Device profile of the Inspiris Resilia valve for aortic valve replacement: overview of its safety and efficacy.

INTRODUCTION: The Inspiris Resilia tissue valve was recently introduced into clinical practice. This review summarizes the pre-clinical and clinical studies leading to this new bioprosthesis. AREAS COVERED: The novel Resilia tissue was tested extensively in a large animal model. The clinical use of the tissue started in 2011 with the European Feasibility study, followed by a North-American multi-center study. Since 2017, the Inspiris Resilia valve has been in full commercial use. Further prospective evaluations and registries are ongoing. EXPERT OPINION: The Inspiris Resilia valve was clinically introduced after pre-clinical tests revealed superiority compared to contemporary therapy such as the Perimount valve. Prospective long-term follow-up studies on Resilia are ongoing since 2011 and reveal no major complications. Full 5-year data show no signs of early degeneration, but longer follow-up is certainly still needed. Several prospective registries are actively monitoring the outcome with the Inspiris Resilia valve now. The novel tissue, designed to mitigate calcification and increase durability, together with the expandable stent, facilitating potential future valve-in-valve (ViV) procedures, are the cutting-edge aspects. Clinical use in younger patients is currently ongoing: their follow-up and outcome will determine the added value of this valve.

Congenital deficiency of vitamin K-dependent coagulation factors and protein C.

A 15-month-old girl from Coimbra (Portugal) had a history of numerous hemorrhagic episodes with multiple bruises, hematomas but not hemarthroses. On serial testing she showed deficiency of factors II, VII, IX, X and protein C. Malabsorption-induced vitamin K deficiency, liver disease or ingestion of a coumarin compound were excluded. An absence of detectable abnormalities was found among her relatives. Consanguinity was not present. The immunologic assay, immunoelectrophoresis or antibody neutralization, revealed much higher levels of these factors than the clotting assay. The non-physiological activator (Echis carinatus venom) produced higher levels of prothrombin activation than those detected by physiological activation. Two-dimensional immunoelectrophoresis of the patient's plasma in calcium showed that prothrombin had the same mobility as acarboxyprothrombin. No significant response to large doses of intravenous vitamin K3 (6 mg) was observed. Transfusion of 120 ml of frozen fresh plasma led to an immediate increase in the procoagulant activities of vitamin K dependent protein, similar to that found after perfusion of plasma plus vitamin K3. The results obtained from this patient suggest a defect in the gammacarboxylation mechanism inside the hepatocyte.

Serial plasmapheresis in a haemophiliac with antibodies to FVIII.

Serial plasmaphereses were performed on a 23-year-old haemophiliac, with antibodies to factor VIII (FVIII), in order to reduce the antibody level before multiple dental extractions. Eight phereses were carried out in which approximately 1.5 litres plasma was exchanged with isotonic saline. By the ninth exchange, which was carried out immediately before the extraction, the antibody level had fallen from 4 u/ml to 0.8 u/ml. On this occasion fresh frozen plasma and FVIII concentrate were infused immediately after the pheresis. A normal post-infusion level of FVIII was achieved with a normal half disappearance time of the infused FVIII.

[Type-II dyserythropoietic anemia. A partial form of the glycoprotein degradation syndrome?]. / Anemia deseritropoiética tipo II. Forma parcial do síndrome de degradação das glicoproteínas?

The authors present a case of a boy, aged 8 years and 11 months, yellow race, with dyserythropoietic anemia type II, diagnosed at two months of age. Screening for partial form of carbohydrate deficient glycoprotein syndrome was normal. This result did not confirm the publication by Fukuda in 1990.

Congenital anemias in Macau.

The implementation of a Primary Health Care system in Macau has created the need to study the prevalence of Congenital Anemias in the population to facilitate the planning of measures needed for their control. Blood samples from 3815 women attending the antenatal clinics were screened for beta-thalassemia using MCH values as the preliminary test, followed by quantitation of Hb A2 and Hb F. Isoelectric-focusing was used to screen 1091 cord blood samples for the presence of Hb Bart's. The prevalence of alpha- and beta-thalassemia carriers was found to be 6.2% and 3.6%, respectively.

Acquired von Willebrand syndrome with inhibitors both to factor VIII clotting activity and ristocetin-induced platelet aggregation.

A case of acquired von Willebrand's syndrome (vWs) is described which appeared to be due to antibodies directed against factor VIII clotting activity (FVIIIC), factor VIII-related antigen (FVIIIRAg) and von Willebrand factor. The antibodies directed against FVIIIRAg was demonstrated by the inhibitory effect of a platelet eluate on Ristocetin-induced aggregation of normal platelets. This effect was not shown by the patient's platelet-poor plasma alone, nor could it be demonstrated in platelet eluates from 13 other patients who had antibodies to FVIIIC but in whom there was no evidence of an acquired vWs.

PK-LR gene mutations in pyruvate kinase deficient Portuguese patients.

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK-LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G --> C on exon 12 and two 5' splice donor site (GT) mutations on intron 8 [IVS8(+2)T --> G] and intron 10 [IVS10(+1)G --> C]. Two previously described missense mutations, 1456C --> T and 993C --> A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G --> C or IVS8(+2)T --> G.

Beta-thalassemia mutations in the Portuguese; high frequencies of two alleles in restricted populations.

We report the characterization of seven different beta-thalassemia mutations in 131 newly diagnosed Portuguese beta-thalassemia heterozygotes. Methodology included the detection of abnormal fragments by agar gel electrophoresis of PCR-amplified DNA fragments after digestion with specific restriction endonucleases, as well as hybridization with synthetic nucleotide probes and sequencing of amplified DNA. Four mutations, including the newly discovered TGG-->TGA change at codon 15, occurred in excess of 10% and accounted for some 90% of the beta-thalassemia alleles in this population. The geographical distribution is uneven; the TGG-->TGA mutation at codon 15 was primarily observed in the coastal region north of Lisbon, while the IVS-I-6 (T-->C) mutation was confined to the central part of the country.

A variant of spectrin low-expression allele alpha LELY carrying a hereditary elliptocytosis mutation in codon 28.

Allele alpha LELY is a low-expression allele of the erythroid spectrin alpha-gene. It carries mutations in exon 40 (alpha V/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of alpha-chains by beta-chains, and would eventually account for the low-expression character. When it occurs in trans to an alpha-allele responsible for hereditary elliptocytosis (alpha HE allele; alpha HE/alpha LELY diplotype), allele alpha LELY enhances the severity of elliptocytosis. Because allele alpha LELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele alpha LELY will be designated alpha HE-LELY allele. The HE component was the known alpha 28 Arg-->His mutation. This alpha HE-LELY allele was investigated within the alpha HE-LELY/alpha LELY diplotype, a diplotype not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the alpha LELY component in cis of the alpha HE mutation counteracted the like component in trans.

Beta + thalassemia--Portuguese type: clinical, haematological and molecular studies of a newly defined form of beta thalassaemia.

We have characterized 14 patients in 10 families with a mild form of homozygous beta thalassemia which has not been previously well defined. As these patients originate from a small area of northern Portugal we propose to call this beta + thalassaemia--Portuguese type. Clinically, the homozygotes range from asymptomatic to thalassaemia intermedia and they are characterized by low levels of HbF, less than 20%, indicating only a mild deficit in beta globin production. Heterozygotes are indistinguishable from those with the more common types of beta thalassaemia as regards red cell morphology, haemoglobin analysis and globin chain synthesis studies. Globin gene mapping excluded the presence of alpha thalassaemia in these patients and demonstrated no abnormalities in the beta-like globin gene cluster. Restriction enzyme site polymorphisms around the beta gene cluster are identical on both chromosomes in all of the homozygotes, confirming their homogeneity.

Dominant beta-thalassaemia trait in a Portuguese family is caused by a deletion of (G)TGGCTGGTGT(G) and an insertion of (G)GCAG(G) in codons 134, 135, 136 and 137 of the beta-globin gene.

We have studied a Portuguese family with a dominant beta-thalassaemia trait that was present in one member of each of three generations. It was characterized by a moderate anaemia, microcytosis and hypochromia, anisopoikilocytosis, Heinz body formation in peripheral red cells, splenomegaly, and a blood transfusion requirement during pregnancy. Sequence analyses of amplified DNA detected a deletion of (G) TG.GCT.GGT.GT(G) at codons 134-137 (Val.Ala.Gly.Val) and the insertion of (G)GC.AG(G) (Gly.Arg) at the same location. Thus, the resulting beta chain has an abnormal structure only at codons 134-137 and is two residues shorter than the normal 146 residues. This chain could not be detected in circulating red cells and must be degraded rapidly by proteolysis because the Heinz bodies consisted mainly of alpha chains.

Hb Hekinan observed in three Chinese from Macau; identification of the GAG----GAT mutation in the alpha 1-globin gene.

Hb Hekinan, an alpha chain variant that is characterized by a Glu----Asp mutation at position alpha 27, was observed in three Chinese females attending a prenatal clinic in Macau. The relative quantities of the stable hemoglobin were 13-14% (average 13.3%); its identification was greatly aided by the separation and purification of the peptides by reversed phase high performance liquid chromatography. Dot-blot analysis of amplified DNA with 32P-labeled probes located the mutation in codon 27 of the minor alpha 1-globin gene; the change involved a GAG (coding for glutamic acid) to GAT (coding for aspartic acid) mutation.

Hb Coimbra or alpha 2 beta (2)99(G1)Asp----Glu, a newly discovered highoxygen affinity variant.

We have identified a new high oxygen affinity hemoglobin variant in members of a Portuguese family; it is characterized by an Asp----Glu replacement at codon 99 of the beta chain which is in the alpha 1 beta 2 interface. The altered functional properties of Hb Coimbra likely result from the inability to form a hydrogen bond between beta 99Glu and alpha 42Tyr; such a bond is formed in deoxy Hb A between the normally occurring beta 99Asp and alpha 42Tyr. The two affected members of the family have a distinct erythrocytosis with hemoglobin levels of 18 to 20 g/dl. The mutation in the beta-globin gene (GAT----GAA at codon 99) resulting in the Asp----Glu replacement is the seventh type at this specific location. A review of the many variants of the alpha and beta chains identifies primarily aspartic acid and glutamic acid residues as being most frequently replaced; it is speculated that codons GAC and GAT (for Asp), and GAG and GAA (for Glu) are most susceptible to mutational events.

Molecular heterogeneity underlying the G6PD Mediterranean phenotype.

As part of a study aiming to define the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency, we analysed a sample from a Portugese boy with a family history of favism. Although the biochemical properties of red-cell G6PD from this subject were similar to those of the common variant G6PD Mediterranean, the corresponding mutation (563 C----T) was not present. Instead, polymerase chain reaction (PCR) amplification and sequencing of the entire gene detected a C----T transition at nucleotide 592 in exon VI, changing an arginine residue to a cysteine residue only 10 amino acids downstream from the Mediterranean mutation. Single-strand conformation polymorphism analysis of a PCR-amplified DNA fragment spanning exons VI and VII of the G6PD gene has detected the same mutation, confirmed by sequencing, in a G6PD-deficient patient from Southern Italy. We name this new variant G6PD Coimbra.

Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure.

Early biochemical studies defined 4 functional domains of the erythroid protein 4.1 (4.1R). From amino-terminal to carboxy-terminal, these are 30 kd, 16 kd, 10 kd, and 22/24 kd in size. Although the functional properties of both the 30-kd and the 10-kd domain have been demonstrated in red cells, no functional activities have been assigned to either the 16-kd or the 22/24-kd domain in these cells. We here describe new mutations in the sequence encoding the C-terminal 22/24-kd domain that are associated with hereditary elliptocytosis. An unusually mild phenotype observed in heterozygous and homozygous members of 1 family suggested heterogeneity in the pattern of expression of 4.1R deficiency. Using a variety of protein and messenger RNA (mRNA) quantification strategies, we showed that, regardless of the alteration in the C-terminal primary sequence, when the protein is produced, it assembles at the cell membrane. In addition, we found that alterations in red cell morphologic features and membrane function correlate with the amount of membrane-associated protein-and therefore with the amount of mRNA accumulated-rather than with the primary structure of the variant proteins. These data suggest that an intact sequence at exons 19 through 21 encoding part of the C-terminal 22/24-kd region is not required for proper protein 4.1R assembly in mature red cells. (Blood. 2000;95:1834-1841)

A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency.

Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.

Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency.

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5' splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor-like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the triangle upEGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.

[Immunologic and enzymatic markers of acute infantile lymphoblastic leukemias]. / Marcadores immunológicos y enzimáticos de las leucemias linfoblásticas agudas infantiles.

Twenty-one childhood acute lymphoblastic leukemias were characterized immunologically by a series of classic markers--EAET rosette and surface and cytoplasm immunoglobulins--, a panel of 14 monoclonal antibodies --OKla 1, OKT3, 4, 6, 8, 9, 10, 11, cALLA, Leu-1, FMC-7, FMC-8 and 3A-1--and by assessment of terminal deoxynucleotidyl transferase enzyme. Patients were distributed in five immunological subgroups: 1) Unclassifiable/null ALL (la +, TdT+, cALLA-), one patient, 2) common ALL (la+, TdT+, cALLA+) 15 patients, 3) pre-B ALL (la+, TdT+, cALLA+, clg+) one patient, 4) B-LL (la+, TdT-, cALLA-, slg+) 3 patients, 5) T-ALL (TdT+, EAET+, la-, cALLA-), one patient, and the clinical, biological, morphological and cytochemical peculiarities characterizing each immunological variant are discussed.

Severe hereditary spherocytosis and distal renal tubular acidosis associated with the total absence of band 3.

Absence of band 3, associated with the mutation Coimbra (V488M) in the homozygous state, caused severe hereditary spherocytosis in a young child. Although prenatal testing was made available to the parents, it was declined. Because the fetus stopped moving near term, an emergency cesarean section was performed and a severely anemic, hydropic female baby was delivered. She was resuscitated and initially kept alive with respiratory assistance and hypertransfusion therapy. Cord blood smears revealed erythroblastosis, poikilocytosis, and red cells with stalk-like elongations. Band 3 and protein 4.2 were absent; spectrin, ankyrin, and glycophorin A were significantly reduced. Renal tubular acidosis was detected by the age of 3 months. Nephrocalcinosis appeared soon thereafter. After 3 years of follow-up the child is doing reasonably well on a regimen that includes regular blood transfusions and daily bicarbonate supplements. The long-term prognosis remains uncertain given the potential for hematologic and renal complications. (Blood. 2000;96:1602-1604)

Modulation of clinical expression and band 3 deficiency in hereditary spherocytosis.

We present two novel alleles of the anion-exchanger 1 (AE1) gene, allele Coimbra and allele Mondego. Allele Coimbra (V488M, GTG --> ATG) affects a conserved position in the putative second ectoplasmic loop of erythrocyte band 3. In 15 simple heterozygotes, it yielded a mild form of hereditary spherocytosis (HS) with band 3 deficiency (-20% +/- 2%) and a reduced number of 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonate (H2DIDS) binding sites (-35%). However, two additional heterozygotes presented with an aggravated HS and a more pronounced reduction of band 3 (-40%) and of H2DIDS binding sites (-48%). They carried, in trans to allele Coimbra, allele Mondego, defined by two mutations: E40K, GAG --> AAG, the known mutation Montefiore, and P147S, CCT --> TCT, a novel mutation, both located in the cytoplasmic domain of band 3. Allele Mondego itself resulted in no clinical or hematologic HS signs in the simple heterozygous state. Yet it yielded a slight decrease in band 3 (-6% to -12%) and in the number of H2DIDS binding sites (-19%). Thus, the more pronounced decrease in band 3 in the two compound heterozygotes derived from the additive effects of two unequally expressed AE1 alleles, resulting in a more severe clinical picture.