Publications by authors named "Cornelis L Harteveld"

111 Publications

Adapting the ACMG/AMP variant classification framework: A perspective from the ClinGen Hemoglobinopathy Variant Curation Expert Panel.

Hum Mutat 2021 Sep 12. Epub 2021 Sep 12.

Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

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.
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http://dx.doi.org/10.1002/humu.24280DOI Listing
September 2021

Recommendations for diagnosis and treatment of methemoglobinemia.

Am J Hematol 2021 Sep 1. Epub 2021 Sep 1.

Hematology, University of Utah & Huntsman Cancer Center, Salt Lake City, Utah, USA.

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.
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http://dx.doi.org/10.1002/ajh.26340DOI Listing
September 2021

The Evolving Role of Next-Generation Sequencing in Screening and Diagnosis of Hemoglobinopathies.

Front Physiol 2021 27;12:686689. Epub 2021 Jul 27.

Department of Clinical Genetics/LDGA, Leiden University Medical Center, Leiden, Netherlands.

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.
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http://dx.doi.org/10.3389/fphys.2021.686689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353275PMC
July 2021

Breakpoint characterization of a rare alpha -thalassemia deletion using targeted locus amplification on genomic DNA.

Int J Lab Hematol 2021 Jul 12. Epub 2021 Jul 12.

Department of Clinical Genetics, Leiden University Medical Centre, Leiden, the Netherlands.

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 -- , but as it was not registered correctly in the available databases, it was not initially recognized as such.
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http://dx.doi.org/10.1111/ijlh.13651DOI Listing
July 2021

Hemoglobin Yamagata [β132(H10)Lys→Asn; (: c.399A>T)]: a mosaic to be put together.

Clin Chem Lab Med 2021 Sep 23;59(10):1670-1679. Epub 2021 Apr 23.

Human Genetics Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Giannina Gaslini, Genova, Italy.

Objectives: Artifactually altered glycated hemoglobin (HbA) 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 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 Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA HA-411V) and enzymatic assay (Abbott Architect 8000 HbA).

Results: Hb Yamagata [β132(H10)Lys→Asn; (: 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 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 values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA determination methods.
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http://dx.doi.org/10.1515/cclm-2021-0376DOI Listing
September 2021

Usefulness of NGS for Diagnosis of Dominant Beta-Thalassemia and Unstable Hemoglobinopathies in Five Clinical Cases.

Front Physiol 2021 5;12:628236. Epub 2021 Feb 5.

Translational Research in Child and Adolescent Cancer - Rare Anemia Disorders Research Laboratory, Vall d'Hebron Research Institute, ERN-EuroBloodNet Member, Barcelona, Spain.

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 . 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.
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http://dx.doi.org/10.3389/fphys.2021.628236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893112PMC
February 2021

Further evaluation of the world health organization international reference reagent for Haemoglobin A measurement.

Int J Lab Hematol 2021 Jun 2;43(3):494-499. Epub 2020 Dec 2.

UK NEQAS Haematology, West Herts Hospitals NHS Trust, Watford, UK.

Introduction: The accurate measurement of HbA 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 (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 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 values with the WHO IRR and to include participants from laboratories outside of the UK.

Method: Three whole blood specimens with HbA 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.
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http://dx.doi.org/10.1111/ijlh.13403DOI Listing
June 2021

A roadmap for the standardization of hemoglobin A.

Clin Chim Acta 2021 Jan 10;512:185-190. Epub 2020 Nov 10.

Dept. of Clinical Genetics/LDGA, Leiden University Medical Center, Leiden, the Netherlands.

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 (HbA), 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 measurements collected in selected laboratories.

Results: Preliminary results are reported concerning the yearly distributions of HbA, 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 worldwide.
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http://dx.doi.org/10.1016/j.cca.2020.11.008DOI Listing
January 2021

A Woman with Missing Hb A Due to a Novel (εγ)δβ-Thalassemia and a Novel δ-Globin Variant Hb A-Gebenstorf (: c.209G>A).

Hemoglobin 2020 May 1;44(3):214-217. Epub 2020 Jul 1.

Analytica Medizinische Laboratorien AG, Zürich, Switzerland.

A woman completely lacking Hb A on the high performance liquid chromatography (HPLC) analysis, presented with a novel deletional (εγ)δβ-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, γ and γ genes. Additional sequencing revealed a novel variant : c.209G>A, p.Gly70Asp in the heterozygous state, called Hb A-Gebenstorf. The combination of the two variants explains the lack of Hb A in this woman.
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http://dx.doi.org/10.1080/03630269.2020.1779739DOI Listing
May 2020

ATR-16 syndrome: mechanisms linking monosomy to phenotype.

J Med Genet 2020 06 31;57(6):414-421. Epub 2020 Jan 31.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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.
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http://dx.doi.org/10.1136/jmedgenet-2019-106528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279195PMC
June 2020

An Unusual Compound Heterozygosity for Hb O-Arab (: c.364G>A) and Hb D-Los Angeles (: c.364G>C).

Hemoglobin 2020 Jan 23;44(1):61-63. Epub 2020 Jan 23.

The Reference Hemoglobinopathies Laboratory, Department of Human and Clinical Genetics, Leiden University Medical Centre, Leiden, The Netherlands.

We report a newborn with a compound heterozygosity for Hb O-Arab (: 364G>A) and Hb D-Los Angeles (: 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.
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http://dx.doi.org/10.1080/03630269.2019.1710530DOI Listing
January 2020

Laboratory quality systems in clinical laboratory practice in Lagos, Nigeria (West Africa): Associated problems and prospects.

Int J Lab Hematol 2019 12 26;41(6):e130-e133. Epub 2019 Apr 26.

Leiden University Medical Center, Leiden, The Netherlands.

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http://dx.doi.org/10.1111/ijlh.13038DOI Listing
December 2019

Evaluation of the suitability of the World Health Organization International Reference Reagent for Hb A quantitation (89/666) for continued use.

Int J Lab Hematol 2019 Apr 20;41(2):262-270. Epub 2019 Jan 20.

Manchester Metropolitan University, Manchester, UK.

Introduction: The accurate determination of Hb A is a key marker when screening for a β-thalassaemia carrier. Data from external quality assessment (EQA) exercises have shown a lack of alignment of Hb A quantitation both within and between methods. The only reference material available for Hb A quantitative assay at the time of writing is the World Health Organization International Reference Reagent (89/666; WHO IRR) prepared in the 1980s and not validated for all current methodologies.

Method: The WHO IRR was analysed for Hb A concentration by 52 laboratories using a representative range of high-performance liquid chromatography and capillary electrophoresis analysers. The results of the analysis were compared to those of a whole blood EQA specimen of similar Hb A concentration, distributed in the same week.

Results: The mean Hb A value obtained for the WHO IRR was 5.17%, compared to the assigned value of 5.3%. The range of results returned was wide (4.0%-6.2%), with differences in the results observed by between and within analyser groups. A similar range of results was seen with the whole blood sample, although the bias observed between analyser types was different from that seen with the WHO IRR.

Conclusion: The results may indicate a lack of commutability of the WHO IRR material, resulting from deterioration, matrix effects or changes in reagent formulation or calibration parameters. Further examination of the suitability of the WHO IRR (89/666) for continued use is required.
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http://dx.doi.org/10.1111/ijlh.12966DOI Listing
April 2019

Characterization of Two Deep Intronic Variants on the β-Globin Gene with Inconsistent Interpretations of Clinical Significance.

Hemoglobin 2018 Mar 26;42(2):126-128. Epub 2018 Jul 26.

a Department of Medical Biochemistry , Oslo University Hospital , Oslo , Norway.

Sequence variants located in the introns of the β-globin gene may affect the mRNA processing and cause β-thalassemia (β-thal). Sequence variants that change one of the invariant dinucleotides at the exon-intron boundaries may have fatal consequences for normal mRNA splicing. Intronic variants located far from obvious regulatory sequences can be more difficult to evaluate. There is a potential for misinterpretation of such sequence variants. Hence, thorough evaluation of patient data together with critical use of databases and in silico prediction tools are important. Here, we describe two rare sequence variants in the second intron of the β-globin gene, HBB: c.316-70C>G and HBB: c.316-125A>G (NM_000518.4), both previously reported as variants causing β-thal, and later as benign sequence variants. Due to the limited number of published cases and inconsistent interpretations, the significance of these sequence variants has been unclear. We have identified these two sequence variants in multiple individuals, alone and in a variety of combinations with other δ- and β-globin defects, and we find no influence of the sequence variants on the phenotype.
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http://dx.doi.org/10.1080/03630269.2018.1473255DOI Listing
March 2018

Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T], A Second and Third Case Described in Two Unrelated Dutch Families.

Hemoglobin 2018 Jan 6;42(1):51-53. Epub 2018 Feb 6.

d Hemoglobinopathies Laboratory, Department of Clinical Genetics (LDGA) , Leiden University Medical Center (LUMC) , Leiden , The Netherlands.

We report two families, members of which are carriers of a hemoglobin (Hb) variant previously described as Hb Nouakchott [α114(GH2)Pro→Leu; HBA1: c.344C>T; p.Pro115Leu]. In the first family of Dutch origin, the proband, a 32-year-old male and his 65-year-old father, were both carriers of Hb Nouakchott. Of the second family we tested, only the proband, a 56-year-old Dutch female was a Hb Nouakchott carrier. Hematological analyses of these cases showed the anomaly behaves as a silent Hb variant without clinical consequences. The Hb variant remained unnoticed using high performance liquid chromatography (HPLC), while an additional peak was detected by capillary electrophoresis (CE). These independent findings of Hb Nouakchott indicate that this Hb variant might not be very rare, but simply remains under diagnosed depending on the Hb separation technique used.
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http://dx.doi.org/10.1080/03630269.2018.1429280DOI Listing
January 2018

Calibration by commutable control materials is able to reduce inter-method differences of current high-performance methods for HbA.

Clin Chim Acta 2018 Feb 5;477:60-65. Epub 2017 Dec 5.

Dip. di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy. Electronic address:

Background: Most of the current methods used for the determination of HbA seem not well aligned. A comparison among the best performing techniques and the commutability of some control materials currently available and under development has been evaluated.

Methods: Forty blood samples were analyzed in duplicate over two separate days by different HPLC and capillary electrophoresis systems. The commutabilities of different control materials (NIBSC WHO reagent, Bio-Rad Lyphochek, and home prepared lyophilized controls RP1-3) have been assessed by analyzing the controls in quadruplicate over two consecutive days together with the blood samples.

Results: The mean within-run imprecision of HbA measurement on blood samples (CV, %) was between 0.6% and 10.1% for HbA values <3.5%, and between 1.1 and 3.1 for HbA≥3.5%. The different methods were highly correlated (r between 0.9941 and 0.9995) although biased each other. The NIBSC WHO reagent was found not commutable in 15 over 28 comparisons, the Lyphochek 2 in 18/28, and RP3 in 4/28. Recalibration of all methods by RP1 and RP2 materials was able to reduce the overall variability from 6.8% to 3.4% at HbA≤3.0% and from 6.7% to 3.0% at HbA≥4.6%.

Conclusion: The use of adequate commutable control materials as calibrators may reduce the inter-method variability of routine methods to an extent closer to the current analytical goals of bias based on biological variability.
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http://dx.doi.org/10.1016/j.cca.2017.12.001DOI Listing
February 2018

Molecular basis of α-thalassemia.

Blood Cells Mol Dis 2018 05 21;70:43-53. Epub 2017 Sep 21.

Dept. of Clinical Genetics, Hemoglobinopathy Expert Center, Leiden University Medical Center, Leiden, The Netherlands. Electronic address:

α-Thalassemia is an inherited, autosomal recessive, disorder characterized by a microcytic hypochromic anemia. It is one of the most common monogenic gene disorders in the world population. The clinical severity varies from almost asymptomatic, to mild microcytic hypochromic, and to a lethal hemolytic condition, called Hb Bart's Hydrops Foetalis Syndrome. The molecular basis are usually deletions and less frequently, point mutations affecting the expression of one or more of the duplicated α-genes. The clinical variation and increase in disease severity is directly related to the decreased expression of one, two, three or four copies of the α-globin genes. Deletions and point mutations in the α-globin genes and their regulatory elements have been studied extensively in carriers and patients and these studies have given insight into the α-globin genes are regulated. By looking at naturally occurring deletions and point mutations, our knowledge of globin-gene regulation and expression will continue to increase and will lead to new targets of therapy.
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http://dx.doi.org/10.1016/j.bcmd.2017.09.004DOI Listing
May 2018

Preconception carrier screening and prenatal diagnosis in thalassemia and hemoglobinopathies: challenges and future perspectives.

Expert Rev Mol Diagn 2017 03 2;17(3):281-291. Epub 2017 Feb 2.

b Department of Clinical Genetics , Laboratory for Diagnostic Genome Analysis (LDGA) , Leiden , The Netherlands.

Introduction: Hemoglobinopathies constitute the most common severe monogenic disorders worldwide, with an increasing global burden each year. The benefit of applying programmes for preconception carrier screening, with the option of prenatal diagnosis, to minimize the incidence of new cases is recognized in many countries. Areas covered: The challenges associated with identifying carrier couples using hematology-based screening, along with DNA diagnosis and prenatal diagnosis were addressed, based on a literature search and the authors expertise. Expert commentary: The hemoglobinopathies are extremely heterogeneous at the haematological, molecular and clinical level, requiring appropriately equipped and staffed laboratories with experience to support comprehensive screening and diagnosis. However complete services with adequate infrastructure to address the associated technical challenges do not exist widely, especially in low-income countries that, coincidentally, are often those with the highest frequency of hemoglobinopathies in their population. Additionally, overcoming limited public awareness, education and absence of systematic dissemination of information also constitutes a challenge. This article aims to highlight these challenges and to evaluate potential future developments that may address at least some of them, focusing mainly on the technical challenges related to molecular diagnostics.
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http://dx.doi.org/10.1080/14737159.2017.1285701DOI Listing
March 2017

Hb Olivet (HBA1: C.40G > A; p.Ala14Thr), a Novel Silent Hemoglobin Variant in Two Families of Distinct Origin.

Hemoglobin 2016 Sep 14;40(5):349-352. Epub 2016 Sep 14.

c Department of Pediatrics , Groene Hart Hospital , Gouda , the Netherlands.

We report two families, members of which are carriers of a novel hemoglobin (Hb) variant that was named Hb Olivet [α13(A11)Ala→Thr (α1) (GCC > ACC); HBA1: c.40G > A; p.Ala14Thr]. The analysis of these cases allowed a clear description of this anomaly that behaves as a silent Hb. In the first family, of Portuguese ethnicity living in France, the proband, a 24-year-old male and his 57-year-old mother, both appeared to be carriers. The son presented with borderline mean corpuscular volume (MCV), while the mother was normocytic and normochromic. Hemoglobin separation on capillary electrophoresis (CE) was normal, while a slightly asymmetric peak was observed on high performance liquid chromatography (HPLC). In a second family, originally from Surinam but living in The Netherlands, the proband, a 6-year-old girl, showed a mild microcytosis at low ferritin levels. The abnormal Hb was inherited from the mother who was clearly iron depleted, was not present in the sister and brother of the proband. The microcytic hypochromic anemia was only shown in two out of a total of four carriers. It therefore seems likely that iron depletion is causative as two carriers are completely normal. Characterization and genotype/phenotype correlation are briefly described.
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http://dx.doi.org/10.1080/03630269.2016.1210160DOI Listing
September 2016

Hb Melusine and Hb Athens-Georgia: potentially underreported in the Belgian population? Four cases demonstrating the lack of detection using common CE-HPLC methods either for glycated hemoglobin (HbA) analysis or Hb variant screening.

Acta Clin Belg 2016 Dec 30;71(6):458-461. Epub 2016 Jun 30.

a Department of Laboratory Medicine , University Hospitals Leuven , Leuven , Belgium.

Objective And Importance: Suspected hemoglobin (Hb) variants, detected during HbA measurements should be further investigated, determining the extent of the interference with each method.

Clinical Presentation: This is the first report of Hb Melusine and Hb Athens-Georgia in Caucasian Belgian patients. Intervention & Technique: Since common CE-HPLC methods for HbA analysis or Hb variant screening are apparently unable to detect these Hb variants, their presence might be underestimated. HbA analysis using CZE, however, alerted for their presence. Moreover, in case of Hb Melusine, even Hb variant screening using CZE was unsuccessful in its detection.

Conclusion: Fortunately, carriage of Hb Melusine or Hb Athens-Georgia variants has no clinical implications and, as shown in this report, no apparent difference in HbA should be expected.
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http://dx.doi.org/10.1080/17843286.2016.1203559DOI Listing
December 2016

A Mosaic Expression of a Hb J-Amiens (HBB: c.54G > T; p.Lys18Asn) and its Interference with Hb A1c Analysis.

Hemoglobin 2015 15;39(6):435-7. Epub 2015 Sep 15.

a Department of Laboratory Medicine , University Hospitals Leuven , Leuven , Belgium.

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1c analysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [β17(A14)Lys→Asn; HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1c measurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1c analysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing.
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http://dx.doi.org/10.3109/03630269.2015.1079218DOI Listing
August 2016

A Novel Targeted Approach for Noninvasive Detection of Paternally Inherited Mutations in Maternal Plasma.

J Mol Diagn 2015 Sep 7;17(5):590-6. Epub 2015 Jul 7.

Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands.

The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. We have developed a sensitive, mutation-specific, and fast alternative for next-generation sequencing-mediated noninvasive prenatal diagnosis using a PCR-based method. For this proof-of-principle study, noninvasive fetal paternally inherited mutation detection was performed using cell-free DNA from maternal plasma. Preferential amplification of the paternally inherited allele was accomplished through a personalized approach using a blocking probe against maternal sequences in a high-resolution melting curve analysis-based assay. Enhanced detection of the fetal paternally inherited mutation was obtained for both an autosomal dominant and a recessive monogenic disorder by blocking the amplification of maternal sequences in maternal plasma.
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September 2015

Hb Lansing (HBA2: c.264C > G) and a new β promoter transversion [-52 (G > T)]: an attempt to define the phenotype of two mutations found in the Omani population.

Hemoglobin 2015 31;39(2):111-4. Epub 2015 Mar 31.

Molecular Genetic Laboratory, National Genetic Centre , Muscat , Sultanate of Oman and.

We report two examples showing how problematic it can be to define the phenotype of new or rare globin genes mutations. We describe two mutations observed for the first time in the Omani population: the first was found in the consanguineous parents of a deceased newborn with hepatomegaly, cardiomegaly and severe hemolytic anemia, putatively homozygous for the rare Hb Lansing (HBA2: c.264C > G) variant. The second is a novel β-globin gene promoter mutation [-52 (G > T)] observed in four independent patients. Two with borderline/elevated Hb A2, α-thalassemia (α-thal) and hypochromic red cell indices, and two heterozygotes for Hb S (HBB: c.20A > T), α-thal and with Hb A/Hb S ratios possibly indicating a very mild β(+)-thalassemia (β(+)-thal) mutation.
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January 2016
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