Thalassemia major phenotype caused by HB Zürich-Albisrieden [α2 59(E8) Gly > Arg (HBA2:C.178G > C)] in a Brazilian child.

Hemoglobin (Hb) Zürich-Albisrieden (ZA) [α2 59(E8) Gly > Arg; HBA2:c.178G > C] is a rare and highly unstable α-chain variant. A few simple and compound heterozygotes (αZA α/αα and -/αZA α, respectively) have been described so far in Switzerland and China. We describe here a case of homozygosity for the Hb ZA mutation (αZA α/αZA α) in a Brazilian child with severe congenital hemolytic anemia and ineffective erythropoiesis.

Coinheritance of Hb Bristol-Alesha [ß67(E11)Val→Met; HBB: c.202G>A] and the α212 Patchwork Allele in a Brazilian Child with Severe Congenital Hemolytic Anemia.

Hb Bristol-Alesha [HBB: c.202G>A; ß 67 Val>Met] is a rare structural variant of hemoglobin (Hb) resulting from a GTG>ATG substitution at codon 67 of the ß-globin gene that leads to the replacement of valine by methionine in the corresponding position of the ß-globin chain. The methionine residue is subsequently modified to aspartic acid [ß67(E11)Val-Met→Asp], possibly by autoxidation mechanisms. This substitution prevents normal non-polar binding of Val67 to the heme group, resulting in molecular instability and severe hemolysis. We identified Hb Bristol-Alesha (in the heterozygous state), as the cause of severe congenital hemolytic anemia in an 11-month-old girl of mixed (native Indian and European) ethnic origin from the Midwestern region of Brazil, whose parents were clinically and hematologically normal. The mutation on the ß-globin gene was found to have been coinherited with the α212 patchwork allele.

Functional characterization of compound heterozygosity Hb S/Hb Deer Lodge in Brazil.

INTRODUCTION: The Hb Deer Lodge (ß2 His>Arg; HBB:c.8A>G) is a structural hemoglobin variant described in some populations around the world, characterized by increased oxygen affinity, but does not confer clinical symptoms to its carriers. The coinheritance of the Hb Deer Lodge with the most common hemoglobin variant, Hb S, has been reported only once; however, functional data were not described. Here we show a case of the Hb S and Hb Deer Lodge carrier in heterozygosity. METHODS: The Hb S and Hb Deer Lodge association was identified by High-Performance Liquid Chromatography (HPLC), reverse phase HPLC and the ß globin gene sequencing. The functional characterization of this interaction was obtained using the O2 dissociation curve, determination of the cooperativity between the globin chains and the Bohr effect in the presence and absence of organic phosphates. RESULTS: When the Hb S and Hb Deer Lodge were associated, there was a decrease in cooperativity, no significant changes in oxygen affinity and no significant Bohr effect changes. CONCLUSION: Despite these genetic variations, the carrier showed no hematological alterations and no clinical symptoms, possibly due to the high oxygen affinity of the Hb Deer Lodge, which interferes with the Hb S polymerization.

Hb S-São Paulo: a new sickling hemoglobin with stable polymers and decreased oxygen affinity.

Hb S-São Paulo (SP) [HBB:c.20A>T p.Glu6Val; c.196A>G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations.

Two new unstable haemoglobins leading to chronic haemolytic anaemia: Hb Caruaru [beta122 (GH5) Phe-->Ser], a probable case of germ line mutation, and Hb Olinda [beta22 (B4) - 25 (B7)], a deletion of a 12 base-pair sequence.

We describe here two new unstable beta-globin variants, Hb Caruaru and Hb Olinda, found in northeastern Brazil, both associated with chronic haemolytic anaemia. Haemoglobin Caruaru is caused by a single base substitution at codon 122 (TTC-->TCC), possibly originating from the germ line cells of the patient's grandmother. Haemoglobin Olinda is also a de novo mutation, caused by a 12 bp deletion leading to the removal of the 22nd to the 25th residues of the normal beta-globin chain.

EGFR Exon 20 Insertion Mutations Display Sensitivity to Hsp90 Inhibition in Preclinical Models and Lung Adenocarcinomas.

PURPOSE: EGFR exon 20 insertions account for up to 10% of all EGFR mutations in lung adenocarcinomas, representing the third most common cluster of mutations. The management of advanced cancers with these mutations remains elusive, without an approved inhibitor. EXPERIMENTAL DESIGN: Preclinical models of a representative set of EGFR exon 20 insertion mutations to evaluate the efficacy of different inhibitors and description of the clinical outcome of an advanced lung cancer. RESULTS: We show that select first-, second-, and third-generation EGFR inhibitors are unable to deter common EGFR exon 20 insertion mutants in concentrations that spare the wild-type kinase. Nonetheless, EGFR exon 20 insertion mutants associate with the Hsp90 chaperone system. We exploit this vulnerability to show that the nongeldanamycin Hsp90 inhibitor luminespib (formerly AUY922) degrades EGFR exon 20 mutations, downstream targets, and induces apoptosis. In addition, a patient whose EGFR inhibitor-insensitive lung adenocarcinoma harbored an EGFR exon 20 insertion mutation had a confirmed radiographic response to luminespib. CONCLUSIONS: The report confirms that EGFR exon 20 mutations are dependent on Hsp90 and are readily inhibited by the Hsp90 inhibitor luminespib; a treatment strategy that has been pursued in a confirmatory clinical trial (NCT01854034) for this group of lung adenocarcinomas that currently represent an unmet clinical need in precision oncology.

Pulse Afatinib for ERBB2 Exon 20 Insertion-Mutated Lung Adenocarcinomas.

INTRODUCTION: Genomic aberrations involving the erb-b2 receptor tyrosine kinase 2 gene (ERBB2) are driver oncogenes in approximately 2% of lung adenocarcinomas. However, the use of daily dosing of ERBB2 tyrosine kinase inhibitors (TKIs)-including afatinib-has been fraught with plasma concentrations that barely achieve preclinical model inhibition, significant patient-reported toxicities, and limited clinical activity. We hypothesized that alternative dosing strategies could improve tolerability and efficacy. METHODS: We profiled lung cancer cell lines against TKIs and retrospectively evaluated the toxicity of and response to pulse afatinib (280 mg once weekly) in lung cancers with ERBB2 mutations. RESULTS: An ERBB2 exon 20 insertion-mutated lung cancer cell line had a 50% inhibitory concentration in response to afatinib that was higher than the reported plasma concentration of afatinib, 40 mg daily. Three patients with advanced ERBB2-mutated lung adenocarcinomas were treated with off-label pulse afatinib. The 280-mg weekly dose was well tolerated with no reported rash and minimal diarrhea. One TKI-naive patient achieved a partial response for 5 months and another achieved stable disease for 11 months. CONCLUSIONS: Pulse afatinib at a weekly dosing scheme induced antitumor activity in ERBB2 exon 20 insertion-mutated lung adenocarcinomas. Future clinical trials of alternative dosing schemes of ERBB TKIs as monotherapy or in combination with other therapies are warranted for ERBB2-mutated tumors.

Responses to the multitargeted MET/ALK/ROS1 inhibitor crizotinib and co-occurring mutations in lung adenocarcinomas with MET amplification or MET exon 14 skipping mutation.

INTRODUCTION: Genomic aberrations involving ALK, ROS1 and MET can be driver oncogenes in lung adenocarcinomas. Identification of tyrosine kinase inhibitors (TKIs) with activity against these tumors and of preclinical systems to model response are warranted. METHODS: We analyzed cases with lung adenocarcinomas for representative genomic aberrations, evaluated the response to the multitargeted MET/ALK/ROS1 crizotinib TKI in cases with MET aberrations and profiled lung cancer cell lines with the aforementioned genomic changes. RESULTS: Lung cancer cell lines with ALK rearrangement, ROS1 rearrangement or MET amplification had expected in vitro responses to crizotinib and the ALK/ROS1 TKI ceritinib. However, a commercially-available cell line with MET exon 14 skipping mutation and co-occurring PIK3CA-p.Glu545Lys mutation did not respond to crizotinib; suggesting the latter abrogated response. 10% of MET exon 14 skipping mutation co-occurred with PIK3CA mutation in the TCGA cohort. Putative crizotinib-responsive somatic mutations (ALK rearrangements, ROS1 rearrangements, high level MET amplification or MET exon 14 skipping mutations) were present in 10% of lung adenocarcinomas analyzed at our service and in 9.5% of the TCGA lung adenocarcinoma database. One patient each whose advanced tumors harbored high level MET amplification with wild-type PIK3CA or MET exon 14 skipping mutation with PIK3CA-p.Glu542Lys had significant responses to crizotinib; suggesting that PIK3CA co-mutation did not affect clinical response. CONCLUSIONS: Approximately 10% of lung adenocarcinomas harbor aberrations that are targetable using the approved multitargeted TKI crizotinib. MET exon 14 skipping mutation predicts for response to MET TKIs in human lung adenocarcinomas but co-occurrence of PIK3CA mutation needs to be better evaluated as a modifier of response to TKI therapy. MET TKIs should not be omitted from MET exon 14 skipping mutated tumors until further preclinical and clinical data can confirm or refute mechanisms of primary or acquired resistance to crizotinib and other MET TKIs in these recalcitrant cancers.

A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation.

Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future.

Haptoglobin genotypes in Chagas' disease.

OBJECTIVES: To investigate the existence of an association between haptoglobin (Hp) genotypes and the severity of heart complications in Chagas' disease. DESIGN AND METHODS: Hp genotyping was performed by PCR in 107 Brazilian patients sub-classified in asymptomatic, with mild heart disease and with severe heart disease. RESULTS: Multiple logistic regression (R(2)=24%) indicated that patients with the Hp1-1 genotype have lower probability of developing the severe heart complications. CONCLUSION: The Hp polymorphism may influence the clinical evolution of Chagas' disease.

Hb H disease resulting from the association of an α-thalassemia allele [-(α)] with an unstable α-globin variant [Hb Icaria]: First report on the occurrence in Brazil.

Hb H Disease is caused by the loss or inactivation of three of the four functional α-globin genes. Patients present chronic hemolytic anemia and splenomegaly. In some cases, occasional blood transfusions are required. Deletions are the main cause of this type of thalassemia ( α-thalassemia). We describe here an unusual case of Hb H disease caused by the combination of a common α(0) deletion [-( α) (20.5) ] with a rare point mutation (c.427T > A), thus resulting in an elongated and unstable α-globin variant, Hb Icaria, (X142K), with 31 additional amino-acid residues. Very high levels of Hb H and Hb Bart's were detected in the patient's red blood cells (14.7 and 19.0%, respectively). This is the first description of this infrequent association in the Brazilian population.

Three new alpha-globin variants: Hb Itapira [alpha30(B11)Glu-->Val (alpha1)], Hb Bom Jesus Da Lapa [alpha30(B11)Glu-->Ala (alpha1)] and Hb Boa Esperança [alpha16(A14)Lys-->Thr (alpha2)].

Three novel alpha-globin variants were found during a screening program for hemoglobinopathies in blood donors at the UNICAMP Hematology and Hemotherapy Center, Campinas, State of São Paulo, Southeastern Brazil. They were named for the town of origin of the carrier as Hb Itapira [alpha30(B11)Glu-->Val], Hb Bom Jesus da Lapa [alpha30(B11)Glu-->Ala] and Hb Boa Esperança [alpha16(A14)Lys-->Thr]. Hb Itapira, like Hb Bom Jesus da Lapa, shows an electrophoretic mobility similar to that of Hb S [beta6(A3)GluVal, GAG-->GTG] at alkaline pH; it is associated with a triplicate alpha-globin allele (alphaalphaalpha(anti 3.7)) and corresponds to only 5.5% of the total hemoglobin (Hb). Hb Boa Esperança, found in two different individuals, moves faster than Hb A and exhibits an abnormal functional performance.

Hb H disease resulting from the association of an αº-thalassemia allele [-(α)20.5] with an unstable α-globin variant [Hb Icaria]: First report on the occurrence in Brazil

Hb H Disease is caused by the loss or inactivation of three of the four functional a-globin genes. Patients present chronic hemolytic anemia and splenomegaly. In some cases, occasional blood transfusions are required. Deletions are the main cause of this type of thalassemia (α-thalassemia). We describe here an unusual case of Hb H disease caused by the combination of a common αº deletion [-(α)20.5] with a rare point mutation (c.427T > A), thus resulting in an elongated and unstable α-globin variant, Hb Icaria, (X142K), with 31 additional amino-acid residues. Very high levels of Hb H and Hb Bart's were detected in the patient's red blood cells (14.7 and 19.0 percent, respectively). This is the first description of this infrequent association in the Brazilian population.

Identificação e caracterização de variantes novas e raras da hemoglobina humana: [revisão] / Identification of characterization of novel and rare variants of human hemoglobin: [review]

As anormalidades estruturais da hemoglobina estão entre as doenças genéticas mais comumente encontradas nas populações humanas. O Laboratório de Hemoglobinopatias do Departamento de Patologia Clínica da Faculdade de Ciências Médicas da Universidade Estadual de Campinas - Unicamp, localizado em Campinas, no estado de São Paulo, região Sudeste do Brasil, realizou, em seus 27 anos de existência, cerca de 130.000 diagnósticos. Entre as variantes estruturais detectadas, as hemoglobinas S, C e D-Punjab foram, como esperado, as mais freqüentes, porém um número expressivo de outras hemoglobinas anômalas, novas e raras, também foi encontrado. Esses achados estão sumarizados no presente artigo.

Hemoglobin structural abnormalities are among the most commonly found human genetic diseases. The Laboratory of Hemoglobinopathies in the Clinical Pathology Department of the Medical Sciences School of the State University in Campinas - Unicamp, São Paulo, Southeastern Brazil, carried out, in its 27 years of activity, about 130,000 diagnoses. As expected, hemoglobins S, C and D were the most frequently observed variants, but an expressive number of other abnormal, novel and rare hemoglobins, was also detected. These findings are summarized in the present article.