The Structure and Organization of the Human NPAT Gene

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, andATM,a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene,NPAT,is located upstream ofATMon human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5′ flanking sequence. The structure and organization ofNPATwere determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22–q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22–q24. For investigation of the role ofNPATin AT and these tumors with allelic loss of 11q22–q24, appropriate primer sequences and PCR conditions for amplification of all theNPATexons from genomic DNA were determined. We previously reported that no recombinations are found amongAtm, Npat,andAcat1(acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis usingNPAT- andACAT-specific primers and human genomic DNA allowed us to mapACAT12 kb centromeric toNPAT.     

The murine homolog of the human breast and ovarian cancer susceptibility geneBrca1 maps to mouse chromosome 11D

The recently cloned human breast and ovarian cancer suseptibility gene,BRCA1, is located on human chromosome 17q21. We have isolated murine genomic clones containingBrca1 as a first step in generating a mouse model for the loss ofBRCA1 function. A mouse genomic library was screened using probes corresponding to exon 11 of the humanBRCA1 gene. Two overlapping mouse clones were identified that hybridized to humanBRCA1 exons 9–12. Sequence analysis of 1.4 kb of the region of these clones corresponding to part of human exon 11 revealed 72% nucleic acid identity but only 50% amino acid identity with the human gene. The longest of the mouseBrca1 genomic clones maps to chromosome 11D, as determined by two-color fluorescence in situ hybridization. The synteny to human chromosome 17 was confirmed by cohybridization with the mouse probe for the NF1-gene. This comparative study confirms that the relative location of theBRCA1 gene has been conserved between mice and humans.     

The Human Obesity Gene Map: The 1998 Update

PÉRUSSE, LOUIS, YVON C. CHAGNON, JOHN WEISNAGEL, AND CLAUDE BOUCHARD. The human obesity gene map: the 1998 update. Obes Res. 1999;7:111–129. An update of the human obesity gene map incorporating published results up to the end of October 1998 is presented. Evidence from the human obesity cases caused by single gene mutations; other Mendelian disorders exhibiting obesity as a clinical feature; quantitative trait loci uncovered in human genome-wide scans and in crossbreeding experiments with mouse, rat, and pig models; association and case-control studies with candidate genes; and linkage studies with genes and other markers is reviewed. The most noticeable changes from the 1997 update is the number of obesity cases due to single gene mutations that increased from three cases due to mutations in two genes to 25 cases due to 12 mutations in seven genes. A look at the obesity gene map depicted in Figure 1 reveals that putative loci affecting obesity-related phenotypes are found on all but chromosome Y of the human chromosomes. Some chromosomes show at least three putative loci related to obesity on both arms (1, 2, 3, 6, 7, 8, 9, 11, 17, 19, 20, and X) and several on one chromosome arm only (4q, 5q, 10q, 12q, 13q, 15q, 16p, and 22q). The number of genes and other markers that have been associated or linked with human obesity phenotypes is increasing very rapidly and now approaches 27.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

High-Resolution Genetic Map of the Human Glutamyl Aminopeptidase Gene (ENPEP)

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (EAP), the gene symbol for which isENPEP.Using genomic DNA encoding for human EAP as a probe, we identified theENPEPgene location on human chromosome 4q25 by polymerase chain reaction analysis of a human/rodent somatic cell hybrid mapping panel and by fluorescencein situhybridization. Using a radiation hybrid panel, the gene order aroundENPEPwas determined to be centromere–D4S1236–(570 kb)–ENPEP–(210 kb)–D4S262–(270 kb)–D4S953–(270 kb)–D4S474–(570 kb)–IF. The linkage ofENPEPto complement factor I (IF) confirms the human chromosome band 4q25 localization predicted from the chromosomal location of murineENPEP.HumanENPEPthus provides an additional marker for the long arm of chromosome 4 that should facilitate studies of this genomic region.     

Genomic structure and expression analysis of the spastic paraplegia gene, SPG7

SPG7 is a newly identified gene involved in an autosomal recessive form of hereditary spastic paraplegia (HSP), a genetically heterogeneous group of neurodegenerative disorders. This gene encodes a protein characterized as a nuclear-encoded mitochondrial metalloprotease. The present report describes the genomic structure of the SPG7 gene. It is organized into 17 exons ranging from 78 to 242 bp and spans approximately 52 kb within three overlapping cosmids. The exon/intron boundaries and all splice junctions are consistent with the published consensus sequences for donor and acceptor sites. The provided genomic structure of SPG7 should facilitate the screening for mutations in this gene in patients with HSP and other related mitochondrial disease syndromes. SPG7 has been mapped to chromosome 16q24.3, a region of frequent loss of heterozygosity (LOH) seen in sporadic breast and prostate cancer. We have performed single-strand conformation polymorphism analysis of ten exons of this gene in a number of sporadic breast cancer samples showing LOH at 16q24.3. No mutations were detected; only single nucleotide polymorphisms were observed in exon 11, intron 7, intron 10 and intron 12. An expression analysis study has revealed the differential expression of SPG7 mRNA in various tissues and at different developmental stages. Electronic Publication     

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.     

Mucopolysaccharidosis IV A: Assignment of the Human N-Acetylgalactosamine-6-Sulfate Sulfatase (GALNS) Gene to Chromosome 16q24

Plasmid clones of three independent genomic fragments of the gene for human N -acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4) were utilized in a fluorescence in situ suppression hybridization study to assign the locus to chromosome 16q24. Enzyme assay for GALNS in a patient with del(16)(q22.1) confirmed this finding.     

Cloning of cDNA and Genomic DNA Encoding Human Type XVIII Collagen and Localization of the α1(XVIII) Collagen Gene to Mouse Chromosome 10 and Human Chromosome 21

Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which we have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, we report the isolation of human cDNAs and genomic DNAs encoding the α1(XVIII) collagen chain. Utilizing a genomic clone as probe, we have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, we have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2.     

The Mouse Region Syntenic for Human Spinal Muscular Atrophy Lies within theLgn1Critical Interval and Contains Multiple Copies ofNaipExon 5

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval forLgn1,a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacteriumLegionella pneumophila.We have generated a mouse YAC contig across theLgn1/Smainterval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to theLgn1critical region, making these two genes candidates for theLgn1phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy ofNaipexon 5. Identification of theLgn1gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.     

Isolation of Two Novel WNT Genes, WNT14 and WNT15, One of Which (WNT15) Is Closely Linked to WNT3 on Human Chromosome 17q21

TheWntgene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of theWntgene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of theWntgene family that also includes theWnt9sequences found in jawless and cartilaginous fishes. AWnt14cDNA was also isolated from chicken and a partialWnt15sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.     

Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG. Received: 4 November 1997 / Accepted: 5 January 1998     

TheMASProto-oncogene Is Imprinted in Human Breast Tissue

The humanMASproto-oncogene is situated at 6q25.3–q26, a region that is homologous to mouse chromosome 17 where two parentally imprinted genes (MasandIgf2r) have previously been identified. We investigated the imprinting status ofMASin adult lesions to establish the imprinting status of this gene in humans, as certain imprinted genes are known to have altered imprinting phenotypes in cancer. Of 14 breast samples demonstrating aMASRT-PCR product, 4 were informative for a polymorphic marker. In all 4 cases, expression of theMASgene was found to be mono-allelic, indicating the presence of a functional imprint at this locus in human breast tissue.     

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.     

Breast Tumors with Elevated Expression of 1q Candidate Genes Confer Poor Clinical Outcome and Sensitivity to Ras/PI3K Inhibition

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA–drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.