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We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

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PURPOSE: We sought to compare patterns of full mutation repeat-length variability in the peripheral blood DNA of patients with fragile X syndrome to determine whether siblings possess mutation patterns more similar than those of unrelated patients. METHODS: Mutation patterns were visualized by Southern blot analysis and captured digitally with a phosphor imager. Novel comparison strategies based on overlapping profile plots and calculation of weighted mean CGG repeat values were used to assess mutation pattern similarity. RESULTS: Within the population that we analyzed of 56 patients with full mutation, mutation patterns were found to be more similar in siblings than in unrelated patients. CONCLUSION: These results indicate that repeat-length variability may be generated in a nonrandom manner and that familial factors influence this process.

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The fragile X syndrome is characterized at the molecular level by expansion and methylation of a CGG trinucleotide repeat located within the FMR1 locus. The tissues of most full mutation carriers are mosaic for repeat size, but these mutational patterns tend to be well conserved when comparing multiple tissues within an individual. Moreover, full mutation alleles are stable in cultured fibroblasts. These observations have been used to suggest that fragile X CGG repeat instability normally is limited to a period during early embryogenesis. DNA methylation of the repeat region is also believed to occur during early development, and some experimental evidence indicates that this modification may stabilize the repeats. To study the behavior of full mutation alleles in mitotic cells, we generated human-mouse somatic cell hybrids that carry both methylated and unmethylated full mutation FMR1 alleles. We observed considerable repeat instability and analyzed repeat dynamics in the hybrids as a function of DNA methylation, repeat length and cellular differentiation. Our results indicate that although DNA methylation does correlate with stability in primary human fibroblasts, it does not do so in the cell hybrids. Instead, repeat stability in the hybrids is dependent on repeat length, except in an undifferentiated cellular background where large alleles are maintained with a high degree of stability. This stability is lost when the cells undergo differentiation. These results indicate that the determinants of CGG repeat stability are more complex than generally believed, and suggest an unexpected role for cellular differentiation in this process.

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The vast majority of fragile-X full mutations are heavily methylated throughout the expanded CGG repeat and the surrounding CpG island. Hypermethylation initiates and/or stabilizes transcriptional inactivation of the FMR1 gene, which causes the fragile X-syndrome phenotype characterized, primarily, by mental retardation. The relation between repeat expansion and hypermethylation is not well understood nor is it absolute, as demonstrated by the identification of nonretarded males who carry hypomethylated full mutations. To better characterize the methylation pattern in a patient who carries a hypomethylated full mutation of approximately 60-700 repeats, we have evaluated methylation with the McrBC endonuclease, which allows analysis of numerous sites in the FMR1 CpG island, including those located within the CGG repeat. We report that the expanded-repeat region is completely free of methylation in this full-mutation male. Significantly, this lack of methylation appears to be specific to the expanded FMR1 CGG-repeat region, because various linked and unlinked repetitive-element loci are methylated normally. This finding demonstrates that the lack of methylation in the expanded CGG-repeat region is not associated with a global defect in methylation of highly repeated DNA sequences. We also report that de novo methylation of the expanded CGG-repeat region does not occur when it is moved via microcell-mediated chromosome transfer into a de novo methylation-competent mouse embryonal carcinoma cell line.

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The fragile X syndrome is predominantly caused by a large expansion of a CGG trinucleotide repeat in the promoter region of the FMR1 gene, which is associated with methylation and downregulation of transcription. The molecular diagnosis of this disorder is based on repeat size and methylation analysis of the FMR1 gene usually by Southern blot analysis. We describe a PCR-based method for the analysis of methylation of the FMR1 gene, which involves bisulfite treatment of DNA prior to amplification. Fifty-two normal and 48 affected, premutation, or mosaic males were analyzed in a blinded study by this method. A prospective study of 30 males suspected of fragile X was also performed. Amplification specific for the methylated FMR1 sequence was readily observed in all individuals with a full mutation, whereas all normal and premutation individuals showed only amplification-specific for the unmethylated sequence, thus, allowing affected and unaffected males to be distinguished. A full mutation in the presence of mosaicism was also detectable by this method. Methylation-specific PCR appears to be a rapid and reliable tool for the diagnosis of fragile X males.

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Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.

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Prader-Willi syndrome (PWS) is most often the result of a deletion of bands q11.2-q13 of the paternally derived chromosome 15, but it also occurs either because of maternal uniparental disomy (UPD) of this region or, rarely, from a methylation imprinting defect. A significant number of cases are due to structural rearrangements of the pericentromeric region of chromosome 15. We report two cases of PWS with UPD in which there was a meiosis I nondisjunction error involving an altered chromosome 15 produced by both a translocation event between the heteromorphic satellite regions of chromosomes 14 and 15 and recombination. In both cases, high-resolution banding of the long arm was normal, and FISH of probes D15S11, SNRPN, D15S10, and GABRB3 indicated no loss of this material. Chromosome heteromorphism analysis showed that each patient had maternal heterodisomy of the chromosome 15 short arm, whereas PCR of microsatellites demonstrated allele-specific maternal isodisomy and heterodisomy of the long arm. SNRPN gene methylation analysis revealed only a maternal imprint in both patients. We suggest that the chromosome structural rearrangements, combined with recombination in these patients, disrupted normal segregation of an imprinted region, resulting in uniparental disomy and PWS.

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Russell-Silver syndrome (RSS) is a pattern of malformation characterized by intrauterine and postnatal growth retardation, limb asymmetry, triangular face, and hypospadias. We report on a patient, from a triplet pregnancy, who was one of identical male twins discordant for RSS. R.B. was a 710-g male born at 33 weeks of gestation, with hypospadias, chordee, and undescended testes. He had a normal 46,XY karyotype and no renal abnormalities. Female triplet A weighed 1,843 g, and male triplet B weighed 1,920 g. Both had normal physical findings and neonatal period. R.B. was first seen by us at age 6 7/12 years with short stature, triangular and asymmetric face, lower limb length discrepancy, and surgically repaired genital anomalies. Growth hormone testing results were normal. At age 8 7/12 years the brothers appeared physically identical except for size, with a height differential of 114.25 vs. 121.5 cm. Testing to establish biological zygosity was performed using VNTR (variable number tandem repeat) DNA probes YNH24 (D2S44), CMM101 (D14S13), EFD52 (D17S26), TBQ7 (D10S28), and 3'HVR (D16S85), PCR loci MCT118 (D1S80), and HLA-DQ alpha. These data indicate a > 99.99% probability of triplets B and C being monozygotic twins. While most occurrences of RSS are sporadic, familial cases suggesting autosomal dominance have been reported. Three other cases of probable monozygotic twins with RSS have been described. The significance of this confirmation of discordance in determining the cause of RSS is discussed.

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Fragile X syndrome is the most common form of inherited mental retardation and results from the transcriptional inactivation of the FMR1 gene. In the vast majority of cases, this is caused by the expansion of an unstable CGG repeat in the first exon of the FMR1 gene. We describe here a phenotypically atypical case of fragile X syndrome, caused by a deletion that includes the entire FMR1 gene and > or = 9.0 Mb of flanking DNA. The proband, RK, was a 6-year-old mentally retarded male with obesity and anal atresia. A diagnosis of fragile X syndrome was established by the failure of RK's DNA to hybridize to a 558-bp PstI-XhoI fragment (pfxa3) specific for the 5'-end of the FMR1 gene. The analysis of flanking markers in the interval from Xq26.3-q28 indicated a deletion extending from between 160-500 kb distal and 9.0 Mb proximal to the FMR1 gene. High-resolution chromosome banding confirmed a deletion with breakpoints in Xq26.3 and Xq27.3. This deletion was maternally transmitted and arose as a new mutation on the grandpaternal X chromosome. The maternal transmission of the deletion was confirmed by FISH using a 34-kb cosmid (c31.4) containing most of the FMR1 gene. These results indicated that RK carried a deletion of the FMR1 region with the most proximal breakpoint described to date. This patient's unusual clinical presentation may indicate the presence of genes located in the deleted interval proximal to the FMR1 locus that are able to modify the fragile X syndrome phenotype.

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Analysis of 84 human X chromosomes for the presence of interrupting AGG trinucleotides within the CGG repeat tract of the FMR1 gene revealed that most alleles possess two interspersed AGGs and that the longest tract of uninterrupted CGG repeats is usually found at the 3' end. Variation in the length of the repeat appears polar. Alleles containing between 34 and 55 repeats, with documented unstable transmissions, were shown to have lost one or both AGG interruptions. These comparisons define an instability threshold of 34-38 uninterrupted CGG repeats. Analysis of premutation alleles in Fragile X syndrome carriers reveals that 70% of these alleles contain a single AGG interruption. These data suggest that the loss of an AGG is an important mutational event in the generation of unstable alleles predisposed to the Fragile X syndrome.

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We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.

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Decreased cardiac performance is a known complication of diabetes mellitus, but the detailed molecular mechanisms that are responsible for this contractile abnormality are only incompletely explored, and cardiac gene products of known function, which are markedly and actively insulin responsive, have not been described. Recently, we found that creatine kinase (CK) enzyme activity and CK-M subunit mRNA levels are decreased in the heart of rats with experimental diabetes mellitus. These abnormalities could be restored to normal with chronic insulin administration. The CK-M and CK-B genes are expressed in the heart, and we wanted to determine whether diabetes also induces a change in CK-B mRNA levels. Quantitation of CK-M and CK-B mRNA levels on Northern blots with specific cDNA probes showed that, in diabetic hearts, CK-B mRNA levels represent only 19.8% of control levels and are more markedly depressed than CK-M mRNA levels, which are 46.5% of control values. Acute injection of insulin led to a significant 1.6-fold increase in CK-M mRNA and a 2.2-fold increase of CK-B mRNA 5 h after insulin injection. CK-M mRNA levels were restored to normal within 12 h, but 48 h were required to restore CK-B mRNA levels to normal values. After 1 mo of insulin therapy, CK-B mRNA levels had risen 9.7-fold, exceeding normal values by 90%, whereas CK-M mRNA levels were at the normal level as previously shown. CK enzyme activity showed only a small response to insulin administration 48 h postinjection. Diabetes leads therefore to a marked lowering of CK-M and CK-B mRNA levels in the rat heart.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have investigated problems encountered when using the polymerase chain reaction (PCR) to detect recombinants in gene targeting experiments in which homologous recombination occurs between incoming DNA and an endogenous target sequence. The targeting system studied was designed to correct a human sickle-cell beta-globin-encoding gene (HBBS) on human chromosome 11 by replacing the defective gene with incoming DNA carrying normal HBB sequences. Two sets of experiments were executed which led to the isolation of a clone of cells having the sickle-cell gene corrected. We found that a positive control system was essential to allow a real targeting event to be distinguished from various types of false positives that arise during the diagnostic PCR.

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As a step toward using gene targeting for gene therapy, we have corrected a human beta S-globin gene to the normal beta A allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the beta S-globin allele. A beta A-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total of approximately 29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the beta S allele to beta A was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5' to the human beta A-globin gene. Thus gene targeting can correct a beta S allele to beta A, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

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The Popovich Scale holds great promise for use in home healthcare. Having established reliability and validity makes the tool valuable in promoting high quality nursing care at home. It is applicable to all older adults and their caregivers regardless of medical problems, nursing diagnoses, and regulatory agencies.

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