Cornelis L. Harteveld

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We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories.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.

2 α-Thalassaemia

α-Thalassaemias are genetic defects extremely frequent in some populations and are characterized by the decrease or complete suppression of α-globin polypeptide chains. The gene cluster, which codes for and controls the production of these polypeptides, maps near the telomere of the short arm of chromosome 16, within a G + C rich and early-replicating DNA region. The genes expressed during the embryonic (ζ) or fetal and adult stage (α2 and α1) can be modified by point mutations which affect either the processing-translation of mRNA or make the polypeptide chains extremely unstable. Much more frequent are the deletions of variable size (from ≈ 3 to more than 100 kb) which remove one or both α genes in cis or even the whole gene cluster. Deletions of a single gene are the result of unequal pairing during meiosis, followed by reciprocal recombination. These unequal cross-overs, which produce also α gene triplications and quadruplications, are made possible by the high degree of homology of the two α genes and of their flanking sequences. Other deletions involving one or more genes are due to recombinations which have taken place within non-homologous regions (illegitimate recombinations) or in DNA segments whose homology is limited to very short sequences. Particularly interesting are the deletions which eliminate large DNA areas 5′ of ζ or of both α genes. These deletions do not include the structural genes but, nevertheless, suppress completely their expression. Larger deletions involving the tip of the short arm of chromosome 16 by truncation, interstitial deletions or translocations result in the contiguous gene syndrome ATR-16. In this complex syndrome α-thalassaemia is accompanied by mental retardation and variable dismorphic features. The study of mutations of the 5′ upstream flanking region has led to the discovery of a DNA sequence, localized 40 kb upstream of the ζ-globin gene, which controls the expression of the α genes (α major regulatory element or HS-40). In the acquired variant of haemoglobin H (HbH) disease found in rare individuals with myelodysplastic disorders and in the X-linked mental retardation associated with α-thalassaemia, a profound reduction or absence of α gene expression has been observed, which is not accompanied by structural alterations of the coding or controlling regions of the α gene complex. Most probably the acquired α-thalassaemia is due to the lack of soluble activators (or presence of repressors) which act in trans and affect the expression of the homologous clusters and are coded by genes not (closely) linked to the α genes. The ATR-X syndrome results from mutations of the XH2 gene, located on the X chromosome (Xq13.3) and coding for a transacting factor which regulates gene expression. The interaction of the different α-thalassaemia determinants results in three phenotypes: the α-thalassaemic trait, clinically silent and presenting only limited alterations of haematological parameters, HbH disease, characterized by the development of a haemolytic anaemia of variable degree, and the (lethal) Hb Barts hydrops fetalis syndrome. The diagnosis of α-thalassaemia due to deletions is implemented by the electrophoretic analysis of genomic DNA digested with restriction enzymes and hybridized with specific molecular probes. Recently polymerase chain reaction (PCR) based strategies have replaced the Southern blotting methodology. The straightforward identification of point mutations is carried out by the specific amplification of the α2 or α1 gene by PCR followed by the localization and identification of the mutation with a variety of screening systems (denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphisms (SSCP)) and direct sequencing.

Multi Centric Origin of Hb D-Punjab [β121(GH4)Glu→Gln, GAA>CAA]

Hb D-Punjab [β121(GH4)Glu→Gln, GAA>CAA], common in the northern Indian province, is often unexpectedly found in other populations. To study the multi centric origin of this variant which is causing sickle cell disease in association with Hb S [β6(A3)Glu→Val, GAG>GTG], we have examined the haplotype of the Hb D allele in different populations. We studied 43 alleles from south Iran (Hormozgan and Fars provinces) and 14 from Holland and Belgium using high performance liquid chromatography (HPLC), capillary electrophoresis, direct sequencing and/or restriction enzyme analysis. In Iranians, four haplotypes were observed at different frequencies: haplotype I [+ − − – −,+ +] at 67.5%, subhaplotype I [+ – – – –,– +] at 17.5%, haplotype V [– + – – +,+ +] at 10.0% and haplotype III [– + – + +,+ +] at 5.0%. All European cases were on haplotype I. The occurrence of high Hb D frequencies on a single haplotype in specific regions can be expected if we consider founder effect and genetic drift mechanisms. However, considering that haplotype I is the most common haplotype worldwide, that Hb D-Punjab is reported in different populations on different haplotypes, and that codon β121 is a site on which six different mutations are reported, we may expect to observe Hb D-Punjab in different populations, possibly because of a relatively higher occurrence of de novo mutations, generating unexpected risk from mixtures of allochtonous Hb S and indigenous Hb D-Punjab or vice versa.

Human mitochondrial ATP-binding cassette transporter ABCB10 is required for efficient red blood cell development

Arnaud, L., Saison, C., Helias, V., Lucien, N., Steschenko, D., Giarratana, M.C., Prehu, C., Foliguet, B., Montout, L., de Brevern, A.G., Francina, A., Ripoche, P., Fenneteau, O., Da Costa, L., Peyrard, T., Coghlan, G., Illum, N., Birgens, H., Tamary, H., Iolascon, A., Delaunay, J., Tchernia, G. & Cartron, J.P. (2010) A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia. American Journal of Human Genetics, 87, 721–727. Cazzola, M. & Invernizzi, R. (2010) Molecular basis of congenital dyserythropoietic anemia type II and genotype-phenotype relationship. Haematologica, 95, 693–695. Claessens, Y.E., Bouscary, D., Dupont, J.M., Picard, F., Melle, J., Gisselbrecht, S., Lacombe, C., Dreyfus, F., Mayeux, P. & Fontenay-Roupie, M. (2002) In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fasdependent apoptosis. Blood, 99, 1594–1601. Dgany, O., Avidan, N., Delaunay, J., Krasnov, T., Shalmon, L., Shalev, H., Eidelitz-Markus, T., Kapelushnik, J., Cattan, D., Pariente, A., Tulliez, M., Cretien, A., Schischmanoff, P.O., Iolascon, A., Fibach, E., Koren, A., Rossler, J., Le Merrer, M., Yaniv, I., Zaizov, R., Ben-Asher, E., Olender, T., Lancet, D., Beckmann, J.S. & Tamary, H. (2002) Congenital dyserythropoietic anemia type I is caused by mutations in codanin-1. American Journal of Human Genetics, 71, 1467–1474. Finch, C. (1994) Regulators of iron balance in humans. Blood, 84, 1697–1702. Renella, R., Roberts, N.A., Brown, J.M., De Gobbi, M., Bird, L.E., Hassanali, T., Sharpe, J.A., SloaneStanley, J., Ferguson, D.J., Cordell, J., Buckle, V.J., Higgs, D.R. & Wood, W.G. (2011) Codanin1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts. Blood, 117, 6928–6938. Schwarz, K., Iolascon, A., Verissimo, F., Trede, N.S., Horsley, W., Chen, W., Paw, B.H., Hopfner, K.P., Holzmann, K., Russo, R., Esposito, M.R., Spano, D., De Falco, L., Heinrich, K., Joggerst, B., Rojewski, M.T., Perrotta, S., Denecke, J., Pannicke, U., Delaunay, J., Pepperkok, R. & Heimpel, H. (2009) Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II. Nature Genetics, 41, 936–940. Tamary, H., Shalev, H., Perez-Avraham, G., Zoldan, M., Levi, I., Swinkels, D.W., Tanno, T. & Miller, J.L. (2008) Elevated growth differentiation factor 15 expression in patients with congenital dyserythropoietic anemia type I. Blood, 112, 5241–5244. Tanno, T., Bhanu, N.V., Oneal, P.A., Goh, S.H., Staker, P., Lee, Y.T., Moroney, J.W., Reed, C.H., Luban, N.L., Wang, R.H., Eling, T.E., Childs, R., Ganz, T., Leitman, S.F., Fucharoen, S. & Miller, J.L. (2007) High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nature Medicine, 13, 1096–1101. Truksa, J., Lee, P. & Beutler, E. (2009) Two BMP responsive elements, STAT, and bZIP/HNF4/ COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness. Blood, 113, 688–695.

A novel 7·9 kb deletion causing α+‐thalassaemia in two independent families of Indian origin

Summary. We describe the characterization of a novel 7·9u2003kb deletion that eliminated one of the duplicated α‐globin genes, causing an α+‐thalassaemia phenotype in two independent carriers of Suriname–Indian origin. The molecular characterization of the deletion breakpoint fragment revealed neither involvement of Alu repeat sequences nor the presence of homologous regions prone to recombination, suggesting a non‐homologous recombination event. This α+‐thalassaemia deletion was found to give rise to an atypical haemoglobin H (HbH) disease characterized by a non‐transfusion‐dependent moderate microcytic hypochromic anaemia in combination with a poly adenylation signal mutation of the α‐globin gene (α2 AATAAAu2003→u2003AATA‐‐u200a‐‐).

Beta thalassaemia intermedia due to co-inheritance of three unique alpha globin cluster duplications characterised by next generation sequencing analysis

Co-inheritance of a thalassaemia reduces chain imbalance and disease severity in b thalassaemia homozygotes, while increasing a globin output in heterozygotes increases chain imbalance, converting a typically asymptomatic carrier state to that of thalassaemia intermedia. The outcome depends on the number of a globin genes inherited as one or two copies of triplicated (/aaa), or quadruplicated (/aaaa) a globin complexes, and the type of b thalassaemia mutation (b or b) (Thein, 2008). Another mechanism of increasing a globin output is through segmental duplication of the whole a globin complex (Harteveld et al, 2008) but breakpoints of the reported duplications have not been fully characterised due to technological limitations. Here, we applied a previously described next generation sequencing (NGS) methodology (Shooter et al, 2015a,b) to characterise three a globin cluster duplications, permitting to-the-base resolution in two of the three cases. Patient samples were referred for work-up of unusually severe phenotype in b thalassaemia carriers. In Family 1 (Fig 1A), the proband was a 54-year-old Chinese male with hypochromic microcytic anaemia since infancy. His partner (Anglo-Saxon English) and two older sons had normal haematological profiles; the youngest son (aged 14 years) had a haematological profile similar to that of the father. Both father and son had

Homozygosity for a Rare β0-Thalassemia Mutation [Frameshift Codons 25/26 (+T)] Causes β-Thalassemia Intermedia in an Iranian Family

The severity of β-thalassemia (β-thal) is remarkable for its variability in different populations, even in different patients. We studied a family from Azerbaijan Province, Northwestern Iran, who had a rare β0-thal mutation, namely the frameshift codons (FSC) 25/26 (+T), originally reported in Tunisia. Unlike the Tunisian family, in our family the mutation was a β0 type and the affected members were dependent and independent of blood transfusions. This mutation was linked to the –158 (C>T) polymorphism on the Gγ-globin gene (XmnI marker) and two other polymorphisms in the Aγ-globin promoter at position +25 (G>A) and –588 (G>A). Deletions in the α- and β-globin gene clusters were excluded in all samples. This is the first description of the FSC 25/26 mutation in Iran. The results of this study emphasize the complexity of genetic interactions that underlie the phenotype of β-thal intermedia and highlight the importance of the regulation of hemoglobin (Hb) F production in the β-thal syndromes. Simultaneous inheritance of some loci that interfere with the elevation of Hb F probably caused them to have high levels of total Hb and to be transfusion independent.

Beta-Globin Gene Cluster Haplotypes in Yemeni Children with Sickle Cell Disease

a Faculty of Medicine and Health Sciences, Aden University, Aden , Yemen; b Child and Reproductive Health Group, Liverpool School of Tropical Medicine, and c Department of Community Child Health, Royal Liverpool Children’s Hospital, NHS Trust, Liverpool , UK; d Emma Kinderziekenhuis, Academic Medical Centre, University of Amsterdam, Amsterdam , and e Department of Human and Clinical Genetics, Leiden University Medical Centre, Leiden , The Netherlands

Molecular spectrum of α-globin gene defects in the Omani population.

Abstract We describe the molecular characterization of α-globin gene defects in a cohort of 634 Omani patients. A total of 21 different α gene mutations were found in 484 subjects. Overall, we identified three different large deletions, three small deletions, 11 point mutations [two on the α2 polyadenylation signal (polyA) (HBA2: c.*94A>G), and nine α chain variants], three αααanti 3.7 triplication, a 21 nucleotide (nt) duplication on the α1 gene and two novel (presumed) polymorphisms on the α 3.7u2009kb hybrid gene, namely −5 (C>T) and +46 (C>A). Of these defects, 15 have not been previously reported in the Omani population. This large heterogeneity of α-thalassemia (α-thal) observed in the Omani population could be expected in neighboring Arab countries. The high frequency of α-thal, solely or in association with β-globin gene defects, emphasize the necessity of adding α-thal testing to pre marital programs for accurate genetic counseling.

Sickle cell anemia and α-thalassemia: A modulating factor in homozygous HbS/S patients in Oman

We report the general phenotype severity and the hematological presentation in a cohort of 125 sickle cell anemia (SCA) patients with identical homozygous HbS/S genotype and categorized by identical β(S) haplotype, both with and without alpha thalassemia. No clear general phenotype correlation was found when patients were compared regardless of the haplotype but overall, patients with homozygous alpha thalassemia (α-/α-) had the highest Hb, HCT, RBC and the lowest MCV, MCH and MCHC levels. When patients with identical haplotype were compared, the mildest hematological and clinical conditions were observed in patients of the Asian/Asian haplotype, also known as Arab-Indian haplotype, and carriers of α-thalassemia, suggesting an additional ameliorating effect of alpha thalassemia. In conclusion, our results show that alpha thalassemia improves the hematological conditions but amelioration of the general disease severity is only noticed when compared in cohorts of the same haplotype.