Pharmacogenetic studies in children with acute lymphoblastic leukemia in Argentina

The aim of this study was to evaluate the influence of the most common genetic variants in methylenetetrahydrofolate reductase (MTHFR), thiopurine methyltransferase (TPMT) and glutathione-S-transferases (GSTs) on the outcome of acute lymphoblastic leukemia (ALL) treatment in Argentinean children. Two hundred and eighty-six patients with ALL treated with two Berlin–Frankfurt–Münster (BFM)-based protocols were analyzed. Ten genetic variants were studied. Toxicity was evaluated during the consolidation phase. Children who received 2 g/m2/day of methotrexate and carried at least one 677T allele in MTHFR showed an increased risk of developing severe leukopenia (p = 0.004) and neutropenia (p = 0.003). Intermediate-risk (IR) patients with a heterozygous TPMT genotype had a higher probability of event-free survival than those with a wild-type genotype. Genotyping of MTHFR polymorphisms might be useful to optimize consolidation therapy, reducing the associated severe hematologic toxicity. Further studies are necessary to establish the usefulness of MTHFR and TPMT variants as additional markers to predict outcome in the IR group.Fil: Aráoz, Hilda Veronica. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; ArgentinaFil: D'Aloi, Karina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); ArgentinaFil: Foncuberta, Maria Eugenia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; ArgentinaFil: Sanchez La Rosa, Christian German. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; ArgentinaFil: Alonso, Cristina Noemi. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; ArgentinaFil: Chertkoff, Lilien Patricia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; ArgentinaFil: Felice, Marisa. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría ; Argentin

Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements

Exonic mutations associated with exon-skipping events.

<p>On the left, semi-quantification of alternative transcripts by QF-PCR on muscle biopsy cDNA. In the centre, schematic representation of the detected transcript species and their relative ratio. On the right, mutation sequence context and predicted ESE motifs: blue bars indicate ESE finder SR protein binding sites; violet bars indicate Rescue-ESE hexamers; red bars indicate PESE octamers. The mutated nucleotide is indicated in red.</p

Splicing pathways in splice site mutations.

<p>Natural (wt) and cryptic splice sites were scored using Human Splicing Finder (HSF) and Maximum Entropy Scan matrices (MaxEnt).</p

Factors determining the main splicing pathway in 5′ ss mutations: cryptic site activation versus exon-skipping.

<p><b>A</b>) Relative 3′ ss strength (MaxEnt score difference with next distal natural 3′ ss) of exons exhibiting mainly cryptic site activation and exon exhibiting mainly exon-skipping. Box plots indicate the lowest and highest observation, lower and upper quartile, and median. <b>B</b>) Mean density of ESE motifs predicted by different matrices. <b>C</b>) Individual exons are plotted by the relative 3′ ss strength and density of Rescue-ESE motifs. Black-filled circles represent exons showing cryptic site activation. Non-filled circles represent exons showing exon-skipping. Exon numbers are indicated beside circles.</p

Relative ESE density versus relative 3′ ss strength in in-frame exons presenting nonsense and frameshift mutations.

<p>On the y axis: difference between exon PESE density and mean density of all <i>DMD</i> exons. On the x axis: difference between 3′ ss MaxEnt score and next distal natural 3′ ss score. Non-filled circles represent exons with DMD/IMD-associated mutations, while grey-filled circles represent exons with BMD-associated mutations. Black bordered circles indicate exons without PESE disruptions. Blue bordered circles indicate exons with PESE disruption. Exon numbers are indicated beside circles.</p

Representative results of dystrophin immunostaining.

<p>A healthy control and five patients presenting different types of <i>DMD</i> point mutations are shown. BMD patient #1665 shows dystrophin reduction. This patient presented a 3′ ss disrupting mutation causing mainly exon 3 in-frame skipping. Patient #1973 presents the rare combination of DMD phenotype and reduction of dystrophin expression. In this patient, a missense mutation in CH1 of ABD1 domain may cause impaired actin-binding activity. DMD patient #1775 carrying a nonsense mutation in exon 26 shows absence of dystrophin (an isolated revertant fibre can be observed in DYS2). In contrast, patient #1472 carrying a nonsense mutation in exon 28 shows reduced dystrophin expression and milder BMD phenotype. mRNA analysis in this patient revealed in-frame exon-skipping due to the disruption of an ESE motif. In the last row, BMD patient #1497 shows a very mild reduction of dystrophin expression. This patient presented a missense mutation in the ZZ domain that may compromise β-distroglycan binding.</p

Summary of <i>DMD</i> point mutations, clinical phenotype and muscle dystrophin immunostaining in 105 dystrophinopathy patients.

<p>Asterisks indicate novel mutations not previously reported in the LOVD (<a href="http://www.dmd.nl" target="_blank">www.dmd.nl</a>). Protein domains: ABD, actin-binding domain; CH1-2, calponin homology; R1-24, spectrin-like repeats; H1-4, hinge regions; CRD, cysteine-rich domain; WW, domain with a signature of two tryptophan that binds to proline-rich proteins; EF-1/2, EF-hand domains found in calcium-binding proteins; ZZ, zinc-finger domain; CTD, c-terminal domain; SBS α1-syntrophin-binding site.</p

Retrospective Multicenter Real-Life Study on the First-Line Treatment of Classical Hodgkin Lymphoma in Argentina

Abstract There are no data in Argentina on the response rates to first-line treatment of classical Hodgkin Lymphoma (cHL) outside clinical trials. A total of 498 patients from 7 public and private hospitals in Argentina were retrospectively examined. The median follow-up was 37.4 months (CI 95% 17.7–63.5). The median time from diagnosis to treatment was 22 days (IQR 14–42), which was significantly longer in public hospitals (49.3 (IC 95% 38.5–60.2) versus 32.5 (IC 95% 27–38); p = 0.0027). A total of 96.8% of patients were treated with ABVD.:84.3% achieved complete remission (CR) and 6.02% partial remission (PR), being the CR rate higher in private hospitals. End-of-treatment metabolic CR was achieved in 85.4% (n = 373). The interim PET scan was widely used in our cohort (70.5%; n = 351), but in only 23.3% (n = 116) was the treatment strategy response-adapted. The 5-year progression-free survival (PFS) was 76% (CI 95% 70–81). The 2 and 5-years-OS rates were 91% (CI 95% 88–94%) and 85% (CI 95% 80–89%), respectively. No differences in OS were found between public and private institutions (p = 0.27). This is one of the largest retrospective cHL cohorts reported. In Argentina ABVD is the chemotherapy regimen of choice and, although it is well tolerated, it is not exempt from toxicity. We showed that early initiation of treatment impacts the induction results. Although the use of PET scan is widespread, only a minority of patients was treated with respons- adapted strategies. The use of PET-guided treatment is strongly encouraged