Induced-Fit Recognition of CCG Trinucleotide Repeats by a Nickel-Chromomycin Complex Resulting in Large-Scale DNA Deformation.

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. NiII (Chro)2 (Chro=chromomycin A3) binds specifically to the minor groove of (CCG)n repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the NiII chelate itself. The DNA deformation of CCG repeats upon binding forms a GGCC tetranucleotide tract, which is recognized by NiII (Chro)2 . The extruded cytosine and last guanine nucleotides form water-mediated hydrogen bonds, which aid in ligand recognition. The recognition can be accounted for by the classic induced-fit paradigm.

EGFR nuclear import in gallbladder carcinoma: nuclear phosphorylated EGFR upregulates iNOS expression and confers independent prognostic impact.

BACKGROUND: The understanding of epidermal growth factor receptor (EGFR) deregulation in carcinogenesis remains incomplete. We investigated the implications of EGFR gene status and EGFR nuclear translocation in gallbladder carcinoma (GBCA). METHODS: Subcellular localization of EGFR and phosphorylated EGFR (pEGFR) was analyzed by fractional immunoblotting and confocal immunofluorescence in GBCA cell lines. pEGFR binding to iNOS promoter was assessed by chromatin immunoprecipitation with iNOS promoter activity evaluated by luciferase assay. EGFR, pEGFR, and iNOS were immunohistochemically assessable for localization and level in the training set of 104 GBCAs on tissue microarrays, with 76 cases analyzed for EGFR gene by chromogenic in situ hybridization (CISH) and mutant-enriched PCR targeting exons 19 and 21. The prognostic impact of nuclear pEGFR (N-pEGFR) immunoexpression was reaffirmed on whole sections of 58 GBCAs in the test set. RESULTS: Nuclear expression of EGFR and pEGFR was substantiated in vitro with augmented activity of iNOS promoter elicited by pEGFR binding upon EGF treatment. Despite no mutation, EGFR amplification, identified in 11 cases (15%) by CISH, strongly correlated with cytoplasmic EGFR expression (P < 0.001) but not with disease-specific survival (DSS). Immunoexpression of nuclear EGFR (N-EGFR), cytoplasmic pEGFR, and N-pEGFR was strongly related to that of iNOS (all ≤0.005). N-pEGFR independently predicted worse DSS in both training (P = 0.0468, HR = 2.024) and test sets (P = 0.0223, HR = 5.573). CONCLUSIONS: N-EGFR and N-pEGFR express in GBCA, conferring clinical aggressiveness partly through iNOS transactivation. Lacking response-predicting mutation, EGFR gene status, albeit amplified in 15% of GBCA, is neither related to nuclear EGFR translocation nor prognostically useful.

Chromosomal imbalances in lung adenocarcinomas with or without mutations in the epidermal growth factor receptor gene.

BACKGROUND AND OBJECTIVE: Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas of Asian patients, implying a good response to treatment with the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. However, the distinct chromosomal imbalances between lung adenocarcinomas with and those without EGFR mutations have not been fully elucidated. METHODS: Seventy-seven patients of surgically resected lung adenocarcinoma were analysed for the EGFR exon 19 deletion and the L858R mutation, using mutant-enriched PCR, and for chromosomal imbalance alterations using comparative genomic hybridization. RESULTS: EGFR mutations were detected in 42 (54.5%) patients, including 22 with the exon 19 deletion and 20 with the L858R mutation. The mean number of chromosomal arms with imbalance alterations was significantly higher in tumours with EGFR mutations than in those lacking these two mutations. The minimal regions with gain on 1q23-q31, 6p12-p21.1 and 7q11.2, and loss on 3p21, 8p22-p23, 9q33, 10q25 and 13q13, differed significantly between lung adenocarcinomas with or without EGFR mutations. However, neither EGFR mutations, nor any of the common chromosomal imbalance alterations alone, exhibited significant associations with tumour stage or disease-specific survival of the patients. CONCLUSIONS: These results indicate that imbalance alterations at several chromosomal regions occur significantly more frequently in lung adenocarcinomas with EGFR mutations than in those without such mutations. Tumour growth-related genes in these chromosomal regions should be further investigated to improve our understanding of the common genetic alterations in lung adenocarcinomas with EGFR mutations.

Wnt-1 protein as a prognostic biomarker for hepatitis B-related and hepatitis C-related hepatocellular carcinoma after surgery.

BACKGROUND: Up-regulation of Wnt-1 protein has been reported in hepatitis B virus (HBV)-related and hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) tissues and cell lines. It is known to play a fundamental role in signaling cancer progression, whereas its prognostic role in HCC remains unexplored. METHODS: As a prognostic biomarker, this study analyzed Wnt-1 protein expression in 63 histology-verified HCC patients receiving curative resection. In each paired tumor and nontumor specimen, Wnt-1 levels were semiquantitatively measured by Western blotting and expressed by tumor/nontumor ratio. The data were further correlated with quantitative real-time PCR as well as with beta-catenin and E-cadherin expression by immunohistochemistry. Cumulative tumor recurrence-free survival curves were constructed using the Kaplan-Meier method and compared by the log-rank test. RESULTS: The results showed that 26 (group I) and 37 (group II) HCC patients had an expression ratio of Wnt-1 > or =1.5 and <1.5, respectively. The amount of Wnt-1 estimated by tumor/nontumor ratio correlated with the results by quantitative real-time PCR. High tumor Wnt-1 expression correlated with enhanced nuclear beta-catenin accumulation, diminished membranous E-cadherin expression, and increased tumor recurrence after curative tumor resection. CONCLUSIONS: These results suggest that Wnt-1 may be used as a predisposing risk factor for HCC recurrence. The use of tumor Wnt-1 as prognostic biomarker may identify patients with HBV- and/or HCV-related HCC patients with a high risk of tumor recurrence who may then benefit from further intensive therapy after surgery.

Methyl-CpG-binding PCR of bloodspots for confirmation of fragile X syndrome in males.

This study demonstrates that methyl-CpG-binding PCR (MB-PCR) is a rapid and simple method for detecting fragile X syndrome (FXS) in males, which is performed by verifying the methylation status of the FMR1 promoter in bloodspots. Proteins containing methyl-CpG-binding (MB) domains can be freeze-stored and used as stocks, and the entire test requires only a few hours. The minimum amount of DNA required for the test is 0.5 ng. At this amount, detection sensitivity is not hampered, even mixing with excess unmethylated alleles up to 320 folds. We examined bloodspots from 100 males, including 24 with FXS, in a blinded manner. The results revealed that the ability of MB-PCR to detect FMR1 promoter methylation was the same as that of Southern blot hybridization. Since individuals with 2 or more X chromosomes generally have methylated FMR1 alleles, MB-PCR cannot be used to detect FXS in females.

Detection of EBV infection and gene expression in oral cancer from patients in Taiwan by microarray analysis.

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis.

Immunohistochemical and biogenetic features of diffuse-type tenosynovial giant cell tumors: the potential roles of cyclin A, P53, and deletion of 15q in sarcomatous transformation.

PURPOSE: Diffuse-type tenosynovial giant cell tumor (D-TSGCT) is an aggressive proliferation of synovial-like mononuclear cells with inflammatory infiltrates. Despite the COL6A3-CSF1 gene fusion discovered in benign lesions, molecular aberrations of malignant D-TSGCTs remain unidentified. EXPERIMENTAL DESIGN: We used fluorescent in situ hybridization and in situ hybridization to evaluate CSF1 translocation and mRNA expression in six malignant D-TSGCTs, which were further immunohistochemically compared with 24 benign cases for cell cycle regulators involving G(1) phase and G(1)-S transition. Comparative genomic hybridization, real-time reverse transcription-PCR, and a combination of laser microdissection and sequencing were adopted to assess chromosomal imbalances, cyclin A expression, and TP53 gene, respectively. RESULTS: Five of six malignant D-TSGCTs displayed CSF1 mRNA expression by in situ hybridization, despite only one having CSF1 translocation. Cyclin A (P = 0.008) and P53 (P < 0.001) could distinguish malignant from benign lesions without overlaps in labeling indices. Cyclin A transcripts were more abundant in malignant D-TSGCTs (P < 0.001). All malignant cases revealed a wild-type TP53 gene, which was validated by an antibody specifically against wild-type P53 protein. Chromosomal imbalances were only detected in malignant D-TSGCTs, with DNA losses predominating over gains. Notably, -15q was recurrently identified in five malignant D-TSGCTs, four of which showed a minimal overlapping deletion at 15q22-24. CONCLUSIONS: Deregulated CFS1 overexpression is frequent in malignant D-TSGCTs. The sarcomatous transformation involves aberrations of cyclin A, P53, and chromosome arm 15q. Cyclin A mRNA is up-regulated in malignant D-TSGCTs. Non-random losses at 15q22-24 suggest candidate tumor suppressor gene(s) in this region. However, P53 overexpression is likely caused by alternative mechanisms rather than mutations in hotspot exons.

Chronic ovarian pregnancy mimicking an ovarian tumor diagnosed by peritoneal washing cytology: a case report.

BACKGROUND: Chronic ectopic pregnancy is an enigma. The clinical presentation can be mild, with absent or subtle symptoms. Ovarian pregnancy usually ends with rupture. We report the first case of unruptured chronic ovarian pregnancy that was initially diagnosed by peritoneal washing cytology. CASE: A 35-year-old woman suffered from low abdominal pain during the presumed menstrual period for 6 months. Abdominal computed tomography revealed a huge cystic mass with intralesional hematoma and soft tissue components located in the pelvic cavity. Mild right hydronephrosis caused by tumor obstruction of the right ureter were noted. Right ovarian cancer was suspected. Peritoneal washing cytology revealed both cytotrophoblasts and syncytiotrophoblasts. Patient received enucleation of the right ovary. Microscopically, the ovarian mass exhibited extensive hemorrhage and necrosis, embedding degenerated chorionic villi. CONCLUSION: When a patient experiences low abdominal pain during a menstrual period, the possibility of ectopic pregnancy should be considered in addition to possible endometriosis. Ovarian or other abdominal pregnancies, even untruptured, may be discriminated from other lesions in cases of abdominal pain by peritoneal washing cytology.

Deletions of chromosome 1p and 15q are associated with aggressiveness of gastrointestinal stromal tumors.

BACKGROUND/PURPOSE: Site-dependent profiles of chromosome imbalances (CIs) have been reported in gastrointestinal stromal tumors (GISTs). However, the role of specific CIs in association with metastasis is not clear. METHODS: Thirteen resected liver metastatic GISTs, including seven from the stomach and six from the small intestine, were analyzed using comparative genomic hybridization (CGH). The CIs associated with metastatic risk were assessed by comparing them with those identified in our previous study of 25 primary GISTs, including 14 from the stomach and 11 from the small intestine. RESULTS: Synchronous detection of liver metastasis was found more often in patients with intestinal than gastric GIST (5/6 vs. 2/7, p = 0.048). When compared with the primary tumors, the CI profile of liver metastases was similar in the intestinal group, but became more complex in the gastric group. Deletions of chromosomes 1p and 15q were very common (> 80%) in primary and metastatic tumors of the intestinal group, and exhibited a trend towards increase in the metastatic tumors of the gastric group. Both groups had a doubling in the frequency of 22q deletion in the liver metastases, which was not significantly different. Other CIs, including 9p deletion, increased significantly in the liver metastases of the gastric group, but not in the intestinal group. CONCLUSION: Our results, together with clinical findings, indicated a CGH profile associated with the intrinsic aggressiveness of the GISTs. Deletion of 1p and 15q play a critical role in the acquisition of aggressiveness during early GIST development.

Aberrant expression of CD19 and CD43 in a patient with therapy-related acute myeloid leukemia and a history of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with frequent involvement of the gastrointestinal tract and peripheral blood (PB). In addition to the B cell markers, the neoplastic cells express CD5 and CD43. In patients with a prior history of MCL with PB involvement, the appearance of leukemic cells after chemotherapy usually heralds a relapse, particularly if the leukemic cells express B cell markers and CD43. We report a patient with MCL who presented with multiple lymphomatous polyposis of the intestine. The staging procedures revealed the involvement of lymph nodes, bone marrow and PB. Three years after chemotherapy, thrombocytopenia with the appearance of rare leukemic cells in the PB was noted. Leukemic cells obtained from bone marrow aspirate expressed CD19 and CD43, suggesting a relapse. Detailed cytomorphological and immunophenotypic studies unveiled the myeloid nature of these leukemic cells, and a diagnosis of therapy-related acute myeloid leukemia was made. This case illustrates the importance of morphologic examination and performing a complete antibody panel in the diagnosis of a suspected relapse in patients with a prior history of lymphoma.

A novel role of thrombospondin-1 in cervical carcinogenesis: inhibit stroma reaction by inhibiting activated fibroblasts from invading cancer.

Thrombospondin (TSP)-1, a potent angiogenesis inhibitor, has been shown to exert different biological functions on various cell types. Here, we investigate the role of TSP-1 in tumor-stroma reaction, which is mainly characterized by fibroblast activation to create a permissive microenvironment for tumor progression. Immunohistochemistry examinations in the human surgical specimens have shown that a downregulation of TSP-1 during the progression of cervical carcinogenesis was accompanied by an emergence in the upregulation of stroma markers, alpha-smooth muscle actin (alpha-SMA) and desmin. Transfection of SiHa cervical cancer cells with a plasmid expressing the TSP-1 protein exhibited antiangiogenic activity in vitro and resulted in reduced tumor growth in severe combined immunodeficiency (SCID) mice, which was accompanied by a decrease in tumor vascularization and lower expressions of alpha-SMA and desmin than those in the vector controls. Transfection with TSP-1 and purified TSP-1 added to NIH3T3 cells did not alter the protein levels of alpha-SMA and desmin but significantly inhibited matrix metalloprotease-2 activity. Transforming growth factor-beta (TGF-beta), a major factor in the activation of fibroblasts, increased alpha-SMA and desmin expression and the ability of cell migration and invasion in NIH3T3 cells. The increased migration ability and the invasive ability into tumor cluster of TGF-beta-treated NIH3T3 cells were dose dependently inhibited by TSP-1. In contrast, ectopic TSP-1 expression in SiHa cells has little effect on the invasive ability of the NIH3T3 cells. Together, our findings demonstrate a novel role of TSP-1 to inhibit tumor-stroma reaction that could be attributed to the blockage of activated fibroblasts from invading cancer cells.

Roles for hypoxia-regulated genes during cervical carcinogenesis: somatic evolution during the hypoxia-glycolysis-acidosis sequence.

OBJECTIVES: Malignant phenotypic traits are caused by microenvironmental selection pressures during carcinogenesis. Hypoxia can drive a tumor toward a more aggressive malignant phenotype. The objective was to better understand the role of the hypoxia-regulated genes in cervical carcinogenesis. METHODS: We analyzed the expression of the hypoxia-regulated genes, including hypoxia-inducible factor-1alpha (HIF-1alpha), erythropoietin (Epo), vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), carbonic anhydrase IX (CAIX), and MET, in cervical cell lines and human tissue samples of cervical intraepithelial neoplasia (CIN I-III) and invasive squamous cell carcinoma (ISCC). RESULTS: CAIX and MET were expressed in cervical carcinoma cell lines, but not in normal or human papillomavirus-immortalized cervical cells. In clinical tissue samples, Epo, VEGF, GLUT1, and CAIX were not detected in normal squamous epithelia. GLUT1 was expressed in nearly all cases of CIN and ISCC, however, CAIX was expressed only in CIN III and ISCC. HIF-1alpha and MET expression was confined to the basal cells in normal squamous epithelia and was detected in the dysplastic cells of CIN and ISCC. CONCLUSIONS: The role of HIF-1alpha and MET changes from response to proliferation to tumor progression during cervical carcinogenesis. GLUT1 expression, a glycolytic phenotype adaptive to glycolysis, occurs early during cervical carcinogenesis and is a specific marker for dysplasia or carcinoma. MET and CAIX may contribute tumor progression in later stage. CAIX expression, an acid-resistant phenotype, may be a powerful adaptive advantage during carcinogenesis. Successful adaptation to the hypoxia-glycolysis-acidosis sequence in a microenvironment is crucial during carcinogenesis.

Floral leukemic cells transformed from marginal zone lymphoma.

There are three clinicopathological entities of marginal zone lymphoma (MZL), including extranodal or mucosa-associated lymphoid tissue (MALT) lymphoma and MZL of nodal (NMZL) or splenic (SMZL) type. Of these, leukemic presentation, usually as small or villous lymphocytes, is more common in SMZL, while leukemic change in NMZL is rare, and the morphology has not been characterized. We present a stage 4 MZL involving lymph node, spleen, and bone marrow with two relapses after chemotherapy. The leukemic cells at the second relapse revealed irregular nuclear contours with multilobated nuclei (so-called flower cells or floral cells) mimicking the neoplastic cells in adult T-cell leukemia/lymphoma (ATLL). The absence of leukemic change and splenic hilar lymphadenopathy at initial presentation, expression of IgD by tumor cells, and cytogenetic changes of +7 suggested that this tumor might be a NMZL. Although the cytomorphologic features of floral leukemic cells might suggest ATLL, thorough clinical and laboratory workup helped to reach a correct diagnosis. Our findings broaden the cytological spectra of leukemic cells in MZL and illustrate the importance of immunophenotyping.

Genomic alterations in human malignant glioma cells associate with the cell resistance to the combination treatment with tumor necrosis factor-related apoptosis-inducing ligand and chemotherapy.

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical development as a cancer therapeutic agent. Many human malignant glioma cells, however, are resistant to TRAIL treatment. We, therefore, investigated the genomic alterations in TRAIL-resistant malignant glioma cells. EXPERIMENTAL DESIGN: Seven glioma cell lines and two primary cultures were first analyzed for their sensitivity to TRAIL and chemotherapy and then examined for the genomic alterations in key TRAIL apoptotic genes by comparative genomic hybridization (CGH), G-banding/spectral karyotyping, and fluorescence in situ hybridization (FISH). RESULTS: CGH detected loss of the chromosomal regions that contain the following genes: 8p12-p23 (DR4 and DR5), 2q33-34 (caspase-8), 11q13.3 (FADD), 22q11.2 (Bid), and 12q24.1-q24.3 (Smac/DIABLO) in TRAIL-resistant cell lines. Spectral karyotyping showed numerical and structural aberrations involving the chromosomal regions harboring these genes. A combination of G-banding/spectral karyotyping and FISH further defined the loss or gain of gene copy of these genes and further showed the simultaneous loss of one copy of DR4/DR5, caspase-8, Bid, and Smac in two near-triploid cell lines that were resistant to the combination treatment with TRAIL and chemotherapy. Loss of the caspase-8 locus was also detected in a primary culture in correlation with the culture resistance to the combined TRAIL and chemotherapy treatment. CONCLUSIONS: The study identifies chromosomal alterations in TRAIL apoptotic genes in the glioma cells that are resistant to the treatment with TRAIL and chemotherapy. These genetic alterations could be used to predict the responsiveness of malignant gliomas to TRAIL-based therapies in clinical treatment of the tumors.

Chromosomal gain of 3q and loss of 11q often associated with nodal metastasis in early stage cervical squamous cell carcinoma.

BACKGROUND/PURPOSE: Cervical cancer remains a health problem among women worldwide. Delineation of genetic changes is critical to understanding the molecular basis of tumor progression, as well as for identifying genetic markers for early identification of patients at high risk for a poor outcome. METHODS: To provide comparative genomic hybridization data for cervical squamous cell carcinoma in Taiwan, and to gain further insight into genetic markers associated with lymph node metastasis of this disease, we performed comparative genomic hybridization analysis of 30 consecutive cases of cervical squamous cell carcinoma (24 stage IB and 6 stage IIB). RESULTS: The results disclosed that higher staged tumors or those with lymph node metastasis had more chromosomal imbalances. The commonly recurrent chromosomal imbalances were gains of 3q (46.7%), 1q (36.7%) and 8q (20.0%) and losses of 11q (36.7%), 3p (33.3%), 6q (23.3%), and 2q (20.0%). The frequencies of these chromosomal imbalances in stage IB and IIB tumors did not differ significantly. However, when compared with tumors without lymph node metastasis, the loss of 11q14-q22 (5/9 vs. 3/21, p = 0.019) and gains of 3q11-q22 and 3q26-qter (6/9 vs. 5/21, p = 0.026) were significantly more prevalent in tumors with lymph node metastasis. CONCLUSION: The results suggest that certain tumor-associated genes residing on 3q and 11q warrant further investigation to elucidate their role in the progression of this disease.

Aurora-A overexpression associates with Ha-ras codon-12 mutation and blackfoot disease endemic area in bladder cancer.

Our data revealed that 59.4% of the bladder cancer specimens showed Aurora-A overexpression, of which 31.8% also had Ha-ras codon-12 mutation; 45.5% were from blackfoot-disease endemic areas in which arsenic exposure is a major environment factor associated with various cancer formation. We further demonstrated that arsenic treatment of the immortalized bladder cell line, E7, increased Aurora-A expression. All together, co-existence of Aurora-A overexpression and Ha-ras mutation suggests a possible additively effect on the tumorigenesis of bladder cancer. In addition, Aurora-A overexpression and up-regulated by arsenic exposure opens a new direction for exploring the occurrence of bladder cancer occurrence in Taiwan.

Establishment of a mini-gene expression database for bladder tumor.

BACKGROUND AND PURPOSE: Transitional cell carcinoma has been diagnosed mostly in urinary bladder in southern Taiwan and has an exceptionally high mortality rate. To identify the genes associated with bladder cancer, we investigated differential gene expression. Six bladder tumor cDNA libraries were constructed and their sequences were compared and analyzed. METHODS: mRNA from bladder tumor cell lines or tissue samples were used to construct two regular cDNA libraries and four subtractive cDNA libraries after subtractive hybridization with cDNA derived from normal bladder epithelial cells. Subsequently, more than 100 cDNA inserts from each library were randomly isolated and sequenced, followed by sequence comparisons with nucleotide and protein sequence databases. RESULTS: After searching the Basic Local Alignment Search Tool (Blast) databases, the cDNA nucleotide sequences were grouped into novel, known, and common gene categories. Since tumor nucleotide sequences are informative and valuable for research, they were organized as a mini-gene expression database (http://bladder.nhri.org.tw/). Interestingly, in one subtractive cDNA library, the ATPase 6 gene was found to be highly expressed in normal bladder epithelial cells and elevated levels of ATPase 6 mRNA were later confirmed by reverse transcription-polymerase chain reaction. However, the role of ATPase 6 in bladder tumorigenesis remains to be investigated. CONCLUSIONS: The establishment of this database is an important step to enable systematic screening for bladder tumor-associated genes and may also be useful in developing diagnostic and/or therapeutic applications.

Feasibility of blood spot PCR in large-scale screening of fragile X syndrome in southern Taiwan.

BACKGROUND AND PURPOSE: Fragile X syndrome (FXS) is the most common form of hereditary mental retardation. Early diagnosis of the disease may lead to better prognosis for children who participate in early intervention programs. This study attempted to evaluate whether screening newborn boys with simple polymerase chain reaction (PCR) assay could be an effective approach for detection of mutation carriers and FXS, a process that may also facilitate detection of young carrier mothers. METHODS: Filter paper blood spot samples of 4843 newborn boys were collected from five hospitals in southern Taiwan. They were tested with a simple non-radioactive PCR for the presence of FMR1 gene mutation by determining the number of FMR1 CGG repeats. By this method, the examined sample can be reliably classified as normal (<40), intermediate (40-54), and mutant group (> 54), according to the number of CGG repeats. RESULTS: The FMR1 CGC repeat number of all but four samples was below 54, with 90 samples (1.8%) between 40 and 54 (the intermediate range). Two of the four abnormal samples were carriers of the premutation. The other two failed repeatedly in PCR amplification for the FMR1 gene, but not for the control K-ras gene. Hence, these samples seemed to be candidate carriers of large premutation or even full mutation, indicating the need for confirmation with standard Southern blot analysis. CONCLUSIONS: This study demonstrated that a simple PCR combined with blood spot sampling is effective and feasible for large-scale screening of newborn boys for fragile X carrier status. The relatively low carrier rate in this population suggests that the cost-effectiveness of implementation of such screening on a population-wide basis would be lower than in the Jewish and Caucasian populations.

Original article pilot screening for fragile X carrier in pregnant women of southern Taiwan.

BACKGROUND: Carrier detection before or at early pregnancy through a wide screening program may be a practical approach to prevent the fragile X syndrome. However, prior to implementation of such a program, the carrier prevalence in a population and availability of effective screening tests should be evaluated. METHODS: One thousand and two pregnant women were randomly selected from our obstetric clinic and screened for FMRI mutation. Each woman was examined individually using a simple non-radioactive PCR, as well as in pool with two other women using high-resolution Southern blot hybridization. RESULTS: One third of women could be excluded from carrier status by PCR alone, while the rest had to be screened using Southern-blot hybridization in pool with two other women. This screening strategy was reliable and efficient, and suitable for large-scale screening. Among the 1002 women, no carrier of either premutation or full mutation was found. Allele with intermediate CGG-repeat between 40 and 52 was found in only 22 women (2.2%). CONCLUSIONS: Estimation of female fragile X carrier rate in Taiwan could not be made in the present study, due to insufficient sample size. However, the results indicated clearly that the rate in Taiwan was significantly lower than that of Israel (0/1002 vs. 1/113, p = 0.003), and also lower than those from other western counties (1/186-259, p = 0.020-0.049). We doubt that the cost-efficiency of such a wide screening program in Taiwan is acceptable. However, the effective screening strategy proposed in this study would be very helpful for women with family history of mental deficiency of undetermined etiology.

A step-wise diagnosis of fragile X syndrome in Taiwan.

Fragile X syndrome (FXS), an X-linked dominant disorder, is one of the common forms of inherited mental retardation. This project aimed at identifying fragile X syndrome patients in schools by a two-step diagnosis with questionnaire and photography followed by molecular analysis. A total of 734 children with mental retardation were enrolled from kindergartens, primary schools, junior high schools, and schools for the mentally retarded. School teachers or nurses administered the questionnaires and took pictures of the faces and hands for of the patients. After viewing of the questionnaire and photos by a geneticist, 145 cases were selected for molecular study and 11 cases were identified as having full mutations in the FMRI gene. The detection rate was 1.5% (11 in 734) in all enrolled cases, and was 7.6% (11 in 145) in those who underwent molecular test. Those affected by FXS were more likely to have simian crease (p<0.001) and a head circumference larger than the 50th percentile (p=0.0295), and those who were not affected by FXS were lower in gestational age (p=0.0243). This screening method is useful for the detection of fragile X syndrome.