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PubMed2003

Multiple myeloma biology: lessons from the 5TMM models.

Multiple myeloma (MM) is a B cell neoplasm characterized by the monoclonal proliferation of plasma cells in the bone marrow, the development of osteolytic lesions and the induction of angiogenesis. These different processes require three-dimensional interactions, with both humoral and cellular contacts. The 5TMM models are suitable models to study these interactions. These murine models originate from spontaneously developed myeloma in elderly mice, which are propagated by in vivo transfer of the myeloma cells into young syngeneic mice. In this review we report on studies performed in the 5TMM models with special emphasis on the homing of the myeloma cells, the characterization of the migrating and proliferating clone and the identification of the isotype switch variants. The bone marrow microenvironment was further targeted with osteoprotegerin (OPG) to block the RANK/RANKL/OPG system and with potent bisphosphonates. Both treatments resulted in a significant protection against myeloma-associated bone disease, and they decreased myeloma disease, as evidenced by a lower tumor load and an increased survival of the mice. These different studies demonstrate the strength of these models, not only in unraveling basic biological processes but also in the testing of potentially new therapeutic targets.

Animals↗
PubMed1998

Association of a leucine(7)-to-proline(7) polymorphism in the signal peptide of neuropeptide Y with high serum cholesterol and LDL cholesterol levels.

High serum levels of total and LDL cholesterol are important risk factors in the development of atherosclerotic coronary artery disease. Cholesterol metabolism is affected by nutritional, environmental and genetic factors. Neuropeptide Y (NPY), which is widely expressed in both the central and peripheral nervous systems, has an important role in the hypothalamic regulation of energy balance by stimulating food intake and favoring energy storage through increased lipoprotein lipase activity in white adipose tissue. As a part of ongoing study of the genetic basis of obesity, we screened the NPY gene for sequence variants. We report here the identification of a common Leu(7)-to-Pro(7) polymorphism in the signal peptide of NPY. Presence of this Pro(7) in NPY was associated with higher serum levels of total and LDL cholesterol in obese subjects participating in two independent Finnish and Dutch studies. Furthermore, normal-weight Finns with Pro(7) also had higher serum levels of total and LDL cholesterol than did subjects with Leu(7)/Leu(7), as analyzed in three subsequent determinations at 5-year intervals during a 10-year follow-up period. The NPY polymorphism was not associated with higher cholesterol levels in normal-weight Dutch. Our study provides evidence that NPY is linked to cholesterol metabolism and that the polymorphism producing Pro(7) in NPY is one of the strongest genetic factors identified thus far affecting serum cholesterol, particularly in obese subjects.

Adult↗
PubMed2005

Mutation screening and association analysis of six candidate genes for autism on chromosome 7q.

Genetic studies have provided evidence for an autism susceptibility locus (AUTS1) on chromosome 7q. Screening for mutations in six genes mapping to 7q, CUTL1, SRPK2, SYPL, LAMB1, NRCAM and PTPRZ1 in 48 unrelated individuals with autism led to the identification of several new coding variants in the genes CUTL1, LAMB1 and PTPRZ1. Analysis of genetic variants provided evidence for association with autism for one of the new missense changes identified in LAMB1; this effect was stronger in a subgroup of affected male sibling pair families, implying a possible specific sex-related effect for this variant. Association was also detected for several polymorphisms in the promoter and untranslated region of NRCAM, suggesting that alterations in expression of this gene may be linked to autism susceptibility.

Autistic Disorder↗
PubMed2006

Psoriasis: genetic associations and immune system changes.

Psoriasis is a common inflammatory skin disease characterized by infiltration of inflammatory cells into the epidermis and altered keratinocyte differentiation. Psoriasis is currently thought of as a T-cell mediated 'Type-1' autoimmune disease. Gene expression changes in psoriasis lesions have been well documented, and strongly support an important role for tumor necrosis factor and interferon gamma signal pathways in its pathogenesis. The strongest genetic determinant of psoriasis identified to date lies within the class I region of the multiple histocompatibility locus antigen cluster, although its low penetrance implicates a requirement for other genetic risk factors. Multiple genome-wide linkage and an increasing number of association studies have been carried out, leading to multiple linkage peaks, and the identification of potential low risk variants. A number of these variants lie within genes encoding components of the immune system. However, the functional relationships between predisposing genetic variation is unclear, and presumably involves genetic susceptibility factors affecting both immune cell activation and keratinocyte differentiation. The interaction of environmental trigger factors with genetic effects is also not understood, but provide further evidence for the complex basis of this disease.

Animals↗
PubMed2003

Molecular cloning and functional expression of the human sodium channel beta1B subunit, a novel splicing variant of the beta1 subunit.

The voltage gated sodium channel comprises a pore-forming alpha subunit and regulatory beta subunits. We report here the identification and characterization of a novel splicing variant of the human beta1 subunit, termed beta1B. The 807 bp open reading frame of the human beta1Beta subunit encodes a 268 residue protein with a calculated molecular mass of 30.4 kDa. The novel human beta1B subunit shares an identical N-terminal half (residues 1-149) with the human beta1 subunit, but contains a novel C-terminal half (residues 150-268) of less than 17% sequence identity with the human beta1 subunit. The C-terminal region of the human beta1B is also significantly different from that of the rat beta1A subunit, sharing less than 33% sequence identity. Tissue distribution studies reveal that the human beta1Beta subunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle. Functional studies in oocytes demonstrate that the human beta1B subunit increases the ionic current when coexpressed with the tetrodotoxin sensitive channel, NaV1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human beta1 and rat beta1A subunits.

Alternative Splicing↗
PubMed2003

Novel family-based approaches to genetic risk in thrombosis.

The genetic basis of thrombosis is complex, involving multiple genes and environmental factors. The field of common complex disease genetics has progressed enormously over the past 10 years with the development of powerful new molecular and analytical strategies that enable localization and identification of the causative genetic variants. During the course of these advances, a major paradigmatic change has been taking place that focuses on the genetic analysis of measurable quantitative traits that are correlated with disease risk vs. the previous emphasis on the analysis of the much less informative dichotomous disease trait. Because of their closer proximity to direct gene action, disease-related quantitative phenotypes represent our best chance to identify the underlying quantitative trait loci (QTLs) that influence disease susceptibility. This approach works best when data can be collected on extended families. Unfortunately, family-based designs are still relatively rare in thrombosis/hemostasis studies. In this review, we detail the reasons why the field would benefit from a more vigorous pursuit of modern family-based genetic studies.

Alleles↗
PubMed1998

The human glycine receptor subunit alpha3. Glra3 gene structure, chromosomal localization, and functional characterization of alternative transcripts.

The neuronal glycine receptor is a ligand-gated chloride channel composed of ligand binding alpha and structural beta polypeptides. Homology screening of a human fetal brain cDNA library resulted in the identification of two alternative splice variants of the glycine receptor alpha3 subunit. The amino acid sequence predicted for the alpha3L variant was largely identical to the corresponding rat subunit. In contrast, the novel splice variant alpha3K lacked the coding sequence for 15 amino acids located within the cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and TM4. Using P1 artificial chromosome (PAC) clones, the structure of the GLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situ hybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to alpha3L and alpha3K, respectively, were identified and found to be widely distributed throughout the human central nervous system. Structural analysis of the GLRA3 gene revealed that the alpha3K transcript resulted from a complex splice event where excision of the novel exon 8A comprising the alternative sequence of 45 base pairs coincides with the persistence of a large intronic sequence in the 3'-untranslated region. Functional expression in HEK 293 cells of alpha3L and alpha3K subunits resulted in the formation of glycine-gated chloride channels that differed significantly in desensitization behavior, thus defining the cytoplasmic loop as an important determinant of channel inactivation kinetics.

Alternative Splicing↗
PubMed2002

Unusual alternative splicing within the human kallikrein genes KLK2 and KLK3 gives rise to novel prostate-specific proteins.

Prostate-specific antigen (PSA) and human kallikrein 2 are closely related products of the human kallikrein genes KLK3 and KLK2, respectively. Both PSA and human kallikrein 2 are produced and secreted in the prostate and have important applications in the diagnosis of prostate cancer. We report here the identification of unusual mRNA splice variants of the KLK2 and KLK3 genes that result from inclusion of intronic sequences adjacent to the first exon. The novel proteins encoded by these transcripts, named PSA-linked molecule (PSA-LM) and hK2-linked molecule (K-LM), share only the signal peptide with the original protein product of the respective gene. The mature proteins are entirely different and bear no similarity to the kallikrein family or to other proteins in the databases. As is the case with PSA, PSA-LM is expressed in the secretory epithelial cells of the prostate and is up-regulated in response to androgenic stimulation. A similar pattern of expression is suggested for K-LM.

Alternative Splicing↗
PubMed2003

Alternative splicing of the actin binding domain of human cortactin affects cell migration.

Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.

Actins↗
PubMed2004

Benign melanocytic lesions mimicking melanomas.

Review of problematic melanocytic consultation cases has enabled identification of the most common variants of benign melanocytic proliferations which can be confused with melanoma. Apart from Spitz naevus, the most common in this group of lesions are pigmented spindle cell naevus and atypical dermal nodule. Potentially the most problematic but less common are pseudomelanoma following incomplete removal of a naevus and 'activated' benign naevus cells in sentinel lymph nodes. By considering the overall architecture and the degree of nuclear atypia of the lesions, all of these problems can usually be resolved. Nuclear or architectural abnormalities by themselves are insufficient and must be assessed together. In addition, the combinations of pattern and cytological change in most of the lesions described here are distinctive and have become recognised as known variations of benign entities.

Diagnosis, Differential↗
PubMedDate not supplied

Lack of mutations of type 1 11beta-hydroxysteroid dehydrogenase gene in patients with abdominal obesity.

There is increasing evidence that in human obesity, particularly the abdominal phenotype, the activity of the hypothalamic-pituitary-adrenal (HPA) axis is disregulated. At least two distinct alterations have been reported: one is characterized by several neuroendocrine abnormalities and hyperresponsiveness of the HPA axis to different neuropeptides, the other is characterized by elevated cortisol traffic and probably by supranormal cortisol production. The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes interconvert cortisol and cortisone in human. Two different isoforms have been identified. A possible modification of the activity of the enzyme 11beta-HSD1 in subjects with abdominal obesity has been described in the literature. We decided to test the hypothesis that mutated isoforms of type 11beta-HSD1 protein could be responsible for alterations of cortisol metabolism in patients with abdominal obesity. A mutational screening of the whole coding sequence and exon-flanking regions of the 11B-HSD1 gene has been performed in 8 patients. The main results of our study are the exclusion of a common association of 11beta-HSD1 mutations to obesity and the identification of two novel allelic variants for the gene 11beta-HSD1 in the Italian population, not previously described in any database.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2006

BRCA1 and BRCA2 genetic testing in Italian breast and/or ovarian cancer families: mutation spectrum and prevalence and analysis of mutation prediction models.

BACKGROUND: Breast cancer is an extremely complex disease, characterized by a progressive multistep process caused by interactions of both genetic and non-genetic factors. A combination of BRCA1 and BRCA2 gene mutations appears responsible for about 20%-30% of the cases with breast cancer familial history. The prevalence of BRCA1/2 pathogenic mutations largely varies within different populations; in particular, the rate of mutations in Italian breast and/or ovarian cancer families is rather controversial and ranges from 8% to 37%. PATIENTS AND METHODS: Of the 152 breast/ovarian cancer families counseled in our centre, 99 were selected for BRCA1/2 mutation screening according to our minimal criteria. The entire coding sequences and each intron/exon boundary of BRCA1/2 genes were screened by direct sequencing (PTT limited to BRCA1 exon 11). For each proband, the a priori probability of carrying a pathogenic BRCA1/2 germline mutation was calculated by means of different mutation prediction models (BRCApro, IC and Myriad Table) in order to evaluate their performances. RESULTS: Our analysis resulted in the identification of 25 and 52 variants in the BRCA1 and BRCA2 genes, respectively. Seventeen of them represent novel variants, including four deleterious truncating mutations in the BRCA2 gene (472insA, E33X, C1630X and IVS6+1G>C). Twenty-seven of the 99 probands harbored BRCA1 (n = 15) and BRCA2 (n = 12) pathogenic germline mutations, indicating an overall detection rate of 27.3% and increasing by more than 15% the spectrum of mutations in the Italian population. Furthermore, we found the lowest detection rate (19.4%) in pure hereditary breast cancer family subset. All of the prediction models showed praises and faults, with the IC software being extremely sensitive but poorly specific, compared to BRCApro. In particular all models accumulated most false-negative prediction in the HBC subset. Interestingly preliminary results of a study addressing the presence of genomic rearrangements in HBC probands with BRCApro or IC prediction scores >/=95%, provided evidence for additional mutations undetectable with our conventional screening for point mutations. CONCLUSIONS: Altogether our results suggest that HBC families, the largest pool in our series, represent an heterogeneous group where the apparently faulty performances of the prediction models might be at least partially explained by the presence of additional kinds of BRCA1/2 alteration (such as genomic rearrangements) or by mutations on different breast cancer related genes.

Adult↗
PubMed1991

Identifying glycoconjugate-binding domains. Building on the past.

The molecular details of how glycoconjugate-binding proteins interact with their ligands have been revealed by a variety of techniques. For example, proteases, chemical-modifying reagents and antibodies have served as effective probes of lectin functional domains. Protein crystallography has providing insight into how lectins are structured, and aided in determining which amino acids in these proteins are positioned appropriately for bond formation with glycoconjugates. In addition, the characterization and sequencing of naturally occurring, non-functional lectin variants have led to the identification of amino acids which play critical roles in a lectin's glycoconjugate-binding domain. Similarly, studies of lectin mutants produced by site-directed mutagenesis, and of synthetic peptides that mimic lectin binding properties, have demonstrated the importance of particular amino acids for glycoconjugate binding. An alternate approach to understanding lectin functional domains has been to compare the primary sequences of these proteins to reveal common sequence elements which allow them to be organized into families. For example, the discovery of amino acid homologies dispersed over long segments of the primary sequences of several lectins has suggested that many of these proteins have a related three-dimensional organization. In addition, the identification of more highly focused regions of sequence homology has indicated that many structures within the lectin glycoconjugate-binding domains themselves may be conserved. Scanning protein data banks for sequences homologous to known lectins has led to the identification of several previously unrecognized lectins, and aided in determining what portions of these proteins function in their glycoconjugate-binding domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Differential binding of transcription factor E2F-2 to the endothelin-converting enzyme-1b promoter affects blood pressure regulation.

The endothelin-converting enzyme (ECE)-1 gene is a candidate for human blood pressure (BP) regulation and we report the identification of the new gene variants T-839G, C-338A, L75F, A677V and C+295T. Transient transfection of the reporter constructs containing the -338A allele showed an increase in promoter activity compared with the wild-type promoter. EMSA revealed the specific binding of E2F-2 to both ECE-1b promoter sequences, with the -338A allele being associated with an increased affinity to E2F-2 compared with -338C. The clinical relevance of this finding was analyzed in 704 hypertensive patients. In untreated hypertensive women, both the -338A and -839G alleles were significantly associated with ambulatory BP values. This study provides the first evidence of a link between the cell-cycle-associated E2F family and BP regulation via a component of the endothelin system.

Adult↗
PubMed1998

Apparent renal cell carcinomas in tuberous sclerosis are heterogeneous: the identification of malignant epithelioid angiomyolipoma.

Renal epithelial tumors (carcinoma and oncocytoma) have been reported with higher a frequency than expected in patients with the tuberous sclerosis complex. However, the recent identification of a monotypic, epithelioid variant of angiomyolipoma, closely simulating renal cell carcinoma, has cast doubt on the real frequency of carcinoma. Immunohistochemical analysis with a panel of antibodies, including melanogenesis marker HMB45, can discriminate between carcinoma and carcinoma-like angiomyolipoma. We studied five tumors previously reported as carcinoma and found that only one of them showed an immunohistochemical phenotype indicative of an epithelial tumor (Ker+, HMB45-). Three tumors exhibited a phenotype compatible with the monotypic epithelioid variant of angiomyolipoma (HMB45+, Ker-), and two of the three patients died of metastatic disease. The last patient had unusual clinical features, and the tumor was positive both for HMB45 and keratin. It is concluded that (1) renal cell carcinoma is less common in tuberous sclerosis complex than previously believed, (2) some cases called renal cell carcinoma probably represent a monotypic, epithelioid variant of angiomyolipoma, and (3) epithelioid angiomyolipoma is a potentially malignant tumor with invasion and metastases. These findings indicate that all reported renal carcinomas in tuberous sclerosis complex, therefore, must be reevaluated.

Adenoma, Oxyphilic↗
PubMed2003

An Ile to Met polymorphism in the catalytic domain of adenylyl cyclase type 9 confers reduced beta2-adrenergic receptor stimulation.

Adenylyl cyclase (AC) mediates signalling following activation of G(alphas)-coupled receptors such as the beta2-adrenergic receptor (beta2AR). Genetic variation in the receptor component of this pathway can alter signal transduction and the response to beta-agonists in asthma, but little is known about downstream effectors. Here, we characterize the population genomics and signalling effects of a polymorphism within the coding region of the AC9 gene that results in an Ile to Met substitution at amino acid 772 within the C1b region of the enzyme. Allele frequencies were 0.300 and 0.375 in Caucasians and Asians but were lower in African-Americans (0.163). The functional effects were studied in stably transfected HEK293 cells recombinantly expressing equivalent levels of wild-type (Ile772) and polymorphic (Met772) AC9. The polymorphic substitution results in a loss of function compared to wild-type AC9. Met772 AC9 has lower basal and beta2AR-mediated adenylyl cyclase activities compared to Ile772 AC9, as well as reduced activity following stimulation of G(alphas) by NaF. Direct stimulation of AC9 activity by Mn2+/- was also depressed in Met772 membranes, indicating decreased catalytic function, consistent with the location of residue 772. AC9 mRNA and protein were expressed in multiple human lung cell-types, including airway smooth muscle and airway epithelium. In the treatment of asthma, there is marked heterogeneity in the response to inhaled beta-agonists which is associated with polymorphisms of the beta2AR. Identification of a common AC9 variant that confers reduced enzyme activity reveals an additional polymorphism that should be considered in pharmacogenetic studies of beta-agonist therapy of asthma.

Adenylyl Cyclases↗
PubMed2006

The use of genomewide ENU mutagenesis screens to unravel complex mammalian traits: identifying genes that regulate organ-specific and systemic autoimmunity.

T-cell development is perhaps one of the best understood processes of mammalian cell differentiation, as many of the genes and pathways have been identified. By contrast, relatively little is known about the genes and pathways involved in immunological tolerance to self-antigens. Here, we describe the challenges associated with a genomewide screen designed at identifying new immune regulatory genes that uses a model of organ-specific autoimmunity leading to type 1 diabetes. The successful propagation and identification of the new gene variants will shed light on the various developmental checkpoints in lymphocyte development that are crucial for establishing tolerance to self-antigens.

Animals↗
PubMed1996

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction.

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

Animals↗