Major insulin resistance syndromes: clinical and physiopathological aspects

Insulin resistance is a common metabolic disorder. It plays an important role in the metabolic syndrome (or syndrome X), type 2 diabetes, obesity and in the lipodystrophic syndromes recently described, associated with treatments of HIV disease and represent a worrying cardiovascular risk. However, its pathophysiology remains poorly understood in these situations. Syndromes of major insulin resistance, although rare, allow investigations of the mechanisms leading to alterations in the insulin transduction pathways. Mutations of the insulin receptor gene have been discovered in rare patients. Therefore alterations at the post-receptor level are probably causative in other cases. Furthermore, the role of body fat repartition seems determinant in the apparition of insulin resistance, as attested by the clinical characteristics of lipodystrophies, either congenital or acquired. The two lipodystrophic syndromes which molecular defect is identified are the familial partial lipodystrophy of the Dunnigan type, due to mutations of the lamin A/C gene, and the congenital generalized lipodystrophy, linked to alterations in the protein seipin. However, their physiopathology remains mysterious. Lamin A/C is indeed an ubiquitous nuclear protein, which is also mutated in a genetic squelettic and/or cardiac myopathy, and seipin is a protein of unknown function mainly expressed in brain. Progresses in the understanding of these syndromes, in particular lipodystrophies which can be considered as caricatural models of the metabolic syndrome, will probably allow to clarify the physiopathology of the more common forms of insulin resistance.     

Molecular genetics of Hirschsprung disease: a model of multigenic neurocristopathy

Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital malformation regarded as a multigenic neurocristopathy. Three susceptibility genes have been recently identified in HSCR, namely the RET proto-oncogene, the endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene mutations were found in significant proportions of familial (50%) and sporadic (15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous EDNRB an EDN3 missense mutations have been reported in isolated HSCR patients. Some of these results were obtained after the identification of mouse genes whose natural or site-directed mutations resulted in megacolon and coat color spotting. There is also conclusive evidence for the involvement of other independent loci in HSCR. In particular, the recent identification of neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide additional candidate genes for HSCR. The dissection of the genetic etiology of HSCR disease may then provide a unique opportunity to distinguish between a polygenic and a genetically heterogeneous disease, thereby helping to understand other complex disorders and congenital malformations hitherto considered as multifactorial in origin. Finally, the study of the molecular bases of HSCR is also a step towards the understanding of developmental genetics of the enteric nervous system giving support to the role of the tyrosine kinase and endothelin-signaling pathways in the development of neural crest-derived enteric neurons in human.     

Molecular genetic analysis of neurologic diseases]

With the recent progress in molecular genetics, our understanding of neurologic diseases on molecular basis has improved tremendously. Molecular analyses of gene mutations in hereditary neurologic diseases bring us not only the identification of mutations but also better understanding of molecular mechanisms involved in the neurologic diseases. We have identified a missense mutation (444Leu----Pro) in glucocerebrosidase gene of a type 2 Gaucher patient. The mutant glucocerebrosidase carrying 444Pro is unstable and degraded rapidly before the mutant protein is targetted to lysosomes. Detailed analysis on the 444Leu----Pro mutations among three phenotypes (types 1, 2 and 3) of Gaucher disease, it has been demonstrated that patients homozygous for the mutation frequently have the neurologic involvement (types 1 and 2). Very recently we have identified a mutation in tRNA(Lys) gene of mitochondrial genome of patients with myoclonus epilepsy associated with ragged-red fibers. The pathophysiologic mechanism with the tRNA(Lys) mutation is considered to be based on the impaired function of tRNA(Lys).     

Molecular genetics of migraine

Migraine is an episodic neurovascular disorder that is clinically divided into two main subtypes that are based on the absence or presence of an aura: migraine without aura (MO) and migraine with aura (MA). Current molecular genetic insight into the pathophysiology of migraine predominantly comes from studies of a rare monogenic subtype of migraine with aura called familial hemiplegic migraine (FHM). Three FHM genes have been identified, which all encode ion transporters, suggesting that disturbances in ion and neurotransmitter balances in the brain are responsible for this migraine type, and possibly the common forms of migraine. Cellular and animal models expressing FHM mutations hint toward neuronal hyperexcitability as the likely underlying disease mechanism. Additional molecular insight into the pathophysiology of migraine may come from other monogenic syndromes (for instance cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, which is caused by NOTCH3 mutations), in which migraine is prominent. Investigating patients with common forms of migraine has had limited successes. Except for 5′,10′-methylenetetrahydrolate reductase, an enzyme in folate metabolism, the large majority of reported genetic associations with candidate migraine genes have not been convincingly replicated. Genetic linkage studies using migraine subtypes as an end diagnosis did not yield gene variants thus far. Clinical heterogeneity in migraine diagnosis may have hampered the identification of such variants. Therefore, the recent introduction of more refined methods of phenotyping, such as latent-class analysis and trait component analysis, may be certainly helpful. Combining the new phenotyping methods with genome-wide association studies may be a successful strategy toward identification of migraine susceptibility genes. Likely the identification of reliable biomarkers for migraine diagnosing will make these efforts even more successful.     

Gene variants, insulin resistance, and dyslipidaemia

PURPOSE OF REVIEW: Both insulin resistance and dyslipidaemia are determined by genetic and environmental factors. Depending on their expression and their function, gene variants (mutations, polymorphisms) can primarily regulate either insulin action or dyslipidaemia. The purpose of this review is to give some examples from recent studies on gene variants regulating primarily insulin signalling or lipoprotein metabolism. RECENT FINDINGS: Common polymorphisms in the PC-1, insulin receptor substrate 1 and 2, and PPAR-gamma 2 genes have been linked to insulin resistance and dyslipidaemia, although the results have not been consistent. However, the Pro12Pro genotype of the PPAR-gamma 2 gene has been consistently associated with insulin resistance and the risk of type 2 diabetes. Promoter polymorphisms in the hepatic lipase gene, the 54Thr allele of the fatty acid binding protein 2 gene, and genes regulating LDL particle size have been associated with lipid metabolism, but on the other hand their association with insulin resistance is not consistent. SUMMARY: Although results have not always been consistent, gene variants affecting primary insulin action or dyslipidaemia, and particularly their interaction with the environment, are important modulators of glucose and lipoprotein metabolism.     

Combined Mutation And Rearrangement Screening by Quantitative PCR High-Resolution Melting: Is It Relevant for Hereditary Recurrent Fever Genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.     

Therapeutics development for triplet repeat expansion diseases

The underlying genetic mutations for many inherited neurodegenerative disorders have been identified in recent years. One frequent type of mutation is trinucleotide repeat expansion. Depending on the location of the repeat expansion, the mutation might result in a loss of function of the disease gene, a toxic gain of function or both. Disease gene identification has led to the development of model systems for investigating disease mechanisms and evaluating treatments. Examination of experimental findings reveals similarities in disease mechanisms as well as possibilities for treatment.     

Review of the molecular characteristics of gene mutations of the germline and somatic cells of the human

Molecular analyses of the limited number of de novo germinal mutations identified in humans indicate that an array of alterations in gene structure can be generated. Similar conclusions are derived from the large data set obtained from molecular analyses of alleles that segregate in the human population and cause genetic diseases. The molecular alterations include nucleotide substitutions as well as insertions, deletions and other rearrangements of the DNA. The lesions may be located in the coding or the noncoding regions of genes or may involve the flanking sequences. The insertions and deletions involve fragments ranging from single nucleotides to many kilobases, and involve both unique sequences and repetitive elements. The nature of the lesions observed to date as either de novo mutations or segregating variants suggests there are locus-specific characteristics of the alterations in DNA structure that are recovered as genetic diseases. Differences in mutation spectra among genetic loci appear to reflect both the structure of the target sequences and the relationship between gene structure and gene function. No induced germinal mutations have been identified, thus no data are available that reveal the relationships between mutagenic exposures and the molecular fingerprints of the lesion induced in the human germ cell and transmitted to the subsequent generations. In contrast, the prospects for analyzing the roles of genetic target, exposure history and individual responsiveness to exposure in creating particular molecular lesions in somatic cells are excellent, both for alterations of single nucleotides and for major alterations of gene structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presenilins: multifunctional proteins involved in Alzheimer's disease pathology

Early-onset aggressive forms of Alzheimer's disease (AD) are of genetic nature and have been linked to inherited mutations located on chromosomes 21, 14, and 1. The gene products of chromosomes 14 and 1, which are responsible for most of these familial forms of the disease (FAD), have been recently identified and referred to as presenilin 1 and 2 (PS1, PS2), respectively. Several lines of evidence derived from neuropathological, cell biology, and transgenesis approaches indicate that PS could interfere with the processing of the beta-amyloid precursor protein (betaAPP). Thus, FAD-linked mutations in PS exacerbate the production of Abeta42, the readily aggregable Abeta species corresponding to one of the main constituents of the senile plaques that invade the cortical areas of affected brains. Recent studies indicate that PS functions could be intimately related with the susceptibility of PS to further processing by caspase-like enzymes and other unknown proteolytic activities. Here I briefly report on the post-translational events undergone by PS and examine recent advances concerning their possible roles in development, cell signaling, and apoptosis. Possible alterations brought by FAD-linked mutations will be discussed.     

Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene: implications for mutation detection.

We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.     

The genetics of complex diseases

Genetic factors influence virtually every human disorder, determining disease susceptibility or resistance and interactions with environmental factors. Our recent successes in the genetic mapping and identification of the molecular basis of mendelian traits have been remarkable. Now, attention is rapidly shifting to more-complex, and more-prevalent, genetic disorders and traits that involve multiple genes and environmental effects, such as cardiovascular disease, diabetes, rheumatoid arthritis and schizophrenia. Rather than being due to specific and relatively rare mutations, complex diseases and traits result principally from genetic variation that is relatively common in the general population. Unfortunately, despite extensive efforts by many groups, only a few genetic regions and genes involved in complex diseases have been identified. Completion of the human genome sequence will be a seminal accomplishment, but it will not provide an immediate solution to the genetics of complex traits.     

The genetics of complex diseases

Genetic factors influence virtually every human disorder, determining disease susceptibility or resistance and interactions with environmental factors. Our recent successes in the genetic mapping and identification of the molecular basis of mendelian traits have been remarkable. Now, attention is rapidly shifting to more-complex, and more-prevalent, genetic disorders and traits that involve multiple genes and environmental effects, such as cardiovascular disease, diabetes, rheumatoid arthritis and schizophrenia. Rather than being due to specific and relatively rare mutations, complex diseases and traits result principally from genetic variation that is relatively common in the general population. Unfortunately, despite extensive efforts by many groups, only a few genetic regions and genes involved in complex diseases have been identified. Completion of the human genome sequence will be a seminal accomplishment, but it will not provide an immediate solution to the genetics of complex traits.     

The genetics of complex diseases

Genetic factors influence virtually every human disorder, determining disease susceptibility or resistance and interactions with environmental factors. Our recent successes in the genetic mapping and identification of the molecular basis of mendelian traits have been remarkable. Now, attention is rapidly shifting to more-complex, and more-prevalent, genetic disorders and traits that involve multiple genes and environmental effects, such as cardiovascular disease, diabetes, rheumatoid arthritis and schizophrenia. Rather than being due to specific and relatively rare mutations, complex diseases and traits result principally from genetic variation that is relatively common in the general population. Unfortunately, despite extensive efforts by many groups, only a few genetic regions and genes involved in complex diseases have been identified. Completion of the human genome sequence will be a seminal accomplishment, but it will not provide an immediate solution to the genetics of complex traits.     

Molecular biology of disorders of sex differentiation.

Sexual ambiguity can be a difficult and sometimes confusing diagnostic problem in children. Recent developments in molecular biology have provided the opportunity to analyze the gene responsible for testicular determination, SRY, the androgen receptor gene and the gene encoding the cP450 enzyme specific for 21-hydroxylation, CYP21B, whose defects are responsible for congenital adrenal hyperplasia. Southern-blotting studies and PCR analyses of SRY, androgen receptor and CYP21B genes can be routinely used for the direct diagnosis of gonadal dysgenesis, androgen insensitivity syndromes and congenital adrenal hyperplasia, respectively. In sex-reversed XY females, several de novo mutations or deletions in the SRY gene have been reported. Defects in the human androgen receptor cause a spectrum of defects in male phenotypic sexual development associated with abnormalities in the receptor protein. Analyses of the androgen receptor gene structure have identified the causative mutation in some families: mutations that result in large-scale alterations of the structure of the androgen receptor, mRNA or gene mutations that alter the primary structure of the androgen receptor protein and mutations that alter the level of mRNA. The diversity of clinical phenotypes, apparent in 21-hydroxylase deficiency, is paralleled by a considerable degree of mutational heterogeneity in the CYP21 gene locus. Various changes causing severe 21-hydroxylase deficiency have been reported: point mutations, gene conversions and gene deletions. In conclusion, substantial progress has been made elucidating genetic defects causing sex reversal in XY females, the androgen insensitivity syndrome and congenital adrenal hyperplasia. Molecular genetics can also be applied for carrier identification and prenatal diagnosis.     

Genetic Mutation Analysis of Human Gastric Adenocarcinomas Using Ion Torrent Sequencing Platform

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.     

The molecular biology of knockdown resistance to pyrethroid insecticides

The term "knockdown resistance" is used to describe cases of resistance to diphenylethane (e.g. DDT) and pyrethroid insecticides in insects and other arthropods that result from reduced sensitivity of the nervous system. Knockdown resistance, first identified and characterized in the house fly (Musca domestica) in the 1950's, remains a threat to the continued usefulness of pyrethroids in the control of many pest species. Research since 1990 has provided a wealth of new information on the molecular basis of knockdown resistance. This paper reviews these recent developments with emphasis on the results of genetic linkage analyses, the identification of gene mutations associated with knockdown resistance, and the functional characterization of resistance-associated mutations. Results of these studies identify voltage-sensitive sodium channel genes orthologous to the para gene of Drosophila melanogaster as the site of multiple knockdown resistance mutations and define the molecular mechanisms by which these mutations cause pyrethroid resistance. These results also provide new insight into the mechanisms by which pyrethroids modify the function of voltage-sensitive sodium channels.     

Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects

Since its identification in 1997, myostatin has been considered as a novel and unique negative regulator of muscle growth, as mstn-/- mice display a dramatic and widespread increase in skeletal muscle mass. Myostatin also appears to be involved in muscle homeostasis in adults as its expression is regulated during muscle atrophy. Moreover, deletion of the myostatin gene seems to affect adipose tissue mass in addition to skeletal muscle mass. Natural myostatin gene mutations occur in cattle breeds such as Belgian Blue, exhibiting an obviously increased muscle mass, but also in humans, as has recently been demonstrated. Here we review these natural mutations and their associated phenotypes as well as the physiological influence of the alterations in myostatin expression and the physiopathological consequences of changes in myostatin expression, especially with regard to satellite cells. Interestingly, studies have demonstrated some rescue effects of myostatin in muscular pathologies such as myopathies, providing a novel pharmacological strategy for treatment. Furthermore, the myostatin pathway is now better understood thanks to in vitro studies and it consists of inhibition of myoblast progression in the cell cycle, inhibition of myoblast terminal differentiation, in both cases associated to protection from apoptosis. The molecular pathway driving the myogenic myostatin influence is currently under extensive study and many molecular partners of myostatin have been identified, suggesting novel potent muscle growth enhancers for both human and agricultural applications.     

The genetic basis of resistance to anticoagulants in rodents

Anticoagulant compounds, i.e., derivatives of either 4-hydroxycoumarin (e.g., warfarin, bromadiolone) or indane-1,3-dione (e.g., diphacinone, chlorophacinone), have been in worldwide use as rodenticides for >50 years. These compounds inhibit blood coagulation by repression of the vitamin K reductase reaction (VKOR). Anticoagulant-resistant rodent populations have been reported from many countries and pose a considerable problem for pest control. Resistance is transmitted as an autosomal dominant trait although, until recently, the basic genetic mutation was unknown. Here, we report on the identification of eight different mutations in the VKORC1 gene in resistant laboratory strains of brown rats and house mice and in wild-caught brown rats from various locations in Europe with five of these mutations affecting only two amino acids (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln, Leu128Ser). By recombinant expression of VKORC1 constructs in HEK293 cells we demonstrate that mutations at Tyr139 confer resistance to warfarin at variable degrees while the other mutations, in addition, dramatically reduce VKOR activity. Our data strongly argue for at least seven independent mutation events in brown rats and two in mice. They suggest that mutations in VKORC1 are the genetic basis of anticoagulant resistance in wild populations of rodents, although the mutations alone do not explain all aspects of resistance that have been reported. We hypothesize that these mutations, apart from generating structural changes in the VKORC1 protein, may induce compensatory mechanisms to maintain blood clotting. Our findings provide the basis for a DNA-based field monitoring of anticoagulant resistance in rodents.