Complex Modes of Inheritance in Hereditary Red Blood Cell Disorders: A Case Series Study of 155 Patients.

Hereditary erythrocytes disorders include a large group of conditions with heterogeneous molecular bases and phenotypes. We analyzed here a case series of 155 consecutive patients with clinical suspicion of hereditary erythrocyte defects referred to the Medical Genetics Unit from 2018 to 2020. All of the cases followed a diagnostic workflow based on a targeted next-generation sequencing panel of 86 genes causative of hereditary red blood cell defects. We obtained an overall diagnostic yield of 84% of the tested patients. Monogenic inheritance was seen for 69% (107/155), and multi-locus inheritance for 15% (23/155). PIEZO1 and SPTA1 were the most mutated loci. Accordingly, 16/23 patients with multi-locus inheritance showed dual molecular diagnosis of dehydrated hereditary stomatocytosis/xerocytosis and hereditary spherocytosis. These dual inheritance cases were fully characterized and were clinically indistinguishable from patients with hereditary spherocytosis. Additionally, their ektacytometry curves highlighted alterations of dual inheritance patients compared to both dehydrated hereditary stomatocytosis and hereditary spherocytosis. Our findings expand the genotypic spectrum of red blood cell disorders and indicate that multi-locus inheritance should be considered for analysis and counseling of these patients. Of note, the genetic testing was crucial for diagnosis of patients with a complex mode of inheritance.

Acute human parvovirus B19 infection triggers immune-mediated transient bone marrow failure syndrome, extreme direct hyperbilirubinaemia and acute hepatitis in patients with hereditary haemolytic anaemias: multicentre prospective pathophysiological study.

A total of 244 patients with hereditary haemolytic anaemias (HHA) were screened for acute symptomatic human parvovirus B19 infection (HPV-B19) in a prospective study. To assess the risks associated with HPV-B19 infection, patients were classified into Group I and Group II according to presence or absence (symptoms, signs and specific serology) of acute HPV-B19 infection respectively. In all, 131 (53·7%) patients had ß-thalassaemia, 75 (30·7%) hereditary spherocytosis (HS), 27 (11·1%) sickle cell anaemia (SCA) and 11 (4·5%) glucose-6-phosphate dehydrogenase (G6PD) deficiency. Of 33 (13·5%) patients who presented with symptomatic HPV-B19 infection, 19 (57·5%) had HS, nine (27·3%) had ß-thalassaemia and five (15·2%) had SCA. In Group I, there were significant differences in the mean white blood cell, red blood cell and platelet counts, haemoglobin concentration, total bilirubin (TB), alanine aminotransferase, aspartate aminotransferase and serum creatinine (all P < 0·001) compared to Group II. In all, 27 (81·8%) patients had arthropathy and bone marrow failure (BMF); 13 (39·4%) had acute kidney injury (AKI), more in SCA (80%); and 12 (36·4%) patients had hepatitis, more in HS (66·8%). Five (15·2%) patients with HS had BMF, AKI, nervous system involvement and extreme hyperbilirubinaemia (TB range 26·3-84·7 mg/dl). Five (15·2%) patients had haemophagocytic syndrome. Two patients with HS combined with Type-I autoimmune hepatitis presented with transient BMF. Complete recovery or stabilisation was noted at 12 months in every patient except for one patient with SCA who died during the infection. HPV-B19 must be suspected and screened in patients with HHA with typical and atypical presentations with careful follow-up.

Hemolysis and Neurologic Impairment in PAMI Syndrome: Novel Characteristics of an Elusive Disease.

PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome is a rare early-onset autoinflammatory disease associated with various hematologic findings, including chronic neutropenia and pancytopenia. We report a unique case of PAMI syndrome in a toddler with transfusion-dependent hemolytic anemia, hepatosplenomegaly, failure to thrive, developmental delay, and multiple malformations. Because of acute inflammatory-driven decompensation, anakinra was started with dramatic improvement of both the hematologic and neurologic involvement. A customized next-generation sequencing panel later identified a de novo pathogenic variant in the PSTPIP1 gene, confirming the diagnosis. Our case illustrates the broad spectrum of phenotypes associated with PAMI syndrome, which should be considered in any case of unexplained cytopenias associated with autoinflammatory stigmata. It is also one of the few reports of neurologic involvement in PSTPIP1-associated inflammatory diseases. Increased awareness of this rare disease and early performance of genetic testing can correctly diagnose PAMI syndrome and prevent disease complications.

Characterization of hereditary red blood cell membranopathies using combined targeted next-generation sequencing and osmotic gradient ektacytometry.

Hereditary red blood cell (RBC) membranopathies are characterized by mutations in genes encoding skeletal proteins that alter the membrane complex structure. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to hereditary hemolytic anemia with a worldwide distribution and an estimated prevalence, in Europe, of about 1:2000 individuals. The recent availability of targeted next generation sequencing (t-NGS) and its combination with RBC deformability measured with a laser-assisted optical rotational ektacytometer (LoRRca) has demonstrated to be the most powerful contribution to lower the percentage of hereditary hemolytic anemia undiagnosed cases. In order to know the kind and frequency of RBC membrane mutations in our geographical area (Catalonia) and to better understand their pathophysiology, 42 unrelated, non-transfusion-dependent (NTD) patients with hereditary hemolytic anemia have been studied by combining t-NGS and LoRRca. The osmoscan module of LoRRca provides three rheological profiles that reflect the maximal deformability (EImax), osmotic fragility (Omin), and hydration state (Ohyper) of RBCs and contribute to a better understanding of the contribution RBC rheology to the severity of anemia. From the 42 patients studied, 37 were suspected to be a RBC membrane defect due to phenotypic characteristics and abnormal RBC morphology and, from these, in 31 patients (83.8% of cases) the mutation was identified by t-NGS. No definite diagnosis was achieved in 11 patients (26.2% of cases), including 6 out of 37 cases, with suspected membranopathy, and 5 with unclassifiable HHA. In all these undiagnosed patients, the existence of hemoglobinopathy and/or enzymopathy was ruled out by conventional methods.

Heme oxygenase-1 deficiency presenting with interstitial lung disease and hemophagocytic flares.

BACKGROUND: Heme oxygenase-1 (HMOX1) catalyzes the metabolism of heme into carbon monoxide, ferrous iron, and biliverdin. Through biliverdin reductase, biliverdin becomes bilirubin. HMOX1-deficiency is a rare autosomal recessive disorder with hallmark features of direct antibody negative hemolytic anemia with normal bilirubin, hyperinflammation and features similar to macrophage activation syndrome. Clinical findings have included asplenia, nephritis, hepatitis, and vasculitis. Pulmonary features and evaluation of the immune response have been limited. CASE PRESENTATION: We present a young boy who presented with chronic respiratory failure due to nonspecific interstitial pneumonia following a chronic history of infection-triggered recurrent hyperinflammatory flares. Episodes included hemolysis without hyperbilirubinemia, immunodeficiency, hepatomegaly with mild transaminitis, asplenia, leukocytosis, thrombocytosis, joint pain and features of macrophage activation with negative autoimmune serologies. Lung biopsy revealed cholesterol granulomas. He was found post-mortem by whole exome sequencing to have a compound heterozygous paternal frame shift a paternal frame shift HMOX1 c.264_269delCTGG (p.L89Sfs*24) and maternal splice donor HMOX1 (c.636 + 2 T > A) consistent with HMOX1 deficiency. Western blot analysis confirmed lack of HMOX1 protein upon oxidant stimulation of the patient cells. CONCLUSIONS: Here, we describe a phenotype expansion for HMOX1-deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares with hemophagocytosis present in the bone marrow.

Differential diagnosis of hereditary hemolytic anemias in a single multiscreening test by TGA/chemometrics.

A multi-screening test based on the coupling of thermogravimetry and chemometrics was optimized for the differential diagnosis of hereditary hemolytic anemias. The novel test is able to simultaneously perform a simple and fast diagnosis of sickle cell anemia, thalassemia, hereditary spherocytosis and hereditary elliptocytosis in a single analysis of a few microliters of non-pretreated whole blood. The thermogravimetric profile of blood from patients affected by such disorders was found to be characteristic of a specific anemic status or a disorder due to membrane defects. In addition, chemometric tools were used to validate a model of prediction to process the thermogravimetric curves and to obtain in 1 hour an accurate diagnosis. The effectiveness of the novel test was evaluated by comparing results with the confirmatory analyses specific for each disorder. The TGA/chemometric test made it possible to perform a first level test of congenital erythrocyte defects, including the hemoglobinopathies and disorders due to membrane defects with the same accuracy of confirmatory analyses obtained by molecular investigation. In addition, the novel test was used for the diagnosis of a number of Italian difficult cases, including neonatal patients for which the conventional screening tests did not manage to obtain a diagnosis confirming the high prediction ability of the single multiscreening test.

Mechanosensitive Piezo1 ion channel protein (PIEZO1 gene): update and extended mutation analysis of hereditary xerocytosis in India.

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.

Next-Generation Sequencing-Based Diagnosis of Unexplained Inherited Hemolytic Anemias Reveals Wide Genetic and Phenotypic Heterogeneity.

Determination of the cause of inherited hemolysis is based on clinical and stepwise conventional laboratory tests. Patients with obscure etiology require genetic diagnosis, which is time-consuming, expensive, and laborious, mainly because of numerous causal genes. This study enrolled 43 patients with clinical and laboratory evidence of unexplained hemolytic anemia. Initially, 13 patients were tested using a commercial (TruSight One) panel, and remaining cases underwent targeted sequencing using a customized 55-gene panel. Pyruvate kinase deficiency was found in eight, glucose-6-phosphate dehydrogenase (G6PD) deficiency in three (G6PD Guadalajara in two and p.Tyr227Ser: novel, named as G6PD Chandigarh), and glucose-6-phosphate isomerase (GPI) deficiency in two (GPI:p.Arg347His and p.Phe304Leu: novel, named as GPI Chandigarh). Three patients had Mediterranean stomatocytosis/macrothrombocytopenia, and two had overhydrated stomatocytosis. Xerocytosis was found in three patients, whereas six had potentially pathogenic variants in membrane protein-coding genes. Overall, 63% cases received a definite diagnosis. Timely determination of etiology was helpful in diagnosis, genetic counseling, and offering a prenatal diagnosis. Therapeutic implications include performing or avoiding splenectomy that may ameliorate the anemia in many but also predispose to thrombosis in other groups of patients. This first study on the genetic spectrum of unexplained hemolytic anemia from the Indian subcontinent also represents, currently, one of the largest cohort worldwide of such patients.

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

Xerocitosis hereditaria. Presentación de dos casos clínicos pediátricos / Hereditary xerocytosis. Presentation of two pediatric cases

La xerocitosis hereditaria es un desorden poco frecuente causado por defectos en la permeabilidad eritrocitaria, que se caracteriza por anemia hemolítica de gravedad variable y sobrecarga de hierro. El diagnóstico suele ser tardío y confundirse con otras anemias hemolíticas, lo que puede llevar a indicaciones de procedimientos, como la esplenectomía, contraindicados en estos pacientes. Se reportan las características clínicas, hematológicas y moleculares de dos pacientes pediátricos no relacionados con diagnóstico de xerocitosis hereditaria. Ambos presentaban eritrocitos deshidratados con alta concentración de hemoglobina corpuscular media, frotis no patognomónico, marcadores de hemólisis y una curva de fragilidad osmótica resistente. El diagnóstico se confirmó por la secuenciación del gen PIEZO.Se resalta la importancia de reconocer la causa de la anemia hemolítica para dar un enfoque terapéutico preciso y dar adecuado consejo genético

Hereditary xerocytosis is a rare disorder caused by defects of red blood cell permeability that are characterized by hemolytic anemia of variable degree and iron overload. Diagnosis is usually late and confused with other hemolytic anemias, which can lead to procedural indications, such as splenectomy, contraindicated in these patients. We report the clinical, haematological, and molecular characteristics of two patients from two unrelated families affected by hereditary xerocytosis. Both patients had dehydrated erythrocytes with a high concentration of mean corpuscular hemoglobin, non-pathognomonic smears, markers of hemolysis and a resistant osmotic fragility curve. The diagnosis was confirmed by the sequencing of the PIEZO gene. We emphasize the importance of recognizing the cause of hemolytic anemia to give an accurate therapeutic approach and give adequate genetic counseling.

Advances in understanding the pathogenesis of red cell membrane disorders.

Hereditary erythrocyte membrane disorders are caused by mutations in genes encoding various transmembrane or cytoskeletal proteins of red blood cells. The main consequences of these genetic alterations are decreased cell deformability and shortened erythrocyte survival. Red blood cell membrane defects encompass a heterogeneous group of haemolytic anaemias caused by either (i) altered membrane structural organisation (hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis and Southeast Asian ovalocytosis) or (ii) altered membrane transport function (overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis or xerocytosis, familial pseudohyperkalaemia and cryohydrocytosis). Herein we provide a comprehensive review of the recent literature on the molecular genetics of erythrocyte membrane defects and their reported clinical consequences. We also describe the effect of low-expression genetic variants on the high inter- and intra-familial phenotype variability of erythrocyte structural defects.

Anemia predicts lower white matter volume and cognitive performance in sickle and non-sickle cell anemia syndrome.

Severe chronic anemia is an independent predictor of overt stroke, white matter damage, and cognitive dysfunction in the elderly. Severe anemia also predisposes to white matter strokes in young children, independent of the anemia subtype. We previously demonstrated symmetrically decreased white matter (WM) volumes in patients with sickle cell disease (SCD). In the current study, we investigated whether patients with non-sickle anemia also have lower WM volumes and cognitive dysfunction. Magnetic Resonance Imaging was performed on 52 clinically asymptomatic SCD patients (age = 21.4 ± 7.7; F = 27, M = 25; hemoglobin = 9.6 ± 1.6 g/dL), 26 non-sickle anemic patients (age = 23.9 ± 7.9; F = 14, M = 12; hemoglobin = 10.8 ± 2.5 g/dL) and 40 control subjects (age = 27.7 ± 11.3; F = 28, M = 12; hemoglobin = 13.4 ± 1.3 g/dL). Voxel-wise changes in WM brain volumes were compared to hemoglobin levels to identify brain regions that are vulnerable to anemia. White matter volume was diffusely lower in deep, watershed areas proportionally to anemia severity. After controlling for age, sex, and hemoglobin level, brain volumes were independent of disease. WM volume loss was associated with lower Full Scale Intelligence Quotient (FSIQ; P = .0048; r2 = .18) and an abnormal burden of silent cerebral infarctions (P = .029) in males, but not in females. Hemoglobin count and cognitive measures were similar between subjects with and without white-matter hyperintensities. The spatial distribution of volume loss suggests chronic hypoxic cerebrovascular injury, despite compensatory hyperemia. Neurocognitive consequences of WM volume changes and silent cerebral infarction were strongly sexually dimorphic. Understanding the possible neurological consequences of chronic anemia may help inform our current clinical practices.

Erythrocyte ion content and dehydration modulate maximal Gardos channel activity in KCNN4 V282M/+ hereditary xerocytosis red cells.

Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.

Molecular diagnostic update in hereditary hemolytic anemia and neonatal hyperbilirubinemia.

Hereditary hemolytic anemia (HHA) is a group of genetically and phenotypically heterogeneous disorders characterized by premature destruction of red blood cells (RBCs) with clinical manifestations ranging from asymptomatic to marked hemolytic anemia. There are three main categories of HHA: (a) RBC membrane defects; (b) hemoglobinopathies/thalassemias; and (c) RBC enzyme deficiencies. Hyperbilirubinemia is a frequent consequence of hemolytic anemia and can lead to bilirubin-associated neurotoxicity in neonates and to jaundice, and formation of gall stones in adults. Hyperbilirubinemia can also be caused by impaired bilirubin conjugation due to polymorphisms and mutations in genes involved in bilirubin metabolism (eg, UGT1A1). Neonates with HHA and co-inherited variants impairing bilirubin conjugation are at increased risk of bilirubin-associated toxicity. Prior to the advent of next-generation sequencing (NGS), molecular diagnosis of these disorders was limited to targeted single gene Sanger sequencing. However, NGS is making its way into the standard diagnostic workup of complex and multigene disorders like HHA. This review will focus on the molecular updates of HHA with particular focus on the neonatal and pediatric population.

Iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation.

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 µg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.