Segregation analysis of thalassemia in Ferrara

Segregation analysis of 996 families in which the gene for beta-thalassemia was segregating showed no distortion of expected Mendelian ratios. No appreciable frequency of sporadic cases was detected. It was suggested that segregation distortion is not a mechanism which contributes to the maintenance of polymorphism in the beta-thalassemia system in the population of the Ferrara area.     

S1 nuclease hybrid analysis of mitochondrial DNA amplified by long-distance PCR: rapid screening for small-scale rearrangements

We report on a method suitable for screening large regions (>3 kb) of mtDNA for structural changes of <500 bp and their localization. Heteroduplexes consisting of a wild-type and a mutant strand are cleaved by S1nuclease when single-stranded loops are present due to deletions or duplications/insertions. This strategy was successfully applied to screen the muscle mtDNA of 20 patients with mitochondrial encephalomyopathies. In three of them, an altered cleavage pattern was observed caused by a homoplasmic 9 bp deletion as shown by subsequent mapping and sequencing studies.     

Efficient identification of recombinant adenoviruses by direct plaque screening

With the increasing use of adenoviral vectors for gene transfer and gene therapy, it is crucial to produce specific recombinant adenoviruses more efficiently. One of the most time-consuming steps is to screen the unique recombinant adenovirus among the plaques, in which each plaque has to be amplified individually in kidney 293 cells in order to obtain enough adenoviruses for DNA extraction and subsequent identification. We have developed a fast and simple way to screen recombinant adenoviruses by direct plaque screening. The direct plaque-screening method employed DNA obtained from the viral plaque itself for PCR amplification and subsequent adenoviral recombinant identification. The time and labor involved in these steps has been significantly reduced.     

Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases

Previous analyses indicated a high level of oxidative base modification in mitochondrial DNA, the extent of which raised questions about the methodological validity and biological implications. In the present study DNA was isolated from rat liver mitochondria under carefully controlled conditions, and the extent of base oxidation, DNA fragmentation, and nuclear DNA contamination were analyzed. DNA isolated from intact mitochondria treated with DNase consisted of 16.3 kilobase pairs, mostly circular, mitochondrial DNA molecules and a mixture of nuclear and mitochondrial DNA fragments, as identified by agarose gel electrophoresis and hybridization. High-performance liquid chromatography in combination with electrochemical detection confirmed that the overall level of 8-hydroxy-2'-deoxyguanosine, a marker commonly used in the analysis of base oxidation, is higher in mitochondrial than in nuclear DNA. Importantly, 8-hydroxy-2'-deoxyguanosine is relatively scarce in the 16.3 kilobase pair mitochondrial DNA molecules (0.051 pmol/microgram) but is present in high levels in mitochondrial DNA fragments (0.741 pmol/microgram). The fragments constitute about 18% of total mitochondrial DNA. The antitumor agent bleomycin, which binds to DNA, forms an iron complex capable of transferring electrons from Fe2+ to molecular oxygen. Exposure of mitochondria to bleomycin and iron resulted in nicking but not in a significant increase in base oxidation of 16.3 kilobase pair mitochondrial DNA, whereas the amount and the oxidation level of fragmented mitochondrial DNA significantly increased. These findings are relevant for a better understanding of the role of mitochondria in aging and various diseases and are consistent with the notion that despite the overall high DNA oxidation level, mitochondria can faithfully proliferate.     

Comparison of different methods to produce single-strand DNA for identification of canned tuna by single-strand conformation polymorphism analysis

By using single-strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single-strand DNA (ssDNA): (i) Denaturation of double-strand DNA (dsDNA) by formamide and alkali, (ii) two-step asymmetrical PCR, (iii) one-step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).     

Population-specific screening by mutation analysis for diseases frequent in Ashkenazi Jews

Three-dimensional (3D) imaging of intracellular rhodamine 123 fluorescence distribution was performed by means of confocal laser scanning microscopy (CLSM). Human IGR melanoma cells grown in monolayer or multicellular spheroid culture were studied for elucidating mitochondrial membrane potential characteristics, and cell and nucleus volume dimensions. Microspheres 6 microns in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sections as obtained by CLSM. Accurate optical slicing is only possible taking into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination or cover slip correction and the temperature dependence of the immersion medium. While 3D imaging of optical slices can be carried out showing the original shape of the object being tested without physical distortion, 3D images of microspheres show well-reproducible structures of rhodamine B fluorescence. These can be explained by a superposition of two effects, namely scattering of the fluorescence light and a gradient of the electromagnetic field strength of the laser beam due to the shape of the object. 3D imaging of optical slices of IGR cells in monolayer or multicellular spheroid culture, which have been loaded with rhodamine 123, show the location of the dye predominantly within the cytoplasm of the cells with a remarkable heterogeneity of fluorescence intensity within and between single cells, indicating differences in the mitochondrial membrane potential and thus in the metabolic activity. Due to the heterogeneity of the cell shape the cell nucleus occupies between 4 and 14% of the total cell volume. These data reveal calibrated 3D imaging as a valuable noninvasive tool to visualize the heterogeneity of cell parameters under different cell culture conditions.     

Sperm DNA analysis in a Friedreich ataxia premutation carrier suggests both meiotic and mitotic expansion in the FRDA gene

BACKGROUND: Puumala virus infection (nephropathia epidemica) is a disease in the group of hemorrhagic fevers with renal syndrome causing ocular manifestations, e.g. transient myopia and changes in intraocular pressure. PATIENT AND METHODS: Comprehensive and repeated ophthalmic examinations of a previously healthy 35-year-old woman with acute Puumala virus infection were performed. Special attention was paid to ophthalmic A-scan ultrasound measurements and simultaneous blood chemistry tests. RESULTS: The ocular manifestations of this patient's illness included transient myopia, low intraocular pressure, conjunctival hemorrhages and changes of intraocular dimensions. There was forward movement of the anterior diaphragm and thickening of the crystalline lens, which occurred simultaneously with prominent fluctuations in the electrolyte balance, especially potassium. CONCLUSIONS: The observed changes in intraocular dimensions may have been caused by simultaneous fluctuations in electrolyte and osmotic balance, which could explain the myopic shift. The symmetry of the ocular measurements implied a systemic infection as the underlying reason for the ophthalmic symptoms and signs.     

A multiple-stage screening procedure for the identification of childhood anxiety disorders.

Examined the usefulness of a multiple-stage screening procedure in the identification of youth experiencing elevated levels of anxiety. 758 children in Grades 4–7 completed a 3-stage screening procedure. During the 1st and 2nd stages, Ss completed the Revised Children's Manifest Anxiety Scale; the 3rd stage involved a clinical interview with the S based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children. Results were similar to those found when the procedure was used to identify children with depression. Nearly 18% of the original sample was identified in the 1st stage. Of the 124 children who completed the 2nd stage, over 69% met the cutoff criteria for the 3rd stage interview, where 41% of that subset exhibited an anxiety disorder. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Gene identification and DNA sequence analysis in the GC-poor 20 megabase region of human chromosome 21

In contrast to the distal half of the long arm of chromosome 21, the proximal half of approximately 20 megabases of DNA, including 21q11-21 bands, is low in GC content, CpG islands, and identified genes. Despite intensive searches, very few genes and cDNAs have been found in this region. Since the 21q11-21 region is associated with certain Down syndrome pathologies like mental retardation, the identification of relevant genes in this region is important. We used a different approach by constructing microdissection libraries specifically for this region and isolating unique sequence microclones for detailed molecular analysis. We found that this region is enriched with middle and low-copy repetitive sequences, and is also heavily methylated. By sequencing and homology analysis, we identified a significant number of genes/cDNAs, most of which appear to belong to gene families. In addition, we used unique sequence microclones in direct screening of cDNA libraries and isolated 12 cDNAs for this region. Thus, although the 21q11-21 region is gene poor, it is not completely devoid of genes/cDNAs. The presence of high proportions of middle and low-copy repetitive sequences in this region may have evolutionary significance in the genome organization and function of this region. Since 21q11-21 is heavily methylated, the expression of genes in this region may be regulated by a delicate balance of methylation and demethylation, and the presence of an additional copy of chromosome 21 may seriously disturb this balance and cause specific Down syndrome anomalies including mental retardation.     

Developmental change in red blood cell volume: implication in screening infants and children for iron deficiency and thalassemia trait

The mean corpuscular volumen when determined by electronic counter is an accurate tool for identification of children with microcytosis due to either iron deficiency or thalassemia trait. The purpose of this report is to describe the normal developmental changes in MCV that occur in children afler 6 months of age. In 211 healthy infants and children screened to exclude those with borderline or overt iron deficiency, thalassemia trait, or hemoglobinopathy, we found that the lower limit of normal for MCV is 70 ft between 10 and 17 months of age and that there is a gradual increase of MCV with age; the lower limit is 74 between 1 1/2 and 4 years and 76 between 4 and 7 years. All of these values are well below the minimum adult level of 80 fl.     

Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.     

A simplified screening test for the identification of individuals with diminished vision in developing countries

The mammalian visual system, particularly retinal ganglion cells, has been used for studying the functions of neurotrophic factors on neurons for many years. The major biological effects of neurotrophic factors on retinal ganglion cells observed so far are the promotion of viability and axonal regeneration. However, there are still some controversies regarding the effects of neurotrophic factors on retinal ganglion cells in the literature. This review is aimed to summarize the available information on the biological actions of these neurotrophic factors on survival and axonal regeneration of retinal ganglion cells and the expressions of neurotrophic factor receptors in the retina. Generally, brain-derived neurotrophic factor, neurotrophin-4/5, fibroblast growth factor and glial cell line-derived neurotrophic factor increase the survival of retinal ganglion cells while the effect of ciliary neurotrophic factor on the viability of adult retinal ganglion cells is controversial. The ciliary neurotrophic factor is the only effective factor in promoting long distance axonal regeneration of retinal ganglion cells whereas brain-derived neurotrophic factor and neurotrophin-4/5 only enhance neurite sprouting within the retina.     

Flow cytometric DNA analysis of invasive carcinomas detected by screening mammography: use of specimen mammography-guided fine-needle aspirates

Clinical studies of flow cytometric DNA analysis of breast carcinoma are often limited by the lack of fresh tissue samples from smaller, nonpalpable carcinomas. In addition, most studies measuring DNA in the current literature focus on larger palpable masses that may have less relevance to the smaller, nonpalpable lesions. A prospective study of flow cytometric DNA analysis of in vitro specimen mammography-guided fine-needle aspirates (FNAs) of 103 consecutive nonpalpable invasive carcinomas detected by screening mammography was performed to determine efficacy and explore associations with mammographic and pathological features. For 62 (60%) lesions for which DNA analysis on both FNA and standard tissue incision samples was performed, there was excellent (89%) agreement for ploidy determinations (kappa=0.77) and poor agreement for S-phase percentage determinations (kappa=0.23). Specimen mammography-guided FNA analysis detected aneuploidy in 36% of lesions overall, including 34% of 41 lesions for which standard tissue procurement was not possible. Mammographic microcalcifications had a higher aneuploid rate (14 of 28 lesions, 50%) as compared with soft tissue masses (22 of 75 lesions, 29%), P < 0.01. Lobulated masses with indistinct margins had a higher aneuploid rate (5 of 6 lesions, 83%) as compared with more irregular, spiculated masses (7 of 27 lesions, 26%), P < 0.01. The aneuploidy rate was independent of specific histological diagnosis, lesion size, nuclear grade, or nodal or estrogen receptor status. Flow cytometric DNA analysis of mammographic lesion-specific, fresh, cellular FNA samples obtained under specimen mammographic guidance can assess early invasive carcinomas when gross fresh tissue procurement is not possible. This technique could be incorporated into larger clinical follow-up studies to determine the prognostic significance of flow cytometric DNA analysis for these very early breast carcinomas.     

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level

The genus Acinetobacter is phenotypically rather homogeneous, but genotypically heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplified large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypically related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.     

High-throughput screening for known mutations by automated analysis of single sequencing reactions

This work shows that single sequencing reactions analyzed on an automated DNA sequencer can be an efficient way of screening PCR products for known mutations. We have analyzed a mutation in exon 10 of the human aromatase gene and show that an unambiguous genotype could be elucidated in more than 90% of the analyzed samples. Compared to analysis by full sequencing, 4 times more samples can be analyzed per gel, so that the sample capacity of the gel is approaching that of alternative gel-based methods for genotype analysis. Unlike many of these, the method offers direct identification of the variant sequence position and on-line analysis without the need of post-electrophoretic processing of the gel.