Monozygotic twins discordant for submicroscopic chromosomal anomalies in 2p25.3 region detected by array CGH.

Although discordant phenotypes in monozygotic twins with developmental disorder are not an exception, underlying genetic discordance is rarely reported. Here, we report on the clinical and cytogenetic details of 4-year-old female monozygotic twins with discordant phenotypes. Twin 1 exhibited global developmental delay, overweight and hyperactivity. Twin 2 had an autistic spectrum disorder. Molecular karyotyping in twin 1 identified a 2p25.3 deletion, further confirmed by Fluorescence in situ hybridization (FISH) analysis on leukocytes. Interestingly, array comparative genomic hybridization was normal in twin 2 but FISH analysis using the same probe as twin 1 showed mosaicism with one-third of cells with a 2p25.3 deletion, one-third of cells with a 2p25.3 duplication, and one-third of normal cells. Genotyping with microsatellite markers confirmed the monozygosity of the twins. We propose that the chromosome imbalance may be due to a mitotic non-allelic recombination occurring during blastomeric divisions of a normal zygote. Such event will result in three distinct cell populations, whose proportion in each embryo formed after separation from the zygote may differ, leading to discordant chromosomal anomalies between twins. We also discuss that the MYTL1L and the SNTG2 genes within the reported region could probably relate to the phenotypic discordance of the monozygotic twins.

Potential of fibrates in the treatment of fatty acid oxidation disorders: revival of classical drugs?

Exposure to fibrates leads to normalization of fatty acid oxidation (FAO) in fibroblasts from patients with myopathic forms of CPT2 deficiency or VLCAD deficiency. Correction of FAO is related to a drug-induced increase of residual enzyme activity, and this could provide a new treatment strategy for these disorders.

Transcriptional control of MHC genes in fetal trophoblast cells.

Tight control of MHC expression is essential for the outcome of a successful pregnancy. The lack of MHC class II and class I mediated antigen presentation by fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Interestingly, the deficient expression of MHC class II molecules (HLA-DR, -DQ and -DP) and of the classical MHC class I molecules HLA-A and HLA-B is also noted after IFN-gamma treatment in trophoblast-derived cell lines. Our studies show that in trophoblast cell lines the IFN-gamma induced transactivation of HLA-A and HLA-B promoters is repressed. Furthermore, it was found that trophoblast cells lacked IFN-gamma mediated induction of the class II transactivator (CIITA). This lack of CIITA expression in trophoblast cells is due to CIITA promoter hypermethylation. In addition to lack of CIITA expression, trophoblast cells also displayed a repressed expression of RFX5. Together, these observations reveal a silencing of multiple activation pathways that are critical to the transcriptional control of MHC class II and class I antigen presentation functions by trophoblast cells.

The MHC-specific enhanceosome and its role in MHC class I and beta(2)-microglobulin gene transactivation.

The promoter regions of MHC class I and beta(2)-microglobulin (beta(2)m) genes possess a regulatory module consisting of S, X, and Y boxes, which is shared by MHC class II and its accessory genes. In this study we show that, similar to MHC class II, the SXY module in MHC class I and beta(2)m promoters is cooperatively bound by a multiprotein complex containing regulatory factor X, CREB/activating transcription factor, and nuclear factor Y. Together with the coactivator class II transactivator this multiprotein complex drives transactivation of these genes. In contrast to MHC class II, the multiprotein complex has an additional function in the constitutive transactivation of MHC class I and beta(2)m genes. The requirement for all transcription factors in the complex and correct spacing of the binding sites within the SXY regulatory module for complex formation and functioning of this multiprotein complex strongly suggests that this complex can be regarded as a bona fide enhanceosome. The general coactivators CREB binding protein, p300, general control nonderepressible-5, and p300/CREB binding protein-associated factor exert an ancillary function in MHC class I and beta(2)m transactivation, but exclusively through the class II transactivator component of this enhanceosome. Thus, the SXY module is the basis for a specific enhanceosome important for the constitutive and inducible transactivation of MHC class I and beta(2)m genes.

Upregulation of transcription factors controlling MHC expression in multiple sclerosis lesions.

The expression of major histocompatibility complex (MHC) class I and class II in the CNS has received considerable interest because of its importance in neurodegenerative or inflammatory diseases, such as multiple sclerosis (MS). However, at the moment nothing is known about the expression patterns of transcription factors controlling MHC expression in MS lesions. Here, we performed an extensive immunohistochemical analysis on MS affected postmortem brain tissue to determine the cellular localization and distribution of different MHC-controlling transcription factors. We show that phagocytic macrophages in active demyelinating MS lesions displayed a moderate to strong immunostaining of the MHC-specific transcription factors RFX and CIITA, as well as the general transcription factors NF-kappaB, IRF1, STAT1, USF, and CREB, which was congruent with a strongly enhanced expression of HLA-DR, HLA-DQ, HLA-DP, and HLA class I. In the normal-appearing white matter (NAWM), clusters of activated microglial cells forming preactive lesions displayed an overall stronger expression level of these transcription factors, combined with a strong to intense level of MHC class I and class II immunostaining. In general, astrocytes and oligodendrocytes either did not express, or weakly expressed, these transcription factors, correlating with a lack of MHC class II and weak MHC class I expression. Together, the elevated expression level of transcription factors governing expression of MHC class I and class II molecules in activated microglial cells and phagocytic macrophages strongly suggests a general state of microglial cell activation in MS lesions.

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter.

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community.

Hepatic carnitine palmitoyltransferase 1 (CPT1A) deficiency is a rare disorder of mitochondrial fatty acid oxidation inherited as an autosomal recessive trait. Symptomatology comprises attacks of hypoketotic hypoglycemia with risk of sudden death or neurological sequelae. Only one CPT1A mutation has been reported so far. Identification of the disease-causing mutations allows both insights into the structure-function relationships of CPT1A and management of the patients and their relatives. The molecular analysis of CPT1A deficiency in a large Hutterite kindred illustrates this point. Both cDNA and genomic DNA analysis demonstrate that the affected patients are homozygous for a 2129G>A mutation predicting a G710E substitution. Studies in fibroblasts from one patient as well as heterologous expression of the mutagenized CPT1A in yeast show that the G710E mutation alters neither mitochondrial targeting nor stability of the CPT1A protein. By contrast, kinetic studies conclusively establish that the mutant CPT1A is totally inactive, indicating that the G710E mutation dramatically impairs the catalytic function of CPT1A. Finally, due to a strongly suspected founder effect for the origin of CPT1A deficiency in this Hutterite kindred, identification of this disease-causing mutation allows the setup of a targeted DNA-based newborn screening in this at-risk population.

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-gamma inducible promoter (PIV) of CIITA.

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

Transcriptional regulation of the MHC class Ib genes HLA-E, HLA-F, and HLA-G.

The restricted tissue expression of the MHC class Ib molecules HLA-E, HLA-F, and HLA-G has suggested specialized functions and tight transcriptional control of their genes. Transactivation of classical MHC class I genes is mediated by two groups of juxtaposed cis-acting elements, which can be viewed as regulatory modules. The most upstream module consists of the enhancer A and ISRE, and mediates constitutive and cytokine induced expression, whereas the SXY module is important for the constitutive and CIITA-mediated transactivation of MHC class I genes. Nucleotide sequence divergence in these regulatory elements in the promoters of HLA-E, HLA-F, or HLA-G determines their differential responsiveness to NF-kappaB, IRF1, and CIITA-mediated induction. HLA-E is not inducible by NF-kappaB or IRF1, but is responsive to IFN-gamma through an upstream STAT1 binding site. Furthermore, HLA-E is inducible by CIITA through the SXY regulatory module. HLA-F is inducible by NF-kappaB through the kappaB1 site of enhancer A, is responsive to IFN-gamma through the ISRE, and is inducible by CIITA. Both regulatory modules are divergent in HLA-G rendering this gene unresponsive to NF-kappaB, IRF1, and CIITA-mediated induction. This implies a unique regulation of HLA-G transcription amongst the MHC class Ib genes.

Transcriptional control of MHC genes and T cell development in MHC class II deficiency.

MHC class II deficiency has proven to be an excellent model to study transcription regulation of MHC genes and T cell development. Cell lines established from MHC class II deficient patients have been of great value for the identification of proteins necessary for MHC expression and their study has resulted in the identification of a common regulatory pathway for MHC class II and class I genes. The lack of MHC class II expression was found to have a profound effect on the development of the CD4+ T cell lineage, in particular on the composition of the T cell receptor repertoire, revealing aberrant thymic selection processes in these patients. Here, we will discuss several aspects of the transcriptional regulation of MHC genes and the impact of deficient MHC class II expression on T cell development.

Discoordinate expression of invariant chain and MHC class II genes in class II transactivator-transfected fibroblasts defective for RFX5.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific transcription factors. In the present study, we show that fibroblasts derived from a recently identified bare lymphocyte syndrome patient, SSI, were mutated for RFX5, one of the DNA-binding components of the RFX complex. Despite the lack of functional RFX5 and resulting MHC class II-deficient phenotype, transfection of exogenous class II transactivator (CIITA) in these fibroblasts can overcome this defect, resulting in the expression of HLA-DR, but not of DP, DQ, and invariant chain. The lack of invariant chain expression correlated with lack of CIITA-mediated transactivation of the invariant chain promoter in transient transfection assays in SSI fibroblast cells. Consequently, these CIITA transfectants lacked Ag-presenting functions.

Transactivation of classical and nonclassical HLA class I genes through the IFN-stimulated response element.

The IFN-stimulated response element (ISRE) is an important conserved cis-acting regulatory element in the promoter of MHC class I genes, but displays considerable locus-specific nucleotide variation. In this report, the putative ISREs of classical and nonclassical HLA class I genes were investigated for their contribution to MHC class I transactivation. It is shown that IFN-gamma induced MHC class I transactivation through the ISRE of HLA-A, HLA-B, HLA-C, and HLA-F. This is congruent with the binding of IFN regulatory factor-1 to the ISREs of these loci upon IFN-gamma treatment. Sp1 was shown to bind to the CG-rich sequences in the ISRE regions of HLA-B, HLA-C, and HLA-G. The putative E box 5' of the ISRE in most HLA-B alleles was shown to bind the upstream stimulatory factors (USF) 1 and 2. The Sp1 and USF binding sites did not influence IFN-gamma-induced transactivation. However, the USF binding site played a suppressive role in the constitutive expression of HLA-B. The locus-specific transcriptional control through the ISRE could be an important mechanism in the differential regulation of classical and nonclassical MHC class I expression, which determines adequate Ag presentation upon pathogenic challenge.

The regulation of HLA class I expression: is HLA-G the odd one out?

The expression of HLA-G in extravillous cytotrophoblast cells coincides with a general lack of classical MHC class I expression in this tissue. This differential expression of HLA-G and classical HLA class I molecules in trophoblasts suggests a tight transcriptional control. Transactivation of classical MHC class I genes is mediated by two groups of juxtaposed cis -acting elements which can be viewed as regulatory modules. The most up-stream module consists of the enhancer A and ISRE, and mediates the constitutive and cytokine-induced expression. The recently identified S-X-Y module is important in the constitutive and CIITA mediated transactivation. Both modules are divergent in HLA-G rendering this gene unresponsive to NF-kappaB, IRF-1, and CIITA mediated induction pathways. However, other known regulatory sequences that could contribute to the tissue-specific expression of HLA-G have so far not been identified in the proximal promoter region (-1500 bp) and in the first five intronic sequences. This implies a unique regulation of HLA-G transcription. Here, the transcriptional control of HLA-G and classical class I molecules in trophoblast cells are discussed.

Role of the interferon-stimulated response element in HLA-B downregulation in human melanoma cell lines.

Tumor cells are thought to escape immune surveillance from T cells by suppressing expression of major histocompatibility complex (MHC) class I molecules at their cell surface. Human MHC class I molecules are encoded by three different loci (HLA-A, -B, and -C). In primary human melanomas as well as melanoma cell lines, HLA class I expression is frequently downregulated in a B locus-specific manner. To study the involvement of promoter elements in HLA-B locus-specific downregulation, a series of reporter constructs containing 5'-flanking sequences of the HLA-A2 and -B7 genes were transfected into melanoma cell lines expressing high and low levels of HLA-B antigens. It is shown that enhancer A, which is generally believed to be a potent enhancer in HLA class I gene transcription, only weakly activates transcription in melanoma cell lines. In contrast, the interferon-stimulated response element (ISRE), known to induce MHC class I expression in response to IFNs, as well as a region comprising site alpha/enhancer B significantly stimulate constitutive transcription of HLA class I genes. Although none of the promoter elements tested could be demonstrated to mediate HLA-B locus-specific downregulation, high and low HLA-B melanoma cell lines do differ in ISRE activity as well as in ISRE-binding nuclear factors. The finding that high and low HLA-B melanoma cell lines contain different transcription factors binding to elements not actively involved in the process of HLA-B locus abrogation suggests that these cell lines originate from distinct types of melanocyte precursor cells expressing a different set of transcription factors.

The RFX complex is crucial for the constitutive and CIITA-mediated transactivation of MHC class I and beta2-microglobulin genes.

In type III bare lymphocyte syndrome (BLS) patients, defects in the RFX protein complex result in a lack of MHC class II and reduced MHC class I cell surface expression. Using type III BLS cell lines, we demonstrate that the RFX subunits RFX5 and RFXAP are crucial for constitutive and CIITA-induced MHC class I and beta2m transactivation. Similar to MHC class II, the promoters of MHC class I and beta2m contain an S-X-Y region of which the X1 box is crucial for constitutive and CIITA-induced MHC class I and beta2m transactivation. Thus, the RFX complex is part of a regulatory pathway linking the transactivation of MHC class I and II and their accessory genes.

The role of enhancer A in the locus-specific transactivation of classical and nonclassical HLA class I genes by nuclear factor kappa B.

HLA class I expression is tightly controlled at the transcriptional level by several conserved regulatory elements in the proximal promoter region. In this study, the two putative kappa B motifs of enhancer A (kappa B1 and kappa B2) of the classical and nonclassical HLA class I genes were investigated for their binding properties of transcription factors and tested for their contribution to the NF-kappa B-induced route of transactivation. It was shown that NF-kappa B-induced transactivation through enhancer A is most important for the HLA-A locus, which contains two NF-kappa B binding sites. Although the enhancer A of HLA-B contains only one NF-kappa B binding site (kappa B1), there was still a moderate transactivation by NF-kappa B. Since HLA-F, which also possesses one NF-kappa B binding site but lacks protein binding to its KB2 site, was not transactivated by NF-kappa B, the NF-kappa B-mediated transactivation through the kappa B1 motif in HLA-B is most probably facilitated by binding of the transcription factor Spl to the upstream kappa B2 site. Thus, transcriptional regulation of HLA class I genes by NF-kappa B is restricted to the HLA-A and HLA-B loci.