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PubMed2024

Association analysis of mitochondrial DNA heteroplasmic variants: Methods and application.

We rigorously assessed a comprehensive association testing framework for heteroplasmy, employing both simulated and real-world data. This framework employed a variant allele fraction (VAF) threshold and harnessed multiple gene-based tests for robust identification and association testing of heteroplasmy. Our simulation studies demonstrated that gene-based tests maintained an appropriate type I error rate at &#x3b1;&#x202f;=&#x202f;0.001. Notably, when 5&#x202f;% or more heteroplasmic variants within a target region were linked to an outcome, burden-extension tests (including the adaptive burden test, variable threshold burden test, and z-score weighting burden test) outperformed the sequence kernel association test (SKAT) and the original burden test. Applying this framework, we conducted association analyses on whole-blood derived heteroplasmy in 17,507 individuals of African and European ancestries (31&#x202f;% of African Ancestry, mean age of 62, with 58&#x202f;% women) with whole genome sequencing data. We performed both cohort- and ancestry-specific association analyses, followed by meta-analysis on both pooled samples and within each ancestry group. Our results suggest that mtDNA-encoded genes/regions are likely to exhibit varying rates in somatic aging, with the notably strong associations observed between heteroplasmy in the RNR1 and RNR2 genes (p&#x202f;<&#x202f;0.001) and advance aging by the Original Burden test. In contrast, SKAT identified significant associations (p&#x202f;<&#x202f;0.001) between diabetes and the aggregated effects of heteroplasmy in several protein-coding genes. Further research is warranted to validate these findings. In summary, our proposed statistical framework represents a valuable tool for facilitating association testing of heteroplasmy with disease traits in large human populations.

Humans↗
PubMedDate not supplied

In vivo measurement of water self diffusion in the human brain by magnetic resonance imaging.

A new pulse sequence for in vivo diffusion measurements by magnetic resonance imaging (MRI) is introduced. The pulse sequence was tested on phantoms to evaluate the accuracy, reproducibility and inplane variations. The sensitivity of the sequence was tested by measuring the self diffusion coefficient of water with different temperatures. This phantom study showed that the water self diffusion could be measured accurately and that the inplane deviation was less than +/- 10 per cent. Seven healthy volunteers were studied with a 10 mm thick slice through the lateral ventricles, clear differences between grey and white matter as well as regional differences within the white matter were seen. In two patients with infarction, alternations in water self diffusion were seen in the region of the infarct. Likewise, pronounced changes in brain water self diffusion were observed in a patient with benign intracranial hypertension. The results indicate that brain water self diffusion can be measured in vivo with reasonable accuracy. The clinical examples suggest that diffusion measurements may be clinically useful adding further information about in vivo MR tissue characterization.

Adolescent↗
PubMed1993

The genes of major lysosomal membrane glycoproteins, lamp-1 and lamp-2. 5'-flanking sequence of lamp-2 gene and comparison of exon organization in two genes.

Human lysosomal membrane glycoproteins lamp-1 and lamp-2 are the major sialoglycoproteins present in lysosomal membranes. The expression of lamp-2 molecules is uniquely regulated, whereas lamp-1 is constitutively synthesized. In order to investigate the unique expression of lamp-2, and the gene evolution of lamp-1 and lamp-2, we isolated genomic phage clones encoding these glycoproteins. Comparison of the genomic and cDNA sequences revealed that the lamp-2 gene consists of nine exons. The transcriptional start site of the lamp-2 gene was determined by primer extension analysis. In order to locate the transcriptional regulatory region of this gene, various regions of 5'-sequences were tested for promoter activity using chloramphenicol acetyltransferase as a reporter molecule. The results revealed that the 5'-flanking sequence from -172 to -20 base pairs has strong promoter activity. In this sequence, potential SP1 and AP-1 binding sites and CAAT boxes are found. Most notably, the promoter activity is suppressed if the 5' farther upstream KpnI repeat sequence is included in the tested 5'-flanking sequence, thus suggesting that the KpnI repeat sequence may have some regulatory function in the lamp-2 gene expression. Comparison of the exon organization of human lamp-2 and lamp-1 genes, or chicken lamp-1 gene, reveals that these two proteins utilize the same exon phase in corresponding introns. Furthermore, each exon encodes almost identical portions of the proteins. On the other hand, the amino acid sequence of human lamp-1 is more homologous to lamp-1 of other species than it is to human lamp-2. These results indicate that lamp-1 and lamp-2 genes were most likely produced by duplication of a primordial gene, which took place early in evolution.

Amino Acid Sequence↗
PubMed1996

Decision-making: a comparison of referral practice and primary care.

BACKGROUND: Should care by subspecialist physicians be more costly than care by primary care physicians? This article addresses diagnostic testing, one element of the answer to this question. METHODS: A theoretical analysis was conducted of the sequences of testing, treatment, or watchful waiting in patients with low, intermediate, or high probabilities of disease. This was followed by a reanalysis of data from a previously published study of patients with chest pain from two referral populations and two primary care populations. The study used a chest pain score as a summary measure of the number of suggestive findings. RESULTS: The analysis of sequences of testing, treatment, and watchful waiting suggests that patients with intermediate probabilities of disease are most likely to be referred. The study of patients with chest pain shows that the probability of disease for a given chest pain history score is higher in referred patients than it is in primary care patients, as is the proportion of patients with intermediate and high chest pain scores. This result is direct evidence that referral physicians get more patients with suspect but often uncertain histories. In general, the probability of disease given a particular history will be lower in primary care patients, and hence testing will be less fruitful. CONCLUSIONS: Subspecialists are more likely to see patients who represent a diagnostic puzzle and have intermediate probabilities of disease. Since patients with intermediate probabilities of disease are most likely to benefit from testing, a per capita rate of testing that is higher than in a primary care practice might be appropriate in a subspecialist's practice.

Adult↗
PubMed1986

Frequent occurrence of short complementary sequences in nucleic acids.

The hypothesis, that nucleic acids which code specifically interacting receptor and ligand proteins contain complementary sequences was tested. Human insulin mRNA (HSINSU) contained 16 sequences which were 23.8 +/- 1.4 nucleotides long and were complementary to the insulin receptor mRNA (HSIRPR, 74.8 +/- 1.9% complementary matches, p less than 0.001 compared to randomly occurring matches). However, when examining 10 different nucleic acids (coding proteins not interacting with the insulin receptor), 81 additional sequences were found which were also complementary to HSIRPR. Although the finding of short complementary sequences was statistically highly significant, we concluded that this is not specific for nucleic acids coding specifically interacting proteins.

Base Sequence↗
PubMed2026

Prospective clinical validation of targeted long-read sequencing for preimplantation genetic testing of &#x3b1;-thalassaemia.

BACKGROUND: Preimplantation genetic testing for monogenic disorders (PGT-M) can prevent transmission of severe &#x3b1;-thalassaemia, but conventional workflows remain limited by family-specific assay design for direct variant detection, dependence on additional family samples for haplotype construction, and labour-intensive multi-step procedures across several platforms. Targeted long-read sequencing-based PGT-M for &#x3b1;-thalassaemia (tlrPGT-&#x3b1;-thal) integrates direct variant detection and haplotype linkage analysis within a single assay, but prospective clinical validation is lacking. METHODS: This prospective clinical study enrolled 103 families at high risk of transmitting &#x3b1;-thalassaemia at a reproductive medicine centre between August 2024 and March 2025. All families underwent blinded parallel analysis using both conventional NGS-based PGT-M (comparator) and tlrPGT-&#x3b1;-thal. RESULTS: In the primary concordance analysis, tlrPGT-&#x3b1;-thal was fully concordant with conventional NGS-based PGT-M (507/507, 100.0%; exact 95% CI, 99.3-100.0). Direct variant detection was successful in 501/507 embryos (98.82%; 95% CI, 97.4-99.6), haplotype linkage was established in 505/507 embryos (99.61%; 95% CI, 98.6-100.0), and one meiotic recombination event was identified. Among 93 families proceeding to embryo transfer, 57 pregnancies underwent invasive prenatal diagnosis, and all were concordant with the corresponding tlrPGT-&#x3b1;-thal results. Of the 26 comparator-inconclusive embryos, tlrPGT-&#x3b1;-thal resolved 6 complex cases, including cases with incomplete pedigrees or insufficient informative SNPs. Among the remaining 20 embryos with HBA-region aneuploidies, genotype and parental origin could be determined in 12. CONCLUSIONS: The findings show that tlrPGT-&#x3b1;-thal enables direct detection of diverse &#x3b1;-thalassaemia-causing variants together with efficient haplotype linkage analysis within a single workflow, without requiring family-specific assay design or additional family samples. The method demonstrated high diagnostic accuracy while providing added value in complex scenarios. Taken together, tlrPGT-&#x3b1;-thal represents a simplified and broadly applicable strategy for &#x3b1;-thalassaemia PGT-M.

Humans↗
PubMed1994

A small test of a sequence-based typing method: definition of the B*1520 allele.

Santamaria et al. (Human Immunology 1993 37: 39-50) describe a method of sequence-based typing (SBT) for HLA-A, B and C alleles said to give "unambiguous typing of any sample, heterozygous or homozygous, without requiring additional typing information". From SBT analysis, which involves determination of partial sequences of mixed alleles, these investigators reported that cell lines KT17 (HLA-B35,62) and OLGA (HLA-B62) from the reference panel of the 10th International Histocompatibility Workshop express novel variants of HLA-B15 (B1501-MN6) and HLA-B35 (B3501-MN7) respectively. To study further the novel alleles, we cloned and sequenced full-length HLA-B cDNA clones isolated from the KT17 and OLGA cell lines. We find that KT17 expresses B*3501, as assigned by SBT, and B*1501, the common allele encoding the B62 antigen. We were unable to confirm that KT17 expresses the novel B1501-MN6 variant identified by SBT. For OLGA our analysis confirms the partial sequences obtained by SBT. Thus OLGA expresses B*1501 and a novel HLA-B allele. The complete sequence of the latter shows it is a hybrid having exons 1 and 2 in common with B*1501 and other B15 subtypes and exons 3-7 in common with B*3501 and related molecules including B*5301 and B*5801. The novel allele has been designated B*1520 because of its sequence similarity with the B15 group; furthermore, serological analysis shows that the B*1520 product does not express epitopes in common with either B35, B53 or B58. The B*1520 heavy chain has a similar isoelectric point to A*3101; B*1520 was undetected by previous applications of isoelectric focusing because B*1520 and A31 are both expressed by OLGA. In conclusion, HLA-B typing of two cell lines by cDNA cloning and sequencing gives concordant results with SBT for three of the four alleles. The cause of the discrepancy for the fourth allele is unknown, however, this finding indicates that the novel HLA-A, B and C sequences emerging from SBT studies need independent verification.

Alleles↗
PubMed1991

Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine.

Production of live attenuated oral poliomyelitis vaccine (OPV) requires rigorous neurovirulence safety testing of each vaccine lot, currently carried out in monkeys. It has been reported that a change from 472-U to 472-C in the type 3 OPV RNA is associated with an increased histologic lesion score produced upon intraspinal inoculation of the mutant virus in monkeys. We have developed a method, based on polymerase chain reaction, for measuring the relative abundance of these mutant sequences directly in vaccine preparations and used this method to evaluate the proportion of 472-C in 40 different lots of type 3 OPV. Six vaccine lots that had failed the intraspinal monkey neurovirulence test contained a higher proportion of 472-C than all other lots that had passed this test. OPV type 3 virus containing 472-C was rapidly selected during serial passages in African green monkey kidney cells that are used for manufacturing of the vaccine. We have also found that the wild-type poliovirus type 3 strain Leon/37, from which the vaccine strain was originally derived, contained a mixture of 472-U and 472-C sequences. No other mutations in OPV type 3 RNA have been detected by similar assays at position 2034, also associated with attenuation, or at several other positions reported to be altered in some vaccine preparations. Our results suggest that molecular diagnostics may provide a supplement or a potential alternative to animal testing of live attenuated vaccines.

Animal Testing Alternatives↗
PubMed1995

The vitamin D3 hydroxylase-associated protein is a propionamide-metabolizing amidase enzyme.

Previously we isolated a novel protein that coimmunoprecipitates with the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase. This kidney-specific protein found in the inner membrane of mitochondria is named the vitamin D3 hydroxylase-associated protein (VDHAP). To determine a putative function for this protein, an extensive computer search of the deduced amino acid sequence of VDHAP was performed. A BLAST homology search identified amino acid residues 133 through 321 in acetamidase from Aspergillus nidulans that exhibit 38% amino acid identify and 65% amino acid similarity to VDHAP. A protein consensus sequence dictionary, MOTIFS, identified an amidase consensus sequence in VDHAP. This sequence, G-G-S-S-G-G-E-G-A-L-I-A-G-G-G-S-L-L-G-I-G-S-D-V-A-G-S-I-R-L-P-S, in VDHAP is located between amino acids 223 and 254. Propionamide, acetamide, and acrylamide were identified as substrates for an amidase activity in soluble chicken kidney mitochondria. Propionamide is the best substrate with a Vmax of 16.7 nmol NH4+/min/mg protein and an apparent Km of 7.9 mM in soluble chicken kidney mitochondria. A VDHAP monoclonal antibody, IVC2G8, immunoprecipitates 78% of the total propionamidase activity in soluble chicken kidney mitochondria. These results suggest that VDHAP is a propionamidase enzyme in soluble chicken kidney mitochondria and a member of the amidase signature gene family.

Amides↗
PubMed1995

Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase, AAT4.

A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency. pSAT2 contained an open reading frame encoding a 47640 Da protein. The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals. It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity. Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide. A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide. Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis. Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria. Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.

Amino Acid Sequence↗
PubMed1991

Magnetic resonance imaging for localizing the nonpalpable undescended testis.

A prospective evaluation of the value of magnetic resonance imaging (MRI) for localizing the nonpalpable undescended testis was performed in 17 boys aged 1 year to 5 years 4 months who had a clinical diagnosis of nonpalpable testes. The results were compared between the MRI, ultrasonographic, clinical, and definitive surgical diagnosis in all patients. Nine nonpalpable undescended testes were identified in the inguinal canal or just proximal to the inguinal ring by MRI, and these were confirmed by surgical findings. Six nonpalpable testes were not observed by MRI, and the absence of these testes was surgically confirmed. Like scrotal testes, all the undescended testes showed a low intensity on sequences with a short repetition time (TR) and echo time (TE), and a high intensity on long TR/TE sequences. Undescended testes were observed better on coronal images than on axial images. MRI promises to become an important diagnostic tool in the detection of nonpalpable undescended testes.

Child, Preschool↗
PubMed1975

Structural genes adjacent to interspersed repetitive DNA sequences.

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. 3H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3-5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80 percent of that mRNA hybridizable with whole DNA (approximately 65 percent) had reacted with the repeat-contiguous DNA fraction (50 percent). This result shows that 80-100 percent of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.

Animals↗
PubMed1990

The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses.

Monoclonal antibodies which reacted with four different epitopes were used to select neutralization-resistant variants of Australian bluetongue virus serotype 1 (BTV1AUS; isolate CS156). Nucleotide sequencing of the VP2 outer coat protein gene of these variants showed that two of them contained alterations within the previously defined neutralization site at amino acids 328 to 335 (Gould et al., 1988). Comparison of VP2 sequences of several BTV serotypes, in addition to nucleotide sequence changes in a number of variants, suggested that this neutralization site was larger and contained 19 amino acids, the conformation of which could be affected by other regions of the VP2 protein. Nucleotide sequencing of neutralization-resistant variants revealed a total of four other regions of VP2 affecting the ability of monoclonal antibodies to neutralize the virus and these results support the notion that the neutralization site in VP2 was conformation dependent. The complete nucleotide sequence of the VP2 gene of virulent BTV1AUS (C5156) was determined directly from viral nucleic acid isolated from the blood of a sheep suffering clinical bluetongue disease. Comparison of the VP2 sequence of this virulent virus with that previously published for an avirulent, laboratory strain (Gould, 1988), indicated that the passage of virulent virus approximately 20 times in tissue culture over the last decade, not only led to attenuation but resulted in the appearance of ten nucleotide changes in the VP2 gene. Six of these nucleotide changes were silent, two resulted in conservative amino acid substitutions and two generated radical amino acid changes. However, in a separate experiment, a single passage of the virulent virus in tissue culture while leading to attenuation did not result in a nucleotide change in the VP2 outer coat protein gene.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a heat-stable enterotoxin of Vibrio mimicus.

A heat-stable enterotoxin produced by Vibrio mimicus (VM-ST) was studied. VM-ST was purified from a culture supernatant of V. mimicus strain AQ-0915 by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatography on both SP-Sephadex C-50 and DEAE-Sephadex A-25, and HPLC, and the recovery rate was about 15%. Purified VM-ST was heat-stable. VM-ST activity was cross-neutralized by anti-STh antiserum. The amino acid composition of the purified VM-ST was determined 17 amino acid residues in the following sequence: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This composition and sequence were identical to those of V. cholerae non-O1-ST. These results clearly demonstrate the production of a characteristic VM-ST by V. mimicus.

Amino Acid Sequence↗
PubMed1974

The use of hybrid somatic cells as an approach to mitochondrial genetics in animals.

We have studied the fate of parental mitochondrial DNA (mtDNA) in hybrid somatic cells derived by Sendai virus-induced fusion of human cells and mouse or rat cells. Many hybrid cell strains were obtained which contained sequences from both human and rodent mtDNA after 40 to 60 population doublings. Some strains were subcloned and cultured further for up to 150 doublings; a large fraction of these strains contained both parental mtDNA sequences at that time. The relation between human and rodent mtDNA sequences was tested in some of the hybrid cell strains. In a high fraction of strains tested the human and rodent mtDNA sequences were linked to each other by what are most likely covalent bonds. This linkage may be described as "recombination" of mtDNA sequences from two different animals.

Animals↗
PubMed1989

A neutralizing epitope of human immunodeficiency virus type 1 has homologous amino acid sequences with the active site of inter-alpha-trypsin inhibitor.

A neutralizing epitope (epitope beta) of the HTLV-IIIB strain of HIV-1 was mapped to 24 amino acids of an external envelope glycoprotein (gp120) using a neutralizing monoclonal antibody (0.5 beta) and hetero-antisera against synthetic peptides encoding gp120. Proteins that have homologous sequences with epitope beta were sought from a databank of protein sequences to assess biological features of epitope beta. The results showed that epitope beta was found to have homologous sequences to inter-alpha-trypsin inhibitor (ITI). The homologous region of ITI included the active site of the protein. Synthesized peptides including epitope beta were good substrates for trypsin, because these peptides inhibited trypsin activities in a competitive manner (Ki = 24.5 microM). Human urinary trypsin inhibitor (UTI), a protein indistinguishable from ITI, as well as synthetic peptides including epitope beta inhibited syncytium formation caused by the LAV-1-infected CCRF-CEM and uninfected Molt-4 cells in a dose-dependent manner (0.1-1 mM). These findings suggest that epitope beta of HIV-1 could be substrate of protease upon HIV-1 infection and also suggest that protease inhibitory activity of epitope beta may play a role in the pathophysiology of HIV-1-infected individuals.

Alpha-Globulins↗
PubMed1991

Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains.

The host-protective antigen VP2 of a variant strain of infectious bursal disease virus (IBDV) which emerged from a vaccinated flock and is able to circumvent vaccination with classic type I strains of IBDV, was cloned and its nucleotide sequence determined. Virus-neutralizing monoclonal antibodies (MAbs) raised against the Australian 002-73 strain of IBDV did not react or reacted only very weakly with the expression product of the variant virus. The deduced amino acid sequence of VP2 from the variant strain differed in 17 residues from that of the Australian strain and in eight positions from a consensus sequence compiled from six type I strains of IBDV. All the amino acid changes mapped within the central, variable region of VP2, which forms the conformational epitope recognized by virus-neutralizing MAbs. Changes in the two hydrophilic regions at either end of this fragment were unique to the variant virus and were crucial for its ability to escape the virus-neutralizing antibodies induced by vaccination with a standard type I vaccine.

Amino Acid Sequence↗