W.J.M. Van de Ven

GATA3 haplo-insufficiency causes human HDR syndrome

Terminal deletions of chromosome 10p result in a DiGeorge-like phenotype that includes hypoparathyroidism, heart defects, immune deficiency, deafness and renal malformations. Studies in patients with 10p deletions have defined two non-overlapping regions that contribute to this complex phenotype. These are the DiGeorge critical region II (refs 1, 2), which is located on 10p13-14, and the region for the hypoparathyroidism, sensorineural deafness, renal anomaly (HDR) syndrome (Mendelian Inheritance in Man number 146255), which is located more telomeric (10p14–10pter). We have performed deletion-mapping studies in two HDR patients, and here we define a critical 200-kilobase region which contains the GATA3 gene. This gene belongs to a family of zinc-finger transcription factors that are involved in vertebrate embryonic development. Investigation for GATA3 mutations in three other HDR probands identified one nonsense mutation and two intragenic deletions that predicted a loss of function, as confirmed by absence of DNA binding by the mutant GATA3 protein. These results show that GATA3 is essential in the embryonic development of the parathyroids, auditory system and kidneys, and indicate that other GATA family members may be involved in the aetiology of human malformations.

Regulation of gene expression by alternative promoters.

Promoters have been defined as modulatory DNA structures containing a complex array of CiS‐acting regulatory elements required for accurate and efficient initiation of transcription and for controlling expression of a gene. It is becoming increasingly evident that they also constitute prime target elements through which diversity and flexibility in the complex patterns of gene expression in multicellular organisms are created. The use of multiple promoters and transcription start sites is apparently a frequently used mechanism, whereas at the same time there is considerable variation and complexity in the patterns of alternative promoter usage. This review discusses the use of alternative promoters as a versatile mechanism to create diversity and flexibility in the regulation of gene expression. Alternative promoter usage can influence gene expression in very diverse ways. The level of transcription initiation can vary between alternative promoters, the turnover or translation efficiency of mRNA isoforms with different leader exons can differ, alternative promoters can have different tissue specificity and react differently to some signals, and finally, alternative promoter usage can lead to the generation of protein isoforms differing at the amino terminus.—Ayoubi, T. A. Y., Van de Ven, W. J. M. Regulation of gene expression by alternative promoters. FASEBJ. 10, 453‐460 (1996)

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endo-proteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the ‘wild type’ precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of prov WF. In contrast, cotransfection of fur cDNA with a recombinat derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.

Transcriptional Activation Capacity of the Novel PLAG Family of Zinc Finger Proteins

We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12. PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA. To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast. Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region. This effect is less profound in epithelial cells. These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes.

The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter

We have previously shown that the PLAG1 gene on chromosome 8q12 is consistently rearranged in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between the PLAG1 gene, which encodes a novel zinc finger protein, and the constitutively expressed gene for β-catenin (CTNNB1), a protein with roles in cell-cell adhesion and the WG/WNT signalling pathway. In order to assess the importance of other translocation partner genes of PLAG1, and their possible relationship to CTNNB1, we have characterized a second recurrent translocation, i.e. the t(5;8)(p13;q12). This translocation leads to ectopic expression of a chimeric transcript consisting of sequences from the ubiquitously expressed gene for the leukemia inhibitory factor receptor (LIFR) and PLAG1. As for the t(3;8), the fusions occurred in the 5′-noncoding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. The results of the current as well as previous studies indicate that ectopic expression of PLAG1 under the control of promoters of distinct translocation partner genes is a general pathogenetic mechanism for pleomorphic adenomas with 8q12 aberrations.

The neurobeachin gene is disrupted by a translocation in a patient with idiopathic autism

Autism is a developmental disorder characterised by a triad of clearly abnormal or impaired development in social interaction and communication, and a markedly restricted repertoire of activity and interests.1 Its incidence is estimated at about 1/1000 to 1/2000.2 Different metabolic and structural brain anomalies have been observed in subjects with autism but these data have not yet led to a single unifying theory on its pathogenesis. In a minority (5–10%) of cases, autism is a symptom of a recognisable disorder such as fragile X syndrome, tuberous sclerosis, or untreated phenylketonuria.3 However, the molecular pathways involved in these disorders have also not contributed to an increased understanding of the pathogenesis of autism. In the majority of cases, the cause of autism is not known but there is strong evidence for a genetic cause. A polygenic inheritance is likely but estimates on the number of interacting genes vary from two to 10.4,5 Moreover, it is likely that different combinations of genes are implicated in unrelated subjects.6 The identification of genes involved in autism is expected to increase our understanding of the pathogenesis of this disorder. Several large scale linkage studies and follow up analyses have yielded suggestive linkage to several different chromosomal regions. However, neither this approach nor the large number of association studies using candidate genes has resulted in the identification of autism susceptibility genes.5 As an alternative approach to identifying candidate genes for autism, we initiated a positional cloning strategy starting with subjects with autism carrying a de novo chromosomal anomaly. In a group of 525 subjects with autism who were karyotyped and had no recognised underlying medical condition, four were found to carry such a de novo chromosomal aberration. In none of them was there a family history of autism. Three had …

The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of synpolydactyly.

Molecular analysis of the reciprocal chromosomal translocation t(12;22)(p11.2;q13.3) cosegregating with a complex type of synpolydactyly showed involvement of an alternatively spliced exon of the fibulin-1 gene (FBLN1 located in 22q13.3) and the C12orf2 (HoJ-1) gene on the short arm of chromosome 12. Investigation of the possible functional involvement of the fibulin-1 protein (FBLN1) in the observed phenotype showed that FBLN1 is expressed in the extracellular matrix (ECM) in association with the digits in the developing limb. Furthermore, fibroblasts derived from patients with the complex type of synpolydactyly displayed alterations in the level of FBLN1-D splice variant incorporated into the ECM and secreted into the conditioned culture medium. By contrast, the expression of the FBLN1-C splice variant was not perturbed in the patient fibroblasts. Based on these findings, we propose that the t(12;22) results in haploinsufficiency of the FBLN1-D variant, which could lead to the observed limb malformations.

PA26 is a candidate gene for heterotaxia in humans: identification of a novel PA26-related gene family in human and mouse

Heterotaxia is an aetiologically heterogeneous condition caused by an abnormal left-right axis formation, resulting in reversed left-right polarity of one or more organ systems. In a patient with heterotaxia and a de novo reciprocal translocation t(6;18)(q21;q21), we found that the PA26 gene was disrupted by the 6q21 breakpoint. Northern blot analysis showed decreased expression of the PA26 gene in an Epstein-Barr virus-transformed cell line of this patient. During early embryogenesis of Xenopus, the orthologue of PA26, XPA26 is exclusively expressed in the notochord, a midline structure. This further supports a possible role of PA26 in human situs determination. Mutation analysis of human PA26 gene in 40 unrelated individuals with unexplained heterotaxia failed to identify mutations, indicating that PA26 mutations are not a frequent cause of heterotaxia in humans. Analysis of the PA26 gene structure resulted in the identification of a novel PA26-related gene family, which we have named the sestrin family, and which comprises three closely related genes in human and in mouse.

Partial DiGeorge syndrome in two patients with a 10p rearrangement

We describe 2 patients with a partial DiGeorge syndrome (facial dysmorphism, hypoparathyroidism, renal agenesis, mental retardation) and a rearrangement of chromosome 10p. The first patient carries a complex chromosomal rearrangement, with a reciprocal insertional translocation between the short arm of chromosome 10 and the long arm of chromosome 8, with karyotype 46, XY ins(8;10) (8pter→8q13::10p15→10p14::8q24.1→8qter) ins(10;8) (10pter→10p15::8q24.1→8q13::10p14→10qter). The karyotype of the second patient shows a terminal deletion of the short arm of chromosome 10. In both patients, the breakpoints on chromosome 10p reside outside the previously determined DiGeorge critical region II (DGCRII). This is in agreement with previous reports of patients with a terminal deletion of 10p with breakpoints distal to the DGCRII and renal malformations/hypoparathyroidism, and thus adds to evidence that these features may be caused by haploinsufficiency of one or more genes distal to the DGCRII.

First insights into the molecular basis of pleomorphic adenomas of the salivary glands.

Pleomorphic adenoma, or mixed tumor of the salivary glands, is a benign tumor originating from the major and minor salivary glands. Eighty-five percent of these tumors are found in the parotid gland, 10% in the minor (sublingual) salivary glands, and 5% in the submandibular gland. It is the most common type of salivary gland tumor, accounting for almost 50% of all neoplasms in these organs. In fact, after the first observation of recurrent loss of chromosome 22 in meningioma, this was the second type of benign tumor for which non-random chromosomal changes were reported. The rate of malignant change with the potential to metastasize has been reported to be only 2 to 3%, and only a few cases of metastasizing pleomorphic salivary gland adenomas have been described to date. The fact that these tumors arise in organs located in an ontogenetic transitional zone, a region where endoderm and ectoderm meet, might be one of the reasons for the often-problematic histopathological classification. This type of benign tumor has been cytogenetically very well-characterized, with several hundreds of tumors karyotyped. In addition to the cytogenetic subgroup with an apparently normal diploid stemline (making up approximately 30% of the cases), three major cytogenetic subgroups can be distinguished. In addition to a subgroup showing non-recurrent clonal abnormalities, another subgroup is composed of tumors with various translocations involving 12ql5. By far the largest cytogenetic subgroup, however, consists of tumors with chromosome 8 abnormalities, mainly showing translocations involving region 8ql2. The most frequently encountered aberration in this group is a t(3;8)(p21;q12).