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.

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.

[Carrier diagnosis of F9 gross deletion by multiple ligation-dependent probe amplification in hemophilia B].

OBJECTIVE: To investigate the application of multiplex ligation-dependent probe amplification (MLPA) in the gene diagnosis of hemophilia B (HB). METHODS: MLPA and linkage analysis of short tandem repeat (STR) were used for gene diagnoses of two HB families with gross deletions of F9 gene, which were negative by sequencing. RESULTS: The MLPA results indicated the loss of one or two exons in the two patients with the ratio lower than 0.10. Their mothers showed a ratio average of 0.50 ± 0.05 for the corresponding probes, which revealed she was carrier of large deletions of the F9 gene. The ratios of three sisters of the HB patients were normal, which indicated they were non-carriers. Linkage analysis was consistent with MLPA, but sequencing was not conclusive. CONCLUSION: This report illustrated that MLPA technique represented an efficient method to screen F9 gene gross deletions in sequencing undiagnosed carriers of hemophilia B.

[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.

[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.

[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.

Identification of seven novel mutations in the factor VIII gene in 18 unrelated Chinese patients with hemophilia A.

BACKGROUND: Hemophilia A (HA) is an X-linked inherited bleeding disorder caused by decreased activity of factor VIII (FVIII) due to heterogenous mutations in the FVIII coding gene (F8). The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA. METHODS: Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTeRS database. A clotting method was used to assay the FVIII activity level and the Bethesda assay was used to detect the FVIII inhibitor. RESULTS: A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation (4382-3 AC deletion) was associated with inhibitor development. CONCLUSION: These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FVIII inhibitor.

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.

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.

[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.

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.

[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.

[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.

[A study on the (CA)n in FVIII gene in Han ethnic group in Guangxi Zhuang Autonomous Region by amplification polymorphisms combined with silver staining].

OBJECTIVE: Hemophilia A is an inherited bleeding disorder caused by defects in factor VIII (FVIII) gene. In the present study, the frequencies of the microsatellite alleles at introns 13 and 22 in the factor VIII gene were analyzed in the group of Han nationality in Guangxi Zhuang Autonomous Region to explore their diagnostic value for hemophilia A. These two sites were also used to detect the carriers in 13 hemophilia A families. METHODS: Ninty-one individuals of Han ethnic group in Guangxi Zhuang Autonomous Region (135 X chromosomes) and 13 HA families were subjected to molecular studies. First, these two fragments were PCR amplified simultaneously. Then, silver staining was used later to show their polymorphisms. The investigators selected one sample at random to obtain its lengths of the PCR products at these two sites by ABI310 PCR amplifier. After counting its repeated numbers of (CA) according to the documents concerned, the repeated numbers of the other samples could be counted easily. RESULTS: In the 91 individuals, 6 and 4 alleles were detected at these two sites, respectively. At intron 13 the allele frequencies ranged from 0.0002 to 0.5408 and polymorphism information content (PIC) was 0.5899. At intron 22 the allele frequencies ranged from 0.0444 to 0.4963 and its PIC was 0.5359. The actual heterozygosity for intron 13 and intron 22 were 0.6364 (28/44) and 0.5227 (23/44), respectively. In 13 hemophilia A families with positive history, 9 of them were diagnosed by this method and the diagnosis rate was 69%. CONCLUSION: With high PICs, (CA)n at intron 13 and intron 22 were two valuable sites in the diagnosis of hemophilia A in the population of Han ethnic group in Guangxi Zhuang Autonomous Region. Compared with some other HA restrictive fragment length polymorphisms (RFLP), intron 22 (GT)n (AG)n was more informative.

[Analysis of X ba I polymorphism of FVIII gene and its application on prenatal diagnosis for hemophilia A].

OBJECTIVE: To establish the linkage methods of X ba I polymorphisms specific for FVIII gene intron 22, and to find a rapid and simple system for haemophilia A (HA) carrier detection and prenatal diagnosis. METHODS: A long PCR to amplify FVIII gene intron 22 followed by X ba I digestion was used to assay the gene rate and heterozygosity rate of 206 unrelated people. Detection of intron 22 inversion by long distance PCR (LD-PCR) and XbaI, BclI, Hind III, DXS52, STR polymorphism within intron 13 and 22 by hereditary linkage analysis were assays in 20 HA pedigrees. RESULTS: The gene rate and polymorphism information contents of 206 people were 0.5475 and 0.4955 respectively, 7 of 20 HA families were diagnosed as intron 22 inversion, 6 of 13 non-inversion HA families were diagnosed by X ba I linkage analysis, 8 of 13 non-inversion HA families were diagnosed by two or more linkage analysis. CONCLUSIONS: The improved X ba I linkage analysis is a specific and useful molecular diagnosis marker. LD-PCR and five-linkage analysis can be used in prenatal HA gene diagnosis.

[High-level expression of phenylalanine ammonia-lyase in Lactococcus Lactis via synthesized sequence based on bias codons].

To construct a safer and more efficient gene engineering Lactococcus Lactis for expressing phenylalaine ammonia lyase (PAL) which will be benefit for PKU therapy, pal cDNA of Parsly and synthesized sequence based on Lactococcus Lactis bias codons were recombined into two Lactococcus Lactis NICE systems. The activities of the expressed PAL were detected, and the effect of Lactococcus Lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using Lactococcus Lactis bias codons in both Lactococcus Lactis NICE systems. Through which several safer andmore efficient strains of the gene engineering Lactococcus Lactis were obtained.