Mutational Analysis Of CHRNB2 Gene In The Southern Chinese ADNFLE Population

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is recognized as a specific entity of inherited partial epilepsy and is characterized by clusters of brief seizures during night sleep that are often misdiagnosed as nightmares. ADNFLE was the first inherited epilepsy in which the underlying genetic abnormality was identified.Clinical onset is usually in childhood and the disorder often persists throughout adult life, with clusters of attacks arising from sleep. ADNFLE patients are often stereotyped and consist of brief stiffening of the limbs, accompanied by gradual turning of the head and dystonic movements of arms or legs. Grunting sounds, screaming and difficulty with breathing often occur with the seizures, and sleep-walking has also been reported. The penetrance in most known ADNFLE families is about 70-80% or much lower, although sporadic cases are also seen. Sporadic and familial forms have similar clinical and EEG features. There is intra and inter familial variability in severity. Usually the nervous system in patients of physical inspection and cranial CT, MRI imaging studies were normal. ADNFLE is genetically heterogeneous despite a relatively homogeneous clinical picture. The diagnosis of ADNFLE is made on clinical grounds. The interictal electroencephalograms (EEG) are seldom showed unormal, and even ictal recordings do not always show definitive epileptiform activity. At present, there is lack of specific diagnostic criteria, so that it severely threatens the health and safety of the children affected with ADNFLE. The chief reason is that the pathogenesis of ADNFLE has not yet been make clear completely. At present, searching for the pathogenic genes in ADNFLE to explore its pathogenesis has become a research focus for the neurologist.ADNFLE was the first human idiopathic focal epilepsy for which specific gene mutations were described. The role of neuronal acetylcholine receptors (nAChRs) coding subunits genes has been clearly established in ADNFLE including the a4, a2 and b2 (CHRNA4, CHRNA2, and CHRNB2) subunits associated. This recent finding strongly reinforces the link between nocturnal frontal lobe epilepsy and neuronal nicotinic receptors. CHRNB2 is the second mutant nAChRs subunit gene associated with ADNFLE. So far there are five missense mutations in CHRNB2 have been found in several different ethnic probands. However, the genetic basis of the disease is still unkown in the majority of ADNFLE families, and nearly all sporadic cases were negative for CHRNB2 mutations. These mutations located the transmembrane domains (TM) 2-3 in the fifth exon of CHRNB2. Research show the mutations exhibit differences in their respective response profile and calcium permeability all mutations found so far cause an increase in receptor sensitivity to Ach. This suggests that the cause triggering seizures in ADFNLE is attributable to the increase in Ach sensitivity.More than a hundred ADNFLE families have been reported to date. There are still no clear association between phenotype and genotype. ADNFLE is genetically heterogeneous despite a relatively homogeneous clinical picture. Clinically, sporadic cases of nocturnal frontal lobe epilepsy with no demonstrable structural brain lesions are more common than those ADNFLE family cases, and there are no phenotypic differences between the sporadic and familial forms. Despite the sporadic cases and family cases have been speculated that perhaps have the same genetic background, but most of the known nAChR gene mutation exists only in family cases. To date there have been two cases of sporadic cases found each gene mutation in CHRNA4, but other mutations are not confirmed in CHRNB2 and CHRNA2 gene in sporadic NFLE.1 sporadic case in which the Lebanese descent carrying S256L, subsequently confirmed to family cases. Recently, China first reported one sporadic case found a novel missense mutation R308H in the CHRNA4. However, domestic ADNFLE pedigrees are rare, the disease-related molecular biology was still weak, and lack of large sample ADNFLE gene screening studies. The genetic background of Chinese ADNFLE population need to be further explored.In the early research we collected southern Chinese Han population of 106 patients, including 74 sporadic cases and 32 patients from eight unrelated familial pedigrees. We failed to find the known mutations in screening CHRNA4 gene. In this study we decided to make a mutation detection by using the direct sequencing (DS) of PCR product in all the exons of CHRNB2 to search the virulence gene and mutation feature in Chinese patients of ADNFLE. Finally, the results of the research may be helpful to interpret the molecular pathogenesis in Chinese patients affected with ADNFLE. The pathogenesis and genetic basis of ADNFLE will provide a scientific basis and new targets establishing a pedigree for the patient to provide genetic counseling and early diagnosis and prenatal diagnosis of genetic diagnostics system, thereby contributing to the early prevention and treatment ADNFLE.[Materials and Methods]1. Subject:The study included 106 patients of the Han-nationality population in southern China with ADNFLE. Among of them 32 familial patients came from 8 unrelated pedigrees and 74 patients were sporadic cases. Two hundred healthy samples from the same area were used as controls. We enrolled those patients who visited the pediatric department of the Guangdong General Hospital between January 2006 and December 2008.2. Clinical data:All patients were diagnosed by the clinical history, EEG/ictal VEEG and cranial magnetic resonance imaging (MRI). Diagnosis of ADNFLE followed the criteria established in the 1989 International Classification of Epileptic Syndromes. All patients underwent clinical and full-night video-polysomnography, as·described.4 A family history was reviewed during counseling and family members were traced back at least three generations. The presence of at least three affected members in a single family was considered evidence of a genetic transmission, while the presence of a single affected individual was considered as a sporadic case.3. Methods:the Screening of the genetic mutation of CHRNB2:Patients and healthy individuals included in this study underwent 200ul peripheral blood sampling for genotype analyses. Genomic DNA was isolated from peripheral blood using a DNA extractor kit. The exons 1 to 6 in CHRNB2 were amplified by the PCR. The amplified products were sequenced and analyzed. Data were analyzed with the chromatogram file editor software Chromas 2.22. The sequences obtained from patients' DNA were compared with the CHRNB2 cDNA sequence.[Result]1. In this study we collected 8 ADNFLE pedigrees, with a total 35 patients, male 17, female 18 cases, male:female was about 1:1; survival in 32 patients, including 8 probands. an average age of onset 5.5±0.5 years, each family in patients≧3, simple segregation analysis indicates that the genetic model are autosomal dominant inheritance. A total of 74 cases of sporadic cases, male 42 cases, female 32 cases, male:female was about 1.3:1, with an average age of onset of 12.7±0.8 years. Clinical manifestations of all patients are in line with nocturnal frontal lobe epilepsy diagnostic criteria. The main manifestations of them involved in the night suddenly eyes open, sit, violent over-activity, limb and trunk dystonia, and dyskinesia kind of uncoordinated movement, abnormal posture, walk, such as fear and involuntary audible. Some patients may be secondary GTCS. Patients within the family there are different types of attack, a handful of automatism. EEG mostly occurred in non-rapid eye movement phase (non-REM) in the second stage, abnormal EEG, mostly one or both frontal epileptiform electrical activity or the limitations of slow-wave increased. In most patients seizures were respond to carbamazepine, oxcarbazepine, valproate and topiramate and other antiepileptic drug treatment.2. All the 6 coding exons of the CHRNB2 were sequenced bidirectionally for muta-tions. None of the five kown missense mutations were found in any coding exons of CHRNB2 either in 32 familial affected individuals or 74 sporadic cases.3. A novel synonymous coding region mutation in one sporadic patient was identifi-ed:c.483C>T, which does not change the encoded His residue in the exon5.This nucleotide variation was present a heterozygous form and absent in 200 control healthy samples. The mutation was not found in his mother who has been classified as unaffected. It was regretted that his father has not been available for blood test in this research.. It didn't make the type of the protein changed. But the substitutions may affect the abundance of the proteins. It may be one of the reasons that induce the disease. Another sporadic case found a coding nucleotide polymorphisms (cSNP) c.1407C>G, which are also heterozygous mutation located in CHRNB2 gene exon 6. It did not change the expression of the amino acid (Val), However, in the 2/200 cases of healthy people wre also available.[Conclusion] 1. We have successfully sequence all the exons 1-6 of CHRNB2 in this study. No gene mutation in kown regions of kown was found. We concluded that the CHRNB2 may not be the chief virulence gene in southern Chinese ADNFLE population.2. In the two sporadic cases we discovered a new synonymous mutation H161H (c.483C>T) and a coding single nucleotide polymorphisms, cSNP (c.1407C>G.), all of them have not been reported before. The novel synonymous mutation H161H (c.483C>T) does not change the encoding amino acid sequence, but the impact on the transcription of mRNA or nAChR receptor function, and pathogenesis of ADNFLE has yet to be further studied. Another sporadic case was found a coding single nucleotide polymorphisms (c.1407C>G). It also showed a heterozygous mutation and was also found in control healthy samples. This means it may not be the underlying factors causing ADNFLE pathogenesis.