More than structural haemoglobin variants have been describe
More than 900 structural haemoglobin variants have been described. Of these more than 100 show high oxygen affinity . Erythrocytosis caused by Hb Ypsilanti was first described in 1967 , and the mutation results in substitution of amino raas inhibitors (β99 Asp→Tyr) involved in the stability of the quaternary structure of the haemoglobin tetramer [2,4]. Hb Ypsilanti is unusual, because the tetramer of the liganded form of Hb Ypsilanti is more stable than the deoxy tetramer. Hb Ypsilanti also exhibits large quaternary enhancement effect, where the binding of all 4 ligands is greater than for the isolated α1β1 dimers .
Since 2008, diagnostic make-up of patients presenting with polycythaemia includes examination for the JAK2 V6217F/exon 12 mutations . The JAK2 V617F mutation is present in more than 95% of patients with polycythaemia vera (PV) and in the small group of patients without this clonal marker a variant mutation in exon 12 is almost always found . Furthermore endogenous erythroid colony (EEC) formation in vitro may be a criterion of PV .
Cases In 2003 a 51-year-old woman (patient 1) was referred due to a B-Hb up to 21g/dl and a haematocrit of 0.67. The patient presented with symptoms of a (transient) cerebral ischaemic attack. Magnetic resonance imaging of the cerebrum showed no apoplexy but hydrocephalus. The patient was later treated with a ventriculo-peritoneal shunt. In 2007 her daughter, a 24-year-old woman, (patient 2), was referred for evaluation due to headaches for 3 weeks and an elevated B-Hb (16g/dl). Both patients were considered as atypical PV (Table 1). From the time of diagnosis the patients have been treated with venesectio, when the haematocrit was above 0.42, or the patient experienced symptoms such as itching and sweating – and almost always followed by a subjective relief and benefit. Patient 1 also received acetylsalicylate 75mg daily and later marcoumar after multiple pulmonary embolisms due to immobilisation after surgery. Patient 2 had no thromboembolic complications. She has received acetylsalicylate prophylaxis, and had two uneventful pregnancies and deliveries. In 2008 and 2010 the JAK2 V617F and exon 12 mutation analyses were performed, and showed a wild type status in both patients. EEC was found normal by in vitro analysis (Table 1). Three other family members have been tested for erythrocytosis, which was not observed (Fig. 1). Further follow-up is not possible due to lack of contact, death or very young age. The two children of patient 2 have not had haemoglobin measured at birth. Besides, for patient 1 thrombosis have occurred in at least 2 other family members and one has been treated with anticoagulants (cause unknown) (Fig. 1). In both patients reported here we have reduced the venesectio frequency, allowing a B-Hb higher in the normal range.
Methods Haemoglobin fractions were obtained by liquid chromatography on a Waters UPLC (Waters, Milford, MA, USA) by using a Cation-exchange PolyCat column (35×4.6mm, 3µm, 1500Å) (PolyLC Inc., Columbia, MD, USA). Genomic DNA was purified from leucocytes using the QIAamp DNA Blood Mini QIAcube Kit (Qiagen, Hilden, Germany) according to the manufacturer′s instructions. Part of the β-globin gene was amplified using the forward primer A (5′–ATA TCT TAG AGG GAG GGC–3′) and the reverse primer B (5′–CCC ATT CTA AAC TGT ACC–3′). Platinum®Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer’s instructions for polymerase chain reaction (PCR) amplification. The following conditions were used: denaturation for 120s at 95°C, 32 cycles of 30s at 9°C, 40s at 60°C and 60s at 72°C followed by a final elongation of 7min at 72°C. The PCR product was purified using QIAquick (Qiagen) PCR purification kit according to the manufacturer’s instructions. Sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) and subsequently purified using the BigDye® XTerminator purification kit according to the manufacturer′s instructions. Primers for sequencing were primers A and B. Sequencing was performed on an Applied Biosystems 3500 Genetic Analyser (Life Technologies, Carlsbad, CA, USA).