Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC.

We have analysed 118 families with inherited medullary thyroid carcinoma (MTC) for mutations of the RET proto-oncogene. These included cases of multiple endocrine neoplasia types 2A (MEN 2A) and 2B (MEN 2B) and familial MTC (FMTC). Mutations at one of 5 cysteines in the extracellular domain were found in 97% of patients with MEN 2A and 86% with FMTC but not in MEN 2B patients or normal controls. 84% of the MEN2A mutations affected codon 634. MEN 2A patients with a Cys634 to Arg substitution had a greater risk of developing parathyroid disease than those with other codon 634 mutations. Our data show a strong correlation between disease phenotype and the nature and position of the RET mutation, suggesting that a simple, constitutive activation of the RET tyrosine kinase is unlikely to explain the events leading to MEN 2A and FMTC.

A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability.

Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07-1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.

Time-cost analysis of a quantum key distribution system clocked at 100 MHz.

We describe the realization of a quantum key distribution (QKD) system clocked at 100 MHz. The system includes classical postprocessing implemented via software, and is operated over a 12 km standard telecommunication dark fiber in a real-world environment. A time-cost analysis of the sifted, error-corrected, and secret key rates relative to the raw key rate is presented, and the scalability of our implementation with respect to higher secret key rates is discussed.

Microgeographic population structure of green swordail fish: genetic differentiation despite abundant migration.

Swordtails (Xiphophorus; Poeciliidae) have figured prominently in research on fish mating behaviours, sexual selection, and carcinogenesis, but their population structures and dispersal patterns have been relatively neglected. Using nine microsatellite loci, we estimated genetic differentiation in Xiphophorus helleri within and between adjacent streams in Belize. The genetic data were complemented by a tagging study of movement within one stream. In the absence of physical dispersal barriers (waterfalls), population structure followed an isolation by distance (IBD) pattern. Genetic differentiation (F(ST) up to 0.07) was significant between and within creeks, despite high dispersal in the latter as judged by the tagging data. Such heterogeneity apparently was a result of genetic drift in local demes, due to small population sizes and highly skewed paternity. The IBD pattern was interrupted by waterfalls, boosting F(ST) above 0.30 between adjacent samples across these barriers. Overall, our results are helpful in understanding the interplay of evolutionary forces and population dynamics in a small fish living in a changeable habitat.

Heterogeneous mutation of the RET proto-oncogene in subpopulations of medullary thyroid carcinoma.

Mutations in the RET proto-oncogene are associated with the pathogenesis of medullary thyroid carcinoma (MTC). In an attempt to understand this process, we examined microdissected subpopulations from MTC and multiple metastases from these tumors. Approximately 80% of sporadic MTC's had at least one subpopulation with the RET codon 918 mutation, which is a mutation previously detected in sporadic MTC as a somatic mutation and in multiple endocrine neoplasia type 2B as a germline mutation. However, the distribution of this mutation was nonhomogeneous, occurring only in subpopulations in most tumors and among subsets of multiple metastases, thus implying that although the codon 918 mutation could be an early event, it is not necessarily an early or essential event in tumorigenesis. This heterogeneity suggests either that the codon 918 mutation can arise as an event in progression within a metastatic clone or within a single tumor, or that MTC can be of polyclonal origin. Of significance, one of two multiple endocrine neoplasia type 2A MTCs carried a somatic mutation at codon 918, in addition to the RET mutation present in the germline. We found no correlation between the presence of other somatic genetic events, such as loss of heterozygosity on chromosome arms 1p and 22q, and RET mutation status in the various subpopulations of MTC.

Multiple mRNA isoforms of the human RET proto-oncogene generated by alternate splicing.

The RET proto-oncogene encodes a receptor tyrosine kinase. We and others have recently shown that distinct germline mutations of the RET proto-oncogene account for the majority of cases of the dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes, and can cause a dominantly inherited form of Hirschsprung disease, a disorder of development of the autonomic innervation of the gut. RET is also oncogenically activated in some sporadic thyroid and adrenal tumours. Here we report the characterisation of multiple mRNA isoforms of RET generated by alternate splicing. Two isoforms are predicted to encode membrane-spanning receptors with a truncated extracellular ligand-binding domain. A third isoform is predicted to encode a soluble, secreted form of the receptor. These mRNA isoforms are expressed in both normal and tumour tissues.

A novel point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC.

Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.

Mutation of the RET proto-oncogene is correlated with RET immunostaining in subpopulations of cells in sporadic medullary thyroid carcinoma.

Mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, are associated with the pathogenesis of medullary thyroid carcinoma (MTC). Somatic mutations in RET, predominantly at codon 918, and very rarely at codon 883, have been found in a proportion of sporadic MTC. We have previously shown that approximately 80% of sporadic MTCs had at least one subpopulation with a somatic RET mutation. Uneven distribution of somatic mutation within a single tumor or among metastases from a single individual was notable. In the present study, we sought to correlate RET expression, as demonstrated by RET immunohistochemistry, with mutation status in sporadic MTC for each tumor. Seventy evaluable subpopulations, belonging to 28 unrelated sporadic cases, comprising primary MTC and metastases, were immunostained with two different polyclonal antibodies raised against the C-terminus of RET. The regional presence of codon 918 or 883 seemed to coincide with increased RET immunopositivity in at least 62 of 70 (89%, P < 0.000001) tumor subpopulations. The reasons for this concordance are not entirely clear but could be related to either RNA or protein stability. Preliminary studies have suggested that the presence of somatic codon 918 mutation in MTC has a prognostic significance. If these preliminary results prove true, then given our data, we can further explore the feasibility of RET immunocytochemistry as a rapid assessment for the presence of somatic codon 918 for molecular diagnostic and prognostic purposes.

A systematic review of genetic polymorphisms and breast cancer risk.

Studies investigating the relationship between common genetic variants and cancer risk are being reported with rapidly increasing frequency. We have identified 46 published case-control studies that have examined the effect of common alleles of 18 different genes on breast cancer risk. Of these, 12 report statistically significant associations, none of which were reported by more than one study. However, many of the studies were small: 10 of the 46 had 80% power or greater to detect a rare allele homozygote relative risk <2.5. We therefore combined the results of individual studies to obtain more precise estimates of risk. Statistically significant differences in genotype frequencies were found in three case-control comparisons of unselected cases. These were for CYP19 (TTTA)n polymorphism [(TTTA)10 carrier odds ratio (OR) = 2.33; P = 0.002], the GSTP1 Ile105Val polymorphism (Val carrier OR = 1.60; P = 0.02), and the TP53 Arg72Pro polymorphism (Pro carrier OR = 1.27; P = 0.03). In addition, the GSTM1 gene deletion was found to be significantly associated with postmenopausal breast cancer (null homozygote OR = 1.33; P = 0.04). There was also some evidence that homozygotes for the PR PROGINS allele are protected against breast cancer, although this result was of borderline statistical significance. For polymorphisms in BRCA1, COMT, CYP17, CYP1A1, NAT1, and NAT2, the best estimate of risk either from the individual studies or the meta-analyses was sufficiently precise to exclude a relative risk of 1.5 or greater. For the polymorphisms in EDH17B2, ER, CYP2D6, CYP2E1, GSTT1, HSP70, and TNFalpha, the risk estimates, although nonsignificant, were insufficiently precise to exclude a moderate risk (>1.5). Precise estimation of the risks associated with these and other as yet untested genes, as well as investigation of more complex risks arising from gene-gene and gene-environment interactions, will require much larger studies.

Factor V Leiden, prothrombin 20210G-->A and the MTHFR C677T mutations in childhood stroke.

Ischaemic stroke is a rare occurrence in children and in a proportion of cases the aetiology remains unknown. We have investigated the role of thrombophilia in the aetiology of this condition. Of 50 cases identified at two centres, 37 were available for detailed haematological analysis. No cases were identified with deficiencies of antithrombin, protein C or protein S. One case had elevated IgG anticardiolipin antibodies at low titre. The prevalence of the prothrombin 20210 G-->A mutation, factor V Leiden (FVL) mutation and the C677T mutation in the MTHFR gene was compared in cases to that observed in random unselected cord blood controls. The odds ratio for stroke was not significantly increased in carriers of the prothrombin mutation (OR 1.2; 95% CI 0.1-10.7), FVL (OR 2.5; 95% CI 0.5-13.5), or the C677T mutation (OR 1.7; 95% CI 0.6-4.5). Our findings suggest that thrombophilia may not play a significant role in the aetiology of stroke in children, although a large prospective study is required to investigate this area further.

Regression of columnar lined (Barrett's) oesophagus with continuous omeprazole therapy.

Twenty-three adult patients with a columnar lined (Barrett's) oesophagus are being treated with long-term omeprazole, 40 mg daily. Twelve had never undergone anti-reflux surgery (Group 1), the other eleven having previously had insertion of an Angelchik anti-reflux prosthesis (Group 2). Endoscopy was carried out six months before, immediately before and six months, one year and two years into treatment. Multiple and standardized biopsies were taken at each endoscopy. Results from the two groups were similar. During the 6-month run-in period there was a statistically non-significant increase in the linear extent of the columnar mucosa, but this showed a progressive, statistically significant decrease during the two years of treatment. Other evidence for regression of the Barrett's mucosa includes the emergence of large numbers of macroscopic squamous islands within the abnormal mucosa, an increase in the number of microscopic squamous islands, and microscopic squamous encroachment of the abnormal mucosa at the squamo-columnar junction. Histological assessment showed a reduction in the proportion of sulphomucin-rich intestinal metaplasia, but this only achieved statistical significance in Group 1. The results substantiate the importance of acid in the pathogenesis of Barrett's oesophagus. Omeprazole may have a therapeutic role in bringing about regression of the metaplastic epithelium.

Development of the Leeds Dependence Questionnaire (LDQ): a questionnaire to measure alcohol and opiate dependence in the context of a treatment evaluation package.

The Leeds Dependence Questionnaire (LDQ) has been developed as part of a treatment evaluation package. The LDQ is a 10-item, self completion questionnaire designed to measure dependence upon a variety of substances; it has been shown to be understood by users of alcohol and opiates. The questionnaire was designed to be sensitive to change over time and to be sensitive through the range from mild to severe dependence; the follow-up data are insufficient to demonstrate change over time, but are encouraging. It is expected that both clinicians and researchers will find it useful to have a single measure relating to substance use, but not limited by specific substances. All items are scored 0-1-2-3; there are no normative data. The procedure for establishing content validity is described and estimates of concurrent, discriminant and convergent validities are reported; these validities are thought to be satisfactory. A principal components analysis produced a single factor accounting for 64% of the variance. Cronbach's alpha was 0.94. Test-retest reliability was found to be 0.95.

Detection of the hepatitis C virus by rt-PCR.

The hepatitis C virus (HCV) is an orgainsm of the age of molecular biology, for its discovery and much of the research into the infection have relied heavily on molecular techniques. The development of molecular cloning enabled a successful strategy that finally identified HCV (1)as the cause of 90% of posttransfusion (2)and > 50% of sporadic non-A, non-B hepatitis (3) after the failure by immunological techniques to discover the responsible agent. It is an important infection as most infected patients developed chronic hepatitis (> 50%) that can progress to cirrhosis and hepatocellular carcinoma (4-6) . Following the identification of the viral genome, antibody tests were developed which could detect exposure to the virus (7). The presence of antibodies to HCV, however, does not distinguish between those with chronic infection and those who had cleared the virus. Chronic HCV infection can be difficult to diagnose as patients may be asymptomatic and have normal liver biochemistry (8),(9) despite abnormal liver histology. Therefore, demonstration of virus RNA (usually from serum samples) is often necessary to confirm Infection. Detection of HCV RNA requires the sensitivity of nucleic acid amplification (e.g., the polymerase chain reaction) as circulating levels of vnus RNA can be very low (10),(11). Such tests are now widely used to confirm infection, monitor the response to anti-viral therapy, and in epidemiological studies of HCV infection.

Perinatal mortality in northern Benin.

PIP: The perinatal mortality rate (PMR) at the Hopital Evangelique in the Borgon region of Benin was 224/1000 in 1984 compared to 164/1000 in 1994. Despite the decrease, the rate was still high. Perinatal deaths for 1994 were reviewed retrospectively from case notes and data routinely recorded (presentation, distance traveled, and prenatal care). The probable causes of death were determined. A total of 511 babies were delivered by 484 women in 1994 at the hospital. There were 62 stillbirths (26 cases of birth asphyxia, 14 cases of antepartum hemorrhage, 7 cases of ruptured uterus, and 6 cases of intrauterine death before labor) and 22 neonatal deaths (7 cases of birth asphyxia and 11 cases of prematurity). Maternal errors accounted for 14 stillbirths and 4 neonatal deaths; logistical difficulties accounted for 8 stillbirths and 4 neonatal deaths; and the errors of management/referring maternity accounted for 4 stillbirths and 1 neonatal death. Some examples of maternal errors included a patient (gravida 6, para 5) who had not received prenatal care and presented with antepartum hemorrhage after being in labor for 72 hours. She had a ruptured uterus and required hysterectomy. Another patient (gravida 6, para 5) who had not received prenatal care presented to a maternity unit in labor with a shoulder presentation. On arrival, and after referral, intrauterine death was diagnosed and the fetus was delivered. Examples of logistical problems included a primigravida referred from a maternity unit more than 50 km away for delay in the second stage of labor. She delivered normally after arrival; however, the baby was asphyxiated and died. A patient who was gravida 2, para 1 was referred from a maternity unit with a cord prolapse. The fetal heart beat was present on leaving the maternity unit but was absent on arrival at the hospital. Bad management cases related to prolonged labor or attempts at a traumatic vaginal delivery. Maternal errors were the main source of deaths; thus, prenatal and intrapartum care should be more accessible to reduce the PMR.^ieng

Semicontinuous cardiac output monitoring using a neural network.

OBJECTIVES: This study compared 2-mL bolus thermodilution cardiac output measurements with standard 10-mL bolus measurements. DESIGN: Cardiac output was measured with the new 2-mL bolus technique and the 10-mL standard thermodilution technique in a perspective series. We describe a system that automatically cools and injects 2-mL boluses of saline into a standard pulmonary artery catheter. It uses a Peltier effect solid-state cooler and pneumatically driven syringe injector to measure cardiac output once per minute. SETTING: Animal laboratory. ANIMALS: Eight adult Duroc swine weighing between 38.0 and 57.5 kg. INTERVENTIONS: Once each minute, 2 mL of cooled 5% dextrose was injected through the pulmonary catheter. Once every 8 mins, four sequential measurements of cardiac output were made using 10-mL injections. MEASUREMENTS AND MAIN RESULTS: A total of 1249 paired waveforms were processed with both a conventional algorithm and with a neural network. For the conventional algorithm, the correlation coefficient was r2 = .92 and the SD of the difference was 1.30 L/min. For the neural network, the correlation coefficient was r2 = .94 and the SD of the difference was 0.88 L/min. Output filtering improved the results in both cases. CONCLUSION: Neural networks accurately derive cardiac output from 2-mL bolus thermodilution injections, allowing cardiac output to be monitored automatically once per minute in many patients. The technique is convenient and uses standard low-cost catheters.