Duchenne muscular dystrophy involving translocation of the dmd gene next to ribosomal RNA genes.

Duchenne muscular dystrophy (DMD) is a severe X-linked disorder leading to early death of affected males. Females with the disease are rare, but seven are known to be affected because of a chromosomal rearrangement involving a site at or near the dmd gene on the X chromosome. One of the seven has a translocation between the X and chromosome 21. The translocation-derived chromosomes from this patient have been isolated, and the translocation is shown to have split the block of genes encoding ribosomal RNA on the short arm of chromosome 21. Thus ribosomal RNA gene probes may be used to identify a junction fragment from the translocation site, allowing access to cloned segments of the X at or near the dmd gene and presenting a new approach to the study of this disease.

Molecular analysis of a constitutional X-autosome translocation in a female with muscular dystrophy.

The gene responsible for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) maps to the X chromosome short arm, band Xp21. In a few females with DMD or BMD, the Xp21 region is disrupted by an X-autosome translocation. Accumulating evidence suggests that the exchange has physically disrupted the DMD/BMD locus to cause the disease. One affected female with a t(X;21)(p21;p12) translocation was studied in detail. The exchange points from both translocation chromosomes were cloned, restriction-mapped, and sequenced. The translocation is reciprocal, but not conservative. A small amount of DNA is missing from the translocated chromosomes; 71 to 72 base pairs from the X chromosome and 16 to 23 base pairs from the 28S ribosomal gene on chromosome 21.

Human ribosomal RNA genes: orientation of the tandem array and conservation of the 5' end.

The multiple copies of the human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters that map to the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. Concerted evolution of the gene family is thought to be mediated by interchromosomal recombination between rDNA repeat units, but such events would also result in conservation of the sequences distal to the rDNA on these five pairs of chromosomes. To test this possibility, a DNA fragment spanning the junction between rDNA and distal flanking sequence has been cloned and characterized. Restriction maps, sequence data, and gene mapping studies demonstrate that (i) the rRNA genes are transcribed in a telomere-to-centromere direction, (ii) the 5' end of the cluster and the adjacent non-rDNA sequences are conserved on the five pairs of chromosomes, and (iii) the 5' end of the cluster is positioned about 3.7 kb upstream from the transcription initiation site of the first repeat unit. The data support a model of concerted evolution by interchromosomal recombination.

Duchenne muscular dystrophy gene expression in normal and diseased human muscle.

A probe for the 5' end of the Duchenne muscular dystrophy (DMD) gene was used to study expression of the gene in normal human muscle, myogenic cell cultures, and muscle from patients with DMD. Expression was found in RNA from normal fetal muscle, adult cardiac and skeletal muscle, and cultured muscle after myoblast fusion. In DMD muscle, expression of this portion of the gene was also revealed by in situ RNA hybridization, particularly in regenerating muscle fibers.

A polymorphism in the NPPA gene associates with asthma.

BACKGROUND: Atrial natriuretic peptide (ANP) plays an important role in the lung and in augmenting allergic inflammation in asthma. The gene encoding ANP, NPPA, is located on chromosome 1p36, a region that has been linked to asthma. OBJECTIVES: Determine associations between asthma and four common SNPs on the NPPA gene: C/G (rs13305986) in the promoter; G/A (rs5063) in Exon 1 resulting in NPPAMet32-->Val substitution; T/C (rs5065) in Exon 3 resulting in an Arg152-->Ter substitution; and T/C in the 3'UT region (rs5067). Methods A case-control design was used in White participants. The screening cohort consisted of 336 asthmatic cases who participated in a large clinical trial and 154, non-asthmatic controls. The replicate cohort consisted of 172 asthmatic cases from a second clinical trial and 115 healthy controls. Demographic characteristics were well matched for cases and controls in the screening cohort. Adjusted (age, gender, body mass index) odds ratio (OR) were calculated by chi(2) and logistic regression; a P-value of 0.0167 defined the threshold of significance. RESULTS: The C allele of rs5067 was associated with asthma in the screening and replicate cohorts: adjusted ORs (95% confidence intervals) 0.5 (0.29-0.84; P=0.009) and 0.24 (0.11-0.53; P<0.0001), respectively. The C allele of rs5065 was associated with asthma in the screening cohort but not in the replicate. The population-attributable risk for asthma in carriers of the C allele for rs5067 was 23.3%. CONCLUSIONS: For rs5067, the risks of asthma in carriers of the C allele in the screening and replicate cohorts were reduced by 50% and 76%, respectively. NPPA may be an important susceptibility gene for asthma.

Sequence and structure correlation of human ribosomal transcribed spacers.

We report the sequences of the transcribed spacers of human rRNA that now allow us to piece together the entire primary transcript sequence of approximately 13.3 x 10(3) base-pairs. Comparison of transcribed spacer sequences with those of variable regions of rRNA and with those of the non-transcribed spacers supports the hypothesis that the variable regions are descended from transcribed spacers. Nucleotide sequence-derived secondary structures for the 5' external transcribed spacer and for internal transcribed spacers 1 and 2 match both the sizes and shapes of the structures that were visualized 15 years ago on electron micrographs. Parts of these structures are conserved in mammals and may be related to transcript processing.

Karyotypic dissection of Hodgkin's disease cell lines reveals ectopic subtelomeres and ribosomal DNA at sites of multiple jumping translocations and genomic amplification.

Although the neoplastic significance of the chromosome changes widespread in Hodgkin's disease (HD) remains obscure, a distinct cytogenetic picture has emerged combining aneuploidy with structural rearrangements clustered at certain breakpoints. Notably absent are the recurrent chromosome translocations which distinguish other hematopoietic neoplasms and serve as clues to underlying oncogene alterations. The paucity of neoplastic cells in HD biopsies hinders detailed chromosome analysis. As an alternative, we investigated a panel of well characterized cell lines by classical and molecular cytogenetics, using single-gene and subtelomeric probes, including three autologous HD examples (HDLM-1/2/3) analyzed by 'spectral karyotyping' - the first complete HD karyotype to be documented. Although complex, most rearrangements in HDLM cells arose in vivo and included few rare but many typical HD breakpoints, notably at the r(ibosomal)DNA regions. Two types of genomic rearrangement involving DNA repeats were conspicuous: insertion and genomic amplification/coamplification of rDNA-the first genomic rDNA rearrangements to be reported in a tumor cell, and the first example of multiple 'jumping translocations' (JT). Of four subtelomeric microsatellite repeats tested in HDLM cells, three exhibited interstitial sites at JT, of which two (at 5qter and 9pter) were respectively associated with deletion of the 5q31-32 myeloid region, and coamplification of a recently described HD-recurrent amplicon at 9p2 together with transcriptionally silent rDNA. Altogether, three out of four HD cell lines carried interstitial 9p subtelomeres and rDNA rearrangements. Taken together, these data suggest tumorigenic rearrangements may be facilitated by 'hitchhiking' along with mobile DNA repeat sequences which may target gene rearrangement at 9p in HD. Southern analysis of parallel rearrangements within rDNA intergenic spacers in HDLM cells highlighted several at, or near, retroposons. As well as validating HD cell lines as cytogenetic models, and resources for identifying genes rearranged in HD, our findings warrant further investigation of the roles of DNA repeat sequences, notably subtelomeric microsatellites, rDNA spacer sequences and retroposons as facilitators and markers of tumor-gene rearrangement.

Human ribosomal DNA: novel sequence organization in a 4.5-kb region upstream from the promoter.

We have investigated the molecular organization of a portion of the human ribosomal nontranscribed spacer, by determining the sequence of 4580 bp of DNA upstream from the promoter. This region contains two pairs of oppositely oriented Alu elements, each of which is separated by dA (or dT)-rich stretches. One dT-rich region extends over 800 bp and is of variable length in different ribosomal genes. This and other portions of the spacer consist of simple sequences reiterated many times: for instance, (TACAA)26, (TTTC)117, and (TTGC)47. We are able to position the distal junction of the ribosomal repeat and sequences that have the propensity to form alternate structures, such as Z-DNA and bent DNA. A complex DNA methylation pattern and the influence on transcription of analogous regions in other species, suggest that this upstream area may be important to the expression of the human gene.

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Human embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene.

A 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.

The use of postoperative irradiation for the prevention of heterotopic bone formation after total hip replacement.

Formation of heterotopic bone (HTB) following total hip replacement may partially or completely ankylose the joint space, causing pain and/or limiting the range of motion. Patients at high risk for formation of HTB postoperatively include those with previous HTB formation, heterotopic osteoarthritis, and active rheumatoid spondylitis. Patients in these high risk groups have a 63-69% incidence of post-operative HTB formation, usually seen radiographically by 2 months post-operation. From 1980-1986 twenty-nine hips in 28 consecutively treated patients were irradiated post-operatively at the UCLA Center for the Health Sciences. The indication for irradiation was documented HTB formation previously in 26 of the 27 hips presented below. From 1980-1982 patients received 20 Gray (Gy) in 2 Gy fractions; from 1982-1986 the dose was reduced to 10 Gy in 2 Gy fractions. Twenty-seven hips in 26 patients completed therapy and were available for evaluation, with a minimum of 2 month follow-up, and a median follow-up of 12 months. Three of 27 hips developed significant HTB (Brooker grade III or IV) post-operatively, whereas 5 of 27 hips developed minor, nonsymptomatic HTB (Brooker grade I). When irradiation was begun by postoperative day 4, 0 of 17 hips formed significant HTB. If irradiation began after post-operative day 4, 3 of 10 hips formed significant HTB (Brooker grade III or IV). These 3 hips received doses of 10 Gy in one hip and 20 Gy in the other 2 hips. There were no differences in the incidence or severity of side effects in the 10 Gy vs. the 20 Gy treatment groups. Eighteen hips received 10 Gy, 8 hips 20 Gy and, 1 hip 12 Gy. In conclusion, 10 Gy in 5 fractions appears as effective as 20 Gy in 10 fractions at preventing post-operative formation of HTB. For optimal results, treatment should begin as early as possible prior to post-operative day 4.

Palladium-103 brachytherapy for prostate carcinoma.

PURPOSE: A report of biochemical outcomes for patients treated with palladium-103 (Pd-103) brachytherapy over a fixed time interval. METHODS AND MATERIALS: Two hundred thirty patients with clinical stage T1-T2 prostate cancer were treated with Pd-103 brachytherapy and followed with prostate-specific antigen (PSA) determinations. Kaplan-Meier estimates of biochemical failure on the basis of two consecutive elevations of PSA were utilized. Multivariate risk groups were constructed. Aggregate PSA response by time interval was assessed. RESULTS: The overall biochemical control rate achieved at 9 years was 83.5%. Failures were local 3.0%; distant 6.1%; PSA progression only 4.3%. Significant risk factors contributing to failure were serum PSA greater than 10 ng/ml and Gleason sum of 7 or greater. Five-year biochemical control for those exhibiting neither risk factor was 94%; one risk factor, 82%; both risk factors, 65%. When all 1354 PSA determinations obtained for this cohort were considered, the patients with a proportion of PSAs < or = 0.5 ng/ml continued to increase until at least 48 months post-therapy. These data conformed to a median PSA half-life of 96.2 days. CONCLUSIONS: Prostate brachytherapy with Pd-103 achieves a high rate of biochemical and clinical control in patients with clinically organ-confined disease. PSA response following brachytherapy with low-dose-rate isotopes is protracted.

10-year biochemical (prostate-specific antigen) control of prostate cancer with (125)I brachytherapy.

PURPOSE: To report 10-year biochemical (prostate-specific antigen [PSA]) outcomes for patients treated with 125I brachytherapy as monotherapy for early-stage prostate cancer. METHODS AND MATERIALS: One hundred and twenty-five consecutively treated patients, with clinical Stage T1-T2b prostate cancer were treated with 125I brachytherapy as monotherapy, and followed with PSA determinations. Kaplan-Meier estimates of PSA progression-free survival (PFS), on the basis of a two consecutive elevations of PSA, were calculated. Aggregate PSA response by time interval was assessed. Comparisons were made to an earlier-treated cohort. RESULTS: The overall PSA PFS rate achieved at 10 years was 87% for low-risk patients (PSA < 10, Gleason Sum 2-6, T1-T2b). Of 59 patients (47%) followed beyond 7 years, 51 (86%) had serum PSAs less than 0.5 ng/mL; 48 (81%) had serum PSAs less than 0.2 ng/mL. Failures were local, 3.0%; distant, 3.0%. No patients have died of prostate carcinoma. The proportion of patients with a PSA < or =0.2 ng/mL continued to increase until at least 7-8 years posttherapy. A plot of PSA PFS against the proportion of patients achieving serum PSA of less than 0.2 ng/mL suggests a convergence of these two endpoints at 10 years. Patients treated in the era of this study (1988-1990) experienced a statistically improved PFS compared with an earlier era (1986-1987). This difference appears independent of patient selection, suggesting that the maturation of the technique resulted in improved biochemical control. CONCLUSION: With modern technique, monotherapy with 125I achieves a high rate (87%) of biochemical and clinical control in patients with low-risk disease at 10 years. The decline of PSA following brachytherapy with low-dose-rate isotopes can be protracted. Absolute PSA and PFS curves merge, and are comparable at 10 years.

Postoperative irradiation for the prevention of heterotopic bone: analysis of different dose schedules and shielding considerations.

Ninety-seven high risk hips were irradiated postoperatively for prevention of heterotopic bone (HTB) in the UCLA Department of Radiation Oncology from 1980 to 1988. Ninety-two hips in 82 patients were eligible for analysis with a minimum follow-up of 2 months and a median follow-up of 10 months. Forty-nine of the hips had porous coated ingrowth prostheses. From 1980 to 1986, 2 Gy fractions were used to deliver 20 Gy (8 hips), 12 Gy (1 hip), and 10 Gy (27 hips). Since December of 1986, 38 hips received 8 Gy in two increments and 18 hips received a single 7 Gy fraction. All porous ingrowth components were shielded with custom blocks. Six out of 92 hips developed clinically significant (Brooker grade 3 or 4 heterotopic bone). There was one clinically significant failure in 78 hips (1.3%) when irradiation was initiated before post-operative day (POD) #6 and shielding was properly placed. One clinical failure occurred in 38 hips which received 8 Gy in two increments. One clinical failure occurred out of the 18 hips treated with 7 Gy in one fraction. This failure could be related to block malposition. There were four clinical failures in the 36 hips treated with 2 Gy fractions to total doses of 10 Gy, 12 Gy, or 20 Gy. Three of these failures were associated with initiation of treatment after POD #5, and the fourth was related to block malposition. Unshielded trochanteric osteotomies resulted in five migrations and seven fibrous unions for a total non-osseous union rate of 12/36 (33%). Shielding of the remaining 28 trochanteric osteotomies resulted in a non-osseous union rate of 7% (0 migrations and 2 fibrous unions). There were no failures of union of components, and the only side effects noted in the series were the five trochanteric migrations. In conclusion, the use of 8 Gy in two increments or 7 Gy in one fraction was found to be as efficacious as conventional 2 Gy fractionation schemes with no increase in side effects. For optimal results, treatment should be implemented prior to POD #5 with shielding of the trochanteric osteotomy. Postoperative irradiation to prevent HTB can be used in hips with porous components using properly placed blocks to shield the porous region.

Determination of human beta(2)-adrenoceptor haplotypes by denaturation selective amplification and subtractive genotyping.

OBJECTIVE: beta(2)-Adrenoceptor haplotype may be better associated with asthma severity and drug response than a polymorphic variant at any single site. Because present methods of haplotype determination are time consuming and impractical for large population studies, we sought to develop a simple and efficient method of determining haplotype of 3 common polymorphisms at codons -19 (Arg/Cys), 16 (Arg/Gly) and 27 (Glu/Gln). DESIGN: Preliminary studies showed that the C/G base pair of the Arg(-19) allele increases the local melting temperature over the T/A base pair of the Cys(-19) allele by 3.6 degrees C and establishes a new local maximum denaturation temperature. By choosing a suitable denaturation temperature and appropriate primers and coupling them with restriction fragment length polymorphism (RFLP) analysis, we hypothesized that the genotype of one separately amplified allele followed by subtraction from the combined genotype of two alleles would yield the beta(2) haplotype in > 99% of the population. RESULTS: Haplotype determined by our method was in complete agreement with haplotype determined by cloning and sequencing in 29 samples. The frequencies of haplotype pairs in 78 healthy adults, according to our method, were in agreement with published values that were inferred, and were: RGE/CRQ, 26.9%; CRQ/CRQ, 25.6%; RGE/CGQ, 16.7%; CRQ/CGQ, 10.3%; RGE/RGE, 11.5%; CGQ/CGQ, 7.7%. The haplotype pair in one individual was RRE/CRQ (1.3%). CONCLUSION: Our method of determining beta(2)-adrenoceptor haplotype is simple, accurate and cost effective for haplotyping large populations.

Should brachytherapy be considered a therapeutic option in localized prostate cancer?

Contemporary prostate brachytherapy incorporates advances in computer analysis, imaging technology, and delivery apparatus, allowing exacting and reproducible results compared with historical approaches. The advances permit brachytherapy to be performed on a cost-effective, outpatient basis with low morbidity in the appropriately selected patient. Although unsettled questions remain regarding dosimetric issues, long-term outcomes, and morbidity, the weight of evidence to date appears to support the use of brachytherapy in selected patients. Brachytherapy may be considered a therapeutic option: as monotherapy for early-stage disease and also a boost following moderate doses of external beam irradiation for locally advanced disease.

Technical considerations in the use of prophylactic radiation therapy to prevent heterotopic bone formation.

The UCLA Department of Radiation Oncology radiated 97 hips at high risk for heterotopic bone (HTB) formation from 1980 through 1988. Adequate follow-up (minimum of 2 months, median, 10 months) is available in 92 hips (82 patients). These hips were treated with a variety of doses, fractionation schedules, and shielding techniques as treatment evolved over time. There was a total of 49 hips with porous coated ingrowth prostheses. These were all shielded with custom blocks. There were no untoward complications in this subgroup. Only 6 of the 92 hips evaluable developed clinically significant HTB. Five of these 6 failures can be attributed to initiating treatment after postoperative day (POD) 5 or block malposition. Of the 78 hips that initiated radiation therapy before POD 6 (with proper shielding), only 1 (1.3%) developed clinically significant HTB. Radiation therapy is very effective at preventing HTB formation following hip surgery when treatment is initiated within 4 days of surgery. To assure an optimal outcome, close attention to the technical aspects treatment is critical.

Complete sequence of the 43-kb human ribosomal DNA repeat: analysis of the intergenic spacer.

We have sequenced the remaining 12.4 kb of the 30-kb human ribosomal DNA intergenic spacer (IGS), which allows us to piece together both a complete IGS and a 43-kb rDNA unit. The sequence of the complete IGS reveals a collection of sequence motifs that can be correlated with functions known or expected to reside in the rDNA repeat: modulation of transcription, recombination, initiation of DNA replication, and chromosomal organization. Finally, we find that IGS accumulates variation at a much higher rate than the transcribed regions. This finding leads us to correlate sequence character with types of mutation and sequence context with rate of mutation.

Beyond ribosomal DNA: on towards the telomere.

We have sequenced and analyzed 8.3 kb of sequence adjacent and distal to the human ribosomal DNA (rDNA); this distal sequence connects to the rDNA cluster just 4 kb upstream of the first promoter and is shared among the acrocentric chromosomes and, at least in part, it is also present in other primates. The sequence differs in character from that of the rDNA intergenic spacer (IGS) in that it does not contain long stretches of either polypyrimidine or polypurine. However, just like the IGS, it contains numerous repetitive elements, including retroposed fragments of 28S rRNA and large pieces of the IGS. In addition, we show that the rDNA clusters are not interrupted by other sequences and do not recombine with this distal segment.