Carrier Screening and Prenatal Gene Diagnosis of β-thalassemia by PCR-RDB Technique

In order to identify the distribution of gene types of β-thalassemia and reduce the birthrates of β-thalassemia major in Guiyang area, 1054 pregnant women and their spouses from Affiliated Hospital, Guiyang Medical College were screened. The positive samples were analyzed with polymerase chain reaction and reverse dot blot method (PCR-RDB). When both partners were heterozygous identified as carriers for β- thalassemia, the risk of having a fetus who was homozygous or compound heterozygous was 2.66 %; the ratio of male to female was 1/1.15. Seven types of mutation were identified. CD17 and CD41-42 were dominant among them. Among the 4 cases subject to prenatal gene diagnosis, one fetus was completely normal and 3 fetuses were diagnosed as having β-thalassemia major (1 homozygous and 2 compound heterozygous). The fetuses diagnosed as β-thalassemia major were selectively terminated within two weeks. It was concluded that the birthrate of β-thalassemia major in Guiyang area was reduced and the target of improving birth outcome and child development has been achieved.

Gene diagnosis and targeted breeding for blast-resistant Kongyu 131without changing regional adaptability

The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131),a leading japonica variety in Northeast China.In this study,two rice lines,KP1 and KP2-Hd1,were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131,respectively.However,both lines headed later than KY131.RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1,and the linkage drag of Hd1 was broken by recombination.On the other hand,no known flowering genes were introgressed into KP1.Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles.Due to its suppression role in heading under long-day conditions,Ghd8 was chosen as the target for gene editing to disrupt its function.Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations,which were easy to detect via agarose gel electrophoresis.All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1,even than KY131,under natural long-day conditions,which ensures its growth in Northeast China.This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.

Rapid genetic diagnosis and prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography

Spinal muscular atrophy （SMA） is a common Pautosomal recessive neuromuscular disorder （1in 6000 to 10 000 births） caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous deletion of SMN1, which has proved to be useful in the diagnosis of SMA. But it is hampered because of the existence of a highly homologous gene, SMN2. Based on nucleotide mismatches between SMN1 and SMN2, the following two DNA tests are usually performed： single-strand conformational polymorphism （SSCP） and polymerase chain reaction （PCR） followed by a restriction enzyme digestion.In this study we developed a new method for rapid genetic diagnosis of SMA by denaturing high-performance liquid chromatography （DHPLC）, which is based on different retention of homoduplexes and heteroduplexes in detecting the homozygous deletion of SMN1. Both genetic and prenatal diagnoses were performed successfully for a SMA family by DHPLC, which was confirmed as a rapid and effective technique for detecting the deletion of SMN1.

Carrier Detection and Presymptomatic Identification of Wilson Disease in Chinese by Non-Isotopic Linkage Analysis with Four Short Tandem Repeat Polymorphisms

Summary: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. To establish an efficient, accurate and fast diagnostic method for carrier detection and presymptomatic identification of WD in Chinese population, we studied haplotypes of short tandem repeat (STR) polymorphisms flanking the WD gene in 40 Chinese WD families. The results suggested that this genetic diagnosis system based on the four STR polymorphisms is of high value for the detection of potential carriers and WD homozygotes in families with at least one previously affected child. It is an efficient, accurate and fast diagnostic method that can be well suited for routine use in clinical laboratories.

ESTABLISHMENT OF IMMUNO－PCR TECHNIQUE FOR THE DETECTION OF TUMOR ASSOCIATED ANTIGEN MG＿7－Ag ON THE GASTRIC CANCER

The gastric cancer associated antigen MG_7-Ag was detected by means of a newly established method,termed Immuno-PCR A McAb-recombinant DNA chimeric molecule was made which possesses bispecific binding affinity for antigen that had been immobilized on microtiter wells and the segment of the attached DNA was amplified by PCR. The antigen of gastric cancer cell line KATO III was monitored by this method. Analysis of PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 20 cells to be detected readily and reproducibly. Immuno-PCR showed a 104 enhancement in detection sensitivity compared with ELISA assay. When the same numbers of cells ( 2×10e/ml ) were immobilized and then the serial diluted chimeric molecule was added. 3.8×10-14 moles and 3.0 × 10-11 moles were needed to give positive results with the Immuno-PCR and ELISA assay. respectively.Therefore, Immuno-PCR could give an enomous amplification capability with good specificity, and has a sensitivity much higher than any existing techniques for antigen detection.

Birth of healthy children after preimplantation diagnosis of β-thalassemia

Background Clinical programs for preventing β-thalassemia are presently based on prospective carrier screening and prenatal diagnosis. This paper report an achievement of a pregnancy with unaffected embryos using in vitro fertilization and embryo transfer (IVF-ET), in combination with preimplantation genetic diagnosis (PGD), for a couple at risk of having children with β-thalassemia. Methods A couple carrying different thalassemia mutations, both a codon 41-42 mutation and the IVS-II-654 mutation, received standard IVF treatment, with intracytoplasmic sperm injection, embryo biopsiy, single cell polymerase chain reaction (PCR) and DNA analysis. Only unaffected or carrier embryos were transferred to the uterine cavity. After confirmation of pregnancy, a prenatal diagnosis was performed. Results Of a total of 13 embryos analyzed for β-globin mutations, PGD indicated that 2 were normal, 3 were affected, and 6 were carriers. Diagnosis could not be made in the other 2 embryos. Three embryos were transferred to the uterus on the third day after oocyte retrieval. Ultrasonography revealed a twin pregnancy with one blighted ovum. The prenatal genetic diagnosis revealed that both fetuses were unaffected, and two healthy boys were born, confirming the results of PGD. Conclusions We developed a single-cell based primer extension preamplification (PEP)-PCR assay for the detection of β-thalassemia mutations. The assays were efficient and accurate at all stages of the procedure, and resulted in the birth of PGD-confirmed β-thalassemia free children in China. PEP was used here in PGD for β-thalassemia.

Frequency analysis of autosomal dominant spinocerebellar ataxias in mainland Chinese patients and clinical and molecular characterization of spinocerebellar ataxia type 6

Background Dominantly inherited spinocerebellar ataxia (SCA) is a clinically and genetically heterogeneous group of neurodegenerative disorders. This study was to further assess the frequency of SCA1 (spinocerebellar ataxia type 1), SCA2, SCA3/MJD (spinocerebellar ataxia type 3/Machado-Joseph disease), SCA6, SCA7, SCA8, SCA10, SCA12, SCA14, SCA17 and DRPLA (dentatorubro-pallidoluysian atrophy) in mainland Chinese, and to specifically characterize mainland Chinese patients with SCA6 in terms of clinical and molecular features.Methods Using a molecular approach, we investigated SCA in 120 mainland Chinese families with dominantly inherited ataxias and in 60 mainland Chinese patients with sporadic ataxias. Clinical and molecular features of SCA6 were further characterized in 13 patients from 4 families. Results SCA3/MJD was the most common type of autosomal dominant SCA in mainland Chinese, accounting for 83 patients from 59 families (49.2%), followed by SCA2[8(6.7%)], SCA1[7(5.8%)], SCA6[4(3.3%)], SCA7[1(0.8%)], SCA8(0%), SCA10(0%), SCA12(0%), SCA14(0%), SCA17(0%) and DRPLA(0%). The genes responsible for 41 (34.2%) of dominantly inherited SCA families remain to be determined. Among the 60 patients with sporadic ataxias in the present series, 3 (5.0%) was found to harbor SCA3 mutations while none was found to harbor SCA6 mutations. In the 4 families with SCA6, significant anticipation was found in the absence of genetic instability on transmission.Conclusion A geographic cluster of families with SCA6 subtype was initially identified in a mainland Chinese population.

PCR assay for the inversion causing severe Hemophilia A and its application

Objective To develop a new technique based on long distance polymerase chain reaction (LD PCR) to replace Southern blotting method to detect Factor Ⅷ (FⅧ) gene inversion leading to severe Hemophilia A (HA) and carrier.Methods Four primers P, Q, A&B were designed and synthesized. P&Q is sp ecific for 5' and 3' flanking regions of F8A 1 respectively. A&B is specific fo r 5' and 3' flanking regions of F8A 2/F8A 3 respectively. LD PCR with 3 p rime rs and 3 temprature was set up, optimized and used to detect the inversion.Results The LD PCR with primers P,Q,A&B, P,Q&B and P,Q&A can be used t o detect the gene inversion and discriminate carrier from wild type. A blind ana lysis of 53 DNA samples from HA families was carried out by the LD PCR and Sout hern blotting respectively. Two sets of the results were completely identical. T hey were 23 cases of inversion, 27 cases of wild type and 3 cases of carriers. T he sensitivity and specificity of LD PCR are both 100%. Three inversion hem izygotes and 4 female carriers were identified from 5 HA families by the LD PCR technology.Conclusions The LD PCR with primer P,Q&B or P,Q,A&B can be used to det ect the gene inversion and the carrier of inversion. Compared with Southern blot ting, this technique is simple, rapid, inexpensive, more sensitive, accurate and non isotopic.

Blood Biomarkers in Minor Stroke and Transient Ischemic Attack

Minor stroke and transient ischemic attack（TIA）are common disorders with a high rate of subsequent disabling stroke, so the early recognition and management of minor stroke and TIA is of great importance. At the moment, the diagnosis of these disorders is based on neurologic deficits in a stroke-clinician＇s examination of the patient, supplemented by the results of acute brain imaging.However, high variability in TIA diagnosis has been reported between physicians, even trained vascular neurologists, and image-based diagnostic confirmation is not always readily available. Some patients still have ischemic events despite sustained standard secondary preventive therapy. Blood biomarkers are promising to aid in the diagnosis, risk stratification, and individual treatment of minor stroke and TIA. Some studies are being conducted in this field. This mini-review aims to highlight potential biomarkers for diagnosis and those helpful in predicting the risk of future stroke and the selection of treatment.