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|Year : 2015 | Volume
| Issue : 2 | Page : 215-219
Genetic analysis of a Chinese family provides further evidence for linkage of familial cortical myoclonic tremor with epilepsy to 5p15.31-p15
Caixia Liu1, Wei Sun2, Qiuhui Chen1, Jia Li1, Guohua Hu1
1 Department of Neurology, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, 130041, Jinlin, China
2 Department of Circulation, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, 130041, Jinlin, China
|Date of Web Publication||5-May-2015|
Dr. Guohua Hu
Department of Neurology, The Second Hospital of Jilin University, 218 Ziqiang Road, ChangChun - 130 041, Jinlin
Source of Support: None, Conflict of Interest: None
Aim: In this study, we genotyped eight microsatellite markers on chromosome 5 and performed linkage analyses. We aimed to establish the pathogenic gene loci in this familial cortical myoclonic tremor with epilepsy (FCMTE) pedigree.
Materials and Methods: Reliable clinical information was obtained on the Chinese family members. Our study performed linkage analysis across these loci to identify and further characterize the pathogenic gene locus underlying FCMTE in
Results: Positive signals (>1) were only obtained for 5p15.31-p15 (Logarithm of Odds (LOD) values 2.16 and 1.34 for D5S1957 and D5S2095, respectively; θ =0.0), supporting involvement of this region in the FCTME pedigree analyzed.
Conclusion: Genetic analysis of a Chinese family provides further evidence for linkage of FCMTE to 5p15.31-p15.
Keywords: Chromosomal location; familial cortical myoclonic tremor with epilepsy; gene; linkage analysis
|How to cite this article:|
Liu C, Sun W, Chen Q, Li J, Hu G. Genetic analysis of a Chinese family provides further evidence for linkage of familial cortical myoclonic tremor with epilepsy to 5p15.31-p15. Neurol India 2015;63:215-9
|How to cite this URL:|
Liu C, Sun W, Chen Q, Li J, Hu G. Genetic analysis of a Chinese family provides further evidence for linkage of familial cortical myoclonic tremor with epilepsy to 5p15.31-p15. Neurol India [serial online] 2015 [cited 2022 Jan 22];63:215-9. Available from: https://www.neurologyindia.com/text.asp?2015/63/2/215/156283
| » Introduction|| |
Familial cortical myoclonic tremor with epilepsy (FCMTE), also known as benign adult familial myoclonic epilepsy (BAFME) or familial adult myoclonic epilepsy (FAME), is an autosomal dominant disorder with its onset primarily in adulthood. A few cases have been known to occur in the adolescent patients also. No significant gender differences have been noted. The pathogenic gene loci have been reported at 5 chromosomal locations: 8q23.3-q24.1; 2p11.1-q12.2; 3q26.32-3q28; 10p15, corresponding to a 2-8Mb region on the short arm of chromosome 10; and, 5p15.31-p15. Our study performed linkage analysis across these loci to identify and further characterize the pathogenic gene locus underlying FCMTE in Chinese patients. Positive signals (>1) were only obtained for 5p15.31-p15 (LOD values 2.16 and 1.34 for D5S1957 and D5S2095, respectively; θ=0.0), supporting the involvement of this region in the FCTME in 9 patients in 4 generations of the family analyzed.
| » Materials and Methods|| |
Pedigree analysis and clinical presentation
The data of nine patients in 4 generations of a family diagnosed with familial tremor of cortical myoclonic epilepsy (FCMTE) were collected by the Department of Neurology of the Liaoning Province People's Hospital in 2011. The clinical features of affected individuals were consistent with FCMTE diagnostic standards:  (1) The incidence of the disease occurred across three generations or more; adult onset occurrence with both genders affected; and, autosomal dominant inheritance; (2) the primary disease manifestations included distal limb tremors and seizures; some patients presented with comprehensive tonic seizures after light and emotional stimuli or startle; (3) the symptoms were non-progressive and effectively managed with oral phenobarbital; and, (4) somatosensory evoked potentials supported a cortical source for the tremors. In the 4 generations of the family used in this analysis, there were 8 confirmed cases (6 female and 2 male patients), and one suspected case [Figure 1]. Four of them (ΙΙ.2, ΙΙ.6, ΙΙ.13, and β.2) had a history of headache, while 4 (ΙΙ.2, ΙΙ.6, ΙΙ.11, and β.2) had tremors. All the 8 patients suffered from tonic-clonic seizures with the onset of manifestations occurring after the age of 30 years. Some patients suffered attacks after a light stimulation, an evoked startle, or an emotional stimuli. The suspected case, IV.13, was young (3 years old), but suffered from numerous convulsive seizures whenever he developed a high fever. The cranial imaging and electroencephalography (EEG) of the 7 surviving patients showed non-specific changes [Figure 2]. Elevated potentials were observed following stimulation of the left and right median nerves to record the somatosensory evoked potential (SEP) [Figure 3].
|Figure 1: The four generations of a Chinese family with familial cortical myoclonic tremor with epilepsy (FCMTE) analyzed in this study. The generations are indicated with roman numerals (I-IV). The identities are marked as follows: Affected male (⋅) or female (·) individuals; Normal male (▫) or female (˚) individuals; Proband (↗); Died (/); Suspected cases (⦿); Abortion(·)|
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|Figure 2: Electroencephalogram (EEG) of the proband. The basic firing rate is 9-10 per second, with medium amplitude, generally regular frequency, poor amplitude modulation, and partial inhibition of the visual reflex Alpha wave. The traces show paroxysmal rate abnormalities of 8 per second with high amplitude and slow activity, especially in the frontal and central regions|
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|Figure 3: Somatosensory evoked potential (SEP) of the proband (a) Left Amplitude is 19.5 μ V. (b) Right Amplitude is 13.5 μ V|
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Sample collection and DNA extraction
After obtaining informed consent, peripheral blood (5 mL) was obtained from the FCMTE patients (7 patients), the suspected case (1 patient) and control individuals (lineal relatives and spouses of patients; 13 cases). Genomic DNA was extracted using the standard phenol chloroform method. Blood genomic DNA extraction kits of Tiangen Biotechnology Company were utilized for this procedure.
Short tandem repeat (STR) selection and amplification
STRs in each of the chromosomal regions of interest for FCMTE, located at the appropriate genetic distances for linkage analysis, were identified from the National Center for Biotechnology Information (NCBI) map-view Atlas More Details of chromosome segments 8q23.3-q24.1, 2p11.1-q12.2, 3q26.32-3q28, 10p15 (corresponding to the 2-8M short arm of chromosome 10) and 5p15.31-p15 in patients with FCMTE as well as controls [Table 1].
The amplification primers for the selected STR sites were synthesized with a 5' FAM label for genotyping. The conditions for polymerase chain reaction (PCR) amplification of STRs obtained from patients with FCMTE as well as controls were as follows: PCR reaction mix (15 µL) containing 5 pmol each of labeled downstream primers and non-labeled upstream primers was added to 60 ng starting genomic DNA (50ng/µl). PCR was performed on a German biometra thermocycler using touch-down PCR with 10 cycles at 95°C for 12 minutes, 94°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds, and then with 20 cycles at 89°C for 15 minutes, 55°C for 15 seconds, 72°C for 30 seconds. The PCR products were analyzed by gel electrophoresis.
Short tandem repeat (STR) genotyping
STR repeat lengths were determined by analyzing PCR products using capillary electrophoresis. Purified PCR products were run on a separation gel composed of POP-4 polymer in the ABI PRISM 3730 DNA Analyzer (Applied Biosystems, USA), at 15 KV, 60°C, for 30 min. The data was analyzed using the ABI PRISM GeneMapper Software (Ver 3.5), which combines both GeneScan and Genetyper software packages and collects experimental data, analyzes the molecular weight of the internal calibration control and the amplified fragments, and provides readout of all the microsatellite marker allele fragment sizes.
Genotypes were determined for each STR marker using GeneMapperV3.5 according to the peak and the retrieved data was analyzed.
According to the pedigree analysis and genetic characteristics, the family inducted in this study demonstrated autosomal dominant genetic inheritance of the disease gene. We utilized the software package Linkage 5.1 to perform linkage analysis for FCMTE in this family. We defined the gene frequency, disease penetrance and phenocopy frequencies in Linkage 5.1, and set the recombination rate at θ =0-0.5 prior to starting the linkage analysis. The calculated LOD (logarithm ratio of probability) score indicates the recombination rate (θ) under different conditions, and reflects the possibility that two loci (such as an STR marker and a disease gene) are inherited together.
| » Results|| |
Linkage analysis using STRs from chromosome segments 8q23.3-q24.1, 2p11.1-q12.2, 3q26.32-3q28, 10p15 (corresponding to the 2-8M short arm of chromosome 10), and 5p15.31-p15, showed that the LOD scores resulting from two point linkage analysis, corresponding to STR loci and the causative genes, were all <1, except at the putative disease causative gene in chromosome 5. Linkage analysis utilizing 8 STRs from within 5p15.31-p15 showed that for D5S1957 and D5S2095, the LOD values were 2.16 and 1.34 at θ =0.0. As these values were >1, the experimental results support a conclusion of positive linkage within the pedigree, and suggest that the pathogenic gene is within the 5p15.31-p15 chromosomal segment [Table 2].
|Table 2: Two-point linkage analysis of STR loci and the disease gene in 5p15.31-p15 in the FCMTE pedigree |
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| » Discussion|| |
Familial cortical myoclonic tremor with epilepsy (FCMTE) has also been described as benign adult familial myoclonic epilepsy (BAFME) and familial adult myoclonic epilepsy (FAME). It shows an autosomal dominant inheritance and usually occurs in adults (although in a few individuals, it may also occur in adolescence). There are no significant gender differences. FCMTE is a non-progressive disease, and the main manifestations are a slight tremor or myoclonic seizures in the distal limbs, with or without epileptic seizures. Seizures may be elicited by sleep deprivation, light stimulation or emotional stress. Antiepileptic drugs are effective, but beta-blockers or alcohol are not. Some reports suggest that FCMTE may be associated with other symptoms, such as migraine, night blindness, or ataxia, and in a small group of families, the elderly patients may also have mild cognitive impairment.  Cranial imaging and EEG are usually unremarkable. An electrophysiological examination shows that the myoclonus or tremor originates from the cerebral cortex. Till date, more than 100 families with FCMTE have been reported worldwide, including those in China, Japan, Holland, Italy, and Turkey. Maximum number of cases have been reported from Japan and Italy; more than 60 cases have been found in Japan alone. The previously reported patients with FCMTE from China have shown cortical tremors and generalized tonic-clonic seizures; however, none of them have had headaches. In this study, we report a family from China who not only exhibited tremor and generalized tonic-clonic seizures in affected members, but also presented with headache, ataxia, and anxiety, in line with the characteristic clinical heterogeneity associated with FCMTE.  The overall clinical characteristics of the family studied corresponded with the diagnostic criteria proposed for FMCTE.
The etiopathogenesis of this disease remains unknown. It has been suggested that the disease may be associated with ion channel defects;  however, defects of GABA receptors signaling in the cerebellar-thalamo-cortical projection loop, leading to increased stimulation of cortical excitability, may also have some contributory role. 
FCMTE not only has clinical heterogeneity but also exhibits genetic heterogeneity. , In the earlier years, the study of Japanese families and an Italian family showed that the pathogenic genes were roughly localized to 8q23.3-24.1 and 2q11.1-q12.2, and the foreign genetic analysis showed that these loci were unlinked, and likely contained separate disease-causing genes. ,, In the recent years, a number of loci related to FCMTE at 5p15.31-p15, 3q26.32-3q28 and 10p15, among other sites using subjects in France, Thailand and China, have also been discovered. ,, In the present study, we selected STR polymorphism markers from the chromosome 5 segments reported at home and abroad, and performed linkage analysis. Multiple STR linkage analysis of 8q23.3-q24.1, 2p11.1-q12.2, 3q26.32-3q28, and 10p15 did not support linkage to these regions, indicating that the pathogenic gene contained in the pedigree we studied is not in these 4 chromosomal segments. Linkage analysis of 5p15.31-p15 using 8 STRs shows potential linkage to D5S1957 and D5S2095, with θ=0.0, and LOD values of 2.16 and 1.34. As these LODs were >1, the experimental results support the pedigree as containing a site of linkage, and suggest that the pathogenic gene is within the 5p15.31-p15 chromosomal segment. The research results are similar to those obtained with the FCMTE family study reported by Jia Li.  We, therefore, conclude that the domestic FCMTE locus is mainly comprised of the 5p15.31-p15 gene locus. We are still searching for other new loci as well. Shimizu et al. in 2003 reported that CSMD3 and ACMSD may be candidate genes for FCMTE. Although their study did not find clear mutations in the coding region of the CSMD3 gene, they found 24 possible single nucleotide polymorphisms, including 18 new polymorphic sites. , But according to reports, the association between the CSMD3 and ACMSD genes and familial FCMTE patients in China has rarely been reported. In future studies, rather than treating the CSMD3 and ACMSD genes as two independent entitites, we will study the two genes together and discuss the site at which CSMD3 and ACMSD genes exist in linkage relationship in FCMTE.
In conclusion, we believe that with continuous progress in genetic decryption and in the application of molecular biological techniques to human genomic science, people will deepen their understanding of the disease and the exact pathogenesis of familial cortical myoclonic termor with epilepsy will be further elucidated.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
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