members by using the primers for the observed mutations. The Sanger sequencing results confirm the compound heterozygous mutation NM_173630 c.5225A > G p.His1742Arg in exon 39 and NM_173630 c.6038G > T p. Cys2013Phe in RTTN gene as one parent was normal at one position and was heterozygous at other position and similarly the other parent was heterozygous at the position where the other parent was normal while both heterozygous mutations were present in the affected boy as shown in Fig. 2. Finally, to find it out that the identified variants are pathogenic for the family and to rule out this possibility the Sanger sequencing for 100 unrelated control people were also done.

RTTN protein docking and alignment of the protein
Swiss docking modeling was done for both wildtype and mutated showed wildtype and mutated structure of Rotatin protein prediction for the position of p.His1742Arg and for the position of p.Cys2013Phe. The automated protein structure homology-modelling model was done using online SWISS-MODEL database as shown in Fig. 3AB. Sequence alignment of different species was also done and highlighted the strong conservation of the two variants at p.His1742Arg and p.Cys2013Phe as shown in Fig. 3C.

Discussion
The RTTN gene is very important gene that is responsible for centrosomal protein translation. The exact function of this gene is still not very clear, but the studies showed that human mutant fibroblasts showed anomalous growth of cilia. Further, Real-Time Quantitative Reverse Transcription qRT-PCR showed the involvement and dysregulations of numerous genes in the signaling pathway for brain development of the Sonic Hedgehog (Kheradmand Table 1 Mutation spectrum in RTTN gene. Compound heterozygous

Saudi Arabia
Microcephaly related craniofacial dysmorphism, axial hypotonia, and limb spasticity Present study Fig. 2. Sanger sequencing of the individuals participated in the study. A novel compound heterozygous mutation was identified in the affected member III-3 where c.5225A > G p.His1742Arg in exon 39 and c.6038G > T p.Cys2013Phe in exon 45 of a consanguineous Saudi family. The father II-1 was homozygous having ''A" allele at the position and the mother II-2 was heterozygous at c.5225A > G at this position while the father was heterozygous at the position c.6038G > T and mother II-2 was homozygous having ''G" allele. The affected member was heterozygous at both positions c.5225A > G p.His1742Arg in exon 39 and c.6038G > T p.Cys2013Phe in exon 45 clearly representing mutation pattern of compound heterozygous manner. et al., 2012). Rttn expression in mesoderm and telencephalon in mice embryos was also observed. If the gene is knockout mortality of embryo occure at the age of E.11.5. Moreover, many other major developmental defects includes an abnormal distinction of the somites notochord degeneration, the neural tube, randomization of heart looping and axial rotation failure was also studied (Faisst et al., 2002).
The RTTN gene has been added in list of gene mutations that are linked with the cortical malformations. Two different consanguineous families reported with mutations in RTTN gene in four patients having intellectual disability, seizures and polymicrogyria in brain MRI (Kheradmand et al., 2012). Polymicrogyria is a condition of abnormal cortical malformation considered by small undulating gyri with combination of the different molecular layer in two sulci. This condition also linked with a normal or irregular gyration that might be the result of brain damage with hypoxic ischemic or may be due to any developmental disorders with different environmental conditions (Squier and Jansen, 2014).
RTTN gene knockdown study in Drosophila ortholog also suggested that the mechanism of RTTN-related with asymmetry that is due to abnormal cilia development along with shortening of the cilia size (Kheradmand et al., 2012;Stevens et al., 2009). The normal development of cilia play very important and critical role in the formation of the nodal flow, that is linked with a primary cue that cause the interruptions the of the left and right symmetry that is a standard condition of primary ciliary dyskinesia (Nonaka et al., 1998;Okada et al., 2005).
In the Murine embryos any genetically abolished in RTTN gene resulted in expression demonstrate delayed neural tube closure, randomized heart looping along with left-right sidedness abnormalities (Faisst et al., 2000;Chatterjee et al., 2007). Different studies suggest species specific changes in the role of RTTN such as fetal mortality linked with genetic dismissal for RTTN encoded functions during cardiac development RTTN-mediated cardiac abnormalities in humans, while in murine model showed different phenotypic conditions in the null alleles along with biallelic splice variants or missense mutations was also observed in the newborns and children. Genetic disorder of RTTN gene and its homolog Ana3 in Drosophila related to the expression in fly models murine further explain the role and the importantce of this important gene for the development of brain (Stevens et al., 2009). Wambach et al. (2018) reported that patients as similar neurologic conditions such as reduced white matter, corpus callosum agenesis, microcephaly, cortical dysplasia, simplified gyral pattern and nervous system (bilateral microphallus, renal pyelocaliectasis and cryptorchidism) conditions as reported by different patients with biallelic RTTN variants (Shamseldin et al., 2015;Kheradmand et al., 2012;Grandone et al., 2016). Whereas some ophthalmologic findings such as optic nerve hypoplasia, rudimentary retinal vasculature, pale optic disks and misshapen orbital globes along with skeletal findings such as thin ribs, hypoplastic mandible, gracile appearing bones, syndactyly and augmented density of the temporal bones were not reported in previous studies (Wambach et al., 2018).
RTTN gene encodes a large 2,226 amino acid protein called Rotatin and the exact function is still unknown as having two domains Armadillo-like that is highly conserved among species. These Armadillo domains control the interaction between proteins by controlling the transducing indications of cell adhesion molecules to the cytoskeleton. (Shamseldin et al., 2015;Kheradmand et al., 2012).
Previously, studies showed that mice with Rttn knockout having the missing the murine ortholog of RTTN, showed many defect such as notochord degeneration, with lacking axial rotation, embryonic lethality, irregular differentiation of the neural tube, severe hydrocephalus along with loss of the left and right requirement of the heart (Faisst et al., 2002). These abnormalities leading to the disorders disturbing primary and motile cilia Goetz and Anderson, 2010;Badano et al., 2006;Gerdes et al., 2009). Furthermore Rttn mouse mutant also showed has anteroposterior and dorsoventral modeling defects as showed in the Kif3a and Kif3b mutant mice, which control the motor subunits of kinesin-II for ciliary transport (Chatterjee et al., 2007;Melloy et al., 1998;Marszalek et al., 1999).

Conclusions
Finally, in this study we reported a novel compound heterozygous mutation in RTTN gene, this will further help in population screening of primary microcephaly families that is required for better understanding and management of this disease for efficient and accurate genetic testing. These type of reports are helpful for patient management and to reduce of the disease risk through genetic counselling in the population. Moreover, our study enlarges the mutation data of RTTN gene related to MCPH and offers new insight into the types and frequencies of the mutations in Saudi population, which will further help to identify innovative strategies to tackle primary microcephaly.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.