The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA)....The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA). The TK6 cell line has the native p53 tumor-suppressor gene, whereas WTK1 cells contain a p53 mutation. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. The impact of irradiation on these two cell lines was investigated using probes that target specific regions on chromosomes associated with subtelomeric regions. Results indicate that WTK1 and TK6 are impacted differently after irradiation, and that each cell line presents its own unique MLPA profile. The most notable differences are the appearance of a number of probes in the post-irradiated MLPA profile that are not present in the controls, and two unique probe signals only seen in WTK1 cells. These results build on our previous studies that indicate how different human cell lines can be affected by radiation in significantly different ways depending on the presence or absence of wild type p53.展开更多
Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counselin...Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counseling and an incorrect prenatal diagnosis.This study was aimed to accurately analyze the genotypes of HKααcarriers and-α3.7/αααanti-4.2.Methods::Samples were collected in our hospital from July 2017 to October 2019.Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of theαααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics and Fisher’s exact tests.Results::Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples.The results showed thatα,β,andαβcompound thalassemia were identified in 1190(46.78%),1286(50.55%),and 68(2.67%)cases,respectively.A total of 227 samples from thalassemia patients were identified as-α3.7/ααby Gap-PCR,and the genotypes of two samples were uncertain.There was a difference between Gap-PCR and combined groups(Gap-PCR combined with nested PCR and MLPA)in detecting HKαα(P<0.05).Among the 229 patients,20 patients were identified as HKααcarriers and one was identified as-α3.7/ααα anti-4.2 by two-round nested PCR and MLPA,including 15 patients with HKαα/αα,three with HKαα/αα and β-thalassemia coinheritance,one with HKαα/-SEA,one with HKαα/-α4.2 andβ-thalassemia coinheritance,and one with-α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions::αααanti-4.2 and HKααgenotypes of patients carrying-α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKααand-α3.7/αααanti-4.2 alleles.More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.展开更多
Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the featu...Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification （MLPA） and array comparative genomic hybridization （array CGH）. Methods： Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results： All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions： The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.展开更多
Multiplex Ligation-Dependent Probe Amplification (MLPA) was used to study the integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiatio...Multiplex Ligation-Dependent Probe Amplification (MLPA) was used to study the integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation. WTK1 cells contain a p53 mutation, whereas the TK6 cell line has the native p53 tumor-suppressor gene. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. Using probes that target specific regions on chromosomes associated with a distinct subset of microdeletions and microduplications either established or thought to be responsible for intellectual disability or developmental delay, we have demonstrated that WTK1 and TK6 are not impacted in the same way by irradiation. Instead, each cell line presents its own unique MLPA profile. The most notable differences are the appearance of nine unique probe signals only seen in WTK1 cells. These results are important in the study of how different cell lines can be affected in significantly different ways depending on the presence or absence of wild type p53.展开更多
目的：探讨多重连接依赖性探针扩增（MLPA）技术在脊髓性肌肉萎缩症（SMA）患者、携带者及产前基因诊断中的应用。方法以临床诊断为 SMA 患儿为研究对象，采用基因组 DNA 多重连接探针扩增（MLPA）技术进行 SMN1基因和 SMN2基因拷贝数...目的：探讨多重连接依赖性探针扩增（MLPA）技术在脊髓性肌肉萎缩症（SMA）患者、携带者及产前基因诊断中的应用。方法以临床诊断为 SMA 患儿为研究对象，采用基因组 DNA 多重连接探针扩增（MLPA）技术进行 SMN1基因和 SMN2基因拷贝数变异检测。结果 MLPA 分析结果显示，9个家系中9例患者及2例胎儿呈 SMN1基因纯合缺失，其父母双方 SMN1基因7、8外显子均为杂合性缺失，均为 SMA 携带者。结论 ML‐PA 技术可以应用于 SMA 患儿的基因诊断，不仅快速、简便，还可辨别携带者致病基因杂合缺失情况，可筛查携带者。展开更多
AIM:To study the characteristics of APC(adenomatous polyposis coli)gene germline mutation in Chinese patients with familial adenomatous polyposis(FAP).METHODS:APC gene from 14 FAP families was amplified by polymerase ...AIM:To study the characteristics of APC(adenomatous polyposis coli)gene germline mutation in Chinese patients with familial adenomatous polyposis(FAP).METHODS:APC gene from 14 FAP families was amplified by polymerase chain reaction(PCR)and underwent direct sequencing to determine the micromutation type.For the samples without micromutation,the large fragment deletion of APC gene was examined by multiplex ligation-dependent probe amplification(MLPA).RESULTS:There were gene micromutations in 9 families with a micromutation detection rate of 64.3%(9/14),including 6 frameshift mutations(66.7%),1 nonsense mutation(11.1%)and 2 splicing mutations(22.2%).Large fragment deletions were detected by MLPA in 2 families.The total mutation detection rate of micromutations and large fragment deletions was 78.6%(11/14).CONCLUSION:The detection rate of APC gene germline mutation can be improved by direct sequencing combined with MLPA large fragment deletion detection.展开更多
Oculocutaneous albinism (OCA)is an autosomal recessive pigmentation abnormality,characterized by variable hair,skin,and ocular hypopigmentation.OCA1 is the most frequent subtype of OCA,caused by mutations in the tyros...Oculocutaneous albinism (OCA)is an autosomal recessive pigmentation abnormality,characterized by variable hair,skin,and ocular hypopigmentation.OCA1 is the most frequent subtype of OCA,caused by mutations in the tyrosinase gene (TYR). In this study,we investigated the genetic mutation of a Chinese family with a female OCA patient who came for genetic counseling before pregnancy.Complete physical examination was performed,and DNA from blood samples was collected from the family members.Mutations of TYR,OCA2,and SLC45A2 genes were examined in the proband, and verified in her parents by Sanger sequencing.Large deletion or duplication of TYR and OCA2 genes was detected by multiplex ligation-dependent probe amplification (MLPA).A homozygous TYR c.307T>C (p.Cys103Arg)missense mutation was identified in the proband,and both parents were heterozygous carriers.No large deletion or duplication was found in the proband.This mutation was absent in 1000G,ExAC,or HGMD database,and multiple lines of in silico tools supported a deleterious effect.These results suggest that TYR c.307T>C mutation might be responsible for OCA1,and our study further expands the mutation spectrum of OCA1 in the Chinese population.展开更多
文摘The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA). The TK6 cell line has the native p53 tumor-suppressor gene, whereas WTK1 cells contain a p53 mutation. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. The impact of irradiation on these two cell lines was investigated using probes that target specific regions on chromosomes associated with subtelomeric regions. Results indicate that WTK1 and TK6 are impacted differently after irradiation, and that each cell line presents its own unique MLPA profile. The most notable differences are the appearance of a number of probes in the post-irradiated MLPA profile that are not present in the controls, and two unique probe signals only seen in WTK1 cells. These results build on our previous studies that indicate how different human cell lines can be affected by radiation in significantly different ways depending on the presence or absence of wild type p53.
基金This work was supported by a grant from the Department of Science and Technology of Sichuan province,China(No.30504010332).
文摘Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counseling and an incorrect prenatal diagnosis.This study was aimed to accurately analyze the genotypes of HKααcarriers and-α3.7/αααanti-4.2.Methods::Samples were collected in our hospital from July 2017 to October 2019.Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of theαααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics and Fisher’s exact tests.Results::Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples.The results showed thatα,β,andαβcompound thalassemia were identified in 1190(46.78%),1286(50.55%),and 68(2.67%)cases,respectively.A total of 227 samples from thalassemia patients were identified as-α3.7/ααby Gap-PCR,and the genotypes of two samples were uncertain.There was a difference between Gap-PCR and combined groups(Gap-PCR combined with nested PCR and MLPA)in detecting HKαα(P<0.05).Among the 229 patients,20 patients were identified as HKααcarriers and one was identified as-α3.7/ααα anti-4.2 by two-round nested PCR and MLPA,including 15 patients with HKαα/αα,three with HKαα/αα and β-thalassemia coinheritance,one with HKαα/-SEA,one with HKαα/-α4.2 andβ-thalassemia coinheritance,and one with-α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions::αααanti-4.2 and HKααgenotypes of patients carrying-α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKααand-α3.7/αααanti-4.2 alleles.More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.
文摘Background： Wolf-Hirschhorn syndrome （WHS） is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification （MLPA） and array comparative genomic hybridization （array CGH）. Methods： Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results： All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions： The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.
文摘Multiplex Ligation-Dependent Probe Amplification (MLPA) was used to study the integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation. WTK1 cells contain a p53 mutation, whereas the TK6 cell line has the native p53 tumor-suppressor gene. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. Using probes that target specific regions on chromosomes associated with a distinct subset of microdeletions and microduplications either established or thought to be responsible for intellectual disability or developmental delay, we have demonstrated that WTK1 and TK6 are not impacted in the same way by irradiation. Instead, each cell line presents its own unique MLPA profile. The most notable differences are the appearance of nine unique probe signals only seen in WTK1 cells. These results are important in the study of how different cell lines can be affected in significantly different ways depending on the presence or absence of wild type p53.
文摘目的：探讨多重连接依赖性探针扩增（MLPA）技术在脊髓性肌肉萎缩症（SMA）患者、携带者及产前基因诊断中的应用。方法以临床诊断为 SMA 患儿为研究对象，采用基因组 DNA 多重连接探针扩增（MLPA）技术进行 SMN1基因和 SMN2基因拷贝数变异检测。结果 MLPA 分析结果显示，9个家系中9例患者及2例胎儿呈 SMN1基因纯合缺失，其父母双方 SMN1基因7、8外显子均为杂合性缺失，均为 SMA 携带者。结论 ML‐PA 技术可以应用于 SMA 患儿的基因诊断，不仅快速、简便，还可辨别携带者致病基因杂合缺失情况，可筛查携带者。
基金Supported by The National Natural Science Foundation of China,No.30940086
文摘AIM:To study the characteristics of APC(adenomatous polyposis coli)gene germline mutation in Chinese patients with familial adenomatous polyposis(FAP).METHODS:APC gene from 14 FAP families was amplified by polymerase chain reaction(PCR)and underwent direct sequencing to determine the micromutation type.For the samples without micromutation,the large fragment deletion of APC gene was examined by multiplex ligation-dependent probe amplification(MLPA).RESULTS:There were gene micromutations in 9 families with a micromutation detection rate of 64.3%(9/14),including 6 frameshift mutations(66.7%),1 nonsense mutation(11.1%)and 2 splicing mutations(22.2%).Large fragment deletions were detected by MLPA in 2 families.The total mutation detection rate of micromutations and large fragment deletions was 78.6%(11/14).CONCLUSION:The detection rate of APC gene germline mutation can be improved by direct sequencing combined with MLPA large fragment deletion detection.
基金This project was supported by the National Natural Science Foundation of China (No.81500925).
文摘Oculocutaneous albinism (OCA)is an autosomal recessive pigmentation abnormality,characterized by variable hair,skin,and ocular hypopigmentation.OCA1 is the most frequent subtype of OCA,caused by mutations in the tyrosinase gene (TYR). In this study,we investigated the genetic mutation of a Chinese family with a female OCA patient who came for genetic counseling before pregnancy.Complete physical examination was performed,and DNA from blood samples was collected from the family members.Mutations of TYR,OCA2,and SLC45A2 genes were examined in the proband, and verified in her parents by Sanger sequencing.Large deletion or duplication of TYR and OCA2 genes was detected by multiplex ligation-dependent probe amplification (MLPA).A homozygous TYR c.307T>C (p.Cys103Arg)missense mutation was identified in the proband,and both parents were heterozygous carriers.No large deletion or duplication was found in the proband.This mutation was absent in 1000G,ExAC,or HGMD database,and multiple lines of in silico tools supported a deleterious effect.These results suggest that TYR c.307T>C mutation might be responsible for OCA1,and our study further expands the mutation spectrum of OCA1 in the Chinese population.
