Nina Horelli-Kuitunen

Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC and PDGFD genes

BackgroundPlatelet-derived growth factor (PDGF), which is a major mitogen for vascular smooth muscle cells and has been implicated in the pathogenesis of arteriosclerosis, is composed of dimers of PDGF-A and PDGF-B polypeptide chains, encoded by different genes. Here, we have analyzed the chromosomal localization, structure, and expression of 2 newly identified human genes of the PDGF family, called PDGFC and PDGFD. Methods and ResultsWe used fluorescence in situ hybridization to locate PDGFC and PDGFD in chromosomes 4q32 and 11q22.3 to 23.2, respectively. Exon structures of PDGFC and PDGFD were determined by sequencing from genomic DNA clones. The coding region of PDGFC consists of 6 and PDGFD of 7 exons, of which the last 2 encode the C-terminal PDGF cystine knot growth factor homology domain. An N-terminal CUB domain is encoded by exons 2 and 3 of both genes, and a region of proteolytic cleavage involved in releasing and activating the growth factor domain is located in exon 4 in PDGFC and exon 5 in PDGFD. PDGF-C was expressed predominantly in smooth muscle cells and PDGF-D in fibroblastic adventitial cells, and both genes were active in cultured endothelial cells and in a variety of tumor cell lines. Both PDGF-C and PDGF-D also stimulated human coronary artery smooth muscle cells. ConclusionsPDGFC and PDGFD have similar genomic structures, which resemble those of the PDGFA and PDGFB genes. Their expression in the arterial wall and cultured vascular cells suggests that they can transduce proliferation/migration signals to pericytes and smooth muscle cells.

Novel Human Vascular Endothelial Growth Factor Genes VEGF-B and VEGF-C Localize to Chromosomes 11q13 and 4q34, Respectively

BACKGROUND Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation, migration, and permeability during embryonic vasculogenesis as well as in physiological and pathological angiogenesis. The recently isolated VEGF-B and VEGF-C cDNAs encode novel growth factor genes of the VEGF family. METHODS AND RESULTS Southern blotting and polymerase chain reaction analysis of somatic cell hybrids and fluorescence in situ hybridization (FISH) of metaphase chromosomes were used to assess the chromosomal localization of VEGF-B and VEGF-C genes. The VEGF-B gene was found on chromosome 11q13, proximal to the cyclin D1 gene, which is amplified in a number of human carcinomas. However, VEGF-B was not amplified in several mammary carcinoma cell lines containing amplified cyclin D1. The VEGF-C gene was located on chromosome 4q34, close to the human aspartylglucosaminidase gene previously mapped to 4q34-35. CONCLUSIONS The VEGF-B locus in 11q13 and the VEGF-C locus in 4q34 are candidate targets for mutations that lead to vascular malformations or cardiovascular diseases.

Distinct differentiation characteristics of individual human embryonic stem cell lines.

BackgroundIndividual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic.ResultsThe cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines.ConclusionhESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

High-resolution Fluorescence In Situ Hybridization: A New Approach in Genome Mapping

Mapping of the human genome has been a global effort utilizing both genetic and physical mapping techniques. One approach which has greatly facilitated the physical mapping of the human genome is fluorescence in situ hybridization (FISH). Although FISH is by now a well-established technology, new recently developed modifications have enabled an easier use and higher resolution. The high-resolution FISH techniques have given a special impact in positional cloning: searching the functional gene from a chromosomal area where the gene has been genetically localized. New high-resolution FISH techniques include hybridization of probes to free chromatin, DNA fibres or mechanically stretched chromosomes. These targets have widened the resolution of FISH to detect distances from the traditional cytogenetic resolution level down to a resolution of a few kilobases. They also have significantly speeded up high-resolution physical mapping and thus made the search of new disease genes easier.

A Microduplication on Chromosome 17p13.1p13.3 Including the PAFAH1B1 (LIS1) Gene

Recently, three children with a microduplication in 17p13 including the PAFAH1B1 gene that encodes LIS1 were reported. LIS1 overexpression has earlier been shown to affect brain development by causing migrational defects and reductions in brain volume [Bi et al., 2009 ]. Here, we report an additional patient with a microduplication on chromosome 17p13.1p13.3 including the PAFAH1B1 gene, that was inserted into the long arm of chromosome 4. The patient had psychomotor and growth retardation, dysmorphic features, small ventricular septal defect (VSD), and immunoglobulin abnormality. Only subtle abnormalities in brain MRI scan were seen. Interestingly, the facial features of our patient closely resemble those previously reported in 17p trisomy patients.

Interstitial deletion of bands 11q21-->22.3 in a three-year-old girl defined using fluorescence in situ hybridization on metaphase chromosomes.

A 3-year-old girl has a de novo deletion of 11q21-22.3. The patient was studied because of minor anomalies, disproportionate short stature, and developmental delay. The deletion was first detected by conventional cytogenetic analysis and defined further by using chromosome 11-specific YAC clones by fluorescent in situ hybridization (FISH) on metaphase chromosomes. Three YAC clones, 11H7, 4A5, and IH4, were lacking from one of the patients chromosome 11. Trigonocepahly, hypertelorism, apparently low-set ears, mild renal abnormality, and delay in speech development found in our patient are similar findings in other published interstitial deletion cases. Our study shows that a molecular cytogenetic approach is useful in defining the specific location and the extent of an interstitial deletion in cytogenetically difficult areas such as 11q.

Assignment of ACVR2 and ACVR2B the human activin receptor type II and IIB genes to chromosome bands 2q22.2-->q23.3 and 3p22 and the human follistatin gene (FST) to chromosome 5q11.2 by FISH.

Supported by The Academy of Finland, Helsinki University Central Hospital Research Funds, the Duodecim Society, Medical Association of Finland (Finska Läkaresällskapet i Finland) and the Maud Kuistila Foundation. We thank Dr. Pieter de Jong, Roswell Memorial Institute, Buffalo, NY, USA, for providing the PAC library. Ms. Ritva Javanainen and Ms. Maritta Putkiranta are thanked for excellent technical assistance.

Chromosomal localization of SLC12A5/Slc12a5, the human and mouse genes for the neuron-specific K+-Cl– cotransporter (KCC2) defines a new region of conserved homology

K+-Cl– cotransporters (KCCs) constitute a branch of the cation-chloride cotransporter (CCC) family. To date, four KCC isoforms (KCC1-KCC4) have been identified and they all mediate obligatorily coupled, electroneutral transmembrane movement of K+ and Cl– ions. KCC2 (gene symbol SLC12A5) is expressed exclusively in neurons within the central nervous system and abnormalities in its expression have been proposed to play a role in pathological conditions such as epilepsy and neuronal trauma. Here we have determined chromosome location of both the human and the mouse genes encoding KCC2, which may assist in future efforts to determine the contribution of KCC2 to inherited human disorders. We assigned human SLC12A5 to 20q12→q13.1 and its murine homolog, Slc12a5, to 5G2–G3 by fluorescence in situ hybridization (FISH). These mapping data are contradictory to the previously reported human-mouse conserved synteny relationships disrupting an exceptionally well-conserved homology segment between human Chr 20 and mouse Chr 2. We hence suggest the first region of conserved homology between human Chr 20 and mouse Chr 5.

The characterization and sequence analysis of thirty CTG-repeat containing genomic cosmid clones.

We have systematically isolated and characterized DNA containing large CTG (n>7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.

Human cationic amino acid transporter gene hCAT-2 is assigned to 8p22 but is not the causative gene in lysinuric protein intolerance.

Abstract Lysinuric protein intolerance (LPI) is a recessively inherited amino acid disorder characterized by defective efflux of cationic amino acids at the basolateral membrane of the intestinal and renal tubular epithelium. Recently, cDNAs encoding the related proteins hCAT-2A and hCAT-2B have been cloned. These two carrier proteins are most likely the product of the same gene, hCAT-2. Using the hCAT-2B cDNA, we assigned the hCAT-2 gene to chromosome 8p22. Furthermore, by linkage analysis in Finnish LPI families, we ruled out that hCAT-2B is involved in LPI disease.