Child inflammatory myofibroblastic tumor of the kidney misdiagnosed as Wilms' tumor: case report.

Inflammatory myofibroblastic tumor (IMT) is a rare mesenchymal tumor with recurrent potential, most commonly occurring in the lung but rarely in the kidney with nonspecific clinical symptoms and radiographic features, thus may be misdiagnosed as primary malignant lesions. We described a 6-year-old boy with renal IMT misdiagnosed as Wilms' tumor and then treated with right nephrectomy. It should be emphasized that in addition to the most common renal tumors in children, IMT should also be taken as a differential diagnosis. It is therefore mandatory to carry out clinical interpretation, careful histologic examination, and immunohistochemical studies collectively to make solid diagnosis.

Asymptomatic adult Wilms' tumor: A case report.

Wilms' tumor, also called nephroblastoma, is an extremely uncommon kidney tumor of adulthood. We reported a adult man with a left kidney mass diagnosed as Wilms' tumor. Case presentation: A 25-year-old man was hospitalized due to injury of the anterior cruciate ligament of the right knee. Preoperative imaging accidentally revealed a mass measuring 53 × 46 mm involving the middle and lower segments of the left kidney without evidence supporting the invasion of the surrounding structures or metastasis. The patient didn't show any symptom commonly occurred in Wilms' tumor, such as flank pain or hematuria. After nephrectomy, the diagnosis of adult Wilms' tumor was confirmed based on the tumor morphology and immunohistochemical findings. Conclusion: In adult patients without any clinical manifestations or favorable imaging findings for low-stage renal cell carcinoma, the diagnosis of Wilms' tumor should be taken into consideration.

[Improvement and application of DXS52(St14) in gene diagnosis of hemophilia A].

OBJECTIVE: To improve the experimental method of DXS52 (St14) and apply it to genetic testing for hemophilia A (HA). METHODS: PCR of DXS52 and agarose gel electrophoresis were performed for genetic testing in 61 non-inversion HA families. Linkage analysis of 7 loci within the FVIII gene including Bcl I, Hind III, Xba I, STR1, STR13, STR22 and STR24 were also carried out for the 61 families. RESULTS: DXS52 can provide information in 43 out of 61 families and the diagnostic rate was 70.5%. Eight families can be diagnosed only by DXS52 locus, accounting for 13.1%. Two families were found to have recombination between DXS52 and FVIII. CONCLUSION: The new experimental conditions can reach accurate and clear results in DXS52 genetic testing. This gene maker has high diagnostic rate, so it is an indispensable linkage analysis method in HA gene diagnosis. More caution should be paid when using the extragenic locus DXS52 to perform gene diagnosis because of its high recombinant rate with FVIII.

[Application of multiplex ligation-dependent probe amplification to diagnosis and prenatal diagnosis of common aneuploidies].

OBJECTIVE: To investigate the application value of the multiplex ligation-dependent probe amplification (MLPA) technique in diagnosis and prenatal diagnosis of chromosomes 13, 18, 21, X and Y aneuploidy. METHODS: Forty-four cases including 30 peripheral blood samples, 10 fetal cord blood samples, and 4 amniotic fluid samples were collected in this study. DNA was isolated from the samples and detected by MLPA, followed by analyzing in ABI310 Genetic Analyzer. Analysis of copy number changes for chromosomes 13, 18, 21, X and Y was carried out with RH-MLPA-analysis software. The routine karyotype analyses were also done for all the samples. RESULTS: Of 44 samples, the results of 42 by MLPA method was consistent with that by chromosome karyotyping. Only one case with trisomy 21 chimerism was failed to reach conclusion. In addition, one case of mark chromosome segment was identified as Y-chromosome segment by MLPA, while karyotyping failed to make judgment. The accurate rate of MLPA was 97.7% (43/44). CONCLUSION: The MLPA technique can simultaneously detect dozens of different target sequences and their copy number changes in a single reaction. It showed high specificity, good reproducibility, was fast and high-throughput. The MLPA technique can be applied to diagnosis and prenatal diagnosis of the common chromosomal aneuploidy.

[Detection of common deletions and mutations causing α-thalassemia in Southeast Asians and Southern Chinese with denaturing high performance liquid chromatography].

OBJECTIVE: To establish a comprehensive and simple assay using denaturing high performance liquid chromatography (DHPLC) for the diagnosis of most common mutations and deletions of α-thalassemia gene in Southeast Asians and Southern Chinese. METHODS: This assay has included a duplex polymerase chain reaction (PCR) followed by DHPLC analysis. An improved PCR was also performed followed by DHPLC analysis. With this assay, a blinded study of 160 samples was screened for three common mutations and three common deletions. RESULTS: The duplex PCR-DHPLC combined with the improved PCR-DHPLC analysis has detected all mutations and the wild-type allele. The results were consistent with those by the original methods. CONCLUSION: This molecular assay may be used for the diagnosis of α-thalassemia patients from this geographical region. The method is accurate, rapid, semi-automatic and cost-effective, which makes it suitable for large-scale screening.

Computed Tomography and Magnetic Resonance Imaging Appearances of Abdomen and Pelvis Gossypibomas at the Varied Durations After Cesarean Section.

The incidence of gossypiboma is considerably higher in open cavity surgeries, among which cesarean section ranks number one. However, it is difficult to diagnose abdomen or pelvic gossypibomas after cesarean section. We retrospectively analyzed the clinical and imaging data of three pathologically confirmed gossypiboma patients at varied durations after cesarean section. In case one, at four months after cesarean section, a gossypiboma near the small intestine caused fistula and intestinal obstruction. Soft tissue density lesion along the intestinal canal made the "segmental honeycomb sign" and "truncation" with metal markings on the edge on computed tomography (CT). Magnetic sensitivity artifacts were demonstrated as hypointensity on T1 weighted image (T1WI) and T2 weighted image (T2WI), while hyperintensity was seen on the diffusion weighted image (DWI). In case two, a gossypiboma in the peritoneal and intestinal space was revealed with MRI at 18 months after cesarean section. It was featured as a cystic and solid lesion, with "vortex like sign" and obvious ring enhancement on contrast-enhanced MRI scan. In case three, five years after cesarean section, a mass was palpated in the right middle and lower abdomen. MRI revealed a round mass of T1 hypointensity with mixed T2 signal, as well as swirling hypointensity in T2WI, T2WI-fat suppression (FS), and DWI. In CT and MRI examinations for suspected gossypiboma after cesarean section, "honeycomb sign" and "vortex like sign" are the characteristic appearances; gauze translocated into the intestine may show the "truncation sign". Accurate diagnosis is based on the surgery history, symptoms, and imaging features.

Prenatal diagnosis of haemophilia A in China.

OBJECTIVES: To develop a one-tube fluorescent multiplexed polymerase chain reaction (PCR) method to perform prenatal diagnosis of haemophilia A (HA). METHODS: Peripheral blood samples were collected from 220 women and from members of five families with proven HA. One-tube fluorescent PCR and capillary electrophoresis were performed to investigate four short tandem repeats (STRs) in intron 1, 13, 22 and 24 (STR1, STR13, STR22 and STR24, respectively) in FVIII. RESULTS: Our analysis revealed 7 different alleles for STR1, 10 for STR13, 7 for STR22 and 9 for STR24. The heterozygosity rate (HR) for STR1, 13, 22 and 24 was 34.6%, 49.6%, 43.6% and 38.2%, respectively. The HR was 75.0% (165/220) when these four markers were combined. Prenatal diagnosis was made for five male foetuses. Four foetuses were identified as affected ones of HA. The STR results were consistent with the data we obtained by PCR of St14 VNTR (DXS52) and DNA sequencing, which showed that one foetus harbours a mutation in exon12 (1804C > T) in FVIII. CONCLUSION: This study demonstrates that multiplex fluorescent analysis of four STRs is a rapid and simple method to perform genetic diagnosis of HA in families with a history of this disorder.

Rapid diagnosis of the alpha-thalassemia-1 Southeast Asian type deletion using a single tube real-time SYBR-polymerase chain reaction combined with dissociation curve analysis.

Dear Sir, A single tube polymerase chain reaction (PCR) with three primers and SYBR GREEN1 combined with dissociation curve analysis was set up that can clearly differentiate between Hb Bart's hydrops fetalis, normal subjects and - -(SEA) heterozygotes. This method seems to be simpler than that using a two-tube real-time SYBR-PCR with two different primer sets followed by analyses of DeltaC(T) and C(T) ratio.

[Detection of factor VIII intron 1 inversion in severe haemophilia A].

OBJECTIVE: Screening the intron 1 inversion of factor VIII (FVIII) in the population of severe haemophilia A(HA) in China and performing carrier detection and prenatal diagnosis. METHODS: Using LD-PCR to detect intron 22 inversions and multiple-PCR within two tubes to intron 1 inversions in severe HA patients. Carrier detection and prenatal diagnosis were performed in affected families. Linkage analysis and DNA sequencing were used to verify these tests. RESULTS: One hundred and eighteen patients were seven diagnosed as intron 22 inversions and 7 were intron 1 inversions out of 247 severe HA patients. The prevalence of the intron 1 inversion in Chinese severe haemophilia A patients was 2.8% (7/247). Six women from family A and 2 from family B were diagnosed as carriers. One fetus from family A was affected fetus. CONCLUSION: Intron 1 inversion could be detected directly by multiple-PCR within two tubes. This method made the strategy more perfective in carrier and prenatal diagnosis of haemophilia A.

Molecular prenatal diagnosis of alpha-thalassemia using real-time and multiplex polymerase chain reaction methods.

Molecular analysis of two fetuses at high risk of alpha-thalassemia (alpha-thal), and their family members, was performed using real-time polymerase chain reaction (PCR) with SYBR Green 1 (SYBR-PCR) dye combined with dissociation curve analysis and multiplex PCR (m-PCR) and DNA sequencing techniques. The genotype of the fetus from one family was --SEA/--SEA (Southeast Asian deletion), which produces hydrops fetalis syndrome. The genotype of the parents was --SEA/alphaalpha. A boy with Hb H disease and his sibling fetus from the other family had the genotype --SEA/alphaCSalpha [the Hb Constant Spring (CS) mutation: alpha142, Term-->Gln (TAA>CAA in alpha2)] and alphaalpha/alphaalpha (normal), respectively. The diagnosis, based on SYBR-PCR combined with dissociation curve analysis, was in agreement with the results from the m-PCR method. This indicates that these are alternative and reliable assays for the molecular diagnosis of deletional alpha-thal.

Detection of alpha-thalassemia in China by using multiplex ligation-dependent probe amplification.

The multiplex ligation-dependent probe amplification (MLPA) method was used to analyze 118 DNA samples from 90 alpha-thalassemia (alpha-thal) patients and 28 normal persons from Southern China, where the main causes of alpha-thal are three large deletions (-alpha3.7, -alpha4.2, and --SEA) and two point mutations in the alpha-globin gene cluster on chromosome 16. The results, detected by the P140B HBA kit, were in complete concordance with the results detected by multiplex polmymerase chain reaction (m-PCR) and real-time PCR. The advantages and limitations of the techniques are discussed. We concluded that MLPA was a rapid and reliable method to determine the cause of both deletional and nondeletional alpha-thal in China.

[Frequency of intron 1 inversion of factor VIII gene in Chinese hemophilia A patients with case report of a female patient with heterozygous intron 1 inversion].

OBJECTIVE: To investigate the frequency of intron 1 inversion (inv1) in FVIII gene in Chinese hemophilia A (HA) patients and to investigate the mechanism of pathogenesis. METHODS: Peripheral blood samples were collected from 158 unrelated HA patients, aged 20 (1 - 73), including one female HA patient, aged 5, and several family members of a patient positive in inv1. One-stage method was used to assay the FVIII activity (FVIII:C). Long distance PCR and multiple PCR in duplex reactions were used to screen for the intron 22 inversion (inv22) and inv1 of the FVIII coding gene (F8). The F8 coding sequence was amplified with PCR and sequenced with an automatic sequencer. RESULTS: Two unrelated patients (pedigrees) were detected as inv1 positive with a positive rate of 1.26%. A rare female HA patient with inv1 was also discovered in a positive family (3 HA cases were found in this family and regarded as one case in calculating the total detection rate). The full length of FVIII was sequenced, and no other mutation was detected. CONCLUSION: There frequency of FVIII inv1 is low in Chinese HA patients compared with other populations. Female HA patients are heterozygous for FVIII inv1 and that may be resulted from nonrandom inactivation of X chromosome.

[Improvement of gene analysis method in hemophilia A and its application of prenatal diagnosis].

OBJECTIVE: To establish a simple and rapid system for carrier detection and prenatal diagnosis in hemophilia A (CHA) family. METHODS: Long distance-polymerase chain reaction (LD-PCR) was selected for detection factor VIII intron 22 inversion. Polymorphism of factor VIII intragenic restriction fragment length polymorphism (RFLP) of Xba I and Hin d III, short tandem repeat (STR) within intron 13 and 22, as well as extragenic DXS52 (ST 14) variable number of tandem repeat (VNTR) were assayed by PCR and linkage analysis. RESULTS: Seventy-one females were diagnosed as carriers within 52 HA families. Twenty-one families were diagnosed to be factor VIII intron 22 inversion and 28 families were diagnosed by linkage analysis, whereas 3 families could not been diagnosed. Seventeen of 18 fetuses at risk were male. Ten of 17 male fetuses were shown to be affected and were subsequently aborted. Seven male fetuses were diagnosed to be not affected. One female fetus was identified to be HA carrier. One-year follow-up study demonstrated that these babies were normal and living well. CONCLUSION: LD-PCR combined with multiple locus linkage analysis enables the direct and indirect detection of HA for carrier testing and prenatal diagnosis.

[Detection of alpha thalassemia using real-time PCR and dissociation curve analysis].

OBJECTIVE: To establish an automatic, high throughput, quick detection method of alpha thalassemia. METHODS: The genotypes of -alpha(4.2) and -alpha(3.7) were detected by two real-time fluorescence PCRs using SYBR-Green1 (SYBR-PCR) with the analysis of dissociation curve (DC) and melting temperature (Tm). The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique. RESULTS: The length of product of -alpha(4.2) and -alpha(3.7) were 1.65 kb and 1.9 kb respectively, and the Tm were (81.5+/-0.5) degrees C and (82.5+/-0.5) degrees C respectively. The detection deadline were 9 x 10(2 ) copies and 4.3 x 10(2) copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR. CONCLUSION: The genotypes of -alpha(4.2), -alpha(3.7), non-deletion alpha alpha/and --(SEA) can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Green1 combined with the dissociation curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe.

[Rapid diagnosis of Down's syndrome by multiplex real-time fluorescence relative quantitative PCR].

To establish a multiplex real-time fluorescence relative quantitative PCR method for diagnosis of Down's syndrome. The fragment from Down's syndrome critical region gene 3 (DSCR3) on chromosome 21 was used as the target gene, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene on chromosome 12 was used as the control gene. The two genes were amplified in the same tube. The relative quantitative index-CT value was used to differentiate trisomy 21 patient from normal person. The peripheral blood sample from a Down's syndrome patient was collected and the B-lymphocytes were transformed by Epstein-Barr virus to establish the immortalized cell lines as standard material. The reaction conditions were optimized to obtain an equal amplification efficiency from both the target and the control genes. The slopes of both genes were almost -3.32, indicating that the efficiencies of the two amplifications were approximately equal. Among a certain range from 3-300 ng/PCR, the variation of detected DeltaCT value were less than 15%, and amplification showed the highest reproducibility when the concentration of DNA template was 30 ng/microL. Then, the variation of DeltaCT value with inter- and intra-assay were 9.8% and 13.3% at this DNA concentration of the templates. Clinical samples, including 20 blood samples from patients and 30 blood samples from normal persons, were detected using the established method. The DeltaCT value from Down's syndrome group were dramatically different from normal group (P < 0.001). The trisomy 21 immortalized cell lines were established and the genetic integrity of the cell lines was stable as evaluated by karyotype and DNA analysis. The relative quantitative PCR with DeltaCT value could be used to rapidly diagnose Down's syndrome.

[Fifteen short tandem repeat polymorphisms in Han race of North China].

OBJECTIVE: To research on the genetic polymorphism distributions of 15 short tandem repeat (STR) loci in Han race of North China and the genetic data of population genetics. METHODS: The capillary electrophoresis and five-color fluorescent multi-amplifying were applied to detect the genotypes of 15 STR loci in 597 unrelated Chinese Han individuals. RESULTS: No significant deviation from the Hardy-Weinberg Equilibrium was observed. High polymorphism was detected in the loci. Statistical analysis was carried out to obtain some parameters of forensic medicine. The heterozygosity of 15 loci was above 0.62. The values of discrimination power (DP) at 15 STRs ranged from 0.802 to 0.967. The values of excluding probability of paternity (EP) ranged from 0.320 to 0.697. The values of probability matching (Pm) ranged from 0.033 to 0.198. The fifteen loci showed an accumulated total discrimination power (TDP) more than 0.999999, a cumulative excluding probability of paternity (CEP) as 0.99999571, and total probability matching to be 8.93 x 10(-18). CONCLUSION: The data indicated that detecting combined 15 STRs is sensitive and reliable, and can be used to forensic and individual identification cases in Chinese group.

Relationship between polymorphisms of genes encoding microsomal epoxide hydrolase and glutathione S-transferase P1 and chronic obstructive pulmonary disease.

BACKGROUND: Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). However, only 10% - 20% of chronic heavy cigarette smokers develop symptomatic disease. COPD is most likely the result of complex interactions between environmental and genetic factors. Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST). In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113-->His), exon 4 (His139-->Arg) and GSTP1 polymorphism in exon 5 (Ile105-->Val) in 100 COPD patients and 100 age- and sex-matched healthy controls. RESULTS: The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarette years was 2.96 (95% CI 1.24 - 7.09). There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls. When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1.00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1.00. There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls. CONCLUSIONS: mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China. The interaction might exist between mEH genotype and smoke. The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population.

[The changes of gene expression in multiple myeloma treated with thalidomide].

OBJECTIVE: To investigate the effect of thalidomide on bone marrow cells gene expression in multiple myeloma (MM) patients with suppression subtractive hybridization (SSH) and explore the molecular mechanism of thalidomide therapy for MM. METHODS: In a MM patient receiving thalidomide therapy and bone marrow cell from himself, total RNA extraction, mRNA isolation and cDNA synthesis were carried out respectively with routine procedures. SSH were performed in A and B group respectively. The subtracted cDNA was selectively amplified by suppression PCR. The product was inserted into T vector, and then transfected into the competent host JM109. So two subtractive libraries were constructed. After blue-white screening, colonies were selected and plasmids extracted. Homologous comparation was conducted in GenBank. RESULTS: In group A, seven clones were isolated, including ribosomal protein L19 (HUMAN), IgG lambda chain v-v region (HUMAN), contains Alu repetitive element, NADH-ubiquinone oxidoreductase chain 2, elongation factor 1-gamma, human beta globin region, and 40S ribosomal protein S4 (HUMAN). In group B, six clones were isolated, including cytochrome B, up-regulated by 1,25-dihydroxyvitamin D(3) (VDUP1), NADH- ubiquinone oxidoreductase 20 Kd subunit, mu-calpain large subunit, tumor protein, translationally-controlled 1 (TPT1) and COATOMER alpha subunit. CONCLUSION: Thalidomide induces apoptosis and antiangiogenesis by down-regulating some genes and up-regulating some others genes.

[Frequent 4 977 bp deletion of mitochondrial DNA in tumor cell lines, solid tumors and precancerous lesions of human stomach].

OBJECTIVE: To clarify the frequency of mtDNA 4 977 bp deletion in tumor cell lines, solid tumors, patient's serum of gastric tumor and to find a easy and exact method to diagnose gastric tumor. METHODS: Primer-shift PCR method was used in 13 gastric tumor cell lines, 52 cases of gastric fresh tumor tissues matched the adjacent normal tissues, 40 cases of patient's serum of gastric tumor and 40 cases of normal serums for analysis of mtDNA deletion. RESULTS: Frequency of 4 977 bp mtDNA deletion was detected in 12 of 13 (92.3%) tumor cell lines, 38 of 52 (73.1%) cases of solid tumor tissues, 27 of 52 (52%) adjacent normal tissues, 17 of 40 (42.5%) patient's serum and 8 of 40 (20%) normal serums. Further more, in 2 of 10 pairs microdissected specimens, we found this deletion occurred not only in primary tumor but also in intestinal metaplasia comparing with no deletion was found in normal tissues. The frequency of this deletion was statistically significantly higher in the gastric tumor tissues and serums than in the adjacent normal tissues and serums. A good correlation between the deletion and young age of patients was representative in our data. CONCLUSION: mtDNA 4 977 bp deletion maybe play an important role in the carcinogenesis of human gastric mucous and maybe has happened in the tumor cell malignant transformation. To detect this deletion in patient's serum is a easy and exact method and maybe become a potential tumor marker.

[Detection of deletional determinants of alpha-thalassemia in Li people in Hainan province by a single tube multiplex polymerase chain reaction method].

OBJECTIVE: To develop a single tube multiplex polymerase chain reaction technique (mPCR) and use it to screen 3 deletional alpha-globin genes, namely -alpha(3.7), -alpha(4.2) (single gene deletion), and --SEA (South East Asia double gene deletion) in the Li people in Hainan province. METHODS: Based on previously developed 3 separate PCR methods used to detect the -alpha(3.7), -alpha(4.2), --SEA respectively, a single tube mPCR was investigated, optimized, and used to detect the genotypes of 40 samples from well-known alpha-thalassemia patients and 116 non-relative individuals of Li nationality in Hainan province. RESULTS: The results of the 40 samples detected by mPCR were the same as those by the 3 separate PCR Methods. Among the 116 sample, the combined incidence of -alpha(3.7) and -alpha(4.2) was found to be as high as 38.0%, and the -alpha(4.2) genotype was more frequent than the -alpha(3.7) genotype in the Li people. No --(SEA) deletion was found in the Li people. CONCLUSION: The mPCR technique shows a good sensitivity and specificity. The Hainan Li people has the highest incidence of -alpha(3.7) and -alpha(4.2) among Chinese.