Lymphocyte profiling is the mainstay of diagnosing and monitoring patients with primary and secondary immune cell dysregulation. We developed a method for lymphocyte quantification based on epigenetic (i.e. DNA methylation) markers specific for various lymphocyte subsets. The method is suitable for the analysis of dried or frozen blood samples. This allows management of immune compromised patients in areas with limited resources for fresh blood sample logistics. It also opens the perspective of near-patient testing using minimally invasive sampling procedures – e.g. via finger prick.
We identified genomic regions of lymphocyte-specific de-methylation. Real-time PCR assays were developed to specifically amplify de-methylated regions after bisulfite conversion of genomic DNA. Inclusion of housekeeping genes as well as calibrators and standards allows for absolute and relative quantification of lymphocyte subsets as well as quality control of the test run.
We demonstrate perfect correlation of epigenetic immune cell quantification of T-/B- and NK cells with flow cytometry in whole blood samples of healthy volunteers and patients after hematopoietic stem cell transplantation. We also demonstrate successful application of the epigenetic method to dried blood samples of HIV patients as well as newborns.
Our data demonstrate the feasibility of molecular immune cell quantification using epigenetic markers specific for various lymphocyte subsets. This allows molecular diagnostic applications in settings where fresh blood samples are not available in sufficient quality or quantity for flow cytometry - e.g. monitoring of immune-compromised patients in resource-limited areas, early detection of immune dysregulation in newborns and near-patient testing using point-of care technology.
The need for accurate metadata to describe clinical information not only in a genetic manner, but also phenotypically is increasingly clear. Data structures such as the Human Phenotype Ontology (HPO) and disease ontologies such as OrphaNet aim to provide standardized vocabularies of phenotypic abnormalities and human diseases.HPO and OrphaNet enable efficient patient data exchangeand facilitate seamless communication between clinicians and researchers todetect novel disease-causing genes and address phenotype-genotype correlations. Despite the ongoing efforts, there are still crucial, disease-specific gaps in HPO and OrphaNet disease ontology when describing rare immune mediated disorders.
Our initiative brings together geneticists, medical doctors, bioinformatics, and immunologists. They are organized into functional working groups, with the aim to systematically re-evaluate and complete HPO and OrphaNet disease ontology terms, and re-annotate diseases.
So far, we have successfully revised four branched of HPO terms: neutrophils, lymphocyte count structure, humoral immunity hierarchy, immune system physiology. Two of the revised branches are already implemented in HPO terminology. In addition, we have fully re-annotated 15 diseases with HPO terms and collected a repository of articles representing a knowledge-base detailing phenotypes of patients for more than 60 diseases.
With the continued systematic re-evaluation, we expect to (i) unify the nomenclature of patient phenotyping (ii) standardize patient characterization: clinician/researcher can characterize patients in a language independent manner (iii) allow for efficient data exchange between clinicians, laboratories and centers (iv) facilitate matching phenotypically similar patients to enable gene discovery (v) allow for similarity measures across diseases/shared phenotypes.