Copy number variations in Saudi family with intellectual disability and epilepsy
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Epilepsy is genetically complex but common brain disorder of the world affecting millions of people with almost of all age groups. Novel Copy number variations (CNVs) are considered as important reason for the numerous neurodevelopmental disorders along with intellectual disability and epilepsy. DNA array based studies contribute to explain a more severe clinical presentation of the disease but interoperation of many detected CNVs are still challenging.
In order to study novel CNVs with epilepsy related genes in Saudi family with six affected and two normal individuals with several forms of epileptic seizures, intellectual disability (ID), and minor dysmorphism, we performed the high density whole genome Agilent sure print G3 Hmn CGH 2x 400 K array-CGH chips analysis. Our results showed de novo deletions, duplications and deletion plus duplication on differential chromosomal regions in the affected individuals that were not shown in the normal fathe and normal kids by using Agilent CytoGenomics 184.108.40.206 softwear. Copy number gain were observed in the chromosome 1, 16 and 22 with LCE3C, HPR, GSTT2, GSTTP2, DDT and DDTL genes respectively whereas the deletions observed in the chromosomal regions 8p23-p21 (4303127–4337759) and the potential gene in this region is CSMD1 (OMIM: 612279). Moreover, the array CGH results deletions and duplication were also validated by using primer design of deleted regions utilizing the flanked SNPs using simple PCR and also by using quantitative real time PCR.
We found some of the de novo deletions and duplication in our study in Saudi family with intellectual disability and epilepsy. Our results suggest that array-CGH should be used as a first line of genetic test for epilepsy except there is a strong indication for a monogenic syndrome. The advanced high through put array-CGH technique used in this study aim to collect the data base and to identify new mechanisms describing epileptic disorder, may help to improve the clinical management of individual cases in decreasing the burden of epilepsy in Saudi Arabia.
KeywordsEpilepsy CNVs Intellectual disability Array-CGH Saudi population
Array comparative genomic hybridization
Central nervous system
Copy number variations
CUB and sushi multiple domains 1
Database of Genomic Variant
Glutathione S-transferase theta 1
Late comified envelope
Real-time quantitative PCR
Epilepsy in human is among one of the utmost common brain disorder with 1 % occurrence and characterized by regular and unprovoked seizures due to irregular electric activity in central nervous system (CNS) . Until now there are more than 50 different epileptic syndromes with wide range of clinical features have been identified. Epilepsy can be divided as idiopathic or symptomatic epilepsies. Infections, metabolic disorders, brain tumors, head trauma, or stroke are the main reasons of symptomatic epilepsy whereas genetic contributions are the main cause of the idiopathic seizure . In human genetics structural changes are largely unexplored area that may be the common factor of epileptic disease . These changes include deletions, duplications insertions, inversions, and translocations of DNA sequences, and includes copy number differences also known as CNVs [4, 5]. Recently the development of advanced technologies for genome wide studied such as array-CGH helped to detect minute amplifications and deletion in the chromosomal region [6, 7]. CNVs are the most important component of genetic variation and act as important players in the genetic etiology of various neurodevelopmental disorders. CNVs are developing as an important genetic contribution to identify a wide range of epilepsies, such as common new discoveries in epilepsies from genetic generalized epilepsies to the individually rare particular epileptic of encephalopathies . The role of CNVs highlighted in the etiology of number of disorders such as intellectual disability (ID)  autism [10, 11], schizophrenia [12, 13] and epilepsy [14, 15]. Now a days various free databases for pathogenic and normal genome variations are available on internet and these are exceptionally valuable tools for interpretation of CNVs identified in normal and patients such as Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources: DECIPHER and Database of Genomic Variants: DGV;).
In this study, we observed de novo CNVs gain and deletion in a large family with affected mother and five affected kids having intellectual disability, epilepsy and some member with mild intellectual disability nd we hypothesized that genes found within those CNVs would be novel candidate for epilepsy. We are reporting de novo CNVs and the genes cluster in that deleted or amplified region only in the affected members of the family that might be helpful to understand the genetic etiology of epilepsy.
Results and discussion
Microarray data showed deleted CNVs
The gene CSMD1 is a unique multiple domain complement-regulatory protein, which is highly expressed in the epithelial tissues and CNS and play a role as important regulator of complement initiation and inflammation in the developing.
CNS and also play role in the context of growth cone function . The deleted region in our patient is at the start of CSMD1 and all the affected member having the language delay, learning difficulties and epilepsy as shown to be totally deleted (haploinsufficiency) and interrupted by the breakpoint patients with autism and ‘severe language delay’ and learning difficulties reported by [21, 22].
Microarray data showed CNVs copy number gain
We also found gain at chromosome 16 that starts from 70,647,078–70,669,681 cytoband 16q22.2 in our all five affected members of the family and the potential gene in this region is HPR gene code haptoglobin protein in plasma acts as a scavenger for free heme, and haptoglobin-related protein (coded by the HPR gene) (Fig. 4a). In African-Americans HPR gene copy number variation has been reported, where extra copies of the HPR gene have been produced by non-allelic homologous recombination .
Previously, duplication of HPR gene showed a slight, non-significant under transmission to human African trypanosomiasis-affected children from normal parents in the Democratic Republic of Congo . There is no study available that link this gene to epilepsy, intellectual disability and autism so we suggest further investigation of this duplication the Saudi population.
Our micro array data showed another copy number gain at chromosome 22 starts 22,686,690–22,735,300 cytoband 22q11.23 and the gene cluster in this region is GSTT1, GSTTP2, GSTT2B, GSTT2, DDT, and DDTL. Additional analysis through qPCR confirmed the duplications that has shown a significant fold increase in the GSTT1, GSTTP2, GSTT2B GSTT2 gene copy number compared to the controls (Fig. 4c). Glutathione S-transferase theta 1 (GSTT1) are a member of superfamily of enzymes that play role in both protection from oxidative damage and detoxification . In liver they play an important role in metabolizing antiepileptic drugs (AEDs) [29, 30]. Many AEDs produce active metabolites, including epoxides, which might have result on suppression of epileptic spike, but inappropriately, also in systemic toxicity through covalent binding to lipids and proteins . Glutathione S-transferase (GSTs) help in catalyzing the conjugation of these metabolites to glutathione, particularly in the elimination of epoxide metabolites that are produced during AEDs metabolism. GSTs not only help in decreasing their toxicity and helping their excretion from the body, it is expected that GSTs may also affect the response to anticonvulsant therapy [29, 31].
In the development of neurological disorders CNVs play important role and may contribute in the genetic etiology of epilepsy. Our results showed novel CNVs/genes for the first time in Saudi family with six affected member with intellectual disability and epilepsy. Our results enhanced the knowledge of the copy number variants underling in epilepsy in Saudi population. This could provide a basis for the understanding of the chromosomal region that will be critical in the genome may be involved in the development of epilepsy, intellectual disability and further linked with schizophrenia. Further detailed studies and complete clinical information of the patients and identification of possible causative CNVs and genes in the CNV regions using array technology will be obligatory to identify novel syndromes that will help to improve the diagnosis of epilepsy.
A large consanguineous pedigree of epileptic’s family with six affected (two female and all four male affected) and two normal individuals along with normal father and affected mother was enrolled for this study as shown in Fig. 1. Written informed consent was obtained from the study participants themselves or from the parents of children. The study was approved by the Center of Excellence in Genomic Medicine and Research King Abdulaziz University, following Helsinki Declaration of research and ethics standards. The family was primarily diagnosed as epileptic family and all the children and mother having epilepsy along with different clinical features such as mother was diagnosed as juvenile myoclonic epilepsy as the seizure started at the age of 16 years whereas the proband IV-1 having epilepsy with myoclonic seizures, muscle twitching along with uncontrolled jerking of the body sickle cell anemia and osteoporosis, proband lV-4 having epilepsy with myoclonic seizures and mild intellectual disability, while proband IV-6 having seizure with muscle twitching and uncontrolled body jerk.with speaking problems and IV-7 and IV-8VIII having seizure with myoclonic jerks with intellectual disability. In all the affected member of the family the seizure was started at different age group such as in mother seizure started when she was 16 years old and still suffering with this disease.
All the family member visited the clinic and blood samples from affected and normal individuals has been taken at King Abdulaziz University Hospital with their informed consent after the approval of the ethical committee. Genomic DNA was obtained from the blood through the QIAamp DNA blood mini kit followed by the protocol from the manufacturer.
Genomic DNA fragmentation
The array-CGH analysis was performed by Agilent sure print G3 Hmn CGH 2x 400 K arrays (Agilent Technologies, USA) followed the protocol as provided by the manufacturer. Briefly, 500 ng of patient’s DNA along reference DNA of the similar sex (Promega, USA). RsaI and AluI enzymes used to digest the DNA and incubated for 2 h at 37 °C. The reference DNA was heat fragmented for 10 min at 95 °C.
Fluorescent labeling, purification and hybridization
The digested DNA from affected and normal family member were labeled via random priming through Agilent labeling kit (Agilent Technologies, USA) following the manufacturer guidelines. Patient’s and reference DNA was labelled with Cy5-dUTPand Cy3-dUTP respectively. For the purification of the labelled samples, the Microcon YM-30 filter units were used (Millipore, USA). The DNAs from patient and reference were mixed with Cot-1 DNA (Invitrogen, USA) used as blocking agent along with hybridization buffer as per manufacturer instructions. Hybridization was done at 65 °C for 40 h, after the denaturation at 95 °C.
Microarray chip washing and scanning
After the hybridization, microarrays and gaskets were disassembled in wash buffer 1 (Agilent Technologies). The slides were shifted to the wash buffer 2 after 05–30 min, (Agilent Technologies) and agitated at 37 °C for 01 min followed by the washing of slides with anhydrous acetonitrile. Scanning and image analysis were performed as per oligonucleotide array-CGH protocol (Agilent, version 4.0). Microarrays were scanned through Agilent Scanner (G2505C) and the data was extracted through Agilent’s Feature Extraction software (V.220.127.116.11).
Agilent CytoGenomic Analytics software (V.18.104.22.168) was used to visualize, detect and analyze the aberrations deletion and duplication from microarray profiles using Tiff images from data.
List of gene primers used in the present study
CSMD1 exon 1
CSMD1 exon 1
GSTTP2 exon 5
GSTTP2 exon 5
LCE3C exon 5
LCE3C exon 5
HPR exon 1
HPR exon 1
This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH) – King Abdulaziz City for Science and Technology - the Kingdom of Saudi Arabia – award number (12-BIO3059-03). The authors also acknowledge with thanks Science and Technology Unit, King Abdulaziz University for technical support.
This article has been published as part of BMC Genomics Volume 17 Supplement 9, 2016: Proceedings of the 3rd International Genomic Medicine Conference: genomics. The full contents of the supplement are available online at http://bmcgenomics.biomedcentral.com/articles/supplements/volume-17-supplement-6.
Availability of data and materials
MIN, MMJ, MR and AGC designed the study. MIN, FTA performed the array CGH experiments, MIN, SKZ, SAA and MR analyzed the data and wrote the manuscript, FA, MA, GK and MHQ edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All authors have read and approved the final manuscript. Authors give BioMed Central the consent to publish this article.
This study was approved by the Research Committee of the Center of Excellence in Genomic Medicine Research (CEGMR) Ethical Unit, King Abdulaziz University, Jeddah, Saudi Arabia.
This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH) – King Abdulaziz City for Science and Technology - the Kingdom of Saudi Arabia award number (12-BIO3059-03).
Publication fees for this article are paid by the Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
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