Physical and genetic characterization of the distal segment of the myotonic dystrophy area on 19q.

The mutation involved in myotonic dystrophy (DM) has been mapped to the region between the ERCC1 DNA repair gene and the anonymous D19S51 locus on 19q13.3. Starting at locus D19S112 (probe pX75b), which served as a novel entry site for this chromosome region, we have established a cosmid contig of approximately 200 kb. In the contig, a gene expressed in the brain and a highly informative, 12-allele (TG)n variable simple sequence motif (VSSM) were identified. With this marker, designated X75b-VSSM, a highly characteristic size distribution of alleles linked with DM, which differed significantly from that on normal chromosomes, was observed. Combining our physical mapping and genetic data, we show that the X75b-VSSM marker is the closest distal to DM, thus excluding the DM mutation from the entire telomeric portion of the ERCC1-D19S51 region.     

Physical and genetic mapping of a novel chromosome 19 ERCC1 marker showing close linkage with myotonic dystrophy.

Recent genetic linkage analyses have mapped the myotonic dystrophy locus to the region of 19q13.2-13.3 lying distal to the gene for creatine kinase subunit M (CKM). The human excision repair gene ERCC1 has also been mapped to this region of chromosome 19. A novel polymorphic DNA marker, pEO.8, has been isolated from a chromosome 19 ERCC1-containing cosmid that maps to a 300-kb NotI fragment encompassing both CKM and ERCC1. Genetic linkage analysis reveals close linkage between pEO.8 and myotonic dystrophy (DM) (zmax = 19.3, theta max = 0.01). Analysis of two key recombinant events suggests a mapping of DM distal to pEO.8 and CKM.     

Physical mapping and cloning of the proximal segment of the myotonic dystrophy gene region.

The myotonic dystrophy (DM) region has been recently shown to be bracketed by two key recombinant events. One recombinant occurs in a Dutch DM family, which maps the DM locus distal to the ERCC1 gene and D19S115 (pE0.8). The other recombinant event is in a French Canadian DM family, which maps DM proximal to D19S51 (p134c). To further resolve this region, we initiated a chromosome walk in a telomeric direction from pE0.8, a proximal marker tightly linked to DM, toward the genetic locus. An Alu-PCR approach to chromosome walking in a cosmid library from flow-sorted chromosome 19 was used to isolate DM region cosmids. This effort has resulted in the cloning of a 350-kb genomic contig of human chromosome 19q13.3. New genetic and physical mapping information has been generated using the newly cloned markers from this study. As a result of this new mapping information, the minimal area that is to contain the DM gene has been redefined. Approximately 200 kb of sequence between pE0.8 and the closest proximal marker to DM, pKEX0.8, that would have otherwise been screened for DM candidate genes, has been eliminated as containing the DM gene.     

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2-13.3.

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.     

Characterization of a YAC and cosmid contig containing markers tightly linked to the myotonic dystrophy locus on chromosome 19.

Myotonic dystrophy (DM) is caused by a defect in an unknown gene that maps to 19q13.3, flanked by the tightly linked markers ERCC1 on the proximal side and D19S51 on the distal side. We report the isolation and characterization of overlapping YAC and cosmid clones around D19S51 for the construction of a physical map around this locus. The resulting contig contains the markers D19S51 and D19S62 (another new marker tightly linked to the DM locus) and the distal breakpoint of a radiation hybrid cell line used in the physical mapping of the DM region. We have compared the restriction maps of the YACs and cosmids with that of the genome to investigate the fidelity of these clones.     

D19S51 is closely linked with and maps distal to the myotonic dystrophy locus on 19q.

Recent genetic linkage studies have mapped the myotonic dystrophy (DM) locus to 19q13.3. All closely linked DM markers identified to date have been located on the centromeric side of the disease locus, with a relatively large genetic interval (9 cM) observed between the nearest distal marker and DM. We show here that the recently described marker p134C is tightly linked to DM (peak lod score 35.8 at peak recombination fraction .006) and confirm the previous suggestion that the p134C locus, D19S51 maps distal to the disease locus. D19S51 and the closest proximal flanking loci, ERCC1 and D19S115 (pE0.8), define a small genetic interval of less than 2 cM that contains the DM locus.     

Recombination events that locate myotonic dystrophy distal to APOC2 on 19q

We previously reported a recombination in an individual with myotonic dystrophy (DM) which placed the markers D19S19 and APOC2 on the same side of the DM locus. Haplotyping of this family with more recently characterized probes which are either tightly linked to DM or distal to the linkage group at q13.2 shows that the DM locus is distal to APOC2. This is confirmed by other recombinants where DM segregates with distal probes. Additional marker to marker recombinations in unaffected individuals are reported and support the order and orientation of the DM linkage group as pter-(INSR, LDLR,S9)-(S19,BCL3,APOC2)-(CKMM,DM)-(S22,+ ++PRKCG)-qter. The data presented here cannot determine whether DM is proximal or distal to CKMM. The consequences of this probe order for antenatal diagnosis and future research aiming to isolate the gene which is affected in DM are discussed.     

Myotonic dystrophy of Steinert

The gene for myotonic dystrophy maps to 19q13.2----19q13.3. The closest proximal marker is the gene for creatine kinase CKMM at a recombination faction of 0-2%. Prenatal diagnosis will be performed with a minimal risk when a distal closer to the gene is available.     

Refined mapping of the three DNA repair genes, ERCC1, ERCC2, and XRCC1, on human chromosome 19

Three DNA repair genes, ERCC1, ERCC2, and XRCC1, have been regionally mapped on human chromosome 19. ERCC2 and XRCC1 have been assigned to bands q13.2----q13.3 by in situ hybridization using fluorescently-labeled cosmid probes. ERCC1 and ERCC2 have been found to be separated by less than 250 kb by large fragment restriction enzyme site mapping.     

Use of variable simple sequence motifs as genetic markers: application to study of myotonic dystrophy

Summary Among the many classes of repetitive elements present in the human genome, the ubiquitous simple sequence motifs (SSMs) composed of [A]n, [TG]n, [AG]n or codon-tandem repeats form a major source of genetic variation. Here we report a detailed molecular-genetic study of a variable simple sequence motif (VSSM) in the apolipoprotein C2 (apoC2) gene, which maps to the 19q13.2 region in the vicinity of the myotonic dystrophy (DM) locus. By combining in vitro DNA-amplification using the polymerase chain reaction and high-resolution gel electrophoresis, we could demonstrate a high degree of allelic variation with at least ten alleles, which differ in the number of repeated [TG] or [AG] dinucleotide units. Similar results were found for the somatostatin I gene locus. To evaluate the usefulness of SSM-length polymorphisms as genetic markers, the apoC2-VSSM was employed for linkage analysis in DM families. Our results establish that the orientation of the apolipoprotein gene cluster on 19q is cenapoE-apoC2-ter and indicate that the many thousands of structurally similar VSSMs in the human genome represent a rich source of highly informative genetic and diagnostic markers.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

The 1.5-Mb region spanning the myotonic dystrophy locus shows uniform recombination frequency.

The myotonic dystrophy (DM) mutation has been identified as a heritable unstable CTG trinucleotide repeat sequence. The intergenerational amplification of this sequence is an example of a new class of dynamic mutations responsible for human genetic diseases. To ascertain whether recombination activity influences, or is affected by, the presence of this unique sequence, a comprehensive study of the physical and genetic mapping data for the 1.5-Mb region of human chromosome 19q13.3, which contains the DM locus, was conducted. The recombination rate for this region was examined by correlating genetic distance to physical distance for six selected marker loci. The following markers span the DM region: 19qCEN-p alpha 1.4 (D19S37)-APOC2-CKM-pE0.8 (D19S115)-pGB2.6 (DM)-p134c (D19S51)-19qTER. Initial linear regression analysis of these two parameters failed to reveal a significant linear correlation (coefficient of determination, r2 = .19), suggesting nonuniform rates of recombination. However, the presence of a recombination hot spot was believed to be unlikely, as the marker-to-marker pairs that showed the greatest deviation in recombination frequency were not restricted to a specific region of the 1.5 Mb studied and had relatively broad confidence intervals, as reflected by low LOD values. A second linear regression analysis using only marker intervals with high LOD scores (Zmax > 22) showed linear correlation (r2 = .68) for the entire 1.5-Mb region. This analysis indicated a relatively uniform recombination frequency in the 1.5-Mb region spanning the DM locus. Furthermore, the recombinations observed were neither under- nor overrepresented on DM chromosomes. Consequently, recombination activity is unlikely to influence, or be affected by, the presence of the DM mutation.     

A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19

Employing 16 polymorphic DNA markers as well as the chromosome 19 centromere heteromorphism, we have performed a genetic linkage study in 26 families with myotonic dystrophy. Fourteen of these markers had been assigned previously to one of five different intervals of the 19cen-19q13.2 segment by using somatic cell hybrids. For the long arm of chromosome 19, a genetic map that encompasses 9 polymorphic markers and the DM gene has been constructed. Our studies indicate that the DM and CKMM genes map distal to the ApoC2-ApoE gene cluster and to the anonymous polymorphic markers D19S15 and D19S16, but proximal to the D19S22 marker. The orientation of DM and CKMM remains to be determined.     

Regional localization of the selenocysteine tRNA gene (TRSP) on human chromosome 19.

The human selenocysteine tRNA gene (TRSP) has been localized on chromosome 19q13.2-->q13.3 by in situ hybridization and ordered with respect to other genes and anonymous DNA markers in this region by linkage analysis in the forty CEPH pedigrees. These loci span only 10 cM in males and about 30 cM in females. The order of the loci is cen ... D19S7-D19S9-D19S47-CYP2A-CYP2F1-APOC2++ +-(TRSP, CKM). CYP2B flanks the CYP2A and CYP2F1 loci, but it cannot be determined whether it is proximal or distal to the other two cytochrome P450 loci with respect to the centromere.     

Human glandular Kallikrein genes: genetic and physical mapping of the KLK1 locus using a highly polymorphic microsatellite PCR marker.

We describe a highly polymorphic microsatellite repeat sequence, KLK1 AC, which is located 3' to the human glandular kallikrein gene (KLK1) at 19q13.3-13.4. A multiplex PCR was developed to simultaneously genotype the KLK1 AC repeat length polymorphism and a similar repeat at the adjacent APOC2 locus at 19q13.2. Genotypes from these two loci in the 40 large kindred pedigrees from the Centre d'Etude du Polymorphisme Humain were used in conjunction with the background genetic map to establish a multipoint linkage map. The KLK1 locus was also localized physically using somatic cell hybrid DNA templates for polymerase chain reaction analysis. Both genetic and physical mapping studies are consistent with the assignment cen-APOC2-KLK1-D19522-qter. The linkage map places KLK1 approximately 10 cM distal to APOC2. These markers therefore flank the myotonic dystrophy gene and may be useful for diagnosis.     

Detection of linkage disequilibrium between the myotonic dystrophy locus and a new polymorphic DNA marker.

We have examined the linkage of two new polymorphic DNA markers (D19S62 and D19S63) and a previously unreported polymorphism with an existing DNA marker (ERCC1) to the myotonic dystrophy (DM) locus. In addition, we have used pulsed-field gel electrophoresis to obtain a fine-structure map of this region. The detection of linkage disequilibrium between DM and one of these markers (D19S63) is the first demonstration of this phenomenon in a heterogeneous DM population. The results suggest that at least 58% of DM patients in the British population, as well as those in a French-Canadian subpopulation, are descended from the same ancestral DM mutation. We discuss the implications of this finding in terms of strategies for cloning the DM gene, for a possible role in modification of risk for prenatal and presymptomatic testing, and we speculate on the origin and number of existing mutations which may result in a DM phenotype.     

Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200–300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.     

A second locus for an axonal form of autosomal recessive Charcot-Marie-Tooth disease maps to chromosome 19q13.3

Autosomal recessive Charcot-Marie-Tooth disease (CMT) represents a heterogeneous group of disorders affecting the peripheral nervous system. The axonal form of the disease is designated as "CMT type 2" (CMT2), and one locus (1q21.2-q21.3) has been reported for the autosomal recessive form. Here we report the results of a genomewide search in an inbred Costa Rican family (CR-1) affected with autosomal recessive CMT2. By analyzing three branches of the family we detected linkage to the 19q13.3 region, and subsequent homozygosity mapping defined shared haplotypes between markers D19S902 and D19S907 in a 5.5-cM range. A maximum two-point LOD score of 9.08 was obtained for marker D19S867, at a recombination fraction of.00, which strongly supports linkage to this locus. The epithelial membrane protein 3 gene, encoding a PMP22 homologous protein and located on 19q13.3, was ruled out as being responsible for this form of CMT. The age at onset of chronic symmetric sensory-motor polyneuropathy was 28-42 years (mean 33.8 years); the electrophysiological data clearly reflect an axonal degenerative process. The phenotype and locus are different from those of demyelinating CMT4F, recently mapped to 19q13.1-13.3; hence, the disease affecting the Costa Rican family constitutes an axonal, autosomal recessive CMT subtype (ARCMT2B).     

Radiation-reduced hybrids for the myotonic dystrophy locus.

The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.     

A chromosome 19 clone from a translocation breakpoint shows close linkage and linkage disequilibrium with myotonic dystrophy

The gene for myotonic dystrophy (DM), the most common form of adult muscular dystrophy, is situated on the proximal long arm of chromosome 19. Although there exist markers that are tightly linked to the DM locus, its precise location is unknown. The identification and characterization of additional DNA probes closely linked to the DM locus continue to be priorities. In this study, we report on the linkage between a new DNA marker, designated p alpha 1.4P, and the DM locus in 50 families. The probe p alpha 1.4P was derived from a cloned breakpoint junction fragment from the chromosomal translocation t(14;19)(q32;q13.1). This translocation has been previously described in some cases of chronic lymphocytic leukemia. We have identified a BanI restriction fragment length polymorphism that is detected by p alpha 1.4P. Segregation analysis between this RFLP and DM revealed close linkage between the two loci (lod = 10.95, theta = 0). Furthermore, statistical evidence for linkage disequilibrium between p alpha 1.4P and the DM locus in a French Canadian population was found. Finally, by means of a somatic cell hybrid mapping panel, p alpha 1.4P was sublocalized to 19q12----19q13.2.