Isolation and characterization of 20 polynucleotide microsatellite markers in a vulnerable Korean snail, Ellobium chinense, using 454 pyrosequencing

현영세,송하연,우지영,오수민,김민섭,안혜석 한국유전학회 2017 Genes & Genomics Vol.39 No.2

A small and air-breathing snail, Ellobium chinense (Ellobiidae), is a vulnerable species by International Union for Conservation of Nature (IUCN). To protect and manage habitat and population of E. chinense, microsatellite markers were developed using 454 pyrosequencing and 20 polymorphic microsatellite markers were identified. A total of 146,704 sequences containing a minimum of four repeat motifs (mean, 631 base pairs) were identified from 499,505 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5 %) produced strong PCR products, of which 20 were polymorphic among 48 samples of E. chinense. All loci exhibited high genetic variability, with an average of 18.9 alleles per locus, and the mean observed and expected heterozygosities were 0.65 and 0.90, respectively. In addition, cross-amplification was tested for all 20 loci in the same family species, Melampus sincaporensis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 20 newly developed microsatellites will be useful in future conservation genetic studies of this species.

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species

현영세,김일훈,송하연,박대식,Mamoru Toda,Tein‑Shun Tai,안혜숙 한국유전학회 2018 Genes & Genomics Vol.40 No.4

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The crossspecies amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.

Novel Compound Heterozygous Nonsense PRX Mutations in a Korean Dejerine-Sottas Neuropathy Family

최예지,현영세,남수현,구혜수,홍영빈,정기화,최병옥 대한신경과학회 2015 Journal of Clinical Neurology Vol.11 No.1

Background Mutations in the gene encoding periaxin (PRX) are known to cause autosomalrecessive Dejerine-Sottas neuropathy (DSN) or Charcot-Marie-Tooth disease type 4F. However, there have been no reports describing Korean patients with these mutations. Case Report We examined a Korean DSN patient with an early-onset, slowly progressive,demyelinating neuropathy with prominent sensory involvement. Whole-exome sequencing andsubsequent capillary sequencing revealed novel compound heterozygous nonsense mutations(p.R392X and p.R679X) in PRX. One mutation was transmitted from each of the patient’s parents. No unaffected family member had both mutations, and the mutations were not found inhealthy controls. Conclusions We believe that these novel compound heterozygous nonsense mutations arethe underlying cause of DSN. The clinical, electrophysiologic, and pathologic phenotypes inthis family were similar to those described previously for patients with PRX mutations. Wehave identified the first PRX mutation in a Korean patient with DSN.

Exome Sequencing Reveals a Novel PRPS1 Mutation in a Family with CMTX5 without Optic Atrophy

박진,현영세,김예진,남수현,김승희,홍영빈,박진모,정기화,최병옥 대한신경과학회 2013 Journal of Clinical Neurology Vol.9 No.4

Background X-linked Charcot-Marie-Tooth disease type 5 (CMTX5) is caused by mutations in the gene encoding phosphoribosyl pyrophosphate synthetase I (PRPS1). There has been only one case report of CMTX5 patients. The aim of this study was to identify the causative gene in a family with CMTX with peripheral neuropathy and deafness. Case Report A Korean family with X-linked recessive CMT was enrolled. The age at the onset of hearing loss of the male proband was 5 months, and that of steppage gait was 6 years; he underwent cochlear surgery at the age of 12 years. In contrast to what was reported for the first patients with CMTX5, this patient did not exhibit optic atrophy. Furthermore, there was no cognitive impairment, respiratory dysfunction, or visual disturbance. Assessment of his family history revealed two male relatives with very similar clinical manifestations. Electrophysiological evaluations disclosed sensorineural hearing loss and peripheral neuropathy. Whole-exome sequencing identified a novel p.Ala121Gly (c.362C>G) PRPS1 mutation as the underlying genetic cause of the clinical phenotype. Conclusions A novel mutation of PRPS1 was identified in a CMTX5 family in which the proband had a phenotype of peripheral neuropathy with early-onset hearing loss, but no optic atrophy. The findings of this study will expand the clinical spectrum of X-linked recessive CMT and will be useful for the molecular diagnosis of clinically heterogeneous peripheral neuropathies.

15 STR 좌위의 동시증폭을 위한 Kplex-15 HID PCR kit의 개발과 동아시아 인류 집단의 적용

김종열,최순성,현영세,이승환,박수정,이숭덕,신경진,정기화 한국법과학회 2015 한국법과학회지 Vol.16 No.1

ABCD1 유전자 Trp77-Leu82del 돌연변이가 확인된 대뇌부신척수신경병증

이규선,박은경,현영세,이혜진,정기화,구혜수,최경규,박기덕,최병옥 대한신경과학회 2011 대한신경과학회지 Vol.29 No.4

Cerebral adrenomyeloneuropathy is a subtype of X-linked adrenoleukodystrophy with a mutation of ABCD1; however,there have been no reported cases of cerebral adrenomyeloneuropathy with myelopathy. Here we report a 20-year-old male cerebral adrenomyeloneuropathy patient with myelopathy harboring a deletion mutation of c.225-242 (Trp77-Leu82del) from exon 1 of ABCD1. His spinal cord MRI revealed high signal intensities in the cervical spinal cord. Electrophysiological and histopathologic studies revealed mixed axonal and demyelinating neuropathy.

인삼 (Panax ginseng C.A. Meyer)의 Microsatellite 마커에 대한 유전적 다형성과 특성 규명

박선화(Sun Wha Park),현영세(Young Se Hyun),정기화(Ki Wha Chung) 고려인삼학회 2009 Journal of Ginseng Research Vol.33 No.3

인삼에 대한 microsatellite 개발은 다른 분자적 마커들에 비해 늦게 이루어져, 최근에 와서야 인삼의 microsatellite 들이 보고되고 있는 실정이다. 본 연구에서는, 분리된 microsatellite들 중에서 5 개의 다형성 마커를 선별하여 국내 경작지나 시장에서 유통되는 인삼을 대상으로 유전적 다형성을 조사하고, 각 마커의 특성을 규명하였다. 유전자형 분석은 변성 PAGE와 silver staining법으로 하거나 형광표지 primer로 표지한 PCR 산물을 자동 염기서열 분석기로 분석하였다. 본 연구에서 개발한 5개의 microsatellite 마커들의 평균 대립유전자 수는 3.2 개였으며, 평균 GD는 0.367 였다. 전체적으로 볼 때, PG1419가 가장 높은 다형성을 보였으며 (PIC: 0.460, GD: 0.543), PG770은 가장 낮은 다형성을 나타내었다 (PIC: 0.070, GD: 0.078). 각 좌위들의 예상 이형 접합도 (Hexp)는 0.077에서 0.541 (mean = 0.313)로 계산되었으나, 관측 이형접합도 (Hobs)는 0.040에서 0.130 (mean = 0.083)으로 훨씬 낮게 관찰되었으며, 유전자형의 분포는 Hardy-Weinberg 평형상태에서 벗어남을 보였다. 본 연구에서 개발한 인삼의 microsatellite 마커들은 인삼의 분자적 마커의 데이터베이스 확립의 기초 자료로 활용될 뿐 아니라, 인삼의 분자적 구별법 및 QTL 좌위의 염색체지도 작성에 유용하게 활용될 것이다. Ginseng (Panax ginseng C.A. Meyer) is one of the most important medicinal plants in East Asia. Microsatellite or simple sequence repeat (SSR) markers are used in obtaining genetic analysis and authentication in many plants. The present study examined five microsatellites in conjunction with P. ginseng in Korea. The total observed allelic number was 17 (mean = 3.4), and gene diversities varied from 0.078 to 0.543 with an average of 0.314. Through a combined analysis of five loci in 100 ginseng samples, 44 different combined genotypes were observed. Expected and observed heterozygosites ranged from 0.077 to 0.541 (mean = 0.313) and 0.040 to 0.130 (0.083), respectively. All examined loci exhibited deficiency of heterozygosity and deviation from the Hardy-Weinberg equilibrium. Such results may be explained by the non-random mating and inbreeding that has occurred for several hundred years. These microsatellite markers could be used for the study of molecular genetics and the establishment of DNA marker database, as well as authentication of ginseng species and chromosomal mapping of QTL loci in P. ginseng.

Characterization of 20 microsatellite loci by multiplex PCR in swimming crab, Portunus trituberculatus

이혜진,이도현,윤승종,김대현,김상규,현영세,민기식,정기화 한국유전학회 2013 Genes & Genomics Vol.35 No.1

The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura), is one of the most important fishery resources in East Asia. The aim of this work is to develop microsatellite multiplex PCR systems for 20 microsatellites which were isolated from a microsatellite-enriched genomic library and to explore the genetic diversity of P. trituberculatus captured in the coastal regions of the Yellow Sea in Korea. We prepared four multiplex PCR systems (two hexplex and tetraplex systems each) which could be used for the genotyping of 20 markers. Most markers in this system are highly informative with mean polymorphism information contents and observed heterozygosity of 0.913 and 0.930. The total number of alleles observed in this study was 724, and the mean allelic number per locus was 36.2. Significant deviations from the Hardy–Weinberg equilibrium (HWE) were observed at six loci in the Wonsando Island population, but all the loci deviated from the HWE in the Jeonjangpo population. The genetic differentiation between the two populations and the phylogenetic tree constructed by the unweighted pair group method with the arithmetic average suggested that there is no significant genetic difference between Wonsando Island and Jeonjangpo populations. The prepared multiplex PCR systems and population genetic data will be helpful to study phylogeographic analysis as well as to prepare strategies of stock management and aquaculture of P. trituberculatus.

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases

최병옥,Jung Hee Hwang,Eun Min Cho,현영세,Hyeon Jeong Jeon,Ki Min Seong,Nam Soo Cho,정기화,Eun Hye Jeong 생화학분자생물학회 2010 Experimental and molecular medicine Vol.42 No.6

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5confirmed pathogenic mutations were identified in 17families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNALeu(UUR) was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNALys of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes,but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases,as well as establishment of mtDNA database in Koreans.

Phylogenetic Analysis of Mitochondrial DNA Control Region in the Swimming Crab, Portunus trituberculatus

Eun Min Cho,민기식,Sumaira Kanwal,현영세,박선화,정기화 한국통합생물학회 2009 Animal cells and systems Vol.13 No.3

The control region of mitochondrial DNA (13516- 14619) is located between srRNA and tRNAlle gene in swimming crab, Portunus trituberculatus. The present study was investigated the genetic polymorphisms of the control region in samples of P. trituberculatus collected at coastal waters of the Yellow Sea in Korea. A total of 300 substitution and indel polymorphic sites were identified. In addition to SNPs and indel variation, a hypervariable microsatellite motif was also identified at position from 14358 to 14391, which exhibited 10 alleles including 53 different suballeles. When the hypervariable microsatellite motif was removed from the alignment, 95 haplotypes were identified (93 unique haplotypes). The nucleotide and haplotype diversities were ranged from 0.024 to 0.028 and from 0.952 to 1.000, respectively. The statistically significant evidence for geographical structure was not detected from the analyses of neighbor-joining tree and minimum-spanning network, neither. This result suggest that population of P. trituberculatus are capable of extensive gene flow among populations. We believed that the polymorphisms of the control region will be used for informative markers to study phylogenetic relationships of P. trituberculatus.