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PubMed1995

Use of maximum length sequence analysis in newborn hearing testing.

The use of maximum length sequence analysis (MLSA) with rapid click rates may be of clinical value in newborn hearing screening because a greater number of individual responses can be signal averaged without adding to test time. To examine the potential clinical value of MLSA in newborn screening, auditory brainstem responses (ABRs) from 50 premature newborns were studied. ABRs were acquired with conventional signal averaging at four stimulus intensity levels (50 dB, 40 dB, 30 dB, and 20 dB nHL) using a click rate of 33.3/sec. These responses were directly compared with the ABRs acquired with MLSA using a rate of 227.3/sec. MLSA and conventional signal averaging yielded similar results with no statistically significant differences in the number of responses detected. Across babies, the overall quality of the tracings slightly favored MLSA, particularly when recording conditions were poor. Though the results of this investigation do not support the use of MLSA as the primary technique in newborn screening in the neonatal intensive care unit (NICU), they do support consideration of the use of MLSA as an alternative technique when the responses obtained with conventional signal averaging are poorly defined.

Acoustic Stimulation↗

PubMed1993

Single-strand conformational polymorphism and direct sequencing applied to carrier testing in families with ornithine transcarbamylase deficiency.

Single-strand conformational polymorphism (SSCP) and direct sequencing were used to confirm or deny carrier status in three families with ornithine transcarbamylase (OTC) enzyme deficiency. Two male probands with "late onset" OTC deficiency, whose "private" mutations were previously characterized, inherited the mutations form their heterozygous mothers. One of the heterozygous mothers had a false negative allopurinol test. Three female siblings of the two male probands were tested, one proved to be a carrier of the respective mutation while the other two were found to have normal alleles. In the third family, the proband was a female with "late onset" presentation of OTC deficiency. We found a new point mutation in this girl consisting of a guanine-to-cytosine transversion at nucleotide 520 resulting in a substitution of proline for alanine at amino acid 142 of the mature OTC protein. We confirmed that this mutation occurred spontaneously and that neither of the two parents carries this mutation. We conclude that SSCP, in conjunction with direct sequencing, is a useful technique that can be practically applied for carrier testing in families with OTC deficiency.

Amino Acid Metabolism, Inborn Errors↗

PubMed1978

The role of frontal and parietal cortex in cognitive processing: tests of spatial and sequence functions.

Normal monkeys and monkeys with resection of anterior frontal or posterior parietal cortex were trained to press a panel next to a green panel as a test of extrapersonal spatial orientation and to press a panel next to their own prior press as a test of personal spatial orientation. All monkeys also learned two sets of sequence problems in which the solutions were made independent of spatial location by randomly shifting the locations of the stimuli after each response within a trial. The Parietal Group was significantly impaired on the extrapersonal 'next-to' task but not the more difficult personal 'next-to' task. The Frontal Group was impaired on both the personal and the extrapersonal 'next-to' tasks but only when the relevant cues shifted spatial locations from trial to trial. The performance of the Parietal Group completely overlapped that of the Normal Group on the sequence problems regardless of the level of testing sophistication the monkeys had attained. In contrast, the Frontal Group demonstrated a significant impairment in learning sequences but only when the monkeys were naive. Once they became sophisticated they learned each sequence at a normal rate. Their poor performance was attributed to the lack of stability in the spatial location of the stimuli. The data support the view that a distinction between personal and extrapersonal spatial orientation is relevant to posterior parietal function but indicate that neither sequencing per se nor personal spatial orientation or spatial memory per se is dependent on intact frontal functioning. Rather, the frontal cortex is involved with a higher-order control essential to allow the monkey to perceive the reliable aspects of stimuli contained in a stimulus context full of unreliable noise and to further allow for flexible response pattern appropriate to the demands of a variable context.

Animals↗

PubMed1989

Optimizing radiologic workup: an artificial intelligence approach.

The increasing complexity of diagnostic imaging is presenting an ever expanding variety of radiologic test options to clinicians. As a result, it is becoming more difficult for referring physicians to select an appropriate sequence of tests. The current economic pressures on medicine make it particularly important that resources be used judiciously. Radiologic workup often involves a sequence of tests that lead from presenting signs and symptoms to a definitive diagnosis or intervention. This sequence ideally begins with simple, inexpensive, safe, non-invasive tests and progresses to more complex, expensive, and hazardous tests only if the simpler tests are insufficient to establish a diagnosis. DxCON is a developmental artificial intelligence-based computer system that gives advice to physicians about the optimum sequencing of radiologic tests. DxCON evaluates basic clinical information and a physician's proposed workup plan. The system then creates an analysis of the strengths and weaknesses of his plan. The domain chosen to explore computer-based workup advice is the radiologic workup of obstructive jaundice.

Cholestasis↗

PubMed2026

Longitudinal surveillance of antibiotic resistance and virulence evolution in Clostridioides difficile: a 4-year retrospective study of hospitalized patients in a tertiary hospital in China.

UNLABELLED: Clostridioides difficile (C. difficile) is the primary pathogen responsible for nosocomial infectious diarrhea and pseudomembranous colitis. In China, metronidazole and vancomycin are the preferred treatments for C. difficile infection (CDI). This study aimed to investigate the evolution of vancomycin (VA) and metronidazole (MTZ) resistance, as well as the longitudinal changes in virulence over time, using next-generation sequencing, drug susceptibility tests, and analysis of resistance and virulence genes. Additionally, we monitored the emergence of the highly virulent C. difficile strain RT027 and the spread and potential outbreak of C. difficile in the hospital setting. A random stratified sampling method was used to select 114 fecal samples from inpatients at Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, between 2021 and 2024. Clinical data from the enrolled patients were also collected. We conducted antigen and toxin protein detection for C. difficile, strain isolation and identification, drug sensitivity tests, whole genome sequencing, and bioinformatics analysis. This included comparisons of drug resistance genes, detection of toxin genes, and the construction of phylogenetic trees based on pan-genome analysis to investigate the resistance and toxin gene variations in C. difficile. Among the 114 samples collected from Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, no vancomycin- or metronidazole-resistant strains were identified. However, the average minimum inhibitory concentration (MIC) of C. difficile to vancomycin increased annually (H = 33.208, P < 0.05). The average MIC of C. difficile to metronidazole was highest in 2022 but decreased in 2023 and 2024 (H = 41.990, P < 0.05). Notably, in 2024, one C. difficile strain exhibited an MIC for metronidazole at the resistance threshold (2.00 μg/mL). Further Spearman correlation analysis of the strain years with drug sensitivity results revealed a positive correlation between strain years and the MIC levels of vancomycin and metronidazole (r = 0.528, P < 0.05; r = 0.377, P < 0.05). The proportion of toxin-producing strains increased annually, with 100% of strains in 2024 producing toxins, representing the highest proportion compared to the previous three years (X² =11.75, P < 0.05). Both vancomycin and metronidazole remain effective for the treatment of CDI in clinical practice. However, the sensitivity of C. difficile to these two drugs is gradually decreasing, and the rate of toxin gene carriage is also rising in clinical cases. No hospital outbreaks of C. difficile infections were identified in this study. IMPORTANCE: Clostridioides difficile has developed resistance to multiple antibiotics, including cephalosporins, clindamycin, and fluoroquinolones. This has exacerbated the global antibiotic resistance crisis. In China, according to current treatment guidelines, vancomycin and metronidazole are the preferred first-line drugs for treating C. difficile infections. However, there are reports indicating the emergence of new resistance to both vancomycin and metronidazole. Although there is extensive research on the long-term antibiotic resistance of C. difficile abroad, research on the continuous monitoring of antibiotic resistance and potential outbreaks of C. difficile in China is relatively limited. To fill this gap, we studied positive C. difficile strains from a tertiary general hospital in China. Through Next-Generation Sequencing (NGS), drug sensitivity testing, and analysis of drug resistance and virulence genes, we revealed the evolution of C. difficile's resistance to vancomycin and metronidazole, as well as changes in virulence, and monitored the spread within the hospital and potential outbreaks of C. difficile.

Humans↗

PubMed1992

Phylogenetic position of phylum Nemertini, inferred from 18S rRNA sequences: molecular data as a test of morphological character homology.

Partial 18S rRNA sequence of the nemertine Cerebratulus lacteus was obtained and compared with those of coelomate metazoans and acoelomate platyhelminths to test whether nemertines share a most recent common ancestor with the platyhelminths, as traditionally has been implied, or whether nemertines lie within a protostome coelomate clade, as suggested by more recent morphological analyses. Maximum-parsimony analysis supports the inclusion of the nemertine within a protostome-coelomate clade that falls within a more inclusive coelomate clade. Bootstrap analysis indicates strong support for a monophyletic Coelomata composed of a deuterostome and protostome-coelomate clade. Support for a monophyletic protostome Coelomata is weak. Inference by distance analysis is consistent with that of maximum parsimony. Analysis of down-weighted paired sites by maximum parsimony reveals variation in topology only within the protostome-coelomate clade. The relationships among the protostome coelomates cannot be reliably inferred from the partial sequences, suggesting that coelomate protostomes diversified rapidly. Results with evolutionary parsimony are consistent with the inclusion of the nemertine in a coelomate clade. The molecular inference corroborates recent morphological character analyses that reveal no synapomorphies of nemertines and flatworms but instead suggest that the circulatory system and rhynchocoel of nemertines are homologous to coelomic cavities of protostome coelomates, thus supporting the corresponding hypothesis that nemertines belong within a protostome-coelomate clade. The sequence data provide an independent test of morphological character homology.

Animals↗

PubMed1979

Regulation of herpesvirus macromolecular synthesis. VIII. The transcription program consists of three phases during which both extent of transcription and accumulation of RNA in the cytoplasm are regulated.

This report concerns the stable viral RNA sequences that accumulate in HEp-2 cells infected with herpes simplex virus type 1. By hybridizing labeled total DNA and restriction endonuclease DNA fragments with excess unlabeled total nuclear and cytoplasmic RNA, we determined the genetic complexity of the RNA and we mapped the regions on the physical map of herpes simplex virus type 1 DNA that are homologous to the RNA. Our results show the following. (i) The viral RNAs accumulating in the nucleus and cytoplasm of cells infected and maintained in the presence of inhibitory concentrations of either cycloheximide or emetine were homologous to 33 and 12% of viral DNA, respectively. All of the fragments tested contained sequences homologous to nuclear RNA. However, only the fragments mapping between 0.00 and 0.18, and 0.53 and 1.00 map units contained sequences homologous to cytoplasmic RNA. (ii) The viral RNAs that accumulate in the nucleus and cytoplasm of cells infected and maintained in the presence of inhibitory concentrations of phoaphonoacetic acid were homologous to 39 and 26% of viral DNA, respectively. In this instance all of the fragments except those mapping between 0.42 and 0.53 map units contained sequences homologous to cytoplasmic RNA. (iii) The viral RNAs that accumulate in the nucleus and cytoplasm 8 h after infection were homologous to greater than 50 and 41%, respectively. All of the fragments tested contained sequences homologous to cytoplasmic RNA. (iv) The viral RNAs that accumulate in the nucleus and cytoplasm of cells infected and maintained in the presence of canavanine are homologous to 33 and 19% of viral DNA, respectively. All of the fragments tested contained sequences homologous to both nuclear and cytoplasmic RNAs. Our results indicate the following. First, there are at least three phases of transcription of viral DNA. Phase 1 does not require the synthesis of host cell or viral proteins. Phase 2 requires the synthesis of viral proteins made before the initiation of viral DNA synthesis. Phase 3 appears to be related to the initiation of viral DNA synthesis. Second, both the extent of transcription and the accumulation of viral RNA in the cytoplasm are tightly regulated. The genetic complexity of total RNA accumulating in infected cells increased in each successive phase. Moreover, the genetic complexity of nuclear RNA was invariably higher than that of cytoplasmic RNA in each phase. Lastly, the results of the studies on viral RNA accumulating in canavanine-treated cells reinforce the hypothesis made previously that more than one polypeptide in each of the alpha and beta polypeptide groups is involved in the transcription preceding the transitions from alpha to beta and beta to gamma polypeptide synthesis, respectively, and that canavanine selectively inactivated subsets of these polypeptides permitting only partial transitions from alpha to beta and beta to gamma to occur.

Base Sequence↗

PubMed1993

Cell-specific expression in the silkmoth follicle: developmental characterization of a major chorion protein, its mRNA and gene.

Choriogenesis (eggshell formation) within the silkmoth Antheraea polyphemus proceeds in parallel for the two major subpopulations of follicle cells, diverging only during the very late period when aeropyle crown surface structures form in one region but not in the other. Correlated with their appearance is the synthesis of a set of region-specific proteins. In this report, aeropyle crowns are physically isolated and their protein composition is shown to consist of those same region-specific proteins. A cDNA clone, called pcvl 16, has been selected and shown to encode a lamellar-forming, aeropyle crown-specific protein, probably of the previously described C3,4 group. These conclusions are based on hybrid-selected translation, Northern analysis, and sequence analysis. pcvl 16 was used to isolate two distinct cloned copies of the 16 gene. Both 16 genes are closely paired with another region-specific gene but the proximity of the two gene pairs to each other is uncertain. Non-region-specific chorion genes expressed at earlier times in choriogenesis surround the 16 gene pairs, suggesting that cis sequences necessary for regionalized expression may be closely linked to coding sequences. To test this hypothesis, 5'-flanking sequences from eight region-specific genes are compared and shown to share two oligonucleotide sequences. One is a known regulatory element found in virtually all moth and fly chorion genes examined. The other, located just upstream from the TATA box, is not found in non-regionally expressed chorion genes and, thus, is a candidate for specifying regional expression.

Amino Acid Sequence↗

PubMed1992

Response of beta-endorphin and estradiol to resistance exercise in females during energy balance and energy restriction.

The acute effect of weightlifting on beta-endorphin and estradiol was studied in experienced female recreational weightlifters. Five eumenorrheic females completed two months of testing, each with a different sequence of testing conditions (SEQ1 and SEQ2). In SEQ1, a week of weight maintenance diet and prescribed exercise (3 d.wk-1, 3 sets, approximately 85% 1 RM, 10-12 reps, eight lifts) beginning on d 11 of their menstrual cycle was followed by measurement of hormone response to a weightlifting bout during energy balance (EBAL) on d 18. This included blood sampling via a catheter before, just after, and at 15 and 30 min of recovery. The women consumed 500 kcal per day for the next 48 hrs and then repeated the weightlifting test during negative energy balance (NEBAL). SEQ2 was similar except that the 48 hrs of NEBAL preceded the EBAL test condition. Estradiol and beta-endorphin increased from baseline to immediately post exercise under both dietary conditions but was significant only during NEBAL. Estradiol increased 1.6 fold and beta-endorphin 3.7 fold by the end of the resistance exercise bout during NEBAL. Both hormones were also elevated for a longer time during recovery in the NEBAL condition. Since estradiol and beta-endorphin can suppress gonadotropin release, it is possible that repeated elevations in these hormones during weightlifting, especially concurrent with energy restriction, could contribute to disruption of the menstrual cycle.

Adult↗

PubMed1985

MR imaging of the acoustic nerves and small acoustic neuromas at 0.6 T: prospective study.

To evaluate the capability of magnetic resonance (MR) in imaging normal acoustic nerves, 12 volunteers without signs or symptoms of intracranial disease were examined using a 0.6 T superconductive system. Several spin-echo (SE) pulse sequences were tested to identify the optimal sequence for demonstration of the acoustic nerve bundle. Repetition times (TRs) varied from 300 to 2000 msec and echo times (TEs) from 30 to 120 msec. A single-slice technique was used with 5 and 8 mm sections, one or two data acquisitions per projection, and axial and coronal imaging. The normal acoustic nerves were demonstrated readily by MR in axial and/or coronal sections. The distal parts of the nerves and tumors were imaged best with SE 1500/60. The medial extremities of the seventh and eighth nerves tended to be obscured in this sequence by brightening the cerebrospinal fluid signal adjacent to the brainstem, but they were demonstrated clearly with 500 or 800 msec TR and 30 msec TE. Five patients were studied who had hearing loss and evidence of retrocochlear disease. In four patients, MR imaging demonstrated five acoustic nerve tumors ranging in size from purely intracanalicular to a 12 mm cisternal component. In the fifth case, no tumor was identified by MR imaging or gas computed tomographic (CT) cisternography. Contrast-enhanced CT using a Siemens Somatom DR 3 or GE CT/T 8800 scanner failed to provide convincing evidence of tumor in any case, while gas CT cisternography was positive in all five tumors. All five acoustic neuromas were identified readily using the SE sequences that proved optimal for demonstration of normal nerves. This experience revealed that MR imaging can demonstrate the eighth nerve complex well and reliably. Single-slice (5 or 8 mm) technique is adequate, but multislice without tissue gaps (used recently) is more efficient. Small, even intracanalicular, acoustic neuromas are imaged effectively, indicating that the method is capable of superseding contrast CT cisternography, particularly with improving technology.

Atrophy↗

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 α = 0.001. Notably, when 5 % 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 % of African Ancestry, mean age of 62, with 58 % 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 < 0.001) and advance aging by the Original Burden test. In contrast, SKAT identified significant associations (p < 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 α-thalassaemia.

BACKGROUND: Preimplantation genetic testing for monogenic disorders (PGT-M) can prevent transmission of severe α-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 α-thalassaemia (tlrPGT-α-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 α-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-α-thal. RESULTS: In the primary concordance analysis, tlrPGT-α-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-α-thal results. Of the 26 comparator-inconclusive embryos, tlrPGT-α-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-α-thal enables direct detection of diverse α-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-α-thal represents a simplified and broadly applicable strategy for α-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↗