MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype-phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C-terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD.
Inherited thrombocytopenias are heterogeneous diseases caused by at least 20 genes playing different role in the processes of megakaryopoiesis and platelet production. Some forms, such as thrombocytopenia 4 (THC4), are very rare and not well characterized. THC4 is an autosomal dominant mild thrombocytopenia described in only one large family from New Zealand and due to a mutation (G41S) of the somatic isoform of the cytochrome c (CYCS) gene. We report a novel CYCS mutation (Y48H) in patients from an Italian family. Similar to individuals carrying G41S, they have platelets of normal size and morphology, which are only partially reduced in number, but no prolonged bleeding episodes. In order to determine the pathogenetic consequences of Y48H, we studied the effects of the two CYCS mutations in yeast and mouse cellular models. In both cases, we found reduction of respiratory level and increased apoptotic rate, supporting the pathogenetic role of CYCS in thrombocytopenia.
© F e r r a t a S t o r t i F o u n d a t i o napproximately 80% of FA patients worldwide, will not be successful in at least 20%. Secondly, there is a wide spectrum of mutations, mainly private, spanning the entire genes, including intragenic deletions, as frequently observed in FANCA (http://www.rockefeller.edu/fanconi/). Since mutational screening would largely benefit from a preliminary knowledge of the candidate gene, complementation by cell fusion or by viral transduction, as well as protein analyses, have been established as screening strategies to identify the putative gene that is defective. 6,7 Thirdly, a false negative or inconclusive chromosomal breakage test might occur in patients who develop hematopoietic mosaicism due to reversion of the cellular FA phenotype. 8,9 This phenomenon arises if a spontaneous genetic event reconstitutes normal protein activity of one allele. Since it is unlikely that the same somatic event will also occur in primary skin fibroblasts, these cells are often used for mutational screening.In this paper, we report the molecular data of 100 FA families enrolled into the National Network of the Marrow Failure Study Group of the Italian Association of Pediatric Hematology and Oncology. This cohort consists of 76 new families, as well as 24 families that have been described in previous reports. 10,11 Methods Patients, cell lines, and DNA samplesPatients with a positive chromosomal breakage test were included in this study for molecular screening of FA genes by the National Network of the Marrow Failure Study Group of the Italian Association of Pediatric Hematology and Oncology. All the subjects or their legal guardians gave written informed consent to the investigation, according to the Declaration of Helsinki. Protocols were approved by the ethics review boards of the institutions that enrolled the patients. DNA was extracted from peripheral blood, lymphoblastoid cell lines and/or primary fibroblasts. Complementation and Western blot analysesLymphoblast cell lines, peripheral blood T lymphocytes or primary fibroblasts were transduced with retroviral expressing the cDNAs for FANCA, FANCC or FANCG as previously reported. 6 Complementation was considered to occur when the viability of the transduced cells increased by more than 20% that of controls at at least three different mitomycin C concentrations. Western blot analysis of FANCD2 using a monoclonal antibody (Santa Cruz, CA, USA; diluted 1:500) was performed as previously described. 10 Sequencing analysis and multiplex ligation-dependent probe amplificationThe coding exons of the FA genes and their flanking regions were sequenced using a set of oligonucleotides (the primer sequences are available upon request) according to standard procedures. 10 Six samples were first analyzed using the Ion PGM TM system for next generation sequencing of the 16 FA genes according to the manufacturer's protocols (Life Technologies). Variants with minor allele frequency less than 1% were then confirmed by Sanger sequencing as above. N...
Metformin (MET) is the drug of choice for patients with type 2 diabetes and has been proposed for use in cancer therapy and for treating other metabolic diseases. More than 14,000 studies have been published addressing the cellular mechanisms affected by MET. However, several in vitro studies have used concentrations of the drug 10-100-fold higher than the plasmatic concentration measured in patients. Here, we evaluated the biochemical, metabolic, and morphologic effects of various concentrations of MET. Moreover, we tested the effect of MET on Fanconi Anemia (FA) cells, a DNA repair genetic disease with defects in energetic and glucose metabolism, as well as on human promyelocytic leukemia (HL60) cell lines. We found that the response of wild-type cells to MET is concentration dependent. Low concentrations (15 and 150 µM) increase both oxidative phosphorylation and the oxidative stress response, acting on the AMPK/Sirt1 pathway, while the high concentration (1.5 mM) inhibits the respiratory chain, alters cell morphology, becoming toxic to the cells. In FA cells, MET was unable to correct the energetic/respiratory defect and did not improve the response to oxidative stress and DNA damage. By contrast, HL60 cells appear sensitive also at 150 μM. Our findings underline the importance of the MET concentration in evaluating the effect of this drug on cell metabolism and demonstrate that data obtained from in vitro experiments, that have used high concentrations of MET, cannot be readily translated into improving our understanding of the cellular effects of metformin when used in the clinical setting.
Fanconi anemia is a rare disease characterized by congenital malformations, aplastic anemia, and predisposition to cancer. Despite the consolidated role of the Fanconi anemia proteins in DNA repair, their involvement in mitochondrial function is emerging. The purpose of this work was to assess whether the mitochondrial phenotype, independent of genomic integrity, could correlate with patient phenotype. We evaluated mitochondrial and clinical features of 11 affected individuals homozygous or compound heterozygous for p.His913Pro and p.Arg951Gln/Trp, the two residues of FANCA that are more frequently affected in our cohort of patients. Although p.His913Pro and p.Arg951Gln proteins are stably expressed in cytoplasm, they are unable to migrate in the nucleus, preventing cells from repairing DNA. In these cells, the electron transfer between respiring complex I–III is reduced and the ATP/AMP ratio is impaired with defective ATP production and AMP accumulation. These activities are intermediate between those observed in wild-type and FANCA−/− cells, suggesting that the variants at residues His913 and Arg951 are hypomorphic mutations. Consistent with these findings, the clinical phenotype of most of the patients carrying these mutations is mild. These data further support the recent finding that the Fanconi anemia proteins play a role in mitochondria, and open up possibilities for genotype/phenotype studies based on novel mitochondrial criteria.
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