Low frequency of oncogenic mutations in the core promoter region of the RB1 gene.
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DNA Mutational Analysis↗
Biomedical subjects
K Minoda
Publications and source records attributed to K Minoda.
Hypermethylation in the retinoblastoma gene is associated with unilateral, sporadic retinoblastoma.
We previously reported 9 unilateral, sporadic retinoblastomas with hypermethylation in the 5' region of the RB gene, and we found that CpG methylation in the RB promoter inhibits the binding of the retinoblastoma binding factor 1 (RBF-1) and the activating transcription factor (ATF)-like factors, thereby resulting in a considerable reduction in RB promoter activity. In this study, we screened for hypermethylation in 121 additional cases of retinoblastoma, and found 5 tumors with hypermethylation, including 4 unilateral, sporadic tumors, and one hereditary tumor. The hereditary tumor had a germline deletion of one allele, and the hypermethylation was an acquired, epigenetic change in the other allele. Another tumor had hypermethylation restricted to approximately 800 base pairs in the RB promoter region including the essential RBF-1 and ATF sites. The frequency of hypermethylation in unilateral, sporadic tumors was 9.3% combining our previous and present examinations (13 among 140), whereas the frequency was 1.0% in bilateral hereditary tumors (one among 101). The statistical analyses using the chi-square test indicated significant correlation between hypermethylation and unilateral, sporadic tumors (p < 0.05). These results suggest that hypermethylation in the RB gene is always an acquired, epigenetic change and causes about 9% of unilateral, sporadic tumors.
Blotting, Southern↗
Exon-by-exon screening for RB germline mutations using Heteroduplex-SSCP analysis.
OBJECTIVE: To disclose the nature of RB1 germline mutations in Chinese and to develop a practical and effective way for mutational screening. METHODS: Leukocyte DNA was prepared from 8 Chinese patients with hereditary retinoblastoma. PCR combined with nonisotopic heteroduplex-SSCP analysis was used to screen leukocyte DNA for RB1 germline mutations, exon-by-exon, without the use of restriction endonuclease digestion. The mutations were finally identified by sequencing. In order to testify the effectiveness of this method, the same method was used to detect other 17 samples which have been previously analyzed by other methods. RESULTS: Heterozygous germline mutations were detected in the leukocyte DNA of 6 out of 8 Chinese patients: G del/codon 46, T del/codon 131, CAGAA del/codon 257-258, GCAgta-->GCAgca/donor of exon 16, C-->T/codon 661, and C-->T/codon 787. Heteroduplex-SSCP analysis may detect RB1 germline mutations in 68% (17/25) unselected patients, which is more effective than SSCP(56%) or heteroduplex analysis(64%) alone. The corrected RB1 mutation detection rate may be 80% if the samples were previously analyzed by Southern blotting, which is much higher than those reported on literature. CONCLUSION: Mutations involving a few base pairs in RB1 gene are common in Chinese. Heteroduplex-SSCP analysis is more useful and effective than SSCP or heteroduplex analysis alone for the rapid screening for unknown mutations.
Female↗
Detection of congenital color vision defects using heteroduplex-SSCP analysis.
Gene deletion and hybrid gene formation result in congenital red and green color vision defects. The innumerable variations in these defects require the development of appropriate methods for detailed investigation. We used heteroduplex-single-strand conformation polymorphism (SSCP) analysis to detect the gene variations in subjects with congenital red and green color vision defects. For comparison, we also used polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) to detect these defects. In all 13 protan and 9 of the 15 deutan subjects, defects of the red or green pigment gene could be identified by either heteroduplex-SSCP or PCR-RFLP analysis. Through heteroduplex-SSCP analysis, sequence polymorphism was detected in exon 5 of the green pigment gene. One polymorphic sequence was present in 8 of 21 trichromats but in only one of 17 dichromats, showing a biased distribution. A specific variation was found in one protan by SSCP analysis. Heteroduplex-SSCP procedures are useful for objective clinical diagnostic testing and for further study of color vision variations because of their simplicity and reliability and because they provide more information.
Blotting, Southern↗
Single 5'green-3'red hybrid gene in protanopia.
PURPOSE: To disclose the structure of visual pigment gene for a protanopia with specific variation. METHODS: Exon 5 fragments of the red and green visual pigment genes from the protanopia with specific variation as well as controls were amplified by polymerase chain reaction (PCR). The PCR products were put through heteroduplex-SSCP analysis and PCR-RFLP (restriction fragment length polymorphism) analysis to clarify the specific variation. The specific variation of the exon 5 DNA fragment from the protanopia was identified by sequencing. RESULTS: A novel 5'green-3'red hybrid gene fragment without the normal red and green visual pigment gene was discovered in the protanopia. He should only have a single visual pigment gene, 5'green-3'red hybrid gene, on his X chromosome. The fusion point is between codon 285 and codon 296 in exon 5. CONCLUSION: Unequal intragenic recombination may occur in exon 5 as well as its upstream. A 5'green-3'red hybrid gene may present independently on the X chromosome without accompanying the red or green visual pigment gene. Single visual pigment gene with 5'green-3'red hybrid fragment of exon 5 could express function which is similar to the green pigment gene. This example also gives in vivo evidence that codons 277 and 285, rather than codon 309, play a major role in tuning the absorption spectra of the red and green visual pigments.
Amino Acid Sequence↗
Detection of RB germline mutations using exon-by-exon heteroduplex analysis compared with SSCP.
PURPOSE: To compare heteroduplex analysis with SSCP and to develop a simple and effective method for mutational screening of RB gene. MATERIALS AND METHODS: Leukocyte DNA was prepared from 12 unrelated Japanese patients with hereditary retinoblstoma. PCR combined with simultaneous nonisotopic heteroduplex and SSCP analysis was used to screen leukocyte DNA for such mutations, exon-by-exon, without the use of restriction endonuclease digestion. PCR was conducted using 28 pairs of primers flanking all 27 exons and the promoter region of the RB gene, with PCR products ranging from 159bp to 326bp. Mutations were identified by sequencing. RESULTS: Heterozygous germline mutations were detected in 8 of 12 Japanese patients. The mutations were identified by sequencing as follows: G-->C/acceptor of exon 11, T insertion/codon 389, C-->T/codon 455, 33bp insertion/codon 455 (C decreases GA), G-->T/codon 533, C-->T/codon 579, C deletion/codon 674, and C-->T/codon 787. CONCLUSION: Our results suggest that small RB gene mutations are common. Heteroduplex analysis has advantage over SSCP and they may complement each other for the rapid detection of unknown mutations.
DNA, Neoplasm↗
[Ectopic ureterocele in a male adult: a case report].
A case of ectopic ureterocele in a 21-year-old male is reported. His chief complaint was right flank pain. We diagnosed ectopic ureterocele in right complete ureteric duplication with upper pole and right heminephrectomy was performed. Postoperative course was uneventful and ureterocele was markedly collapsed. The 10 reported cases with adult male ectopic ureteroceles including our case in Japan are reviewed and some characteristics of this entity are discussed.
Adult↗
[Genetics of retinoblastoma].
Recent advancement of molecular genetics has enabled us to perform presymptomatic prediction for hereditary retinoblastoma, based on RB gene diagnosis. We used PCR combined with SSCP and heteroduplex analysis to screen leukocyte DNA, exon by exon, in patients with bilateral retinoblastoma. Germline mutations were detected in the 22 of the 33 cases, and, in 16 cases, the mutations were identified by sequencing. Among 2 families with those hereditary retinoblastoma presymptomatic prediction by the method described above was applied for 2 newborn babies, resulting in both success. It is expected that gene diagnosis will be applied for not only bilateral but also unilateral cases for genetic counseling.
Base Sequence↗
Mutation detection and genetic counseling in retinoblastoma using heteroduplex analysis.
Gene diagnosis is essential for confident presymptomatic prediction, genetic counseling, and early management of hereditary retinoblastoma. In screening the leukocyte DNA of three patients with bilateral retinoblastoma for RB1-gene heterozygous germline mutations, we identified mutations involving exon 3 or 18 of the RB1 gene by using heteroduplex analysis and sequencing. In one case the mutation was a 2 bp GT deletion resulting in the loss of the exon 18 splicing-donor; another mutation was a G-to-T transversion at codon 580 in exon 18, which converts Arg to a stop codon. The third mutation involved in 1 bp deletion at codon 96 in exon 3, which leads to a premature stop codon at codon 110. We used information from this heteroduplex technique for genetic counseling and presymptomatic prediction. A newborn was identified as normal, using gene diagnosis; his 15-month follow-up confirmed our prediction.
Base Sequence↗
Influence of exercise-induced coronary artery spasm on thallium-201 initial distribution and washout kinetics in patients with variant and classic angina pectoris.
Thallium-201 single-photon emission computed tomography was performed immediately, and 2 and 4 hours after exercise-induced anginal attack in 2 groups of patients with either exercise-induced coronary spasm or severe fixed stenosis on the isolated proximal left anterior descending coronary artery. All patients with variant angina had transient ST-segment elevation during the exercise-induced attack for thallium-201 scintigraphic study. Both perfusion defects and 4-hour washout abnormalities were significantly greater in patients with variant angina than in those with stable effort angina (p < 0.01). In patients with stable effort angina, thallium-201 activity in ischemic regions (as a percentage of initial count in the normal region) progressively decreased, whereas in patients with variant angina it increased from 38% (initial) to 48% (2 hours), and then declined to 42% (4 hours). The initial normalized thallium-201 activity in the ischemic regions was significantly lower in patients with variant angina than in those with stable effort angina (p < 0.001). In conclusion, perfusion and washout abnormalities during exercise-induced angina are greater in patients with variant angina than in those with stable effort angina. Exercise-induced coronary spasm seems to contribute to the profound reduction in initial thallium-201 distribution and delayed thallium-201 accumulation in the ischemic region.
Adult↗
Comparison of the distribution of myocardial blood flow between exercise-induced and hyperventilation-induced attacks of coronary spasm: a study with thallium-201 myocardial scintigraphy.
Exercise and hyperventilation tests are often used as the provocative tests for coronary artery spasm. To examine the distribution of myocardial blood flow during exercise-induced and hyperventilation-induced attacks of coronary spasm, thallium-201 myocardial scintigraphy was performed in 47 patients with variant angina. The extent and severity scores and severity index (severity score/extent score) of scintigraphic perfusion defect were calculated. In 32 patients, anginal attack associated with ST elevation on the electrocardiogram was induced by exercise; in 23 patients, the attack was induced by hyperventilation. In patients with either anterior or inferior wall ischemia, both the extent and severity scores and severity index in the scintigram were significantly greater in exercise-induced anginal attack than those in hyperventilation-induced attack. In patients with simultaneous anterior and inferior wall ischemia, they tended to be greater in exercise-induced attack. In eight patients in whom anginal attack was induced by both exercise and hyperventilation, these scores and index were significantly greater in exercise-induced attack than those in hyperventilation-induced attack. These data suggest that myocardial ischemia during exercise-induced attack is enhanced as compared with that during hyperventilation-induced attack. Thallium-201 scintigraphy combined with exercise testing seems to be more sensitive for detecting myocardial ischemia induced by coronary spasm.
Aged↗
Photoreceptor differentiation of retinoblastoma: an electron microscopic study of 29 retinoblastomas.
Retinoblastomas exhibit a unique form of differentiation to produce cell elements similar to those seen in a photoreceptor cell. An ultrastructural study was performed on 29 cases of retinoblastoma to further clarify the cytologic characteristics of the tumor cells. The age of the retinoblastomas averaged 17.1 months and the tumor cells showing photoreceptor differentiation were demonstrated in 10 cases (35%). The findings were especially notable in retinoblastomas with Flexner-Wintersteiner rosette formation (seven cases, 28%). Similar photoreceptor differentiation was also evident in solid cell clusters without rosette formation (four cases, 14%). The presence of photoreceptor elements was assumed to be significantly frequent both in Flexner-Wintersteiner rosettes and in the solid cell clusters. The cell cytoplasm also showed proliferation of long mitochondria and microtubules, reflecting photoreceptor differentiation. The hereditary-type retinoblastoma showed more advanced cell differentiation than the non-hereditary type. Photoreceptor differentiated retinoblastoma showed rather indolent growth compared with the undifferentiated type, and the former can expect a curative treatment by operation. These observations provide additional findings of the biological nature of retinoblastomas.
Cell Differentiation↗
[Diagnosis and management of intraocular tumors].
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Adult↗
Occurrence of sequential intraocular tumors: malignant medulloepithelioma subsequent to retinoblastoma.
A 13-year-old boy, whose bilateral retinoblastoma had been treated by enucleation (OD) and irradiation (OS), developed anterior staphyloma (OS) necessitating the second enucleation. Histopathological, immunohistochemical and electron microscopic studies revealed malignant teratoid medulloepithelioma of the ciliary body, with demonstrated multipotential histogenesis. This is the first report which demonstrates the development of a second malignant intraocular tumor in an eye previously affected by hereditary retinoblastoma. Careful monitoring of patients who have had hereditary retinoblastoma is necessary because of the potential for a second malignancy.
Adolescent↗
Advances in management of retinoblastoma.
Retinoblastoma is a highly malignant intraocular tumor of children that requires accurate diagnosis to prompt treatment. This article reviewed clinical, pathological and follow-up data on 1 147 cases of retinoblastoma registered in Japan from 1975 to 1982. It is obvious that the prognosis of children with retinoblastoma has improved remarkably in recent years. The current advances in the management of the retinoblastoma were discussed.
Eye Neoplasms↗
Usefulness of reinjection image for evaluating viable myocardium in the infarcted zone on exercise thallium-201 SPECT.
Reinjection images were obtained in 23 patients with myocardial infarction by the additional injection of 37 MBq of thallium-201 after obtaining 4 hour delayed images on exercise thallium-201 SPECT (TSPECT). A redistribution index (RI) was derived of the changes in perfusion defects between immediate and 4 hour delayed images as well as immediate and reinjection images on polar bull's eye maps. The RI of reinjection images (46 +/- 27%) was significantly greater than that of 4 hour delayed images (26 +/- 26%) in patients with myocardial infarction (p less than 0.01). Significant redistribution after reinjection occurred in 4 of 9 patients (44%) without significant redistribution on 4 hour delayed images. Improvement in redistribution on reinjection images correlated significantly to the small extent of coronary artery disease and collateral development. The appearance of redistribution from 4 hour delayed imaging to reinjection imaging also might reflect the function of collateral development in the resting state in patients without significant redistribution on 4 hour delayed images. It has been demonstrated that underestimated viable myocardium on 4 hour delayed images in the infarcted zone can be better assessed on reinjection images. This reinjection technique is recommended in patients with no or partial redistribution on 4 hour delayed images.
Adult↗
Nodoventricular Mahaim fiber connecting to the left ventricle.
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Adult↗
Prognosis of orbital rhabdomyosarcoma in children in Japan.
Retrospective studies were made of patients with orbital rhabdomyosarcoma (RMS) who were registered in the Japan Children's Cancer Registry and the National Registry of Ocular Tumors in Japan during 1974-1989. Thirty-five children under 15 years of age were registered as orbital RMS patients. The median age of onset was 5 years and 2 months; lid swelling was the most frequent initial symptom and finding. Histologically, 79% were classified as the embryonal type. Orbital exenteration was the standard treatment until the mid-1970's, but all the 5 patients who had only surgical therapy experienced relapses. Since the mid-1970's, a combined regimen of surgery, radiation and chemotherapy has been used. Chemotherapy has recently been considered to play the most important role in this combined therapy. Among the 35 patients studied, three patients with only biopsy as the surgical procedure have had no relapses up to the present time. Recurrent tumors developed in 15 of the remaining 32 patients: 7 of those were local, 8 were distant. All relapses (10 of the 14 patients in the 1970's, and 5 of the 18 in the 1980's) occurred within 1 year and 11 months after initial treatment. The 3-year survival rate of these 32 patients, estimated by the Kaplan-Meier method, was 70% (52% in the 1970's, 86% in the 1980's).
Adolescent↗