Further clinical evaluations elicited by functional biological investigations in childhood autism

Data on the protective role of antioxidants in models of atherosclerosis are only partially confirmed in man. Observational and epidemiological data, as well as randomized trials, provide no clear cut indications, because of positive and disappointing results on the use of antioxidants in cardiovascular protection. Despite the lack of a general consensus, recent data reinforce the concept that the regular intake of antioxidants present in food limits the progression of atherosclerosis. When it is possible to monitor the efficacy of any antioxidant therapy with validated markers of oxidation, the potential influence of vitamins and antioxidants on coronary artery disease may eventually be resolved.     

Molecular genetic studies of epilepsies]

The heart is frequently involved in the acquired immunodeficiency syndrome (AIDS). This study was planned to assess the prevalence of cardiac involvement in a large and selected population of patients who died of AIDS. Of 440 AIDS patients who underwent autopsy, cardiac involvement was documented in 82 patients. Dilated cardiomyopathy was found in 12 patients; lymphocytic interstitial myocarditis was documented in 30 patients, and in 10 of 12 patients with dilated cardiomyopathy. Inflammatory infiltrate was predominantly composed by CD3+ and CD8+ with a positive staining for major histocompatibility class I in 70% of the cases. Infective endocarditis was documented in 28 patients, pericardial effusion in 53 patients, myocardial Kaposi's sarcoma in 2 patients, myocardial B-cell immunoblastic lymphoma in 1 patient. Sequences of human immunodeficiency virus (HIV) nucleic acid were detected using the technique of in situ hybridization in the myocytes of 29 autopsy patients and in 25 of 29 patients with a positive hybridization signal an active myocarditis was documented. Among them, 7 presented a coinfection with Coxsakievirus group B, 2 with Epstein-Barr virus, and 1 with cytomegalovirus. HIV-associated cardiomyopathy may be related either to a direct action of HIV on the myocardial tissue or to an autoimmune process induced by HIV even in association with other cardiotropic viruses.     

Feasibility of collecting disease reports from relatives for genetic epidemiologic investigations

BACKGROUND: For many years, the treatment of facial wrinkles has been performed exclusively by dermabrasion, chemical peeling, or surgical face lifting. However, the recently introduced carbon dioxide lasers which emit ultrashort coherent light beams enable the cosmetic surgeon to ablate superficial epidemic layers with absent or very limited side effects. The purpose of this paper is to compare laser skin resurfacing with classical face lifting and discuss the potentials and limitations of each method. METHODS AND PATIENTS: Three patients suffering from facial wrinkles on photoaged skin were treated with the ultrapulsed CO2 laser (UltraPulse 5000 C; wavelength 10,600 nm, pulse duration 0.6 to 0.9 ms, maximum pulse energy 500 mJ). This laser guarantees vaporization of very thin superficial skin layers without scarring and with minimizing lateral thermal injury due to extreme short pulse duration. A special handpiece (CPG) permits an exact approach and a bloodless ablation of relatively large areas of facial skin. The fourth patient underwent a surgical face lift due to the depth of wrinkles. RESULTS: Excellent cosmetic results were achieved in all three patients with superficial wrinkles who were treated by laser skin resurfacing. When treating deeper wrinkles, e.g., glabella or nasolabial fold, the surgical face lift is the preferred method. CONCLUSION: Ultrapulsed CO2 laser treatment expands the therapeutic options for superficial facial wrinkles, especially for perioral, periorbital, forehead, and cheeks wrinkles. It proves to be a safe and effective method with very limited if any side effects. Nevertheless, deeper wrinkles are still a domain of the classical face lift. The combination of both methods may improve the overall outcome in the future.     

Molecular genetic insights into cardiovascular disease

The recent application of molecular genetic tools to inherited forms of cardiovascular disease has provided important insight into the molecular mechanisms underlying cardiac arrhythmias, cardiomyopathies, and vascular diseases. These studies point to defects in ion channels, contractile proteins, structural proteins, and signaling molecules as key players in disease pathogenesis. Genetic testing is now available for a subset of inherited cardiovascular diseases, and new mechanism-based therapies may be available in the near future. This remarkable progress and the implications it may have for more common forms of cardiovascular disease are reviewed here.     

Molecular genetic evidence for subtypes of oligoastrocytomas

Because of inherent genetic and physiological characteristics, the natural concentration of cadmium in the kernels of sunflowers grown in uncontaminated soils of the northern Great Plains region of the United States is higher than in most other grains. We tested the hypothesis that a habitual consumption of sunflower kernels will increase the body burden and health effects of cadmium in humans. Sixty-six men and women who reported consuming various amounts of sunflower kernels were recruited and divided by sex and kernel consumption: those who consumed less than or equal to 1 ounce(oz)/week and those who consumed more than 1 oz/week. Cadmium intake was assessed by calculation from 7-day food diaries, cadmium burden by whole blood cadmium, red blood cell (RBC) cadmium and urine cadmium concentrations, and health effects by urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) activity and beta2-microglobulin (beta2MG). The results showed that high intakes of sunflower kernels (>1 oz/day) significantly increased the intake of cadmium (p<0.004). However, the amount of cadmium in whole blood or RBCs was not affected by cadmium intake. Urinary excretion of cadmium also was not affected by cadmium intake. Urine NAG activity and the amount of urinary beta2MG were significantly elevated in the urine of high sunflower kernel consumers when the values were expressed on a urine volume basis (p<0.03), but not when expressed on a creatinine basis (p>0.05). Because normal ranges for the excretion of these protein markers have not been established, it was not possible to determine if these elevated values were meaningful. However, given the knowledge that habitual consumption of sunflower kernels with natural cadmium concentrations higher than most other food products will increase the average intake of dietary cadmium, the potential exists for an increased body burden of cadmium. Controlled feeding studies in humans should be pursued in order to determine if the body burden does indeed increase and, if so, is it a cause for concern.     

Molecular genetic characterization of XRCC4 function

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.     

Molecular genetic analysis of giant cell glioblastomas

Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.     

Molecular genetic studies of sporadic pituitary tumors

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.     

Molecular genetic diagnosis of leukemias and lymphomas

Leukemias and lymphomas are genetically characterized by pathogenetically important, disease-specific mutations in the malignant cells. Many of the mutations arise through balanced chromosomal rearrangements, often translocations, that fuse oncogenes with other gene loci. In some instances, this leads to the formation of a qualitatively new hybrid gene encoding a leukemogenic or lymphomogenic protein product, whereas on other occasions the result is deregulation of an otherwise normal gene. The demonstration of such gene rearrangements is of both diagnostic and prognostic value and hence serves as an important supplement to the more traditional phenotype-based investigative techniques. All available methods to detect neoplasia-specific, acquired mutations have both advantages and disadvantages, depending partly on the level of resolution at which they operate (chromosomes, nucleic acids, proteins), but also on the specific characteristics of each individual technique. Ideally, the initial genetic diagnosis of a given tumor should be reached by means of an open-framed screening technique, whereas less labour-intensive, specific techniques may be used during the subsequent follow-up of the patient. The most reliable and complete information is always obtained by combining cytogenetic, molecular cytogenetic, and strictly molecular genetic investigations. However, such an approach may be too cumbersome and expensive to be taken routinely.     

Molecular genetic analysis of von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is a dominantly inherited multisystem family cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal carcinoma, phaeochromocytoma, pancreatic islet cell tumours and endolymphatic sac tumours. In addition, renal, pancreatic and epididymal cysts occur. Morbidity and mortality from VHL disease can be reduced by the identification and surveillance of affected individuals and at-risk relatives so that complications are diagnosed at an early presymptomatic stage. The detailed mapping and subsequent isolation of the VHL tumour suppressor gene has enabled molecular genetic analysis in families and patients with definite or possible VHL disease. Initially, linked DNA markers were used in informative families to modify individual risks and then to make appropriate alterations in surveillance programs. However, currently most DNA analysis involves the characterisation of germline mutations. World-wide, mutations have been identified in almost 500 families (including 132 in our laboratory). These studies have revealed considerable heterogeneity both in the type and in the location of mutations within the VHL gene. In our experience, most recurrent mutations result from de novo mutations at hypermutable sequences, although a founder effect for the Tyr98His ('Black Forest') mutation has been reported in German and American families. Although many mutations are predicted to impair the ability of pVHL to combine with the elongin regulatory subunits, analysis of genotype-phenotype relationships suggests that the VHL protein has multiple and tissue specific functions. Calculation of tumour risks for different classes of VHL mutations has provided important prognostic information especially with respect to the likelihood of phaeochromocytoma. However, there is evidence that retinal involvement does not correlate with allelic heterogeneity, but that the variability in retinal angiomatosis is influenced by modifier gene effects. VHL gene mutation analysis also provides a basis for investigating the genetic basis of familial phaeochromocytoma and renal cell carcinoma, and apparently isolated retinal angiomas. Results to date suggest that a substantial proportion of patients with familial pheochromocytoma have VHL gene mutations but in contrast, most familial clusters of clear cell renal cell carcinoma (RCC) without evidence of VHL do not have germline VHL mutations.     

Molecular genetic study in congenital myotonic dystrophy

Congenital myotonic dystrophy (CMD) is the neonatal form of Steinert's myotonia. However, the symptoms and neuro-physiological findings are different from the classical adult form, there is a high mortality and early diagnosis of the condition is difficult. CMD occurs as a result of abnormal expansion of CTG triplets on chromosome 19. There is dominant autosomal transmission of this multi-systemic disorder, although when it occurs in children, it is the mother who is always the affected parent. Molecular genetic techniques enable unequivocal diagnosis of the condition, evaluation of anticipation and the possibility of offering genetic counselling to the families involved.     

Molecular and genetic features of fragile X syndrome

This study explored the claim that superior disembedding performance in autism reflects "less capture by meaning" and/or reduced "central coherence" [Shah & Frith, Journal of Child Psychology & Psychiatry, 24, 613-620 (1983); Shah & Frith, Journal of Child Psychology & Psychiatry, 34, 1351-1364 (1993)]. Meaningless as well as meaningful disembedding contexts were used, and memory for contextual information was examined. Neither qualitative (search strategy) nor quantitative (RT or accuracy) data indicated that high-functioning individuals with autism/PDD were superior to younger, developmentally matched controls. For both groups, disembedding was slowest from meaningful contexts, which generally were remembered best. No evidence was provided for "less capture by meaning" or reduced "central coherence" in autism/PDD, raising the possibility that earlier findings reflect a developmental, rather than a stable autism-specific, phenomenon.     

Molecular genetic detection of female carriers of protan defects

Females heterozygous for congenital colour vision defects are of interest because they are believed to have cone photoreceptor ratios and cone photopigments that differ from normal. We describe a molecular genetic method to identify protan carriers that involves characterizing the genes that occur in the most upstream position in each of the X-chromosome photopigment gene arrays.     

Molecular genetic analysis of McArdle''s disease in Spanish patients

As the extracardiac Fontan operation evolves, a reliable method for creating and subsequently closing communications between the systemic and pulmonary venous chambers would be useful. We describe a surgical technique for creating this "fenestration" and a complementary transcatheter technique that allows safe and reliable closure of these communications regardless of size and position.     

Molecular genetic analysis of plastocyanin biosynthesis in Chlamydomonas reinhardtii

Five plastocyanin-deficient mutants were identified from a population of UV-mutagenized Chlamydomonas reinhardtii cells. Genetic complementation experiments indicated that four mutants represented alleles at the PCY1 locus (pcy1-2, pcy1-3, pcy1-4, and pcy1-5). Sequence analysis confirmed that two strains, pcy1-2 and pcy1-3, carry a frameshift (-1) and a nonsense mutation, respectively, while strains pcy1-4 and pcy1-5 synthesize an extended protein as a result of read-through mutations at the stop codon. The C-terminal extension does not affect synthesis or processing of the pre-proteins, but the polypeptides are rapidly degraded after the second (lumenal) processing event. The frameshift mutation in pcy1-2 results in loss of Pcy1 mRNA, as noted previously for strain ac208 (pcy1-1), but the abundance of Pcy1 mRNA in strain pcy1-3, which carries a nonsense mutation at codon 26, is unaffected relative to wild-type cells. The decreased abundance of frameshifted Pcy1 mRNA is attributed to increased degradation rather than decreased synthesis, since the mRNAs can be stabilized by treatment of cells with cycloheximide or anisomycin. The fifth strain has a wild-type plastocyanin-encoding gene, but the strain accumulates apoplastocyanin at the expense of holoplastocyanin. We suggest that the mutation identifies a new locus (PCY2) whose function is required for normal holoplastocyanin accumulation. Like ac208 (pcy1-1), several of the new mutants were suppressed spontaneously owing to accumulation of cytochrome c6 (a functional substitute for plastocyanin). The suppressor mutation(s) displayed Mendelian inheritance and segregated independently from the PCY1 locus, which confirms that regulation of Cyc6 expression is not tightly linked to plastocyanin function.     

Molecular genetic diagnosis of von Hippel-Lindau disease in familial phaeochromocytoma

Inherited predisposition to phaeochromocytoma is seen in multiple endocrine neoplasia type 2 syndromes, von Hippel-Lindau (VHL) disease, and neuro-fibromatosis type 1. In addition familial phaeochromocytoma alone has been reported. To investigate the genetic basis for familial phaeochromocytoma alone, we screened three affected kindreds for mutations in the RET proto-oncogene and the VHL tumour suppressor gene. We did not detect MEN 2 associated RET mutations in any family, but missense VHL gene mutations (V155L and R238W) were identified in two kindreds with no clinical evidence of VHL disease. Patients with familial, multiple, or early onset phaeochromocytoma should be investigated for germline VHL and RET gene mutations as the molecular diagnosis of multisystem familial cancer syndromes enables appropriate counselling and screening to be provided.     

Molecular dynamics investigations of DNA triple helical models: unique features of the Watson-Crick duplex

We have built computer models of triple helical structures with a third poly(dT) strand Hoogsteen base paired to the major groove of a poly(dA).poly(dT) Watson-Crick (WC) base-paired duplex in the canonical A-DNA as well as B-DNA. For the A-DNA form, the sugar-phosphate backbone of the third strand intertwines and clashes with the poly(dA) strand requiring a radical alteration of the duplex to access the hydrogen bonding sites in the major groove. In contrast, when the duplex was in the canonical B-DNA form, the third strand was readily accommodated in the major groove without perturbing the duplex. The triple helical model, with the duplex in the B-DNA form, was equilibrated for 400ps using molecular dynamics simulations including water molecules and counter-ions. During the entire simulations, the deoxyriboses of the adenine strand oscillate between the S-type and E-type conformations. However, 30% of the sugars of the thymine strands-II & III switch to the N-type conformation early in the simulations but return to the S-type conformation after 200ps. In the equilibrium structure, the WC duplex portion of the triplex is unique and its geometry differs from both the A- or B-DNA. the deoxyriboses of the three strands predominantly exhibit S-type conformation. Besides the sugar pucker, the major groove width and the base-tilt are analogous to B-DNA, while the X-displacement and helical twist resemble A-DNA, giving a unique structure to the triplex and the Watson & Crick and Hoogsteen duplexes.