Physical mapping of chromosome 3p25-p26 by flourescence in situ hybridisation (FISH)

As part of our effort to isolate and characterise the von Hippel-Lindau (VHL) disease gene, we constructed a physical map of chromosome 3p25-26 by fluorescence in situ hybridisation (FISH) studies on a panel of cytogenetic rearrangements involving this region. Biotinylated cosmid and lambda probes were hybridised to metaphase chromosome spreads and positioned with respect to each cytogenetic breakpoint. These studies unequivocally established the order of five loci linked to the VHL disease gene: cen-(RAF1,312)-D3S732-D3S1250-D3S601-D3S18-pter and determined the position of three other probes within this map. These results ordered RAF1 and D3S732 for the first time, confirmed the localisation of D3S1250 between RAF1 and D3S601 and determined the position of D3S651 with respect to other chromosome 3p25-p26 loci. The establishment of an ordered set of cytogenetic aberrations will enable the rapid assignment of polymorphic and nonpolymorphic cloned sequences within the chromosome region 3p25-p26.     

Localization of the von Hippel-Lindau disease gene to a small region of chromosome 3

We studied 25 families with von Hippel-Lindau disease (VHL) to locate VHL more precisely on chromosome 3. We found that VHL was linked to RAF1, confirming previous observations, and to two polymorphic DNA markers, D3S18 and D3S191. Multipoint linkage analysis indicated that the most likely location for VHL was in the interval between RAF1 and D3S18. D3S18 was located at 3p26. Genetic heterogeneity was not detected in this panel of von Hippel-Lindau disease families. The polymorphic markers RAF1, D3S18, and D3S191 should be useful in identifying asymptomatic gene carriers in VHL families and in guiding efforts at gene isolation.     

Genetic Linkage Mapping of the Dehydroepiandrosterone Sulfotransferase (STD) Gene on the Chromosome 19q13.3 Region

In the human liver and adrenal, there is a single hydroxysteroid sulfotransferase, which catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundantly circulating steroid in humans, and also catalyzes the sulfation of a series of other 3β-hydroxysteroids as well as cholesterol. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in several peripheral tissues, indicating that hydroxysteroid sulfotransferase plays a pivotal role in controlling the hormonal action of sex steroids by regulating their bioavailability. We recently elucidated the structure of the gene encoding hydroxysteroid sulfotransferase (STD), also designated dehydroepiandrosterone sulfotransferase, which spans 17 kb and contains six exons. The STD gene was preliminarily assigned to chromosome 19 by polymerase chain reaction (PCR) amplification of DNA from a panel of human/rodent somatic cell hybrids. To locate the STD gene, the novel biallelic polymorphism found in intron 2 was genotyped in eight CEPH reference families by direct sequencing of PCR products. Two-point linkage analysis was first performed between the latter polymorphism and chromosome 19 markers from Généthon and NIH/CEPH. The closest linkage was observed with D19S412 (Z_max= 9.23; θ_max0.038) and HRC (Z_max= 5.95; θ_max0.036), located on the 19q13.3 region. A framework map including six Généthon markers flanking the polymorphic STD gene was created by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the region was constructed, yielding to the following order: qter–D19S414–D19S224–D19S420–D19S217–(APOC2–D19S412)–(STD–HRC)– KLK–D19S22–D19S180–PRKCG–D19S418–tel.     

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.     

Refined physical map of the spinal muscular atrophy gene (SMA) region at 5q13 based on YAC and cosmid contiguous arrays

The gene for the autosomal recessive neurodegenerative disorder spinal muscular atrophy has been mapped to a region of 5q13 flanked proximally by CMS-1 and distally by D5S557. We present a 2-Mb yeast artificial chromosome (YAC) contig constructed from three libraries encompassing the D5S435/D5S629/CMS-1—SMA—D5S557/D5S112 interval. The D5S629/CMS-1—SMA—D5S557 interval is unusual insofar as chromosome 5-specific repetitive sequences are present and many of the simple tandem repeats (STR) are located at multiple loci that are unstable in our YAC clones. A long-range restriction map that demonstrates the SMA-containing interval to be 550 kb is presented. Moreover, a 210-kb cosmid array from both a YAC-specific and a chromosome 5-specific cosmid library encompassing the multilocus STRs CATT-1, CMS-1, D5F149, D5F150, and D5F153 has been assembled. We have recently reported strong linkage disequilibrium with Type I SMA for two of these STRs, indicating that the gene is located in close proximity to or within our cosmid clone array.     

Mapping of the HSD17B2 gene encoding type II 17β-hydroxysteroid dehydrogenase close to D16S422 on chromosome 16q24.1–q24.2

The enzymes of the 17β-hydroxysteroid dehydrogenase (17β-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17β-hydroxysteroid counterparts. The structure of human type II 17β-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of Δ⁴-androstenedione and testosterone, androstanedione and dihydrotestosterone, and estrone and 17β-estradiol, whereas type I 17β-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 bp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Généthon. The maximal pairwise lod score (Z_max = 33.3) with a maximal recombination fraction (θ_max) of 0.008 was obtained with the marker D16S422 located on 16q24.1–q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Généthon markers. The order of the HSD17B2 gene and markers is qter-D16S516 — D16S504 — D16S507 — D16S505 — D16S511 — [HSD17B2—D16S422]—D16S520—D16S413—tel.     

Autistic Disorder and Chromosome 15q11–q13: Construction and Analysis of a BAC/PAC Contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2–10/10,000 individuals. Chromosome 15q11–q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the γ-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11–q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11–q13.     

Mapping of the von Hippel-Lindau disease locus to a small region of chromosome 3p by genetic linkage analysis.

Genetic linkage studies were performed in 22 families with von Hippel-Lindau (VHL) disease by using polymorphic DNA markers from distal chromosome 3p. Linkage was detected between VHL disease and the markers D3S18 (Zmax = 6.6 at theta = 0.0, confidence interval (CI) 0.00-0.06), RAF1 (Zmax = 5.9 at theta = 0.06, CI 0.01-0.16), and THRB (Zmax 3.4 at theta = 0.11). Multipoint linkage analysis localized the VHL disease gene within a small region (approximately 8 cM) of 3p25-p26 between RAF1 and (D3S191, D3S225) and close to the D3S18 locus. There was no evidence of locus heterogeneity, and families with and without pheochromocytoma showed linkage to D3S18. The identification of DNA markers flanking the VHL disease gene allows reliable presymptomatic and prenatal diagnosis to be offered to informative families.     

The age of human mutation: genealogical and linkage disequilibrium analysis of the CLN5 mutation in the Finnish population.

Variant late infantile neuronal ceroid lipofuscinosis (vLINCL) is an autosomal recessive progressive encephalopathy of childhood enriched in the western part of Finland, with a local incidence of 1 in 1500. We recently assigned the locus for vLINCL, CLN5, to 13q21.1-q32. In the present study, the haplotype analysis of Finnish CLN5 chromosomes provides evidence that one single mutation causes vLINCL in the Finnish population. Eight microsatellite markers closely linked to the CLN5 gene on chromosome 13q were analyzed, to study identity by descent by shared haplotype analysis. One single haplotype formed by flanking markers D13S160 and D13S162 in strong linkage disequilibrium (P < .0001) was present in 81% of disease-bearing chromosomes. Allele 4 at the marker locus D13S162 was detected in 94% of disease-bearing chromosomes. To evaluate the age of the CLN5 mutation by virtue of its restricted geographical distribution, church records were used to identify the common ancestors for 18 vLINCL families diagnosed in Finland. The pedigrees of the vLINCL ancestors merged on many occasions, which also supports a single founder mutation that obviously happened 20 to 30 generations ago (i.e., approximately 500 years ago) in this isolated population. Linkage disequilibrium was detected with seven markers covering an extended genetic distance of 11 cM, which further supports the young age of the CLN5 mutation. When the results of genealogical and linkage disequilibrium studies were combined, the CLN5 gene was predicted to lie approximately 200 - 400 kb (total range 30 - 1360 kb) from the closest marker D13S162.     

A sublocus of the multicopy microsatellite marker CMS1 maps proximal to spinal muscular atrophy (SMA) as shown by recombinant analysis

The critical region containing the spinal muscular atrophy (SMA) gene is flanked by the 5q11–q13 markers, D5S435 and D5S557, as determined by linkage analysis. Here we present the results of an analysis of a Dutch SMA family with the multicopy microsatellite marker CMS1. A crossover is revealed in the critical SMA region. We conclude that at least one of the CMS1 subloci maps proximal to the SMA gene. This reduces the minimal SMA region from approximately 1.4 Mb to 600–700 kb.     

A new dinucleotide repeat polymorphism at the telomere of chromosome 21q reveals a significant difference between male and female rates of recombination.

We have used a half-YAC containing the human chromosome 21 long-arm telomere to clone, map, and characterize a new dinucleotide repeat polymorphism (D21S1575) close to 21qter. This marker is < 120 kb from the telomeric (TTAGGG)n sequences and is the most distal highly polymorphic marker on chromosome 21q. This marker has a heterozygosity of 71% because of a variable (TA)n repeat embedded within a long interspersed element (LINE) element. Genotyping of the CEPH families and linkage analysis provided a more accurate determination of the full length of the chromosome 21 genetic map. A highly significant difference was detected between male and female recombination rates in the telomeric region: in the most telomeric 2.3 Mb of chromosome 21q, recombination was only observed in male meioses.     

Genetic and physical mapping of the GLUR5 glutamate receptor gene on human chromosome 21

Glutamate receptors (GluRs) mediate excitatory neurotransmission and may have important roles in central nervous system disorders. To characterize the human GLUR5 gene, which is located on human chromosome 21q22.1, we isolated cDNAs, genomic phage lambda clones, and yeast artificial chromosomes (YACs) and developed sequence tagged sites (STSs) and simple sequence length polymorphisms (SSLPs) for GLUR5. Genetic mapping with a tetranucleotide AGAT repeat named GLUR5/AGAT (six alleles observed, 70% heterozygosity) placed GLUR5 5 cM telomeric to APP (D21S210) and 3cM centromeric to SOD1 (D21S223). The humanGLUR5 gene is located near the familial amyotrophic lateral sclerosis (FALS) locus; linkage analysis of GLUR5 SSLPs in FALS pedigrees yielded negative lod scores, consistent with the recent association of the FALS locus with the SOD1 gene. Physical mapping of GLUR5 using a YAC contig suggested that the GLUR5 gene spans approximately 400–500kb, and is within 280kb of D21S213. The large size of the GLUR5 gene raises questions regarding its functional significance. Our GLUR5 YAC contig includes clones found in the Genethon chromosome 21 YAC contig, and reference to the larger contig indicates the orientation centromere — D21S213 — GLUR5 5 end-GLUR5/ AGAT — GLUR5 3 end — SODI. The development of GLUR5/AGAT should permit rapid determination of the status of the GLUR5 gene in individuals with partial trisomy or monosomy of chromosome 21. Such studies may provide insights concerning the possible role of GLUR5 in Down syndrome.     

An Integrated Genetic and Physical Map of the Autosomal Recessive Polycystic Kidney Disease Region

Autosomal recessive polycystic kidney disease is one of the most common hereditary renal cystic diseases in children. Genetic studies have recently assigned the only known locus for this disorder, PKHD1, to chromosome 6p21–p12. We have generated a YAC contig that spans 5 cM of this region, defined by the markers D6S1253–D6S295, and have mapped 43 sequence-tagged sites (STS) within this interval. This set includes 20 novel STSs, which define 12 unique positions in the region, and three ESTs. A minimal set of two YACs spans the segment D6S465–D6S466, which contains PKHD1, and estimates of their sizes based on information in public databases suggest that the size of the critical region is <3.1 Mb. Twenty-eight STSs map to this interval, giving an average STS density of <1/150 kb. These resources will be useful for establishing a complete trancription map of the PKHD1 region.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

Five Subunit Genes of the Human Muscle Nicotinic Acetylcholine Receptor Are Mapped to Two Linkage Groups on Chromosomes 2 and 17

RFLPs were detected in the five subunit genes of the human muscle nicotinic acetylcholine receptor (nAChR) using genomic DNA or cDNA probes from the homologous mouse loci. The RFLPs at the alpha-, beta-, gamma-, delta-, and epsilon-subunit gene loci were analyzed for genetic linkage in 16 families (n = 188). Significant evidence was obtained for close linkage of the β- and ε-nAChR genes and much greater genetic distance between the α-nAChR gene and the γ/δ-nAChR gene complex. The linkage analysis program CRI-MAP was used to map the positions of the β- and ε-nAChR genes relative to seven markers on chromosome 17. The results indicate the β- and ε-nAChR genes are separated by about 5 cM and located in the region of chromosome 17p occupied by D17S1, D17S31, TP53, and D17S513. The statistical evidence was confirmed by hybridization of the β- and ε-nAChR probes to a panel of human-hamster somatic cell hybrids. The α-, γ-, and δ-nAChR genes were placed on a map of 13 chromosome 2 markers. The linkage analysis placed the nAChR genes at two sites on chromosome 2q about equidistant from the marker CRYGP1, with the α-nAChR gene about 27 cM proximal and the γ/δ-nAChR gene complex about 31 cM distal to CRYGP1.     

A 370-kb Cosmid Contig of the Serpin Gene Cluster on Human Chromosome 14q32.1: Molecular Linkage of the Genes Encoding α1-Antichymotrypsin, Protein C Inhibitor, Kallistatin, α1-Antitrypsin, and Corticosteroid-Binding Globulin

The human genes encoding α1-antitrypsin (α1AT, gene symbol PI), corticosteroid-binding globulin (CBG), α1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen–CBG–ATR–α1AT–KAL–PCI–AACT–tel. The genes occupy 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is 12 kb of intergenic DNA between KAL and PCI and 19 kb between PCI and AACT. The proximal subcluster includes α1AT, ATR, and CBG; it occupies 90 kb of genomic DNA, with 12 kb of DNA between α1AT and ATR and 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.     

Affected-sib-pair mapping of a novel susceptibility gene to insulin-dependent diabetes mellitus (IDDM8) on chromosome 6q25-q27.

Affected-sib-pair analyses were performed using 104 Caucasian families to map genes that predispose to insulin-dependent diabetes mellitus (IDDM). We have obtained linkage evidence for D6S446 (maximum lod score [MLS] = 2.8) and for D6S264 (MLS = 2.0) on 6q25-q27. Together with a previously reported data set, linkage can be firmly established (MLS = 3.4 for D6S264), and the disease locus has been designated IDDM8. With analysis of independent families, we confirmed linkage evidence for the previously identified IDDM3 (15q) and DDM7 (2q). We also typed additional markers in the regions containing IDDM3, IDDM4, IDDM5, and IDDM8. Preliminary linkage evidence for a novel region on chromosome 4q (D4S1566) has been found in 47 Florida families (P < .03). We also found evidence of linkage for two regions previously identified as potential linkages in the Florida subset: D3S1303 on 3q (P < .04) and D7S486 on 7q (P < .03). We could not confirm linkage with eight other regions (D1S191, D1S412, D4S1604, D8S264, D8S556, D10S193, D13S158, and D18S64) previously identified as potential linkages.     

Localization of the human caveolin-3 gene to the D3S18/D3S4163/D3S4539 locus (3p25), in close proximity to the human oxytocin receptor gene. Identification of the caveolin-3 gene as a candidate for deletion in 3p-syndrome.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Recently, we identified an autosomal dominant form of limb girdle muscular dystrophy in humans that is due to mutations within exon 2 of the caveolin-3 gene (3p25). However, the detailed location of the human caveolin-3 gene and its position with regard to neighboring genes remains unknown. Here, we have isolated three independent BAC clones containing the human caveolin-3 gene. Using a PCR-based approach, we determined that these clones contain both exons 1 and 2 of the human caveolin-3 gene. In addition, we performed microsatellite marker analysis of these BAC clones, using a panel of 13 markers that are known to map within the 3p25 region. Our results indicate that these BAC clones contain the following three markers: D3S18, SHGC-1079 (also known as D3S4163) and D3S4539. Interestingly, D3S18 is a marker for two known human diseases, von Hippel-Lindau disease and 3p-syndrome. As D3S4163 and D3S4539 are known to map in the vicinity of the 3' end of the human oxytocin receptor gene, we determined if these caveolin-3 positive BACs also contain the oxytocin receptor gene. We show that (i) these BACs contain all four exons of the oxytocin receptor gene and (ii) that the genes encoding caveolin-3 and the oxytocin receptor are located approximately 7-10 kb apart and in the opposite orientation. As 3p-syndrome is characterized by cardiac septal defects and caveolin-3 is expressed primarily in the heart and skeletal muscle, caveolin-3 is a candidate gene that may be deleted in 3p-syndrome.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19–63. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.