Correlation of chromosome 17p loss with clinical outcome in medulloblastoma.

Medulloblastomas are primitive neuroectodermal tumors that arise in the cerebella of children. Cytogenetic and loss of heterozygosity (LOH) studies have shown that deletions on the short arm of chromosome 17 occur in 25-50% of cases, suggesting that loss of a tumor suppressor gene located on 17p plays a role in the genesis or progression of medulloblastoma. We report here on an LOH analysis of 21 patients with medulloblastoma using markers for 15 polymorphic loci previously ordered on chromosome 17 by meiotic break point mapping. Although the frequency of LOH in our patients (48%) was consistent with previous reports, our maps showed much larger deletions, the smallest spanning a 38-cM genetic distance distal to marker UT49 (D17S731). Survival analysis did not show a significant association between LOH on 17p and survival, although the sample size was too small to rule out a difference of less than 10-fold. Clinical risk factors were better prognostic indicators than LOH, with significantly prolonged survival in patients free of craniospinal metastasis following gross total tumor resection.

Precise mapping of t(12;14) leiomyoma breakpoint on chromosome 14 between D14S298 and D14S540.

Uterine leiomyoma is a common tumor of smooth muscle cell origin often characterized by the presence of a balanced t(12;14)(q13-15;q24.1) chromosomal translocation. This breakpoint on chromosome 14 had previously been placed between the markers SPTB and D14S77, a region estimated to span 7 cM. In this study we have used a meiotic breakpoint mapping panel to construct a high resolution genetic map of this interval. Markers that mapped within this interval were used to analyze DNA from a somatic cell hybrid containing the t(12;14) translocated chromosome. The results of this analysis localize the t(12;14) breakpoint on chromosome 14 between D14S298 and D14S540, between which no meiotic recombination was detected. This sets the stage for identifying the gene(s) disrupted by the chromosomal translocation by defining the markers that flank the translocation breakpoint.

A strategy for constructing high-resolution genetic maps of the human genome: a genetic map of chromosome 17p, ordered with meiotic breakpoint-mapping panels.

Genetic linkage analyses with genotypic data obtained from four CEPH reference families initially assigned 24 new PCR-based markers to chromosome 17 and located the markers at specific intervals of an existing genetic map of chromosome 17p. Each marker was additionally genotyped with an ordered set of obligate, phase-known recombinant chromosomes. The breakpoint-mapping panels for each family consisted of two parents, one sib with a nonrecombinant chromosome, and one or more sibs with obligate recombinant chromosomes. The relative order of markers was determined by sorting segregation patterns of new markers and ordered anchor markers and by minimizing double-recombination events. Consistency of segregation patterns with multiple flanking loci constituted support for order. A genetic map of chromosome 17p was completed with 39 markers in 23 clusters, with an average space of 3 cM between clusters. The collection of informative genotypes was highly efficient, requiring fivefold fewer genotypes than would be collected with all the CEPH families. Given the availability of large numbers of highly informative PCR-based markers, meiotic breakpoint mapping should facilitate construction of a human genomic map with 1-cM resolution.

Breakpoint analysis: precise localization of genetic markers by means of nonstatistical computation using relatively few genotypes.

Placing new markers on a previously existing genetic map by using conventional methods of multilocus linkage analysis requires that a large number of reference families be genotyped. This paper presents a methodology for placing new markers on existing genetic maps by genotyping only a few individuals in a selected subset of the reference panel. We show that by identifying meiotic breakpoint events within existing genetic maps and genotyping individuals who exhibit these events, along with one nonrecombinant sibling and their parents, we can determine precise location for new markers even within subcentimorgan chromosomal regions. This method also improves detection of errors in genotyping and assists in the observation of chromosome behavior in specific regions.

Physical localization of microsatellite markers at the ataxia-telangiectasia locus at 11q22-q23.

The autosomal recessive disorder ataxia-telangiectasia (A-T) is genetically heterogeneous, with four complementation groups. The genes for the two major groups (ATA and ATC) have been mapped to 11q22-q23. Genetic analysis of the disease has been conducted to date using biallelic polymorphisms. We have physically mapped to this region eight new microsatellite markers that were generated by three laboratories that construct whole-genome linkage maps. These markers should be valuable for refined localization and positional cloning of the A-T genes and for diagnostic purposes. The results demonstrate the value of integrating genetic and physical maps generated by different laboratories.

The CEPH consortium linkage map of human chromosome 13.

The CEPH consortium map of chromosome 13 is presented. This map contains 59 loci defined by genotypes generated from CEPH family DNAs with 94 different probe and restriction enzyme combinations contributed by 9 laboratories. A total of 25 loci have been placed on the map with likelihood support of at least 1000:1. The map extends from loci in the centromeric region of chromosome 13 to the terminal band of the long arm. Multipoint linkage analyses provided estimates that the male, female, and sex-averaged maps extend for 158, 203, and 178 cM respectively. The largest interval is 24 cM and is between D13Z1 (alpha RI) and ATP1AL1. The mean genetic distance between the 25 uniquely placed loci is 7 cM.

Identification of mutations in the COL4A5 collagen gene in Alport syndrome.

X-linked Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by progressive loss of hearing. Ultrastructural defects in glomerular basement membranes (GBM) of Alport syndrome patients implicate an altered structural protein as the cause of nephritis. The product of COL4A5, the alpha 5(IV) collagen chain, is a specific component of GBM within the kidney, and the gene maps to the same X chromosomal region as does Alport syndrome. Three structural aberrations were found in COL4A5, in intragenic deletion, a Pst I site variant, and an uncharacterized abnormality, which appear to cause nephritis and deafness, with allele-specific severity, in three Alport syndrome kindreds in Utah.

Threonyl-tRNA synthetase gene maps close to leucyl-tRNA synthetase gene on human chromosome 5.

The structural gene for threonyl-tRNA synthetase was mapped to human chromosome 5 by an analysis of the isoelectric focusing patterns of this enzyme from human X Chinese hamster interspecific somatic cell hybrids. The threonyl-tRNA synthetase gene is the fourth of seven aminoacyl-tRNA synthetase genes mapped in humans to be assigned to this chromosome. Regional mapping studies showed that the threonyl-tRNA synthetase gene is on the short arm of chromosome 5, p13-cen, and is close to, but separable from, the gene for leucyl-tRNA synthetase which maps to 5cen-5q11.

Histidyl-tRNA synthetase of Chinese hamster ovary cells contains phosphoserine.

The Chinese hamster ovary cell line 2H2-5, which expresses elevated levels of histidyl-tRNA synthetase, was used to demonstrate that histidyl-tRNA synthetase contains phosphoserine. After growth of the cells in medium containing [32P]orthophosphate, histidyl-tRNA synthetase was isolated by partial purification and immunoprecipitation and shown to be a phosphoprotein. Phosphoamino acid analysis showed that histidyl-tRNA synthetase is phosphorylated on serine, as has previously been shown for threonyl-tRNA synthetase of CHO cells.

Threonyl-tRNA synthetase from Chinese hamster ovary cells is phosphorylated on serine.

A Chinese hamster ovary cell line, 2000A, in which threonyl-tRNA synthetase accounts for 1.5% of the total soluble protein, was used to demonstrate that this enzyme is a phosphoprotein. Threonyl-tRNA synthetase was isolated by immunoprecipitation from cells labeled with 32Pi for 18 h. Phosphoamino acid analysis of radiolabeled threonyl-tRNA synthetase showed that phosphorylation occurs on serine.

Chinese hamster ovary cells resistant to borrelidin overproduce threonyl-tRNA synthetase.

Chinese hamster ovary cell lines that are 1000-fold more resistant to the threonyl-tRNA synthetase inhibitor borrelidin than the sensitive parental cells were isolated after stepwise selection for growth in increasing concentrations of the drug. These cells show a 10-20-fold increase in threonyl-tRNA synthetase activity. Quantitation of the amount of threonyl-tRNA synthetase protein by immunological techniques indicated a 60-100-fold increase compared to sensitive cells. No significant changes in the Km for substrates, inhibition by borrelidin or thermal stability were found for the threonyl-tRNA synthetase of resistant cells. These data suggest that the resistant cell lines may have amplified the gene encoding threonyl-tRNA synthetase, but no evidence of homogeneously staining regions or double minute chromosomes was found. The resistant cell lines should prove useful for the study of the regulation of threonyl-tRNA synthetase.

Correlation of a bioassay with the clinical status of patients with rheumatoid arthritis.

Joint synovia and fluids taken from patients with various stages of rheumatoid arthritis (RA) were injected into embryonated chicken eggs, resulting in characteristic joint abnormalities ("crooked toes") in the extremities of the embryos and chicks. There was a direct correlation between the number of the induced lesions and the stages of the human RA disease. The percentage of abnormality of the bioassays was consistent with acute, subacute, chronic or remission phases in the clinical course of the RA disease. Specimens from chronic or remission RA joints produced few or no experimental abnormalities, suggesting few or no infectiojs RA particles available to the bioassay. Likewise, dilution of acute RA joint fluids of 1 : 100 by 0.15M NaCl resulted in a characteristic extinction of the bioassay test, suggesting that specimens could lose their reactivity in the bioassay because of too great dilution in the preparation of homogenates of the synovia. Numerical changes in the bioassay consistently followed the changes in the clinical courses of the RA during waning and waxing episodes. A useful numerical scale (0--100%) of the 10-day embryonated egg bioassay as an indication or measurement of the severity of the RA in the patient is proposed on the basis of these observations.

Further studies on the specific infectious agent isolated in rheumatoid arthritis.

The active agent in crude or extracted materials from patients with acute rheumatoid arthritis (RA), when injected into embryonated eggs, produced an increased percentage of characteristic "crooked toe" defects in the bioassay. Experimental RA chick materials also produced these defects. A protein-free ribonucleic acid extract from RA synovia and joint fluids of patients with acute or subacute classical RA produced the same experimental RA lesions as did the crude materials, but non-RA materials and controls did not. The injected RA material produced the "crooked toe" disease in chicks in direct proportion to the clinically diagnosed severity of the RA in the patient, and inversely with dilution.