Whole exome screening of neurodevelopmental regression disorders in a cohort of Egyptian patients

Developmental regression describes a child who begins to lose his previously acquired milestones skills after he has reached a certain developmental stage and though affects his childhood development. It is associated with neurodegenerative diseases including leukodystrophy and neuronal ceroid lipofuscinosis diseases (NCLs), one of the most frequent childhood-onset neurodegenerative disorders. The current study focused on screening causative genes of developmental regression diseases comprising neurodegenerative disorders in Egyptian patients using next-generation sequencing (NGS)-based analyses as well as developing checklist to support clinicians who are not familiar with these diseases. A total of 763 Egyptian children (1 to 11 years), mainly diagnosed with developmental regression, seizures, or visual impairment, were studied using whole exome sequencing (WES). Among 763 Egyptian children, 726 cases were early clinically and molecularly diagnosed, including 482 cases that had pediatric stroke, congenital infection, and hepatic encephalopathy; meanwhile, 192 had clearly dysmorphic features, 31 showed central nervous system (CNS) malformation, 17 were diagnosed by leukodystrophy, 2 had ataxia telangiectasia, and 2 were diagnosed with tuberous sclerosis. The remained 37 out of 763 candidates were suspected with NCLs symptoms; however, 28 were confirmed to be NCLs patients, 1 was Kaya-Barakat-Masson syndrome, 1 was diagnosed as infantile neuroaxonal dystrophy, and 7 cases required further molecular diagnosis. This study provided an NGS-based approach of the genetic causes of developmental regression and neurodegenerative diseases as it comprised different variants and de novo mutations with complex phenotypes of these diseases which in turn help in early diagnoses and counseling for affected families.


Introduction
Developmental regression is a complex phenomenon that has been described in different developmental disorders and defined as the early absence of acquired milestones skills which affects the brain and behavioral development [1]. It differs from developmental delay that children suffering developmental delay do not achieve developmental milestones in comparison to those of the same age range, while a child experiencing developmental regression refers to a normal developmental phase, followed by loss of previously acquired skills [2]. To date, there is a limited knowledge about the onset or the divergent pathways of developmental regression, due to a complex interaction between biological and environmental factors which in turn presenting many clinical challenges [3]. Children with developmental regression are characterized by neurological disabilities such as ataxia, epilepsy, vision or hearing impairment, movement disorders, and sleeping and behavioral problems [4]. Developmental regression is associated with diagnoses of childhood disintegrative disorders, Landau Kleffner syndrome, Phelan McDermid syndrome, and neurodegenerative diseases such as neuronal ceroid lipofuscinoses (NCLs) [5]. It is 1 3 extremely important to make a precise diagnosis of developmental disorders to allow appropriate and specific therapies, which may change disease progression and improve quality of life in some cases. Loss of motor, language, and social skills can be treated with occupational physical and speech therapies [6]. Neurodegenerative disorders of childhood are complicated diseases, and their diagnosis signifies a great challenge to clinicians. They include a heterogeneous group of diseases that result from specific genetic and biochemical defects such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and the spinocerebellar ataxias. Also, many inherited metabolic disorders with neural regression especially NCLs [7]. The clinical approach of these disorders counts on the age of onset and developmental abnormalities in the brain. Early clinical diagnosis leads to better management and accurate genetic counseling [8]. NCLs are a group of autosomal recessive hereditary lysosomal storage disorders that cause progressive neurodegenerative diseases, with an incidence of about 1-3/100.000 and a prevalence of about 2-4/1.000.000. They are associated with motor and cognitive regression, cerebellar atrophy, vision loss, ataxia, epilepsy, seizures, and a shortened lifespan. According to genetic classification, NCLs are 14 types [9]. As rare disease entities, NCLs comprise the most common cause for progressive neurodegenerative disease in children. NCLs are clinically classified into four major types based on the age of onset of the disease: infantile (6-24 months), late infantile (2-4 years), juvenile (5-10 years), and adult-onset (> 18 years) [10]. Biochemical and genetic studies especially whole exome sequencing (WES) are considered to be the main diagnostic tools of NCLs. Over the last 2 decades, knowledge of the molecular basis of NCLs has been achieved, but the precise pathomechanisms leading to cell and neuronal death have not yet been illuminated. A few targeted therapies for the NCLs, including enzyme replacement and gene therapies, have become available for human study recently, but only one product is commercially available at present for type NCL2. There is an urgent need to find safe and effective treatments for rare neurodegenerative diseases, such as the NCLs, as well as mutual agreements between families of patients and health systems together with pharmaceutical companies [11]. Due to the complexity of genetic diseases, next-generation sequencing (NGS) technology had led to great advances in understanding the causes of Mendelian and neurological diseases. To improve clinical and genetic diagnosis as well as providing significant treatments, a precise genetic test should be selected depending on the rapid quality of time, cost-effectiveness, coverage area, and sequencing range. WES is an appropriate method for finding new mutations and thousands of variants, including missense variants, protein-truncating variants, and large structural variants (SVs); however, whole genome sequencing (WGS) is suitable for exploring the roles of specific and de novo genes in neurodegenerative disorders. A combination of clinical and molecular analysis has become a more effective diagnosis approach [12]. We aimed to highlight genes causing developmental regression and neurodegenerative disorders in Egypt using NGS-based analyses, including interpretations of different variants and mutations.

Participants
The present study was performed through years from 2017 to 2022 on 763 Egyptian children, most of them from consanguineous families (78%) of age ranged from 1 to 11 years who were presented with seizures, ataxia, visual impairment, and developmental regression. Affected individuals were subjected to detailed clinical evaluation, family history, and specific neurological examination; an MRI brain scan was requested when needed. Participants were recruited from the Centre of Excellence of Medical Research, National Research Centre, Cairo, Egypt. An informed consent form was signed from parents or guardian that was approved by the Medical Research Ethics Committee, NRC.

Clinical investigations
The inclusion criteria were seizures, developmental regression, myoclonus, visual failure, and ataxia. However, exclusion criteria include brain trauma and medication intake.

Molecular analysis
DNA from blood samples was extracted from each sample of 763 participants using Thermo Scientific Gene JET Genomic DNA Purification Kit (#K0721, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The concentration and purity of DNA were quantified using a nano-drop spectrophotometer device kit (Thermo Scientific, USA) and stored in aliquots at − 20 °C.

Whole exome sequencing
A total of 50 ng of genomic DNA of 763 children is fragmented to target regions using DNA capture probes. These regions include approximately 41 Mb of the human coding exome (targeting > 98% of the coding RefSeq from the human genome build GRCh37/hg19). The generated library is sequenced on an Illumina platform to obtain at least 20 × coverage depth for > 98% of the targeted bases. An in-house bioinformatics pipeline including read alignment to GRCh37/hg19 genome assembly and revised Cambridge Reference Sequence (rCRS) of the Human Mitochondrial DNA (NC_012920), variant calling, annotation, and comprehensive variant filtering is applied. All variants with minor allele frequency (MAF) of less than 1% in gnomAD database and disease-causing variants reported in HGMD®, in ClinVar, or in CentoMD® are evaluated. The investigation for relevant variants is focused on coding exons and flanking + / − 10 intronic nucleotides of genes with clear gene-phenotype evidence (based on OMIM® information). Variants with low sequencing quality and/or unclear zygosity are confirmed by orthogonal methods. Consequently, a specificity of > 99.9% for all reported variants is warranted. The copy number variation (CNV) detection software has a sensitivity of more than 95% for all homozygous/hemizygous and mitochondrial deletions, as well as heterozygous deletions/duplications and homozygous/hemizygous duplications spanning at least three consecutive exons. For the uniparental disomy (UPD) screening, a specific algorithm is used to assess the well-known clinically relevant chromosomal regions (6q24, 7, 11p15.5, 14q32, 15q11q13, 20q13, and 20). Selected variations were obtained from WES with minor allele frequencies < 0.05 using the following databases (dbSNP, 1000 Genomes Project). Effects of single nucleotide variants (SNVs) were predicted by SIFT, Polyphen-2, PROVEAN, and Mutation Taster programs. The novel mutation was confirmed using Gnomad and Novel Taster.

Sanger sequencing
Sanger sequencing was performed to confirm identified mutations of candidates. Polymerase chain reaction (PCR) of genomic DNA was performed in thermal cycler (Perkin-Elmer; USA) using specific primers that were designed referring to genomic sequence (GenBank accession numbers and Qiagen Taq PCR Core kit: USA). PCR primers were designed using the Primer3 program, and all sequences of the primers are available on request. PCR amplicons were purified using enzymatic Exonuclease/Shrimp Alkaline phosphatase treatment (Sigma, USA) that was held in (PerkinElmer). Relevant PCR products were covered by both forward and reverse strand sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and analyzed on the system (ABI 3130 Genetic Analyzer). Mutational analysis was carried using FinchTV 1.4.0 software.

Participants and ethics statement
The current study comprised 763 Egyptian children (their age ranged from 1 to 11 years), were collected from years 2017 to 2022; 595 (78%) of them were offspring of consanguineous marriages. Affected individuals were subjected to clinical details including, family history, pedigree analysis, neurological examination, EEG, and MRI brain scan that was requested when needed. Patients were recruited from the Centre of Excellence of Medical Research, National Research Centre, Cairo, Egypt, and their parents or guardian signed an informed consent approved by the Medical Research Ethics Committee at NRC for the patient that was approved by the Medical Research Ethics Committee, NRC. All data were anonymous and coded to assure the confidentiality of participants.

Molecular analysis
Molecular screening using whole exome sequencing and Sanger sequencing of 37 Egyptian children revealed 30 mutations in different causative genes with autosomal recessive inheritance pattern. A total of 28 mutations have been investigated in genes (CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN10, CLN13, CLN14, and CLCN2) in 28 NCL patients; one mutation was detected in YIF1B gene in one patient who showed symptoms of Kaya-Barakat-Masson syndrome, and one was demonstrated in PLA2G6 gene of one patient who was suspected to have infantile neuroaxonal dystrophy disease. Seven cases required further molecular studies using the whole genome sequencing technique (Table 2).

Sanger sequencing
In the present study, we started with applying the direct Sanger sequencing technique of CLN6 and CLN7 genes as the most common genes of NCLs disease, on the recent 37 Egyptian children with suspected neurodegenerative symptoms. It revealed 6 pathogenic missense mutations in 6 unrelated candidates. Two reported mutations c.406C > T (p.Arg136Cys) and c.896C > T (p.Pro299Leu) were detected in CLN6 gene, and three mutations were described in CLN7 gene; one of them was novel mutation c.600 G > A (p.Trp200Ser), and the other two mutations were reported c.416G > A (p.Arg139His) and c.881C > A (p.Thr294Lys). One mutation c.789G > C (p. Trp263Cys) was investigated in CLN8 gene in patient who had symptoms and age of onset of suspected CLN8-type (Table 3).

Whole exome sequencing
Whole exome sequencing was carried out on remained 31 affected probands. No variants were detected in the genes    (Table 4). All novel mutations were confirmed using dbSNP, 1000 Genomes Project, PROVEAN, PolyPhen2, SIFT, Mutation Taster, Gnomad, and Novel Taster. All variants detected in whole exome sequencing were confirmed together with those of their parents using the Sanger sequencing technique (Fig. 3). Both parents of probands were identified as carriers of the mutations.

Discussion
Developmental regression (DR) is the progressive loss of previously acquired skills after normal developmental phase especially in children. It is considered as an ominous sign for a genetic disorder, associated with neurodegenerative conditions including neuronal ceroid lipofuscinosis disease and though presenting many clinical challenges [13]. Children diagnosed with developmental regression are suffering from epilepsy, ataxia, vision loss, movement disabilities, sleeping impairment, and behavioral problems [14]. The present study enrolled 763 Egyptian children of age ranged from 1 to 11 years. They were clinically diagnosed with neurodevelopmental regression symptoms as physical disability, congenetive defects, seizures, ataxia, and visual impairment [15]. A total of 726 (95%) cases of 763 Egyptian candidates included different neurodegenerative diseases as 482 (66%) cases demonstrated stroke, congenital infection, and hepatic  [17]; one mutation was found in YIF1B gene in one patient with symptoms of Kaya-Barakat-Masson syndrome, and one was illustrated in PLA2G6 gene of one patient with suspected to have infantile neuroaxonal dystrophy disease, and 7 cases required whole genome sequencing for further molecular investigation [18]. Two pathogenic homozygous mutations of 2 different neurodevelopmental diseases were reported in two unrelated patients; the first one is a novel mutation c.626A > C (p.Tyr209Ser) in gene YIF1B in one patient of 6 years old with suspected symptoms: developmental regression, hypotonia, spasticity, central hypoventilation, poor eye contact, absence of speech, seizures, and brain abnormalities of Kaya-Barakat-Masson syndrome. The novel mutation was confirmed using PolyPhen2, SIFT, Mutation Taster, Gnomad, and Novel Taster software [19]. The second one is a reported mutation c.1039G > A (p.Gly347Arg) in PLA2G6 gene in one patient of age 3 years old; he was pre-diagnosed with symptoms of classical infantile neuroaxonal dystrophy [INAD] such as psychomotor regression, ataxia, visual loss, abnormality of movement, and hypotonia, and his MRI showed cerebellar atrophy. Both mutations were probably damaging with a score of 1 according to PolyPhen2 software and disease   [32]. All missense mutations were predicted to be deleterious and would alter protein structure and function which results causing disease [11].

Conclusions
This study provided NGS-based approach of the genetic causes of neurodevelopmental regression diseases and focused on issues related to NGS-based analyses, including interpretations of different variants and de novo mutations of congenital genetic diseases with complex phenotypes, which in turn contributes to genetic early diagnoses and counseling of families with neurodegenerative diseases. We investigated 30 different mutations in 30 Egyptian children molecularly diagnosed with WES that mainly comprised 6 novel mutations and highlighted 3 rare neurodevelopmental diseases (Kaya-Barakat-Masson syndrome, classical infantile neuroaxonal dystrophy [INAD], and CLCN2-related leukoencephalopathy). We assumed that this study would be a part of demographic screening and a platform for better data communication and diagnostic experience sharing between clinicians for further investigation.

Declarations
Ethical approval Participants were recruited from the Centre of Excellence of Medical Research, National Research Centre, Cairo, Egypt, and an informed consent was signed from the accompanying parents or guardian and approved by the Medical Research Ethics Committee, NRC. The current study was granted by an ethical committee of the Medical Research of Excellence Centre, National Research Centre (NRC), Cairo, Egypt.

Competing interests
The authors declare no competing interests.
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