Upregulation of microRNA-23a regulates proliferation and apoptosis by targeting APAF-1 in laryngeal carcinoma.

MicroRNA-23a (miR-23a) is a potential biomarker for laryngeal cancer. Apoptotic protease activating factor 1 (APAF-1) was recently demonstrated to be a target of miR-23a. However, whether miR-23a exerts its effects via APAF-1 in laryngeal cancer, remains unknown. In the present study, miR-23a expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). APAF-1 mRNA and protein expression levels were assayed by RT-qPCR and western blotting, respectively. Binding of miR-23a to APAF-1 was monitored by a luciferase reporter assay. Gain-of-function and loss-of-function studies were performed in order to investigate the roles of miR-23a and APAF-1 in Hep2 cell proliferation and apoptosis. miR-23a and APAF-1 were found to be significantly upregulated and downregulated, respectively, in laryngeal cancer tissues, and there was a significant negative correlation between APAF-1 and miR-23a expression. The results of the luciferase reporter assay demonstrated that miR-23a bound directly to the APAF-1 mRNA 3'-untranslated region. Ectopic expression of miR-23a and knockdown of APAF-1 significantly promoted cell proliferation and colony formation, and inhibited early apoptosis in Hep2 cells. In conclusion, miR-23a acts as an oncogenic regulator in laryngeal carcinoma by directly targeting APAF-1, and may be a useful biomarker in the diagnosis and treatment of laryngeal carcinoma.

MicroRNA-27a promotes proliferation and suppresses apoptosis by targeting PLK2 in laryngeal carcinoma.

BACKGROUND: miRNA-27a has been confirmed as an important regulator in carcinogenesis and other pathological processes. Whether and how it plays a role in the laryngeal carcinoma is unknown. METHODS: Mature miRNA-27a expression in laryngeal cancer was detected by qRT-PCR. Gain-of-function studies using mature miR-27a were performed to investigate cell proliferation and apoptosis in the Hep2 cells. In silico database analysis and luciferase reporter assay were applied to predict and validate the direct target, respectively. Loss-of-function assays were performed to investigate the functional significance of the miR-27a target gene. qRT-PCR and Western blot were used to evaluate mRNA and protein levels of the target, respectively. RESULTS: miR-27a was significantly up-regulated in the laryngeal tumor tissues compared to the adjacent non-tumor tissues. In silico database analysis result revealed that PLK2 is a potential target of miR-27a. luciferase reporter assay result showed the direct inhibition of miR-27a on PLK2-3'UTR. In the cases with miR-27a up-regulation, PLK2 protein expression level was significantly lower in cancer tissues than that in the adjacent non-tumor tissues, which showed a negative correlation with miR-27a expression level. Both miR-27a and knockdown of PLK2 caused the increase of the cell viability and colony formation and inhibition of the late apoptosis in the Hep2 cell lines. Moreover, miR-27a but not PLK2 also repressed the early apoptosis in the Hep2 cells. Additionally, no alteration of the Hep2 cell cycle induced by miR-27a was detected. CONCLUSIONS: miR-27a acts as an oncogene in laryngeal squamous cell carcinoma through down-regulation of PLK2 and may provide a novel clue into the potential mechanism of LSCC oncogenesis or serve as a useful biomarker in diagnosis and therapy in laryngeal cancer.

Dynamic expression of molecules that control limb muscle development including Fhl1 in hind limbs of different gestational age.

Muscle abnormality could be a key reason for congenital clubfoot (CCF) deformity, which manifests itself during fetal development. FHL1 down-regulated expression is involved in the formation of skeletal muscle abnormalities in CCF and FHL1 gene mutations contribute to the development of some kinds of myopathies. Therefore, detecting dynamic expression of Fhl1 and other molecules (Hgf, MyoD1, Myogenin, and Myh4) that control limb muscle development in hind limbs of different gestational age will provide a foundation for further research on the molecular mechanism involves in the myopathies or CCF. The dynamic gene expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 in the lower limbs of E16, E17, E19, and E20 rat embryos were examined by real-time RT-PCR. Immunofluorescence was used to detect formation of specific muscle fibers (fast or slow fibers) in distal E17 hind limbs. The expression levels of Fhl1, Hgf, MyoD1, Myogenin, and Myh4 were varying in hind limbs of different gestational age. Real-time PCR results showed that all the genes that control skeletal muscle development except for Fhl1 exhibited a peak in E17 lower limbs. Immunofluorescence results showed obviously positive fast-myosin in the distal E17 lower limbs and meanwhile slow-myosin had no apparently signals. E17 was a critical time point for terminal skeletal muscle differentiation in the lower limbs of rat embryos.

S100A4 is upregulated via the binding of c-Myb in methylation-free laryngeal cancer cells.

DNA hypomethylation is correlated with the overexpression of the S100A4 gene in several types of cancers including laryngeal cancer, but the molecular mechanism is unknown. We speculated that the methylation status of the promoter affects its binding to the corresponding transcription factors. In the present study, luciferase reporter assay results indicated that the sequences -485 - +73 and -486 - -530 of the S100A4 promoter may harbor the positive and negative cis-acting elements, respectively; and moreover, the luciferase activity promoted by the sequence -485 - +73 increased and the S100A4 gene was significantly upregulated in 5-Aza-induced HEp2 cells. This implies that the methylation status of the sequence is important in regulating the expression of S100A4. Four transcription factor binding motifs including c-Myb, C/EBpα, Ap2 and Msx-1 in the region were predicted by P-Match software. c-Myb and C/EBpα but not Ap2 and Msx-1 were confirmed by EMSA and ChIP as transcription factors of S100A4. The decreased luciferase activity in methylation-free HEp2 cells transfected by the mutant c-Myb motif related to the methylated cytosine suggests that the hypomethylation of the c-Myb motif upregulates the S100A4 expression in laryngeal cancer.

Overexpression of DICER1 induced by the upregulation of GATA1 contributes to the proliferation and apoptosis of leukemia cells.

Dicer, a member of the RNase III family, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs. Recent evidence indicates that DICER1 expression levels vary among different solid tumors and decreased or increased DICER1 expression has been associated with aggressive cancers. In this study, we assessed DICER1 expression levels in acute myeloid leukemia (AML) and investigated its biological effects and transcriptional regulation in leukemia cell lines. We demonstrated that DICER1 was overexpressed in AML patients and leukemia cell lines by real-time quantitative PCR and western blot analysis. A functional assay demonstrated that the silencing of DICER1 inhibited cell proliferation and promoted apoptosis in leukemia cell lines. We also demonstrated that DICER1 was upregulated by the hematopoietic transcription factor, GATA1, through luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays. These data suggest that DICER1 plays an important role in AML and the finding that the upregulation of DICER1 is induced by GATA1 may provide a framework for the understanding of differential DICER1 expression levels in multiple types of cancer.

Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1 in laryngeal squamous cell carcinoma.

BACKGROUND: MYCT1, previously named MTLC, is a novel candidate tumor suppressor gene. MYCT1 was cloned from laryngeal squamous cell cancer (LSCC) and has been found to be down-regulated in LSCC; however, the regulatory details have not been fully elucidated. METHODS: Here, we sought to investigate the methylation status of the CpG islands of MYCT1 and mRNA levels by bisulfite-specific PCR (BSP) based on sequencing restriction enzyme digestion, reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR). The function of specific sites in the proximal promoter of MYCT1 in LSCC was measured by transient transfection, luciferase assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). RESULTS: The results suggested hypermethylation of 12 CpG sites of the promoter in both laryngeal cancer tissues and the laryngeal cancer line Hep-2 cell. The hypermethylation of the site CGCG (-695 to -692), which has been identified as the c-Myc binding site, was identified in laryngeal cancer tissues (59/73) compared to paired mucosa (13/73); in addition, statistical analysis revealed that the methylation status of this site significantly correlated with cancer cell differentiation(p < 0.01). The mRNA level of MYCT1 increased in Hep-2 cells treated with 5-aza-C (p < 0.01). The luciferase activity from mutant transfectants pGL3-MYCT1m (-852/+12, mut-695-C > A, mut-693-C > G) was significantly reduced compared with the wild type pGL3-MYCT1 (-852/+12), while the luciferase activity from wild transfectants pGL3-MYCT1 (-852/+12) rose after 5-aza treatment in Hep-2 cells. Finally, EMSA and ChIP confirmed that the methylation of the CGCG (-695 to -692) site prevented c-Myc from binding of the site and demethylation treatment of the 5' flanking region of MYCT1 by 5-aza induced the increased occupation of the core promoter by c-Myc (p < 0.01). CONCLUSION: In summary, this study concluded that hypermethylation contributed to the transcriptional down-regulation of MYCT1 and could inhibit cancer cell differentiation in LSCC. DNA methylation of the CGCG site (-695 to -692) of MYCT1 altered the promoter activity by interfering with its binding to c-Myc in LSCC. Epigenetic therapy of reactivating MYCT1 by 5-aza should be further evaluated in clinical trails of LSCC.

MYCT1-TV, a novel MYCT1 transcript, is regulated by c-Myc and may participate in laryngeal carcinogenesis.

BACKGROUND: MYCT1, a putative target of c-Myc, is a novel candidate tumor suppressor gene cloned from laryngeal squamous cell carcinoma (LSCC). Its transcriptional regulation and biological effects on LSCC have not been clarified. METHODOLOGY/PRINCIPAL FINDINGS: Using RACE assay, we cloned a 1106 bp transcript named Myc target 1 transcript variant 1 (MYCT1-TV) and confirmed its transcriptional start site was located at 140 bp upstream of the ATG start codon of MYCT1-TV. Luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed c-Myc could regulate the promoter activity of MYCT1-TV by specifically binding to the E-box elements within -886 to -655 bp region. These results were further verified by site-directed mutagenesis and RNA interference (RNAi) assays. MYCT1-TV and MYCT1 expressed lower in LSCC than those in paired adjacent normal laryngeal tissues, and overexpression of MYCT1-TV and MYCT1 could inhibit cell proliferation and invasion and promote apoptosis in LSCC cells. CONCLUSIONS/SIGNIFICANCE: Our data indicate that MYCT1-TV, a novel MYCT1 transcript, is regulated by c-Myc and down-regulation of MYCT1-TV/MYCT1 could contribute to LSCC development and function.

The involvement of CHD5 hypermethylation in laryngeal squamous cell carcinoma.

Chromodomain helicase DNA-binding protein 5 (CHD5) has been found to be a candidate tumor suppressor gene (TSG) in malignant neural tumors. In mice heterozygous for chd5 deficiency, the first tumor observed was pathological squamous cell carcinoma. More than 95% of primary laryngeal cancer is squamous cell carcinoma. Thus, we explored the expression of CHD5 in 65 patients with laryngeal squamous cell carcinoma (LSCC) using real-time PCR, immunohistochemistry and Western blotting. DNA methylation was detected using bisulfate-specific sequencing. The potential function of CHD5 was determined using MTT, apoptosis and transwell migration assays in CHD5-transfected Hep-2 cells. Our results revealed that the mRNA and protein expression levels of CHD5 in LSCC tissues were significantly lower than those in clear surgical margin tissues (p<0.05), and there is a significant correlation between the mRNA and protein expression levels of CHD5 (p<0.01). In addition, there were significant differences in CHD5 mRNA and protein levels with respect to the patient's clinical stage (p<0.05). Aberrant methylation of the CHD5 promoter was frequently found in the Hep-2 cell line and LSCC tumor tissues, especially tumor tissues from advanced TNM (p<0.05) or older patients (p<0.05). Finally, ectopic expression of CHD5 in laryngeal cancer cells led to significant inhibition of growth and invasiveness. Our data suggest that CHD5 is a tumor suppressor gene that is epigenetically downregulated in LSCC.

Reduced ACTC1 expression might play a role in the onset of congenital heart disease by inducing cardiomyocyte apoptosis.

BACKGROUND: The Cardiac α actin 1 gene (ACTC1) has been related to familial atrial septal defects. This study was set to explore a potential role of this gene in the formation of sporadic congenital heart disease (CHD). METHODS AND RESULTS: Assessment of cardiac tissue samples from 33 patients with sporadic CHD (gestational age (GA) 18 weeks-49 months) with real-time RT-PCR, Western blotting and immunohistochemistry has revealed a markedly decreased ACTC1 expression in the majority of samples (78.8%) compared with autopsied normal heart tissue from aged-matched subjects (GA 17 weeks-36 months). Also, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, the proportion of apoptotic cardiomyocytes in samples featuring down-regulated ACTC1 expression (Group 1) was significantly greater than those with normal expression (Group 2) and the controls (P<0.01). The proportion of apoptotic cells strongly correlated with the expression of ACTC1 (r=-0.918, P<0.01). A study of 2 essential genes involved in apoptosis, Caspase-3 and Bcl-2, confirmed that the former has significantly increased expression, whilst the latter has decreased expression in Group 1 than in the other groups (P<0.01). Transfection of a small interfering RNA targeting, Actc1 (Actc1-siRNA), to a cardiomyocyte cell line, H9C2, also detected more apoptotic cells. CONCLUSIONS: Reduced ACTC1 expression might play a role in the onset of CHD through induction of cardiomyocyte apoptosis.

14-3-3epsilon contributes to tumour suppression in laryngeal carcinoma by affecting apoptosis and invasion.

BACKGROUND: 14-3-3epsilon regulates a wide range of biological processes, including cell cycle control, proliferation, and apoptosis, and plays a significant role in neurogenesis and the formation of malignant tumours. However, the exact function and regulatory mechanism of 14-3-3epsilon in carcinogenesis have not been elucidated. METHODS: The expression of 14-3-3epsilon was assessed by RT-PCR and western blotting. The invasiveness and viability of Hep-2 cells were determined by the transwell migration assay and MTT assay, respectively. Cell cycle and apoptosis of Hep-2 cells were detected by flow cytometry. RESULTS: The mRNA and protein expression of 14-3-3epsilon in larynx squamous cell carcinoma (LSCC) tissues were significantly lower than those in clear surgical margin tissues. Statistical analysis showed that the 14-3-3epsilon protein level in metastatic lymph nodes was lower than that in paired tumour tissues. In addition, the protein level of 14-3-3epsilon in stage III or IV tumours was significantly lower than that in stage I or II tumours. Compared with control Hep-2 cells, the percentages of viable cells in the 14-3-3epsilon-GFP and negative control GFP groups were 36.68 +/- 14.09% and 71.68 +/- 12.10%, respectively. The proportions of S phase were 22.47 +/- 3.36%, 28.17 +/- 3.97% and 46.15 +/- 6.82%, and the apoptotic sub-G1 populations were 1.23 +/- 1.02%, 2.92 +/- 1.59% and 13.72 +/- 3.89% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The percentages of the apoptotic cells were 0.84 +/- 0.25%, 1.08 +/- 0.24% and 2.93 +/- 0.13% in the control, negative control GFP and 14-3-3epsilon-GFP groups, respectively. The numbers of cells that penetrated the filter membrane in the control, negative control GFP and 14-3-3epsilon-GFP groups were 20.65 +/- 1.94, 17.63 +/- 1.04 and 9.1 +/- 0.24, respectively, indicating significant differences among the different groups. CONCLUSIONS: Decreased expression of 14-3-3epsilon in LSCC tissues contributes to the initiation and progression of LSCC. 14-3-3epsilon can promote apoptosis and inhibit the invasiveness of LSCC.

[Teaching experience in integrated course of human development and genetics].

Establishment of integrated course system in human development and genetics is an important part of course reformation, and the improvement of this system is achieved by integrating the content of course, stabilizing teaching force, building teaching materials and applying problem-based learning. Integrity-PBL teaching model is founded and proved to be feasible and effective by teaching practice. Therefore, it maybe play an important role in improving teaching effect and cultivating ability of students to analyse and solve problems.

Hypomethylation-induced expression of S100A4 increases the invasiveness of laryngeal squamous cell carcinoma.

The present study aimed to identify genes related to 5AZA-CdR in laryngeal squamous cell carcinoma (LSCC) and to investigate the role of S100A4 in the development and aggression of LSCC. Differentially expressed proteins were identified in Hep-2 cells treated with 5AZA-CdR by two-dimensional gel electrophoresis combined with MALDI-TOF-MS. mRNA, protein levels and DNA methylation status of S100A4 were assessed by RT-PCR, Western blotting and methylation-specific PCR, respectively. The invasiveness of Hep-2 cells transfected by siRNA S100A4 was determined by transwell migration assay. Protein profiles from Hep-2 cells treated with 5AZA-CdR were obtained, and several differentially expressed proteins such as S100 calcium-binding protein A4 (S100A4) were identified. Results of RT-PCR and Western blotting revealed that both mRNA and protein levels of S100A4 were significantly higher in the metastatic lymph nodes than those in paired adjacent normal laryngeal (PANL) or tumor tissues. The DNA methylation status displayed significant differences between the LSCC and the PANL tissues. The expression level of S100A4 decreased in Hep-2 cells undergoing RNA interference of S100A4. The number of cells which crossed the basement membrane filter was significantly lower in the RNAi S100A4 group when compared with the number in the control group. The abnormal expression of S100A4 identified in Hep-2 cells treated with an inhibitor of DNA methyltransferase appeared to result from the aberrant DNA methylation status of S100A4. The abnormal expression of S100A4 altered the invasiveness of LSCC.

Transcriptional regulation of Fhl1 by estradiol in rat myoblastocytes.

Fhl1 (Four and a Half LIM domain 1) regulates muscle growth and development. In addition, skeletal myoblast growth is significantly affected by gender differences, implicating estrogen in the regulation of muscle development. We sought to determine if estrogen influences Fhl1 gene expression levels in rat L6GNR4 myoblastocytes that express the estrogen receptor beta (ERbeta), while luciferase assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were employed to confirm the interaction between ERbeta and Fhl1. Treatment of L6GNR4 cells with physiological levels of 17beta-estradiol (E2) results in markedly decreased endogenous Fhl1 expression. Tamoxifen, an ER antagonist, partially reverses E2-mediated Fhl1 down-regulation in L6GNR4 cells. Furthermore, luciferase assay and EMSA identified a novel promoter region of Fhl1 that directly interacts with ERbeta. ChIP of the ERbeta-Fhl1 promoter complex from L6GNR4 cells confirmed that endogenous ERbeta interacts with this region. These data indicate that E2 down-regulates Fhl1 expression through its binding to the ERbeta. This is the first report of a small molecule that can affect Fhl1 expression. E2 may therefore be useful in developing new strategies for regulating Fhl1 expression and understanding the influence of estrogen on muscle growth and development.

[The mechanism of TBX5 abnormal expression in simple congenital heart disease].

To explore the mechanism of TBX5 abnormal expression in simple congenital heart disease (CHD), 100 CHD venous blood, 50 CHD heart tissues, and 5 non-CHD heart tissues were involved in this study. The mutation and methylation in the 1 200 bp region upstream of TBX5 gene were detected by high-performance liquid chromatography (DHPLC) and methylation-sensitive restriction endonuclease (MS-RE), respectively. The binding site of NKX2-5 to Tbx5 predicted by P-MATCH software was validated by EMSA (Electrophoretic mobility shift assay). Tbx5 gene expression in mouse cardiac muscle cell H9C2(2-1) transfected with NKX2-5 expression vector was evaluated. No mutation was found in all patients. Both non-CHD and CHD heart tissues had the same methylation in the two CpG islands. Exogenous Nkx2-5 efficiently activated the transcription of the endogenous Tbx5 gene in H9C2 (2-1) cells. EMSA showed that the special binding band appeared when Nkx2-5 existed. These results indicates that the down expression of TBX5 might not be caused by mutation and methylation in the 1 200 bp region upstream of gene, and might be regulated by abnormal expression of NKX2-5 gene in heart muscle of CHD.

[Effect of cobalt chloride on S100A4 expression in human gastric cancer cell BGC823].

S100A4 is an important metastasis-associated gene. Researches have confirmed the close correlation between overexpression of S100A4 gene and gastric cancer's infiltration, lymph node metastasis and in vitro invasiveness of gastric cancer cells. In order to investigate the mechanism of overexpression of S100A4 gene, hypoxia mimetic cobalt chloride (CoCl2) was used to treat gastric cancer cell BGC823, and then the expression of S100A4 mRNA and protein in BGC823 cells were detected by RT-PCR, immunohistochemistry, immunofluorescence, and Western blotting analysis. After treatment with CoCl2, the expression of S100A4 mRNA and protein in BGC823 cell was increased. These results suggested that hypoxia mimetic cobalt chloride could increase the expression of S100A4 gene in gastric cancer cell BGC823.

Derivation of embryonic stem cell line from frozen human embryos and neural differentiation.

We established a human embryonic stem cell line derived from frozen human embryos of Chinese origin. The cell line expressed the pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and alkaline phosphatase. The pluripotency of the cell line was also demonstrated in vivo by teratoma formation in severe combined immunodeficiency mice. The embryonic stem cells formed embryoid bodies after culturing in suspension for 7 days. The embryoid bodies were transferred to an adherent culture system in serum-free medium. The differentiating cells derived from the embryoid bodies expressed Nestin and Sox2, markers of neural progenitor cells. After the induction of cyclic AMP for 7 days, the neural progenitor cells had differentiated into neurons and glial cells.

[Association study between mutations of transcription regulator sequences of COL1A1 gene and idiopathic con-genital talipes equinovarus].

RT-PCR was used to detect the expressions of COL1A1 mRNA in 20 patients with idiopathic congenital talipes equinovarus (ICTEV). The primers were designed by Primer 5 according to sequences of -1 031 bp~ +30 bp and the first intron of COL1A1. PCR-DGGE was used to screen the mutations in COL1A1 gene. Expression of COL1A1 on mRNA levels showed significantly higher in patients with ICTEV than in normal persons (t=12.680, P < 0.05). By DNA sequencing, a -161(T--> C) heterozygous mutation and a+ 274(C-->G) homozygous mutation were detected, and both were new identified mutations. These results indicated that the mutations in transcription regulator sequences of COL1A1 could cause ICTEV.

[Research of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus].

To investigate the relationship of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus(ICTEV), 84 samples from patients with ICTEV were used in the study. Mutation in the coding region of HOXD13 was detected by denaturing gradinent electrophoresis. The mRNA and protein levels of HOXD13 and FHL1 were evaluated by RT-PCR and immunohistochemistry, respectively. The binding site of FHL1 to HOXD13 predicted by PMATCH software was validated by EMSA( Electrophoretic mobility shift assay,EMSA).No mutation was found in the coding region of HOXD13 in 84 samples from patients with ICTEV. Both HOXD13(33.3%) and FHL1(46.6%) were down-regulated in ICTEV muscle tissue. The result of EMSA showed that the special binding band appeared when HOXD13 existed. The results shows that HOXD13 gene mutation was not involved in outbreak in idiopathic congenital talipes equinovarus, but changes of HOXD13 and FHL1 gene expression related to the development of talipes equinovarus malformation. HOXD13 might play an role in ICTEV through regulating FHL1 expression.

[Application study on inversion diagnosis of F8 gene in hemophilia A].

OBJECTIVE: To establish an effective method of genetic diagnosis on hemophilia A (HA) by detecting the inversion mutation in intron 22 of F8 gene. METHODS: Intron 22 inversion mutation in F8 gene was detected by using long distance-polymerase chain reaction (LD-PCR) and inversion-PCR (I-PCR) in 31 HA patients. The mothers of HA patients with intron 22 inversion mutation were selected to carrier diagnosis and amniotic fluid of the pregnant women with inversion mutation was collected at intermediate stage of gestation, and used to prenatal genetic diagnosis. RESULTS: Seven patients showed F8 gene inversion mutation in thirty-one patients. Three in four mothers of HA patients with intron 22 inversion mutation were diagnosed as carriers. The prenatal diagnosis result indicated that the fetus conceived in the HA-carrier woman was normal individual. CONCLUSION: The detection of intron 22 inversion mutation by LD-PCR and I-PCR is time-saving, and can be used in prenatal diagnosis on HA.