Three Haemoglobin Variants for Congenital Erythrocytosis and Sickle Cell Disease HbSC

The inherited haemoglobin disorders and the clinical laboratory is essential in the diagnosis. Phlebotomy is the therapeutic procedure indicated to reduce blood viscosity and decreasing the risk of thrombotic events in the haemoglobin variants with high affinity for O2 or vaso-occlusive crises in Sickle Cell Disease (SCD) with double heterozygosity HbSC. We describe how therapeutic bleeding remains the treatment in two variant haemoglobinopathies, Haemoglobin Regina and the Haemoglobin Trollhattan, reported for the first time in the Italian population and a double heterozygosity HbSC. In patients with high haematocrit level when other secondary conditions of polyglobulia are excluded, a High-Performance Liquid Chromatography (HPLC) analysis for haemoglobin study should be suggested. Also in children with erythrocytosis frequently, such as the finding of Haemoglobin Okazaky.

The inherited haemoglobin disorders, including thalassemias and haemoglobinopathies, are the most common genetic diseases and the clinical laboratory is essential in the diagnosis. Haemoglobinopathies are monogenic disorders in the genes that encode globin chains that can lead to a defective globin production or a variant in the haemoglobin structure.

The replaced amino acid nature and its location are responsible for the stability, solubility and function of the molecule that could lead to clinical manifestations. Some haemoglobinopathies are clinically silent and are discovered accidentally [1]. Congenital erythrocytosis is a clinical manifestation that can be found in patients with structural haemoglobin variants with a high oxygen affinity [2]. Otherwise, some structural alterations of the haemoglobin such as in the Sickle Cell Disease (SCD), in particular in the double heterozygosity HbSC, could have a high haematocrit level with increased risk of haemoglobin polymerization and subsequently falcemic crisis.

Phlebotomy is the therapeutic procedure highly indicated to reduce blood viscosity and improve clinical conditions, decreasing the risk of thrombotic events in the hemoglobin variants with high affinity for O2 or vaso-occlusive crises in SCD with double heterozygosity HbSC.

In this report we describe three different haemogobinopathies found during a haematologic counselling, two of them with high oxygen affinity [3] and one double heterozygosity HbSC disease that needed phlebotomy treatments to reduce hyperviscosity [4]. The rarity of these haemoglobinopaties in our country proved that a HPLC analysis for haemoglobin study should be required in the diagnostic tests of patients with high haematocrit level in absence of other secondary conditions of polyglobulia.

Hb setting tests performed in chromatography by High Performance Liquid Chromatography (HPLC) β Thal. Short, Variant II Bio-Rad.

Molecular genetic analysis in Devyser Thalassemia kit, NGS sequencing by HBA1 (7 amplicons), HBA2 (7 amplicons) Hemoglobin Genes Amplicon Library and HBBs (13 amplicons), including the promoter, exon and intron regions of these genes. In addition, another 18 amplicons on the Alpha-globin gene cluster and 10 on the Betaglobin gene cluster. Sequencing on Miseq platform.

The method allows the analysis of:

• Promoter, exon and intron regions of these genes.
• Large deletions/duplications (CNV) also with Direct Detection (DD) method analytical sensitivity and specificity of the test: > 99 %.

A 68 years old patient born in Italy was treated for a polyglobulia with quarterly phlebotomies of 450 ml. per year without a significant improvement of the hematocrit level. The patient was a non-smoker without symptoms of hyperviscosity. At the diagnosis the haemochromocytometric analysis showed haemoglobin 19.2 gr/dl, haematocrit 61.5%, normal levels of leucocytes and platelets. JAK2 test resulted negative whereas erythropoietin level was increased. Other cardiac, nephrological and pneumological causes were excluded. Iron balance was normal. The patient revealed that the mother was affected by an erythrocyte plethora that was never investigated in the past. Therefore at the counselling, an HPLC analysis was performed to investigate haemoglobin variants as a possible etiology. The HPLC chromatogram showed an abnormal peak in the P3 area of a 39.7%, HbA 48.8%, HbA2 2.6% and HbF less 0.8%. The HPLC result was suggestive of haemoglobinopathy and in subsequent molecular analysis of nucleotide sequence of the beta globin gene from nucleotide -190 to +530 and from +1120 to +1500; it revealed a mutation C > G at the nucleotide +469 (codon96), in a heterozygous state that was responsible for an amino acid substitution [ß 96 Leu > Val] characteristic of the variant Haemoglobin Regina.

Clinical Cases 2 & 3

Two brothers 57 and 58 years old born in Italy were blood donors in the past, but they were respectively deferred from the donation for repeated values of high haematocrit level. Other cardiac, nephrological and pneumological causes were excluded. Both brothers were non-smoker and were asymptomatic. They were introduced into a therapeutic phlebotomy treatment regime to reduce the haematocrit and put into an anti-aggregation therapy. The haemochromo showed erythrocytosis RBC 5.45 106/uL, haemoglobin 18-19 gr/dl, haematocrit of about 58%, normal levels of leucocytes and platelets. JAK2 analysis resulted negative. During counselling, they referred different personal histories of phlebotomy: the first was in therapy since 2004 with phlebotomies of 400 ml with a two months interval, while the second since 2006 with quarterly intervals. During familial anamnesis the patients reported a non-specific polyglobulia in the parents and daughter.

An HPLC analysis was performed and the chromatogram showed a haemoglobin variant represented by 40-44%, HbA2 2.3-2.2%, HbF < 1.0%. The chromatograms required identification by genetic testing that revealed heterozygosity for Haemoglobin Trollhattan. The analysis of the nucleotide sequence of the beta globin gene from nucleotide 190 to +530 and from +1120 to +1500 showed a mutation T > A at the nucleotide 112 (codon 20), that was responsible for an amino acid substitution [ß 20 Val > Glu].

Clinical Cases 4 & 5

Father and son 34 and 3 years old respectively. Presence of hemoglobin variant in heterozygosity, HbX 39.7% - HbA 48.7%, results in HPLC, HbA2 4.20 > %, HbF 0.20%. Family origin from Romania father and Neapolitan mother with normal hemoglobin structure. Blood tests son: MCV 79.6 fL, haemoglobin 13.5 gr/dL, erythrocytosis RBC 5.41 > 106/μL, haematocrit 43.1 > % in 2022 and 40.0 > % in 2021, possibly after fever episodes. Anamnesis: nocturnal snoring, in 2020 hospitalization for hypoxia with oxygen dependence for SpO2 max of 95% in AA. In 03/2022 hospitalization for suboptimal SpO2, mild hypercapnia EGAv, normal ECG and eco-cardio. Hospitalization 06/2022 for polysomnography, 96% SpO2 in AA, with a polygraphic score of mild OSAS in probable obstructive hypoventilation, due to adenoid hypertrophy. Father normal blood test and asymptomatic. The molecular hemoglobinopathies analysis shows in both cases, the HBB variant: c.280T > C p. (Cys94Arg) Haemoglobin Okazaky, in heterozygous carrier. Hb-Okazaky is described in the literature as a hemoglobin variant defined phenotypically silent, but unstable variant with increased affinity for oxygen.

Clinical Case 6

In 2013 a 29 year old young man born in Ghana was known to be affected by a sickle cell disease with double heterozygosity HbSC. He was admitted to the hospital six times in a year with a painful vaso-occlusive crisis, due to constant hyperviscosity, but in the absence of anemia and pathologies [5,6]. Due to an unacceptable response to hydroxyurea [7], he was maintained solely folic acid treatment. Prebleed values were: haemoglobin 14.5-16.0 g/dL, iron dosage normal. An HPLC study was performed to evaluate the pathologic haemoglobin. The chromatogram showed two haemoglobin variants HbS 46%, HbC 43.7%, HbA2 4%, HbF 0.9% and HbA 3% that did not need a molecular biology study. The patient started a treatment of phlebotomies of about 400 ml every three months to reduce blood viscosity achieving and maintaining haemoglobin value of 10-12 gr/dl. The clinical condition of the patient improved and no longer required hospital admission for pain due to vaso-occlusive crisis.

The HBA gene mutation is included and all patients tested negative for mutations in the HBA1-HBA2 alpha globin genes absent.

In our report we describe how therapeutic bleeding remains the treatment of choice to reduce hyperviscosity that manifests itself in pathological situations with different etiopathogenesis such as haemoglobinopathies.

Haemoglobin Regina and the Haemoglobin Trollhattan, first described in a Scandinavian family and in a Swedish one respectively, are identified among the variants of about 100 haemoglobins; they are characterized by a compensatory erythrocytosis of genetic origin, related to oxygen release by haemoglobin. To our knowledge this is the first report in the Italian population; individuals with haemoglobin disorders were observed in various countries, however finding them outside the first described geographic area shows that the people left the original regions for various reasons in the past [8-10].

The reduction of the haematocrit remains effective with phlebotomy. The experience of congenital erythrocytosis suggests that it is sufficient to reduce the haematocrit to 52% with quarterly blood drawning; in the case of comorbidity for thrombotic risk it is suggested to maintain the haematocrit value at 50%.

The first evidence of Haemoglobin Okazaky to 1984 taking name from the Japanese city of origin’s patient. Found in Italy in a Neapolitan family of Italian origin. The cases in literature present normal blood tests, even if the instability and affinity tests for O2 are positive [11-13].

Interestingly, in the patient with double heterozygosity HbSC the sickle cell crises are due to hyperviscosity in the absence of anemia: an unusual clinical picture compared to homozygous sickle cell disease (HBSS) in which phlebotomy therapy is necessary to maintain haemoglobin levels between 10-12 g/dL for reducing vasoocclusive crisis [14].

In these cases beyond phlebotomies also erythrocytapheresis techniques are to consider, such as Red Blood Cell (RBC) depletion, RBC exchange transfusions, or RBC depletion/exchange procedures. In particular RBC exchange transfusions are standard treatment in SCD patients with a history of or a risk for acute stroke and are clinical options for other acute complications of SCD, like the chest syndrome, serious clinical eventuality. Also about the double heterozygosity HbSC beyond phlebotomies for to reduce the sickle cell crises due to hyperviscosity, but it is recommended for risk stroke or chest syndrome or pregnancy to execute erythrocytapheresis for RBC depletion or RBC exchange transfusions, or RBC depletion/exchange procedures also manual with phlebotomy and transfusion subsequently.

To date patients with haemochromatosis are not denied as blood donors. Persons with high haematocrit levels and carrying a hemoglobin variant with a high oxygen affinity with congenital erythrocytosis are unlikely to be suitable for blood donation Indeed, they will not be able to maintain a good therapeutic haemaatocrit range with regular donation intervals of 90 days and it is strongly possible that transfusion of blood cells with hemoglobin variant having a high oxygen affinity would not have any clinical benefit.

Our report wants to focus the attention on these rare haemoglobinopaties in our country; a HPLC analysis for Haemoglobin study should be suggested in the diagnostic tests of patients with high haematocrit level when other secondary conditions of polyglobulia are excluded, Also in children with erythrocytosis frequently. The reduction of the haematocrit remains effective with phlebotomy, to think about in some genotypes of SCD like HbSC or in rare Haemoglobinopaties with high oxygen affinity.

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