[Methylation analysis of CpG island DNA of FMR1 gene in the fragile X syndrome].

OBJECTIVE: To establish a method of methylation-sensitive restriction enzymes based quantitative PCR (MSRE-qPCR) for analysis of CpG island DNA of FMR1 gene, and to assess its value for molecular diagnosis of fragile X syndrome. METHODS: Thirty boys with mental retardation and abnormal repeats of 5'(CGG)n in the FMR1 gene and 20 mothers were analyzed by conventional PCR screening. Eag I was used to digest genomic DNA, and qPCR was performed to amplify CpG island in the FMR1 gene using both undigested and digested templates. Raw Ct values were obtained through quantitative PCR amplification. The degree of CpG island methylation was calculated by 2 - U+0394 U+0394 Ct. The result of MSRE-qPCR was verified by Southern blotting. 30 healthy females and 30 healthy males were used as controls to optimize the established MSRE-qPCR method. RESULTS: The ranges of 2 - U+0394 U+0394 Ct value for normal methylation, partial methylation and full methylation were determined. Among the 30 patients, 3 were found to have partial methylation of CpG island of the FMR1 gene, and 27 were found to have full methylation (3/30 results were verified by Southern blotting). Only 7 mothers were found abnormal methylation of CpG island of FMR1 gene, whilst the remaining 13 mothers were normal. CONCLUSION: MSRE-qPCR is a quick and reliable method for quantitative analysis of CpG island methylation status in FMR1 gene, which may provide a new strategy for the diagnosis of fragile X syndrome.

[Clinical Features and Prognostic Factors of Patients with Primary Cutaneous Lymphoma].

OBJECTIVE: To retrospectively analyze the clinical characteristics and prognostic factors of patients with primary cutaneous lymphoma. METHODS: The clinical data of 22 patients with primary cutaneous lymphoma admitted to Xinjiang Hotan District People's Hospital, Heji Hospital affiliated to Changzhi Medical College and the Fifth Medical Center of PLA General Hospital from January 2013 to June 2021 were retrospectively analyzed. RESULTS: The incidence of primary cutaneous T cell and NK/T cell lymphoma was about 91.9/100 000, and the incidence of primary cutaneous B cell lymphoma was about 14.5/100 000. The overall survival (OS) of patients aged ≥65 years was significantly shorter than that of patients younger than 65 years (P <0.05). Patients with elevated ß2-microglobulin (ß2-MG) had shorter OS and progression-free survival (PFS) (both P <0.05). Patients who achieved complete/partial response after initial treatment had longer OS than those with stable or progressive disease (P <0.05). There were significant differences in OS and PFS among patients with different pathological types of primary cutaneous lymphoma that originated from T and NK/T cells, the OS and PFS of patients with mycosis fungoides were longer than those of patients with other pathological types (both P <0.05). In addition, disease stage might also affect the PFS of the patients (P =0.056). CONCLUSION: The age, disease stage, ß2-MG level, pathological type and remission state after treatment of the patients were related to the clinical prognosis.

Effects of Rhythmic Auditory Stimulation on Gait and Motor Function in Parkinson's Disease: A Systematic Review and Meta-Analysis of Clinical Randomized Controlled Studies.

Objective: This study aimed to summarize the effectiveness of rhythmic auditory stimulation (RAS) for the treatment of gait and motor function in Parkinson's disease (PD) through a systematic review and meta-analysis. Methods: All studies were retrieved from eight databases. The effects of RAS on PD were determined using the following indicators: gait parameters including step length, stride width, step cadence, velocity, stride length; motor function including 6 min walk test (6MWT) and timed up-and-go test (TUGT); the Unified Parkinson's Disease Rating Scale (UPDRS); and the Berg Balance Scale (BBS). The risk map of bias of the quality of the studies and the meta-analysis results of the indicators was prepared with RevMan 5.2 software. Results: Twenty-one studies were included in the systematic review, and 14 studies were included in the meta-analysis. In the meta-analysis, the results of gait parameters, namely, velocity, step length, and stride length, were statistically significant (P < 0.05), whereas the results of cadence and stride width were not statistically significant (P ≧ 0.05). The results of 6MWT and TUGT for motor function as well as UPDRS-II, UPDRS-III, and BBS were statistically significant (P < 0.05). Conclusions: RAS could improve gait parameters, walking function, balance function, and daily living activities of individuals with PD. The application of RAS in conventional rehabilitation approaches can enhance motor performance in PD. Future studies should use a large sample size and a rigorous design to obtain strong conclusions about the advantages of RAS for the treatment of gait and motor function in PD.

[Evaluation of detection and analysis of chromosome 22q11.2 microdeletion by multiple ligation-dependent probe amplification assay].

OBJECTIVE: To evaluate multiplex ligation-dependent probe amplification (MLPA) assay detection in analysis of chromosome 22q11.2 microdeletion. METHODS: Between March 2008 and September 2009, thirty-two patients including 10 males and 16 females aged between years (3.6±3.1) were selected and evaluated by history, physical examination and medical records. Of these patients, sixteen patients who were previous diagnostic as 22q11.2 microdeletion were in positive control group, the other 16 healthy children were in negative control group. All the patients were detected by MLPA and fluorescence in situ hybridization (FISH) for the presence of a 22q11.2 microdeletion after informed consent. Diagnostic efficacy was assessed by sensitivity, specificity and Kappa analysis. RESULTS: We have applied the two assays of detection of chromosome 22q11.2 microdeletion in 32 patients. Sixteen patients in positive control group were found to have a 22q11.2 deletion and, with the deletion size of 3-Mb. However, as expected, chromosome 22q11.2 deletion was not found in negative control group. The MLPA results were in good agreement with that by FISH. Therefore, MLPA has high sensitivity and specificity. CONCLUSION: MLPA is a rapid, reliable, high-throughput and relatively economical alternative to FISH technology for the diagnosis of 22q11.2 microdeletion. It can provide reliable and helpful information for clinical diagnosis of 22q11.2 microdeletion syndrome.

[Relationship between 22q11 microdeletion syndrome and congenital heart disease].

OBJECTIVE: To investigate the relationship between 22q11 microdeletion syndrome and congenital heart disease. METHODS: Clinical screening assessment and genetic testing using standard fluorescence in-situ hybridization (FISH) were applied in 207 subjects suspected for 22q11 microdeletion syndrome. Patients with 22q11 microdeletion syndrome were examined by echocardiography, patients with complicated congenital heart disease were examined further by cardiac catheterization. RESULTS: 22q11 microdeletion syndrome was detected in 39 subjects. The incidence of 22q11 microdeletion syndrome was 1.6% in suspects with simple congenital heart disease without extracardiac manifestations, 53.0% in suspects with congenital heart disease combined with at least two extracardiac manifestations, 3.8% in suspects without congenital heart disease. The incidence of congenital heart disease in 22q11 microdeletion syndrome patient and non 22q11 microdeletion syndrome patient was 94.9% and 54.2% (P < 0.01). The incidence of congenital heart disease combined with at least two extracardiac manifestations in 22q11 microdeletion syndrome patient and non 22q11 microdeletion syndrome patient was 89.7% and 18.5% (P < 0.01). In 22q11 microdeletion syndrome patients, Tetralogy of Fallot was the most common type of congenital heart disease. Dysmorphic faces, learning difficulties and retarded physical development were the most common extracardiac manifestations of the congenital heart disease patients. CONCLUSION: 22q11 microdeletion syndrome is related to congenital heart disease.

Patent ductus arteriosus and pulmonary valve stenosis in a patient with 18p deletion syndrome.

We report on a patient with a partial deletion on the short arm of chromosome 18 (del 18p), who presented with dysmorphic features and delayed developmental milestones as well as with a patent ductus arteriosus (PDA) and pulmonary valve stenosis (PS). Several forms of congenital heart disease (CHD) are found in about 10% of patients with del (18p), but coexisting PDA and PS have not been reported. Del (18p) must be considered in patients with characteristic phenotypic abnormalities and congenital heart disease, including a combination of PDA and PS.

[Visual search attention in children with Williams syndrome].

OBJECTIVE: To explore the characteristics of visual search attention in children with Williams syndrome (WS). METHODS: Twenty-two children with WS, 13 male and 9 female, aged 12 (6-16), 25 chronological age (CA) and mental age (MA)-matched children with Down's syndrome (DS), 45 biological age-matched children, and 43 mental age-matched children, aged 4 +/- 2 (2-8) underwent Peabody picture vocabulary test (PPVT). Visual search attention including selective, switch, and sustained attention, was tested using a set of computerized visual search tasks via a touch-screen. In the dual target tasks, participants were required to alternate between two different targets. Sustained attention was investigated with vigilance task test. RESULTS: (1) In the single-target searching task with no target similar distractor, the accurate response rate of the WS patients was 71% +/- 25%, significantly lower than that of the CA-matched children (87% +/- 14%, P = 0. 001). The searching time of the WS children was 5 s, significantly longer than that of the CA-matched children (3 s, P = 0.000). The distance for touch of the WS children was 25 relative units, significantly longer than that of the CA-matched children (18 relative units, P = 0.000). The mean reaction time of the WS children was 5 s, significantly longer than that of the children with Down's syndrome (3 s, P = 0.022). The shape error rate of the WS children was 15%, significantly higher than that of the CA-matched children (0, P = 0.000). When non-targets similar to the targets were added, the accurate response rate of the WS group was 39% +/- 20%, significantly lower than those of the CA-matched and MA-matched children (77% +/- 23% and 66% +/- 23% respectively, both P = 0.000); the mean searching time of the WS children was 13 s, significantly longer than those of the CA-matched and MA-matched children (3 s and 5 s respectively, both P = 0. 000); and the distance per touch of the WS children was 41 relative units, significantly longer than those of the CA-matched and MA-matched children (20 and 27 relative units, P = 0.000 and P = 0.004). (2) The results of the dual target tasks showed that the accurate response rate of the WS children was 52% +/- 28%, significantly lower than that of the CA-matched children (78% +/- 22%, P = 0. 000), the mean searching time of the WS children was 11 s, significantly longer than that of the CA-matched children (4 s, P = 0.000); and the distance per touch of the WS children was 54 +/- 27 relative units, significantly longer than that of the CA-matched children (31 +/- 13 relative units, P = 0.000). However, there were not significant differences in the accurate response rate, mean searching time, and distance per touch between the WS and DS children. The switching error rate of the WS children was 13%, significantly higher than those of the CA and MA-matched children (0 and 4% respectively, P = 0.000 and P = 0.004). (3) The vigilance task test showed that the accurate response rate of the WS children was 52% +/- 25%, significantly lower than that of the CA-matched children (80% +/- 21%, P = 0.000); the mean searching time of the WS children was 4 s +/- 1 s, significantly longer than that of the CA-matched children (3 s +/- 1 s, P = 0.000); and the error hit number of the WS children was 8, significantly more than that of the CA-matched children (3, P = 0.000). However, there were not significant differences in the accurate response rate, mean searching time, and error hit number between the WS and DS children. Conclusion Distinct visual search deficits exist in WS children.

[Rapid detection of chromosomes X and Y using dual-color primed in situ labeling technique].

OBJECTIVE: To develop a rapid method for detection of chromosomes, and to present a feasible method for rapid detection of the interphase nuclei of uncultured amniocytes using modified dual-color primed in situ labeling (PRINS) technique. METHODS: Chromosomes X and Y were detected and analyzed by the modified dual-color PRINS in 205 amniotic fluid samples. RESULTS: The specific chromosomes were obtained on both metaphase and interphase nuclei. In more than 98% of the samples PRINS reactions with primers X and Y were successfully induced. One sample was properly identified and correctly scored as 47, XXY. CONCLUSION: The results suggest that PRINS technique is rapid, simple and cost-effective. It is also sensitive and specific, and thus useful for rapid detection of chromosome abnormalities.

[Study on clinical features and fluorescence in situ hybridization detections of 22q11 microdeletion syndrome].

OBJECTIVE: To investigate clinical features and the diagnosis by fluorescence in situ hybridization (FISH) of 22q11 microdeletion syndrome (22q11 DS). METHODS: The clinical data of suspects were analyzed, and their peripheral blood samples were tested by FISH for microdeletion of 22q11. The diagnosis and correlated clinical factors of 22q11 DS were investigated by using the multiple factor Logistic regression analysis and Chi-square test. RESULTS: In 64 suspects, 14 were shown to have 22q11 microdeletion with many different types of malformation, and the percentage was 21.9%. The Logistic regression predictive equation for 22q11 DS was: y=-8.206+2.324x1+2.725x2+1.674x3, P=exp(y)/[1+exp(y)], in which the concomit ant variables were facial dysmorphic features (x1), congenital heart defects (x2), thymus scarcity/infection problem (x3), the P value meant the probability of diagnosis of 22q11 DS. CONCLUSION: Accurate clinical evaluation is just as preliminary screening to patients at risk for del22q11. The results of FISH test can be predicted by using the suitable Logistic regression equation.

[Cardiovascular manifestations in 40 patients with Williams syndrome].

OBJECTIVE: To evaluate the cardiovascular manifestations of Williams syndrome (WS) confirmed by fluorescence in situ hybridization (FISH). METHODS: Between July 2004 and January 2007, FISH was used to confirm diagnosis in 71 suspected WS cases by detecting chromosome 7q microdeletion. Cardiovascular abnormalities were assessed by echocardiography and Doppler echocardiography. RESULTS: Forty out of 71 patients were detected to have Elastin gene locus microdeletion, 25 patients (25/40, 62.5%) had at least one cardiac anomaly; among these patients, supravalvular aortic stenosis (SVAS) was diagnosed in 18 patients (18/25, 72%) and 6 of them had complex abnormalities. Patent ductus arteriosus was diagnosed in 3 patients (3/25, 12%, 1 was associated with other malformations), isolated pulmonary stenosis in 1 patient (1/25, 4%), isolated coarctation of aorta in 2 patients (2/25, 8%), and hypertension in 2 patients (2/25, 8%), mild aortic regurgitation in 2 patients, mild mitral regurgitation and moderate mitral regurgitation in 3 patients respectively. CONCLUSION: A detailed cardiac evaluation should be performed in all patients with Williams syndrome due to the high frequency of cardiovascular abnormalities.

[Rapid detection of 18-trisomy syndrome using primed in situ labeling technique].

OBJECTIVE: To develop a rapid method for the detection of chromosomes and try to verify the feasibility of using modified primed in situ labeling (PRINS) technique for rapid detection of the interphase nuclei of uncultured amniocytes. METHODS: Chromosome 18 was detected and analyzed by the modified PRINS in 262 amniotic fluid samples. RESULTS: The specific chromosomes were obtained on both metaphase and interphase nuclei. In more than 95% of the samples, PRINS reactions with primer 18cen were successfully induced. Two samples were properly identified and correctly scored as trisomic 18. CONCLUSION: The results suggest that PRINS technique is simple, rapid and cost-effective. It is sensitive, specific, and thus can enhance the accuracy of standard cytogenetic analysis.

Effect of ethanol extract of Selaginella doederleinii Hieron on the proliferation of nasopharyngeal carcinoma CNE-1 and C666-1 cells.

This paper mainly studied the effect of ethanol extract of Selaginella doederleinii Hieron on the proliferation of two kinds of nasopharyngeal carcinoma cell lines, CNE-1 and C666-1, and their mechanisms of action. Extract was obtained by heat reflux extraction with ethanol, and the effect of extract on the extracellular matrix adhesion of the cells, on their proliferation process, as well as on their colony-forming ability were tested using MTT assay. The results showed that the yield of dry extract was 36.4%. 2.5 g/ml extract in the high concentration group exhibited inhibitory activity that was directly proportional to the concentration on CNE-1 cells, while not exhibiting obvious proportional trend in respect with C666-1 cells. However, the inhibition rates against two types of cells can both reach between 30% ~ 50%. Under the effect of ethanol extract of Selaginella doederleinii Hieron, proliferative capacities of C666-1 and CNE-1 cells were affected to some extent.

[Effect of brucine on secretion function and proliferation capability of T lymphocytes in patients with aplastic anemia].

The aim of this study was to investigate the effect of brucine on secretion of TNF-α, IFN-γ, IL-4 and proliferation of T lymphocytes in patients with aplastic anemia (AA), and to explore its mechanism. Peripheral blood T lymphocytes from 10 patients with AA and 10 healthy volunteers were isolated, purified and cultured. T lymphocytes from the patients were divided into 0, 100, 200 and 400 µg/ml brucine-treated groups. T lymphocytes from healthy volunteers were used as control group. After being cultured for 72 hours, the levels of TNF-α, IFN-γ, IL-4 in the supernatant of cultured T lymphocytes from AA patients were detected by ELISA, and the proliferation of T lymphocytes from AA patients was detected by MTT. The results showed that compared with the normal control group, the levels of TNF-α and IFN-γ in the culture supernatant significantly increased, and IL-4 was significantly decreased. The levels of TNF-α and IFN-γ in the culture supernatant of brucine treated groups were lower, and were dependent on the concentration of brucine. However, the levels of IL-4 were found to be not obviously changed. The inhibition rate of T lymphocytes in 100, 200 and 400µg/ml brucine-treated groups were (13.61 ± 4.31)%, (14.28 ± 4.31)% and (15.12 ± 4.56)% respectively, among which the differences were not statistically significant. It is concluded that the brucine can reduce the levels of TNF-α and IFN-γ through inhibiting the proliferation of T lymphocytes in AA patients, which provides experimental basis for therapy of AA patients.

[Screening for neonatal inborn errors of metabolism by electrospray ionization-tandem mass spectrometry and follow-up].

OBJECTIVE: To determine the impact of expanded newborn screening using tandem mass spectrometry (MS/MS) on the overall detection rate of inborn errors of metabolism in Zhejiang province and to assess the outcome of the patients who were diagnosed. METHOD: Blood spots were collected between days 3 and 6 of life from the newborns. All samples were subjected to MS/MS analysis using Waters Quattro API. Confirmation tests included amino acid analysis, urinary organic acids by GC-MS, routine blood analysis, biochemistry, blood gas analysis, blood glucose and ammonia tests, blood homocysteine, lactate and pyruvate tests, urine acetone tests, biotin and biotin enzyme profile and DNA analysis. Standard treatment protocol was given to the patients. Protein restricted diet, special powdered formula and medicines recommended for the patients with amino acidemias. Protein restricted diet and L-carnitine, folic acid and Vitamin B12 supplementation were given for the patients with organic acidemia. L-carnitine was given to the patients with primary carnitine deficiency. The overall epidemiology, prognosis, follow-up of the screening program were also investigated in the neonates. RESULT: A total of 129 415 neonates were investigated for 26 inborn errors of metabolism during the period. Twenty-three newborns were confirmed as having inborn errors of metabolism, including 13 with amino acidemias, 6 with organic acidemias and 4 with fatty acid oxidation disorders. The prevalence was 1:5626. Positive predictive value was 2.10%, specificity was 99.72% and sensitivity 100%. Seventeen children remain asymptomatic during the follow-up. Five patients had motor and mental developmental delay. One patient presented metabolic disorders during the follow-up. No death occurred in this series of patients. CONCLUSION: This strategy represents a valuable preventive medicine approach by enabling diagnosis and treatment before the onset of symptoms.