The Research On NAChR Gene And CSNP Of Chinese Patients With ADNFLE

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is recognized as a idiopathic focal epilepsy syndrome, it is a specific entity of inherited partial epilepsy and often onset during light sleep.Most patients are children.The age of onset was from two months to fifty-two years and the symptoms can often persists throughout adulthood.Male and female incidence rates were similar. ADNFLE patients are often twitching, dystonic movements of arms or legs screaming and grunting sounds, with clusters of attacks arising from sleep.The disease is often mistaken for sleep disorders. The penetrance in most ADNFLE families is about70-80%and sporadic cases can be seen occasionally.The diagnosis of ADNFLE is often made on clinical grounds and there are lack of specific diagnostic criterias, The interictal electroencephalograms (EEG) and cranial CT or MRI imaging studies were normal.Sporadic cases and familial forms have similar clinical features and EEG characteristics. But the complexity and severity of clinical symptoms were different in each patients.At present, the diagnosis of ADNFLE have no specific diagnostic criterias,so there were great difficulty in diagnosis and differential diagnosis.ADNFLE have a serious threatens on children,So to investigate its pathogenesis and searching for the new genes in ADNFLE has more practical significance.The ADNFLE first described by Ingrid E. Scheffer in1994, with clusters of attacks arising from sleep. Steinlein was the first person to discover the pathogenic genes, ADNFLE was the first human idiopathic focal epilepsy with specific gene mutations.Many studies reported the nocturnal frontal lobe epilepsy and neuronal nicotinic receptors have a close relationship,but there are some racial differences.At present,most scientists believe this disease is caused by the gene mutation:CHRNA4、 CHRNB2and CHRNA2, CHRNA4which locate in the chromosome20q13.2is the main mutation related with ADNFLE, Ser248Phe、263insL. S256L and T265I have been confirmed with ADNFLE. CHRNB2which locate in the chromosome Iq21and encoding the receptor β2subunit of nAChRs is the second reported gene mutation with ADNFLE, So far there are six missense mutations in CHRNB2have been reported in different pedigrees by scholars:V287L(Italy), V287M(Scotland,Spain), I312M (Switzerlang,South Korea), L301V (Turkey), V308A (Scotland,England), V337G (China),All five kinds of mutations are located in the fifth exon of CHRNB2.CHRNA2which locate in the chromosome8p12.3-q12.3is the third gene associated with ADNFLE and only I279N(Italy)was reported.Most scholars believe that the gene mutations of nAChR cause this diease by changing the channel function.ADNFLE has significant genetic heterogeneity,each members of families has different clinical manifestations.There are no phenotypic differences between the familial forms and sporadic cases,So we speculated that sporadic and familial forms have the same genetic background. but most of the reported gene mutation exists only in family patients,only a few sporadic cases have these gene mutations.The research of ADNFLE is still at the clinical level in domestic.However, domestic studies of ADNFLE were in the comprehensive analysis of the EEG and clinical assistant examination.The pathogenesis of Chinese ADNFLE population need to be further studies.In this experiment, the total number of the ADNFLE patients is two hundreds and fifty-seven,including eleven family forms and two hundreds and fifiteen cases of sporadic patients.In the early research,we have made a mutation detection by the PCR with all the exons of CHRNA4and CHRNB2,We failed to find the known mutations.In this study,we decided to make a mutation gene screening by the PCR with all the exons of CHRNA2、CHRNB2and CHRNA4to search the new mutations with ADNFLE in Chinese patients.Finally, the results of the studies may be helpful to provide the new directions in pathogenesis and genetics research of Chinese patients with ADNFLE.The studies of ADNFLE will provide new drugs to control the onset of this disease and improve the quality of life in children,so this experiment have a great significance.[Subjects and Methods]1. Subjects:We collected Chinese Han population of two hundreds and fifty-seven patients who visited in Guangdong people’s Hospital between January2006and December2012,including eleven families and two hundreds and fifiteen cases of sporadic,The diagnosis of ADNFLE followed the criteria established in the2001International Classification of Epileptic Syndromes.Two hundred cases of healthy people were used as controls.2. Clinical data:All patients underwent the cranial magnetic resonance imaging (MRI)and full-night video-polysomnography,we create the database of all patients.3. the Screening of the genie mutation of CHRNA4、CHRNA2and CHRNB2(1) the genomic DNA was extracted from peripheral blood of two hundreds and fifty-seven patients and two hundreds healthy controls.(2) According to standard genome sequence of CHRNA4、CHRNA2and CHRNB2,we designed primers for all exons.The exons were amplified by the PCR.(3) The PCR products were sequenced and analyzed by ABI3730,Data were analyzed by Vector NTI8.0analysis software and compared with the standard genome sequence.The mutation gene need to be validated in control group to get rid of cSNP.[Result]1. In this study, we collected two hundreds and fifty-seven patients of ADNFLE, male to female ratio was about1:1, including eleven probands and wo hundreds and fifiteen cases of sporadic,According with the relevant information and analysis for all the families indicates that the genetic model are autosomal dominant inheritance.all patients were diagnosed as nocturnal frontal lobe epilepsy. 2. In the early research,we have made a mutation detection by the PCR with all the exons of CHRNA4and CHRNB2,We failed to find the known mutations. All the exons of three genes have been amplified by PCR successfully in two hundreds and fifty-seven patients of ADNFLE. And the product of PCR have been sequenced.The result shows that the hot spot mutation of CHRNA4(S248F、S284L、263insL、T265I)、 CHRNB2(V287L、V287M、I312M、L301V、V308A、V337G) and CHRNA2(I279N)which have been reported do not exist.3.We find three base substitutions in exon2and exon5of CHRNA2(c.66C>T, c.249C>T, c.375A>G)and eight base substitutions in exon5of CHRNA4(c.639T>C, c.678T>C, c.1209G>T, c.1227T>C, c.1659G>A, c.1629C>T, c.57C>T).4. A novel synonymous coding region mutation which is located in the exon5of CHRNA4was identified, This mutation does not change the amino acid (D190D),It is not found in the control group.[Conclusion]1. All the exons of three genes have been amplified successfully in vitro by PCR with all the exons of CHRNA4. CHRNB2and CHRNA2in257ADNFLE patients.The sequencing result shows that the hot spot mutation which have been reported do not exist.We speculated that the CHRNA4、CHRNB2andCHRNA2may not be the chief mutation in Chinese population with ADNFLE.2. We find a mutation in exon5of CHRNA4(c.570C>T), The mutation does not change the encoding amino acid, but whether the impact on the transcription of mRNA has need to be further studied in the large number of samples with ADNFLE.