A Study of Radiation-Induced Telomere Instability Using 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.

Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction

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.

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

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.

A Study of Radiation-Induced Instability for the Gene Locus Associated with Intellectual Disorders or Developmental Delays

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.

APC gene mutations in Chinese familial adenomatous polyposis patients

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.

Identification of a Homozygous Missense Mutation in the TYR Gene in a Chinese Family with OCA1

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.

Coexistent Charcot-Marie-Tooth type 1A and type 2 diabetes mellitus neuropathies in a Chinese family

Charcot-Marie-Tooth disease type 1A（CMT1A） is caused by duplication of the peripheral myelin protein 22（PMP22） gene on chromosome 17. It is the most common inherited demyelinating neuropathy. Type 2 diabetes mellitus is a common metabolic disorder that frequently causes predominantly sensory neuropathy. In this study, we report the occurrence of CMT1 A in a Chinese family affected by type 2 diabetes mellitus. In this family, seven individuals had duplication of the PMP22 gene, although only four had clinical features of polyneuropathy. All CMT1 A patients with a clinical phenotype also presented with type 2 diabetes mellitus. The other three individuals had no signs of CMT1 A or type 2 diabetes mellitus. We believe that there may be a genetic link between these two diseases.

多重连接探针扩增技术及其应用

多重连接探针扩增是一种建立在PCR技术上的新型相对定量检测技术，该技术具有高效率、高分辨率、操作简便、设备要求低等诸多优势，目前已被广泛应用于基因片段缺失与重复、点突变及甲基化检测等相关疾病的分子生物学研究。其与基因芯片结合而建立的多重连接探针扩增微阵列芯片技术不但真正具备了高通量的检测能力，还使该技术的可靠性获得了很大的提高。本文就多重连接探针扩增的技术方法、应用进展及目前还存在的局限性作一综述。

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China

Background： Duchenne muscular dystrophy （DMD） and Becker muscular dystrophy （BMD） are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction （multiplex PCR） and multiplex ligation-dependent probe amplification （MLPA） are the most common methods for detecting dystrophin gene mutations, This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. Methods： We collected 121 probands, 64 mothers ofprobands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. Results： We found that 61.98% of the subjects had genetic mutations including deletions （50.41%） and duplications （11.57%）. There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency ofexons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. Conclusions： MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DM D high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

THREE NOVEL FOXL2 GENE MUTATIONS IN CHINESE PATIENTS WITH BLEPHAROPHIMOSIS-PTOSIS-EPICANTHUS INVERSUS SYNDROME

Blepharophimosis-ptosis-epicanthus inversus Psyndrome （BPES, OMIM # 110100） is a rare autosomal dominant disorder affecting the eyelid and ovarian development. When co-occurred together, it is type Ⅰ and when only the eyelid abnormalities are present, it is type Ⅱ. Both types had been mapped to the same locus 3q23 on the basis of cytogenetic rearrangements 1-3 and linkage analyses. 4-6 Subsequently,

A novel large deletion(exons 12,13)and a missense mutation(p.G46R)in the PAH in a Japanese patient with phenylketonuria

Background:Phenylketonuria(PKU)is caused by a defect in phenylalanine hydroxylase(PAH).More than 500 mutations have been reported for the gene encoding PAH.However,approximately l%-5%of these include large deletions and large duplications that cannot be detected by conventional methods.Methods:In this report we tried to fully characterize a PAH-deficient patient.The patient was a 2-year-old Japanese boy who was diagnosed with classical PKU at the time of neonatal screening,which was confirmed by the tetrahydrobiopterin-loading test.PCR-related direct sequencing and multiplex ligation-dependent probe amplification(MLPA)were used to analyze of the PAH of the patient.Results:Using PCR-related direct sequencing method,we could detect only a heterozygous novel missense mutation:p.136G>C(p.G46R).A second mutation was detected by MLPA.The patient was heterozygous for a novel large deletion of exons 12 and 13:c.1200-?_1359+?del(EX12_13del).For genetic counseling,an accurate genetic diagnosis is often necessary.Conclusions:Through a combination of MLPA and conventional methods,the success rate of PAH mutation identification can be close to 100%.