Molecular genetics and pathogenic mechanisms of hereditary anemias due to altered permeability of erythrocyte membrane

Genetic defects of erythrocyte transport proteins cause disorders of red blood cell volume that are characterized by abnormal permeability to the cation and, consequently, by changes in red cell hydration. Within this group of hereditary anemias we focused on familial pseudohyperkalemia and dehydrated hereditary stomatocytosis. The main aims of the project thesis are to study both the molecular genetics and the pathogenic mechanisms of these two disorders. Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced ‘passive leak’ of red cell K+ into plasma. The causative gene of this condition is ABCB6, encoding an erythrocyte membrane ABC transporter protein bearing the Langereis blood group antigen system. Dehydrated hereditary stomatocytosis (DHS) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Red cells are characterized by cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHS cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. Our study started with the recruitment of 97 patients affected by both FP and DHS from 41 unrelated families of Italian and foreign countries. Regarding familial pseudohyperkalemia, analyzing three new families, we reported the first functional characterization of ABCB6 mutants, including homozygous mutation V454A, heterozygous mutation R276W, and compound heterozygous mutations R276W and R723Q. All these mutations are annotated in public databases, suggesting that FP could be common in the general population. Indeed, we identified variant R276W in one of 327 random blood donors (0.3%). Measurement of cation flux demonstrated greater loss of K+ or Rb+ from HEK-293 cells expressing ABCB6 mutants than from cells expressing ABCB6 WT. The R276W/R723Q mutations elicited greater cellular K+ efflux than did the other mutants tested. Regarding dehydrated hereditary stomatocytosis by whole exome sequencing analysis of two previously undiagnosed DHS families we identified the second causative gene of DHS, the KCNN4 gene, encoding the Gardos channel (KCa3.1), the erythroid Ca2+-sensitive K+ channel of intermediate conductance. We characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of hematopoietic progenitor cell CD34+ and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development. Finally, we demonstrated that the mutations in KCNN4, as for PIEZO1, cause a gain of function, by increasing potassium efflux. Moreover, by analysing the genotype of the patients here collected, we characterized a new interesting mutation in PIEZO1, that is a duplication of two aminoacids localized in the pore of the channel, found in two families with different phenotype. We further analysed the modified effect of an additional PIEZO1 missense variant carried by the family exhibiting the more severe phenotype. We found that the missense variant co-inherited with the duplication cause an augmented potassium efflux. In conclusion, ABCB6 missense mutations in FP erythrocytes show elevated K+ efflux. The patients are present at moderate frequency in the blood donor population. Storage of blood of these patients leads to significantly increased K+ levels, with serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Genetic tests for FP could be added to blood donor pre-screening. Further study of ABCB6 function and trafficking could be informative for the study of other pathologies of red blood cell hydration. The identification of KCNN4 mutations in DHS patients supports recent studies that indicate it plays a critical role in normal erythrocyte deformation in the microcirculation and participates in maintenance of erythrocyte volume homeostasis. The characterization of PIEZO1 and KCNN4 mutations in DHS has contributed to the understanding of DHS pathogenesis that will be useful for the prognosis, the management, the follow-up, and the treatment of these patients

MicroRNAs and Cancer Stem Cells in Medulloblastoma

In this chapter, we are describing the biology of medulloblastoma influenced by several genes/pathways which concur to its pathogenesis. Of note, several levels of regulation are mediated by miRNAs functions, which we dissect their “state of art” to underline their crucial roles on controlling cancer development. In brain tumours, literatures data, are supporting the values of Cancer Stem Cells (tumor propagating cells) and their functions for tumour recurrence for future therapeutic treatments. Thus, we link the potential use of miRNAs as “shuttle” to impair Cancer Stem Cells in medulloblastoma

The frameshift Leu220Phefs*2 variant in KRIT1 accounts for early acute bleeding in patients affected by cerebral cavernous malformation

Abstract Background and Objectives Cerebral cavernous malformation (CCM) is a neurovascular disease characterized by abnormally expanded and tortuous microvessels with increased predisposition to thrombosis and focal hemorrhage. Its incidence is estimated to range between 0.4% and 0.8%. Sporadic and familial forms of CCM are described. The first one is characterized by single lesion, while the familial form is defined by multiple malformations. In this scenario, more than 300 mutations affecting the CCM genes have been described to date, but the exact pathogenic mechanism is yet unknown. Most of the causative variants of KRIT1 gene are frameshift but there are many missense and nonsense variants and they have been found some splicing mutations. The diagnosis is based on magnetic resonance images (MRI) and genetic testing. Case report A 15-year-old male presented with a two weeks duration worsening headache accompanied by vomiting and three months behavioral changes. Computer tomography revealed a large right temporal lesion with other smaller in left parietal and left cerebellar region. At the time of diagnosis, the two siblings of the proband were asymptomatic. Nevertheless, four months later, the 7-years-old brother was admitted to the emergency room for balance deficit, diplopia, right-hitting nystagmus and stiff neck with deviation of the head. A cerebral CT revealed polylobate hyperdense mass of the middle cerebral pedicle associated to acute bleeding. A genetic testing for hereditary cavernous brain malformation was carried out. Results The molecular analysis identified a 2-bp duplication (NM_194456.1:c.658_659dupTT) as heterozygous within the exon 8 of CCM1/KRIT1 gene (Fig. 1C). This duplication leads to a frameshift variant, resulting in a premature stop codon (p.Leu220Phefs*2). Discussion The clinical data collected confirm the variable phenotypic expression of CCM and suggest a greater severity of symptoms in the youngest patients

Characterization of Two Cases of Congenital Dyserythropoietic Anemia Type I Shed Light on the Uncharacterized C15orf41 Protein

CDA type I is a rare hereditary anemia, characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in one of the two genes: (i) CDAN1, encoding Codanin-1, which is implicated in nucleosome assembly and disassembly; (ii) C15orf41, which is predicted to encode a divalent metal ion-dependent restriction endonuclease with a yet unknown function. We described two cases of CDA type I, identifying the novel variant, Y94S, in the DNA binding domain of C15orf41, and the H230P mutation in the nuclease domain of the protein. We first analyzed the gene expression and the localization of C15orf41. We demonstrated that C15orf41 and CDAN1 gene expression is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Moreover, we functionally characterized the two variants, establishing that the H230P leads to reduced gene expression and protein level, while Y94S induces a slight decrease of expression. We demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. However, no altered nuclear-cytosolic compartmentalization of mutated C15orf41 was observed. Both mutants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle in these cells. Our functional characterization demonstrated that the two variants have different effects on the stability of the mutated mRNA, but both resulted in impaired erythroid maturation, suggesting the block of cell cycle dynamics as a putative pathogenic mechanism for C15orf41-related CDA I

PIEZO1 Hypomorphic Variants in Congenital Lymphatic Dysplasia Cause Shape and Hydration Alterations of Red Blood Cells

PIEZO1 is a cation channel activated by mechanical force. It plays an important physiological role in several biological processes such as cardiovascular, renal, endothelial and hematopoietic systems. Two different diseases are associated with alteration in the DNA sequence of PIEZO1: (i) dehydrated hereditary stomatocytosis (DHS1, #194380), an autosomal dominant hemolytic anemia caused by gain-of-function mutations; (ii) lymphatic dysplasia with non-immune fetal hydrops (LMPH3, #616843), an autosomal recessive condition caused by biallelic loss-of-function mutations. We analyzed a 14-year-old boy affected by severe lymphatic dysplasia already present prenatally, with peripheral edema, hydrocele, and chylothoraces. By whole exome sequencing, we identified compound heterozygosity for PIEZO1, with one splicing and one deletion mutation, the latter causing the formation of a premature stop codon that leads to mRNA decay. The functional analysis of the erythrocytes of the patient highlighted altered hydration with the intracellular loss of the potassium content and structural abnormalities with anisopoikolocytosis and presence of both spherocytes and stomatocytes. This novel erythrocyte trait, sharing features with both hereditary spherocytosis and overhydrated hereditary stomatocytosis, complements the clinical features associated with loss-of-function mutations of PIEZO1 in the context of the generalized lymphatic dysplasia of LMPH3 type

Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue

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The metallophosphodiesterase Mpped2 impairs tumorigenesis in neuroblastoma

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Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of \u3b2-thalassemia

Anemia of \u3b2-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of \u3b2-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free \u3b1-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2\u3b1 phosphorylation. The improvement of \u3b2-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of \u3b2-thalassemia

Common variants at 21q22.3 locus influence MX1 and TMPRSS2 gene expression and susceptibility to severe COVID-19

The established risk factors of coronavirus disease 2019 (COVID-19) are advanced age, male sex and comorbidities, but they do not fully explain the wide spectrum of disease manifestations. Genetic factors implicated in the host antiviral response provide for novel insights into its pathogenesis. We performed an in-depth genetic analysis of chromosome 21 exploiting the genome-wide association study data, including 6,406 individuals hospitalized for COVID-19 and 902,088 controls with European genetic ancestry from the COVID-19 Host Genetics Initiative. We found that five single nucleotide polymorphisms within TMPRSS2 and near MX1 gene show associations with severe COVID-19. The minor alleles of the five SNPs correlated with a reduced risk of developing severe COVID-19 and high level of MX1 expression in blood. Our findings demonstrate that host genetic factors can influence the different clinical presentations of COVID-19 and that MX1 could be a potential therapeutic target