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

Sequence variants located in the introns of the ß-globin gene may affect the mRNA processing and cause ß-thalassemia (ß-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 ß-globin gene, HBB: c.316-70C>G and HBB: c.316-125A>G (NM_000518.4), both previously reported as variants causing ß-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 ß-globin defects, and we find no influence of the sequence variants on the phenotype.

Towards a prevention program for ß-thalassemia. The molecular spectrum in East Java, Indonesia.

Defining the spectrum of specific thalassemia mutations is an important issue when planning prevention programs in large multi ethnic countries as is Indonesia. In a first attempt to define the prevalence of the common mutations in East Java we selected a cohort of 17 transfusion-dependent patients attending the Dr. Soetomo Hospital, Surabaya, Indonesia. After basic diagnostics we performed direct DNA sequencing for all ß-globin genes. The results obtained on 34 independent chromosomes revealed the following prevalence rates: c.79 G>A p. Glu27Lys (Hb E) 47.0%; c.92+5G>C (IVS-I-5 G>C) 20.6%; c.109_110 delC p.Pro37Leu fs X7 [codon 35 (-C)] 17.6%; c.46del T p.Trp16Gly fsX4 [codon 15 (-T)] 5.9%; c.126_129delCTTT p. Phe42Leu fs X19 (codons 41/42) 2.9%; c.316-197 C>T [IVS-II-654 (C>T)] 2.9%; c*112 A>G (PolyA) 2.9%. Our preliminary results show that the distribution of the prevalent mutations in our cohort is quite homogeneous but with different forms than previously reported. This indicates that more studies on a larger scale and in different geographical areas are needed to refine our provisional results and to characterize the molecular background of the disease in the whole country.

Hb Boskoop [HBA2c.112C>T p.Pro38Ser]: a new α2 chain variant observed in a Morrocan family.

We describe a new nondeletional α-thalassemia (α-thal) determinant found in a Moroccan infant and in two members of his family. The new mutation generates an abnormal hemoglobin (Hb) as a consequence of a Pro→Ser amino acid substitution at codon 37 (old nomenclature) of the α2 gene. The new Hb variant is barely separable on high performance liquid chromatography (HPLC) but the expression of the α chain mutant measured on reversed phase chromatography is one-third of that expected from a stable α2 variant, which explains the mild α-thal phenotype observed in the carriers. As shown for other mutations described in our laboratory (i.e., Hb Gouda), this variant could also be common in the North African population, overlooked because of the mild phenotype and silent behavior on HPLC. Nevertheless, these silent variants could generate intermediate Hb H diseases in association with Mediterranean α(0)-thal deletion defect.

A new alpha(0)-thalassemia deletion found in a Dutch family (--(AW)).

Alpha-thalassemia is an inherited hemoglobin disorder characterized by a microcytic hypochromic anemia caused by a quantitative reduction of the alpha-globin chain. The majority of the alpha-thalassemias is caused by deletions in the alpha-globin gene cluster. A deletion in the alpha-globin gene cluster, which was found in a Dutch family, was characterized by MLPA, long-range PCR and direct sequencing. We describe the molecular characterization of a novel 8.2kb deletion (--(AW)), involving both alpha-globin genes in cis. The deletion is caused by a non-homologous recombination event between an Alu and an L1-repeat sequence. This deletion is the third example of a non-homologous recombination event involving an Alu and an L1 repeat, and the first described in the human alpha-globin gene cluster. Because of a 25% risk of Hb Bart's with hydrops foetalis in the offspring when in combination with another alpha(0)-thalassemia allele, it is important to diagnose this deletion.

alpha-thalassaemia masked by beta gene defects and a new polyadenylation site mutation on the alpha2-globin gene.

We report three examples of chronic anaemia involving complex combinations of alpha- and beta-globin gene defects. The first case had a potential Hb H disease caused by the classic SEA/RW deletions masked by Hb E [beta26(B8)Glu-->Lys] in the homozygous state. The second had an unusual Hb H disease caused by compound heterozygosity for two different alpha2 polyadenylation site mutations masked by a beta-thalassaemia heterozygosity. The third had an intermediate alpha-thalassaemia with considerable anaemia caused by an as yet unknown polyadenylation site (AATAAA>AATAAC) mutation in combination with a common RW deletion masked by a common Hb C [beta6(A3)Glu-->Lys] heterozygosity. Diagnostic methods, genotype/phenotype correlations and the chance of overlooking these combinations during risk assessment in a multiethnic society are discussed.

Two new alpha1-globin gene point mutations: Hb Nedlands (HBA1:c.86C>T) [alpha28(B9)Ala-->Val] and Hb Queens Park (HBA1:c.98T>A) [alpha32(B13)Met-->Lys].

We report two new point mutations of the alpha1-globin gene found in a Greek and a Burmese patient, both living in Western Australia. The patients were initially selected for their microcytic hypochromic parameters as belonging to a group suspected for uncommon (deletion) defects. Gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA) technologies were applied, and in those cases not showing deletions, direct sequencing was performed. We have found 1) HBA1:c.86C>T, Hb Nedlands [alpha28(B9)Ala-->Val] which, based on the red cell indices and phenotype prediction scores, is presumed to be clinically silent, and 2) HBA1:c.98T>A, Hb Queens Park [alpha32(B13)Met-->Lys] which seems to be associated with a mild alpha-thalassemia (alpha-thal) phenotype. The phenotype/genotype correlation is briefly described.

Hb St. Truiden [α68(E17)Asn→His] and Hb Westeinde [α125(H8)Leu→Gln]: two new abnormalities of the α2-globin gene.

We report two new abnormal hemoglobins (Hbs) caused by mutations on the α2 gene. One resulted into an Asn→His substitution at position 68, the other in a Leu→Gln substitution at position 125. The first mutation was observed in a 61-year-old North European Belgian male during Hb A(1c) analysis and subsequently in other members of his family. The variant was expressed at a normal level and caused no hematological abnormalities in the carriers. The second was found in a 27-year-old Turkish male living in The Hague, The Netherlands, who presented with microcytic hypochromic parameters without iron deficiency and was also carrier of the common α2 IVS-I (-5 nt) deletion.

New and known ß-thalassemia determinants masked by known and new δ gene defects [Hb A(2)-Ramallah or δ6(A3)Glu→Gln, GAG>>CAG].

We report a novel thalassemia determinant found in a Nigerian woman living in the Netherlands, resulting from a 2 bp insertion at codons 9/10 of the ß-globin gene (HBBc.28_29insTA p.Ser10LeufsX11). The novel defect causes a frameshift with a consequent premature TGA stop codon, located at 11 positions downstream from the mutated codon. The phenotype was typical of a ß-thalassemia (ß-thal), trait with high RBC counts and compensated mild microcytic anemia. However, the Hb A(2) level was reported to be normal due to the presence of the common Hb A(2)' or Hb B2 [δ16(A13)Gly→Arg, GGC>CGC] variant that was not taken into account. We also present the opposite but comparable situation found in an a Palestinian man living in the USA. He was a carrier of a common ß-globin gene defect [codon 6 (-A), HBB:c.20delA] in combination with a novel δ-globin gene defect at codon 6 [HBD. c.19G>C, Glu6Gln] that we have named Hb A(2)-Ramallah. In both cases, the provisional diagnosis could have been compromised when based on the measurement of the normal Hb A(2) fraction only.

Hb Den Haag [beta45(CD4)Phe-->Tyr]. A new hemoglobin variant observed during early pregnancy diagnostics.

During a second pilot study, intended to explore the possibility of a country wide implementation of carrier diagnostics for hemoglobinopathies in The Netherlands, we observed a new abnormal hemoglobin (Hb) variant in three members of a family of Scandinavian origin living in the Dutch city of The Hague (Den Haag). The proband, a 34-year-old female presented with low Hb, packed cell volume (PCV) and red blood cell (RBC) values but was normocytic and normochromic. High performance liquid chromatography (HPLC) analysis revealed a partially separated fraction following Hb A. Molecular diagnostics disclosed a TTT>TAT transversion at HBB:c.137 causing a Phe-->Tyr single amino acid substitution at position 45 of the beta-globin gene. Previously described heterozygous mutations at the same position [Hb Cheverly (Phe-->Ser) and Hb Arta (Phe-->Cys)] were reported to be associated with mild chronic hemolysis similar to this case. We describe the hematological features of the six family members, the biochemical and molecular data and we discuss the possible consequences in combination with the common beta-thalassemia (beta-thal) trait.

Codon 24 (TAT>TAG) and codon 32 (ATG>AGG) (Hb Rotterdam): two novel alpha2 gene mutations associated with mild alpha-thalassemia found in the same family after newborn screening.

We report two novel alpha2-globin gene mutations found in the same Surinamese family. The proband, a newborn presenting during neonatal screening with 21.3% Hb Bart's (gamma4), proved to be a carrier of the common -alpha(3.7) deletion and a novel codon 32 (ATG>AGG) transversion that we named Hb Rotterdam. The father carried the same point mutation with borderline hemoglobin (Hb), MCV and low MCH values. The mother presented with a significant microcytic hypochromic anemia and also carried the -alpha(3.7) deletion and a second novel TAT>TAG transversion generating a stop codon at position 24. Shortly thereafter, Hb Rotterdam was again found in two unrelated adult females and in a Canadian newborn, all of African origin, suggesting that Hb Rotterdam could be a frequently occurring alpha(T) determinant in the Black population. Screening and characterization of the mutations, phenotype/genotype correlation and the issue of reporting newborn carriers of alpha-thalassemia (alpha-thal) are discussed.

Hb Lepore-Leiden: a new delta/beta rearrangement associated with a beta-thalassemia minor phenotype.

The Lepore hemoglobins (Hbs) are a group of structural defects resulting from different recombination events between the delta- and beta-globin genes. They may come with different beta-thalassemia (beta-thal) minor-like phenotypes in the carrier and with variably severe phenotypes in the rare homozygote, and in the common compound heterozygote with beta-thal. The most seriously affected patients are those of Yugoslavian origin presenting with severe transfusion-dependent hemolytic anemia, dyserythropoiesis, hepatosplenomegaly and skeletal malformations. Because of genetic risk, couples where both partners are carriers of these combinations may require prognosis and prenatal diagnosis. In these cases, recognition of the defect must be done with particular care. We report a case of Hb Lepore induced by a yet unknown crossover event found in a 24-year-old Turkish male and compare the novel mutation with those previously reported.

The first case of Hb Groene Hart [alpha119(H2)Pro-->Ser, CCT-->TCT (alpha1)] homozygosity confirms that a thalassemia phenotype is associated with this abnormal hemoglobin variant.

Hb Groene Hart [alpha119(H2)Pro-->Ser, CCT-->TCT (alpha1)] has been reported in heterozygotes of Moroccan origin and also in association with the common -alpha(3.7) deletion. In all cases, the mutated protein was not detectable but was apparently associated with a mild alpha-thalassemia (thal) phenotype, presumably due to a modification of the alpha-globin chain domain that is recognized by the a hemoglobin stabilizing protein (AHSP). The present case of Hb Groene Hart homozygosity, confirms that the alpha-thal phenotype is associated with this alpha-globin chain. Hb Groene Hart must be quite frequent not only in Morocco but probably also among the northern African coastal population.