B. Wieringa

Evidence for linkage of the central core disease locus to the proximal long arm of human chromosome 19

Central core disease of muscle (CCD; MIM 117000) is a rare inheritable myopathy that is frequently found in association with susceptibility to malignant hyperthermia (MHS). This observation has prompted us to perform a linkage study in CCD families using various chromosome 19q probes that are linked to the MHS locus and map close to the ryanodine receptor gene (RYR1), a strong MHS candidate gene. Our genetic linkage data support a location of the CCD gene on proximal 19q13.1 and thus suggest that CCD and MHS may be allelic.

Presymptomatic diagnosis of myotonic dystrophy.

The discovery of an expanded (CTG)n repeat sequence in myotonic dystrophy (DM) has greatly improved our ability to detect DM gene carriers who have few or none of the classical signs of this disorder. We report here our experience with two such groups of gene carriers. We used a PCR based protocol that should be especially sensitive to small increases in CTG triplet number which might escape detection by conventional Southern blot analysis. Our analyses show that on 100 non-DM chromosomes the number of CTG triplets ranged from five to 37. We then studied 17 obligate gene carriers aged 55 years and over who showed no muscle weakness. All of the gene carriers in this group showed a relatively small increase in the number of CTG triplets (52 to 90 CTG triplets) with limited somatic mosaicism. We subsequently studied 11 subjects (aged 19 to 36 years) who had previously been identified as gene carriers by genetic linkage studies, but who lacked diagnostic signs. In this prospectively studied group, nine subjects showed an expanded allele, confirming the earlier prediction from linked genetic markers. The other two subjects had only two normal alleles and no expanded allele. Revision of the clinical data casts doubt on the original diagnosis of DM in their families. Preferential amplification of the normal non-expanded allele was noted in three asymptomatic gene carriers in this study (as well as in two of their clinically affected relatives). We caution that, at least in our hands, the DM mutation can be confidently excluded by this PCR based method only if both normal alleles have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

New Polymorphic DNA Marker Close to the Fragile Site FRAXA

DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.

Assignment of the human gene for the water channel of renal collecting duct Aquaporin 2 (AQP2) to chromosome 12 region q12→q13

The chromosomal localization of the gene encoding Aquaporin 2 (previously called WCH-CD), which acts as a water channel in the collecting tubules of the kidney, was determined. Southern blot hybridizations of chromosomal DNA from a panel of 25 different human-rodent hybrid cell lines assigned AQP2 to the q-arm of human chromosome 12. Additionally, in situ hybridization on R-banded metaphase chromosomes localized AQP2 to the q12-->q13 region of this chromosome.

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.

High-resolution physical mapping of four microsatellite repeat markers near the RYR1 locus on chromosome 19q13.1 and apparent exclusion of the MHS locus from this region in two malignant hyperthermia susceptible families

Malignant hyperthermia susceptibility (MHS) is a potentially lethal, hereditary disorder of skeletal muscle that may be triggered by inhalation anesthetics and depolarizing muscle relaxants. Defects in the gene encoding the ryanodine receptor (RYR1) localized on human chromosome 19q13.1 have been proposed to be responsible for MHS. Using a chromosome 19-specific human/hamster somatic cell hybrid mapping panel, we were able to determine that four closely linked microsatellite repeat markers bracket RYR1 with the order 19cen-D19S75-D19S191-RYR1-(D19S47, D19S190)-19ter. Application of the four markers to genetic studies of MHS showed recombination between the markers and MHS in two families, with linkage analysis apparently excluding the MHS locus from the RYR1 region of 19q13.1. These results therefore support the recent observations of genetic heterogeneity in MHS.

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.

The human gene for water channel aquaporin 1 (AQP1) is localized on chromosome 7p15→p14

The chromosomal localization of the gene encoding aquaporin 1 (previously called CHIP28), which acts as a water channel in erythrocytes and in the renal proximal tubules and descending limbs of Henle, was determined. Southern blot hybridizations on chromosomal DNA of a panel of 25 different human x rodent hybrid cell lines localized the aquaporin 1 gene to human chromosome 7. Additionally, in situ hybridization on R-banded metaphase chromosomes sublocalized the aquaporin 1 gene (AQP1) to 7p15-->p14, and some sequence-tagged sites in this region were identified.

Assignment of the human gene for receptor-type protein tyrosine phosphatase IA-2 (PTPRN) to chromosome region 2q35-q36.1 and identification of an intragenic genetic marker

Using a mouse protein tyrosine phosphatase cDNA fragment as a probe, cosmid clones containing segments of the human IA-2 PTPase gene (PTPRN) were isolated. The gene was assigned to chromosome region 2q35 --> q36.1 by fluorescence in situ hybridization. In an intronic region of the IA-2 gene a polymorphic microsatellite sequence was found, which will be useful as a genetic marker for the 2q35 --> q36 region.

The gene (PTPN13) encoding the protein tyrosine phosphatase PTP-BL/PTP-BAS is located in mouse chromosome region 5E/F and human chromosome region 4q21.

Both mouse and human genomic clones were isolated for protein tyrosine phosphatase PTP-BL/PTP-BAS (HGM approved gene symbols Ptpn13 and PTPN13, respectively). Using these clones as a probe, PTPN13 was assigned to human chromosome region 4q21 and mouse chromosome region 5E/F by fluorescence in situ hybridization (FISH).