Anne S. Olsen

Positionally Cloned Gene for a Novel Glomerular Protein—Nephrin—Is Mutated in Congenital Nephrotic Syndrome

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier.

A serine/threonine kinase gene defective in Peutz-Jeghers syndrome

Studies of hereditary cancer syndromes have contributed greatly to our understanding of molecular events involved in tumorigenesis. Here we investigate the molecular background of the Peutz–Jeghers syndrome, (PJS), a rare hereditary disease in which there is predisposition to benign and malignant tumours of many organ systems. A locus for this condition was recently assigned to chromosome 19p (ref. 3). We have identified truncating germline mutations in a gene residing on chromosome 19p in multiple individuals affected by PJS. This previously identified but unmapped gene, LKB1 (ref. 4), has strong homology to a cytoplasmic Xenopus serine/threonine protein kinase XEEK1 (ref. 5), and weaker similarity to many other protein kinases. Peutz–Jeghers syndrome is therefore the first cancer-susceptibility syndrome to be identified that is due to inactivating mutations in a protein kinase.

Comparison of human genetic and sequence-based physical maps

Recombination is the exchange of information between two homologous chromosomes during meiosis. The rate of recombination per nucleotide, which profoundly affects the evolution of chromosomal segments, is calculated by comparing genetic and physical maps. Human physical maps have been constructed using cytogenetics, overlapping DNA clones and radiation hybrids; but the ultimate and by far the most accurate physical map is the actual nucleotide sequence. The completion of the draft human genomic sequence provides us with the best opportunity yet to compare the genetic and physical maps. Here we describe our estimates of female, male and sex-average recombination rates for about 60% of the genome. Recombination rates varied greatly along each chromosome, from 0 to at least 9 centiMorgans per megabase (cM Mb-1). Among several sequence and marker parameters tested, only relative marker position along the metacentric chromosomes in males correlated strongly with recombination rate. We identified several chromosomal regions up to 6 Mb in length with particularly low (deserts) or high (jungles) recombination rates. Linkage disequilibrium was much more common and extended for greater distances in the deserts than in the jungles.

The DNA sequence and biology of human chromosome 19

Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

Physical maps of human α(1,3)fucosyltransferase genes FUT3–FUT6 on chromosomes 19p13.3 and 11q21

Sialyl Lewis x and related fucosylated glycans are differentially expressed in human cells and form ligands for selectin adhesion receptors. alpha(1,3)Fucosyltransferases (FUTs) that complete their biosynthesis also show tissue specificity. We have established physical maps of the FUT3-6 loci to study regulation of this gene family. FUT4 has previously been localized to chromosome 11q21; FUT3, FUT6, and now FUT5 are localized to chromosome 19p13.3. Conventional and pulsed-field gel electrophoresis mapping of total genomic DNA and large genomic clones were used to generate a fine map of both loci, defining the order, orientation, and distances between FUTs. A P1 clone with all three 19p FUT genes in tandem orientation was isolated and used to study regions flanking FUT3, -5, and -6. Our studies provide preliminary information to study regulation of human FUT genes.

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.

Structure of the human amyloid-precursor-like protein gene APLP1 at 19q13.1

Abstract Amyloid-precursor-like protein 1 (APLP1) is a membrane-associated glycoprotein, whose gene is homologous to the APP gene, which has been shown to be involved in the pathogenesis of Alzheimer’s disease. APLP1 is predominantly expressed in brain, particularly in the cerebral cortex postsynaptic density. The genomic organization of mouse APLP1 has been determined, and the human gene has been mapped to chromosomal region 19q13.1. In the present study, the entire sequence of human APLP1 has been determined from a cosmid clone, and the genomic structure has been determined. The gene is 11.8 kb long and contains 17 exons. We have previously mapped the gene for congenital nephrotic syndrome (CNF) to the APLP1 region, to the vicinity of marker D19S610 located between markers D19S191 and DS19608. APLP1 is the only known gene in the vicinity of the marker D19S610. Because of its location and the proposed interference of amyloid with basement membrane assembly, APLP1 has been considered a candidate gene for CNF. All exon regions of the gene were amplified by the polymerase chain reaction and sequenced from DNA of CNF patients. No differences were observed between CNF patients and controls, suggesting that mutations in APLP1 are not involved in the etiology of CNF.

LOCATION OF MOUSE AND HUMAN GENES CORRESPONDING TO CONSERVED CANINE OLFACTORY RECEPTOR GENE SUBFAMILIES

Olfactory receptors are G protein-coupled, seven-transmembrane-domain proteins that are responsible for binding odorants in the nasal epithelium. They are encoded by a large gene family, members of which are organized in several clusters scattered throughout the genomes of mammalian species. Here we describe the mapping of mouse sequences corresponding to four conserved olfactory receptor genes, each representing separate, recently identified canine gene subfamilies. Three of the four canine genes detected related gene clusters in regions of mouse Chromosomes (Chrs) 2, 9, and 10, near previously mapped mouse olfactory genes, while one detected a formerly unidentified gene cluster located on mouse Chr 6. In addition, we have localized two human gene clusters with homology to the canine gene, CfOLF4, within the established physical map of Chr 19p. Combined with recently published studies, these data link the four conserved olfactory gene subfamilies to homologous regions of the human, dog, and mouse genomes.

A fine physical map of the CACNA1A gene region on 19p13.1-p13.2 chromosome

The P/Q-type Ca(2+) channel alpha(1A) subunit gene (CACNA1A) was cloned on the short arm of chromosome 19 between the markers D19S221 and D19S179 and found to be responsible for Episodic Ataxia type 2, Familial Hemiplegic Migraine and Spinocerebellar Ataxia type 6. This region was physically mapped by 11 cosmid contigs spanning about 1. 4Mb, corresponding to less than 70% of the whole region. The cosmid contig used to characterize the CACNA1A gene accounted only for the coding region of the gene lacking, therefore, the promoter and possible regulation regions. The present study improves the physical map around and within the CACNA1A by giving a complete cosmid or BAC contig coverage of the D19S221-D19S179 interval. A number of new STSs, whether polymorphic or not, were characterized and physically mapped within this region. Four ESTs were also assigned to cosmids belonging to specific contigs.

Physical mapping of EMR1 and CD97 in human Chromosome 19 and assignment of Cd97 to mouse Chromosome 8 suggest an ancient genomic duplication.

EMR1 and CD97 belong to the EGF-TM7 family of nonclassicalseven-span transmembrane receptors, members of which are ex-pressed primarily in the immune system. The membrane-spanningregions of CD97, EMR1, and other EGF-TM7 proteins show sig-nificant homology to the secretin receptor superfamily. However,unlike this group of peptide hormone receptors, EMR1 and CD97have extended extracellular portions that possess several EGF do-mains at the N-terminus (McKnight and Gordon 1998). The EGFdomain region can function as a ligand-binding site, as demon-strated by the interaction of CD97 with CD55 (Hamann et al.1996). EMR1 and CD97 show a high degree of structural similar-ity despite the fact that they share only about 31% amino acidsequence identity. The similarity between these two proteins sug-gests that CD97 and EMR1 coding sequences arose through an-cient duplication of a common ancestral gene. CD97 has beenassigned to human Chromosome (Chr) 19p13.12–p13.2 by fluo-rescence in situ hybridization (FISH; Hamann et al. 1995), andEMR1 has been mapped to Chr 19p13.3 through a combination ofFISH and somatic cell hybrid analysis (Baud et al. 1995). Thesedata indicate that the two related genes are linked but separated bya significant distance in the human genome.Emr1 has been mapped to distal mouse Chr 17 within a regionrelated to human 19p13.3, tightly linked to the gene encodingtranscription factor Rfx2 (Lin et al. 1997; McKnight et al. 1997).Interestingly, RFX1, which encodes a transcription factor proteinrelated to RFX2 in both structure and immune-system function,has been mapped to 19p13.1 (Doyle et al. 1996), suggesting thathuman RFX1 and CD97 might also be close neighbors. RFX1 andRFX2 encode site-specific DNA binding proteins that serve criti-cal immune-system functions (Reith et al. 1994) and are alsothought to have arisen from a common ancestral gene sequence indistant evolutionary time. These data suggested that the tight link-age of RFX2 to EMR1, and RFX1 to CD97, respectively, mightreflect an ancient duplication encompassing the predecessors ofboth sets of immunologically active genes.To investigate this possibility, we set out to define physicallocations of CD97 and EMR1 genes in the human and to determinethe location of Cd97 in mice. We localized the mouse Cd97 geneby following the segregation of variant M. musculus and M. spre-tus alleles of the gene in an interspecific backcross (Doyle et al.1996; Stubbs et al. 1996). The results confirmed the tight linkageof Cd97 and Rfx1 in central mouse Chr 8 (Fig. 1). To define thepositions of human CD97 and EMR1, we hybridized probes rep-resenting the two genes to a Chr 19 cosmid library (Olsen et al.1994), and positive cosmids were ordered within the Chr 19 metricphysical map (Ashworth et al. 1995). The human CD97 probeidentified several overlapping cosmids located in 19p13.1 betweenthe RFX1 and NOTCH3, approximately 700 kb and 400 kb awayfrom those two genes, respectively (Fig. 2). The EMR1 probedetected two cosmid clones, 31568 and 34349; positive hybridiza-