Structure and Chromosomal Localization of the GPI-Anchor Synthesis GenePIGFand Its Pseudogene ψPIGF

Posttranslational modification by the GPI glycolipid anchor is essential for the surface expression of many membrane proteins. Defect of GPI biosynthesis due to somatic mutation in the hematopoietic stem cell is the basis for an acquired genetic disease, paroxysmal nocturnal hemoglobinuria (PNH). Previously, an X-linked genePIGA(phosphatidylinositol glycan class A), which participates in the first step of the biosynthesis, was shown to be mutated in abnormal cells from all 60 patients with PNH. The cDNA of another GPI synthesis genePIGFwas previously cloned, but it is not involved in pathogenesis of PNH. In the present study, we have analyzedPIGFgenomic clones. ThePIGFgene contained six exons spanning about 40 kb and was located to the short arm of chromosome 2 at 2p16–p21. The frequency of mutations on both alleles ofPIGFshould be much lower than that of mutation in the X-linkedPIGA,accounting for a lack of involvement ofPIGFin PNH. We also identified the processed pseudogene ofPIGF(ψPIGF) and mapped it to 5q35.     

Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

The Mouse Glutathione PeroxidaseGpx2Gene Maps to Chromosome 12; Its PseudogeneGpx2-psMaps to Chromosome 7

TheGPX2gene codes for GSHPx-GI, a glutathione peroxidase whose mRNA is readily detectable in the gastrointestinal tract. AlthoughGPX2is a single gene in humans, there are two genes in the mouse genome with homology toGPX2.By analyzing a panel of mouse interspecies DNA from the Jackson Laboratory's backcross resource, we have chromosomally mapped these two genes. One was mapped to the central region of mouse chromosome 12 betweenD12Mit4andD12Mit5,nearfosandTgfb3.This region is homologous to human 14q24.1, where humanGPX2has been mapped, and most likely represents the functional mouseGpx2gene. The otherGpx2-like gene was mapped to mouse chromosome 7 betweenPcsk3andHbb.We have isolated the latter gene from a P1 phage library. Its pseudogene nature is revealed by the sequence analysis: (a) it is intronless; (b) it has a single nucleotide deletion in the coding region; and (c) it has a poly(A) tail at its 3′-untranslated region.     

The phenotype of a germline mutation in PIGA: the gene somatically mutated in paroxysmal nocturnal hemoglobinuria

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412^∗. This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412^∗ PIGA has residual function. Transfection of a mutant p.Arg412^∗ PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412^∗) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in PIGA results in the lethal X-linked phenotype recognized in the reported family.     

Chromosomal Locations of Three Human Nuclear Genes (RPSM12, TUFM, and AFG3L1) Specifying Putative Components of the Mitochondrial Gene Expression Apparatus

We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination ofin situhybridization and interspecies hybrid mapping. The genesRPMS12(mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center),TUFM(mitochondrial elongation factor EF-Tu), andAFG3L1(similar to the yeast genesAfg3andRca1involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing.RPMS12maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. TheTUFMgene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17.AFG3L1is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy.     

The human phosphoglycerate kinase multigene family. HLA-associated sequences and an X-linked locus containing a processed pseudogene and its functional counterpart

Several phosphoglycerate kinase genes were previously detected in the human genome by blot hybridization with a phosphoglycerate kinase cDNA probe. Using subcloned fragments of the cDNA we estimate the presence of four independent phosphoglycerate kinase genes. These genes have been mapped to both the human X chromosome (band q13) and chromosome 6 (p12-21.1) using a panel of human-rodent somatic cell hybrids and by chromosomal in situ hybridization. The genomic distribution of phosphoglycerate kinase sequences is conserved in man and mouse, not only for the X chromosome, but also for linkage to the respective major histocompatibility complexes. Molecular cloning of X-linked phosphoglycerate kinase sequences led to the identification of a novel intronless phosphoglycerate kinase pseudogene which is localized proximal to the active gene on the X chromosome.     

Mapping of Aldose Reductase Gene Sequences to Human Chromosomes 1, 3, 7, 9, 11, and 13

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, we mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q42, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other active genes, non-aldose reductase homologous sequences, or pseudogenes.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

YAC Contigs of theRab1andwobbler(wr) Spinal Muscular Atrophy Gene Region on Proximal Mouse Chromosome 11 and of the Homologous Region on Human Chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized thewobblerspinal atrophy genewrto proximal mouse Chr 11, tightly linked toRab1,a gene coding for a small GTP-binding protein, andGlns-ps1,an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of theRab1region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescencein situhybridization (FISH), and sequence-tagged site (STS) isolation and mapping.Rab1andGlns-ps1were found to be only 200 kb apart. A potential CpG island near a methylatedNarI site and a trapped exon,ETG1.1,were found between these loci, and a new STS,AHY1.1,was found over 250 kb fromRab1.Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising theRAB1locus,AHY1.1,and the human homologue ofETG1.1,indicating a high degree of conservation of this region in the two species. We mappedAHY1.1and thus humanRAB1on Chr 2p13.4–p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the geneLMGMD2Bfor a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13–p16. The conservation between the mouseRab1and humanRAB1regions will be helpful in identifying candidate genes for thewobblerspinal muscular atrophy and in clarifying a possible relationship betweenwrandLMGMD2B.     

An expressed β-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13

The chromosomal assignments of an expressed β-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human X Chinese hamster somatic cell hybrids cleaved with Hind III or EcoR I. Probes containing the 3′ untranslated regions of the expressed gene M40 and of pseudogene 21β were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21 → 6pter, the 21β pseudogene (TUBBP1) to chromosome 8 region 8q21 → 8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families.     

Localization of Human Flavin-Containing Monooxygenase GenesFMO2andFMO5to Chromosome 1q

The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1toFMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3,andFMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human–rodent somatic cell hybrids demonstrates that the two remaining identified members of theFMOgene family,FMO2andFMO5,also are located on chromosome 1q.     

Chromosomal Mapping ofTmp(Emp1),Xmp(Emp2), andYmp(Emp3), Genes Encoding Membrane Proteins Related toPmp22

We have recently characterized a novel mammalian gene family, encoding membrane glycoproteins with four trans-membrane domains. This gene family includes the previously studiedPMP22,which is involved in the Charcot–Marie–Tooth neuropathy, and three novel genes:TMP, XMP,andYMP(HGMW-approved symbolsEMP1, EMP2andEMP3,respectively). TheTmp(tumor-associated membrane protein) gene was isolated from a c-mycinduced mouse brain tumor and is expressed in several highly proliferative cell types. We have now isolated cDNAs of the mouseXmpandYmpgenes and determined the chromosomal localization of mouseTmp, Xmp,andYmp. Tmpwas mapped to mouse chromosome 6,Xmpwas mapped to chromosome 16, andYmpwas mapped to chromosome 7.TmpandYmpmap to paralogous chromosomal regions, whereasXmpmaps to a chromosomal region that is putatively paralogous to a region on chromosome 11, to whichPmp22was previously mapped. These data suggest that this family of membrane glycoproteins evolved as a result of chromosomal duplications.     

The CCAAT/enhancer binding protein (C/EBP alpha) gene (CEBPA) maps to human chromosome 19q13.1 and the related nuclear factor NF-IL6 (C/EBP beta) gene (CEBPB) maps to human chromosome 20q13.1.

The CEBPA gene encoding CCAAT/enhancer binding protein (C/EBP alpha) has been mapped to human chromosome 19 and the CEBPB (formerly TCF5) gene encoding NF-IL6 (C/EBP beta) to human chromosome 20 by Southern blot analysis of Chinese hamster x human and mouse x human somatic cell hybrids. CEBPA has been further mapped to 19q13.1 between the loci GPI and TGFB using human x hamster somatic cell hybrids containing restricted fragments of human chromosome 19. This position was confirmed by fluorescence in situ hybridization. Furthermore, CEBPB has been mapped to 20q13.1 by fluorescence in situ hybridization.     

The Evolutionarily Conserved CKAP2 Gene Is Located in Human Region 13q14.3 Often Rearranged in Various Tumors

The human CKAP2 gene, which is involved in diffuse large B-cell lymphomas, was localized by screening the GeneBridge 4 somatic cell radiation hybrid panel by means of the polymerase chain reaction (PCR). The CKAP2 gene was mapped between the WI-15460 and WI-3673 markers at the boundary between regions 13q14.3 and 13q21.1, at the distance of 14.39 cR (with 4.8 cR per cM) from the WI-5867 framework marker (lod score > 2.26). The human CKAP2 gene displayed high homology to mouse and rat expressed orthologs. A CKAP2-like sequence was found in human chromosome 14 and assumed to be a pseudogene resulting from duplication and subsequent mutations of the CKAP2 gene on chromosome 13. A possible role of the CKAP2 gene in oncogenesis associated with deletions and rearrangements of region 13q14.3–21.1 is discussed.     

Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.