Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation

Two thirds of the Duchenne muscular dystrophy population haveeither gene deletions or duplications. The nondeletion/duplicationcases are most likely the result of point mutations or smalldeletions and duplications that cannot be easily identifiedby current strategies. The major obstacle in identifying smallmutations is due to the large size of the dystrophin gene. Weselectively screened 5 DMD exons containing CpG dinucleotidesin 110 DMD patients without detectable deletions or duplications.Nonsenses mutations are frequently due to a C- to -T transitionwithin a CG dinucleotide pair. To screen for the nonsense mutations,we used the heteroduplex method. Utilizing this approach, weidentified 2 different nonsense mutations and a single basedeletion all occurring in exon 19. This is the first reportof a clustering of small mutations in the the dystrophin gene.     

Direct comparison of umbilical cord blood versus bone marrow-derived endothelial precursor cells in mediating neovascularization in response to vascular ischemia.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.     

Review of Technology: Planning for the Development of Telesonography

Teleradiology allows contemporaneous interpretation of imaging exams performed at some distance from the interpreting radiologist. The transmitted images are usually static. However, there is benefit to real-time review of full-motion ultrasound (US) exams as they are performed. Telesonography is transmission of full-motion sonographic data to a remote site. We hypothesize that US exams, read after having been compressed utilizing Motion Picture Experts Group version 4 (MPEG-4) compression scheme, transmitted over the Internet as streaming multimedia, decompressed, and displayed, are equivalent in diagnostic accuracy to reading the examinations locally. MPEG-4 uses variable compression on each image frame to achieve a constant output bit rate. With less compression, the bit rate rises, and the only way the encoder can contain bit rate within the set bandwidth is by lowering frame rate or reducing image quality. We review the relevant technologies and industry standard components that will enable low-cost telesonography.     

Porphyrin Loading of Lipofuscin Granules in Inflamed Striated Muscle

To further the understanding of oxidative effects on inflammation injury to muscle fiber structure, fluorescent imaging analysis of human striated muscle tissues from a variety of inflammatory or postinflammatory etiologies was undertaken in a search for accumulated coproporphyrin, a red autofluorescent byproduct of heme biosynthesis that would theoretically be formed under oxidative insult. Using a differential excitation method of in situ analysis, porphyrin autofluorescence was detected in intact fibers within the context of the yellow autofluorescent subsarcolemmal lipofuscin granules. Relative measurements of porphyrin concentration in the granules from different patients indicated that the acute/subacute inflammatory specimens grouped significantly higher than the more chronic inflammatory and nonpathological specimens. Myoglobin was also found to be associated with the granules. Myoglobin heme iron could potentially serve as a Fenton reagent for the intracellular generation of hydroxyl radicals, which are responsible for the oxidation of the porphyrinogens. High-performance liquid chromatography analysis of extracted dense particles revealed coproporphyrin as the sole porphyrin present. The observation of coproporphyrin within lipofuscin granules, previously unreported, suggests that lipofuscin accumulation in striated muscle may begin under conditions of acute oxidative stress, as marked by the oxidation of extramitochondrial porphyrinogens that are immediately incorporated into the granules.     

CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae.

A genetic screen was devised to identify genes important for spindle pole body (SPB) and/or microtubule functions. Four mutants defective in both nuclear fusion (karyogamy) and chromosome maintenance were isolated; these mutants termed cik (for chromosome instability and karyogamy) define three complementation groups. The CIK1 gene was cloned and characterized. Sequence analysis of the CIK1 gene predicts that the CIK1 protein is 594 amino acids in length and possesses a central 300-amino-acid coiled-coil domain. Two different CIK1-beta-galactosidase fusions localize to the SPB region in vegetative cells, and antibodies against the authentic protein detect CIK1 in the SPB region of alpha-factor-treated cells. Evaluation of cells deleted for CIK1 (cik1-delta) indicates that CIK1 is important for the formation or maintenance of a spindle apparatus. Longer and slightly more microtubule bundles are visible in cik1-delta strains than in wild type. Thus, CIK1 encodes a SPB-associated component that is important for proper organization of microtubule arrays and the establishment of a spindle during vegetative growth. Furthermore, the CIK1 gene is essential for karyogamy, and the level of the CIK1 protein at the SPB appears to be dramatically induced by alpha-factor treatment. These results indicate that molecular changes occur at the microtubule-organizing center (MTOC) as the yeast cell prepares for karyogamy and imply that specialization of the MTOC or its associated microtubules occurs in preparation for particular microtubule functions in the yeast life cycle.     

Adaptive changes in early and late blind: a FMRI study of verb generation to heard nouns

Literacy for blind people requires learning Braille. Along with others, we have shown that reading Braille activates visual cortex. This includes striate cortex (V1), i.e., banks of calcarine sulcus, and several higher visual areas in lingual, fusiform, cuneus, lateral occipital, inferior temporal, and middle temporal gyri. The spatial extent and magnitude of magnetic resonance (MR) signals in visual cortex is greatest for those who became blind early in life. Individuals who lost sight as adults, and subsequently learned Braille, still exhibited activity in some of the same visual cortex regions, especially V1. These findings suggest these visual cortex regions become adapted to processing tactile information and that this cross-modal neural change might support Braille literacy. Here we tested the alternative hypothesis that these regions directly respond to linguistic aspects of a task. Accordingly, language task performance by blind persons should activate the same visual cortex regions regardless of input modality. Specifically, visual cortex activity in blind people ought to arise during a language task involving heard words. Eight early blind, six late blind, and eight sighted subjects were studied using functional magnetic resonance imaging (fMRI) during covert generation of verbs to heard nouns. The control task was passive listening to indecipherable sounds (reverse words) matched to the nouns in sound intensity, duration, and spectral content. Functional responses were analyzed at the level of individual subjects using methods based on the general linear model and at the group level, using voxel based ANOVA and t-test analyses. Blind and sighted subjects showed comparable activation of language areas in left inferior frontal, dorsolateral prefrontal, and left posterior superior temporal gyri. The main distinction was bilateral, left dominant activation of the same visual cortex regions previously noted with Braille reading in all blind subjects. The spatial extent and magnitude of responses was greatest on the left in early blind individuals. Responses in the late blind group mostly were confined to V1 and nearby portions of the lingual and fusiform gyri. These results confirm the presence of adaptations in visual cortex of blind people but argue against the notion that this activity during Braille reading represents somatosensory (haptic) processing. Rather, we suggest that these responses can be most parsimoniously explained in terms of linguistic operations. It remains possible that these responses represent adaptations which initially are for processing either sound or touch, but which are later generalized to the other modality during acquisition of Braille reading skills.     

Characterization of translational frame exception patients in Duchenne/Becker muscular dystrophy

The clinical progression of Duchenne muscular dystrophy (DMD)patients with deletions can be predicted in 93% of cases bywhether the deletion maintains or disrupts the translationalreading frame (frameshift hypothesis). We have identified andstudied a number of patients who have deletions that do notconform to the translational frame hypothesis. The most commonexception to the frameshift hypothesis is the deletion of exons3 to 7 which disrupts the translational reading frame. We identifieda Becker muscular dystrophy (BMD) patient, an intermediate,and a DMD patient with this deletion. In all three cases, dystrophinwas detected and localized to the membrane. One DMD patientwith an inframe deletion of exons 4–18 produced no dystrophin.One patient with a mild intermediate phenotype and a deletionof exon 45, which shifts the reading frame, produced no dystrophin.Two patients with large inframe deletions had discordant phenotypes(exons 3–41, DMD; exons 13–48, BMD), but both produceddystrophin that localized to the sarcolemma. The DMD patient,113, indicates that dystrophin with an intact carboxy terminuscan be produced in Duchenne patients at levels equivalent tosome Beckers. The dystrophin analysis from these patients, togetherwith patients reported in the literature, indicate that morethan one domain can localize dystrophin to the sarcolemma. Lastely,the data shows that although most patients show correlationof clinical severity to molecular data, there are rare patientswhich do not conform.     

BRCA1 and pancreatic cancer: pedigree findings and their causal relationships

Anecdotal reports and series studies indicate that 5-10% of pancreatic cancer (PC) cases are familial. In addition, PC is associated with a variety of hereditary cancer syndromes. PC appears to be an integral cancer in the hereditary breast-ovarian cancer (HBOC) syndrome, with most notice given to the role of BRCA2. Our purpose is to call attention to BRCA1, which also predisposes to PC. Using data from our familial breast cancer registry, we identified 19 BRCA1/2 families that contain PC affecteds in the pedigrees, 15 with BRCA1 mutations and 4 with BRCA2 mutations. The association between BRCA2 and pancreatic cancer is well established; however, a definite link with pancreatic cancer in families carrying a BRCA1 mutation has been far less studied. Thus, the focus of this report is on 9 of the 15 BRCA1 families, in which PC affecteds were either confirmed carriers of the BRCA1 mutation or were inferred as probable obligate BRCA1 mutation carriers. The numbers are small, but nevertheless illustrate the finding of others of an apparent association between PC and BRCA1-mutation-bearing families. Given the dismal prognosis of PC, with the only current hope for survival being through surgical extirpation of the pancreas prior to metastasis, it is prudent that we realize the potential predisposition toward PC via BRCA1, in the hope of early diagnosis and prevention.