Association of a c.1084A>G (p.Thr362Ala)Variant in the DCTN4 Gene with Wilson Disease

Robin Dong-Woo Lee,Jae-Jung Kim,Joo-Hyun Kim,Jong-Keuk Lee,Han-Wook Yoo 대한의학유전학회 2011 대한의학유전학회지 Vol.8 No.1

목적: 윌슨병은 간조직에 구리의 과도한 침착으로 발병하는 상염색체 열성 유전질환이다. 지금까지 ATP7B 유전자가 유일한 원인유전자로 알려져 왔다. 그러나, 약 15%의 환자에서는 ATP7B 유전자 돌연변이가 발견되지 않는다. 본 연구는 ATP7B 유전자의 돌연변이가 발견되지 않은 윌슨병 환자를 대상으로 새로운 원인 유전자를 발견하기 위하여 시행되었다. 대상 및 방법: ATP7B 돌연변이가 발견되지 않은 12명의 윌슨병 환자를 대상으로 ATP7B 와 상호작용을 하는 것으로 알려진 ATOX1, COMMD1, GLRX, DCTN4와 ZBTB16 유전자의 전사부위와 엑손-인트론 경계부위의 염기서열을 분석하였다. 결과: DCTN4 유전자의 12번 엑손에 존재하는 c.1084A>G(p.Thr362Ala)를 포함하는 3가지의 변이가 환자에서 발견되었다. in silico 분석을 통해 3가지 변이 중 c.1084A>G가 유일하게 단백질 기능 변화를 일으킬 것으로 예측되었다. 176명의 윌슨병 환자와 414명의 정상인을 대상으로 이 변이의 빈도를 조사한 결과, 정상인보다 윌슨병 환자에서 더 높은 빈도를 나타내었다(상대비, odds ratio [OR]=3.14, 95% 신뢰도=1.36-7.22, P=0.0094). 결론: 본 연구의 결과는 ATP7B 와 상호작용하는 DCTN4 유전자의 c.1084A>G (p.Thr362Ala) 다형성이 윌슨병의 발병과 연관이 있음을 시사한다. Purpose: Wilson disease is an autosomal recessive disorder which causes excessive copper accumulation in the hepatic region. So far, ATP7B gene is the only disease-causing gene of Wilson disease known to date. However, ATP7B mutations have not been found in ~15% of the patients. This study was performed to identify any causative gene in Wilson disease patients without an ATP7B mutation in either allele. Materials and Methods: The sequence of the coding regions and exon-intron boundaries of the five ATP7B-interacting genes, ATOX1, COMMD1, GLRX, DCTN4, and ZBTB16, were analyzed in the 12 patients with Wilson disease. Results: Three nonsynonymous variants including c.1084A>G (p.Thr362Ala) in the exon 12 of the DCTN4 gene were identified in the patients examined. Among these, only p.Thr362Ala was predicted as possibly damaging protein function by in silico analysis. Examination of allele frequency of c.1084A>G (p.Thr362Ala) variant in the 176 patients with Wilson disease and in the 414 normal subjects revealed that the variant was more prevalent in the Wilson disease patients (odds ratio [OR]=3.14, 95% confidence interval=1.36-7.22, P =0.0094). Conclusion: Our result suggests that c.1084A>G (p.Thr362Ala) in the ATP7B-interacting DCTN4 gene may be associated with the pathogenesis of Wilson disease.

Association of a c.1084A>G (p.Thr362Ala)Variant in the DCTN4 Gene with Wilson Disease

Lee, Robin Dong-Woo,Kim, Jae-Jung,Kim, Joo-Hyun,Lee, Jong-Keuk,Yoo, Han-Wook Korean Society of Medical Genetics and Genomics 2011 대한의학유전학회지 Vol.8 No.1

목 적: 윌슨병은 간조직에 구리의 과도한 침착으로 발병하는 상염색체 열성 유전질환이다. 지금까지 ATP7B 유전자가 유일한 원인유전자로 알려져 왔다. 그러나, 약 15%의 환자에서는 ATP7B 유전자 돌연변이가 발견되지 않는다. 본 연구는 ATP7B 유전자의 돌연변이가 발견되지 않은 윌슨병 환자를 대상으로 새로운 원인 유전자를 발견하기 위하여 시행되었다. 대상 및 방법: ATP7B 돌연변이가 발견되지 않은 12명의 윌슨병 환자를 대상으로 ATP7B 와 상호작용을 하는 것으로 알려진 ATOX1, COMMD1, GLRX, DCTN4와 ZBTB16 유전자의 전사부위와 엑손-인트론 경계부위의 염기서열을 분석하였다. 결 과: DCTN4 유전자의 12번 엑손에 존재하는 c.1084A>G(p.Thr362Ala)를 포함하는 3가지의 변이가 환자에서 발견되었다. in silico 분석을 통해 3가지 변이 중 c.1084A>G가 유일하게 단백질 기능 변화를 일으킬 것으로 예측되었다. 176명의 윌슨병 환자와 414명의 정상인을 대상으로 이 변이의 빈도를 조사한 결과, 정상인보다 윌슨병 환자에서 더 높은 빈도를 나타내었다(상대비, odds ratio [OR]=3.14, 95% 신뢰도=1.36-7.22, P=0.0094). 결 론: 본 연구의 결과는 ATP7B 와 상호작용하는 DCTN4 유전자의 c.1084A>G (p.Thr362Ala) 다형성이 윌슨병의 발병과 연관이 있음을 시사한다. Purpose: Wilson disease is an autosomal recessive disorder which causes excessive copper accumulation in the hepatic region. So far, ATP7B gene is the only disease-causing gene of Wilson disease known to date. However, ATP7B mutations have not been found in ~15% of the patients. This study was performed to identify any causative gene in Wilson disease patients without an ATP7B mutation in either allele. Materials and Methods: The sequence of the coding regions and exon-intron boundaries of the five ATP7B-interacting genes, ATOX1, COMMD1, GLRX, DCTN4, and ZBTB16, were analyzed in the 12 patients with Wilson disease. Results: Three nonsynonymous variants including c.1084A>G (p.Thr362Ala) in the exon 12 of the DCTN4 gene were identified in the patients examined. Among these, only p.Thr362Ala was predicted as possibly damaging protein function by in silico analysis. Examination of allele frequency of c.1084A>G (p.Thr362Ala) variant in the 176 patients with Wilson disease and in the 414 normal subjects revealed that the variant was more prevalent in the Wilson disease patients (odds ratio [OR]=3.14, 95% confidence interval=1.36-7.22, P=0.0094). Conclusion: Our result suggests that c.1084A>G (p.Thr362Ala) in the ATP7B-interacting DCTN4 gene may be associated with the pathogenesis of Wilson disease.

Distinct clinical courses according to presenting phenotypes and their correlations to <i>ATP7B</i> mutations in a large Wilson's disease cohort

Lee, Beom H.,Kim, Joo H.,Lee, Sun Y.,Jin, Hye Y.,Kim, Kwi‐,Joo,Lee, Jin‐,Joo,Park, Jung‐,Young,Kim, Gu‐,Hwan,Choi, Jin‐,Ho,Kim, Kyung M.,Yoo, Han‐,Wook Blackwell Publishing Ltd 2011 Liver International Vol.31 No.6

<P><B>Abstract</B></P><P><B>Introduction and aims: </B> Wide phenotypic and genotypic heterogeneities in Wilson's disease (WD) have been reported, hampering the study of their correlations. The goal of this study was to identify the factors related to these diversities.</P><P><B>Methods: </B> Clinical courses and molecular genetic characteristics were analysed in 237 unrelated Korean WD families. The average follow‐up period was 8.2 ± 5.8 years.</P><P><B>Results: </B> Presenting phenotypes were classified as H1 (12.2%), H2 (42.4%), N1 (21.6%), N2 (0.4%), NX (0.4%), presymptomatic (22.4%) and other (0.4%), modifying the guidelines by Ferenci and colleagues. Age at presentation was youngest and cirrhosis was rarest in the presymptomatic group. Decompensated cirrhosis was the highest in the H1 group. Favourable outcome was rarest in the N1 group. Forty‐seven (11 novel) <I>ATP7B</I> mutations were identified in 85% of the 474 alleles. Multiplex ligation‐dependent probe amplification assays in <I>ATP7B</I> and analyses of <I>ATOX1</I> and <I>COMMD1</I> genes identified no additional mutations. Yeast complementation assays demonstrated functional perturbation of the seven novel missense mutants. Five major mutations, p.Arg778Leu, p.Ala874Val, p.Asn1270Ser, p.Lys838SerfsX35 and p.Leu1083Phe, accounted for 63% of the alleles. H1 was more common, age at presentation was younger and N1+N2+NX tended to be less common in patients with nonsense, frame shifting or splicing mutations than in those with missense mutations alone. Patients with both mutations in the transduction (Td) or the ATP hinge domain showed presymptomatic or hepatic manifestations but no neurological manifestation.</P><P><B>Conclusions: </B> The presenting phenotype strongly affects the clinical outcome of WD, and is related to the <I>ATP7B</I> mutation type and location, providing an evidence for genotype–phenotype correlations in WD.</P>

한국인 윌슨병 유전자의 일배체형 분석과 R778L 돌연변이에서 창시자효과에 대한 연구

배선환 ( Sun Hwan Bae ),김종원 ( Jong Won Kim ),서정기 ( Jeong Kee Seo ) 대한간학회 2009 Clinical and Molecular Hepatology(대한간학회지) Vol.15 No.3

배경/목적: 윌슨병은 ATP7B 유전자의 돌연변이에 의하여 구리대사에 이상이 발생하는 상염색체열성 질환이다. 윌슨병 유전자의 돌연변이가 300여종이 넘고, 인종별, 지역별 차이가 뚜렷하여, 한국인 윌슨병 유전자 연구는 중요성을 갖는다. 본 연구는 한국인 윌슨병 환자에서 빈발하는 돌연변이를 확인하고, 처음으로 한국인 윌슨병 유전자의 일배체형(haplotype)을 분석하며, 나아가 한국인 윌슨병 유전자에 창시자효과(founder effect)가 존재하는지를 확인하고자 시행되었다. 대상과 방법: 21명의 윌슨병 환아와 환아의 부모 42명, 총 63명을 대상으로 혈액을 채취하여 중합효소연쇄반응(PCR, polymerase chain reaction)을 시행한 후, 직접 염기서열분석방법(direct sequencing method)을 사용하여 돌연변이를 분석하였으며, 발광체가 붙여진 시발체를 사용하여 D13S315, D13S1325 및 D13S316 초위성체(microsatellies)에 대한 분석을 시행하였다. 윌슨병 유전자에 존재하는 창시자효과를 확인하기 위하여, 부모의 정상 DNA 42개를 대조군으로 이용하였다. 연쇄비평형(linkage disequilibrium)을 확인하기 위한 통계적 분석은 Chi-square검사를 이용하였으며, 일배체형 사이의 빈도를 비교한 통계적 분석은 Fisher`s Exact검사를 이용하였고, P 값이 0.05 미만일 때 통계적으로 유의하다고 정의하였다. 결과: 21명의 윌슨병 환아에서 모두 23개의 돌연변이가 발견되었으며, 8번 exon에 존재하는 R778L 돌연변이가 15개(65.2%), 11번 exon에 존재하는 A874V 돌연변이가 6개(26.1%), 18번 exon에 존재하는 N1270S 돌연변이가 2개(8.7%)의 DNA에서 발견되었다. R778L 돌연변이군을 대상으로 초위성체를 분석하였을 때, D13S315좌의 5번 대립유전자의 빈도가 대조군에 비해 통계적으로 유의한 차이를 보여(10/15 대 11/41, P=0.0157) 연쇄비평형을 나타내었으며, D13-S316좌의 4번 대립유전자 또한 대조군에 비해 유의한 차이를 보여(12/15 대 7/42, P=0.0001) 연쇄비평형을 나타내었다. 돌연변이 분석과 초위성체 분석결과를 바탕으로 한국인 윌슨병 유전자의 일배체형을 구성한 결과, 가장 빈발하는 일배체형은 5-3-R778L-4(D13S315-D13S1325-돌연변이-D13S316)로, 대조군과 비교하여 통계적으로 유의한 빈도차를 보였다(P=0.0213). 통계적 의미가 없는 것으로 나타난 D13S1325를 배제하고 구성한 5-R778L-4(D13S315-Mutation-D13S316) 일배체형의 빈도는 대조군과 비교하여 더욱 유의한 차이를 나타내었다(P=0.0018). 결론: D13S315-R778L-13S316(5-돌연변이-4) 일배체형이 한국인 윌슨병 유전자의 원시 일배체형(arche haplotype)으로 추정되며, 한국인 윌슨병은 R778L 돌연변이에 창시자효과가 존재하는 것으로 생각된다. Background/Aims: Wilson`s disease (WD) is an inherited disorder of copper metabolism caused by alteration of the P-type adenosine triphosphatase (ATP) 7B gene. In this study, we analyzed the frequency of well-known mutations and constructed the first haplotypes for Koreans. In addition, we evaluated whether a founder effect existed in Korean patients with WD. Methods: We obtained DNA samples from 21 patients with WD and their parents (total cohort n=63). ATP7B gene mutations were identified by direct sequencing methods, and microsatellite typing was performed at D13S315, D13S1325, and D13S316 with fluorescent dye-labeled primers. Any founder effect was identified by using 42 normal alleles from parents with a normal phenotype as a control group. The x2 test and Fisher`s exact test were used for statistical analysis. Results: Three common mutations were found in 23 chromosomes obtained from 21 patients: the R778L mutation at exon 8 (15/23, 65.2%), the A874V mutation at exon 11 (6/23, 26.1%), and the N1270S mutation at exon 18 (2/23, 8.7%). D13S315 and D13S316 showed linkage disequilibrium at alleles 5 and 4, respectively, in patients with the R778L mutation (P=0.0157 and 0.0001, respectively). The haplotype made up of these two alleles occurred significantly more frequently in patients with the R778L mutation (5-R778L-4, D13S315-mutation-D13S316) than in the controls (P=0.0018). Conclusions: The arche haplotype of the ATP7B gene in Korean patients with WD may be 5-R778L-4 (D13S315-mutation-D13S316), and it might illustrate a founder effect. (Korean J Hepatol 2009,15:309-319)

Cisplatin 내성 난소암 세포주로부터 2차 내성 아세포 수립과 성격규명

안춘산 ( Chun San An ),김성엽 ( Sung Yob Kim ) 대한산부인과학회 2007 Obstetrics & Gynecology Science Vol.50 No.1

Objective: Multidrug resistance to chemotherapy is a major obstacle in attempts to improve the clinical outcome of ovarian carcinoma patients. The aim of this study is to establish secondary anticancer drug resistant cell line from original SNU-8/WT resistant to cisplatin and characterize it. Methods: After establishing secondary drug resistant cell line (SNU-8/Fac), drug sensitivity was measured by MTT assay. Multidrug resistance (MDR) was analyzed by RT-PCR and western blotting analysis. Results: MTT assay data demonstrated that MDR was expressed in SNU-8/Fac. In addition, mRNA expression of MDR1, ATP7B in SNU-8/Fac was increased. However, overexpression of MRP1, BCRP, TS and MT mRNA was not observed. At the protein level, protein P-gp, ATP7B were overexpressed in SNU-8/Fac. Conclusion: We established a new anticancer drug resistant cell line from original SNU-8/WT. Overexpression of P-gp and ATP7B was observed in SNU-8/Fac.

Diagnosis of Wilson Disease in Young Children: Molecular Genetic Testing and a Paradigm Shift from the Laboratory Diagnosis

Jeong Kee Seo 대한소아소화기영양학회 2012 Pediatric gastroenterology, hepatology & nutrition Vol.15 No.4

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism that results in accumulation of copper primarily in the liver, brain and cornea. Mutations in the WD gene, ATP7B, cause failure of copper excretion from hepatocyte into bile and a defective synthesis of ceruloplasmin. More than 500 mutations are now recognized, scattered throughout the ATP7B gene. Since WD has protean clinical presentations, awareness of WD in clinical practice is important for the early diagnosis and prevention of accumulated copper toxicity. Molecular genetic testing is playing an increasingly important role in the diagnosis of WD in uncertain cases and family screening. Siblings should be screened for WD once an index case has been diagnosed. Discrimination of heterozygotes from asymptomatic patients is essential to avoid inappropriate lifelong therapy for heterozygotes. Genetic testing, either by haplotype analysis or by mutation analysis, is the only definite solution for differentiating heterozygote carriers from affected asymptomatic patients. Routine genetic testing, because of the multitude of documented mutations, has been thought to be impractical until recently. However, genetic testing is now being more actively applied to the diagnosis of WD, particularly in young children in whom conventional biochemical diagnosis has much limitation and only genetic testing is able to confirm WD. Because advancement of modern biochemical technology now allows more rapid, easier, and less expensive mutation detection, direct DNA sequencing could be actively considered as the primary mode of diagnostic investigation rather than a supplementary test to the conventional biochemical tests. This review will focus on the recent advancement of molecular genetics and genetic diagnosis of WD in very young children on the basis of research data of the Seoul National University Children’s Hospital and recent literature.

PARALLEL SYMPOSIUM 2 : Wilson`s Disease: Molecular Genetics and a Paradigm Shift of the Diagnostic Strategy

서정기 ( Jeong Kee Seo ) 대한간학회 2013 춘·추계 학술대회 (KASL) Vol.2013 No.1

Wilson`s disease (WD) is the most common inherited liver disease with a prevalence of 1: 37,000 in Korean population. Mutations in the WD gene, ATP7B, cause the functional loss as a copper transporter and result in impairment of hepatic biliary copper excretion and also copper incorporation into apo-ceruloplasmin. Lifelong accumulations of copper lead to fatal hepatic failure or severe neurologic deterioration and death. More than 500 ATP7B mutations are now recognized. There seems to be some differences among various mutations in the failure of copper dependent trafficking pathway and copper transport defects to apo-ceruloplasmin at the molecular level. Further studies are needed to confirm the correlation of diverse manifestations of WD with these molecular level differences. Low serum ceruloplasmin concentrations, increased 24 hour urinary copper excretion, increased hepatic copper concentrations and the presence of Kayser-Fleischer rings in the cornea are major diagnostic points. Because of the absence of single laboratory test for the definite diagnosis and protean clinical presentations of WD, early diagnosis is not easy particularly in patients who do not meet the diagnostic criteria of the copper related conventional laboratory tests. Recently, molecular genetic testing is playing an increasingly important role in the diagnosis of WD in uncertain cases and family screening. Genetic testing, either by haplotype analysis or by mutation analysis, is the only definite solution for differentiating heterozygote carriers from affected asymptomatic patients. Because advancement of modern biochemical technology now allows more rapid, easier, and less expensive mutation detection, direct DNA sequencing could be actively considered as the primary mode of diagnostic investigation rather than a supplementary test to the conventional laboratory tests.

논평 : 월슨병의 한국인 특이 ATP7B 유전자의 R778L 돌연변이에 대한 일배체형 연구

진현석 ( Hyun Seok Jin ),오범석 ( Berm Seok Oh ) 대한간학회 2009 Clinical and Molecular Hepatology(대한간학회지) Vol.15 No.3

Clinical Characteristics, Genetic Features, and Long-Term Outcome of Wilson’s Disease in a Taiwanese Population: An 11-Year Follow-Up Study in a Taiwanese Population: An 11-Year Follow-Up Study

Chin-Hsien Lin 대한파킨슨병및이상운동질환학회 2023 Journal Of Movement Disorders Vol.16 No.2

Wilson’s disease (WD) is a rare genetic disorder of copper metabolism, and longitudinal follow-up studies are limited. We performed a retrospective analysis to determine the clinical characteristics and long-term outcomes in a large WD cohort.