Efficient Molecular Diagnostic Strategy for ABCC6 in Pseudoxanthoma Elasticum

2004 ◽  
Vol 8 (3) ◽  
pp. 292-300 ◽  
Author(s):  
Xiaofeng Hu ◽  
Astrid Plomp ◽  
Theo Gorgels ◽  
Jacoline Ten Brink ◽  
Willem Loves ◽  
...  
Keyword(s):  
Pseudoxanthoma Elasticum ◽  
Molecular Diagnostic ◽  
Diagnostic Strategy

Novel NGS-Based Platforms For Molecular Diagnosis Of Severe Congenital Neutropenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1034-1034
Author(s):  
Tomas Racek ◽  
Jacek Puchalka ◽  
Naschla Kohistani ◽  
Christoph Klein
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Molecular Diagnostic ◽  
Compound Heterozygous ◽  
Neutrophil Granulocytes ◽  
Diagnostic Strategy ◽  
Congenital Neutropenia ◽  
Coding Regions ◽  
Whole Exome ◽  
Custom Made

Abstract Congenital neutropenia (CN) is a heterogeneous disorder. More than 30 distinct genetic defects have been discovered in patients with genetic diseases associated with decreased numbers of peripheral neutrophil granulocytes. Currently, most molecular diagnostic laboratories use Sanger-based sequencing techniques to define disease-causing mutations in patients with CN. In approximately 50% of patients no known genetic disorder can be found. To identify novel genes that can be causative for unexplained CN cases we embarked on next-generation whole-exome sequencing using SOLiD 5500™ and Ion Proton™ sequencers. Up to date we sequenced whole exomes of 49 families, in which children were diagnosed with CN. The fragment libraries were constructed using the SureSelect™ V4+UTRs System (Agilent) allowing us to target whole coding sequence and the majority of UTRs of human genome (approx. 71 Mb). The vast majority of the families were analysed in the “Trio” approach and suitable homozygous or compound heterozygous rare variations (frequency below 1%) in protein coding regions or in splice sites were chosen for further validations. In seven cases mutations previously described as causative for neutropenia were identified including G6PC3, HAX1, and ELANE. Four other rare variants are currently being analysed for their potential to cause CN. In 35 patients, no plausible candidate could be identified so far. When we assessed variants within the genes related to CN, our data revealed unequal coverage pattern over these genes. Around 10% of the exons were insufficiently covered (coverage of less than 10) to allow for reliable variant and genotype call. These facts limit the power of whole exome sequencing as a diagnostic tool, as mutations at the non-covered positions cannot be ruled out, and demonstrate the need of an alternative comprehensive approach. We are currently assessing sensitivity and specificity of a robust, rapid, and cost-effective approach that comprehensively analyses the sequence of 34 CN-relevant genes. Our approach is based on enrichment of specific exon regions by amplification using custom made AmpliSeq™ (Life Technologies) panel. For 25 genes we are able to sequence coding region as well as both UTR sequences, for 9 genes sequencing is limited to coding regions. This approach will provide a reliable, quick, and inexpensive diagnostic strategy for CN patients which will be offered free-of-charge to patients worldwide, independent of ethnic, national, or financial considerations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IgE-binding residues analysis of the house dust mite allergen Der p 23

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sze Lei Pang ◽  
Sri Anusha Matta ◽  
Yang Yie Sio ◽  
Yu Ting Ng ◽  
Yee-How Say ◽  
...  
Keyword(s):  
Allergic Rhinitis ◽  
House Dust ◽  
Major Allergen ◽  
House Dust Mites ◽  
Molecular Diagnostic ◽  
Diagnostic Strategy ◽  
Dot Blot ◽  
Ige Binding ◽  
Atopic Individual ◽  
Binding Residues

AbstractHouse dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P < 0.001). Atopic individuals who were only sensitized to HDM have a significantly higher number of IgE-binding residues than the individuals who were polysensitized to HDM and other crude allergens (P < 0.05). Individuals with allergic multimorbidity and moderate-to-severe allergic rhinitis also have a higher number of IgE-binding residues compared to those with single allergic disease and mild allergic rhinitis. The results prompt us to hypothesize that the individuals who have a higher number of IgE-binding residues may face a bigger challenge to be treated through immunotherapy due to the complexity in designing an effective hypoallergen with a high number of IgE-binding residues. We propose that the development of a refined molecular diagnostic assay, which includes alanine substitution of surface-exposed residues could be a more precise diagnostic strategy to identify all the IgE-binding residues of a major allergen for an atopic individual and the development could be another new dimension in allergy diagnosis and allergen immunotherapy treatment.

Genotyping simplified: Rationally designed antisense oligonucleotide-mediated PCR amplification-free colorimetric sensing of viral RNA in HCV genotypes 1 and 3

The Analyst ◽  
2021 ◽  
Author(s):  
Almas Shamaila Mohammed ◽  
Aniket Balapure ◽  
Aleem Ahmed Khan ◽  
Mahammad Nanne Khaja ◽  
Ramakrishnan Ganesan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Diagnosis ◽  
Antisense Oligonucleotide ◽  
Pcr Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Strategy ◽  
Colorimetric Sensing ◽  
Chain Reaction ◽  
Hcv Genotypes ◽  
Polymerase Chain

Molecular diagnosis of viral genotyping devoid of polymerase chain reaction (PCR) amplification in clinical cohorts has hitherto been challenging. Here we present a simplified molecular diagnostic strategy for direct genotyping...

scholarly journals Optimization of a molecular diagnostic strategy to verify SARS-CoV-2 infections by RT-qPCR

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Marcel Noßmann
Keyword(s):  
Nucleic Acid ◽  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Diagnostic Strategy ◽  
The Novel ◽  
Rna Molecules ◽  
Rna Purification ◽  
Nucleic Acid Purification ◽  
Negative Impacts ◽  
The Individual

AbstractObjectivesFast and precise detection of SARS-CoV-2 RNA in infected patients is essential for treatment decisions.MethodsA diagnostic strategy by analyzing nasopharyngeal swabs to detect SARS-CoV-2 RNA in individuals was established. The negative impacts of the individual buffer components on RT-qPCR analysis was reviewed and overcome by RNA purification. To investigate the functionality of the improved protocol we compared the novel diagnostic strategy to a Bead-based RNA extraction method using previously positive tested samples.ResultsA method to extract purify RNA molecules from SARS-CoV-2 was established. We examined the significance of nucleic acid purification and the need for an RNase inhibitor. Evaluation of 3,664 samples from March 23rd until May 18th in 2020 showed the incidence of COVID-19 infections in Thuringia, Germany. Additionally, benefits and limits of three RT-qPCR kits were assessed.ConclusionsOur study suggests that virus capsid lysis combined with nucleic acid purification enables a viable alternative for the molecular diagnostics of SARS-CoV-2 infections. Due to the current delivery delays from different companies, this method offers the possibility to continue diagnosis and to handle the large number of samples.

scholarly journals A Feasible Molecular Diagnostic Strategy for Rare Genetic Disorders Within Resource- Constrained Environments

2021 ◽  
Author(s):  
Maria Mabyalwa Mudau ◽  
Heather Seymour ◽  
Patracia Nevondwe ◽  
Robyn Kerr ◽  
Careni Spencer ◽  
...  
Keyword(s):  
Genetic Disorders ◽  
African Ancestry ◽  
Mutation Screening ◽  
African State ◽  
Genetic Diagnosis ◽  
Cost Effective ◽  
Molecular Diagnostic ◽  
Diagnostic Strategy ◽  
Resource Constrained ◽  
Wide Range

Abstract BackgroundThe timeous and accurate diagnosis of rare genetic disorders is critical, as it enables a better understanding of patient management, prognosis and more personalized treatment. A confirmed genetic diagnosis also enables accurate genetic counselling for affected individuals and their relatives by offering options for prenatal and cascade testing. In a resource-constrained environment such as the South African State healthcare system, the challenge is to design appropriate and cost-effective assays that will enable accurate genetic diagnostic services in patients of African ancestry across a broad disease spectrum. Next-generation sequencing (NGS) has transformed testing approaches for many Mendelian disorders, but this technology is still relatively expensive to implement. The paucity of baseline genetic data on African populations adds to the complexity of implementing an appropriate NGS-based service and interpretation of results.ResultIn the current study, the approach taken aimed at balancing NGS cost, efficiency, data quality as well as diagnostic utility of results. As a proof of concept, we describe a feasible diagnostic strategy for genetic disorders frequently seen in our genetics clinics (RASopathies, Cornelia de Lange syndrome, Treacher Collins syndrome, CHARGE syndrome and other phenotypically overlapping syndromes. The custom-designed targeted NGS gene panel enabled mutation screening for these disorders that range in incidence from 1 in 1000 to 1 in 100 000 newborns. Samples were batched during sequencing and analyzed selectively based on the clinical phenotype. This study had an overall diagnostic yield of 54.5%. ConclusionThe strategy employed is cost-effective as it allows batching of samples from patients with different diseases in a single run, an approach that can be utilized with rare and less frequently ordered molecular diagnostic tests. The subsequent selective analysis pipeline allowed for timeous reporting back of patients results. This is feasible and can be employed for the molecular diagnosis of a wide range of rare monogenic disorders in resource-constrained environment.

scholarly journals RHD Genotypes in a Chinese Cohort of Pregnant Women

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianjun Zhang ◽  
Yan Zeng ◽  
Yuefeng Wang ◽  
Jiaming Fan ◽  
Haijiang Chen ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Chinese Women ◽  
Maternal Plasma ◽  
Molecular Diagnostic ◽  
Diagnostic Strategy ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Chinese Cohort ◽  
Polymerase Chain ◽  
Intron 2

RHD variants in D¯ Chinese pregnant women arose difficulties in management during pregnancy. Therefore, this study aims to precisely manage D¯ pregnant women by evaluating the spectrum of RHD mutations in D¯ pregnant women and getting insight into the possible rare alleles of RHD. A total of 76 D¯ pregnant women were analyzed by performing polymerase chain reactions with sequence-specific primers (PCR-SSP), the 10 RHD exons Sanger sequencing, RHD zygosity detection, and mRNA sequencing (mRNA-seq). About 40% of alleles are variations of RHD, including RHD 1227A homozygous, RHD-CE(2-9)-D, et al. Therefore, we developed a molecular diagnostic strategy for Chinese women, and most D¯ pregnant women can be diagnosed with this simple decision tree. After RHD genotyping for D¯ pregnancy women, we eliminated at least 15% unnecessary ante- and postpartum injections of Rh immunoglobulin (RhIG). As the first pedigree study and the first functional analysis under physiological conditions, mRNA-seq revealed that c.336-1G&gt;A mutation mainly led to the inclusion of the intron 2, which indirectly explained the D¯ phenotype in this family. We also developed a robust protocol for determining fetal RhD status from maternal plasma. All 31 fetuses were predicted as RhD positive and confirmed the RhD status after birth.

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