Detection Of Aberrant Expression Research Articles

Transcriptomic Approaches to Diagnosis of Inborn Errors of Immunity

Molecular diagnosis of inborn errors of immunity (IEI) leads to many benefits including more accurate prognosis, access to molecularly targeted therapies, and better informed genetic counseling for relatives and offspring. However, in practice, diagnostic testing is often limited to gene panels and clinical exome sequencing or rarely genome sequencing. As a result, treating physicians remain unable to identify a specific cause in the majority of suspected IEI patients using currently established technologies, even when a genetic cause is suspected based on similarly affected family members. Even once a diagnosis is made, selection of appropriate treatments provides additional challenges as response to immunomodulatory therapy is heterogeneous. Better strategies are needed to identify which groups of patients are most likely to benefit from different therapies. Determining the optimal diagnostic and therapeutic strategy requires a personalized approach bringing together expertise from multiple clinical specialties and basic scientists to apply new discoveries to patient care and improve outcomes for these complex patients. We hypothesize that adding transcriptomic profiling to existing genomic diagnostic approaches could help close these gaps, facilitating identification of the molecular diagnosis in a larger fraction of patients and monitoring the gene expression response to treatment. To test this, we plan to (1) deploy RNA sequencing of peripheral blood paired with DNA sequencing in a cohort of IEI patients with known (N = 10) IEIs representing a spectrum of disorders as classified by IUIS (e.g. X-linked SCID due to ILR2g, deficiency of adenosine deaminase 2, CD40L deficiency) to validate the pipeline for detecting known disorders, (2) apply the pipeline to a pilot cohort of participants with unknown disorders (N = 10) to assess diagnostic efficacy, and (3) perform longitudinal RNA sequencing in IEI patients undergoing treatment to understand predictors of treatment response. To date, we have obtained whole-blood total RNA from 5 participants (3 known, 2 unknown and pretreatment). In parallel, we have adapted the detection of RNA outliers pipeline (DROP) for local use to identify novel monogenic causes of IEI through detection of aberrant expression, aberrant splicing, and mono-allelic expression, and have tested the pipeline using publicly available, well-characterized datasets.

Clinical implementation of RNA sequencing for Mendelian disease diagnostics

BackgroundLack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics.MethodsWe implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.ResultsWe detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.ConclusionTogether, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Minimal residual disease: optimal methods, timing, and clinical relevance for an individual patient.

After approximately 20 years of development and after several prospective clinical trials, the detection of minimal residual disease (MRD) has emerged as part of state-of-the-art diagnostics to guide the majority of contemporary treatment programs both in pediatric and adult acute lymphoblastic leukemia (ALL). For ALL, several methods of MRD analysis are available, but 2 are widely applicable. One is based on the detection of aberrant expression of leukemia specific antigens by flow cytometry and the other one uses the specific rearrangements of the TCR or Ig genes, which can be detected by quantitative PCR in the DNA of leukemic cells. In some cases with known fusion genes such as BCR/ABL, RT-PCR can be used as a third method of identifying leukemic cells by analyzing RNA in patient samples. Clinical application of such sophisticated tools in the stratification and treatment of ALL requires reliable, reproducible, and quality-assured methods to ensure patient safety.

Urticaria pigmentosa and mastocytosis: The role of immunophenotyping in diagnosis and determining response to treatment

Recent advances in our understanding of mast cell biology and disease resulted in identification of important differences in expression of mast cell surface antigens in normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts leads to the inclusion of this immunophenotypic abnormality in the World Health Organization's diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients with limited disease that lacks histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. Flow cytometric analysis of bone marrow mast cells is therefore a sensitive method of diagnosis of mast cell disease and is expected to find increasing use in determining response to emerging mast cell cytoreductive therapies.

CD44 expression in normal, hyperplastic, and malignant endometrium.

A total of 140 endometrial carcinomas (endometrioid type), as well as 72 hyperplasias (41 of simple or complex and 31 of atypical type) and 141 normal endometria (35 in the proliferative and 106 in the secretory phase), were immunohistochemically investigated for expression of the standard and variant-3 and -6 isoforms of CD44, and the results compared with several known prognostic factors. A combination of the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization (SBH) for CD44 mRNA levels was also carried out on 27 endometrial carcinomas. In normal endometrium, the expression of standard and variant CD44 forms was remarkably higher in the secretory than in the proliferative phase, with immunoreactivity scores being inversely correlated with numbers of oestrogen and progesterone receptors. Significantly elevated levels of CD44 expression in endometrial carcinomas compared with the proliferative phase and hyperplasia were also revealed by both the immunohistochemical and the RT-PCR/SBH assays, while no association was noted with any prognostic factors. The results indicate that CD44 expression in the normal menstrual cycle is closely related to the secretory differentiation of the glandular epithelium. Moreover, detection of aberrant expression may be useful for the early diagnosis of endometrial carcinoma, but not as an indicator of tumour progression.