Viscoelasticity in trapezius myofascial pain syndrome: quantitative assessment using Real-Time Shear-Wave Elastography.

OBJECTIVE: To investigate the differences in the viscoelastic properties between normal trapezius muscles and those in patients with trapezius myofascial pain syndrome (MPS) using real-time shear-wave elastography (SWE). MATERIALS AND METHODS: This study included 31 patients with trapezius MPS and 31 volunteers. Sixty-one trapezius muscles (41 and 20 on the affected and non-affected side, respectively) of patients with MPS and 62 normal trapezius muscles in volunteers were assessed. Conventional ultrasonic parameters, including skeletal muscle thickness, resistance index (RI), and mean shear wave velocity (SWVmean) of trapezius muscles, were obtained in the seated position with the shoulders and neck relaxed. The daily neck leaning time (unit:hours) of all participants was obtained using a questionnaire. RESULTS: Ultrasound showed no statistically significant differences in thickness or RI of the trapezius muscles of the affected and non-affected sides in MPS patients versus normal trapezius muscles (p = 0.976 and 0.106, respectively). In contrast, the SWVmean of trapezius muscles in patients with MPS was significantly higher than that of normal trapezius muscles in both the affected and non-affected sides (4.41 ± 1.02 m/s vs. 3.35 ± 0.79 m/s, p < 0.001; 4.05 ± 0.63 m/s vs. 3.35 ± 0.79 m/s, p = 0.002). There was no significant difference between the SWVmean of the trapezius muscles on the affected and non-affected sides in patients with MPS (4.41 ± 1.02 m/s vs. 4.05 ± 0.63 m/s, p = 0.225). Correlation analysis showed that daily neck forward time was positively correlated with the SWVmean of the trapezius muscles on the affected and non-affected sides in patients with MPS (r = 0.635, p < 0.001; r = 0.576, p = 0.008). CONCLUSION: SWE can quantitatively evaluate stiffness of trapezius muscles in patients with trapezius MPS. The stiffness of both affected and non-affected trapezius muscles increased in patients with trapezius MPS, and the degree of increase positively correlated with the time of cervical forward leaning.

Upper trapezius muscle elasticity in cervical myofascial pain syndrome measured using real-time ultrasound shear-wave elastography.

Background: Myofascial pain syndrome (MPS) is a common cause of neck pain, which is a global public health problem. Because MPS does not present morphological changes within lesioned muscles, there are no imaging diagnostic criteria for this condition. In this study, we evaluate elasticity changes in upper trapezius muscles most frequently involved in cervical MPS using real-time ultrasound shear-wave elastography, and we examine their potential diagnostic value. Methods: We consecutively enrolled 109 right posterior neck pain patients for this prospective study. Of these, 51 were diagnosed with MPS and 58 with non-MPS in the right side of their neck. Among MPS patients, 19 fell into the 1-3 range (mild pain) for pain scores on the visual analog scale (VAS), 25 fell into the 4-6 range (moderate pain), and 7 into the 7-10 range (severe pain). MPS was diagnosed by two independent clinicians using the diagnostic criteria proposed by Simons et al. Using real-time ultrasound shear-wave elastography, we measured right trapezius mean shear-wave velocity (SWVmean). The midpoint of the line between the foramen magnum and the end of the right acromion served as measuring point. Regions of interest were scaled to span 0-8.0 m/s. Results: Trapezius SWVmean was significantly higher in MPS patients compared with non-MPS patients (P<0.001). Stratified analysis of MPS patients according to pain severity revealed similar trapezius SWVmean between mild pain and non-MPS patients (P=0.324), however SWVmean was higher in moderate and severe pain MPS patients compared with non-MPS patients (P<0.001). The area under the curve (AUC) value for upper trapezius SWVmean in MPS patients was 0.791 (95% CI: 0.703-0.863). Corresponding sensitivity and specificity values were 86.27% (95% CI: 73.7-94.3%) and 62.07% (95% CI: 48.4-74.5%). Stratified analysis of MPS patients by pain severity produced the following AUC values for trapezius SWVmean in MPS patients with mild, moderate, and severe pain: 0.578 (95% CI: 0.460-0.690), 0.899 (95% CI: 0.814-0.955), and 0.983 (95% CI: 0.914-0.999), respectively. Conclusions: Elasticity changes and increased stiffness in the trapezius occur in cervical MPS patients with moderate and severe pain. The SWVmean parameter reflecting trapezius muscle elasticity may be valuable for successful screening of cervical MPS, especially in patients with moderate and severe pain.

Functional Liver Imaging in Radiotherapy for Liver Cancer: A Systematic Review and Meta-Analysis.

Backgrounds: Functional liver imaging can identify functional liver distribution heterogeneity and integrate it into radiotherapy planning. The feasibility and clinical benefit of functional liver-sparing radiotherapy planning are currently unknown. Methods: A comprehensive search of several primary databases was performed to identify studies that met the inclusion criteria. The primary objective of this study was to evaluate the dosimetric and clinical benefits of functional liver-sparing planning radiotherapy. Secondary objectives were to assess the ability of functional imaging to predict the risk of radiation-induced liver toxicity (RILT), and the dose-response relationship after radiotherapy. Results: A total of 20 publications were enrolled in descriptive tables and meta-analysis. The meta-analysis found that mean functional liver dose (f-MLD) was reduced by 1.0 Gy [95%CI: (-0.13, 2.13)], standard mean differences (SMD) of functional liver volume receiving ≥20 Gy (fV20) decreased by 0.25 [95%CI: (-0.14, 0.65)] when planning was optimized to sparing functional liver (P >0.05). Seven clinical prospective studies reported functional liver-sparing planning-guided radiotherapy leads to a low incidence of RILD, and the single rate meta-analysis showed that the RILD (defined as CTP score increase ≥2) incidence was 0.04 [95%CI: (0.00, 0.11), P <0.05]. Four studies showed that functional liver imaging had a higher value to predict RILT than conventional anatomical CT. Four studies established dose-response relationships in functional liver imaging after radiotherapy. Conclusion: Although functional imaging modalities and definitions are heterogeneous between studies, but incorporation into radiotherapy procedures for liver cancer patients may provide clinical benefits. Further validation in randomized clinical trials will be required in the future.

Research on human glioma stem cells in China.

Research on human glioma stem cells began early in the 21st century and since then has become a rapidly growing research field with the number of publications increasing year by year. The research conducted by our diverse group of investigators focused primarily on cell culture techniques, molecular regulation, signaling pathways, cancer treatment, the stem cell microenvironment and the cellular origin and function of glioma stem cells. In particular, we put forward our view that there are inverse or forward transformations among neural stem cells, glial cells and glioma stem cells in glioma tissues under certain conditions. Based on the background of the progress of international research on human glioma stem cells, we aim to share our progress and current findings of human glioma stem cell research in China with colleagues around the world.

Doppler Echocardiographic Measurements in Normal Chinese Adults (EMINCA): a prospective, nationwide, and multicentre study.

AIMS: Currently, available Doppler echocardiographic reference values are derived mainly from North American and European population studies, which may not applicable to the Chinese population. We aimed to establish normal reference values of Doppler echocardiographic parameters in a nationwide, population-based cohort of healthy Han Chinese adults. METHODS AND RESULTS: A total of 1394 qualified healthy subjects (mean age 47.3 ± 16.0 years, 678 men) were enrolled at 43 collaborating laboratories, 37 transvalvular flow and tissue Doppler parameters were obtained, and the impacts of gender and age on each parameter were analysed. Significant differences were found between men and women in 48.6% (18/37) of the parameters analysed, and among different age groups in 83.8% (31/37) of the parameters in men and in 86.5% (32/37) of the parameters in women. CONCLUSIONS: Normal reference values of Doppler echocardiographic parameters were established for the first time in a nationwide, population-based cohort of healthy Han Chinese adults. Since most of these parameters differed by gender and/or age, reference values specified for gender and age should be recommended in clinical practice.

Echocardiographic measurements in normal chinese adults focusing on cardiac chambers and great arteries: a prospective, nationwide, and multicenter study.

BACKGROUND: Currently available echocardiographic reference values are derived mainly from North American and European population studies, and no echocardiographic reference values are available for the Chinese population. The aim of this study was to establish normal values of echocardiographic measurements of the cardiac chambers and great arteries in a nationwide, population-based cohort of healthy Han Chinese adults. METHODS: A total of 1,586 healthy Han Chinese volunteers aged 18 to 79 years were screened at 43 collaborating laboratories throughout China. Standard M-mode and two-dimensional echocardiography was performed to obtain measurements of the cardiac chambers and great arteries. The impacts of gender and age on all echocardiographic measurements were analyzed. RESULTS: A total of 1,394 qualified healthy subjects (mean age, 47.3 ± 16.0 years; 678 men) were ultimately enrolled. Except for left ventricular ejection fraction, values of cardiac chamber and great arterial dimensions were significantly higher in men than in women. Most measurements of the atrial and great arterial dimensions, left ventricular wall thickness, and left ventricular mass increased with age in both men and women. CONCLUSIONS: Normal reference values of cardiac dimensional parameters were established for the first time in a nationwide, population-based cohort of healthy Han Chinese adults. Because most of these parameters were found to vary with gender and age, reference values stratified for gender and age should be used in clinical practice.

[Surface-enhanced Raman spectra analysis of trace degradation products from goat horn].

Nano-silver colloid was synthesized by using microwave method on the mixtures of sodium citrate solution and silver nitrate solution. The method has advantages of fast heating speed, uniform temperature distribution and easily controlled reaction conditions. The sizes and size distributions of the silver particles were characterized by means of quasi-elastic laser scattering (QLS). The average particles size was (53.27 +/- 2.65) nm and the size of the particles was mainly distributed around 56 nm. Surface-enhanced Raman spectra of the degradation products from goat horn were obtained with silver colloid as active substrate. It was observed that the Raman signal of SERS was enhanced significantly compared with that of regular Raman spectrum, especially at the Raman bands of 659, 830, 850, 929, 999, 1 028, 1 280, 1 439 and 1 599 cm(-1) which reflect the biochemical components in degradation products. The characteristic Raman bands of degradation products from goat horn were preliminary assigned. The assignments showed that the main constituents of the degradation products from goat horn were amino acids and polypeptides. It was for the first time that Surface-enhanced Raman spectroscopy was used to detect trace degradation products from the horns. Raman signal enhancement can be obtained with high sensitivity for the trace concentrations as low as ppm level. It is concluded that surface-enhanced Raman spectroscopy can provide a fast, direct and precise detecting method for the detection of trace degradation solution from horns.

Establishment of a green fluorescent protein tracing murine model focused on the functions of host components in necrosis repair and the niche of subcutaneously implanted glioma.

Due to progress in the research of glioma stem cells and the glioma niche, development of an animal model that facilitates the elucidation of the roles of the host tissue and cells is necessary. The aim of the present study was to develop a subcutaneous xenograft green fluorescent protein nude mouse model and use this model to analyze the roles of host cells in tumor necrosis repair. Tumors derived from the human glioma stem/progenitor cell line SU3 were subcutaneously implanted in green fluorescent protein nude mice. The implanted tumors were then passed from animal to animal for 10 generations. Finally, subcutaneous xenografts were assayed with traditional pathology, immunopathological techniques and fluorescence photography. For each generation, the tumorigenicity rate was 100%. Subcutaneous xenografts were rich in blood vessels, and necrotic and hemorrhagic foci, which highly expressed hypoxia-inducible factor-1α, tumor necrosis factor, Ki-67, CD68 and CD11b. In the interstitial tissue, particularly in old hemorrhagic foci, there were numerous cells expressing green fluorescent protein, CD68 and CD11b. Green fluorescent protein nude mouse subcutaneous xenografts not only consistently maintained the high invasiveness and tumorigenicity of glioma stem/progenitor cells, but also consisted of a high concentration of tumor blood vessels and necrotic and hemorrhagic foci. Subcutaneous xenografts also expressed high levels of tumor microenvironment-related proteins and host-derived tumor interstitial molecules. The model has significant potential for further research on tumor tissue remodeling and the tumor microenvironment.

Microstructure and mechanical properties of horns derived from three domestic bovines.

The microstructure and mechanical properties of horns derived from three domestic bovines (buffalo, cattle and sheep) were examined. The effects of water content, sampling position and orientation of three bovid horns on mechanical properties were systematically investigated by uniaxial tension and micron indentation tests. Meanwhile, the material composition and metal element contents were determined by Raman spectroscopy and elemental analysis respectively, and the microstructures of the horns were measured by scanning electron microscopy (SEM). Results show that the mechanical properties of horns have negative correlation with water contents and depend on sampling position and orientation. The spatial variations of the mechanical properties in horns are attributed to the different keratinization degrees in the proximal, middle and distal parts. And the mechanical properties of horns in the longitudinal direction are better than those in transverse. Among the three kinds of horns, the mechanical properties of buffalo horn are the best, followed by cattle horn, and those in sheep horn are the worst. This is due to the differences in material composition, metal element, and the microstructures of the horns. But the mechanical properties of buffalo horns are not dependent on the source of the buffalo. Therefore, regular engineered buffalo keratinous materials with standard mechanical properties can be obtained from different buffalo horns by using proper preparation methods.

Feasibility of single-beat full-volume capture real-time three-dimensional echocardiography for quantification of right ventricular volume: validation by cardiac magnetic resonance imaging.

BACKGROUND: The lack of an accurate noninvasive method for assessing right ventricular (RV) volume and function has been a major deficiency of two-dimensional (2D) echocardiography. The aim of our study was to test the feasibility of single-beat full-volume capture with real-time three-dimensional echo (3DE) imaging system for the evaluation of RV volumes and function validated by cardiac magnetic resonance imaging (CMRI). METHODS: Sixty-one subjects (16 normal subjects, 20 patients with hypertension, 16 patients with pulmonary heart disease and 9 patients with coronary heart disease) were studied. RV volume and function assessments using 3DE were compared with manual tracing with CMRI as the reference method. RESULTS: Fifty-nine of 61 patients (96.7%; 36 male, mean age, 62 ± 15 years) had adequate three-dimensional echocardiographic data sets for analysis. The mean RV end diastolic volume (EDV) was 105 ± 38 ml, end-systolic volume (ESV) was 60 ± 30 and RV ejection fraction (EF) was 44 ± 11% by CMRI; and EDV 103 ± 38 ml, ESV 60 ± 28 ml and RV EF 41 ± 13% by 3DE. The correlations and agreements between measurements estimated by two methods were acceptable. CONCLUSION: RV volumes and function can be analyzed with 3DE software in most of subjects with or without heart diseases, which is able to be estimated with single-beat full-volume capture with real-time 3DE compared with CMRI.

[Host glial cell canceration induced by glioma stem cells in GFP/RFP dual fluorescence orthotopic glioma models in nude mice].

OBJECTIVE: During the process of tissue remodeling in human tumor transplantation models, the roles of the inoculated tumor cells and host tissue in tumor progression is still largely unknown. The aim of this study was to investigate the relationships and interactions between these two sides using GFP-RFP double fluorescence tracing technique. METHODS: Red fluorescence protein (RFP) gene was stably transfected into glioma stem cell line SU3, then SU3-RFP cells were transplanted into the brain of athymic nude mice with green fluorescence protein (GFP) expression. After the intracerebral tumors were formed, the relationship and interaction between GFP cells and RFP cells were analyzed. Highly proliferative GFP cells were screened out, and monocloned with micro-pipetting. DNA content assay, chromosome banding and carcinogenicity test of the GFP cells were performed to observe the GFP cells' cancerous phenotype in nude mice. RESULTS: In the transplantable tumor tissue, besides a great quantity of RFP cells, there were still a proportion of GFP cells and GFP/RFP fusion cells. The proportion of RFP cells, GFP cells and GFP/RFP cells were (88.99 ± 1.46)%, (5.59 ± 1.00)%, and (4.11 ± 1.020)%, respectively. Two monoclonal host GFP cells (H1 and H9) were cloned, which demonstrated the properties of immortality, loss of contact inhibition, and ultra-tetraploid when cultured in vitro. Both H1 and H9 cells expressed CNP, a specific marker of oligodendrocytes. The GFP cells also demonstrated 100% tumorigenic rate and high invasive properties in vivo. CONCLUSIONS: In this glioma transplantation model, the transplanted tumor tissues contained not only transplanted glioma stem cells but also cancerous host GFP cells. Our findings offer important clues to further research on the relationships among different members in the tumor microenvironment.

[Screening for EXT1 and EXT2 gene mutations in a ethnic Han Chinese family from Shanxi with hereditary multiple exostoses].

OBJECTIVE: To screen for potential mutations in an ethnic Han Chinese family from Shanxi with hereditary multiple exostoses. METHODS: Polymerase chain reaction and DNA sequencing were used to screen potential mutations in EXT1 and EXT2 genes. RESULTS: For EXT1 gene, two synonymous mutations (P477P and E587E), three intronic mutations (c.1537 -48A>G, c.1721 +203A>G and c.1722 -103C>G) were detected. For EXT2 gene, five intronic mutations (c.-29 -148A>T, c.1080 -18T>A, c.1336 -93C>T, c.1526 -166C>T, and c.1526 -195C>T) were identified. Among these, EXT1 P477P, EXT1 E587E and EXT2 c.1080 -18T>A are polymorphisms listed by Multiple Osteochondroma Mutation Database, whilst the other 7 sites have not been reported. CONCLUSION: No mutations have been found among all exons of the EXT1 and EXT2 genes in this family. Linkage analysis is necessary for identifying the cause of this disease.

[Correlations of integrons and resistance gene cassettes in Gram-negative bacteria with multi-drug resistance].

OBJECTIVE: To explore the correlations of integrons, gene cassettes and drug resistance phenotypes in 90 multi-drug resistant Gram-negative bacteria. METHODS: Class I/II/III integron and variable region of positive strains of 90 Gram-negative bacteria were amplified by PCR and types of integron variable region gene cassettes analyzed by DNA sequence. And the resistant rates of integron positive and negative strains were tested by drug susceptibility. RESULTS: The detection rate of integron was 81.1% (73/90) in 90 Gram-negative bacteria. The integron types were class I (n = 70), class II (n = 3) and class III (n = 0). Based on the BLAST analysis by GenBank database, in the amplified fragments of Class I integron positive strains variable region gene ranging from 730 to 3300 bp, 8 types of integron structure were identified. And there were aadB (n = 11), aac (6')-II (n = 7), aadA5 (n = 10), dfrA17-aadA5 (n = 14), dfrA12-OrfF-aadA2(n = 1), aacA4-catB8-aadA1(n = 24), aacC1-OrfA-OrfB-aadA1 (n = 3), catB3-aadB-dhfrV-aacA4-nit1-nit2 (n = 1), in which catB3-aadB-dhfrV-aacA4-nit1-nit2 was a new resistance gene cassette; the variable region fragment of class II integron positive strain was 1600 bp, with 3 carrier strains of sat2-aadA1 gene cassette.Susceptibility testing showed that the antimicrobial resistance rate of integron positive strains to aminoglycosides and sulfa were significantly higher than those of integron negative strains and accorded with the results of integration variable region gene cassettes; the positive strains were more sensitive to amikacin with a resistance rate of 32.9% (24/73); and the drug resistance rates of all beta-lactam strains were ≥ 80%. CONCLUSIONS: There is a higher carrier rates of classI integron in Gram-negative bacteria. And the resistant phenotype is related with the types of resistance gene cassettes of integron variable region.

Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi, China.

The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH) gene was investigated in 59 children with phenylketonuria (PKU) and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%), followed by Ivs7 + 2 T > A (5.1%) and T278I (2.5%). G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.

Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling.

BACKGROUND: The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells. However, these tumor cells are hard to be visualized directly in histopathological preparations, or in experimental glioma models. Therefore, we developed an experimental human dual-color in vivo glioma model, which made tracking solitary invasive glioma cells possible, for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells. This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling. METHODS: Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein (EGFP) were crossed with male Balb/c nude mice. Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive. Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein (RFP) gene, and a rat C6 glioma cell line was stained directly with CM-DiI, to establish three glioma cell lines emitting red fluorescence (SU3-RFP, U87-RFP, and C6-CM-DiI). Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice. Tumor-bearing mice were sacrificed when their clinical symptoms appeared, and the whole brain was harvested and snap frozen for further analysis. Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells. RESULTS: Almost all the essential tissues of the established EGFP athymic Balb/c nude mice, except hair and erythrocytes, fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm, approximately 50% of the offsprings were nu/nu EGFP+. SU3-RFP, U87-RFP, and C6-CM-DiI almost 100% expressed red fluorescence under the fluorescence microscope. Under fluorescence microscopic view, RFP+ cells were observed growing wherever they arrived at, locating in the brain parenchyma, ventricles, and para-vascular region. The interactions between the transplanted tumor cells and host adjacent cells could be classified into three types: (1) interweaving; (2) mergence; and (3) fusion. Interweaving was observed in the early stage of tumor remodeling, in which both transplantable tumor cells and host cells were observed scattered in the tumor invading and spreading area without organic connections. Mergence was defined as mutual interactions between tumor cells and host stroma during tumorigenesis. Direct cell fusion between transplantable tumor cells and host cells could be observed occasionally. CONCLUSIONS: This study showed that self-established EGFP athymic nude mice offered the possibility of visualizing tumorigenesis of human xenograft tumor, and the dual-color xenograft glioma model was of considerable utility in studying the process of tumor remodeling. Based on this platform, mutual interactions between glioma cells and host tissues could be observed directly to further elucidate the development of tumor microenvironment.

[Detection of exon 7 mutations of PAH gene in classical phenylketonuria by high-resolution melting analysis].

OBJECTIVE: To establish a simple, rapid, inexpensive and sensitive method for detecting hot region for mutations in exon 7 of PAH gene. METHODS: High-resolution melting (HRM) technology was used to detect a c.728G>A mutation in exon 7 in 88 patients with classical type phenylketonuria. Suspected mutations were validated by direct DNA sequencing. RESULTS: The results detected by HRM are in good agreement with the results obtained by direct sequencing. CONCLUSION: HRM analysis is a simple, rapid, inexpensive and sensitive method for detecting hot mutational region in exon 7 of PAH gene.

[Study on three common mitochondrial DNA mutations in Leber's hereditary optic neuropathy].

OBJECTIVE: To screen for genetic mutations in 35 patients with Leber's hereditary optic neuropathy (LHON). METHODS: Polymerase chain reaction and DNA sequencing were used to screen for the presence of mitochondrial DNA mutations. RESULTS: The total detection rate of top 3 common LHON mutations were 20.0%, which included 6 cases of ND4 11778 G to A, 1 case of ND1 3460 G to A. No ND6 14484 T to C mutation was detected. A ND4 G11719A synonymous mutation was found in all patients. In addition, 21 other mutations were discovered among 23 patients, among which 13 had a single mutation, 8 had a second mutations, and 2 had a third mutation. Among the 21 mutations, ND4 11778 G to A had a frequency of 28.6%(6/21). ND1 3552 T to A, ND6 14470 T to C, ND4 11794 T to C, ND1 3497 C to T and 3644 T to C respectively had a frequency of 19.0% (4/21), 19.0%(4/21), 14.3%(3/21), 9.5%(2/21) and 9.5%(2/21). Among the 3 patients who harbored a ND4 11794 T to C mutation, 2 were heteroplasmic and one was homoplasmic in nature. CONCLUSION: The ND4 11778 G to A mutation is common in the Top "3" primary mutations of patients with LHON. Candidate LHON mutation ND1 3552 T to A or ND1 3644 T to C resulted in LHON pathogenesis as single or synergistic effect. The visual impairment at onset of the disease with candidate mutation were better than the eyes with the ND4 11778 G to A mutation.