Number Tandem Repeat Research Articles

Molecular Typing of Pseudomonas aeruginosa Isolates Collected in Abidjan Hospitals (Côte d'Ivoire) Using the Multiple-Locus Variable Number of Tandem Repeats Method.

Background/objectives:Pseudomonas aeruginosa can cause community-acquired infections affecting various body sites. The present retrospective study investigated the genetic diversity of 173 isolates (166 clinical, 7 environmental) of P. aeruginosa collected from clinical pathology laboratories in Abidjan, Côte d'Ivoire (2001-2011). Methods: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 13 loci was applied to all isolates and compared to published MLVA data. The antibiotics status of the isolates was compiled when available and compared to published profiles. Results: Among 95 isolates analyzed for their antibiotics status, 14 displayed concerning resistance profiles: five multidrug-resistant (MDR) and nine extensively drug-resistant (XDR). MLVA typing revealed a high genetic diversity (>130 genotypes), with many genotypes represented by a single strain. Notably, thirteen clusters (≥4 related isolates) were observed. Some clusters displayed close genetic relatedness to isolates from France, Korea, and well-studied strains (ST560, LES and PA14). Comparative analysis suggested the presence of international high-risk MDR clones (CC233, CC111) in Côte d'Ivoire. Importantly, MLVA clustering revealed a close relationship of CC235-MDR strains with a locally identified cluster (group 9). Conclusions: These findings support MLVA as a reliable and cost-effective tool for low-resource settings, allowing the selection of relevant strains for future whole genome sequence analyses. This approach can improve outbreak investigations and public health interventions aimed at curbing MDR P. aeruginosa transmission within hospitals and at the national level.

Dopamine Transporter and CYP2D6 Gene Relationships with Attention-Deficit/Hyperactivity Disorder Treatment Response in the Methylphenidate and Atomoxetine Crossover Study.

Background: Few biological or clinical predictors guide medication selection and/or dosing for attention-deficit/hyperactivity disorder (ADHD). Accumulating data suggest that genetic factors may contribute to clinically relevant pharmacodynamic (e.g., dopamine transporter-SLC6A3 also commonly known as DAT1) or pharmacokinetic (e.g., the drug metabolizing enzyme Cytochrome P450 2D6 CYP2D6) effects of methylphenidate (stimulant) and atomoxetine (non-stimulant), which are commonly prescribed medications. This is the first study of youth with ADHD exposed to both medications examining the clinical relevance of genetic variation on treatment response. Methods: Genetic variations in DAT1 and CYP2D6 were examined to determine how they modified time relationships with changes in ADHD symptoms over a 4-week period in 199 youth participating in a double-blind crossover study following a stepped titration dose optimization protocol. Results: Our results identified trends in the modification effect from CYP2D6 phenotype and the time-response relationship between ADHD total symptoms for both medications (atomoxetine [ATX]: p = 0.058, Methylphenidate [MPH]: p = 0.044). There was also a trend for the DAT1 3' untranslated region (UTR) variable number of tandem repeat (VNTR) genotype to modify dose relationships with ADHD-RS total scores for atomoxetine (p = 0.029). Participants with DAT1 9/10 repeat genotypes had a more rapid dose-response to ATX compared to 10/10, while those with 9/9 genotypes did not respond as doses were increased. Regardless of genotype, ADHD symptoms and doses were similar across CYP2D6 metabolizer groups after 4 weeks of treatment. Conclusions: Most children with ADHD who were CYP2D6 normal metabolizers or had DAT1 10/10 or 9/10 genotypes responded well to both medications. While we observed some statistically significant effects of CYP2D6 and DAT1 with treatment response over time, our data indicate that genotyping for clinical purposes may have limited utility to guide treatment decisions for ATX or MPH because both medications were generally effective in the studied cohort after 3 weeks of titration to higher doses. The potential DAT1 association with ATX treatment is a novel finding, consistent with prior reports suggesting an association of the DAT1 in 9/9 genotypes with lower responsive rates to treatment at low and moderate doses.

Nanopore sequencing with unique molecular identifiers enables accurate mutation analysis and haplotyping in the complex lipoprotein(a) KIV-2 VNTR.

Repetitive genome regions, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR), are major constituents of the uncharted dark genome and evade conventional sequencing approaches. The protein-coding LPA kringle IV type-2 (KIV-2) VNTR (5.6kb per unit, 1-40 units per allele) is a medically highly relevant example with a particularly intricate structure, multiple haplotypes, intragenic homologies, and an intra-VNTR STR. It is the primary regulator of plasma lipoprotein(a) [Lp(a)] concentrations, an important cardiovascular risk factor. Lp(a) concentrations vary widely between individuals and ancestries. Multiple variants and functional haplotypes in the LPA gene and especially in the KIV-2 VNTR strongly contribute to this variance. We evaluated the performance of amplicon-based nanopore sequencing with unique molecular identifiers (UMI-ONT-Seq) for SNP detection, haplotype mapping, VNTR unit consensus sequence generation, and copy number estimation via coverage-corrected haplotypes quantification in the KIV-2 VNTR. We used 15 human samples and low-level mixtures (0.5 to 5%) of KIV-2 plasmids as a validation set. We then applied UMI-ONT-Seq to extract KIV-2 VNTR haplotypes in 48 multi-ancestry 1000 Genome samples and analyzed at scale a poorly characterized STR within the KIV-2 VNTR. UMI-ONT-Seq detected KIV-2 SNPs down to 1% variant level with high sensitivity, specificity, and precision (0.977 ± 0.018; 1.000 ± 0.0005; 0.993 ± 0.02) and accurately retrieved the full-length haplotype of each VNTR unit. Human variant levels were highly correlated with next-generation sequencing (R2 = 0.983) without bias across the whole variant level range. Six reads per UMI produced sequences of each KIV-2 unit with Q40 quality. The KIV-2 repeat number determined by coverage-corrected unique haplotype counting was in close agreement with droplet digital PCR (ddPCR), with 70% of the samples falling even within the narrow confidence interval of ddPCR. We then analyzed 62,679 intra-KIV-2 STR sequences and explored KIV-2 SNP haplotype patterns across five ancestries. UMI-ONT-Seq accurately retrieves the SNP haplotype and precisely quantifies the VNTR copy number of each repeat unit of the complex KIV-2 VNTR region across multiple ancestries. This study utilizes the KIV-2 VNTR, presenting a novel and potent tool for comprehensive characterization of medically relevant complex genome regions at scale.

8182 New Mouse Model Mirrors Human MODY8: Opening Doors to Understanding Exocrine-Endocrine Crosstalk in the Diabetic Pancreas

Abstract Disclosure: K. El Jellas: None. V. Salerno: None. L.S. Katz: None. J. Hu: None. G. Fogarty: None. A. Mandal: None. A. DiStefano-Forti: None. D. Scott: None. M. Lowe: None. A. Molven: None. R.N. Kulkarni: None. Maturity-onset diabetes of the young, type 8 (MODY8) is a dominantly inherited form of monogenic diabetes, caused by heterozygous mutations in the carboxyl ester lipase (CEL) gene expressed in pancreatic acinar cells. MODY8 is characterized by chronic pancreatitis and exocrine dysfunction. Despite the intimate anatomical relationship between the endocrine and exocrine pancreas, little is known about the biological components mediating exocrine-endocrine communication in regulating glucose homeostasis. Here, we report a refined mouse model that bears the mutation present in MODY8 patients on one allele, and the humanized normal variable number of tandem repeat (VNTR) of CEL with 16 repeats (16R) on the other, mimicking the genetic make-up seen in MODY8 patients. Examination of glucose homeostasis revealed that inducing stress (e.g. by high-fat diet) leads to glucose intolerance in MODY8/16R mice and a blunted glucose-stimulated insulin-secretion compared to their control littermates. Histopathological analyses demonstrated features of chronic pancreatitis in some pancreatic lobes, including inflammation, acinar atrophy, acinar-to-ductal metaplasia, fibrosis and fat infiltration, mirroring the morphology of the human disease. Moreover, we applied immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) to intact pancreata and found that beta-cell mass was significantly decreased in the MODY8/16R mice, with a large number of islets located within fatty lobes. These results are coupled with comparative proteomics and lipidomic profiling with the aim of understanding the molecular signatures of exocrine and endocrine cells during pathogenesis. In conclusion, we have generated a mouse that recapitulates several features of the exocrine and endocrine phenotype characteristic of human MODY8. This novel model provides a unique opportunity, not possible in humans, to characterize pancreatic endocrine-exocrine communication in a longitudinal manner as a response to diverse environmental stressors. Presentation: 6/2/2024

Systematic Screening of Autosomal Dominant Tubulointerstitial Kidney Disease- MUC1 27dupC Pathogenic Variant through Exome Sequencing.

The MUC1 gene is associated with autosomal dominant tubulointerstitial kidney disease (ADTKD), leading to CKD. Current methods of sequencing, like exome sequencing, rarely detect MUC1 pathogenic variants because of the variable number of tandem repeats (VNTR) in MUC1 exon2. We demonstrated that combining fast read filtering with a sensitive VNTR genotyping strategy enables systematic screening of 27dupC pathogenic MUC1 variant from exome data. We initially validated our bioinformatics pipeline in a proof-of-concept cohort incorporating exome data from 33 participants with a known MUC1 pathogenic variant identified by Snapshot PCR and confirmed by 54 MUC1-negative individuals for negative control. We then retrospectively analyzed exome sequencing data from January 2019 to October 2023 from 3512 adult participants with nephropathy of unknown origin. Finally, we prospectively validated our pipeline in 825 additional participants enrolled from November 2023. SharkVNTyper accurately identified MUC1 variants in 32 out of 33 participants and excluded its presence in all the 54 negative controls in the proof-of-concept cohort (sensitivity of 97%, specificity of 100%). Integration of Shark tool with VNTyper significantly reduced running time from 6-12 hours to 5-10 minutes per sample, allowing both retrospective and prospective analysis. In the retrospective cohort, SharkVNTyper identified 23 additional positive cases that were not suspected clinically and had been missed in the initial exome analysis; 18 of these cases were confirmed as carrying the MUC1 27dupC mutation by low-throughput Snapshot PCR. In the prospective cohort of 825 CKD cases, systematic screening discovered 13 positive cases, with 12 confirmed by PCR. Overall, of 63 participants (1.4% of 4653) with molecularly confirmed ADTKD-MUC1, comprehensive diagnoses and descriptions of the disease were available for 24 participants. Median age of kidney failure was 50 years, 38% exhibited bilateral multiple kidney cysts, 8% had early-onset gout, and 58% had arterial hypertension. SharkVNTyper enables the analysis of highly repeated regions, such as the MUC1 VNTR, and facilitates the systematic screening of ADTKD-MUC1 from exome data, fostering 27dupC variation identification.

Development of a method for the imputation of the multi-allelic serotonin-transporter-linked polymorphic region (5-HTTLPR) in the Japanese population.

Serotonin-transporter-linked polymorphic region (5-HTTLPR), a variable number of tandem repeats in the promoter region of serotonin transporter gene, is classified into short (S) and long (L) alleles. Initial case-control association studies claiming the risks of the S allele in depression and anxiety were not completely supported by recent studies. However, most studies, especially those on East Asian populations, have overlooked the complexity of 5-HTTLPR, which involves multiple different alleles with distinct functional properties. To address this issue, distinguishing multiple 5-HTTLPR alleles is essential. Here, using the 5-HTTLPR genotypes previously determined by exhaustive Sanger sequencing of approximately 1,500 Japanese subjects and their comprehensive SNP data, we constructed a method for 5-HTTLPR genotype imputation. We identified 28 tag SNPs for the imputation of four major 5-HTTLPR alleles, which collectively account for 97.6% of 5-HTTLPR alleles in the Japanese population. Our imputation method, achieved an accuracy of 0.872 in cross-validation, will contribute to association analysis of 5-HTTLPR in the Japanese subjects.

Whole Mitochondrial Genome Sequencing Analysis of Canine Testicular Tumours.

Currently, the molecular background based on mitochondrial DNA (mtDNA) analysis of canine testicular tumours is underestimated. The available data mostly focus on histopathological evaluations, with a few reports of nuclear genome (nDNA) studies. Tumourigenesis represents a highly complex and diverse genetic disorder, which can also encompass defects in mtDNA. The aim of this study was to identify molecular changes in whole mitochondrial genome sequences obtained from dogs affected by testicular tumours. Samples of blood, tumour, and healthy tissue were collected from each animal, and mtDNA (ultimately 45 samples) was subsequently sequenced. Thereafter, protein analyses were performed to assess the impact of the identified molecular alterations on the amino acid level. The total number of observed changes included 722 SNPs, 12 mutations, 62 indels, 5 indel mutations, and 35 heteroplasmic sites. The highest number of mtDNA variants in protein-coding genes COX1, COX3, ATP6, ND1, ND4, and ND5 was observed. Interestingly, SNPs were found in 10 out of 22 tRNA genes. Most of the identified mtDNA defects were synonymous changes at the amino acid level. Also, polymorphisms and heteroplasmy were frequently observed in the variable number of tandem repeat (VNTR) regions, especially in its fragment spanning 16,138-16,358 bp. Based on the obtained results, it was possible to select 11 polymorphisms that occurred in all the tested samples (benign, malignant) and an additional five SNPs identified only in benign neoplasms. The comprehensive analysis of malignant testicular tumours demonstrated a significant diversity in their molecular profiles, with changes ranging from 17 to 101 per sample.

Genetic variability of 23 autosomal STRs in Austroasiatic-speaking populations from Thailand.

Austroasiatic (AA) speakers constitute around 4% of the population of Thailand, while the majority (89.4%) speak Kra-Dai (KD) languages. Previous forensic and population genetic studies in various Thai populations have employed a limited number of short tandem repeats (STRs). This study aims to expand the investigation of the genetic makeup of AA populations in Thailand and their relationship to KD populations using a larger number of autosomal STRs with the VeriFiler™ Plus PCR Amplification Kit. We generated 593 new genotypes from AA-speaking groups and combined them with previously reported data from AA and KD groups. A total of 1,129 genotypes across 23 STR loci were used to construct the largest allelic frequency profile for Thai and Lao populations. However, several loci deviated from Hardy-Weinberg equilibrium, likely due to the reduced genetic diversity in some highland populations, which should be considered in forensic investigations. Beyond forensic applications, our findings reveal genetic differences between AA-speaking groups in Northern and Northeastern Thailand. The AA groups from Northeastern Thailand exhibit greater genetic homogeneity and diversity, likely due to population interactions. In contrast, reduced diversity and increased heterogeneity in AA groups from Northern Thailand are possibly driven by genetic drift and cultural and geographic isolation. In conclusion, we emphasize the usefulness of increasing the number of autosomal STRs in forensic and anthropological genetic studies. Additional Y-STR and X-STR data from various AA-speaking groups in Thailand would further enhance and strengthen forensic STR databases in the region.

Correlations of PSGL-1 VNTR polymorphism with the susceptibility to severe HFMD associated with EV-71 and the immune status after infection

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71–induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71–associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71–induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases.

Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.

The evaluation of IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms in Egyptian patients with alopecia areata: a case–control study

BackgroundAlopecia areata (AA) is a non-scarring hair loss condition that usually affects the scalp. The exact pathogenesis is poorly understood; however, multiple factors like genetics, environmental, psychological, and immunological factors may have a role. The purpose of this study was to look into possible links between the functional interleukin-4 (IL-4) gene intron 3 variable number of tandem repeats (VNTR) and TNF-(rs1799964) gene polymorphism and AA susceptibility. This case–control study consisted of 79 unrelated patients and 156 age- and sex-matched healthy individuals as a control group. The Severity of Alopecia Tool was used to assess the extent of hair loss from the scalp. Polymerase chain reaction (PCR) with specific primers was used to determine IL-4 gene 70-bp VNTR polymorphism while polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) was used to investigate TNF-α (rs1799964) gene polymorphism.ResultsNone of the selected polymorphisms for both genotypes and alleles had statistical significance when patients and controls were compared with each other (p-values for IL-4 VNTR were 0.11, 0.74, 0.052 and 0.27 and for TNF-α polymorphism was 0.71, 0.43, 0.65 and 0.55, respectively, for codominant, dominant, recessive and overdominant models of inheritance, respectively). Furthermore, the same results were retrieved when the genotypes were compared with the patient’s clinical and demographic data (p-value > 0.05).ConclusionThe findings indicate that IL-4 VNTR intron 3 and TNF-α (rs1799964) gene polymorphisms are not linked to the development of AA in the Egyptian population.

Exploring Gene Polymorphisms Associated with Otitis Media through Variable Number Tandem Repeats (VNTR) Region

Background: Otitis is an inflammation and infection of the inner lining of the middle ear. The problem known as suppurative otitis media if there are droplets and holes in the tympanic membrane due to this inflammation and infection of the mucous membrane. The present study focused on specific genes suspected to be associated with otitis media in 60 subjects clinically diagnosed with OM The study focuses on two genes, MUC5B and IL-1RN, whose genes mutations by variable number of tandem repeat (VNTR) regionsMethods: Variable numbers of tandem repeats (VNTR) regions were used to genotype 60 patients with otitis media and 30 healthy individuals for genotypic polymorphisms for the MUC5B and IL-1RN genes by number of tandem repeats (VNTR) regions on a variable basis Blood samples were collected from participants, and three milliliters were placed directly into EDTA tubes. Subsequently, PCR products were detected by agarose gel electrophoresis and visualized by ethidium bromide staining. The size of amplified DNA fragments was determined by comparison with a 100-bp DNA ladder molecule size markerResults: About the same, the paper represents the association of gene polymorphisms with otitis media by genotyping a total of 60 patients as OM, and, on the other hand, 30 individuals were noted to be healthy. This was to determine genotypic polymorphisms for the two genes, that is, MUC5B and IL1RN, within the VNTR region. The results showed that the carriers of allele 2 in the genotypic position had a risk of nearly twofold getting otitis media.Conclusion: Polymorphism of the MUC5B and IL-1RN genes can be responsible for the severity of the inflammatory process in the middle ear, complicated by otitis media or hearing loss.Keywords: Otitis media; Gene polymorphism; MUC5B; IL-1RN; VNTR

The influence of PER3 VNTR genotypes on the age of onset in a group of bipolar I disorder patients: an exploratory study

BackgroundPER3 is a circadian gene that contains a variable number of tandem repeats (VNTR) which codifies for three genotypes: 4/4; 4/5; and 5/5 and is involved in non-visual response to light, a critical process associated with bipolar disorder onset. Benedetti et al. (Neurosci Lett 445(2):184–7) related this VNTR with bipolar disorder age of onset and linked genotype 5/5 with an earlier onset. In this study, we aimed to investigate these associations of PER3 VNTR genotypes with age of onset in a homogenous sample of German patients with bipolar I disorder through Kaplan-Meier curves.Methods45 patients were enrolled and divided into three groups according to PER3 VNTR genotypes. Recognizing common biological features, we built a combined group of -5 allele carriers (4/5 + 5/5). As a primary outcome, Kaplan-Meier analysis was conducted to delineate the three genotypes’ influence on age of onset. The secondary Kaplan-Meier analysis aimed to evaluate the relation between the 4/4 homozygotes group and the combined group (4/5 + 5/5) with age of onset. Finally, we proceeded to compare groups through a Log Rank Test and performed an analysis of covariance (ANCOVA).ResultsThe Kaplan-Meier analysis with three separate genotypes didn’t replicate the findings of Benedetti’s study. The analysis comparing genotype 4/4 with the combined group showed the influence of PER3 VNTR variants on the age of onset and relates genotype 4/4 to an earlier onset. ANCOVA between the combined and the 4/4 genotype groups, correlated genotype 4/4 with an increased number of depressive episodes.ConclusionThis study showed no significant effect of PER3 VNTR genotypes on the age of onset and in linking genotype 5/5 with an earlier onset age. Contrasting results may arise from intrinsic differences between the two studies but also shed light on hypothetically different levels of functioning of PER3 VNTR genotypes in the context of bipolar pathology. Further studies will require bigger and more homogeneous clinical samples.

A study of association of the VNTR MIR-137 rs58335419 with schizophrenia

The MIR137 gene encodes microRNA-137 (miR-137), which is a brain-enriched miR that is highly expressed in various brain regions. miR-137 has been identified as a modulator of processes involved in the pathogenesis of neuropsychiatric disorders. Functional polymorphism of variable number of tandem repeats (VNTR) rs58335419 was found in the regulatory region of the MIR137 gene. It is associated with a change in the expression of miR-137 and, as a result, with an increased risk of developing psychopathologies, including schizophrenia. In this study, we for the first time have analyzed the distribution of frequencies of alleles and genotypes of VNTR MIR137 in a large sample from the Russian population. The association of VNTR with the risk of schizophrenia has been studied. It was found that the presence of VNTR alleles with more than three repeats, as well as a genotype homozygous for such alleles, is associated with an increased risk of developing schizophrenia (OR = 1.4, 95% CI: 1.01-1.95).

The role of immune checkpoint inhibitors: Variable number of tandem repeat (VNTR) polymorphism in the second exon of the P-selectin glycoprotein ligand-1 (PSGL-1) gene polymorphism in multiple myeloma.

Somatic mutations and polymorphisms may play a role in multiple myeloma (MM) susceptibility and survival. One of the immune checkpoint inhibitors is P-selectin glycoprotein ligand-1 (PSGL-1); the majority of tumor-infiltrating leukocytes express PSGL-1, causing T cell and immune inhibition via PSGL-1 mediator molecules. We aimed to investigate the effect of variable number of tandem repeat (VNTR) polymorphism in the second exon of the PSGL-1 gene on MM susceptibility, response to treatment and survival in our patient group. A total of 238 patients diagnosed with MM between January 2010 and January 2021 and 162 healthy individuals as a control group were included in this cross-sectional study. The genotypes of the VNTR polymorphism in the second exon of the PSGL-1 gene were statistically compared between patients and healthy controls; the statistically significant effects of the genotypes on response to first-line treatment and survival were examined. The AC genotype was significantly higher in healthy controls compared to patients diagnosed with MM (p < 0.001). The median PFS in patients with AA/AB/AC was 56 months, while it was 100 months in patients with BB/CC. The hazard ratio of 1.34 for PFS was found to be clinically significant and having the BB/CC genotype could provide a longer PFS compared to others, but it was not statistically significant due to the sample size. Our study results will shed light on new study plans in terms of immune checkpoint target therapies among conventional treatment preferences in MM.

Association of NLRP3 and IL-4 VNTR polymorphisms and genetic susceptibility to preeclampsia: A case-control study

IntroductionAbnormalities in the maternal immune system and insufficient gestational immune tolerance may significantly contribute to the development of preeclampsia (PE). The NLR family pyrin domain containing 3 (NLRP3) functions as a pattern recognition receptor that identifies pathogen-associated molecular patterns. Interleukin-4 (IL-4) is a potent anti-inflammatory cytokine that modulates the immune response. Therefore, this study aims to elucidate the impact of NLRP3 and IL-4 variable number of tandem repeats (VNTR) polymorphisms on susceptibility to PE. Materials and MethodsA total of 1,018 patients with PE and 1,007 normal pregnant women were recruited as the case group and the control group, respectively. Peripheral blood DNA was extracted, and NLRP3 and IL-4 VNTR polymorphisms were genotyped using polymerase chain reaction and gel electrophoresis. Genotypes and allele frequencies of pregnant women were assessed in both cohorts. ResultsThe NLRP3 VNTR 9–7 genotype in the PE group was significantly lower than that in the control group, but 9 and 14 allele frequencies were significantly higher in patients with PE. Individuals with IL-4 VNTR genotypes 1–2 had a lower risk of PE than controls, and the IL-4 VNTR 2 allele frequency was significantly lower in patients with PE. ConclusionsThis study, the first of its kind in the literature, evaluates the impact of NLRP3 VNTR and IL-4 VNTR polymorphisms on PE, revealing a significant correlation with PE susceptibility. This investigation contributes to understanding the pathogenesis of PE and provides a reference point for developing strategies to prevent and treat the disease in the future.

Evaluation of a Multilocus Variable-Number Tandem-Repeat Analysis Scheme for Typing Ochrobactrum anthropi.

Ochrobactrum anthropi (O. anthropi) is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with low immunity. The identification of O. anthropi mainly uses biochemical methods, such as the API 20NE or Vitek-2. The typing studies of O. anthropi have mainly utilized PFGE, rep-PCR, AFLP, 16s rDNA sequencing, RecA-PCR RFLP, and MALDI-TOF MS. This study aims to evaluate the polymorphisms of variable-number tandem-repeats (VNTRs) within genomic DNA of O. anthropi strains. The tandem repeats (TRs) in genomic DNA are discovered using Tandem Repeat Finder software (version 4.09). Twelve different VNTRs are designated and assigned to the nomenclature. The primers for PCR of 12 loci are designed. The PCR product size is converted to the number of tandem repeats in every locus. The relatedness of 65 O. anthropi strains from geographically different countries are analyzed by means of 12-variable-number tandem-repeat analysis(MLVA-12). A total of 51 different genotypes are found in 65 O. anthropi strains. These strains, which were collected from the same environmental samples, hospitals, and countries, are clustered within the same or closely genotypes. The MLVA-12 assay has a good discriminatory power for species determination, typing of O. anthropi, and inferring the origin of bacteria.

Mini-heterochromatin domains constrain the cis-regulatory impact of SVA transposons in human brain development and disease.

SVA (SINE (short interspersed nuclear element)-VNTR (variable number of tandem repeats)-Alu) retrotransposons remain active in humans and contribute to individual genetic variation. Polymorphic SVA alleles harbor gene regulatory potential and can cause genetic disease. However, how SVA insertions are controlled and functionally impact human disease is unknown. Here we dissect the epigenetic regulation and influence of SVAs in cellular models of X-linked dystonia parkinsonism (XDP), a neurodegenerative disorder caused by an SVA insertion at the TAF1 locus. We demonstrate that the KRAB zinc finger protein ZNF91 establishes H3K9me3 and DNA methylation over SVAs, including polymorphic alleles, in human neural progenitor cells. The resulting mini-heterochromatin domains attenuate the cis-regulatory impact of SVAs. This is critical for XDP pathology; removal of local heterochromatin severely aggravates the XDP molecular phenotype, resulting in increased TAF1 intron retention and reduced expression. Our results provide unique mechanistic insights into how human polymorphic transposon insertions are recognized and how their regulatory impact is constrained by an innate epigenetic defense system.

#276 ADTKD genetic diagnostics in Europe: where do we stand and what is needed?

Abstract Background and Aims We are the first European patient organization to pursue the goal of raising awareness of the monogenic kidney disease ADTKD (autosomal dominant tubulointerstitial kidney disease) and supporting its research (www.adtkd.de/en-gb). To date, causative mutations are known in at least five disease genes (UMOD, MUC1, REN, HNF1B, SEC61A1) [1] and one can expect that even more disease genes exist. There are already strong international collaborations to define the clinical course of ADTKD [2] and to evaluate a targeted therapy in clinical trials [3]. Clinical registries form the basis for the inclusion of patients, preferably at an early stage of the disease. Such a registry already exists in the USA and is currently being established in Europe (ADTKD-Net) as part of the European Joint Program on Rare Diseases (EJP RD). Due to its non-specific and variable clinical characteristics, ADTKD is often misdiagnosed or not diagnosed at all. Experts therefore suspect a high number of unreported cases. There are also challenges in genetic diagnostics. While next generation sequencing (NGS) panels have been established for ADTKD, they are usually unable to detect pathogenic MUC1 variants due to their specific location in the genome. This is the insertion of an additional cytosine (dupC) in a VNTR (Variable Number of Tandem Repeats) domain of the MUC1 gene, which cannot be detected by NGS and therefore requires special procedures such as SnaPshot Minisequencing [4] or VNtyper [5]. For instance, there are only two laboratories that can perform this specific MUC1 testing in Germany to date. The aim of this study is to assess the status quo of ADTKD diagnostics in Europe. We hypothesize that there is a lack of appropriate structures to adequately and reliably diagnose ADTKD, notably ADTKD-MUC1. The study is intended to help identify deficits and obstacles in order to address these in a targeted manner. Methods We used Survey Monkey to create a digital survey, which we sent by email to around 50 European ADTKD experts. They were asked to answer 7 key questions (Fig. 1). For the selection of suitable ADTKD experts, we use various sources such as publications on ADTKD in PubMed, the Expert Finder and the European Patient Advocacy Groups (ePAG) in the European Reference Network for Rare Kidney Diseases (ERKNet) and our own network. Our aim is to cover at least the 30 largest countries in Europe in terms of population. Results Results will be presented at the ERA conference. Conclusion With our survey we would like to substantiate the hypothesis that the possibilities of molecular diagnostics of ADTKD in Europe are currently insufficient. Based on our results, short and long-term measures should be taken to remedy potential deficits so that as many patients as possible can be identified and have access to therapeutic options in the future. Another interesting aspect is the awareness of ADTKD among European nephrologists. This could be a future activity of our patient organization.

Attention-deficit/hyperactivity disorder and dopamine receptor D4 (DRD4) exon 3 variable number of tandem repeats (VNTR) 2-repeat allele.

To investigate the association of attention-deficit/hyperactivity disorder (ADHD) with the 48-base pair (bp) variable number of tandem repeats (VNTR) in exon 3 of the dopamine receptor D4 (DRD4) gene, we genotyped 240 ADHD patients and their parents from Hong Kong. The 4R allele was most common, followed by 2R. We examined association between the 2R allele (relative to 4R) and ADHD by Transmission Disequilibrium Test (TDT). The odds ratio (OR) (95% confidence interval) was 0.90 (0.64-1.3). The p-value was 0.6. Examining subgroups revealed nominally significant association of 2R with inattentive ADHD: OR=0.33 (0.12-0.92) and p=0.03. Because our study used TDT analysis, we meta-analyzed the association of 2R with ADHD in Asians (1329 patient alleles), revealing results similar to ours: OR=0.97 (0.80-1.2) and p=0.8. To examine the association of 2R with inattentive ADHD, we meta-analyzed all studies (regardless of analysis type or ethnicity, in order to increase statistical power): 702 patient alleles, 1420 control alleles, OR=0.81 (0.57-1.1) and p=0.2. Overall, there is no evidence of association between ADHD and the 2R allele, but the suggestive association with the inattentive type warrants further investigation.