Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases

Mutations in the paralogous human α-globin genes yielding identical hemoglobin variants

The human α-globin genes are paralogues, sharing a high degree of DNA sequence similarity and producing an identical α-globin chain. Over half of the α-globin structural variants reported to date are only characterized at the amino acid level. It is likely that a fraction of these variants, with phenotypes differing from one observation to another, may be due to the same mutation but on a different α-globin gene. There have been very few previous examples of hemoglobin variants that can be found at both HBA1 and HBA2 genes. Here, we report the results of a systematic multicenter study in a large multiethnic population to identify such variants and to analyze their differences from a functional and evolutionary perspective. We identified 14 different Hb variants resulting from identical mutations on either one of the two human α-globin paralogue genes. We also showed that the average percentage of hemoglobin variants due to a HBA2 gene mutation (α2) is higher than the percentage of hemoglobin variants due to the same HBA1 gene mutation (α1) and that the α2/α1 ratio varied between variants. These α-globin chain variants have most likely occurred via recurrent mutations, gene conversion events, or both. Based on these data, we propose a nomenclature for hemoglobin variants that fall into this category

FINDbase: a relational database recording frequencies of genetic defects leading to inherited disorders worldwide

Frequency of INherited Disorders database (FINDbase) () is a relational database, derived from the ETHNOS software, recording frequencies of causative mutations leading to inherited disorders worldwide. Database records include the population and ethnic group, the disorder name and the related gene, accompanied by links to any corresponding locus-specific mutation database, to the respective Online Mendelian Inheritance in Man entries and the mutation together with its frequency in that population. The initial information is derived from the published literature, locus-specific databases and genetic disease consortia. FINDbase offers a user-friendly query interface, providing instant access to the list and frequencies of the different mutations. Query outputs can be either in a table or graphical format, accompanied by reference(s) on the data source. Registered users from three different groups, namely administrator, national coordinator and curator, are responsible for database curation and/or data entry/correction online via a password-protected interface. Databaseaccess is free of charge and there are no registration requirements for data querying. FINDbase provides a simple, web-based system for population-based mutation data collection and retrieval and can serve not only as a valuable online tool for molecular genetic testing of inherited disorders but also as a non-profit model for sustainable database funding, in the form of a ‘database-journal’

The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases

HBB mutations and HbA2 level: Escaping the carrier screening programs

HbA2 level alone for beta thalassemia trait may not be accurate and reliable even without iron deficiency so molecular genetic testing is important and should be considered for some individuals. © 2020 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd

The development of mass spectrometry-based approaches for the diagnosis of hemoglobinopathies

Hemoglobinopathies are disorders of the protein hemoglobin (Hb), one of the most common inherited disorders, and pose an increasing healthcare problem. Worldwide, approximately 200 million people have hemoglobinopathies (Hartwell et al. 2005), and whilst the majority of them are clinically silent, some are life-threatening. Screening programs for hemoglobinopathies traditionally employ chromatography and electrophoretic techniques, which only provide presumptive identification of clinically important hemoglobin disorders. The definitive characterisation of these disorders requires protein sequence elucidation or DNA analysis. The development of a rapid population screening method for hemoglobinopathies, which could also provide definitive diagnosis of a disorder, is of significant interest to the healthcare profession. Mass spectrometry (MS) is currently used to routinely characterise variants but not as a validated clinical diagnostic tool. The work undertaken addresses the increasing demand for population screening. Different mass spectrometry-based approaches to hemoglobinopathy detection have been developed based on previously described procedures found in literature. The methods, for intact globin chain analysis and targeted variant specific peptide analysis, have been optimised and evaluated using clinical blood samples on a triple quadrupole system. Intact globin chain analysis and targeted tryptic peptide screening methods have been evaluated in a clinical trial analysing 2017 blood samples to identify clinically significant variants routinely screened as a requirement by the NHS Screening programme and to quantify minor globin chains (HbA2 and HbF) suitable for the diagnosis of thalassemic and other disorders. This work was carried out in a three month mirrored testing experiment against existing clinical laboratory methods. The results obtained showed an excellent correlation to values from current clinical methods, as all clinically significant variants were detectable by MS methods. In addition, MS was able to detect Hb variants undetected by electrophoretic or chromatographic methods and highlighted some issues with the current phenotypic methods which can lead to misdiagnosis. A top-down ETD method utilising an iontrap mass spectrometer has been developed and applied to provide fragmentation for positive identification of known hemoglobin variants. This method was evaluated by analysing 1000 samples in a pilot study as a sideproject to the clinical trial. The generation of an automated report with the detection of clinically significant variants and minimal sample preparation are some of the benefits associated with this approach. This method has been shown to meet the majority of requirements for a rapid diagnostic screening method and has the potential to become a validated tool in the clinical laboratory screening in the near future

The development of molecular assays for the detection and identification of inherited disorders of haemoglobin in a highly heterogeneous population

Inherited disorders of haemoglobin are the most common monogenic diseases in the world. These conditions arise as a result of deletions or point mutations affecting the globin genes. Gap polymerase chain reaction (Gap-PCR) is commonly used to detect globin gene deletions, however this technique is not ideal since it is time consuming and only able to detect known deletions with well-defined breakpoints. To address these issues an alternative technique called Gene Ratio Assay Copy Enumeration (GRACE) PCR was developed. In contrast to Gap-PCR, GRACE-PCR is a rapid, closed tube technique, requiring no further hands on time after the initial PCR setup. Furthermore, since it is based on copy number determination, GRACE-PCR does not require prior knowledge of the breakpoints and can thus be used for the detection of both known and novel deletions. GRACE-PCR methods were validated for the most clinically significant globin genes, HBA1, HBA2 and HBB. The large number of point mutations associated with the HBB gene make gene scanning by High Resolution Melting (HRM) PCR a potentially attractive diagnostic method. HRM-PCR assays have been previously described, however interference from certain Single Nucleotide Polymorphisms (SNPs) limited their ability to detect some mutations. Through the use of unlabelled probes and primers incorporating universal bases, it was possible to develop a more universally applicable HRM-PCR assay that was not affected by common SNPs. Additionally, a number of examples of two very rare haemoglobins, Hb Fontainebleau and Hb Handsworth were encountered during the course of this work. Each of these variants had previously been reported in just ten individuals worldwide. The diagnostic features of these two rare variants are described

FINDbase: a worldwide database for genetic variation allele frequencies updated

Frequency of INherited Disorders database (FIND base; http://www.findbase.org) records frequencies of causative genetic variations worldwide. Database records include the population and ethnic group or geographical region, the disorder name and the related gene, accompanied by links to any related external resources and the genetic variation together with its frequency in that population. In addition to the regular data content updates, we report the following significant advances: (i) the systematic collection and thorough documentation of population/ethnic group-specific pharmacogenomic markers allele frequencies for 144 markers in 14 genes of pharmacogenomic interest from different classes of drug-metabolizing enzymes and transporters, representing 150 populations and ethnic groups worldwide; (ii) the development of new data querying and visualization tools in the expanded FINDbase data collection, built around Microsoft’s PivotViewer software (http://www.getpivot.com), based on Microsoft Silverlight technology (http://www.silverlight.net) that facilitates querying of large data sets and visualizing the results; and (iii) the establishment of the first database journal, by affiliating FINDbase with Human Genomics and Proteomics, a new open-access scientific journal, which would serve as a prime example of a non-profit model for sustainable database funding