A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Genetics and race variability of the lucerne - Colletotrichum trifolii pathosystem in Australia

The Australian-bred lucerne cultivars, Trifecta and Sequel, were found to possess useful levels of resistance to both Colletotrichum trifolii races 1 and 2. Race 2 has only been previously observed in the United States and surveys did not reveal its presence in Australia. Multilocus fingerprinting using random amplified polymorphic DNA (RAPDs) analysis revealed low diversity (<10% dissimilarity) within Australian C. trifolii collections, and between the Australian race 1 isolates and a US race 2 isolate. Studies on the inheritance of resistance to C. trifolii race 1 in individual clones from Trifecta and Sequel revealed the presence of 2 different genetic mechanisms. One inheritance was for resistance as a recessive trait, and the other indicated that resistance was dominant. The recessive system has never been previously reported, whereas in the US, 2 completely dominant and independent tetrasomic genes Anl and Ant have been reported to condition C. trifolii resistance. It was not possible to fit the observed segregations from our studies to a single-gene model. In contrast to US studies, clones of cv. Sequel exhibiting the recessive resistance reacted differently to spray and stem injection with C. trifolii inoculum, being resistant to the former and susceptible to the latter, providing additional evidence for the presence of a different genetic mechanism conditioning resistance to those previously reported in the US. As C. trifolii is one of the most serious diseases of lucerne worldwide, the future development of molecular markers closely linked to the dominant and recessive resistances identified in these studies, and the relationships between these resistances and Anl and Ans as determined by genetic mapping, appear to be useful areas of future study.

Clinical and molecular genetics of myoclonic-astatic epilepsy and severe myoclonic epilepsy in infancy (Dravet syndrome)

The majority of severe epileptic encephalopathies of early childhood are symptomatic where a clear etiology is apparent. There is a small subgroup, however, where no etiology is found on imaging and metabolic studies, and genetic factors are important. Myoclonic-astatic epilepsy (MAE) and severe myoclonic epilepsy in infancy (SMEI), also known as Dravet syndrome, are epileptic encephalopathies where multiple seizure types begin in the first few years of life associated with developmental slowing. Clinical and molecular genetic studies of the families of probands with MAE and SMEI suggest a genetic basis. MAE was originally identified as part of the genetic epilepsy syndrome generalized epilepsy with febrile seizures plus (GEFS(+)). Recent clinical genetic studies suggest that SMEI forms the most severe end of the spectrum of the GEFS(+). GEF(+) has now been associated with molecular defects in three sodium channel subunit genes and a GABA subunit gene. Molecular defects of these genes have been identified in patients with MAE and SMEI. Interestingly, the molecular defects in MAE have been found in the setting of large GEFS(+) pedigrees, whereas, more severe truncation mutations arising de novo have been identified in patients with SMEI. It is likely that future molecular studies will shed light on the interaction of a number of genes, possibly related to the same or different ion channels, which result in a severe phenotype such as MAE and SMEI. (C) 2001 Elsevier Science B.V. All rights reserved.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Primary immunodeficiencies unravel critical aspects of the pathophysiology of autoimmunity and of the genetics of autoimmune disease

Background Primary Immunodeficiencies (PIDs) represent unique opportunities to understand the operation of the human immune system. Accordingly, PIDs associated with autoimmune manifestations provide insights into the pathophysiology of autoimmunity as well as into the genetics of autoimmune diseases (AID). Epidemiological data show that there are PIDs systematically associated with AID, such as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), Omenn syndrome, autoinunune polyendocrinopathy-candidiasis-ectodertnal dystrophy (APECED), autoinumine lymphoproliferative syndrome (ALPS), and C1q deficiency, while strong associations are seen with a handful of other deficits. Conclusion We interpret such stringent disease associations, together with a wealth of observations in experimental systems, as indicating first of all that natural tolerance to body components is an active, dominant process involving many of the components that ensure responsiveness, rather than, as previously believed, the result of the mere purge of autoreactivities. More precisely, it seems that deficits of Treg cell development, functions, numbers, and T cell receptor repertoire are among the main factors for autoimmunity pathogenesis in many (if not all) PIDs most frequently presenting with autoimmune features. Clearly, other pathophysiological mechanisms are also involved in autoimmunity, but these seem less critical in the process of self-tolerance. Comparing the clinical picture of IPEX cases with those, much less severe, of ALPS or APECED, provides some assessment of the relative importance of each set of mechanisms.

Gastrointestinal stromal tumor in Brazil: Clinicopathology, immunohistochemistry, and molecular genetics of 513 cases

The aim of the present study was to evaluate the clinicopathological, immunohistochemical, and molecular genetic features of gastrointestinal stromal tumors in Brazil and compare them with cases from other countries. Five hundred and thirteen cases were retrospectively analyzed. HE-stained sections and clinical information were reviewed and the immunohistochemical expression of CD117, CD34, smooth-muscle actin, S-100 protein, desmin, CD44v3 adhesion molecule, p53 protein, epidermal growth factor receptor, and Ki-67 antigen was studied using tissue microarrays. Mutation analysis of KIT and platelet-derived growth factor receptor-alpha genes was also performed. There was a slight female predominance (50.3%) and the median age at diagnosis was 59 years. The tumors were mainly located in the stomach (38.4%). Immunohistochemistry showed that CD117 was expressed in 95.7% of cases. Epidermal growth factor receptor expression was observed in 84.4% of tumors. p53 protein expression was found only in 2.6% of cases but all belonged to the high-risk group for aggressive behavior according to the National Institutes of Health consensus approach. No CD44v3 adhesion molecule expression was detected. KIT exon 11 mutations were the most frequent (62.2%). The present data confirm that gastrointestinal stromal tumors in Brazilian patients do not differ from tumors occurring in other countries.

Natural selection and quantitative genetics of life-history traits in Western women: A twin study

Whether contemporary human populations are still evolving as a result of natural selection has been hotly debated. For natural selection to cause evolutionary change in a trait, variation in the trait must be correlated with fitness and be genetically heritable and there must be no genetic constraints to evolution. These conditions have rarely been tested in human populations. In this study, data from a large twin cohort were used to assess whether selection Will cause a change among women in contemporary Western population for three life-history traits: age at menarche, age at first reproduction, and age at menopause. We control for temporal variation in fecundity (the baby boom phenomenon) and differences between women in educational background and religious affiliation. University-educated women have 35% lower fitness than those with less than seven years education, and Roman Catholic women have about 20% higher fitness than those of other religions. Although these differences were significant, education and religion only accounted for 2% and 1% of variance in fitness, respectively. Using structural equation modeling, we reveal significant genetic influences for all three life-history traits, with heritability estimates of 0.50, 0.23, and 0.45, respectively. However, strong genetic covariation with reproductive fitness could only be demonstrated for age at first reproduction, with much weaker covariation for age at menopause and no significant covariation for age at menarche. Selection may, therefore, lead to the evolution of earlier age at first reproduction in this population. We also estimate substantial heritable variation in fitness itself, with approximately 39% of the variance attributable to additive genetic effects, the remainder consisting of unique environmental effects and small effects from education and religion. We discuss mechanisms that could be maintaining such a high heritability for fitness. Most likely is that selection is now acting on different traits from which it did in pre-industrial human populations.

The impact of the Human Genome Project on medical genetics

The near completion of the Human Genome Project stands as a remarkable achievement, with enormous implications for both science and society. For scientists, it is the first step in a complex process that will lead to important advances in the diagnosis and treatment of many diseases. Society, meanwhile, must prevent genetic discrimination, and protect genetic privacy through appropriate legislation.

Genetics of brain function and cognition

There is overwhelming evidence for the existence of substantial genetic influences on individual differences in general and specific cognitive abilities, especially in adults. The actual localization and identification of genes underlying variation in cognitive abilities and intelligence has only just started, however. Successes are currently limited to neurological mutations with rather severe cognitive effects. The current approaches to trace genes responsible for variation in the normal ranges of cognitive ability consist of large scale linkage and association studies. These are hampered by the usual problems of low statistical power to detect quantitative trait loci (QTLs) of small effect. One strategy to boost the power of genomic searches is to employ endophenotypes of cognition derived from the booming field of cognitive neuroscience This special issue of Behavior Genetics reports on one of the first genome-wide association studies for general IQ. A second paper summarizes candidate genes for cognition, based on animal studies. A series of papers then introduces two additional levels of analysis in the ldquoblack boxrdquo between genes and cognitive ability: (1) behavioral measures of information-processing speed (inspection time, reaction time, rapid naming) and working memory capacity (performance on on single or dual tasks of verbal and spatio-visual working memory), and (2) electrophyiosological derived measures of brain function (e.g., event-related potentials). The obvious way to assess the reliability and validity of these endophenotypes and their usefulness in the search for cognitive ability genes is through the examination of their genetic architecture in twin family studies. Papers in this special issue show that much of the association between intelligence and speed-of-information processing/brain function is due to a common gene or set of genes, and thereby demonstrate the usefulness of considering these measures in gene-hunting studies for IQ.

Genetics of cognition: Outline of a collaborative twin study

Amultidisciplinary collaborative study examining cognition in a large sample of twins is outlined. A common experimental protocol and design is used in The Netherlands, Australia and Japan to measure cognitive ability using traditional IQ measures (i.e., psychometric IQ), processing speed (e.g., reaction time [RT] and inspection time [IT]), and working memory (e.g., spatial span, delayed response [DR] performance). The main aim is to investigate the genetic covariation among these cognitive phenotypes in order to use the correlated biological markers in future linkage and association analyses to detect quantitativetrait loci (QTLs). We outline the study and methodology, and report results from our preliminary analyses that examines the heritability of processing speed and working memory indices, and their phenotypic correlation with IQ. Heritability of Full Scale IQ was 87% in the Netherlands, 83% in Australia, and 71% in Japan. Heritability estimates for processing speed and working memory indices ranged from 33–64%. Associations of IQ with RT and IT (−0.28 to −0.36) replicated previous findings with those of higher cognitive ability showing faster speed of processing. Similarly, significant correlations were indicated between IQ and the spatial span working memory task (storage [0.31], executive processing [0.37]) and the DR working memory task (0.25), with those of higher cognitive ability showing better memory performance. These analyses establish the heritability of the processing speed and working memory measures to be used in our collaborative twin study of cognition, and support the findings that individual differences in processing speed and working memory may underlie individual differences in psychometric IQ.

Telemedicine and clinical genetics: establishing a successful service

There is a surprising lack of published experience on the use of videoconferencing in clinical genetics. Patients were randomly allocated to either a telegenetic (cases) or face-to-face (control) conventional clinic. The telegenetic consultation was done by videoconferencing, using ISDN lines at 384 kbit/s. Evaluation by the doctor and counsellor took place immediately after each appointment. The patient was asked to evaluate the appointment by telephone questionnaire about four weeks after the event. Forty-two patients were invited to participate and 33 (79%) returned their consent forms. Four patients declined to participate and were seen in ordinary face-to-face clinics. Preliminary results showed that the assessment of the telegenetics consultations by doctors, counsellors and patients was very favourable, and they responded positively when asked if they would be happy to use telemedicine in the future. For use in selected consultations, videoconferencing does appear to fulfil a useful role in clinical genetics.