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We report some observations from our laboratory practice that might be important for the treatment of sickle cell disease (SCD). We describe data from two cases indicating that iron depletion might have a beneficial effect diminishing the formation of HbS in favor of HbF, possibly reducing the severity of the disease. We believe that it would be worthwhile to monitor the course of the disease comparing cases with identical genotypes with and without iron depletion, and we advise to consider chelation therapy to reduce iron overload in patients with SCD. [ABSTRACT FROM AUTHOR]

Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and a clinical phenotype varying from almost asymptomatic to a lethal haemolytic anaemia.It is probably the most common monogenic gene disorder in the world and is especially frequent in Mediterranean countries, South-East Asia, Africa, the Middle East and in the Indian subcontinent. During the last few decades the incidence of alpha thalassaemia in North-European countries and Northern America has increased because of demographic changes. Compound heterozygotes and some homozygotes have a moderate to severe form of alpha thalassaemia called HbH disease. Hb Bart's hydrops foetalis is a lethal form in which no alpha-globin is synthesized. Alpha thalassaemia most frequently results from deletion of one or both alpha genes from the chromosome and can be classified according to its genotype/phenotype correlation. The normal complement of four functional alpha-globin genes may be decreased by 1, 2, 3 or all 4 copies of the genes, explaining the clinical variation and increasing severity of the disease. All affected individuals have a variable degree of anaemia (low Hb), reduced mean corpuscular haemoglobin (MCH/pg), reduced mean corpuscular volume (MCV/fl) and a normal/slightly reduced level of HbA2. Molecular analysis is usually required to confirm the haematological observations (especially in silent alpha-thalassaemia and alpha-thalassaemia trait). The predominant features in HbH disease are anaemia with variable amounts of HbH (0.8-40%). The type of mutation influences the clinical severity of HbH disease. The distinguishing features of the haemoglobin Bart's hydrops foetalis syndrome are the presence of Hb Bart's and the total absence of HbF. The mode of transmission of alpha thalassaemia is autosomal recessive. Genetic counselling is offered to couples at risk for HbH disease or haemoglobin Bart's Hydrops Foetalis Syndrome. Carriers of alpha+- or alpha0-thalassaemia alleles generally do not need treatment. HbH patients may require intermittent transfusion therapy especially during intercurrent illness. Most pregnancies in which the foetus is known to have the haemoglobin Bart's hydrops foetalis syndrome are terminated due to the increased risk of both maternal and foetal morbidity. [ABSTRACT FROM AUTHOR]

Aim: In this article, we present two cases of severe fetal hemolytic anemia based on a beta-thalassaemia trait inherited from a single parent., Results: These cases, presented at 20 and 28 weeks' gestation, necessitated intra-uterine blood transfusions. This occurrence is remarkable because it challenges the common assumption that beta-thalassaemia typically has no prenatal implications regarding fetal anemia. Both fetuses inherited a rare heterozygous mutation from their mother, resulting in gamma-thalassaemia-related anemia. In the first case, the anemia was related to a deletion in the beta locus control region (βLCR) and in the second case, a deletion on chromosome 11p15.4 was the cause. These mutations not only affect the beta chain production, but also the gamma chain production, leading to a reduction in the synthesis of HbF, ineffective erythropoiesis and consequently, perinatal hemolytic anemia., Conclusion: Clinicians should be vigilant regarding these rare mutations in families with a history of beta-thalassaemia as the fetal clinical consequences can be severe and intra-uterine blood transfusions may prove life-saving for these fetuses., (© 2024 The Author(s). Prenatal Diagnosis published by John Wiley & Sons Ltd.)

It is well known that modifiers play a role in ameliorating or exacerbating disease phenotypes in patients and carriers of recessively inherited disorders such as sickle cell disease and thalassemia. Here, we give an overview of the literature concerning a recently described association in carriers of SUPT5H Loss-of-Function variants with a beta-thalassemia-like phenotype including the characteristic elevated levels of HbA 2 . That SUPT5H acts as modifier in beta-thalassemia carriers became evident from three reported cases in whom combined heterozygosity of SUPT5H and HBB gene variants was observed to resemble a mild beta-thalassemia intermedia phenotype. The different SUPT5H variants and hematologic parameters reported are collected and reviewed to provide insight into the possible effects on hematologic expression, as well as potential disease mechanisms in carriers and patients.

Background: Hemoglobinopathies, the most common inherited blood disorder, are frequently underdiagnosed. Early identification of carriers is important for genetic counseling of couples at risk. The aim of this study was to develop and validate a novel machine learning model on a multicenter data set, covering a wide spectrum of hemoglobinopathies based on routine complete blood count (CBC) testing., Methods: Hemoglobinopathy test results from 10 322 adults were extracted retrospectively from 8 Dutch laboratories. eXtreme Gradient Boosting (XGB) and logistic regression models were developed to differentiate negative from positive hemoglobinopathy cases, using 7 routine CBC parameters. External validation was conducted on a data set from an independent Dutch laboratory, with an additional external validation on a Spanish data set (n = 2629) specifically for differentiating thalassemia from iron deficiency anemia (IDA)., Results: The XGB and logistic regression models achieved an area under the receiver operating characteristic (AUROC) of 0.88 and 0.84, respectively, in distinguishing negative from positive hemoglobinopathy cases in the independent external validation set. Subclass analysis showed that the XGB model reached an AUROC of 0.97 for β-thalassemia, 0.98 for α0-thalassemia, 0.95 for homozygous α+-thalassemia, 0.78 for heterozygous α+-thalassemia, and 0.94 for the structural hemoglobin variants Hemoglobin C, Hemoglobin D, Hemoglobin E. Both models attained AUROCs of 0.95 in differentiating IDA from thalassemia., Conclusions: Both the XGB and logistic regression model demonstrate high accuracy in predicting a broad range of hemoglobinopathies and are effective in differentiating hemoglobinopathies from IDA. Integration of these models into the laboratory information system facilitates automated hemoglobinopathy detection using routine CBC parameters., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The prevalence of hemoglobinopathies in The Netherlands is increasing due to migration. Hemoglobinopathies are severe hereditary diseases. An informed reproductive choice by at-risk couples, such as pre-implantation diagnosis or termination of affected pregnancies, can be made if carriers are detected prior to conception. Using a qualitative design, the needs and wishes of patients, carriers and general practitioners were evaluated regarding carrier detection of hemoglobinopathies in primary care practice. 30 semi-structured interviews were established with 10 general practitioners, 10 patients and 10 carriers. The interviews were audio-recorded, transcribed verbatim and analysed using content analysis to identify recurring themes. Three themes were generated regarding carrier detection of hemoglobinopathies: (1) a need for more information about hemoglobinopathy, (2) a need for indications when to refer for analysis (carrier diagnostics) and (3) insight concerning organization and roles in care for hemoglobinopathy carriers and patients. These themes reflected a need to increase awareness of hemoglobinopathy, improve competences among general practitioners through better education and improvement of communication with patients and their unidentified family members. This study shows the scope of the problem and the critical need for action to improve informed reproductive decision making for the at-risk population., (© 2022. The Author(s).)

Several types of haemoglobinopathies are caused by copy number variants (CNVs). While diagnosis is often based on haematological and biochemical parameters, a definitive diagnosis requires molecular DNA analysis. In some cases, the molecular characterisation of large deletions/duplications is challenging and inconclusive and often requires the use of specific diagnostic procedures, such as multiplex ligation-dependent probe amplification (MLPA). Herein, we collected and comprehensively analysed all known CNVs associated with haemoglobinopathies. The dataset of 291 CNVs was retrieved from the IthaGenes database and was further manually annotated to specify genomic locations, breakpoints and MLPA probes relevant for each CNV. We developed IthaCNVs, a publicly available and easy-to-use online tool that can facilitate the diagnosis of rare and diagnostically challenging haemoglobinopathy cases attributed to CNVs. Importantly, it facilitates the filtering of available entries based on the type of breakpoint information, on specific chromosomal and locus positions, on MLPA probes, and on affected gene(s). IthaCNVs brings together manually curated information about CNV genomic locations, functional effects, and information that can facilitate CNV characterisation through MLPA. It can help laboratory staff and clinicians confirm suspected diagnosis of CNVs based on molecular DNA screening and analysis., Competing Interests: The authors declare no conflict of interest.

Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1 , HBA2, and HBB . By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework., Competing Interests: ST, MX, AM, CS, CH, CB, JT, IF, NY, FA, EE, HH, BZ, AK, MK, PK No competing interests declared, (© 2022, Tamana, Xenophontos et al.)

Hemoglobinopathies are the most common monogenic disorders in the world with an ever increasing global disease burden each year. As most hemoglobinopathies show recessive inheritance carriers are usually clinically silent. Programmes for preconception and antenatal carrier screening, with the option of prenatal diagnosis are considered beneficial in many endemic countries. With the development of genetic tools such as Array analysis and Next Generation Sequencing in addition to state of the art screening at the hematologic, biochemic and genetic level, have contributed to the discovery of an increasing number of rare rearrangements and novel factors influencing the disease severity over the recent years. This review summarizes the basic requirements for adequate carrier screening analysis, the importance of genotype-phenotype correlation and how this may lead to the unrevealing exceptional interactions causing a clinically more severe phenotype in otherwise asymptomatic carriers. A special group of patients are β-thalassemia carriers presenting with features of β-thalassemia intermedia of various clinical severity. The disease mechanisms may involve duplicated α-globin genes, mosaic partial Uniparental Isodisomy of chromosome 11p15.4 where the HBB gene is located or haplo-insufficiency of a non-linked gene SUPT5H on chromosome 19q, first described in two Dutch families with β-thalassemia trait without variants in the HBB gene., (© 2022 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on a set of shared standards for the classification of variants in Mendelian diseases. The generality of these standards and their subjective interpretation between laboratories has prompted efforts to reduce discordance of variant classifications, with a focus on the expert specification of the ACMG/AMP guidelines for individual genes or diseases. Herein, we describe our experience as a ClinGen Variant Curation Expert Panel to adapt the ACMG/AMP criteria for the classification of variants in three globin genes (HBB, HBA2, and HBA1) related to recessively inherited hemoglobinopathies, including five evidence categories, as use cases demonstrating the process of specification and the underlying rationale., (© 2021 The Authors. Human Mutation published by Wiley Periodicals LLC.)

As in most Northern European countries, the prevalence of hemoglobinopathies in The Netherlands is increasing due to migration. Although hemoglobinopathies are severe chronic diseases with few treatment options, timely detection of carriers allows at-risk couples to make informed reproductive choices such as pre-implantation diagnosis, prenatal diagnosis or termination of affected pregnancies. Using a quantitative design, we evaluated the prevalence of hemoglobinopathies in The Hague region, The Netherlands. Patient and carrier registries from hospital, laboratory and general practitioners allowed this quantitative analysis. The highest prevalence of hemoglobinopathies was seen in immigrant neighborhoods, and a large gap was noted between estimated carrier prevalence and the actual registration of carriers in electronic patient records. Carrier prevalence was estimated to be 13,704; however, the ELAN database contains only 1542 cases with ICPC codes for sickle cell disease or thalassemia. Although more research is needed to define the requirements of the healthcare system to address this challenge, this study clearly shows the gap between estimated carrier prevalence and registration and thereby the pressing need for action., (© 2022. The Author(s).)

We describe a first Dutch case of Hb M Saskatoon (HBB:c.190C > T p.His64Tyr) in a 47-year-old female Dutch patient who presented with cyanosis, hemolysis, and abnormal co-oximetry. A mean corpuscular volume (MCV) of 105 fL caused by reticulocytosis (160 × 10 9 /L) and low red blood cell count (3.6 × 10 12 /L) suggested an increased erythrocyte turnover. An HPLC glyco-globin analysis revealed a decreased HbA1c fraction of 12.3 mmol/mmol, HbA0 of 93.3% and an additional unidentified fraction at 1.2 min. DNA sequencing revealed a missense mutation in the HBB gene, (HBB:c.190C > T p.His64Tyr), known as Hb M Saskatoon, a variant which has been previously identified as an unstable hemoglobin variant leading to methemoglobinemia and anemia. In this report, we describe the clinical and remarkable laboratory aspects of our patient with Hb M Saskatoon, and the consequences for treatment and drug use.

Introduction: The high-sequence homology of the α-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site., Methods: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing., Results: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results., Conclusions: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the -- gb , but as it was not registered correctly in the available databases, it was not initially recognized as such., (© 2021 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro-iron of hemoglobin (Hb) to ferri-iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi-like approach and the experts panel reached a final consensus >75% of agreement for all the questions., (© 2021 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

During the last few years, next-generation sequencing (NGS) has undergone a rapid transition from a research setting to a clinical application, becoming the method of choice in many clinical genetics laboratories for the detection of disease-causing variants in a variety of genetic diseases involving multiple genes. The hemoglobinopathies are the most frequently found Mendelian inherited monogenic disease worldwide and are composed of a complex group of disorders frequently involving the inheritance of more than one abnormal gene. This review aims to present the role of NGS in both screening and pre- and post-natal diagnostics of the hemoglobinopathies, and the added value of NGS is discussed based on the results described in the literature. Overall, NGS has an added value in large-scale high throughput carrier screening and in the complex cases for which common molecular techniques have some inadequacies. It is proven that the majority of thalassemia cases and Hb variants can be diagnosed using routine analysis involving a combined approach of hematology, hemoglobin separation, and classical DNA methods; however, we conclude that NGS can be a useful addition to the existing methods in the diagnosis of these disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Achour, Koopmann, Baas and Harteveld.)

Introduction: The accurate measurement of HbA 2 is essential for the detection of β-thalassaemia carriers and as no single calibrant is used by the various manufacturers of analysers, differences are seen in results obtained. The World Health Organization International Reference Reagent for HbA 2 (WHO IRR 89/666) was made available to diagnostic laboratories in the 1980s and remains the only international reference material available. A previous study (2015) demonstrated that the WHO IRR remained suitable for use as an HbA 2 standard as tested by 52 participants in the UK NEQAS Haematology Abnormal Haemoglobins Programme. This study was undertaken to include simultaneous analysis of three whole blood specimens over a range of HbA 2 values with the WHO IRR and to include participants from laboratories outside of the UK., Method: Three whole blood specimens with HbA 2 levels ranging from 2.4% to 5.7% and the WHO IRR were distributed to 56 laboratories located in 14 different countries. Participants were requested to test the specimens at defined intervals and return results accompanied by chromatograms or electropherograms produced., Results: Differences found in results from different analyser groups reflect the bias found in the 2015 study in that bias is seen according to the methodology used and also varies in relation to the level of analyte being measured., Conclusion: Results of measurements from whole blood specimens and the lyophilized WHO IRR standard did not show any deterioration of the IRR, and it remains suitable for use. Linearity and calibration of analysers remain a problem., (© 2020 John Wiley & Sons Ltd.)

Objectives: Artifactually altered glycated hemoglobin (HbA 1c ) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis., Methods: Cation exchange high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II; Trinity Biotech Premier Hb9210 Resolution), capillary electrophoresis (CE) (Sebia Capillarys 2 Flex Piercing) and mass spectrometry (MS) (Waters) were used for variant detection; Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) were used for DNA analysis; HbA 1c was measured with cation exchange HPLC (Bio-Rad Variant™ II; Arkray Adams HA-8180V; Tosoh HLC-723 G7), CE (Sebia Capillarys 2 Flex Piercing), boronate affinity HPLC (Trinity Biotech Hb9210 Premier), immunoassay (Cobas c501 Tina-quant HbA 1c Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA 1c HA-411V) and enzymatic assay (Abbott Architect c 8000 HbA 1c )., Results: Hb Yamagata [β132(H10)Lys→Asn; ( HBB : c.399A>T)] was identified in the proband by MS after the observation of an abnormal peak in HPLC and CE. A mosaic expression of this variant was detected by NGS (mutant: 8%; wild type: 92%), after negative results in Sanger sequencing. Hb Yamagata interfered with HbA 1c measurements by cation exchange HPLC and CE whereas immuno and enzymatic assay values showed good agreement with boronate affinity HPLC measurement., Conclusions: A mosaicism of Hb Yamagata was found in a patient with altered HbA 1c values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA 1c determination methods., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Unstable hemoglobinopathies (UHs) are rare anemia disorders (RADs) characterized by abnormal hemoglobin (Hb) variants with decreased stability. UHs are therefore easily precipitating, causing hemolysis and, in some cases, leading to dominant beta-thalassemia (dBTHAL). The clinical picture of UHs is highly heterogeneous, inheritance pattern is dominant, instead of recessive as in more prevalent major Hb syndromes, and may occur de novo . Most cases of UHs are not detected by conventional testing, therefore diagnosis requires a high index of suspicion of the treating physician. Here, we highlight the importance of next generation sequencing (NGS) methodologies for the diagnosis of patients with dBTHAL and other less severe UH variants. We present five unrelated clinical cases referred with chronic hemolytic anemia, three of them with severe blood transfusion dependent anemia. Targeted NGS analysis was performed in three cases while whole exome sequencing (WES) analysis was performed in two cases. Five different UH variants were identified correlating with patients' clinical manifestations. Four variants were related to the beta-globin gene (Hb Bristol-Alesha, Hb Debrousse, Hb Zunyi, and the novel Hb Mokum) meanwhile one case was caused by a mutation in the alpha-globin gene leading to Hb Evans. Inclusion of alpha and beta-globin genes in routine NGS approaches for RADs has to be considered to improve diagnosis' efficiency of RAD due to UHs. Reducing misdiagnoses and underdiagnoses of UH variants, especially of the severe forms leading to dBTHAL would also facilitate the early start of intensive or curative treatments for these patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rizzuto, Koopmann, Blanco-Álvarez, Tazón-Vega, Idrizovic, Díaz de Heredia, Del Orbe, Pampliega, Velasco, Beneitez, Santen, Waisfisz, Elting, Smiers, de Pagter, Kerkhoffs, Harteveld and Mañú-Pereira.)

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zittersteijn, Harteveld, Klaver-Flores, Lankester, Hoeben, Staal and Gonçalves.)

Background: Standardization of laboratory tests can be a long process, and this is the case with regards to the methods used to measure hemoglobin A 2 (HbA 2 ), an important marker for beta-thalassemia and other thalassemic conditions. The IFCC standardization project started in 2004, and it took at least 15 years before developing a reference measurement procedure, defining and producing calibrators and certified reference materials., Methods: A series of steps have to be undertaken in order to promote the standardization in the field, a process involving a number of stakeholders (manufacturers, scientific societies, national health bodies, laboratory professionals, clinicians). In this work we describe some possible process indicators, in order to assure that the standardization will have internal and external validity and be effective for a long time. These indicators concern the inter-method studies, elaboration of External Quality Assessment Schemes, and the evaluation of the yearly distributions of HbA 2 measurements collected in selected laboratories., Results: Preliminary results are reported concerning the yearly distributions of HbA 2 , collected in two different locations, and using different analytical methods. Median yearly values were found very constant over the years, but different between methods. On the other side, results obtained on the same specimens using two different techniques, proved that results by capillary electrophoresis in 2 out of the 3 years of observation, were significantly lower than those by HPLC., Conclusion: In this document we report what has been done so far, and what has to be done to achieve the standardization of the measurement of HbA 2 worldwide., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individual's clinical phenotype is challenging., Methods: Here, as an example of this common phenomenon, we analysed 41 patients with simple deletions of ~177 to ~2000 kb affecting one allele of the well-characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised deletion extents and screened for genetic background effects, telomere position effect and compensatory upregulation of hemizygous genes., Results: We find the risk of developmental and neurological abnormalities arises from much smaller distal chromosome 16 deletions (~400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes; however, genetic background effects substantially modify phenotypic abnormalities., Conclusions: Using ATR-16 as a general model of disorders caused by CNVs, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

A woman completely lacking Hb A 2 on the high performance liquid chromatography (HPLC) analysis, presented with a novel deletional (εγ)δβ 0 -thal and a δ-globin gene variant. This combination causes a β-thalassemia (β-thal) minor phenotype. The woman was referred by a hematologist due to abnormal blood counts. Multiplex ligation-dependent probe amplification (MLPA) and microarray analysis showed a heterozygous, 177 kb long deletion that removed the locus control region enhancer plus the ε, G γ and A γ genes. Additional sequencing revealed a novel variant HBD : c.209G>A, p.Gly70Asp in the heterozygous state, called Hb A 2 -Gebenstorf. The combination of the two variants explains the lack of Hb A 2 in this woman.

We report a newborn with a compound heterozygosity for Hb O-Arab ( HBB : 364G>A) and Hb D-Los Angeles ( HBB : 364G>C). To the best of our knowledge, the combination of these two hemoglobin (Hb) variants has not been identified and reported before. The variants of the proband and parents were identified by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). DNA analysis was performed to confirm the variants. The levels of Hb variants of the proband were determined post-partum, at 3 months and 1 year after birth. Blood count analysis after 1 year revealed that the proband had a mild microcytic anemia. Furthermore, HPLC and CE analysis revealed an equal distribution of Hb D-Los Angeles compared to Hb O-Arab at the age of 1 year. The follow-up of the patient, suggested that the Hb combination is clinically silent or mild.