Capture And Identification Of New Pathogenic Genes In Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

Epilepsy is one of the most common disease of the central nervous system. According to the WHO data, there are9million epilepsy patients in China.2/3of this patients is children. Repeated seizures not only affect children’s health, but also threaten patients’life. The etiopathogenisis of epilepsy is complex. Genetics play a important role in pediatric patients. The International League Against Epilepsy makes genetic epilepsy to be a new categorization in2010. Pathogenesis of epilepsy is caused by the abnormally synchronized discharge of cerebral neural cells. Any gene mutants which are likely to change ion channel function will cause epilepsy. So we consider those genes which encode the ion channel as the candidate pathogenic genes. The animal model studies such as tottering mouse model show that mutations or deletions of the encoding gene can induce seizures. And some of idiopathic epilepsies are caused by mutations of ion channel. For example, mutations in sodium and chloride ions channel result in generalized epilepsy with febrile seizures plus disease （GEFS+）.In addition, mutations in potassium ion channel lead to benign familial neonatal convulsions （BFNC）. This evidences show that the change of ion channels is one of the most important pathogenesis of epilepsy.Nocturnal frontal lobe epilepsy （NFLE） is an idiopathic partial epilepsy. In 1994, Scheffer reported a family had several NFLE patients. He found that all the patients in this family was inherited follow autosomal dominant model. And then Scheffer named it as autosomal dominant nocturnal frontal lobe epilepsy（ADNFLE）. ADNFLE was the first human idiopathic focal epilepsy with specific gene mutations. Most ADNFLE patients often onset during light sleep in children. The patients start this symptom from two months to fifty-two years old and ninety patients often onset before20years old. The incidence rates are similar between male and female. ADNFLE is characterized by clusters of brief seizures during light sleep.This symptom are often misdiagnosed as nightmares. Patients’nervous system physical examination, imaging studies and interictal EEG is always normal. The ictal EEG always shows Sharpe wave, spike wave or slow-sharp wave in frontal lobe. Sporadic cases have similar clinical and EEG features with familial cases. But the complexity and severity of clinical symptoms is different in each patients. The penetrance in most ADNFLE families is about70-80%.In contrast, the incidence of sporadic cases is very low. ADNFLE is often diagnosed by the clinical grounds for there are no specific diagnostic criterias for this disease. So, it is greater difficult to diagnose ADNFLE and finding a effective way to diagnose this disease is urgent.Over the past decade, several researches have showed that genes encoding subunit of neuronal nicotinic receptors（nAChRs） have a close relationship with ADNFLE. But there are some differences in different races. CHRNA4gene encondinga4subunit of nAChRs was the first gene identified as ADNFLE pathogenic gene by Steinlein. Then other encoding subunit of nAChRs like CHRNB2and CHRNA2succession are considered as the ADNFLE disease genes. It has been speculated that the sporadic cases and family cases have the same genetic background, but most of the known nAChR gene mutation exists only in the patients of the family, while sporadic cases rarely found. The Lebanese sporadic patient who carries a mutation of CHRNA4（S256L） has been identified as a family case later. China have reported a missense mutation R308H in CHRNA4in a sporadic case. The role of neuronal acetylcholine receptors （nAChRs） coding subunits genes has been clearly established in ADNFLE. Scientist speculate ADNFLE is genetically heterogeneous for only about10%of the ADNFLE family, and there must be another pathogenic gene.There is still no clear association between phenotype and genotype. The clinical symptoms are different between families, and even among patients in the same family. Patients who have same phenotype may carry different gene mutations.It is difficult to look for new pathogenic gene because ADNFLE has significant phenotype heterogeneity and genetic heterogeneity. The traditional method is time-consuming, and needs many human and material resources. It is difficult to meet the current requirements of the large-scale and high-throughput sequencing. Whole genome sequencing become the main technology to identificate pathogenic gene. Whole exome sequencing is a high-throughput method which can capture and enrich the whole genome exon regions. It only sequence1%of the whole genome. Heron identified a new pathogenic gene in three ADNFLE families and a sporadic case by whole genome sequencing technology.We have collected two hundreds and fifty-seven ADNFLE patients, including eleven family forms and two hundreds and fifteen sporadic cases. We seek for mutation by sequencing all the exons of CHRNA4, CHRNB2and CHRNA2, but we failed to find the known mutations. In this study, we plan to capture and identified a new pathogenic gene in Chinese ADNFLE families by perform whole exome sequencing. Finally, the results of the studies may be helpful to provide the new directions in pathogenesis and genetics research of Chinese patients with ADNFLE.[Subjects and Methods] 1. Subjects:In the early research we collected Chinese Han populations a total of257cases who was diagnosised with ADNFLE, including42cases from11unrelated familial pedigrees and215sporadic cases. The diagnosis of ADNFLE followed the criteria established in the2001International Classification of Epileptic Syndromes.500healthy people were used to be a control groups. All patients were asked for medical and family history, and performed the cranial MRI and video-polysomnography. Then we recorded the database of all patients. We extracted DNA from Peripheral venous blood in all cases. All the subjects signed informed consent by themselves or their legal guardians, the study was approved by the Ethics Committee of the Guangdong Provincial People’s Hospital.2. Whole exome sequencingIn the early research, we failed to find the known mutations of CHRNA4,CHRNB2and CHRNA2in all cases, we only find a novel synonymous mutation （D190D） in the exon5of CHRNA4. In this study, we conducted whole exome sequencing in four affected individuals from the family1.3.Verified mutation by Sanger sequencing3.1At first we verified the mutation harboring in four affected individuals. Data were analyzed with the chromatogram file editor software Chromas2.22, and compared with the standard sequence of the gene in order to rule out false positive variants, positive variants for further verification3.2The positive variants in other member of family1were verified. We excluded the variant which don’t exist in patients because of ADNFLE significant genetic heterogeneity.3.3We sequenced all exons of the gene got from step3.2in all cases. If a mutation was found, we verified it by comparing with NCBI SNP database and500healthy controls.3.4At first we verified the mutation harboring in eight sporadic affected individuals, then the positive variants were identified in215sporadic ADNFLE patients. If there is not positive variant, we choose the variants which have been reported associated with epilepsy to verify.[Result]1. We obtained SNP and Indels of12samples （including four pedigree member and eight sporadic cases） by whole exome sequencing;2. We test seven affected members and one unaffected individual of family1and identify a novel mutation in the third exon of CaBP4gene(（c.464G>T, p.G155D）. Comparing with the NCBI database, this mutations are highly conserved region. SIFT predicts the score is0.00, this result means that the mutations have great effect on protein structure. By analyzing the secondary structure of mutation point, the position is close with CaBP4protein binding ion domain; This mutation is not found in the control group.3. We failed to discover any mutations in all exons of CaBP4in other ten families and215sporadic cases.4. We failed to discover any mutation in all exons of CHRNA7in215sporadic ADNFLE patients, and find three coding nucleotide polymorphisms （cSNP） in exon5, exon6and exon7of CHRNA7（c.370G>A, c.698A>G, c.497₄98delTG） and two nucleotide polymorphisms （SNP） in exon7of CHRNA7（c.654>T、c.690G>A）。[Conclusion]1. We discover a novel missense mutation （p.G155D） in the third exon of CaBP4gene in a ADNFLE pedigree have seven affected individuals. This discover implicate CaBP4gene may be the new pathogenic gene of ADNFLE.2. The mutation in CaBP4gene were discovered first time in Chinese ADNFLE pedigree and it never been reported.3. We speculated that the CHRNA7gene may not be the chief mutation in Chinese population with ADNFLE.