Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel β-globin mutation

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with β-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the β-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality

A Family Affected by a Life-Threatening Erythrocyte Defect Caused by Pyruvate Kinase Deficiency With Normal Iron Status: A Case Report

Background: Red cell pyruvate kinase deficiency (PKD) is a defect of glycolysis causing congenital non-spherocytic hemolytic anemia. PKD is transmitted as an autosomal recessive trait. The clinical features of PKD are highly variable, from mild to life-threatening anemia which can lead to death in the neonatal period. Most patients with PKD must receive regular transfusions in early childhood and as a consequence suffer from iron overloading. Patient: Here, we report a Polish family with life-threatening hemolytic anemia of unknown etiology. Whole exome sequencing identified two heterozygous mutations, c.1529 G > A (p.R510Q) and c.1495 T > C (p.S499P) in the PKLR gene. Molecular modeling showed that the both PKLR mutations are responsible for major disturbance of the protein structure and functioning. Despite frequent transfusions the patients do not show any signs of iron overload and hepcidin, a major regulator of iron uptake, is undetectable in their serum. The patients were homozygous for the rs855791 variant of the TMPRSS6 gene which has earlier been shown to down-regulate iron absorption and accumulation. Conclusion: The lack of iron overload despite a reduced level of hepcidin in two transfusion-dependent PKD patients suggests the existence of a hepcidin-independent mechanism of iron regulation preventing iron overloading

Two novel C-terminal frameshift mutations in the β-globin gene lead to rapid mRNA decay

BACKGROUND: The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. METHODS: Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. RESULTS: We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. CONCLUSION: Our results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway

Coexistence of Gilbert syndrome with hereditary haemolytic anaemias.

Genotyping of the UGT1A1 gene showed distinct distribution of the common A(TA)(n)TAA polymorphism relative to other European populations. Because of a greater risk of hyperbilirubinaemia due to hereditary haemolytic anaemia, the diagnosis of Gilbert syndrome in this group of patients is very important

Modern diagnosis of thalassemia in children — own experience

Wstęp: Talasemie to grupa genetycznie uwarunkowanych niedokrwistości hemolitycznych, spowodowanych ilościowym zaburzeniem syntezy łańcuchów globiny, zaliczana do grupy hemoglobinopatii ilościowych. Niniejsza praca, jako jedna z niewielu w polskim piśmiennictwie, zwraca uwagę na problem występowania talasemii w populacji dzieci polskich. Celem pracy była ocena poszczególnych parametrów hematologicznych w rozpoznaniu różnicowym niedokrwistości mikrocytowych u dzieci, określenie roli badań genetycznych w rozpoznawaniu talasemii u dzieci oraz opracowanie optymalnego algorytmu postępowania diagnostycznego przy podejrzeniu talasemii. Materiał i metody: Badaniami objęto grupę 52 dzieci (31 chłopców i 21 dziewcząt) w wieku od 1–15 lat bezskutecznie leczonych preparatami żelaza z powodu niedokrwistości mikrocytowej. Badanie uzyskało zgodę Komisji Bioetycznej WUM Nr 65/2007. Po analizie wywiadu i podstawowych badań hematologicznych u wszystkich chorych wykonano elektroforezę hemoglobin, oznaczenie poziomu HgbA2 i HgbF, w trzech przypadkach oznaczenie enzymów krwinek czerwonych, w dwóch badanie w kierunku sferocytozy wrodzonej. Ponadto u wszystkich pacjentów wykonano badania genetyczne w Zakładzie Genetyki PAN w Warszawie. Wyniki: Spośród 52 dzieci objętych badaniem u 29 rozpoznano talasemię-b minor. U 28 wystarczające okazały się rutynowe badania diagnostyczne oraz wykonanie elektroforezy hemoglobin i oznaczenia stężeń HgbA2 i HgbF. U jednego pacjenta rozpoznanie ustalono po badaniu genetycznym. U 6 dzieci dzięki badaniom genetycznym rozpoznano talasemię-a minor. U 17 dzieci pomimo badań genetycznych nie udało się ustalić rozpoznania. U 6 z nich badanie wykazało mutację promotora genu g globiny (Xmn1) odpowiedzialną za podwyższenie stężenia HgbF. Badanie pozwoliło na stworzenie algorytmu postępowania diagnostycznego u chorych na niedokrwistość mikrocytową. Wnioski: Z uwagi na dużą liczbę fałszywie dodatnich i ujemnych wyników, wskaźniki matematyczne nie wydają się mieć wielkiego znaczenia w diagnostyce mikrocytozy. Erytrocytoza, mikrocytoza, obniżenie MCH przy prawidłowych parametrach gospodarki żelazem, krwinki tarczowate w rozmazie są wskazaniem do przeprowadzenia diagnostyki w kierunku talasemii-α, rzadszych postaci talasemii-β lub ich współwystępowania. Rola badań genetycznych w diagnostyce talasemii a jest nieoceniona, choć nie zawsze daje ostateczną odpowiedź. J. Transf. Med. 2011; 3: 105–114Background: There has been a consensus that thalassemias are typical of the Mediterranean region, North Africa, and Far and Middle Asia. However, recent progress in diagnostic methods has led to the detection of thalassemia in Poland. Routinely employed diagnostics often turn out insufficient to diagnose thalassemia, substantiating the need to use modern molecular biological techniques. This paper aimed at assessing the utility of some so far infrequently employed molecular genetic tests for facilitating diagnosis of rare non-typical cases of thalassemia in Polish children. Against this background, the significance of hematological parameters in the differential diagnosis of microcytic anemias was evaluated. Based on the above, an attempt has been made to establish an optimal algorithm for diagnosis of suspected thalassemia. Material and methods: The study comprised a group of 52 children (31 boys and 21 girls) at the age of 1–15 years, who were subjected to long-term treatment with iron preparation due to microcytic anemia, with no subsequent improvement. A permission of the local Bioethical Committee was obtained for this study. After taking the history, basic hematologic tests were performed. The tests were then extended to electrophoretic separation of hemoglobins, determination of HgbA2 and HgbF. In three cases erythrocyte enzymes were tested, and in two examination towards hereditary spherocytosis was carried out. Finally, genetic tests were performed. Results: Of the 52 children examined, 29 cases were diagnosed as b-thalassemia minor. Routine diagnostic tests (blood morphology, serum iron and ferritin, hemoglobin elctrophoresis, HgbA2 and HgbF assay) were enough to ascertain diagnosis in 28 children. Diagnosis of 1 case was only possible on the basis of genetic tests. Alpha-thalassemia minor was diagnosed in 6 children, and the diagnosis was only possible due to genetic tests. No diagnosis was ascertained in 17 children. Within this group, in 6 children only mutation in the g globin gene promoter region (Xmn1) was held responsible for increased HgbF. Conclusions: Considering numerous false positive and false negative results, mathematical indexes as the initial screening in microcytic anemia seem not to have significant impact on differential diagnosis. Genetic tests play essential role in diagnosing thalassemia although they are not always effective. Changes such as erythrocytosis, microcytosis, hypochromy as well as the presence of shield erythrocytes in peripheral blood smears indicating to do examinations. We presented the practical diagnostic algorithm in microcytosis. J. Transf. Med. 2011; 3: 105–11

Erythropoietin — the alternative treatment of anemia in infants

Niedokrwistość to jeden z najczęstszych problemów hematologicznych u niemowląt. Okresem szczególnej podatności na tę chorobę są pierwsze 3 miesiące życia, w których może się ona rozwijać na podłożu tak zwanej fizjologicznej niedokrwistości. Grupą niemowląt o zwiększonym ryzyku niedokrwistości są wcześniaki oraz niemowlęta urodzone o czasie, które są obarczone dodatkowymi czynnikami pogarszającymi parametry czerwonokrwinkowe, takimi jak konflikt serologiczny, przebycie zakażenia czy utrata krwi w okresie okołoporodowym. Wśród czynników determinujących wystąpienie niedokrwistości w pierwszych miesiącach życia kluczową rolę odgrywa niedobór erytropoetyny (Epo). Do niedawna jedynym dostępnym sposobem leczenia opisanych pacjentów były uzupełniające transfuzje koncentratu krwinek czerwonych (kkcz). Od lat 90. XX wieku leczeniem, które w wielu przypadkach może stanowić alternatywę dla kkcz, stała się ludzka rekombinowana erytropoetyna (rHuEpo). Powstałe w ciągu 20 lat bogate piśmiennictwo, dokumentujące liczne wieloośrodkowe badania, potwierdza wysoką skuteczność i bezpieczeństwo rHuEpo w terapii niemowląt z niedokrwistością. Hematologia 2011; 2, 1: 71–82Anemia is one of the most common hematological problems in infants. The first 3 months of life are the period of special susceptibility to anemia development, because it may occur on the basis of a physiological decrease in morphological parameters of blood. The risk group of anemia are premature infants and term infants with additional complaints which have a negative influence on hematological parameters, for instance presence of serological incompatibility, infection or bleeding at birth or just after birth. Reduced production of erythropoietin has a key role in determining the occurrence of anemia the decrease in blood morphology in the first 3 months of life. Up to the recent years the only available treatment for this type of anemia was supplementary transfusion of packed red blood cells. Since the 1990s recombinant human erythropoietin (rHuEpo) has, in many cases, become an alternative treatment in such cases. The rich literature, created on the basis of researches carried out in many centres all over the world, confirms high effectiveness and safety of rHuEpo in the treatment of anemia in the first 3 months of life. Hematologia 2011; 2, 1: 71–8

Additional file 1: Table S1. of Two novel C-terminal frameshift mutations in the β-globin gene lead to rapid mRNA decay

Sequences of the primers. (DOCX 13 kb