A novel α(0) -thalassemia deletion in a Greek patient with HbH disease and ß-thalassemia trait.

OBJECTIVES: To determine the molecular basis in a Greek child suspected of having HbH disease and ß-thalassemia trait. METHODS: Standard hematology, Hb electrophoresis, and HPLC. Multiplex ligation-dependent probe amplification (MLPA), direct sequencing, and breakpoint characterization by NimbleGen fine-tiling array analysis. RESULTS: The index patient showed a moderate microcytic hypochromic anemia with normal ZPP and elevated HbA(2) , indicative for ß-thalassemia trait. However, the moderate microcytic hypochromic anemia along with the observation of HbH inclusions in occasional red blood cells suggested a coexisting α-thalassemia. Molecular analysis indicated that the propositus inherited the ß(+) -thalassemia mutation IVS2-745 (c>g) and a novel α(0) -thalassemia deletion from the mother, and the common non-deletion α-thalassemia allele α(2) (-5nt)α from the father. The α(0) -thalassemia deletion, named - -(BGS) , is approximately 131.6 kb in length. It removes the major regulatory elements along with the functional α-globin genes but leaves the theta-gene intact. CONCLUSIONS: The compound interaction of a ß-thalassemia defect along with a single functional α-globin gene is quite rare. Although patients with HbH/ß-thal and simple HbH disease have comparable levels of Hb, the absence of free ß-globin chains and thus detectable non-functional HbH means that in HbH/ß-thal, the levels of functional Hb are higher, resulting in a better compensated functional anemia. Rare large deletions as the one described here remain undetected by gap-PCR in routine molecular screening. The introduction of MLPA as a diagnostic screening tool may improve laboratory diagnostics for these defects. The use of NimbleGen fine-tiling arrays may give additional information about the precise location of breakpoints.

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.

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.

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.

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.

Known and new delta globin gene mutations and their diagnostic significance.

Mutations in the delta-globin gene (HBD, MIM# 142000) are not pathologically relevant. However, since high HbA2 levels are diagnostic for beta-thalassemia trait and a lowered level for an alpha- or delta-mutation, co-inheritance of delta- and beta-gene defects may lead to misinterpretation of diagnostic results. We examined 29 cases with low HbA2 level diagnosed in our laboratory, in the presence or absence of a second HbA2 fraction. We found a delta globin gene mutation in 20 cases. In total four different known mutations were found, three structural and one expressional. Moreover, two new defects were observed, one causing a structural abnormality and one a beta-thalassemia. The structural abnormality HBD c.431A->G (p.His144Arg)(dcd 143 CAC->CGC) was homologous to the beta-globin gene variant called Hb-Abruzzo and we have named this mutation HbA2 -Abruzzo. The new delta-thalassemia defect HBD c.-118C->T (d -68 C->T) has no homology on the beta-globin gene (HBB, MIM# 141900). All mutations caused a low HbA2 level and through this could lead to misdiagnosis when inherited together with a beta-thalassemia.

Hb Bronovo, a new globin gene mutation at alpha2 103 (His->Leu) associated with an alpha thalassemia phenotype.

Mild alpha-thalassemia, a common condition in many ethnic groups, presents with hematologic abnormalities almost identical to those found in iron deficiency. We report a new alpha globin chain variant associated with an alpha-thalassemia phenotype in two members of a Turkish family.

Beta thalassemia IVS-I-5(G-->C) heterozygosity masked by the presence of HbJ-Meerut in a Dutch-Indian patient.

We describe the genotype/phenotype correlation in a 35 year old anemic female referred to our laboratory because a fast eluting minor fraction on HPLC, mild hemolysis and hematological parameters suggesting a Thalassemia trait, eventually in combination with iron depletion. Direct sequencing of the alpha globin genes revealed heterozygosity for HbJ-Meerut, a Glu-->Ala substitution at residue 120 not justifying the hematological parameters. No other point mutations were found on the alpha genes and Gap-PCR excluded the 6 common deletion defects. Direct sequencing of the beta-globin genes revealed the IVS-I-5 (G-->C) transversion in absence of the elevated HbA2 levels usually measured in carriers of this beta-Thalassemia mutation. The HbA2 tetramer in the presence of HbJ-Meerut divides in two parts. One alphaN2/delta2 migrating on the right spot on HPLC. The other alphaJ2/delta2 migrating under the HbA fraction. Classic alkaline electrophoresis and the modern capillary electrophoresis CE showed these two tetramers and the reduction of the elevated HbA2 level of the beta-Thalassemia trait by at least 20% due to HbA2 Meerut.

A new polyadenylation site mutation associated with a mild beta-thalassemia phenotype.

At least 180,000 autochthonous and allochthonous people are carriers of a large spectrum of hemoglobinopathies in The Netherlands. We describe two cases, the first, a 33-year old Surinamese Creole woman, studied because of an intermediate hemolytic anemia; the second, a couple requesting analysis because of a previously diagnosed carrier state in the male partner, while the carrier state in the pregnant female was uncertain.

Hb Zoetermeer: a new mutation on the alpha2 gene inducing an Ala-->Ser substitution at codon 21 is possibly associated with a mild thalassemic phenotype.

A 52-year-old Dutch male was referred to our laboratory for hemoglobinopathy analysis because of persistent microcytic hypochromic parameters and moderate erythrocytosis in the absence of iron deficiency. The hemoglobin (Hb) pattern was normal and breakpoint polymerase chain reaction (PCR) excluded the six common deletion defects of the alpha gene cluster. Direct sequencing revealed a GCT-->TCT transversion at codon 21 of the alpha2 gene generating an Ala-->Ser single amino acid substitution. The hematological parameters observed in the presence of this mutation are consistent with a compensated heterozygous alpha(+)-thalassemia (thal). However, the neutral mutation and the external position of the residue do not explain an association with this phenotype. Nevertheless, we cannot exclude that the mutation could induce the observed hematological abnormalities and could eventually be considered as a mutation associated with a mild alpha-thalassemic phenotype.

The rare Hb Showa-Yakushiji [beta110(G12)Leu-->Pro, CTG-->CCG] in combination with an alpha gene triplication found in a Dutch patient during her first pregnancy examination.

We report a semi dominant beta-thalassemia (thal) phenotype caused by the rare Hb Showa-Yakushiji [beta110(G12)Leu-->Pro, CTG-->CCG] mutation in combination with an alpha gene triplication. This combination of two rare mutations was observed during hemoglobinopathy carrier diagnostics in a 26-year-old Dutch female at 9 weeks gestation, at the first pregnancy examination in the midwives practice. The partner was promptly examined and no abnormalities were found. The beta-thal trait was diagnosed by a standard high performance liquid chromatography (HPLC) procedure showing a normal separation but an elevated Hb A(2) level of 5.9% in the presence of pronounced hypochromic microcytic parameters and mild chronic hemolysis. Direct sequencing of the beta-globin genes was subsequently performed revealing a CTG-->CCG transition at codon 110. This rare mutation was previously described as two independent events in a few Japanese and Indian individuals. The mutation induces a Leu-->Pro substitution and the gene product is highly unstable. Gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the alpha gene triplication as well. The excess of alpha-globin chains contributed only marginally to the hematological abnormalities of the patient and did not aggravate the phenotype to an intermediate level.

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.

Hb St. Jozef, A Val-->Leu N-terminal mutation leading to retention of the methionine, and partial acetylation found in the globin gene in Cis with a -alpha3.7 thalassemia deletion.

We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion.

Ggamma -37 (A-->T): a new nondeletional hereditary persistence of fetal hemoglobin determinant associated with the rare codon 91 (+T) delta0-thalassemia.

We recently described a rare frameshift mutation in the delta-globin gene in a Dutch patient, in association with a new mutation of the Ggamma-globin gene promoter [Ggamma -37 (A-->T)] with a moderately elevated Hb F level of 2.3%. The delta mutation at codon 91 (+T) has been described once before in our laboratory in 1989, in a complex Belgian family with Ggamma (Agammadeltabeta)0-thalassemia (thal) and moderately elevated Hb F levels, without the Ggamma (Agammadeltabeta)0-thal deletion in some individuals. Analysis of the patients from 1989 revealed the presence of the same Ggamma-globin gene mutation and moderately elevated Hb F in all patients, who were also carriers of the delta-globin gene frameshift. Further analysis demonstrated that the two mutations were in linkage with the same haplotype in both the Belgian family and the recently found patient, confirming the association of the elevated Hb F expression with the new Ggamma-globin gene mutation.

Hb Bleuland [alpha108(G15)Thr-->Asn, ACC-->AAC (alpha2)]: a new abnormal hemoglobin associated with a mild alpha-thalassemia phenotype.

We report a new structural defect of the alpha2-globin chain, not detectable on high performance liquid chromatography (HPLC) or electrophoresis, characterized in a 12-year-old boy of Surinamese-Hindustani origin. The child was suspected to be a carrier of alpha-thalassemia (thal) because of microcytic hypochromic parameters in the absence of iron depletion. Gap-polymerase chain reaction (gap-PCR) revealed only normal fragments in the proband, and the pattern of a -alpha4.2 (leftward) deletion in his father and sister. Direct sequencing of the alpha-globin genes revealed an ACC-->AAC transversion at codon 108 of the alpha2-globin gene in the proband, in his mother and in a younger sister. The new mutation predicts a Thr -->Asn amino acid substitution at the corresponding residue. Threonine, a covalent binder with an R-active OH group, situated in the G helix of the alpha-globin chain, is involved in alpha1beta1 contacts. Asparagine, being an equally covalent binder but with a different R-active H2N-C=O group, could make the mutated chain less suitable for tetramer cooperation. Alternatively, an absent or reduced interaction with the alpha hemoglobin (Hb) stabilizing protein (AHSP) could lead to loss of alpha chains. Hb Bleuland is the first mutation described at codon 108 and is therefore interesting in regard to the possible effects and genetic risk. The nearest variant, Hb Suan-Dok [alpha109(G16)Leu -->Arg, CTG-->CGG (alpha2)] was originally observed in a Thai patient affected with Hb H, in combination with an alpha0-thal allele. The same Hb Suan-Dok mutation, recently described in our laboratory in a carrier of African ancestry, was also not detectable as a protein and presented with an alpha-thal phenotype similar to Hb Bleuland.

Alpha-thalassemia phenotype induced by the new IVS-II-2 (T --> A) splice donor site mutation on the alpha2-globin gene.

We present a family of North European extraction referred for a refractory non iron depleted microcytic anemia. The proband, a 36 year-old male, presented with chronic borderline anemia and microcytic hypochromic parameters. No abnormal hemoglobin (Hb) fractions were observed on high performance liquid chromatography (HPLC) or on alkaline electrophoresis. Gap-polymerase chain reaction (gap-PCR) excluded the seven common alpha-thalassemia (thal) deletion defects. However, the beta/alpha-globin chain synthesis ratio measured in vitro was unbalanced, indicating a reduced expression of the alpha-globin genes. Direct sequencing of the alpha-globin genes revealed heterozygosity for a T --> A transversion at the IVS-II-2 position of the alpha2 gene. This is the first IVS-II splice donor site mutation described on the alpha2-globin gene.

Hb Amsterdam [alpha32(B13)Met--Ile (alpha2)]: a new unstable variant associated with an alpha-thalassemia phenotype and a new African polymorphism.

We have characterized a new abnormal hemoglobin (Hb) at position 32 of the alpha-globin chain. The proband, a 38-year-old woman of Surinamese Black ancestry, was referred to the Academic Hospital in Amsterdam, The Netherlands, after 3 years of Prednisone treatment in Surinam. Kidney failure was diagnosed at the Nephrology Department, Free University Medical Center, Amsterdam, The Netherlands; the cortisone treatment was interrupted and dialysis was started. At this stage, a microcytic hypochromic anemia was observed with high reticulocyte (40%) and ferritin (500 microg/L) levels, and hemoglobinopathy was suspected. No abnormal bands were visible on alkaline electrophoresis and high performance liquid chromatography (HPLC). The Hb A2 level was normal (2.7%) and the erythrocyte count was low (3.59 x 10(12)/L) with a normal haptoglobin level (68 mg/100 mL). None of the common alpha-thalassemia (thal) deletion defects were present. The beta-globin gene sequence was normal but the alpha2-globin gene sequence revealed an ATG-->ATA transition at codon 32, changing the methionine into an isoleucine residue. The mutation, called Hb Amsterdam, was observed in the mother of the proband, who was also heterozygous for the--alpha3.7-thal deletion and affected by a moderate microcytic hypochromic anemia. Both Hb Amsterdam and the--alpha(-3.7) allele were found in association with a new polymorphism, IVS-I-39 (C-->T), previously observed in our laboratory in seven patients of African origin, on both the alpha1 and alpha2 genes. In addition, Hb Amsterdam was also associated with the common African alpha2 polymorphism (G-->CTCGGCCC at position 7238 and T-->G at position 7174). Hb Amsterdam is the first mutation ever described at codon alpha32, a position involved in alpha1/beta1 interaction. The possibility of a contribution of this mutation to the nephropatic state of the proband is discussed.

Hb Oegstgeest [alpha104(G11)Cys-->Ser (alpha1)]. A new hemoglobin variant associated with a mild alpha-thalassemia phenotype.

A microcytic hypochromic anemic state was observed in an 8-year old Black female of Surinam origin during pre-operative Hb S [beta6(A3)Glu-->Val] screening. Her high zinc protoporphyrin (ZPP) level suggested a chronic iron depletion but, in contrast, the high red blood cell (RBC) count (5.85 x 10(12)/L) was indicative of a possible coexisting thalassemia. No abnormal hemoglobin (Hb) bands were present on high performance liquid chromatography (HPLC) or alkaline electrophoresis and the Hb A2 level was normal. Break point polymerase chain reaction (PCR) failed to reveal any of the common alpha-thalassemia (thal) mutations but selective DNA sequencing of both alpha-globin genes disclosed a TGC-->AGC transversion at codon 104 of the alpha1 gene. Cystine at codon 104 is involved in alpha/beta globin contact and has been described to be a critical amino acid of the alpha2 chain when substituted by a tyrosine (Hb Sallanches), inducing Hb H (beta4) disease in the homozygous state. Our heterozygous patient had a moderate anemia of 12.2 g/dL and a borderline haptoglobin suggesting some degree of hemolysis.