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PubMed2026

Clinical implementation of next-generation sequencing in tertiary health system: the Rijeka retrospective study.

OBJECTIVE: This study aimed to assess the diagnostic yield, clinical indications, and utility of next-generation sequencing (NGS) testing since its implementation through collaboration between the University of Rijeka Faculty of Medicine and the Clinical Hospital Centre Rijeka. MATERIALS AND METHODS: This retrospective study included patients referred between 2018 and 2023 from the Clinical Hospital Centre Rijeka to the University of Rijeka Faculty of Medicine for genetic testing, primarily using exome sequencing. RESULTS: Between April 2018 and December 2023, 412 patients were referred for exome sequencing, of whom 353 (85.7%) underwent diagnostic genetic testing. A notable increase in tests ordered was observed over time. Patients were most frequently referred from Pediatrics (55.0%), Neurology (29.5%), Cardiology (7.4%), Ophthalmology (3.4%), and others (4.7%). A diagnosis was confirmed in 103/353 patients, corresponding to an overall diagnostic yield of 29.2%, and an adjusted diagnostic yield of 27.2% after collapsing related individuals into single family units. In these confirmed cases, 83 distinct disorders involving 71 unique genes were identified, with most patients showing heterozygous variants and several recurrent disorders and genes. Variants of uncertain significance were reported in 35/353 (9.9%) patients. CONCLUSION: The 27.2% diagnostic yield demonstrates effective integration of NGS into tertiary clinical practice. The recent introduction of medical genetics specialization is expected to further improve referral quality, variant interpretation, and overall diagnostic outcomes.

exome sequencing↗
PubMed1988

Sequencing of trimodality therapy [cis-diamminedichloroplatinum(II)/hyperthermia/radiation] as determined by tumor growth delay and tumor cell survival in the FSaIIC fibrosarcoma.

In order to improve local control of tumors over that achievable with local hyperthermia and radiation, we are testing the use of systemic cis-diamminedichloroplatinum(II) (CDDP) in conjunction with the other two modalities. In the FSaIIC fibrosarcoma, growth delay experiments indicated that the use of any two modalities resulted in at least additive effects on growth delay. When the trimodality treatment was tested, the sequence CDDP followed by hyperthermia followed by X-ray produced a growth delay of approximately 25 days which was superior to the growth delay produced by the sequences CDDP, X-ray, and hyperthermia (19 days) and X-ray, CDDP and hyperthermia (14 days). In excision experiments, also performed in the FSaIIC tumor system, we again observed clearly superior cytotoxicity in the sequence CDDP, hyperthermia, and X-rays over the other sequences tested. Our results indicate that scheduling CDDP just prior to heating and following the heat treatment with the radiation fractions results in the best tumor cell kill, probably because this sequence takes maximum advantage of the radiosensitizing properties of the combined heat-CDDP treatment. In addition, the strong cytotoxic interaction between CDDP and hyperthermia is also optimized by this scheduling. We believe these results have significant clinical implications.

Animals↗
PubMedDate not supplied

Picture and motor sequencing in Parkinson's disease.

A recent report found that the discrepancy between scaled scores on the Vocabulary and Picture Arrangement subtests of the Wechsler Adult Intelligence Scale-Revised (WAIS-R) was much larger for patients with Parkinson's disease than for healthy controls or patients with Alzheimer's disease. On this basis, it was argued that Parkinson's disease causes a specific deficit in cognitive sequencing that occurs even when the memory and speed requirements of the task are minimal. However, other work indicated that Parkinson's disease patients are almost as severely impaired on other Performance subtests from the WAIS-R that do not require sequencing. In the present study, an extremely simple, untimed test of picture sequencing and a version of the Luria three-step test of motor sequencing were employed. Parkinson's disease patients were impaired on both tasks. The extent of their impairments on picture and motor sequencing were positively correlated, and the severity of deficits on both sequencing tests was related to global mental status and to performance on the Wisconsin Card Sorting Test, but not to neurologic measures of disease severity. However, the patients' performance on the picture sequencing test but not on the motor sequencing test was related to performance on Benton's Facial Recognition Test. These results demonstrate the existence of a generalized sequencing deficit in Parkinson's disease that appears dissociable from impairment in performing simple motor acts.

Aged↗
PubMed1989

A statistical test of phylogenies estimated from sequence data.

A simple approach to testing the significance of the branching order, estimated from protein or DNA sequence data, of three taxa is proposed. The branching order is inferred by the transformed-distance method, under the assumption that one or two outgroups are available, and the branch lengths are estimated by the least-squares method. The inferred branching order is considered significant if the estimated internodal distance is significantly greater than zero. To test this, a formula for the variance of the internodal distance has been developed. The statistical test proposed has been checked by computer simulation. The same test also applies to the case of four taxa with no outgroup, if one considers an unrooted tree. Formulas for the variances of internodal distances have also been developed for the case of five taxa. Conditions are given under which it is more efficient to add the sequence of a fifth taxon than to do 25% more nucleotide sequencing in each of the original four. A method is presented for combining analyses of disparate data to get a single P value. Finally, the test, applied to the human-chimpanzee-gorilla problem, shows that the issue is not yet resolved.

Animals↗
PubMed1995

Transcription and processing of the rodent ID repeat family in germline and somatic cells.

ID elements comprise a rodent SINE (short interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected in various rat tissues, including brain and testes. We have analysed the nucleotide sequences of testes- and brain-derived ID transcripts isolated by size-fractionation, C-tailing and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA-transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable in both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification.

Animals↗
PubMed1990

Interrelationships among major protistan groups based on a parsimony network of 5S rRNA sequences.

To test the validity of the maximum parsimony approach to discern protistan interrelationships, we have derived an optimal network of 16S-like rRNA sequences using our parsimony algorithm and compared it with those reported using the distance matrix method. We have also derived an optimal network topology of 50 5S rRNA sequences through an interactive search using our algorithm. In both these networks, the kinetoplastids and euglenoids form a linkage group with Dictyostelium emerging from its neighbourhood. The cryptophytes, dinoflagellates and chromophytes and green algae emerge as independent lines suggesting that plastids arose more than once during protistan evolution. The large 5S rRNA tree further indicates independent origins of mesozoa and metazoa; kinetoplastids and ciliates; and diphyletic origin of fungi. Comparatively close positions of charales and land plants, chytrids and Zygomycetes, Physarum and amoeba, and red algae and green algae are also seen in this network.

Animals↗
PubMed1992

Molecular typing of HLA-B27 alleles.

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701--B*2706). These changes are clustered in alpha 1 and alpha 2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoelectric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.

Alleles↗
PubMed1992

Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB.

The sequence of the Escherichia coli OsmB protein was found to resemble that of the C-terminal region of the beta amyloid protein of Alzheimer's disease, which seems to be the major determinant of its unusual structural and solubility properties. A peptide corresponding to residues 28-44 of the OsmB protein was synthesized, and its conformational properties and aggregation behavior were analyzed. The peptide OsmB(28-44) was shown to form amyloid fibrils, as did two sequence analogs designed to test the sequence specificity of fibril formation. These fibrils bound Congo red, and two of the peptides showed birefringence. The peptide fibrils were analyzed by electron microscopy and Fourier transform infrared spectroscopy. Subtle differences were observed which were not interpretable at the molecular level. The rate of fibril formation by each peptide was followed by monitoring the turbidity of supersaturated aqueous solutions. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, during which the solution became viscous and turbid due to the presence of insoluble fibrils. The observation of a kinetic barrier to aggregation is typical of a crystallization event. The delay period could be eliminated by seeding the supersaturated solution with previously formed fibrils. Each peptide could be nucleated by fibrils formed from that same peptide, but not by fibrils from closely related sequences, suggesting that fibril growth requires specific hydrophobic interactions. It appears likely that this repeated sequence motif, which comprises most of the OsmB protein sequence, dictates the structure and possibly the function of that protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1978

RNA sequencing with radioactive chain-terminating ribonucleotides.

A rapid method for determining nucleotide sequences in RNA is described. It employs the 3'-deoxy analogues of the ribonucleoside triphosphates as specific chain terminators during RNA synthesis. For example, the inclusion of 3'-deoxyuridine 5'-triphosphate in an RNA synthesis reaction in addition to the four usual ribonucleoside triphosphate precursors results in the synthesis of a set of different-length product strands that terminate in a 3'-deoxyuridine that has been incorporated in place of uridine. To sequence an RNA, four separate reactions are run, each employing a different 3'-deoxy terminator. Parallel electrophoretic analysis of the resulting four sets of specifically terminated product chains leads to a direct reading of the nucleotide sequence. We tested this method by sequencing MDV-1 (-) RNA, a molecule that is synthesized in vitro by phage Qbeta replicase. The sequence read from the resulting gels agreed completely with the known sequence of MDV-1 (-) RNA. The bands in some regions of the sequencing gels were unusually close to one another, as has also been observed in other rapid sequencing procedures, making order assignment in these regions very difficult. Because the secondary structure of MDV-1 (-) RNA was known, it was shown that the compression of the bands is due to the persistence of secondary structures during electrophoresis. Thus, structured regions of nucleic acids may introduce difficulties for sequencing techniques that employ the currently available methods of gel electrophoresis.

Base Sequence↗
PubMed1988

The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen.

The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus. The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAGQAKKKKLD, and the sequence AVKRPAATKKAGQAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism.

Amino Acid Sequence↗
PubMed1982

Selective binding of actinomycin D and distamycin A to DNA.

The exact sites at which a number of drugs inhibit the nick translation of DNA by E.coli DNA polymerase-I have been pinpointed. In order to do this, a method has been developed for sequencing double-stranded plasmid DNA from the site of a specifically induced nick. The initial experiments have concentrated on analysis of drug inhibition of nick translation in a 200 nucleotide region near the Eco Rl origin of pBR313. Many drugs were found to inhibit nick translation in a highly sequence specific manner. For actinomycin D, significant inhibition occurred at just four sites in the nucleotide sequence under test and only one sequence (pGpCpGpCpGpGp) gave really strong inhibition. Distamycin A gave a different pattern of inhibition with particularly strong stops in just two of the many A-T rich regions in the DNA. Experiments with caffeine suggest that factors in addition to primary sequence are important in determining where major inhibition occurs.

Adenoviruses, Human↗