Novel variants underlying autosomal recessive neurodevelopmental disorders with intellectual disability in Iranian consanguineous families

Abstract Background Intellectual disability (ID) is a heterogeneous group of neurodevelopmental disorders that is characterized by significant impairment in intellectual and adaptive functioning with onset during the developmental period. Whole‐exome sequencing (WES)‐based studies in the consanguineous families with individuals affected with ID have shown a high burden of relevant variants. So far, over 700 genes have been reported in syndromic and non‐syndromic ID. However, genetic causes in more than 50% of ID patients still remain unclear. Methods Whole‐exome sequencing was applied for investigation of various variants of ID, then Sanger sequencing and in silico analysis in ten patients from five Iranian consanguineous families diagnosed with autosomal recessive neurodevelopmental disorders, intellectual disability, performed for confirming the causative mutation within the probands. The most patients presented moderate‐to‐severe intellectual disability, developmental delay, seizure, speech problem, high level of lactate, and onset before 10 years. Results Filtering the data identified by WES, two novel homozygous missense variants in FBXO31 and TIMM50 genes and one previously reported mutation in the CEP290 gene in the probands were found. Sanger sequencing confirmed the homozygote variant's presence of TIMM50 and FBXO31 genes in six patients and two affected siblings in their respective families. Our computational results predicted that the variants are located in the conserved regions across different species and have the impacts on the protein stability. Conclusion Hence, we provide evidence for the pathogenicity of two novel variants in the patients which will expand our knowledge about potential mutation involved in the heterogeneous disease.


| INTRODUC TI ON
Next-generation sequencing (NGS) methods have revolutionized the neurodevelopmental disorder diagnosis, including intellectual disability (ID). These methods have accelerated the identification of causative genes and variants involved in the ethology of disease. 1 ID is a heterogeneous neurodevelopmental disorder characterized by significant impairment in intellectual and adaptive functioning with onset during the developmental period. By a total prevalence of 1%-3%, the disease has extensive phenotypic variability and genetic heterogeneity worldwide. 2 As of 2016, some papers showed that over 700 genes were reported in syndromic and non-syndromic ID. 1 Ghandil et al reported a novel homozygous variant in an Iranian family with syndromic ID. 3 Therefore, NGS has increased the number of causative genes liked to ID facilitating in the diagnosis of the patients. 4 The clinical features of syndromic ID include mild-to-severe intellectual disability, seizure, microcephaly, neuropsychiatric disorders, epilepsy, and motor dysfunction, and dysmorphic features. 5 The clinical and molecular heterogeneity can pose a major challenge in the molecular diagnosis of ID. Many studies showed that the diagnostic yield of causative variants in ID patients with variable severity is low and changeable, ranging from 16% to 68% using whole-exome sequencing (WES). 6 Several factors such as the reduced penetrance, variable phenotype and syndromic nature of the disease, and lack of data in other family's members are the causes of the low-yield of diagnostic tests. In contrast, WES-based studies in consanguineous families with individuals affected with ID showed a high diagnosis rate of potential variants. For example, one paper posited that the WES approach's diagnostic yield in Middle East Asia was up to 90%. 7 Using WES, we found two novel homozygous variants in FBXO31 and TIMM50 genes in four Iranian consanguineous families diagnosed with autosomal recessive neurodevelopmental disorders with intellectual disability. Besides, one known homozygous variant, which were previously reported, was identified in another family.
These results were confirmed by Sanger sequencing in each family.

| Editorial policies and ethical considerations
The study was approved by the ethics committee of Shahid Sadoughi University of Medical Sciences department (IR.SSU.MEDICINE. REC.1399.199). Written informed consent forms for publishing and participating were obtained from all family members before the study. Five families with ten patients from Baluch region were recruited in Ali Asghar hospital, Zahedan, Iran.

| Whole-exome sequencing
According to the manufacturer's instructions, after collecting blood samples from all family members, DNA extraction was carried out by the QIAamp DNA Mini Kit. We performed library preparation and sequencing on probands of each family (V-6) using the SureSelect Human All Exon V6 kit (Agilent Technologies) and HiSeq4000 machine sequencer from Illumina with the coverage and sensitivity of 100X and >99%, respectively. IlluQC.pl (SCR_005461) and Cutadapt software 8 were applied for filtering the raw data. Then, Burrows Wheeler Aligner (BWA) tool was used to align reads to the reference human genome. 9 We perform post-alignment and variant calling using Picard (SCR_006525) (http://broad insti tute.github.io/ picar d/), the Genome analysis tool kit (GATK, RRID:SCR_001876), 10 SAM tools (SCR_005227), 11 HaplotypeCaller, 12 and ANNOVAR (SCR_012821) software. 13 In filtering strategy, we run a list of the related genes of ID on TSV files, which was provided the published data, gene panels, and DisGenet database. Then, the synonymous, intronic, and benign variants were removed from the filtered files, and the functional variants were prioritized according to their function and inheritance mode. The shortlisted variants' pathogenicity was checked using Varsome, InterVar, and ClinVar (SCR_006169) databases. Also, the candidate variants were compared with Iranome project, which is a catalog of genomic variations on 800 individuals from eight major Iranian ethnic groups. So, the project is useful to filter out the population-specific variants. 14

| Sanger sequencing
Gene Runner software was used for designing the proper sequencing primers for confirming the variants in the five families (Table 1). DNA was amplified with PCR by designed primers for shortlisted variants, and Sanger sequencing was carried out by BigDye™ Terminator v3.1 Cycle Sequencing Kit and ABI-3700 DNA analyzer (Thermo Fisher).

| Bioinformatics analysis
As the 3-D structures of TIMM50 and CEP290 proteins have not been identified in the protein data bank (PDB, SCR_012820), Swiss-

| Pedigree I
There was a history of six members affected with ID in three families born from healthy and consanguineous parents (V-1, V-2, V-3, V-6, IV-15, and IV-16 in Figure 1A,B). The proband (V-2) is an 18-year-old male referred to the genetic counseling center with various clinical features, including seizure, severe intellectual disability, lack of speech, delayed psychomotor development, failure to thrive, delayed walking, absent speech, aggressive behavior, and increased activity serum lactate (Table 2). Therefore, other affected family members developed growth delay, recurrent episodes of seizures within the first month of life, failure to thrive, and increased serum lactate. Thus, they could not raise their head at 7-8 months of age and walk or sit when they are 3-4 years old. Laboratory results like karyotype and brain MRI tests were all normal.

| Molecular analysis
To identify the disease-causing variants, WES was performed on the proband (V-2) and the family. Our results revealed a novel homozy-  Figure 2B). Therefore, it could decrease the stability of protein according to DynaMut, mCSM, SDM, and DUET algorithms (Table 3). Patient Age at last exam    (Figure 2A). Moreover, the variant segregated among all members of family ( Figure 4C).

| DISCUSS ION
Next-generation sequencing was applied with some success to identify the rare causative variants of ID in consanguineous families, associated with the phenotype of the families. 19 We identified two novel variants in FBXO31 and TIMM50 genes and one previously reported mutation in CEP290 gene using WES in five consanguineous families diagnosed with autosomal recessive neurodevelopmental disorders with intellectual disability, confirmed through Sanger sequencing in their respective families.  Table 4.
We found a novel homozygous missense variant (c.1532G>A) in FBXO31 gene in pedigree I, which replaces the arginine residue with glutamine at position 511. FBXO31 (F-Box Protein 31) is a component of the SCF (SKP1-cullin-F-box) complex which ligated the ubiquitin molecule to phosphorylated cyclin-D1. 23 Then, the complex acts as a cell cycle suppressor following DNA damage by an arrest at G1-S checkpoint. 24,25 Some papers reported that the complex is essential for neuronal morphogenesis and axonal identification in the cerebellar cortex by the ubiquitination of the Par6c protein during brain development. 26 Moreover, functional studies of FBXO31 expression in mouse hippocampal neurons showed that the protein is located in axons and soma. 26  So far, our reassessment confirmed a consistent phenotype with Joubert Syndrome in the patient, segregating in the family. A brain MRI of his sister (with the same genotype) did not show the cerebellar vermis aplasia phenotype. Hence, it seems to be the main challenge in the diagnosis of patients.
In conclusion, we found two novel variants in the ID-related genes FBXO31 and TIMM50 in the consanguineous families. The variants were confirmed in all affected and unaffected members of the families via Sanger sequencing, and their potential relevance to the disease was investigated using prediction tools. Therefore, the large and rare pedigrees can be helpful for us to identify ID diagnosis in the recruited families and expand our knowledge about potential mutation involved in the heterogeneous disease. To further confirm and asses the pathogenicity of the aforementioned variants, functional studies will be needed.

ACK N OWLED G EM ENTS
We would like to gratitude the affected individuals and their families for their participation in the study. We thank the technical operators of Ali Asghar Hospital in Zahedan.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

PU B LI S H
Informed consent was obtained from all family members before the study. Also, written informed consent was obtained from their parent for publishing their data and photographs.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data to support the findings in the study are available on request from the corresponding author.