Minor Deletions Research Articles

Genetic characteristics and pathogenicity of variant porcine parvovirus 1 in northern China

Porcine parvovirus type 1 (PPV1) can lead to reproductive disorders in pregnant sows, including stillbirth, mummification, embryonic death, and infertility (SMEDI syndrome). In this study, we isolated and identified 10 PPV1 strains in northern China, with genomes around 5 kb long and minor deletions in the 127-nt repeat region. The sequence analysis results showed that compared with strain NADL-2 (Reference strain), eight amino acid substitutions on the NS1 protein and fourteen amino acid substitutions on the VP2 protein were found in the ten isolates. Because the JX strain exhibited reduced neutralizing activity induced by commercially available vaccines in vitro, it was selected for challenge experiments in sows at 35 days of gestation. Mutant strain JX not only caused viremia, but also mild edema and mild inflammation in the trachea, lungs, lymph nodes, reproductive organs, and some intestines of the pregnant sows. Strain JX also caused fetal congestion and organ infection after penetrating the placental barrier. In conclusion, this study focused on the variation and evolution of PPV1 in northern China, screened a strain with reduced neutralizing activity, and determined that it has a certain degree of pathogenicity.

Reflections on Engaging Pragmatically with Generative AI to Augment Research and Education Practice

Generative AI tools enable researchers to generate multimedia content, including human-like visuals, voice and text. This novel paper’s purpose is to use generative AI to create a short paper, and to critically evaluate the process and implications of the material generated. Firstly, the capabilities of AI to generate multimedia content is summarised with an example of an AI generated professor. Both the opening section, and concluding reflections, are written without the use of AI. Perplexity.ai, an AI tool based on a large language model is used, through a series of prompts, to generate a short paper on AI in the animation industry. The generated paper emerged through a series of prompts written during a 90-minute session. The generated text is presented as is, modified only with minor deletions. In the concluding section, the impact of generative AI tools on researching are reflected upon with some positive and negative insights outlined.

COVID-19 vaccine coverage and the necessity of its urgent development towards Omicron the new SARS CoV-2 B.1.1. 529 variant

A SARS-CoV-2 variant belonging to Pango lineage B.1.1.529 with a significant number of S-gene mutations compared to the original virus was found in early November 2021. On 26th November 2021, the World Health Organization (WHO) designated the mutation as a variant of concern and assigned it the name Omicron. The difference is identified by 30 changes in the spike protein, three minor deletions, and one minor insertion, 15 of which are in the receptor-binding area. The Omicron variant is the most diverging variety discovered in substantial numbers so far during the pandemic, raising significant concerns that it may be associated with significant reductions in vaccination efficacy and an increased risk of reinfections. Omicron pseudo- or live virus isolates are urgently needed to understand better the virus's escape potential against both vaccination, and infection-acquired immunity is urgently required.

Two novel mouse models mimicking minor deletions in 22q11.2 deletion syndrome revealed the contribution of each deleted region to psychiatric disorders

22q11.2 deletion syndrome (22q11.2DS) is a disorder caused by the segmental deletion of human chromosome 22. This chromosomal deletion is known as high genetic risk factors for various psychiatric disorders. The different deletion types are identified in 22q11.2DS patients, including the most common 3.0-Mb deletion, and the less-frequent 1.5-Mb and 1.4-Mb deletions. In previous animal studies of psychiatric disorders associated with 22q11.2DS mainly focused on the 1.5-Mb deletion and model mice mimicking the human 1.5-Mb deletion have been established with diverse genetic backgrounds, which resulted in the contradictory phenotypes. On the other hand, the contribution of the genes in 1.4-Mb region to psychiatric disorders is poorly understood. In this study, we generated two mouse lines that reproduced the 1.4-Mb and 1.5-Mb deletions of 22q11.2DS [Del(1.4 Mb)/+ and Del(1.5 Mb)/+] on the pure C57BL/6N genetic background. These mutant mice were analyzed comprehensively by behavioral tests, such as measurement of locomotor activity, sociability, prepulse inhibition and fear-conditioning memory. Del(1.4 Mb)/+ mice displayed decreased locomotor activity, but no abnormalities were observed in all other behavioral tests. Del(1.5 Mb)/+ mice showed reduction of prepulse inhibition and impairment of contextual- and cued-dependent fear memory, which is consistent with previous reports. Furthermore, apparently intact social recognition in Del(1.4 Mb)/+ and Del(1.5 Mb)/+ mice suggests that the impaired social recognition observed in Del(3.0 Mb)/+ mice mimicking the human 3.0-Mb deletion requires mutations both in 1.4-Mb and 1.5 Mb regions. Our previous study has shown that Del(3.0 Mb)/+ mice presented disturbance of behavioral circadian rhythm. Therefore, we further evaluated sleep/wakefulness cycles in Del(3.0 Mb)/+ mice by electroencephalogram (EEG) and electromyogram (EMG) recording. EEG/EMG analysis revealed the disturbed wakefulness and non-rapid eye moving sleep (NREMS) cycles in Del(3.0 Mb)/+ mice, suggesting that Del(3.0 Mb)/+ mice may be unable to maintain their wakefulness. Together, our mouse models deepen our understanding of genetic contributions to schizophrenic phenotypes related to 22q11.2DS.

Molecular Configuration of Human Genome Neighboring Megabase-Sized Large Deletions Induced by X-Ray Irradiation.

The genomic landscape neighboring large deletions including the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on human X chromosome in 6-thioguanine-resistant mutants originating from immortalized human fibroblast cells exposed to X rays was characterized by real-time quantitative PCR (qPCR)-based analyses. Among the 13 mutant clones with large deletions extending over several Mb, including the HPRT locus, revealed by 10 conventional sequence-tagged site (STS) markers, three clones bearing the largest deletions were selected for further qPCR analysis using another 21 STS markers and 15 newly designed PCR primer pairs. The results indicated that the major deletions were in very specific regions between the 130-Mb and 140-Mb positions containing the HPRT locus on the X chromosome and, contrary to our initial expectations, additional minor deletions were distributed in a patchwork pattern. These findings strongly indicate that the complex deletion patterns in the affected chromosome are related to the radiation track structure with spatially heterogeneous energy deposition and the specific structure of the chromatin-nuclear membrane complex. The uncovered complex deletion patterns are in agreement with the idea of complex chromatin damage, which is frequently associated with carcinogenesis.

Y chromosome copy number variation and its effects on fertility and other health factors: a review.

The Y chromosome is essential for testis development and spermatogenesis. It is a chromosome with the lowest gene density owing to its medium size but paucity of coding genes. The Y chromosome is unique in that the majority of its structure is highly repetitive sequences, with the majority of these limited genes occurring in 9 amplionic sequences throughout the chromosome. The repetitive nature has its benefits as it can be protective against gene loss over many generations, but it can also predispose the Y chromosome to having wide variations of the number of gene copies present in these repeated sequences. This is known as copy number variation. Copy number variation is not unique to the Y chromosome but copy number variation is a well-known cause of male infertility and having effects on spermatogenesis. This is most commonly seen as deletions of the AZF sequences on the Y chromosome. However, there are other implications for copy number variation beyond just the AZF deletions that can affect spermatogenesis and potentially have other health implications. Copy number variations of TSPY1, DAZ, CDY1, RBMY1, the DYZ1 array, along with minor deletions of gr/gr, b1/b3, and b2/b3 have all be implicated in affecting spermatogenesis. UTY copy number variations have been implicated in risk for cardiovascular disease, and other deletions within gr/gr and the AZF sequences have been implicated in cancer and neuropsychiatric diseases. This review sets out to describe the Y chromosome and unique susceptibility to copy number variation and then to examine how this growing body of research impacts spermatogenesis and other health factors.

Современные аспекты генетической диагностики недостаточности 21-гидроксилазы

Objective of the Review: To discuss the current peculiarities of 21-hydroxylase insufficiency diagnostics. Key Points. Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive pathologies, associated with a defective enzyme or transport protein participating in cortisol biosynthesis. Currently, there is information on CAH genetics including information on 21-hydroxylase insufficiency. Neonatal screening, antenatal and postnatal methods to diagnose 21-hydroxylase deficit have been developed. Timely diagnosis and correct therapy have been found to facilitate normal physical and mental development of patients. Molecular genetic testing for CAH associated with 21-hydroxylase deficit is widely used both in Russia and globally and is of importance for differential diagnosis, identification of pathogenic CYP21A2 gene carriers and adequate genetic counselling. The best strategy to diagnose 21-hydroxylase insufficiency is 2-stage CYP21A2 gene analysis. The test should include both gene sequence analysis and identification of point replacements, minor deletions and duplication (e.g., dideoxynucleotide chain-termination method or Next Generation Sequencing), and identification of extended deletions and duplication (multiplex ligation dependent probe amplification, real-time polymerase chain reaction). Such a comprehensive approach can help finding a majority of types of possible changes. Conclusion. Correct genetic testing methods ensure detection of pathogen variants in CYP21A2 gene; evaluation of the possible rate of clinical manifestations of the disease, both during antenatal testing and if an unknown clinical CAH form is encountered; prescribing an adequate therapy; and ensuring genetic counselling in order to develop a management strategy, including when planning for pregnancy in a woman with confirmed CAH. Keywords: congenital adrenal hyperplasia, CYP21A2 gene, genetic counselling, 21-hydroxylase

Synthetic Deletion of the Interleukin 23 Receptor (IL-23R) Stalk Region Led to Autonomous IL-23R Homodimerization and Activation.

Interleukin 23 (IL-23) regulates the development of TH17 cells, which are important for antimicrobial and antifungal responses and autoimmune and chronic inflammatory diseases. IL-23-induced Jak/STAT signaling is mediated via the heterodimeric IL-23 receptor (IL-23R)-IL-12 receptor β1 (IL-12Rβ1) complex. The typical signal-transducing receptor of the IL-6/IL-12 family contains three extracellular-membrane-proximal fibronectin type III (FNIII) domains, which are not involved in cytokine binding but are mandatory for signal transduction. In place of FNIII-type domains, IL-23R has a structurally undefined stalk. We hypothesized that the IL-23R stalk acts as a spacer to position the cytokine binding domains at a defined distance from the plasma membrane to enable signal transduction. Minor deletions of the murine, but not of the human, IL-23R stalk resulted in unresponsiveness to IL-23. Complete deletion of the human IL-23R stalk and the extended murine IL-23R stalk, including a 20-amino-acid-long duplication of domain 3, however, induced ligand-independent, autonomous receptor activation, as determined by STAT3 phosphorylation and cell proliferation. Ligand-independent, autonomous activity was caused by IL-23R homodimers and was independent of IL-12Rβ1. Our data show that deletion of the stalk results in biologically active IL-23R homodimers, thereby creating an as-yet-undescribed receptor complex of the IL-6/IL-12 cytokine family.

TMOD-04. A UNIQUE PEDIATRIC IDH1 MUTANT GLIOBLASTOMA IN VITRO AND IN VIVO MODEL

AbstractThe isocitrate dehydrogenase 1 (IDH1) mutation serves as a hallmark for secondary high-grade gliomas, occurring in approximately 80% of all secondary glioblastomas (GBM) in adults. IDH1 mutations in pediatric high-grade gliomas are much rarer, and its role is still largely unknown. The lack of biologically representative in vitro and in vivo IDH1 mutant models makes it difficult to study this malignancy. Here we describe the establishment of a pediatric endogenous IDH1 mutant GBM primary cell line (VUMC-HGG-09) derived from an 11 year old female patient. We effectively cultured the VUMC-HGG-09 cells both as neurospheres in a serum-free environment, and as an adherent monolayer in serum enriched medium. VUMC-HGG-09 cells were orthotopically injected into brains of athymic nude mice, which caused successful engraftment and aggressively growing tumors in all the animals. Hematoxylin and vimentin staining showed a diffuse GBM like growth pattern through the whole brain. Furthermore, presence of the human R132H IDH1 mutation was confirmed by immunohistochemistry of the resected mouse brains. Shallow sequencing, whole chromosome painting FISH, and SNP-array were used to map chromosomal aberrations. These data revealed a rarely found chromothripsis like pattern within chromosome 7 and several minor deletions and translocations among other chromosomes. The VUMC-HGG-09 is a unique model that to our knowledge represents the first in vitro and in vivo pediatric IDH1 mutant glioma model. A growing body of evidence suggests the IDH1 mutation to play a vital role in the process of tumorigenesis as well as its value as a clinical prognostic marker. Therefore, VUMC-HGG-09 could serve both as a unique pediatric GBM model, as well as a pivotal in vitro and in vivo model to study IDH1 mutant tumors in both pediatric and adult neuro-oncology.

OP46 – 2969: Novel phenotypes of childhood encephalomyopathies with mitochondrial DNA depletion or deletions

Objective To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases, in order to estimate the role of mtDNA rearrangements in pathogenesis of these diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions. Methods Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of diseases. Clinical and laboratory findings were collected. Results Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. MtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase and multiorgan disease without mutations in any of the known mtDNA maintenance genes; muscle electron micrograph of one patient revealed disordered myofibrillar structure, pathological endoplasmic reticulum membranes and accumulation of glycogen and extracellular collagen fibres. The third patient with mtDNA depletion and severe myopathy was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content (4.9 and 6.9-fold increase relative to the median of age-matched controls). Conclusion We describe two novel early-onset phenotypes associated with mtDNA depletion, one of them presenting with pathologic endoplasmic reticulum membranes. Furthermore, mtDNA depletion can be a secondary finding in hereditary muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

P.4.13 Central core disease (CCD): Improving the screening for mutations in RYR1 gene

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients with RYR-related CCD usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with susceptibility to malignant hyperthermia (MH), and both conditions have been linked to mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). RYR1 mutational spectrum linked with CCD includes more than 200 described mutations mostly heterozygous dominant missense mutations and minor deletions or duplications. Definite diagnosis of CCD is done by clinical history and evaluation, histopathological analyses of muscle biopsy and, recently, genetic testing of gene’s “hot spots”, because RYR1 is a large gene and direct sequencing of each exon is laborious. Here, we tested Multiplex Ligation-dependent Probe Amplification (MLPA) as an alternative and simple method for screening patients with histological diagnosis or family history of CCD. This technique uses high-performance PCR to quantify up to 45 transcripts in small human DNA samples. It is based on annealing of two hemi-probes into its complementary sequence and was designed to detect the 33 most common mutations in RYR1 gene. From 41 patients diagnosed with CCD in three different institutions, 24 (58,5%) had mutations found in direct sequencing and 11 (26,8%) in MLPA. Seventeen patients (41,5%) had consistent results: 6 (14,6%) had their mutation diagnosed with sequencing and MLPA and neither method was able to find mutations in 11 patients (26,8%). Mutations in exon 101 were the most prevalent in sequencing (11 patients) and MLPA (4 patients). MLPA had shown to be a relevant tool to help diagnosing CCD; moreover, it is especially useful in diagnosing family members of patients with known mutations detectable by MLPA. Financial support: FAPESP-CEPID, CNPQ-INCT, FINEP.

Primary hyperoxaluria type 1: update and additional mutation analysis of theAGXTgene

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, inherited disorder of glyoxylate metabolism arising from a deficiency of the alanine:glyoxylate aminotransferase (AGT) enzyme, encoded by the AGXT gene. The disease is manifested by excessive endogenous oxalate production, which leads to impaired renal function and associated morbidity. At least 146 mutations have now been described, 50 of which are newly reported here. The mutations, which occur along the length of the AGXT gene, are predominantly single-nucleotide substitutions (75%), 73 are missense, 19 nonsense, and 18 splice mutations; but 36 major and minor deletions and insertions are also included. There is little association of mutation with ethnicity, the most obvious exception being the p.Ile244Thr mutation, which appears to have North African/Spanish origins. A common, polymorphic variant encoding leucine at codon 11, the so-called minor allele, has significantly lower catalytic activity in vitro, and has a higher frequency in PH1 compared to the rest of the population. This polymorphism influences enzyme targeting in the presence of the most common Gly170Arg mutation and potentiates the effect of several other pathological sequence variants. This review discusses the spectrum of AGXT mutations and polymorphisms, their clinical significance, and their diagnostic relevance.

Functional characterization of ribosomal protein L15 from Saccharomyces cerevisiae

In this study we provide general information on the little studied eukaryotic ribosomal protein rpL15. Saccharomyces cerevisiae has two genes, YRPL15A and YRPL15B that could potentially code for yeast rpL15 (YrpL15). YRPL15A is essential while YRPL15B is dispensable. However, a plasmid-borne copy of the YRPL15B gene, controlled by the GAL1 promoter or by the promoter controlling expression of the YRPL15A gene, can functionally complement YrpL15A in yeast cells, while the same gene controlled by the authentic promoter is inactive. Analysis of the levels of YrpL15B-mRNA in yeast cells shows that the YRPL15B gene is inactive in transcription. The function of YrpL15A is highly resilient to single and multiple amino acid substitutions. In addition, minor deletions from both the N- and C-terminal ends of YrpL15A has no effect on protein function, while addition of a C-terminal tag that could be used for detection of plasmid-encoded YrpL15A is detrimental to protein function. YrpL15A could also be replaced by the homologous protein from Arabidopsis thaliana despite almost 30% differences in the amino acid sequence, while the more closely related protein from Schizosaccharomyces pombe was inactive. The lack of function was not caused by a failure of the protein to enter the yeast nucleus.

High‐resolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence

The potential use of human mesenchymal stem cells for therapeutic applications implies large scale in vitro culture, increasing the probability of genetic instability and transformation. We examine here the incidence of unbalanced and balanced chromosome rearrangements in polyclonal and single cell-derived cultures of human adipose stem cells to senescence. G-banding karyotyping of the polyclonal cultures shows a normal karyotype. In addition, high-resolution microarray-based comparative genomic hybridization analyses relative to uncultured adipose stem cells from the same donors reveal overall genomic stability in long-term (∼6 months) polyclonal and clonal culture. One adipose stem cell clone displayed minor deletions in gene-rich telomeric and sub-telomeric regions on three chromosomes in early passage. This however, was detected only in a sub-population of cells that was subsequently spontaneously eliminated from the culture. Apparent pericentromeric instabilities are also occasionally detected in specific chromosomes. Our results indicate that clonal chromosomal aberrations may arise transiently in early passage adipose stem cells (ASC) cultures. Nonetheless, incidence of these aberrations seems to be negligible in the majority of long-term ASC cultures, at least under the culture conditions used here.

Localization of Transgenes and Genotyping of H-2K b -tsA58 Transgenic Mice

Localization of Transgenes and Genotyping of <i>H-2K</i> ^b -tsA58 Transgenic Mice

Genetic insights into the clinical diversity of beta thalassaemia.

Genetic insights into the clinical diversity of beta thalassaemia.

Molecular analysis of the glyoxylate reductase (GRHPR) gene and description of mutations underlying primary hyperoxaluria type 2.

Primary hyperoxaluria type 2, an inherited autosomal recessive disorder of endogenous oxalate overproduction, is caused by mutations in the GRHPR gene encoding the glyoxylate/hydroxypyruvate reductase enzyme. The GRHPR genes from nineteen unrelated patients with PH2 were analysed for mutations using a combination of PCR-SSCP and sequence analysis of genomic and cDNA. Eleven mutations were identified, seven of which are novel. The mutations included five point mutations: c.84-2A>G, c.295C>T (R99X), c.494G>A (G165D), and c.904C>T (R302C) as well as six minor deletions: c.103delG, c.375delG, c.403_405+2 delAAGT, c.540delT, c.608_609delCT and a more complex mutation in intron 1: c.84-13_c.84-12del; c.84-8_c.84-5del. Aberrant transcripts were demonstrated in hepatic mRNA as a result of the c.403_405+2 delAAGT and c.84-2A>G mutations. In addition, a splice variant lacking 28 bp of exon 1 was expressed in a number of tissues but is of unknown function. Two polymorphisms, c.579A>G in exon 6 and a (CT)(n) microsatellite in intron 8 were identified. Expression studies showed that the G165D and R302C mutants had glyoxylate reductase activity 1.5 and 5.6% respectively of the wild type protein. Both mutant proteins were unstable on purification. Although there is wide expression of the GRHPR mRNA demonstrated by northern blot analysis, our study shows that GRHPR protein distribution is predominantly hepatic and concludes that PH2, like the related type 1 disease, is primarily a disorder affecting hepatic glyoxylate metabolism.

Polyphenotypic Expression of Mitochondrial Toxicity Caused by Nucleoside Reverse Transcriptase Inhibitors

An HIV-infected man taking long-term zidovudine and didanosine presented with a polyphenotypic expression of nucleoside reverse transcriptase inhibitor (NRTI)-induced mitochondrial toxicity. Clinical features included lactic acidosis, myopathy, Fanconi-type proximal tubulopathy, pancreatic dysfunction, pseudo-obstruction, mega-oesophagus, peripheral sensory neuropathy and osteoporosis. A muscle biopsy showed morphologically abnormal mitochondria and respiratory chain biochemistry revealed marked reductions in the activity of respiratory chain enzymes containing mitochondrial DNA-encoded subunits. Southern blotting showed no mitochondrial DNA depletion and long PCR revealed only minor deletions. Following withdrawal of NRTI therapy, the lactic acidosis, pancreatic dysfunction and Fanconi's tubulopathy rapidly improved. Over the next 6 months there was marked improvement in osteoporosis, myopathy and neuropathy. At this stage, dual protease inhibitors and nevirapine were started. A repeat muscle biopsy 14 months after presentation showed normal morphology and respiratory chain biochemistry was almost normal.

The use of yeast artificial chromosomes in transgenic animals: expression studies of the tyrosinase gene in transgenic mice

Variegation and inherited somatic mosaicism has been observed in transgenic mice carrying yeast artificial chromosomes (YACs) in which a DNAse I hypersensitive site (HS) located −12 kb upstream of the mouse tyrosinase gene had been deleted. At present, we are generating new transgenic animals with minor deletions of the HS.

Structural polymorphism of the luciferase gene in the firefly, Luciola lateralis.

To study the structural features of genes for luciferase in Luciola lateralis, two different, functional luciferase genes, Luc1 and Luc2, were cloned and their nucleotide sequences were determined. The two genes were nearly identical, but a deletion of 128 bp was found in the 5' upstream region of Luc2, and minor deletions and additions were also found in the 3' downstream region. Seven base substitutions in the coding region produced two amino acid residue differences between the two genes. Southern blot analysis of genomic DNA isolated from 33 individual fireflies revealed that 26 were homozygous for the Luc1 gene and one was homozygous for Luc2. The others were heterozygotes with two, or even three, different alleles for the luciferase gene.