Prediction of the CYP2D6 enzymatic activity based on investigating of the CYP2D6 genotypes around the vivax malaria patients in Yunnan Province, China

Abstract Background In recent years, the incidence rate of vivax malaria recurrence still had 3.1% in Yunnan Province population after eradication therapy using primaquine (PQ). In order to understand the specific failure reasons for preventing vivax malaria relapses, a preliminary exploration on the CYP2D6 enzyme activity was carried out in the vivax malaria patients in Yunnan Province population by analysing mutational polymorphism in the coding region of CYP2D6 gene. Methods Blood samples were collected from vivax malaria patients with suspected relapse (SR) and non-relapsed (NR) malaria in Yunnan Province. The DNA fragments containing 9 exons regions of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were aligned by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape. The alleles of CYP2D6 gene were identified according to the criteria prescribed by Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database and the CYP2D6 enzyme activity was predicted based on diploid genotype. Results A total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1,457 (rs1135840) were observed on the 640 CDS chains (including 320 maternal and 320 paternal chains). The high-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group ($${x}^{2}$$ x 2 = 4.468, 5.889, P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency (36.9%, 236/640), and Hap_9 had the most complex sequence structure, containing 7 loci mutations. Allele *10 was the most frequent among these genotypes (0.423). Among the allele *10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group ($${x}^{2}$$ x 2 = 3.911, P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group ($${x}^{2}$$ x 2 = 10.050, P < 0.05), and genotype *4/*4was detected only in the SR group. Conclusion In the patients receiving PQ dosage in Yunnan Province population, both rs1135840 single nucleotide polymorphism and *10 allele form was common in the CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse.

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The association of CYP2D6 gene polymorphisms in the full-length coding region with higher recurrence rate of vivax malaria in Yunnan Province, China

Malaria Journal ◽

10.1186/s12936-021-03685-3 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Herong Huang ◽

Ying Dong ◽

Yanchun Xu ◽

Yan Deng ◽

Canglin Zhang ◽

...

Keyword(s):

Enzyme Activity ◽

Gene Mutation ◽

Dna Sequences ◽

Vivax Malaria ◽

Yunnan Province ◽

Reference Sequence ◽

Radical Cure ◽

Coding Region ◽

Cyp2d6 Gene ◽

Genotype 4

Abstract Background Accumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene. Methods Blood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation “reference” sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes (star alleles), genotypes and odds ratios (OR) of all the CDSs were analysed. Results Of the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria (OR = 2.167, 95% CI 1.104–4.252, P < 0.05). Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, *10 and *39, were confirmed by comparison, and CYP2D6*10 allele accounted for the largest percentage (45.4%, 108/238). The frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (Χ2 = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group. Conclusion Mutation of CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D6*2 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.

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Genetic Polymorphism of human CYP2D6 gene coding region from vivax malaria cases in Yunnan Province, China

10.21203/rs.3.rs-19379/v4 ◽

2020 ◽

Author(s):

Herong Huang ◽

Ying Dong ◽

Yanchun Xu ◽

Yan Deng ◽

Canglin Zhang ◽

...

Keyword(s):

Enzyme Activity ◽

Vivax Malaria ◽

Yunnan Province ◽

Gene Mutations ◽

World Health ◽

Cyp2d6 Genotype ◽

Radical Cure ◽

Coding Region ◽

Cyp2d6 Gene ◽

Blood Samples

Abstract Background: Primaquine is one of two medications able to effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for the treatment of vivax patients. However, 5-hydroxy-primaquine, the presumed active component, must be generated by CYP2D6 enzyme in human hepatocytes and reduced CYP2D6 enzyme activity caused by gene mutations has been considered cause of primaquine failure. Based on the analysis of CYP2D6 gene polymorphisms in suspected relapsed vivax malaria patients, the current study aims to determine the association between human CYP2D6 genotype and the efficacy of primaquine. Methods: Blood samples from vivax patients in Yunnan Province who received "chloroquine/primaquine eight-day therapy" and experienced suspected relapses were collected from 2014 to 2018. Human genomic DNA was extracted, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and were then sequenced. The mutation types of CDS and their association with CYP2D6 enzyme activity changes were analyzed. Results: A total of 156 blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected and two nested-PCR products, the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp), were obtained from the sample. After splicing and combining these two amplification products, we found that the CDS of CYP2D6 gene in each sample was 1491bp in length. Moreover, we identified 24 haplotypes (Hap_1~Hap_24) in the Clustered CYP2D6 full-CDS of 150 patients, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients (8 haplotypes co-existed in two groups of patients). In addition, we identified 33 diplotypes (A_1~A_33) in 150 patients. The A_10 and A_12 diplotypes showed higher frequency in suspected relapsed group.Conclusion: Among the numerous mutations of CYP2D6 gene， Hap_2 accounted for the largest proportion in 24 haplotypes. And the combined homozygous mutations at c. 408, c. 1457, especially the two loci combined mutations with c. 886, may be related to the poor efficacy of primaquine in the radical cure P. vivax in Yunnan, however, greater sample size is required confirm these findings.

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Genetic Polymorphism of Cyp2d6 Gene Coding Region and Association Between Them With Vivax Malaria Relapse in Yunnan Province, China

10.21203/rs.3.rs-77457/v1 ◽

2020 ◽

Author(s):

Herong Huang ◽

Ying Dong ◽

Yanchun Xu ◽

Yan Deng ◽

Canglin Zhang ◽

...

Keyword(s):

Enzyme Activity ◽

Vivax Malaria ◽

Yunnan Province ◽

World Health ◽

Reference Sequence ◽

Radical Cure ◽

Cyp2d6 Gene ◽

Genotype 4 ◽

Blood Samples ◽

Pcr Products

Abstract BackgroundPrimaquine is one of two medications able to effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for vivax malaria patients. However, the reduced CYP2D6 enzyme activity caused by gene mutations has been considered to result in the failure of primaquine relapse vivax malaria. Based on the analysis of CYP2D6 gene polymorphisms in vivax malaria cases the current study aims to determine the association between human CYP2D6 polymorphism and the relapse of vivax malaria. MethodsBlood samples of vivax malaria cases who received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The suspected relapsed cases were determined by epidemiology and gene sequence alignment. Human genomic DNA was extracted from the blood samples, nine exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and PCR products were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning with the non-mutation reference sequence. The mutation loci, haplotypes (star alleles) and genotypes of CYP2D6 gene were inferred and their association with vivax malaria relapse were analyzed. ResultsA total of 120 blood samples from 45 suspected relapsed cases of vivax malaria and 75 non-relapsed cases were collected and two nested-PCR products (2411bp and 2388bp) containing exon1-4 and exon5-9, were obtained from 119 samples. 119 CDS chains (1491bp length) were formed by splicing and combining sequencing sequences, A total of 12 mutation loci were identified in 238 CDS chains (including 119 chains from direct sequencing and another 119 chains identified from sister chromosome). Among them, the mutation locus at c.886 has the closest relationship with the relapse of vivax malaria (OR=2.167, 95%CI: 1.104~4.252, P<0.05). There were 23 haplotypes (Hap_1~Hap_23) to be identified and be defined as 23 alleles of CYP2D6 gene, Among them, the 5 star alleles (*1, *2, *4, *10 and *39) were confirmed by comparison, and the CYP2D6*10 allele accounted for the most (45.4%, 108/238). the frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (P<0.05). However, among the defined 24 genotypes, there were 8 genotypes (*4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10) appeared only in the SR group.ConclusionAmong the numerous mutations of CYP2D6 gene，CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutation at c. 886 locus is most closely related to the relapse of vivax malaria. In addition, there was the genotype *4/*4 with null CYP2D6 enzyme function. These results suggest that Yunnan Province should pay attention to the risk of reduced CYP2D6 enzyme activity on the radical cure therapeutic effect of primaquine

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Polymorphism in coding region of human CYP2D6 gene contributes to failed radical cure of vivax malaria using primaquine

10.21203/rs.3.rs-19379/v2 ◽

2020 ◽

Author(s):

Herong Huang ◽

Ying Dong ◽

Yanchun Xu ◽

Yan Deng ◽

Canglin Zhang ◽

...

Keyword(s):

Enzyme Activity ◽

Nested Pcr ◽

Vivax Malaria ◽

Critical Role ◽

World Health ◽

Reference Sequence ◽

Cyp2d6 Genotype ◽

Radical Cure ◽

Cyp2d6 Gene ◽

Blood Samples

Abstract Background: So far, it has been confirmed that primaquine is the only medicine could effectively kill the hypnozoites of P. vivax. Therefore, World Health Organization (WHO) recommends primaquine as the only anti-relapsing drug for the treatment of vivax malaria patient. However, due to the active component of primaquine for killing P. vivax hypnozoite is the 5-hydroxy-primaquine, which has to be catalyzed by the Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocytes, the decrease of CYP2D6 enzyme activity caused by CYP2D6 gene mutation has been considered as an risk factor for the use of primaquine. Based on the analysis of CYP2D6 gene polymorphisms in relapsed vivax malaria patients, this study preliminarily revealed the genetic association between human CYP2D6 genotype and the curative effect of primaquine in vivax malaria. Methods: Blood samples of vivax malaria cases treated with "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. Then, the relapsed cases were determined by identifying the homogeneity of P. vivax circumsporozoite protein（pvcsp）gene sequences between the Plasmodiums isolated from patients’ first and second infected blood samples. Human genomic DNA was extracted from blood samples, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing after alignment with the wild-type reference sequence. The mutation types of CDS and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by the allelic form of CYP2D6 gene. Results: One hundred and fifty-six blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected for nested-PCR amplification. Two kinds of nested-PCR products，the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp)，were obtained from every sample. After splicing and combining these two amplification products, the complete CDS of CYP2D6 (1491 bp) of each sample could be gotten. Clustered CYP2D6 full-CDS of 150 patients, 24 haplotypes (Hap_1~Hap_24) were defined, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients. The Hap_6 showed G>C, C>T and G>C base substitutions at c.408, c.886 and c.1457, resulting in V136V invariance and the variations of R296C and S486T at 136th, 296th and 486th amino acid sites. The odds ratio of Hap_6 sequence type was 5.615 (P˂0.05) as compared with the relapse of vivax malaria. For the relapsed cases, the mutation at c.408 of CYP2D6 gene is 100% homozygous allele (C/C, 10/10), while at c.886 is 80% homozygous allele (T/T, 8/10) and 20% heterozygous allele (C/T, 2/2). But the non-relapsed cases only showed mutation heterozygote allele (C/T, 2/2) at c.886 locus. Moreover, most of mutant allele types in relapsed patients were CYP2D6 *2, and the CYP2D6 enzyme activity of them was predicted as normal metabolizer (NM) by scoring the diploid allele of Hap_6 sequence. Conclusion: Among the numerous mutations of CYP2D6 gene, the triple mutations at three loci (c.408, c.886, c.1457) are most closely related to the decreased CYP2D6 enzyme activity, while whether the c.886 locus mutation plays a critical role needs to be further verified by expanded sample size.

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Genetic Polymorphism of human CYP2D6 gene coding region results in failed radical cure of vivax malaria using primaquine

10.21203/rs.3.rs-19379/v3 ◽

2020 ◽

Author(s):

Herong Huang ◽

Ying Dong ◽

Yanchun Xu ◽

Yan Deng ◽

Canglin Zhang ◽

...

Keyword(s):

Enzyme Activity ◽

Nested Pcr ◽

Vivax Malaria ◽

Critical Role ◽

World Health ◽

Reference Sequence ◽

Cyp2d6 Genotype ◽

Radical Cure ◽

Cyp2d6 Gene ◽

Blood Samples

Abstract Background: Primaquine is the traditional medicine could effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for the treatment of vivax malaria patient. However, 5-hydroxy-primaquine, the active component in primaquine to kill P. vivax hypnozoite has to be catalyzed by Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocytes. Nevertheless, suppressed CYP2D6 enzyme activity caused by CYP2D6 gene mutation has been considered as a risk factor for the efficacy of primaquine. Based on the analysis of CYP2D6 gene polymorphism in relapsed vivax malaria patients, the current study aims to preliminarily reveal the genetic association between human CYP2D6 genotype and the curative effect of primaquine in vivax malaria. Methods: Blood samples of vivax malaria cases received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The relapsed cases were determined by identifying the homogeneity of P. vivax circumsporozoite protein (pvcsp) gene sequences between the Plasmodiums isolated from patients’ infected blood samples. Human genomic DNA was extracted from the blood samples, and 9 exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning with the wild-type reference sequence. The mutation types of CDS and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by the allelic form of CYP2D6 gene. Results: A total of 156 blood samples from 75 relapsed cases of vivax malaria and 75 non-relapsed cases were collected for nested-PCR amplification. Two nested-PCR products, the CYP2D6 gene fragments containing exon1-4 and exon5-9 (2411bp and 2388bp), were obtained from the sample. After splicing and combining these two amplification products, we found that the CDS of CYP2D6 gene in each sample was 1491bp in length. Moreover, we identified 24 haplotypes (Hap_1~Hap_24) in the Clustered CYP2D6 full-CDS of 150 patients, including 17 haplotypes in relapsed patients and 15 haplotypes in non-relapsed patients (8 haplotypes co-existed in two groups of patients). Hap_6 showed G>C, C>T and G>C base substitutions at c.408, c.886 and c.1457, resulting in V136V invariance, and variations of R296C and S486T at 136th, 296th and 486th amino acid sites, respectively. The risk of relapsing vivax malaria in Hap_6 was about 5.615-fold higher than in other haplotypes. In the relapsed cases, c.408 locus mutation of CYP2D6 gene was 100% homozygous allele (C/C, 10/10), whereas c.886 locus mutation was 80% homozygous allele (T/T, 8/10) and 20% heterozygous allele (C/T, 2/2). However, the non-relapsed cases only showed mutation heterozygote allele (C/T, 2/2) at c.886 locus. Moreover, most of mutant allele types in relapsed patients were CYP2D6 *2, and the CYP2D6 enzyme activity was predicted as normal metabolizer (NM) according to the scoring of diploid allele of Hap_6 sequence.Conclusion: Among the numerous mutations of CYP2D6 gene, triple mutations at three loci (c.408, c.886, c.1457) are most closely related to impaired CYP2D6 enzyme activity, and greater sample size should be applied to ascertain whether c.886 locus mutation plays a critical role.

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Polymorphism in coding region of human CYP2D6 gene contributes to failed radical cure of vivax malaria using primaquine

10.21203/rs.3.rs-19379/v1 ◽

2020 ◽

Author(s):

Herong Huang ◽

Ying Dong ◽

Yanchun Xu ◽

Yan Deng ◽

Canglin Zhang ◽

...

Keyword(s):

Enzyme Activity ◽

Dna Sequences ◽

Vivax Malaria ◽

Critical Role ◽

Reference Sequence ◽

Cyp2d6 Genotype ◽

Radical Cure ◽

Allelic Form ◽

Cyp2d6 Gene ◽

Blood Samples

Abstract Background: Recent studies found that damaged enzyme activity of cytochrome P450 isoenzyme 2D6 (CYP2D6) can lead to compromised efficacy of primaquine in killing Plasmodium during liver dormancy and consequently relapsed malaria. Therefore, WHO has listed the decrease enzyme activity of CYP2D6 as one of the four scenarios that are not suitable for primaquine therapy. Based on analysis of CYP2D6 gene polymorphisms in relapsed vivax malaria patients, this study preliminarily revealed the genetic association between human CYP2D6 genotype and the declined efficacy of primaquine treatment in vivax malaria. Methods: Blood samples of vivax malaria cases treated with "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected, and the vivax malaria relapsed cases after clinical curing were determined by identifying the infected Plasmodium isolates through gene traceability analysis. Human genomic DNA was extracted from blood samples, and 9 exons of CYP2D6 gene were amplified by PCR and then sequenced. The CDS of CYP2D6 gene was obtained by alignment and splicing with the wild-type reference sequence. The mutation types of coding DNA sequences and their association with vivax malaria relapse were analyzed, and the activity phenotype of CYP2D6 isozyme was predicted by its allelic form. Results: One hundred and fifty-six blood samples from 75relapsed cases of vivax malaria and the same number of 75 non-relapsed vivax malaria cases were collected for PCR amplification. Two amplification products (2411bp and 2388bp) containing exon1-4 and exon5-9 of CYP2D6 gene were obtained from every sample. The 1491-bp-length CDS chains of CYP2D6 gene were obtained from those 150 samples, and were defined as 24 haplotypes (Hap_1~Hap_24).17 haplotypes were determined from the sequences of vivax malaria relapsed patients and 15 haplotypes were from those of non-relapsed patients.Hap_6 sequencing showed G>C, C>T and G>C base substitutions at three loci of c.408, c.886 and c.1457, resulting in V136V invariance and R296C and S486T variation at 136th, 296th and 486th amino acid, and the odds ratio of Hap_6 to vivax malaria relapse was 5.615 (P˂0.05).The diploids of CYP2D6 gene in relapsed cases were 100% mutation homozygous (C/C, 10/10) at c.408, 80% (8/10) at c.1457 and 20% (2/10) at c.886, respectively. However, the coding DNA sequences from non-relapsed cases only showed mutation heterozygote (C/T, 2/2) at c.886 locus. The allelic form of relapsed patients was CYP2D6 *2, and diploid score was used to indirectly predict the CYP2D6 enzyme activity of Hap_6, and the relapsed patients belong to normal metabolizer (NM).Conclusion: Among the numerous mutations of CYP2D6 gene, the joint mutations at three loci (c.408, c.886, c.1457) are most closely related to the damagedCYP2D6 enzyme activity. Whether c.886 locus mutation plays a critical role contributing to such damage warrants further verification with expanded sample size.

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Low frequency of mutations in theWT1 coding region in Wilms' tumor

Genes Chromosomes and Cancer ◽

10.1002/gcc.2870080203 ◽

1993 ◽

Vol 8 (2) ◽

pp. 74-79 ◽

Cited By ~ 34

Author(s):

Keith W. Brown ◽

Helen P. Wilmore ◽

Joanne E. Watson ◽

Martin G. Mott ◽

P. Jeremy Berry ◽

...

Keyword(s):

Wilms Tumor ◽

Low Frequency ◽

Coding Region

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Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene

Molecular and Cellular Biology ◽

10.1128/mcb.3.4.643-653.1983 ◽

1983 ◽

Vol 3 (4) ◽

pp. 643-653

Author(s):

G M Santangelo ◽

C N Cole

Keyword(s):

Gene Expression ◽

Thymidine Kinase ◽

Low Frequency ◽

Simian Virus 40 ◽

Simian Virus ◽

African Green Monkey ◽

Dna Fragments ◽

Coding Region ◽

Kinase Gene ◽

Green Monkey

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.

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Genetic Variation in the Syntaxin-Binding Protein STXBP5 in Type 1 von Willebrand Disease Patients

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661352 ◽

2018 ◽

Vol 118 (08) ◽

pp. 1382-1389 ◽

Cited By ~ 3

Author(s):

Christina Lind-Halldén ◽

Eric Manderstedt ◽

Daniel Carlberg ◽

Stefan Lethagen ◽

Christer Halldén

Keyword(s):

Genetic Variation ◽

Protein Function ◽

Low Frequency ◽

Von Willebrand Disease ◽

Nucleotide Polymorphisms ◽

Coding Region ◽

Swedish Population ◽

Messenger Ribonucleic Acid ◽

Von Willebrand

Abstractvon Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, for example, VWF, ABO and STXBP5. Here, we comprehensively screen for STXBP5 variants and investigate their association with type 1 VWD in Swedish patients and controls. The coding region of the STXBP5 gene was re-sequenced in 107 type 1 VWD patients and the detected variants were genotyped in the type 1 VWD population and a Swedish control population (464 individuals). The functional effects of missense alleles were predicted in silico and the pattern of genetic variation in STXBP5 was analysed. Re-sequencing of 107 type 1 VWD patients identified three missense and three synonymous variants in the coding sequence of STXBP5. The low-frequency missense variants rs144099092 (0.005) and rs148830578 (0.029) were predicted to be damaging, but were not accumulated in patients. No other rare candidate mutations were detected. STXBP5 showed a high level of linkage disequilibrium and a low overall nucleotide diversity of π = 3.2 × 10−4 indicating intolerance to variants affecting protein function. Three previously type 1 VWD-associated single nucleotide polymorphisms were located on one haplotype that showed an increased frequency in patients versus controls. No differences in messenger ribonucleic acid abundance among haplotypes could be found using Genotype-Tissue Expression project data. In conclusion, a haplotype containing the STXBP5 Asn436Ser (rs1039084) mutation is associated with type 1 VWD and no rare STXBP5 mutations contribute to type 1 VWD in the Swedish population.

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Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3463 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3463-3469 ◽

Cited By ~ 12

Author(s):

D Ayusawa ◽

H Koyama ◽

K Shimizu ◽

S Kaneda ◽

K Takeishi ◽

...

Keyword(s):

Genetic Marker ◽

Thymidylate Synthase ◽

Mammalian Cells ◽

Genetic Recombination ◽

Low Frequency ◽

Coding Region ◽

Physiological Factor ◽

X Ray ◽

Alu Sequence ◽

Specific Manner

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.

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