Partial characterization and assignment of the gene for protoporphyrinogen oxidase and variegate porphyria to human chromosome 1q23

Protoporphyrinogen oxidase (PPO) catalyses the conversion ofprotoporphyrinogen IX to protoporphyrin IX, Variegate porphyria(VP), a low-penetrant, autosomal dominant disorder characterizedclinically by skin lesions and neurovisceral attacks, is causedby partial deficiency of this enzyme. Linkage between VP andthe alpha-1-antitrypsin gene on chromosome 14 has been reportedin VP families from South Africa, where the condition occursat high frequency due to a founder effect. We have cloned a4.5 kb genomic DNA fragment containing the entire coding sequencefor human PPO. This clone has been used to localize the humanPPO gene to chromosome 1q23 by fluorescence in situ hybridizationanalysis. The VP gene was mapped by linkage analysis, usingmicrosatellite markers spanning the region 1q21–q25.1,in seven British VP families. Multipoint analysis between VP,SPTA1, APOA2 and D1S194 gave a maximum LOD score of 6.62 atAPOA2, which has been physically mapped to 1q21–q23. Evidencefor significant linkage between VP and markers in the alpha-1-antitrypsinregion of chromosome 14 was not obtained. Our results assignthe genes for PPO and VP to the same region of chromosome 1,indicate that the PPO and VP loci are likely to be the same,and provide evidence against locus heterogeneity in VP.     

A second autosomal split hand/split foot locus maps to chromosome 10q24-q25

Ectrodactyly (split hand/split foot malformation, SHSF) is ahuman limb malformation characterized by absent central digitalrays, deep median cleft, and syndactyly of remaining digits.The disorder is genetically heterogeneous, with at least twoloci thus far determined: an autosomal locus at 7q21 designatedSHFM1 and an X-linked locus at Xq26 designated SHFM2. Cytogeneticanalysis of sporadic SHSF patients and linkage studies in extendedpedigrees both suggest more than one autosomal locus exists.We report a novel SHSF locus suggested by a stillborn infantwith ectrodactyly and other malformations who inherited an unbalancedtranslocation resulting in monosomy 4p15.1–4pter and trisomyfor 10q25.2-qter. To investigate 10q25 as a possible split hand/splitfoot locus, microsatellite markers spanning 52 cM of 10q wereutilized for linkage analysis of a large autosomal dominantSHSF pedigree in which the region encompassing SHFM1 previouslywas excluded as containing the causative mutation. The markerD10S583 was fully informative in the family, giving a maximumLOD score of 4.21 at recombination     

The tuberous sclerosis gene on chromosome 9q34 acts as a growth suppressor

We have previously demonstrated allele loss in hamartomas frompatients with tuberous sclerosis for markers spanning the tuberoussclerosis gene on chromosome 16p13.3 (TSC2). Germline deletionsin the TSC2 gene have been shown in 5% of patients with tuberoussclerosis (TSC). These data support our hypothesis that theTSC2 gene acts as a growth suppressor gene, analogous to thetraditional tumour suppressor gene. We now report a TSC hamartomashowing allele loss for markers on chromosome 9q34 in the regionof the TSC1 gene. We studied six hamartomas from four sporadicand two familial cases of TSC, none of which showed allele lossfor markers on chromosome 16p13.3. The hamartomas were paraffinembedded sections of three renal angiomyolipomas, two giantcell astrocytomas, and a cardiac rhabdomyoma. Eight markerswere analysed, comprising from centromeric to telomeric ASS– D9S64 – D9S149 – ABO – D9S150 –DBH – D9S66 – D9S67. One angiomyolipoma showed alleleloss for the markers ABO, DBH and D9S66, but not for D9S149or D9S67. The patient was not informative for D9S150. The familystructure did not permit the phase of the disease and markeralleles to be determined. These finding support the hypothesisthat the TSC1 gene on 9q34, like the TSC2 gene, acts as a growthsuppressor. The data would place the TSC1 gene between D9S149and D9S67. Mapping of allele loss in hamartomas may help inthe refinement of the location of the TSC1 locus.     

Procarbazine is a potent mutagen at the heterozygous thymidine kinase (tk+/-) locus of mouse lymphoma assay

Procarbazine (Natulan®) is a potent inducer of gene mutationsat the heterozygous tk^+/– locus in L5178Y mouse lymphomacells in the presence of Aroclor-induced rat liver s9 metabolicactivation ({small tilde} 10^–3 mutant frequency at 10µg/ml) while exerting a far weaker effect in the absenceof s9. This mutagenicity is fairly robust with respect to thequantitative composition of the s9 mix and to variations inmouse lymphoma assay protocols (soft agar cloning versus ‘microwell’assays). The high proportion of small colony tk^–/–mutants induced by procarbazine together with the far weakermuta-genic response at the hemizygous hgprt locus in these samecells is interpreted in terms of a chromosomal or mufti-genemutational mechanism. Although procarbazine is clastogenic invivo, it does not appear to be so under standard protocols usingcultured human lymphocytes (±s9). It is not yet clearwhy this should be so, especially in light of its apparent clasto-genicityin mouse lymphoma cells.     

Refined localization and yeast artificial chromosome (YAC) contig--mapping of genes and DNA segments in the 7q21-q32 region

The chromosome localizations for 159 gene and DNA segments havebeen refined to one of five intervals in the 7q21–q32region through hybridization analysis with a panel of somaticcell hybrid lines. Seventy-two of these chromosome 7 markersare also mapped on common or overlapping yeast artificial chromosome(YAC) clones. In addition, the breakpoints of chromosome rearrangmentcontained in five of the somatic cell hybrid lines have beendefined by flanking probes within YAC contigs. To provide aframework for further mapping of the 7q21–q32 region,we have established the physical order of a set of referencemarkers: cen-(COL1A2-D7S15-CYP3A4-PON)-D7S456-(breakpoint containedin cell hybrid 1EF2/3/K017)-GUSB-D7S186-ASL-(PGY1-PGY3-GNB2-EPO-ACHE)-D7S238-(proximalbreakpoint in GM1059-Rag5)-D7S240-(CUTL1-PLANHI)-(breakpointsin 1CF2/5/K016 and 2068Rag22–2)-(PRKAR2B-D7S13)-LAMB1-(breakpointin JSR-17S)-DLD-D7S16-MET-WNT2-CFTR-D7S8-tel.     

A third locus for hereditary haemorrhagic telangiectasia maps to chromosome 12q

Hereditary haemorrhagic telangiectasia (HHT) or Rendu-Osler-Weberdisease is an autosomal dominant vascular disorder which associatesepistaxis, mucocutaneous and visceral telangiectases, and recurrenthaemorrhage with chronic anaemia and visceral shuntings. Recently,the tumour growth factor (TGF)ß binding protein endoglinlocalized to 9q33–34 was identified as responsible forHHT in several large kindreds with pulmonary arteriovenous malformations(PAVMs). Additional linkage studies demonstrated that HHT isa genetically heterogeneous disorder with families unlinkedto this region of 9q. In the families in which HHT was not linkedto chromosome 9, less PAVMs were present. Furthermore, in oneof these families, HHT was found linked to 3p22, where the TGFßII receptor is located. In this linkage study, we have analysed DNA from two families,In which HHT was unlinked to chromosome 9q and 3p, and PAVMswere absent, with a series of genetic markers on the centromericregion of chromosome 12. Using two-point linkage analysis, asignificant lod score of 2^zmx = 7.86 at     

Evidence of a locus for orofacial clefting on human chromosome 6p24 and STS content map of the region

Orofacial clefting is genetically complex, no single gene beingresponsible for all forms. It can, however, result from a singlegene defect either as part of a syndrome (e.g. van der Woudesyndrome, Treacher—Colllns syndrome, velo–cardio–facialsyndrome) or as an Isolated phenotypic effect (e.g. X–linkedcleft palate; non–syndromlc, autosomal dominant orofacialclefting). Several studies have suggested that chromosome 6pis a candidate region for a locus involved in orofacial clefting.We have used YAC clones from contigs in 6p25–p23 to investigatethree unrelated cases of cleft lip and palate coincident withchromosome 6p abnormalities. Case 1 has bilateral cleft lipand palate and a balanced translocation reported as 46, XY,t(6, 7)(p23; q36.1). Case 2 has multiple abnormalities Includingcleft lip and palate and was reported as 46, XX, del(6)(p23;pter). Case 3 has bilateral cleft lip and palate and carriesa balanced translocation reported as 46, XX, t(6; 9)(p23;q22.3).We have Identified two YAC clones, both of which cross the breakpointin cases 1 and 3 and are deleted in case 2. These clones mapto 6p24.3 and therefore suggest the presence of a locus fororofacial clefting in this region. The HGP22 and AP2 genes,potentially involved in face formation, have been found to flankthis region, while F13A maps further telomeric in 6p24.3/25.     

A combined genetic and radiation hybrid map surrounding the Treacher Collins syndrome locus on chromosome 5q

The distal region of chromosome 5q contains a large number ofgenes, including those implicated in a variety of Mendeliandisorders. One of these, Treacher Collins syndrome (TCOF1),is an autosomal dominant disorder of craniofacial developmentthe features of which include conductive hearing loss and cleftpalate. Previous studies have localized the TCOF1 locus betweenD5S519 (proximal) and SPARC (distal). To more accurately definethe genetic distance between these markers, and to extend ahigh resolution genetic map of 5q31 – 33 to include additionalhighly informative markers, 15 loci (including polymorphismsfor 4 known genes) were mapped through the Centre d'Etude duPolymorphisme Humain reference pedigrees. The resulting geneticmap encompasses 29 cM on the sex-averaged map. To help integratethis linkage map with a physical map of the region, 13 locifrom 5q31 – 33, including 6 genes, were used to constructa radiation hybrid map. As eight of the loci are common to bothmaps this has allowed us to combine the maps. The most likelylocation for the TCOF1 locus within this marker framework isin the D5S519–SPARC interval; a region estimated to beapproximately 880 kb.     

Linkage of a new locus for autosomal dominant familial spastic paraplegia to chromosome 2p

Autosomal dominant familial spastic paraplegia (ADFSP) is agenetically heterogeneous neurodegenerative disorder characterizedby a spasticity of the lower limbs. A locus causing AD-FSP (FSP1)has been previously mapped to chromosome 14q. We now reportlinkage of a second AD-FSP locus (FSP2) to chromosome 2p21 –p24 In five of seven French families and one large Dutch pedigree.The analysis of recombination events and multipoint linkageplace FSP2 within a 4 cM interval flanked by loci D2S400 andD2S367.     

Identification and chromosomal assignment of two heterozygous mutations in the Trp53 gene in L5178Y\Tk +\--3.7.2C mouse lymphoma cells

The thymidine kinase locus (Tk1) in Tk ^+\–-3.7.2C mouselymphoma cells is widely used to identify mutagenic agents.Because Trp53 (the mouse homolog of human TP53) is located withTk1 on chromosome 11 and is critical in regulating cellularresponses following exposure to DNA damaging agents, we wantedto determine if these mouse lymphoma cells harbor mutationsin Trp53. Single-stranded conformation polymorphism (SSCP) analysisof PCRamplified exons 4–9 of Trp53 indicated mutationsin both exons 4 and 5. We sequenced exons 4–9 from isolatedclones of Tk^+\–-3.7.2C cells and a Tk^–\– mutant(G4). Mutant G4 has two copies of the chromosome arrying theTk1^– allele and no copy of the chromosome carrying theTk1⁺ allele and thus could establish linkage of the individualTrp53 and Tk1 alleles. DNA sequence analysis revealed no mutationsin exons 6–9 in any Tk^+\–-3.7.2C or G4 clones. Assuggested by SSCP, there was a nonsense mutation in exon 4 atbp 301 (codon 101) in one Trp53 allele. Tk^+\–-3.7.2C cloneshave both mutant and wild-type sequences at bp 301; G4 cloneshave wild-type exon 4 sequence. These data allow assignmentof the Trp53 exon 4 mutated allele to chromosome 11 carryingthe Tk1+ allele. The exon 4 mutation leads to a stop codon earlyin translation, thus functionally deleting the Trp53 alleleon the ^+\–bearing chromosome. As previously reported,we find a missense mutation in exon 5 at bp 517 (codon 173)in one Trp53 allele. Using the G4 clones we determined thatthe exon 5 mutation is linked to the Tk1^– allele. Thusthe Tk^+\–-3.7.2C mouse lymphoma cells have two mutantTrp53 alleles, likely accounting for their rapid cell growthand contributing to their ability to detect the major typesof mutational damage associated with the etiology of tumor development.This ability to integrate across the mutational events seenin the multiple stages of tumor development further supportsthe use of the assay in chemical and drug safety studies andits recommendation as part of the required screening batteryfor regulatory agency submissions. ⁵To whom correspondence should be addressed at: MD–68, Genetic and Cellular Toxicology Branch, Environmental Carcinogenesis Division, US EPA, Research Triangle Park, NC 27711, USA. Tel: +1 919 541 2248; Fax: +1 919 541 0694; Email: clark.scott{at}epamail.epa.gov     

Angelman syndrome associated with a maternal 15q11-13 deletion of less than 200 kb

Angelman syndrome (AS) is a neurogenetic disorder arising froma lack of genetic contribution from the maternal chromosome15q11–13. To date, the AS critical region has been definedby an inherited deletion of approximately 1.5Mb, spanning the3–21 (D15S10), LS6–1 (D15S113) and GABRB3 loci.We have Identified an individual with the typical features ofAS who has a deletion of the maternal chromosome which encompassesLS6–1, but does not extend to either flanking marker.This deletion, initially detected by (CA)n repeat analysis,was further characterised by fluorescence In situ hybridisation(FISH) using cosmids derived from a 260 kb LS6–1 yeastartificial chromosome (YAC). Neither end cosmid from this YACclone falls within the deletion, suggesting that the minimalAS region Is less than 200 kb. We also studied three loci within15q11–13 which detect parent-of-origin specific DNA methylationimprints, and found that both normal maternal and paternal patternswere present in this patient.     

Assignment of a second locus for multiple exostoses to the pericentromeric region of chromosome 11

Hereditary multiple exostoses (EXT) is an autosomal dominantdisorder of enchondral bone formation characterized by multiplebony outgrowths (exostoses), with progression to osteosarcomain a minority of cases. The exclusive involvement of skeletalabnormalities distinguishes EXT from the clinically more complexLanger – Giedion syndrome (LGS), which is associated withdeletions at chromosome 8q24. Previously, linkage analysis hasrevealed a locus for EXT in the LGS region on chromosome 8q24.However, locus heterogeneity was apparent with 30% of the familiesbeing unlinked to 8q24. We report on two large pedigrees segregatingEXT in which linkage to the LGS region was excluded. To localizethe EXT gene(s) in these families we performed a genome searchincluding 254 microsatellite markers dispersed over all autosomesand the X chromosome. In both families evidence was obtainedfor linkage to markers from the proximal short and long armsof chromosome 11. Two-point analysis gave the highest lod scorefor D11S554 (Z_max = 7.148 at theta = 0.03). Multipoint analysisindicated a map position for the EXT gene between D11S905 andD11S916, with a peak multipoint lod score of 8. 10 at 6 cM fromD11S935. The assignment of a second locus for EXT to the pericentromericregion of chromosome 11 implicates an area that is particularlyrich in genes responsible for developmental abnormalities andneoplasia.     

Modification of 15q11 -- q13 DNA methylation imprints in unique Angelman and Prader -- Willi patients

The clearest example of genomic Imprinting in humans comes fromstudies of the Angelman (AS) and Prader—Wil (PWS) syndromes.Although these are clinically distinct disorders, both typicallyresult from a loss of the same chromosomal region, 15q11 - q13.AS usually results from either a maternal deletion of this region,or paternal uniparental disomy (UPD; both chromosomes 15 Inheritedfrom the father). PWS results from paternal deletion of 15q11- q13 or maternal UPD of chromosome 15. We have recently describeda parent-specific DNA methylation imprint in a gene at the D15S9locus (new gene symbol, ZNF 127), within the 15q11 - q13 region,that identifies AS and PWS patients with either a deletion orUPD. Here we describe an AS sibship and three PWS patients inwhich chromosome 15 rearrangements alter the methylation stateat ZNF127, even though this locus is not directly involved inthe rearrangement. Parent-specific DNA methylation imprintsare also altered at ZNF127 and D15S63 (another locus with aparent-specific methylation imprint) in an AS sibship whichhave no detectable deletion or UPD of chromosome 15. These uniquepatients may provide insight into the imprinting process thatoccurs in proximal chromosome 15 in humans.     

The gene for hereditary breast-ovarian cancer, BRCA1, maps distal to EDH17B2 in chromosome region 17q12-q21

A gene for hereditary breast and ovarian cancer, BRCA1, hasbeen mapped to chromosome 17q12–q21. This gene is responsiblefor cancer susceptibility in the majority of families with multiplecases of ovarian cancer and early-onset breast cancer. We reportlinkage results of a family with 10 cases of breast cancer anda single case of ovarian cancer. A recombinant event in thisfamily places BRCA1 distal (telomeric) to the locus EDH17B2,which codes for the enzyme estradiol 17ß-dehydrogenaseII. This recombinant is based on the appearance of breast cancerin a 45 year old woman. Under our genetic model, we estimatethe probability that this woman carries a BRCA1 mutation tobe 94%. These data further reduce the region of assignment ofBRCA1 on chromosome 17q12–q21 and should expedite positionalcloning of this important gene.     

Further characterization of the autism susceptibility locus AUTS1 on chromosome 7q

Autism is a neurodevelopmental disorder that usually ariseson the basis of a complex genetic predisposition. The most significantsusceptibility region in the first whole genome screen of multiplexfamilies was on chromosome 7q, although this linkage was evidentonly in UK IMGSAC families. Subsequently all other genome screensof non-UK families have found some evidence of increased allelesharing in an overlapping 40 cM region of 7q. To further characterizethis susceptibility locus, linkage analysis has now been completedon 170 multiplex IMGSAC families. Using a 5 cM marker grid,analysis of 125 sib pairs meeting stringent inclusion criteriaresulted in a multipoint maximum LOD score (MLS) of 2.15 atD7S477, whereas analysis of all 153 sib pairs generated an MLSof 3.37. The 71 non-UK sib pairs now contribute to this linkage.Linkage disequilibrium mapping identified two regions of association—onelying under the peak of linkage, the other some 27 cM distal.These results are supported in part by findings in independentGerman and American singleton families. ⁺ http://www.well.ox.ac.uk/~maestrin/iat.html MRC Child PsychiatryUnit and Centre for Social, Genetic and Developmental Psychiatry,Institute of Psychiatry, London, UK: Sarah Palferman, Nicola Matthews,Martha Turner, Janette Moore, Amaia Hervas, Anne Aubin, SimonWallace, Janine Michelotti, Catherine Wainhouse, Alina Paul,Elaine Thompson, Marianne Murin, Ramyani Gupta, ClaireGarner, Andrew Pickles, Michael Rutter and Anthony Bailey     

The oculopharyngeal muscular dystrophy locus maps to the region of the cardiac {alpha} and {beta} myosin heavy chain genes on chromosome 14q11.2-q13

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomaldominant muscular dystrophy which presents typically after theage of 50 with progressive eyelid drooping and an increasingdifficulty in swallowing. Though OPMD has a world-wide incidence,it is more common in the French Canadian population. We haveidentified a homogeneous group of families and studied 166 polymorphicmarkers as part of a genome search before establishing linkageto chromosome 14. We determined that the OPMD locus maps toa less than 5 cM region of chromosome 14q11.2–q13. Themaximum two—point lod score in three French Canadian familiesof 14.73 (     

The organization and conservation of the human Surfeit gene cluster and its localization telomeric to the c-abl and can proto-oncogenes at chromosome band 9q34.1

The mouse Surfeit locus contains an unusually tight clusterof six housekeeping genes (Surf-1 to -6) which are unrelatedby sequence homology. Using a mouse Surfeit locus probe, a 16kb clone has been isolated which contains the human Surf-1 andSurf-3 genes and regions of the human Surf-2 and Surf-5 genes.The organization and juxtaposition of these human Surfeit locusgenes are the same as found in the mouse. Using the human cloneas a biotinylated probe for fluorescence in situ hybridization(FISH) we have confirmed the location of the human Surfeit locusto chromosome band 9q34. Metaphase spreads of human chronicmyeloid leukemic cells containing the t(9;22)(q34;q11) translocationinvolving the c-abl gene at 9q34.1 and acute nonlymphocyticleukemic cells containing the t(6;9)(q34;p23) translocationinvolving the can gene at 9q34.1 were analyzed by FISH usingthe human Surfeit clone as a probe. These analyses locate thehuman Surfeit locus telomeric to the c-abl and can genes atchromosome band 9q34.1.     

Localization to chromosome 7q36.1 of the human XRCC2 gene, determining sensitivity to DNA-damaging agents

The Identification of genes controlling cellular response toDNA damage is of considerable importance, and cell lines shsowinghypersensitivity to DNA–damaging agents can be used asvehicles to map and clone these genes. In this study the hamstercell line irs1, showing hypersensitivity to a number of differentDNA–damaging agents, was fused to normal human cells tocomplement the defect. The resultant hybrids were analysed byAlu–PCR, chromosome painting, and with DNA markers tomap the complementing gene (named XRCC2) to a specific chromosomalregion. These hybrids showed correction of sensitivity to bothX-rays and to mitomycin–C, and contained human chromosome7, often as their only human component. Hybrids showing unstableretention of human chromosomes were sub-cloned to show thatloss of chromosome 7 and loss of resistance to mitomycin–Coccurred concordantly. Two separate hybrids were found to havea smaller piece of chromosome 7, and specific DNA probes andmicrosatellite markers defined this as a contiguous region at7q35–p36. Hybrid irradiation–fusion methods wereused to further reduce the size of the complementing genomicregion and to localize the gene to an approximately 3–5Mb region at 7q36.1.     

DFNB79: reincarnation of a nonsyndromic deafness locus on chromosome 9q34.3

Genetic analysis of an inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at θ=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23–q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.     

A novel locus for autosomal dominant nonsyndromic hearing loss identified at 5q31.1-32 in a Chinese pedigree

 Hearing impairment is an extremely heterogeneous disorder. A total of 35 loci and 17 related genes for autosomal dominant nonsyndromic hearing loss have been identified. In a Chinese pedigree characterized by autosomal dominant inheritance with bilateral, postlingual, progressive, and sensorineural nonsyndromic hearing impairment, the putative disease gene locus was localized to chromosome 5q31.1-32 by a genome-wide scan. Fine mapping indicated that the disease gene was located within an 8.8-cM region between markers D5S2056 and D5S638, with a maximum two-point logarithm of differences (LOD) score of 6.89 (θ = 0) at D5S2017. By the candidate gene approach, mutation screening of the DIAPH1 and POU4F3 genes at 5q31 was performed. No mutation was found, suggesting that this is a novel deafness locus, which has been named DFNA42. Received: May 8, 2002 / Accepted: October 1, 2002