Fully Automatic Fine-Grained Grading of Lumbar Intervertebral Disc Degeneration Using Regional Feature Recalibration Network.

Accurate fine-grained grading of lumbar intervertebral disc (LIVD) degeneration is essential for the diagnosis and treatment design of high-incidence low back pain. However, the grading accuracy is still challenged by lacking the fine-grained degenerative details, which is mainly due to the existing grading methods are easily dominated by the salient nucleus pulposus regions in LIVD, overlooking the inconspicuous degeneration changes of the surrounding structures. In this study, a novel regional feature recalibration network (RFRecNet) is proposed to achieve accurate and reliable LIVD degeneration grading. Detection transformer (DETR) is first utilized to detect all LIVDs and then input to the proposed RFRecNet for the fine-grained grading. To obtain sufficient features from both the salient nucleus pulposus and the surrounding regions, a regional cube-based feature boosting and suppression (RC-FBS) module is designed to adaptively recalibrate the feature extraction and utilization from the various regions in LIVD, and a feature diversification (FD) module is proposed to capture the complementary semantic information from the multi-scale features for the comprehensive fine-grained degeneration grading. Extensive experiments were conducted on a clinically collected dataset, which consists of 500 MR scans with a total of 10225 LIVDs. An average grading accuracy of 90.5%, specificity of 97.5%, sensitivity of 90.8%, and Cohen's kappa correlation coefficient of 0.876 are obtained, which indicate that the proposed framework is promising to provide doctors with reliable and consistent fine-grained quantitative evaluation results of the LIVD degeneration conditions for the optimal surgical plan design.

Detection of a novel large deletion causing α-thalassemia in South China.

α-Thalassemia is an inherited autosomal recessive disorder. It is one of the most common monogenic abnormalities known in the world and is prevalent in tropical and subtropical regions. α-Thalassemia is more frequently caused by deletional type than non-deletional type. Recently, we identified a novel large deletional type of α-thalassemia named --(FZ)/αα from a family in South China. Multiplex ligation-dependent probe amplification was used for diagnosing the carrier and prenatal diagnosing for a fetus. Real-time PCR was employed for characterizing the deletion breakpoints and the deletional segment was determined as 300 kb in length extending from the telomere to AXIN1 gene on the short arm of chromosome 16. The carriers in the family members were detected by real-time PCR using designed primers.

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.

A rapid detection for α-thalassemia by PCR combined with dissociation curve analysis.

Deletion mutations of 3.7 kb and 4.2 kb of α-globin gene are the most common causes of α-thalassemia (-α(3.7)/, -α(4.2)/). A simple, rapid assay by using a single-tube PCR to detect the two deletions has been needed. In this study, a pair of shared primers was designed for α2 and α1 gene but with length-different amplicons (159 bp and 409 bp). On the dissociation curve analysis profile after PCR, there shows two obvious peaks which represent the two different amplicons. Relative copy number of α2 and α1 gene can be deduced from the ratio of the two peaks. A comprehensive diagnosis for α-thalassemia 10 genotypes of deletions can be achieved when combined with a single-tube duplex PCR for detecting --SEA and non-deletional alleles of αα or α(T)α. Besides, a single-tube multiplex PCR, which is a cost-effective version of dual-priming-oligonucleotide based system, was designed for two common mutations of α-thalassemia in China (Hb Constant Spring and Hb Quong Sze), and these two mutations can be identified in samples by use of dissociation curve analysis. In all, using above three PCRs followed by dissociation curve analysis, three deletions and two mutations of α-thalassemia in the populations of southern China and Southeast Asia can be detected for molecular diagnosis or prenatal diagnosis. A blinded study of 163 samples was performed using this new assay and it was concordant with the original methods. This comprehensive molecular assay is simple, rapid, automatic and cost-effective, and can be used to diagnose α-thalassemia in this geographical area.

An assay of gene copy number and its application based on heteroduplex products.

The aim of this study was to set up a simple and efficient method for detecting gene copy number, based on heteroduplex products from single-tube PCR/DHPLC. Single-nucleotide polymorphisms (SNPs) on the α-globin gene and chromosome 21 were used as examples. And the formula for quantitative calculation of gene copy number was deduced-based on the peak heights of homoduplexes and heteroduplexes on the DHPLC pattern. 27 samples (14 normal DNA and 13 cases of trisomy-21) were assessed with this method, and 160 samples (48 normal DNA and 112 α-thalassemia samples) were assessed with this method combined with a duplex PCR/DHPLC. Results for 184 of 187 cases were concordant with the known genotypes; three cases of trisomy-21 could not be detected because the target SNPs were homozygous. In conclusion, quantitative assessment of heteroduplex products from single-tube PCR/DHPLC is simple and rapid, and can be used to detect α-thalassemia gene deletions (α(-3.7), α(-4.2)) and trisomy-21.

Detection of α-globin gene deletions using denaturing high-performance liquid chromatography and multiplex ligation-dependent probe amplification.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) has been used to detect deletions and mutations of the α-globin gene for diagnosis of α-thalassemia. MLPA reaction products are usually separated and analyzed by high-voltage capillary gel electrophoresis (CGE). The goal of this study was to find and use a cost-effective method to separate and analyze MLPA products. METHODS: Blood samples were collected from China. DNA was extracted and amplified by PCR using fluorescently labeled primers. In this study, denaturing high-performance liquid chromatography (DHPLC) was used to separate and analyze the reaction products. And the optimal separation conditions were determined using nondenaturing columntemperature. RESULTS: The DHPLC conditions were optimized and have been applied to separate MLPA products and 27 of the MLPA products from 50 to 320 bp were well separated. DHPLC was able to separate up to 37 reaction products that differed by 4-12 base pairs and detected target gene deletions by differences in peak size. Compared with CGE, both the specificity and sensitivity of DHPLC for the 107 DNA samples were 100%. CONCLUSIONS: DHPLC could be used to test routinely for α-globin gene mutations and deletions. Combined with MLPA, DHPLC is a low-cost, simple to use, accurate technique with practical value.

[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.

Improvement in the detection of alpha0- and deletional alpha-thalassemia by real-time PCR combined with dissociation curve analysis.

The prevailing cause of alpha-thalassemia in Southeast Asia is the presence of 3 deletion mutations in the alpha-globin genes (-SEA, -alpha(3.7) and -alpha(4.2)). Current detection methods include gap polymerase chain reaction (PCR), multiplex PCR and real-time PCR with SYBR Green 1 combined with dissociation curve analysis. To improve and simplify a previously published method that requires 4 separate reactions, a duplex PCR assay was designed to detect both the nondeletional and the -SEA alleles. This duplex PCR can successfully identify the nondeletional allele and both the -SEA carrier and homozygous genotypes. The combination of the duplex PCR and 2 gap PCRs (for detection of -alpha(3.7) and -alpha(4.2)) can diagnose all types of deletional alpha-thalassemia. Our method was validated by analysis of 195 DNA samples, the results of which were consistent with prior diagnoses. The developed assay can reliably diagnose alpha0-thalassemia and all types of deletional alpha-thalassemia. The diagnostic method is simple, rapid, accurate, automated, inexpensive and has a high throughput.

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.