Allele-specific Oligonucleotide Research Articles

High-Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis.

Candida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene. In this study, we have developed an assay based on real-time PCR and high-resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S). We designed allele-specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high-resolution melting (HRM) analysis. The designed oligonucleotides to detect the Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real-time PCR combining oligonucleotides for the wild-type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach. This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

109P Preserve sense by antisense – a novel allele-specific antisense oligonucleotide therapy for SPTLC1-related hereditary sensory neuropathy

109P Preserve sense by antisense – a novel allele-specific antisense oligonucleotide therapy for SPTLC1-related hereditary sensory neuropathy

Antisense oligonucleotide therapy in an individual with KIF1A-associated neurological disorder.

KIF1A-associated neurological disorder (KAND) is a neurodegenerative and often lethal ultrarare disease with a wide phenotypic spectrum associated with largely heterozygous de novo missense variants in KIF1A. Antisense oligonucleotide treatments represent a promising approach for personalized treatments in ultrarare diseases. Here we report the case of one patient with a severe form of KAND characterized by refractory spells of behavioral arrest and carrying a p.Pro305Leu variant in KIF1A, who was treated with intrathecal injections of an allele-specific antisense oligonucleotide specifically designed to degrade the mRNA from the pathogenic allele. The first intrathecal administration was complicated by an epidural cerebrospinal fluid collection, which resolved spontaneously. Otherwise, the antisense oligonucleotide was safe and well tolerated over the 9-month treatment. Most outcome measures, including severity of the spells of behavioral arrest, number of falls and quality of life, improved. There was little change in the 6-min Walk Test distance, but qualitative changes in gait resulting in meaningful reductions in falls and increasing independence were observed. Cognitive performance was stable and did not degenerate over time. Our findings provide preliminary insights on the safety and efficacy of an allele-specific antisense oligonucleotide as a possible treatment for KAND.

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5′-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

Carrying SNP rs17506395 (T > G) in TP63 gene and CCR5Δ32 mutation associated with the occurrence of breast cancer in Burkina Faso.

Genetic alterations in the TP63 (GenBank: NC_000003.12, ID: 8626) and CCR5 (receptor 5 chemokine co-receptor) (GenBank: NC_000003.12, ID: 1234) genes may increase the risk of developing breast cancer. The aim of this study was to investigate the probable involvement of polymorphisms rs17506395 in the TP63 (tumour protein 63) gene and the CCR5Δ32 mutation in the occurrence of breast cancer in Burkina Faso. This case-control study included 72 patients and 72 controls. Genotyping of SNP rs17506395 (TP63) was performed by polymerase chain reaction-restriction fragment length polymorphism, and genotyping of the CCR5Δ32 mutation was performed by allele-specific oligonucleotide polymerase chain reaction. For SNP rs17506395 (TP63), the genotypic frequencies of wild-type homozygotes (TT) and heterozygotes (TG) were, respectively, 27.72 and 72.22% in cases and 36.11 and 63.89% in controls. No mutated homozygotes (GG) were observed. For the CCR5Δ32 mutation, the genotypic frequencies of wild-type homozygotes (WT/WT) and heterozygotes (WT/Δ32) were 87.5 and 13.5%, respectively, in the cases and 89.29 and 10.71%, respectively, in the controls. No mutated homozygotes (Δ32/Δ32) were observed. None of the polymorphisms rs17506395 of the TP63 gene (OR = 1.47, 95% CI = 0.69-3.17, P = 0.284) and the CCR5Δ32 mutation (OR = 1.32, 95% CI = 0.46-3.77; P = 0.79) were associated with the occurrence of breast cancer in this study.

Allele-specific antisense oligonucleotides for the treatment of BEST1-related dominantly inherited retinal diseases: An in vitro model

Retinal dystrophies are a common health problem worldwide that are currently incurable due to the inability of retinal cells to regenerate. Inherited retinal diseases (IRDs) are a diverse group of disorders characterized by progressive vision loss caused by photoreceptor cell dysfunction. The eye has always been an attractive organ for the development of novel therapies due to its independent access to the systemic pathway. Moreover, anti-sense oligonucleotides (ASOs), which facilitate manipulation of unwanted mRNAs via degradation or splicing, are undergoing rapid development and have been clinically deployed for the treatment of several diseases. The primary aim of this study was to establish a reliable in vitro model utilizing induced photoreceptor-like cells (PRCs) for assessing the efficacy and safety of ASOs targeting the BEST1 gene. Despite advances in gene therapy, effective treatments for a broad range of IRDs remain limited. An additional aim was to develop an in vitro model for evaluating RNA-based therapeutics, specifically ASOs, for the treatment in IRDs.Firstly, a cell culture model was established by induction of PRCs from dermal fibroblasts via direct programming. The induced PRCs were characterized at both the transcriptomic and protein level. Then, a common single nucleotide polymorphism (SNP) was identified in the BEST1 gene (rs1800007) for targeting with ASOs. ASOs were designed using the GapmeR strategy to target multiple alleles of this SNP, which is potentially suitable for a large proportion of the population. The efficacy and possible off-target effects of these ASOs were also analyzed in the induced PRC model.The findings show that the selected ASOs achieved allele-specific mRNA degradation with virtually no off-target effects on the global transcriptome profile, indicating their potential as safe and effective therapeutic agents. The presented in vitro model is a valuable platform for testing personalized IRD treatments and should inspire further research on RNA-based therapeutics. To the best of our knowledge this study is the first to test RNA-based therapeutics involving the use of ASOs in an induced PRC model. Based on the present findings, it will be possible to establish an ex vivo disease model using dermal fibroblast samples from affected individuals. In other words, the disease model and the ASOs that were successfully designed in this study can serve as a useful platform for the testing of personalized treatments for IRDs.

Association between Genetic Variants Linked to Premature Ovarian Insufficiency and Inflammatory Markers: A Cross-Sectional Study.

Premature menopause (PM) is the cessation of ovarian function before age 40. PM women are more likely to have cardiovascular diseases (CVDs), diabetes, and mental disorders. This is the first study that assessed the association of single nucleotide polymorphisms (SNPs) with anti-heat shock protein 27 (Hsp27), High-sensitivity C-reactive protein (hs- CRP), and PM and serum pro-oxidant-antioxidant balance (PAB), as putative risk factors for CVDs. We aimed to explore the association of oxidative stress markers with eight different SNPs shown to be related to premature menopause. In this cross-sectional research, we included 183 healthy women and 117 premature menopausal women. We determined baseline characteristics for all participants and measured serum hs-CRP, anti-HSP-27 antibody titer, and PAB levels using the established methods. Genotyping for eight SNPs was done using the tetra amplification refractory mutation system polymerase chain reaction (Tetra-ARMS PCR) and allele-specific oligonucleotide PCR (ASO-PCR) methods. We found a significant difference between mean serum PAB levels and the genetic variant of rs16991615 (P=0.03). ANCOVA showed a significant effect of the genotypes rs4806660 and rs10183486 on hs-CRP serum levels in the case and control groups, respectively (P=0.04 and P=0.007). ANCOVA also showed an association between rs244715 genotypes and anti-hsp27 serum levels in the case group (P=0.02). There was a significant effect of the genotypes of rs451417 on the serum hs-CRP level in the control group (P=0.03). There was a significant association of the genetic variants related to PM with oxidative stress and inflammatory markers (serum PAB, anti-hsp27 antibody, and hs-CRP). Accordingly, this seems to be an effective approach to predicting susceptible subjects for cardiovascular and mental disorders as well as various cancers.

Современные подходы к оценке минимальной остаточной болезни при множественной миеломе (плазмоклеточной миеломе)

The treatment of multiple myeloma is inextricably linked to the need for assessment and monitoring of the minimal residual disease (MRD). Assessment of the MRD allows evaluating the efficacy of therapy and obtaining significant prognostic information; it is an indicator of the degree of eradication of the tumor clone. The methods for detecting residual tumor cells evolve constantly, which translates into updates of the criteria reflecting the scale of response to therapy. There is no single MRD detection technique; common recommendations suggest seeking for pathological cells both intramedullary and extramedullary. This review describes current MDR determination methods, including imaging, next generation multiparametric flow cytometry, and methods based on DNA analysis — allele-specific oligonucleotide polymerase chain reaction and next generation sequencing. We compare their advantages, limitations, disadvantages, clinical significance, and show the necessary sensitivity thresholds of the described methods and the conditions that make this or that approach ideal in the context of detection of MRD.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones >5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Association of Measurable Residual Disease with Progression-Free Survival in Patients with Chronic Lymphocytic Leukemia: A Systematic Review and Meta-Analysis in the Era of Novel Agents

Introduction: Achieving undetectable measurable residual disease (uMRD) following treatment has shown significant prognostic value in several hematological malignancies. In chronic lymphocytic leukemia (CLL), uMRD is associated with improved outcomes in the era of chemoimmunotherapy (CIT). However, the role of measurable residual disease (MRD) in the context of novel agents such as Bruton tyrosine kinase inhibitors (BTKi) and/or BCL2 inhibitors (e.g. venetoclax) is uncertain. To address this question, we conducted a systematic review of the literature and performed a comprehensive meta-analysis of trials that included novel agents to evaluate the association between MRD status after treatment and PFS in patients with CLL. Methods: We conducted a comprehensive search of PubMed, EMBASE, Scopus, and Web of Science focusing on CLL trials reporting MRD and its association with PFS. Results were independently screened by two reviewers. Inclusion criteria included prospective phase 3 trials involving a novel agent in one of the treatment arms, while studies with incomplete information on MRD status related to PFS, reviews, abstracts, and retrospective analyses were excluded. The initial search identified 185 studies in PubMed and 402 studies in EMBASE. After applying prespecified criteria, a total of 10 phase 3 trials were included in our analysis. We extracted sample size, patient age, follow-up time, time point and tissue of MRD assessment, MRD cut-off and rate, performance status and molecular status when available. We only considered treatment arms with complete MRD information. The comparison involved patients who achieved uMRD with those exhibiting detectable MRD. When available, hazard ratios (HR) and confidence intervals (CI) were used to evaluate the relationship between MRD status and PFS. If not, we used Kaplan-Meier (KM) curves from both uMRD and MRD subgroups and extracted the coordinates points of the curves using DigitizeIt software (version 2.5.10, I. Bormann) and using the number at risk provided we estimated the HRs and CIs. A meta-analysis was performed using a random effects model from the R package metafor (Viechtbauer, W. (2010). Conducting meta-analyses in R with the metafor package. Journal of Statistical Software, 36(3), 1-48). The pooled hazard ratio estimates, and the corresponding 95% CI and prediction intervals (PI) were reported. Funnel and Baujat plots were generated to assess study heterogeneity and bias. All analysis was performed using R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria). Results: A total of 10 phase 3 trials, encompassing 14 treatment arms and involving 3152 patients, met the stringent inclusion criteria. All but one study used a uMRD cut-off of < 0.01%. Detailed information on the treatment arms and key trial characteristics is presented in Table 1. Notably, ten treatment arms were derived from first-line treatment trials, while the remaining arms pertained to the relapsed/refractory population. Only two trials (4 treatment arms) assessed MRD using next generation sequencing (NGS), while the rest used multicolor flow cytometry (MFC) (9 treatment arms) and allele-specific oligonucleotides polymerase chain reaction (ASO-PCR) (2 treatment arms). All but 3 trials assessed MRD in both bone marrow (BM) and peripheral blood (PB). One trial (2 treatment arms) was excluded from the met-analysis due to higher MRD cut-off. All studies included in the meta-analysis measured uMRD (<0.01) versus MRD. The PFS pooled HR estimate for achieving uMRD at 0.01 cut off from the meta-analysis was 0.32 (95% CI [0.22, 0.45]), with minimal bias observed on the Funnel Plot. Conclusions: In this comprehensive meta-analysis, comprising 9 phase 3 trials, achieving uMRD was strongly associated with PFS. These findings further establish the prognostic role of MRD in CLL management and its potential use as a surrogate marker in clinical trials. As the body of evidence on MRD in CLL continues to grow, it becomes imperative to standardize the optimal time point, methodology, and tissue for MRD assessment. Substantial heterogeneity in these aspects underscores the necessity for unified guidelines to ensure the accurate and consistent evaluation of MRD status in clinical practice.

Heterogeneity of Mycobacterium tuberculosis Strains Circulating in Panama's Western Region.

Tuberculosis remains a challenge in both rural and urban areas. Although a majority of countries display a higher burden in urban areas compared with rural areas, Panama continues to report the highest mortality rate in Central America. Urban areas, such as Panama City, report a high tuberculosis burden, whereas Panama's western region, including the provinces of Chiriquí, Bocas del Toro (both semiurban) and Ngäbe-Bugle (rural), show a lower burden. We aimed to identify highly transmitted Mycobacterium tuberculosis strains within rural and semiurban settings of Panama's western region during a 3-year period (2017, 2019, 2021). We randomly selected 87 M. tuberculosis isolates from a biobank from Panama's western region and analyzed them using allele-specific oligonucleotide polymerase chain reaction and 24-mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Our results show only 11.7% (10/85) of M. tuberculosis strains identified as prevalent A-Beijing, B-Haarlem, or C-LAM Strains. We found a low prevalence of A, B, and C M. tuberculosis strains in both rural and semirural settings compared with isolates collected from the Eastern Colon Province. MIRU-VNTR genotyping revealed a high degree of diversity with no clusters with single loci variation of ≥ 2 loci. These results support the notion that tuberculosis prevalence in the rural and semiurban western region of Panama are not due to previously described highly transmitted strains but is influenced instead by other health determinants, including poor health system access and a lack of systematic transmission chain monitoring. For remote rural and semiurban settings, we recommend allocating resources to reinforce efforts to prevent tuberculosis spread.

New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil

This study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.

Involvement of p.R72P and PIN3 Ins16bp (TP53) Polymorphisms and the I157T (CHEK2) Mutation in Breast Cancer Occurrence in Burkina Faso

Introduction: The TP53 and CHEK2 genes have been described as breast cancer susceptibility genes and some of their polymorphisms have been associated with an increased risk of breast cancer in certain populations.Aim: The objective of this study was to investigate the p.R72P and PIN3 Ins16bp (TP53) polymorphisms and the I157T (CHEK2) mutation developping of breast cancer. Methods: This case-control study had enrolled 144 participants including 65 cases (breast cancer patients) and 79 controls (women without breast abnormalities) in the city of Ouagadougou in Burkina Faso. The DNA was extracted using the method of “salting out” and the genotyping of polymorphisms was performed by ASO-PCR (Allele Specific Oligonucleotides - Polymerase Chain Reaction), conventional PCR and PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) techniques. Results: The heterozygous genotype (RP) of the p.R72P polymorphism of TP53 gene was in the majority in cases (73.85%) and controls (73.42%). Regarding to the PIN3 Ins16bp polymorphism of TP53 gene, the homozygous wild type (A1A1) was the most represented in both cases (53.85%) and controls (60.76%). Concerning the I157T mutation of CHEK2 gene, only one (01) patient was homozygous mutant (TT) and no controls had the mutation. This study found no association between these polymorphisms and the risk of breast cancer occurrence (p.R72P (OR=0.96; 95%IC (0.59-1.56); p=0.471), PIN3 Ins16bp (OR= 1.1; 95%IC (0.61-1.98); p=0.420)). Conclusion: This study showed that the P allele of the p.R72P polymorphism and the wild-type allele (A1) of the PIN3 Ins16bp polymorphism were in the majority. The I157T mutation was very rare. These polymorphisms were not associated with the risk of developing breast cancer in this study.

Apolipoprotein E polymorphism in a Romanian population

Abstract Background: Apolipoprotein E (ApoE) is a ubiquitous protein involved in maintaining cholesterol homeostasis and lipoprotein clearance from circulation. It is coded by three alleles (ε2, ε3, ε4) with six genotypes (ε3/ε3, ε3/ε4, ε2/ε3, ε4/ε4, ε2/ε4, and ε2/ε2). Several studies have shown a relationship between ApoE-specific isoforms and different diseases. There are few data about the prevalence of ApoE polymorphism in the Romanian population. We aimed to assess the prevalence of Apolipoprotein E alleles in a healthy population from Romania and compare it with the data found in other countries of Europe. Methods: ApoE genotype was examined in 187 unrelated healthy volunteers (131 males and 56 females) from the region of southwest Romania (mean age 48.04 ± 8.35 years). Analysis of ApoE genotype variants was done using the PCR method with allele-specific oligonucleotide primers. Results: The frequency of heterozygote genotypes ε2/ε3, ε2/ε4, and ε3/ε4 was 4.8%, 1.6%, and 24.73%, respectively, while the frequency of homozygote genotypes ε2/ε2, ε3/ε3, ε4/ε4 was 0.5%, 66.12%, and 2.1%, respectively, with a mean frequency of ε2, ε3 and ε4 alleles of 6.9%, 96.25%, and 28.49%, respectively. East European countries have the lowest mean frequency of the ε4 allele (9.96%), and the highest frequency of the ε4 allele (23.38%) is in the Nordic Countries. Conclusions: The ε4 allele prevalence in this Romanian population is higher in comparison with other European countries, similar to Nordic countries of Europe.

Minimal Residual Disease Detection Using Gene Scanning Analysis, Fluorescent Fragment Analysis, and Capillary Electrophoresis for IgH Rearrangement in Adult B-Lineage Acute Lymphoblastic Leukemia: A Cross-Sectional Study.

Minimal residual disease (MRD) is considered the greatest prognostic factor in acute lymphoblastic leukemia (ALL). MRD is a valuable tool for anticipating impending relapse and treatment response assessment. The objective of the present study was to investigate whether the detection of IgH gene rearrangement using polymerase chain reaction (PCR)-based GeneScan analysis could be a complementary method to monitor MRD along with the quantitative realtime PCR (qPCR). In this cross-sectional study, we valued the MRD levels, based on the GeneScanning analysis (GSA), and then compared the data with quantitative real-time polymerase chain reaction at different time points in peripheral blood (PB) samples of adult B-lineage ALL patients (n=35). The specific polymerase chain reaction (PCR) primers for IGH gene FR-1 and fluorescence-labeled J-primer were used and analyzed by capillary gel electrophoresis on a sequencer. The results of this study were compared with the previously reported MRD results obtained by the IGH rearrangements allele-specific oligonucleotide (ASO) -qPCR methods. The total concordance rate was 86.7%, with a P<0.001. MRD results obtained by GSA and ASO-qPCR methods were concordant in all diagnostic samples and samples on the 14th and 28th days of induction therapy. The results of these 2.5 years' follow-ups demonstrated a significant correlation between the two techniques (r=0.892, P<0.001). It seems that the PCR-based GeneScan analysis of IGH gene rearrangement detection may be a valuable molecular technique to distinguish monoclonality from polyclonality. And also, it may be a precise tool to detect the residual leukemic DNA in the PB follow-up samples of patients.

Frequency of de novo variants and parental mosaicism in families with inactivating PTH/PTHrP signaling disorder type 2.

iPPSD2 (which includes PHP1A and PPHP/POH) is a rare inherited autosomal dominant endocrine disorder caused by inactivating GNAS pathogenic variants. A high percentage of de novo cases has been suggested. In rare cases, parental mosaicism has been described, but its real frequency is unknown. A retrospective study including a series of 95 genetically confirmed iPPSD2 probands. The frequency of de novo cases was evaluated and the distribution of the type of variants was compared according to the type of inheritance. The putative involved allele was determined by reverse transcriptase PCR (RT-PCR) or allele specific oligonucleotide RT-PCR (ASO-RT-PCR). The possibility of GNAS mosaicism was studied by next-generation sequencing (NGS) on the corresponding parental DNA. In 41 patients the variant was of de novo origin and in 24 the origin could not be established. In both cases 66.67% of variants generated a truncated or absent protein whereas the rest of the variants were missense or in-frame deletion/duplication. Parental origin was studied in 45 of those patients and determined in 35. Curiously, the percentage of de novo variants at the paternal allele was higher than when paternally inherited (31.1% vs 6.67%). NGS detected mosaicism in three independent families: one from paternal DNA (allelic ratio 10%) and two from maternal DNA (allelic ratio 10% and 2%). De novo pathogenic variants are frequent in iPPSD2 (around 45%). Parental mosaicism is infrequent (8.11%) but should be analyzed with NGS, taking into account its importance in genetic counselling.

The association between hemostatic and genetic parameter variation and the susceptibility to develop vascular complications in Egyptian Bechet’s disease patients

Abstract Objectives Bechet’s disease (BD) is a multisystem inflammatory disorder with predominant and frequent vascular complications. The pathogenesis of thrombosis in BD is not clearly known, vasculitis and/or multifactorial procoagulant activities are the cornerstone histopathological features of the disease. Patients and methods In this study, we investigated the role of hypercoagulable state and the hemostatic alteration in BD patients and their susceptibility to develop vascular thrombotic complications using ViennaLab CVD StripAssay for molecular detection of thrombophilia gene mutation by PCR and reverse hybridization to allele-specific oligonucleotide probes immobilized on test strip. Natural anticoagulant levels were also assessed to trace the association between activated coagulation system and increased susceptibility of BD patients to develop vascular complication. Results The study revealed a significant association between human platelet antigen-1 polymorphism and the presence of vascular thrombosis, either arterial or venous, in BD patients. Also, there was a significant relationship between mutant MTHFR genotype and venous thrombosis. But, there was an insignificant association between vascular complications in BD patients from one side and the levels of natural anticoagulants (protein C, protein S, and antithrombin III) and mean platelet volume (MPV) level from the other side. Conclusion Despite the association of some thrombophilic factors with the thrombotic tendency seen in BD, the usage of anticoagulants in BD patients with thrombosis may be unnecessary.

Implications and Prognostic Impact of Mass Spectrometry in Patients with Newly-Diagnosed Multiple Myeloma

Background: The minimally invasive technology mass spectrometry (MS) is emerging as a promising approach for detecting and monitoring monoclonal proteins in the peripheral blood of multiple myeloma (MM) patients. We aimed to define implications and prognostic value of MS in a large cohort of transplant-eligible patients with newly-diagnosed MM treated within the phase 3 multicenter GMMG-MM5 trial (EudraCT No. 2010-019173-16). Patients and Methods: For quantitative immunoprecipitation MS (QIP-MS), we included peripheral blood serum samples from 480 patients at baseline, from 444 of these patients after 3 cycles induction therapy, from 305 patients prior to lenalidomide maintenance treatment (max. 2 years) or observation (in case of complete response [CR] in arm B of the trial), and from 227 patients after 1 year (+/- 3 months) of lenalidomide maintenance / observation. QIP-MS was carried out using the automated EXENT® assays and system (in development; The Binding Site Group Ltd., UK). Minimal residual disease (MRD) in the bone marrow was detected using an allele-specific oligonucleotide (ASO) PCR with a sensitivity of at least 1x10-6. Kaplan-Meier method was used for survival analyses. Progression-free survival (PFS) time was measured from the respective landmark to progression or death from any cause, whichever occurred first. For analyses of sustained MS from start of maintenance / observation until 1 year (+/- 3 months), PFS was calculated from the second time point (after 1 year) and only patients who did not have a prior progression event were included. Results: MS negativity rates increased from 6% post induction to 31% and 44% prior to and after 1 year of maintenance / observation, respectively. MS negativity was associated with improved PFS at all investigated timepoints (post induction: hazard ratio [HR]=0.41; p=0.003), prior to maintenance / observation: HR=0.66, p=0.01 and after 1 year of maintenance / observation: HR=0.37, p<0.001. Next, we evaluated the prognostic value of MS in a multivariable model, which included age at diagnosis, gender, treatment arm, conventional response (CR vs. no CR), revised International Staging System (R-ISS) stages and gain1q21. MS negativity was an independent prognostic factor for PFS (prior to maintenance / observation: HR=0.60, p=0.01, Figure 1A; and after 1 year of maintenance / observation: HR=0.28, p<0.001, Figure 1B). The prognostic impact of MS could be improved by baseline disease features, such as high-risk cytogenetics. Combining the FISH high-risk markers of the R-ISS del(17p13), t(4;14), and t(14;16) as well as gain1q21 with MS before maintenance / observation, patient groups with excellent or dismal outcomes could be defined. The best outcome was seen for patients without high-risk cytogenetics and MS negativity (median PFS: 4.8 years), while patients with high-risk FISH and MS positivity had a median PFS of just 1.9 years from start of maintenance / observation. Next, we aimed to determine the prognostic value of sequential MS testing and chose the time points prior to maintenance / observation and after 1 year. The best outcome was seen for patients who converted from MS positivity to negativity (median PFS not reached). The worst outcome was seen for patients who converted from MS negativity to positivity with a median PFS of only 0.6 years. Sustained MS negativity was associated with improved PFS (median PFS: 3.5 years) as compared to sustained MS positivity (median PFS: 1.9 years, p=0.007). In a pilot study, we combined MS with ASO-PCR for detection of MRD in the bone marrow in 45 patients with suspected CR. Double (MS / MRD) positive patients had the worst outcome (median PFS: 2.2 years) whereas double-negative patients had a median PFS of 3.3 years and no overall survival events. Conclusions: Our study provides strong evidence that MS is superior to conventional response monitoring and should become a new standard to monitor monoclonal protein in MM. We recommend performing MS testing sequentially and combined with baseline disease features to improve prognostication. Disease relapse in MS / MRD double-negative patients in our study probably occurred due to limited duration of lenalidomide maintenance or observation, but also highlights that even a combined approach with a highly sensitive bone-marrow MRD tool was not sufficient to identify disease-free MM patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Digital Droplet PCR for Minimal Residual Disease Assessment in Philadelphia-Positive Acute Lymphoblastic Leukemia Using IG/TR Genes and BCR/ABL1 as Markers. Preliminary Results of a Comparative Analysis

Beldinanzi M, Della Starza I, contributed equally Background. The Philadelphia (Ph) chromosome is the most frequent genetic abnormality in adult acute lymphoblastic leukemia (ALL). BCR/ABL1 fusion transcript levels and clonal immunoglobulin/T-cell receptor (IG/TR) gene rearrangements are used as biomarkers for the study of minimal residual disease (MRD) in Ph+ ALL, with IG/TR used mainly in pediatric cohorts. There is evidence suggesting a poor concordance between these two molecular markers and there is a growing interest in evaluating MRD using a "double-hit strategy", especially in the adult setting where data are lacking. The real-time quantitative polymerase chain reaction (RQ-PCR) represents the current gold standard for MRD monitoring. It carries, however, some intrinsic limitations that digital droplet PCR (ddPCR), an innovative evolution of standard method, could overcome. Aim of this study. To carry out in adult Ph+ ALL patients a parallel MRD monitoring at early time points by both molecular targets - BCR/ABL1 gene fusion and clonal IG/TR gene rearrangements - by ddPCR. For BCR/ABL1 this was carried out by both RQ-PCR and ddPCR, in order to determine the concordance between the MRD values. Materials and Methods. The study comprised samples derived from 17 adults with newly diagnosed Ph+ ALL enrolled in the ongoing phase III GIMEMA LAL2820 clinical trial. Bone marrow (BM) samples were collected at the end of the induction phase (day +70) with ponatinib in the experimental arm or with a chemotherapy backbone plus imatinib in the control arm. At diagnosis, all patients underwent also a IG/TR gene rearrangements screening and MRD monitoring was performed by RQ-PCR and ddPCR for the quantification of BCR/ABL1 transcript levels and by ddPCR for IG/TR rearrangements, as described. MRD results were considered discordant in case of a difference greater than one log10 or in case of positivity/negativity. Results. The comparison between MRD values obtained in all samples by RQ-PCR and ddPCR using BCR/ABL1 mRNA level as biomarker showed an excellent correlation degree (R2=0.99). At variance, by IG/TR screening, it was possible to identify a clonal rearrangement only in 11/17 cases (65%), while 6/17 (35%) had no marker (NM) (3 p190, 3 p210; 2 IKZF1plus, 2 IKZF1 loss only and 2 IKZF1 WT). Moreover, of the 11 patients with a clonal rearrangement, in 3 (27%) (3 p190; 1 IKZF1plus, 2 IKZF1 WT) it was not possible to design a sensitive allele-specific oligonucleotide (ASO) for MRD monitoring. Overall, 8 samples were evaluated using both molecular markers by ddPCR, showing a poor correlation (R2=-0.21) and a low concordance rate. Indeed, 3/8 (37.5%) samples were concordant by both targets, either having highly positive (2/3) or negative (1/3) MRD values, while 5/8 (62.5%) were discordant; among the discordant cases, 1/5 was MRD-positive by both targets but outside the concordance range, while in 4/5 IG/TR MRD monitoring was negative, while BCR/ABL1 MRD monitoring was positive (range 10-3-10-1). Finally, in the 9 patients who failed IG/TR MRD monitoring, the BCR/ABL1 RQ-PCR analysis revealed a quantifiable MRD positivity in 6 cases (ranged 10-1-10-3), 2 proved "positive non-quantifiable" (PNQ) and 1 was MRD-negative (Figure 1). Conclusions. In adult Ph+ ALL, ddPCR with BCR/ABL1 as marker proved to be a sensitive and reliable tool for MRD monitoring at least comparable to RQ-PCR, showing a very high concordance rate. At variance, at least at early time points, the degree of concordance between BCR/ABL1 fusion transcript levels and IG/TR clonal rearrangements appeared suboptimal. Although IG/TR gene rearrangements are the most widely used biomarker for MRD monitoring in pediatric patients, the rate of failure in IG/TR marker identification in the adult Ph+ ALL setting is higher than in other subtypes. These findings suggest that the BCR/ABL1 fusion transcript remains the main sensitive target in adult Ph+ ALL and lead to hypothesize that in in this setting IG/TR clonal rearrangements represent a surrogate marker. Cohort expansion is warranted and clinical correlations are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal