Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT)

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.      

Evaluation of maternal plasma creatine kinase activity as a marker of abnormal early pregnancy

We have tested the value of maternal plasma creatine kinase activity for diagnosing ectopic pregnancies obtained after in-vitro fertilization and embryo transfer. Plasma creatine kinase was assayed in 57 patients: 20 normal, 23 miscarriages and 14 ectopic pregnancies, for a total of 240 samples. All values were in the lower part of the normal range except only one in a miscarrying patient. A statistically significant difference was observed for a cut-off value of 45 IU/l between normal and ectopic pregnancies. However, for this cut-off point, the measurement of plasma creatine kinase activity had a sensitivity of 0.50 and a specificity of 0.76 for the diagnosis of ectopic pregnancy. The positive predictive value was 0.69. Creatine kinase activity measurements are thus of no practical value in this particular population, in which an early and specific marker of ectopic implantation would be of paramount interest. The association of human chorionic gonadotrophin (HCG) determinations and ultrasound scanning of the pelvis still remain the best paraclinical support for an early diagnosis of ectopic implantation.      

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Dietary factors in oral leukoplakia and submucous fibrosis in a population-based case control study in Gujarat, India

OBJECTIVES: To investigate the relationship of specific nutrients and food items with oral precancerous lesions among tobacco users.
DESIGN: A population-based case-control study. SETTING: Villages in Palitana taluk of Bhavnagar district, Gujarat, India.
SUBJECTS AND METHODS: An interviewer-administered food frequency questionnaire, developed and validated for this population, was used to estimate nutrient intake in blinded, house-to-house interviews. Among 5018 male tobacco users, 318 were diagnosed as cases. An equal number of controls matched on age (±5 years), sex, village, and use of tobacco were selected.
MAIN OUTCOME MEASURES: Odds ratios (OR) from multiple logistic regression analysis controlling for relevant variables (type of tobacco use and economic status).
RESULTS: A protective effect of fibre was observed for both oral submucous fibrosis (OSF) and leukoplakia, with 10% reduction in risk per g day^-1 ( P < 0.05). Ascorbic acid appeared to be protective against leukoplakia with the halving of risk in the two highest quartiles of intake (versus the lowest quartile: OR = 0.46 and 0.44, respectively; P < 0.10). A protective effect of tomato consumption was observed in leukoplakia and a suggestion of a protective effect of wheat in OSF.
CONCLUSION: In addition to tobacco use, intake of specific nutrients may have a role in the development of oral precancerous lesions.     

The Relationship Between Sinoatrial Conduction Time and Sinus Cycle Length Revisited

In 1974 we reported an inverse relationship between sinoatrial conduction time (SACT) and sinus cycle length (SCL) during sinus arrhythmia utilizing the indirect atrial premature stimulation technique for estimating SACT, However, this behavior seemed anomalous try analogy with the AV node. Subsequent to 1974, methodological considerations about and limitations of the indirect techniques for estimating SACT became apparent, making us question our former impression. When the capability to directly record sinus node electrograms was developed and established in the 1980s, we had the means to reevaluate the SACT/SCL relationship. This report presents our findings in 40 patients: the SACT/SCL relationship is direct, not inverse. Moreover, we also show that during the phasic fluctuations of sinus arrhythmia, the P-P alterations are initiated more frequently by changes in sinoatrial conduction time than by changes in sinus cycle length.     

DFP Labeling of Platelets During Recovery from Thrombocytopenia

Rat platelets were labeled with tritiated diisopropylfluorophosphate(³H-DFP) during recovery from acute thrombocytopenia. The results indicated that there was significant labeling of megakaryocytes by ³H-DFPwhich, in the presence of an increased rate of platelet production, resulted inmaintenance of relatively constant values for platelet-bound radioactivity during the period of maximum platelet production and reactive thrombocytosis.Significant random loss of platelets was apparent, and, when a cohort ofyoung platelets was transfused to normal recipients, they were destroyed at anormal rate.

     

Establishment of erythropoiesis following bone marrow transplantation in a patient with congenital hypoplastic anemia (Diamond-Blackfan syndrome)

Marrow transplantation was attempted in a 13-yr-old boy with congenital hypoplastic anemia who had never responded to corticosteroid therapy. Prior to the transplant, he had received 238 transfusions, at least 12 of which were from his father. He was prepared for grafting with antilymphocyte globulin, procarbazine, and total body irradiation (1000 rads). The patient, whose red cells were Group B, then received marrow cells from his Group O, histocompatible, sister. Thereafter, reticulocytes, Group O erythrocytes, and female leukocytes appeared in the peripheral blood. Erythroid precursors were seen in the patient's marrow for the first time in his life, and all lacked fluorescent Y chromosomes. Dividing cells were all female. After initially progressing well, the patient developed interstitial pneumonia and died 55 days after the transplant. The successful erythroid graft suggested that this patient's failure to produce red blood cells was due to a defective stem cell rather than to a humoral defect, plasma inhibitor, or abnormal marrow microenvironment. It suggested further that sibling marrow may be engrafted in patients who have received multiple transfusions, even from a parent.     

Massive Cerebral Edema Associated with Fulminant Hepatic Failure in Acetaminophen Overdose

Cerebral edema may complicate the course of fulminant hepatic failure. Response to conventional therapy has been disappointing. We present a patient with fatal acetaminophen-induced fulminant hepatic failure, with signs and symptoms of cerebral edema, unresponsive to conventional medical therapy. Cranial decompression was carried out. A justification of the need for further evaluation of cranial decompression in such patients is presented.