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Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

2013-04-26T14:11:10Z

Title: Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples Authors: Ribeiro, Diogo; Cardoso, Ana; Duarte, Ana Joana; Vieira, Luís; Amaral, Olga Abstract: Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. This PCR-based dHPLC method proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. Description: DR and AJD were FCT research grantees (PIC/IC/82822/2007)

Methionine Adenosyltransferase I/III Deficiency in Portugal: High Frequency of a Dominantly Inherited Form in a Small Area of Douro High Lands

2013-03-25T16:07:34Z

Title: Methionine Adenosyltransferase I/III Deficiency in Portugal: High Frequency of a Dominantly Inherited Form in a Small Area of Douro High Lands Authors: Martins, E.; Marcão, A.; Bandeira, A.; Fonseca, H.; Nogueira, C.; Vilarinho, L. Abstract: Methionine adenosyltransferase deficienc(MAT I/III deficiency) is an inborn error of metabolism resulting in isolated hypermethioninemia, and usually inherited as an autosomal recessive trait, although a dominant form has been reported in several families. During the last 6 years, approximately 520,000 newborns were screened in the Portuguese Newborn Screening Laboratory by MS/MS, and 21 cases of persistent hypermethioninemia were found. One case was confirmed to be a deficiency of cystathionine b-synthase and 20 cases were confirmed by MAT1A gene analysis to have an elevation of methionine due to MAT I/III deficiency, which indicates an incidence for this condition of 1/26,000. Twelve of the MAT I/III deficient newborns, belonging to 11 families, were identified in the northern region of Portugal and sent to the same treatment center, where they are under follow-up. Clinical, biochemical, and genetic characteristics of individuals from these 11 families are presented. Plasma methionine and homocysteine concentrations were found to be moderately increased in all newborns, and molecular analysis revealed that they all were heterozygous for R264H mutation. Normal growth,development, and neurological examination were observed in all cases, and cerebral MRI performed in six cases revealed myelination abnormalities in one case. Plasma methionine concentration for all 12 cases was always below 300 mM, and they are all on a normal diet for their age.

Measurements of CFTR-Mediated Cl- Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

2013-03-22T12:05:45Z

Title: Measurements of CFTR-Mediated Cl- Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis Authors: Sousa, Marisa; Servidoni, Maria F.; Vinagre, Adriana M.; Ramalho, Anabela S.; Bonadia, Luciana C.; Felício, Verónica; Ribeiro, Maria A..; Uliyakina, Inna; Marson A, Fernando; Kmit, Arthur; Cardoso, Silvia R.; Ribeiro, José D.; Bertuzzo, Carmen S.; Sousa, Lisete; Kunzelmann, Karl; Ribeiro, Antônio F.; Amaral, Margarida D. Abstract: BACKGROUND: Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51), individuals with clinical CF suspicion (n=49) and age-matched non-CF controls (n=18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(-) secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl(-) secretion (10-57%) and non-CF controls show CFTR-mediated Cl(-) secretion ≥ 30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(-) secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(-) secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.

BAG-1 stabilizes mutant F508del-CFTR in a Ubiquitin-Like-Domain-Dependent Manner

2013-03-22T11:59:51Z

Title: BAG-1 stabilizes mutant F508del-CFTR in a Ubiquitin-Like-Domain-Dependent Manner Authors: Mendes, Filipa; Farinha, Carlos M.; Felício, Verónica; Alves, Paula C.; Vieira, Isabel; Amaral, Paulo C. Abstract: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the dysfunctional Cl- channel in Cystic Fibrosis, undergoes complex biosynthesis at the endoplasmic reticulum involving several molecular chaperones including Hsp70 and many co-chaperones. Bcl-2-associated athanogenes (BAGs) constitute a protein family sharing an Hsc70-binding domain. BAG-1 possesses an ubiquitin-like domain (Ub-LD) responsible for proteasomal association and for promoting substrate release from Hsc70/Hsp70 in vitro by accelerating the chaperone ATP/ADP exchange rate. Methods: Herein, we studied the in vivo effect of BAG-1 on the turnover and processing of wild type (wt)- and F508del-CFTR, the most frequent mutation in CF patients. Results: Results show that BAG-1 associates with both wt- and F508del-CFTR (in higher yields with the latter) through its Ub-LD and independently of Hsc70. Moreover, the immature form of F508del-CFTR (but not of wt-CFTR) is stabilized by BAG-1 overexpression, albeit in a cell-type specific way, without detectable maturation. Data also show that BAG-1 and the proteasome inhibitor ALLN are not additive on stabilizing F508del-CFTR and this effect depends on BAG-1 Ub-LD. Moreover, under BAG-1 overexpression, a reduction in ubiquitinylated-CFTR occurs suggesting that BAG-1 competes with Ub. Conclusion: Overall, data are compatible with a mechanism in which BAG-1 stabilizes F508del-CFTR by direct binding, probably competing out ubiquitin to partially avoid its proteasomal degradation.