Subclinical atherosclerosis and genetic risk markers in healthy offspring of patients with premature myocardial infarction.

AIM: Healthy young subjects with parental history of premature myocardial infarction (PHPMI) might constitute a privileged population for the study of genetic risk markers (GRM) for atherosclerosis. Aim of this study was to evaluate which, if any, GRM atherosclerosis-associated in previous studies has increased prevalence in a selected population. METHODS: Twenty-four healthy young subjects (12 males and 12 females; mean age 18.0±8.0 years) with PHPMI and 24 age- (±1 year), sex-matched healthy subjects without PHPMI were enrolled in the study. They underwent: 1) fasting measurement of lipid profile, resting blood pressure and body mass index; 2) high resolution B-mode ultrasonographic evaluation of common carotid artery intima-media thickness (IMT); 3) evaluation of Single Nucleotide Polymorphisms (SNPs) for six candidate genes associated with preclinical atherosclerosis. RESULTS: Compared to controls, subjects with PHPMI had increased IMT of common carotid arteries (mean of combined sites: 0.535±0.171 mm versus 0.432± 0.133 mm in controls, P=0.017). Offspring of coronary patients showed an increased prevalence of the unfavourable chemochine (C-X-C motif) ligand 12 (CXCL12) SNP risk genotype (P=0.047). CONCLUSION: In healthy young subjects with PHPMI there is an increased prevalence of the unfavorable CXCL12 SNP risk genotype.

Homeobox genes in normal and malignant cells.

Homeobox genes are transcription factors primarily involved in embryonic development. Several homeobox gene families have so far been identified: Hox, EMX, PAX, MSX as well as many isolated divergent homeobox genes. Among these, Hox genes are most intriguing for having a regulatory network structure organization. Recent indications suggest the involvement of homeobox genes in (i) crucial adult eukariotic cell functions and (ii) human diseases, spanning from diabetes to cancer. In this review we will discuss the mechanisms through which homeobox genes act, and will propose a model for the function of the Hox gene network as decoding system for achieving specific genetic programs. New technologies for whole-genome RNA expression will be crucial to evaluate the clinical relevance of homeobox genes in structural and metabolic diseases.

A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

Homeobox genes and cancer.

Homeobox-containing genes are a family of regulatory genes encoding transcription factors that primarily play a crucial role during development. Several indications suggest their involvement in the control of cell growth and, when dysregulated, in oncogenesis. We will describe the implications, in tumor origin and evolution, of members of the homeobox gene families HOX, EMX, PAX, and MSX as well as of other divergent homeobox genes. We will also propose a model for the function of the HOX gene network in controlling cell identity to account for the involvement of some HOX genes in both normal development and oncogenesis.

Expression and modulation of homeobox genes from cluster B in endothelial cells.

Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.

Cytomegalovirus infection and schizencephaly: case reports.

Two children with left open-lip schizencephaly are reported. Cytomegalovirus (CMV) infection was demonstrated on the second day of life in Patient 1; in the other patient CMV infection was observed at the age of 4 years. Magnetic resonance imaging revealed polymicrogyric cortex lining the clefts. In Patient 2 polymicrogyria was also present in the corresponding area contralateral to the schizencephaly. Computed tomography revealed the presence of subependymal periventricular calcifications. Genetic analysis did not demonstrate the presence of EMX2 homeobox gene. The possible role of CMV infection in the complex multifactorial pathogenesis of schizencephaly is therefore suggested.

The rhGM-CSF-EPO hybrid protein MEN 11300 induces anti-EPO antibodies and severe anaemia in rhesus monkeys.

A recombinant human GM-CSF-EPO hybrid protein named MEN 11300 was administered biweekly for a total of 6 weeks to rhesus monkeys in order to evaluate its pharmacokinetic behaviour, tolerability and immunogenicity. In this primate species a strong antibody response was induced which neutralized the in vitro biological activity of human EPO while no antibody response could be detected against human GM-CSF. A severe drop in reticulocyte counts at approximately 2 weeks after initiation of treatment was followed by a dramatic decrease in the number of erythrocytes. No effects were observed on GM-CSF-dependent hematopoietic lineages and the clinical chemistry analyses did not reveal signs of general toxicity. Reticulocyte and erythrocyte counts started to recover 3-4 weeks after discontinuation of treatment in concert with a decline in anti-EPO antibody titres. Nevertheless, cell numbers remained below basal levels up to 50 days after the last MEN 11300 administration. Haematological impairment indicates that the administration to non-human primate of human EPO fused to human GM-CSF, induces neutralizing autoantibodies to the self EPO. Present data do not allow prediction of the immunogenic potential of the fusion protein in humans and a dose-escalating phase I study should be addressed to investigate the safety of the product.

A genetic polymorphism in the human HOXB1 homeobox gene implying a 9bp tandem repeat in the amino-terminal coding region. Mutations in brief no. 200. Online.

In man there are 39 homeobox genes of the HOX family in four loci, HOXA, HOXB, HOXC and HOXD on chromosomes 7, 17, 12, and 2. We discovered the existence of two major alleles, termed a and b, of gene HOXB1. They differ at a specific position in the 5' portion of the coding region. Sequencing the two alleles revealed that the allele HOXB1A, contains two copies of the 9bp sequence 5'ACAGCGCCC3', starting at position 65 of the coding region, whereas the allele HOXB1b contains three copies of this sequence (Fig. 1). As a consequence, the allele HOXB1b encodes a homeoprotein containing two copies of the tripeptide HisSerAla, starting at amino acid residue 25, which is present in only one copy in allele HOXB1a. We analyzed 250 individuals and found that the allelic frequencies of HOXB1a and HOXB1b were 78.8% and 21.2%. The murine homologue contains only one copy of the 9bp repeat (Fig. 1). 7 mouse strains, namely 129, BALB/c, C57BL/6, C57BL/10, CAST/Ei, C3H and SPRET/Ei, are homozygous for this allele. The allele present in gibbon and rhesus monkey appears to be identical to the human HOXB1b (Fig. 1).

A number of schizencephaly patients including 2 brothers are heterozygous for germline mutations in the homeobox gene EMX2.

We report here that some patients affected by schizencephaly are heterozygous for mutations in EMX2, a homeobox gene implicated in the patterning of the developing forebrain. Schizencephaly is a very rare human congenital disorder characterized by a full-thickness cleft within the cerebral hemispheres. Large portions of these may be absent and replaced by cerebrospinal fluid. We previously reported the presence of EMX2 mutations in 7 out of 8 sporadic cases of schizencephaly. We now extend this analysis to 10 additional patients, including 2 brothers. Six patients were found to be heterozygous for de novo mutations in EMX2. In particular, the 2 brothers show the same mutation affecting the splicing of the first intron, while this mutation is absent in their parents and in the 2 unaffected siblings.

Familial schizencephaly associated with EMX2 mutation.

We describe two brothers aged 8 and 10 affected by severe bilateral schizencephaly, carrying an identical point mutation of the homeobox gene EMX2. Both children had severe neurologic deficits and mental retardation, although they differed in the anatomic extent of the brain malformation and in the severity of the clinical picture. The present findings, together with the reported cases of schizencephaly associated with EMX2 mutations, support the hypothesis that, at least in some cases, schizencephalies are determined by deleterious mutations of this homeobox gene. The different morphoclinical pictures suggest that, besides the EMX2 mutation, other factors are relevant in determining the severity of the brain malformation and clinical picture.

Schizencephaly: surgical features and new molecular genetic results.

Schizencephaly is a rare developmental disorder characterized by a full thickness cleft within the cerebral hemispheres. Large portions of the cerebral hemispheres may be missing and are replaced by cerebrospinal fluid (CSF). The walls of the clefts are lined by polymicrogyric grey matter and are covered by the so-called "pialependymal seam". The cleft may be unilateral or bilateral, and if bilateral are fairly symmetrical. Their dimensions can be small or large. The clinical features may vary from a normal to a severe development delay. 13 patients with this anomaly have been evaluated. Using SSCP (single strand conformation polymorphism) analysis, as previously described (2), they were found to have a mutant homeobox gene, Emx2.

Expression of HOXC4 homeoprotein in the nucleus of activated human lymphocytes.

We have analyzed the expression of homeoproteins of the HOX family in resting and activated lymphoid cells and in neoplastic lymphoid cell lines by the use of monoclonal antibodies (MoAbs) already shown to react with the homeoproteins HOXA10, HOXC6, and HOXD4, respectively. Anti-HOXA10 and C6 MoAbs DIDi not show any reactivity with the lymphoid cells tested, whereas anti-HOXD4 MoAb stained few resting peripheral blood lymphocytes (PBLs) and most phytohemagglutinin (PHA)-stimulated PBLs as early as 6 hours after stimulation. The pattern of staining of PHA-activated PBLs is reminiscent of the stages of nucleolar fragmentation in different phases of the cell cycle. The MoAb reacted also with activated or Epstein-Barr virus-transformed B cells, with clonal or polyclonal T and natural killer (NK) cells, with leukemic T-cell lines, and with a Burkitt's lymphoma cell line. RNAse protection experiments, per formed with probes specific for HOXD4 or for the highly homologous HOXA4, HOXB4, and HOXC4, belonging to the same paralogy group, indicated that only HOXC4 mRNA is present in resting or activated PBLs. Northern blot analysis on polyA+ RNA from activated PBLs or Raji cells showed the presence of two different HOXC4 transcripts of 2.8 and 1.9 kb. Gel retardation and Southwestern blot assays showed the presence of a 32-kD homeoprotein with DNA-binding properties typical of a HOX4 homeoprotein in nucleolar extracts of PHA-activated, but not of resting, lymphocytes. Taken together, these data indicate that the HOXC4 homeoprotein is expressed in activated and/or proliferating lymphocytes of the T-, B-, or NK-cell lineage, whereas it is weakly expressed in a minority of resting cells. The early expression and the nucleolar localization suggest an involvement of HOXC4 in the regulation of genes controlling lymphocyte activation and/or proliferation.

Order of six loci at 2q24-q31 and orientation of the HOXD locus.

HOXD, a gene cluster of 9 homeobox genes of the Antennapedia class; EVX2, a homeobox gene related to Drosophila-even-skipped gene; DLX1 and DLX2, two homeobox genes related to the Drosophila distal-less gene; and TTN and NEB, the genes for the two giant molecules titin and nebulin, both involved in the sarcomere structure, have been previously mapped to human 2q31-q32 and to mouse chromosome 2. We studied their relative order in human by applying FISH to three balanced chromosome rearrangements each with a breakpoint at 2q31. Unambiguous results led us to map these genes and to orient the HOXD locus along chromosome 2 according to the following order: cen, NEB, DLX1-DLX2, EVX2, HOXD (5'-3'), TTN, tel. All of these genes are part of a syntenic region covering 5-10 cM and conserved since the divergence of humans and rodents, and thus the same loci order should be present in mouse. FISH in metaphases of approximately 500 bands localized NEB to 2q24.1-q24.2, while HOXD and TTN were localized to 2q31.

Balanced translocation (t 2q; 10p) and ocular anomalies. A possible HOX gene defect.

The authors report a child with a phenotype typical of a first branchial arch defect. The patient has a balanced translocation involving chromosome 2. They propose a defect that has occurred during the translocation in a gene mapped to chromosome 2 and belonging to the HOXD family. HOX gene defects can perturb the expression of other genes important for head development.

Chromosome locations of human EMX and OTX genes.

We have determined the chromosomal localization of four human homeobox-containing genes, EMX1, EMX2, OTX1, and OTX2, related to Drosophila genes expressed in the developing head of the fly. Murine homologs of these genes are expressed in specific nested domains in the developing rostral brain of midgestation embryos. DNAs from a panel of 19 rodent-human hybrids, each carrying one or a few human chromosomes such that most human chromosome regions were represented, were tested for the presence of the four gene loci by filter hybridization to radiolabeled probes. Regional chromosomal localization was determined by similarly testing DNAs from hybrid mapping panels for each of the candidate chromosomes. Finally, fluorescence in situ hybridization of cosmid clones for these loci refined the locations, two of which were in the vicinity of previously mapped orphan homeobox genes and two of which were near each other. OTX2, the earliest and most widely expressed gene, maps to chromosome region 14q21-q22; the OTX1 locus maps to 2p13; EMX2 maps to 10q26.1; and EMX1, the most narrowly and lately expressed, maps to 2p14-p13. Thus, these homeobox-containing genes involved in brain development are not linked to any of the four HOX clusters on 7p15-p14, 17q21-q22, 12q12-q13, and 2q31. However, the OTX1 and EMX1 loci may be closely linked on or near 2p13, prompting speculation that a clustered gene structure could have functional significance, as is presumably the case for the HOX clusters.

Inhibition of retinoic acid-induced activation of 3' human HOXB genes by antisense oligonucleotides affects sequential activation of genes located upstream in the four HOX clusters.

Most homeobox genes belonging to the Hox family are sequentially activated in embryonal carcinoma cells upon treatment with retinoic acid. Genes located at the 3' end of each one of the four Hox clusters are activated first, whereas upstream Hox genes are activated progressively later. This activation has been extensively studied for human HOX genes in the NT2/D1 cell line and shown to take place at the transcriptional level. To understand the molecular mechanisms of sequential HOX gene activation in these cells, we tried to modulate the expression of 3' HOX genes through the use of antisense oligonucleotides added to the culture medium. We chose the HOXB locus. A 5- to 15-fold reduction of the expression of HOXB1 and HOXB3 was sufficient to produce a significant inhibition of the activation of the upstream HOXB genes, as well as of their paralogs in the HOXA, HOXC, and HOXD clusters. Conversely, no effect was detectable on downstream HOX genes. The extent of this inhibition increased for progressively more-5' genes. The stability of the corresponding mRNAs appeared to be unaffected, supporting the idea that the observed effect might be mediated at the transcriptional level. These data suggest a cascade model of progressive activation of Hox genes, with a 3'-to-5' polarity.

Isolation and mapping of EVX1, a human homeobox gene homologous to even-skipped, localized at the 5' end of HOX1 locus on chromosome 7.

We isolated and mapped the human homeobox gene EVX1. This gene encodes a protein of 407 amino acid residues containing a homeodomain closely related to the Drosophila even-skipped (eve) segmentation gene of the pair-rule class. EVX1 belongs to a small family of vertebrate eve-related homeobox genes including human EVX1 and EVX2 genes, their murine homologs, Evx 1 and Evx 2, and the frog Xhox-3 gene. We previously reported that EVX2 is localized at the 5' end of the HOX4 locus on chromosome 2. We show here that EVX1 is localized at the 5' end of the HOX1 locus on chromosome 7, 48 kb upstream from the most 5' of the eleven HOX1 genes, namely HOX1J. Both EVX genes are transcribed in an opposite orientation as compared to that of adjacent HOX genes. Human HOX1 and HOX4 complex loci appear to be both closely linked to a homeobox gene of the EVX family.

A distinct Hox code for the branchial region of the vertebrate head.

The branchial region of the vertebrate head forms through complex interactions involving rhombomeric segments, neural crest and branchial arches. It is though that aspects of their patterning mechanisms are linked and involve Hox-2 genes, whose overlapping and spatially restricted expression domains represent a combinatorial code for generating regional diversity. Vertebrates possess four Hox clusters of Antennapedia class homeobox genes, related to each other by duplication and divergence from a common ancestral complex. In consequence, at equivalent positions in different clusters there are highly related genes known as subfamilies or paralogous groups. As Hox-2 genes cannot fully account for patterning individual rhombomeres, we investigated whether offsets in expression limits of paralogous genes could account for the generation of regional diversity. We report here that, with the exception of the labial subfamily, paralogues show identical expression limits in rhombomeres, cranial ganglia and branchial arches, providing a combinatorial Hox code for the branchial region that seems to be different in organization to that of the trunk.