Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia.

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.

Aroma volatiles obtained at harvest by HS-SPME/GC-MS and INDEX/MS-E-nose fingerprint discriminate climacteric behaviour in melon fruit.

BACKGROUND: Melon aroma volatiles were extracted at harvest from juice of a climacteric near-isogenic line (NIL) SC3-5-1 with two quantitative trait loci (QTLs) introgressed which produced climacteric behaviour and its non-climacteric parental (PS) using two methodologies of analysis: static headspace solid phase micro-extraction (HS-SPME) by gas chromatography-mass spectrometry (GC-MS) and inside needle dynamic extraction (INDEX) by MS-based electronic nose (MS-E-nose). RESULTS: Of the 137 volatiles compounds identified, most were found at significantly higher concentrations in SC3-5-1 than in PS in both seasons. These volatiles were mostly esters, alcohols, sulfur-derived esters and even some aldehydes and others. The number of variables with high correlation values was reduced by using correlation network analysis. Partial least squares-discriminant analysis (PLS-DA) achieved the correct classification of PS and SC3-5-1. The ions m/z 74, 91, 104, 105, 106 and 108, mainly volatile derivatives precursor phenylalanine, were the most discriminant in SC3-5-1 and PS. As many as 104 QTLs were mapped in season 1 and at least 78 QTLs in each season with an effect above the PS mean. CONCLUSION: GC-MS gave better discrimination than E-nose. Most of the QTLs that mapped in both seasons enhanced aroma volatiles associated with climacteric behaviour.

Savior siblings and Fanconi anemia: analysis of success rates from the family's perspective.

PURPOSE: The current curative treatment of Fanconi anemia is hematopoietic stem cell transplantation; this treatment has a higher rate of successful outcome when donors are compatible siblings. Therefore some families opt to have a healthy and compatible baby after selecting an embryo using preimplantation genetic diagnosis with human leukocyte antigen (HLA) typing. This study aims to estimate the success rate of this procedure from the family's perspective. METHODS: Genetic and embryology data were collected from genetic reports provided by the families. RESULTS: A total of 524 oocytes (14.1 oocytes/cycle) and 299 embryos were generated (8.0 embryos/cycle) after 38 in vitro fertilization cycles. Sixteen embryos were transferred to the uterus because they were non-Fanconi anemia and HLA matched. One baby was born. A younger couple delivered a healthy and HLA-compatible baby after four cycles. Therefore, the success rate per cycle is less than 5% (two babies from 42 trials). CONCLUSION: While Fanconi anemia per se does not worsen the probability of success, a critical factor is advanced maternal age; a late diagnosis leads to few transferrable embryos and high rates of aneuploidy. Families should be informed in advance of the many trials that they will probably need to undergo even if a haploidentical younger relative is available as an oocyte donor.

On the role of FAN1 in Fanconi anemia.

Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1 and 6 additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3'- and 5'-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell survival and G(2) phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand crosslink repair compared with true FA genes and exclude FAN1 as a novel FA gene.

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.

Bound aroma compounds of Gual and Listán blanco grape varieties and their influence in the elaborated wines.

Listán blanco and Gual are two white grape varieties grown in Spain (Canary Islands) and Gual is also grown in Portugal (Madeira Island). Wine quality could be improved by exploiting the varietal characteristics present in grapes as aroma precursors. At the present time, the enologic potential of these varieties has not yet been studied. Aroma precursors in musts and skins, and free volatiles in wines were determined in samples of both varieties. Aroma precursors of must and skins were isolated by absorption on XAD-2 resin and the aglycone liberated by means of acid hydrolysis. Free volatiles from wines and from aroma precursors were extracted with dichloromethane and then analysed by gas chromatography-mass spectrometry (GC-MS). Among the aroma precursors analysed, only α-terpineol, linalool and linalool oxides, 4-allyl-2,6-dimethoxyphenol, 2-methoxy-4-vinylphenol, 4-hydroxybenzaldehyde, vanillin and benzyl alcohol showed contents in grapes comparable to the levels observed in wine volatile compounds.

Effect of skin contact on bound aroma and free volatiles of Listán blanco wine.

Listán blanco is the major grape variety of the Canary Islands. Aroma precursors in musts and free volatiles in wines and their variations due to the skin contact process (10°C and 0-12h of maceration) were determined in samples of the Listán blanco grape variety. Aroma precursors of must were isolated by absorption on XAD-2 resin and the aglycone liberated by means of acid hydrolysis. Free volatiles from wines and from aroma precursors were extracted with dichloromethane and then analysed by GC-MS. The results of two consecutive vintages show that only aroma precursors of some terpenes and phenols may affect the final wine aroma. However, the majority of the free compounds of the wines did not present differences in content that could be related to the skin contact process.

Truncating FLNC Mutations Are Associated With High-Risk Dilated and Arrhythmogenic Cardiomyopathies.

BACKGROUND: Filamin C (encoded by the FLNC gene) is essential for sarcomere attachment to the plasmatic membrane. FLNC mutations have been associated with myofibrillar myopathies, and cardiac involvement has been reported in some carriers. Accordingly, since 2012, the authors have included FLNC in the genetic screening of patients with inherited cardiomyopathies and sudden death. OBJECTIVES: The aim of this study was to demonstrate the association between truncating mutations in FLNC and the development of high-risk dilated and arrhythmogenic cardiomyopathies. METHODS: FLNC was studied using next-generation sequencing in 2,877 patients with inherited cardiovascular diseases. A characteristic phenotype was identified in probands with truncating mutations in FLNC. Clinical and genetic evaluation of 28 affected families was performed. Localization of filamin C in cardiac tissue was analyzed in patients with truncating FLNC mutations using immunohistochemistry. RESULTS: Twenty-three truncating mutations were identified in 28 probands previously diagnosed with dilated, arrhythmogenic, or restrictive cardiomyopathies. Truncating FLNC mutations were absent in patients with other phenotypes, including 1,078 patients with hypertrophic cardiomyopathy. Fifty-four mutation carriers were identified among 121 screened relatives. The phenotype consisted of left ventricular dilation (68%), systolic dysfunction (46%), and myocardial fibrosis (67%); inferolateral negative T waves and low QRS voltages on electrocardiography (33%); ventricular arrhythmias (82%); and frequent sudden cardiac death (40 cases in 21 of 28 families). Clinical skeletal myopathy was not observed. Penetrance was >97% in carriers older than 40 years. Truncating mutations in FLNC cosegregated with this phenotype with a dominant inheritance pattern (combined logarithm of the odds score: 9.5). Immunohistochemical staining of myocardial tissue showed no abnormal filamin C aggregates in patients with truncating FLNC mutations. CONCLUSIONS: Truncating mutations in FLNC caused an overlapping phenotype of dilated and left-dominant arrhythmogenic cardiomyopathies complicated by frequent premature sudden death. Prompt implantation of a cardiac defibrillator should be considered in affected patients harboring truncating mutations in FLNC.

Determination of major compounds in sweet wines by headspace solid-phase microextraction and gas chromatography.

Headspace solid-phase microextraction (HS-SPME) was studied by high resolution gas chromatographic analysis of major compounds (ethyl acetate, methanol, 1-butanol, 2-butanol, 1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol) in sweet wines. Five different SPME fibres were tested and the influence of different factors such as temperature and time of desorption, extraction time, stirring, sample and vial volume, sugar and ethanol content were studied and optimized using model solutions. The SPME method was validated with the direct injection method. The proposed HS-SPME-GC method is an appropriate technique for the quantitative analysis of the mentioned analytes in real sweet wines.

Targeted gene therapy and cell reprogramming in Fanconi anemia.

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.

Dysfunctional telomeres in primary cells from Fanconi anemia FANCD2 patients.

BACKGROUND: Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models. RESULTS: We analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis. CONCLUSION: The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further.

A new method for the determination of short-chain fatty acids from the aliphatic series in wines by headspace solid-phase microextraction-gas chromatography-ion trap mass spectrometry.

A new analytical method for the determination of nine short-chain fatty acids (acetic, propionic, isobutyric, butyric, isovaleric, 2-methylbutyric, hexanoic, octanoic and decanoic acids) in wines using the automated HS/SPME-GC-ITMS technique was developed and optimised. Five different SPME fibers were tested and the influence of different factors such as temperature and time of extraction, temperature and time of desorption, pH, strength ionic, tannins, anthocyans, SO(2), sugar and ethanol content were studied and optimised using model solutions. Some analytes showed matrix effect so a study of recoveries was performed. The proposed HS/SPME-GC-ITMS method, that covers the concentration range of the different analytes in wines, showed wide linear ranges, values of repeatability and reproducibility lower than 4.0% of RSD and detection limits between 3 and 257 µgL(-1), lower than the olfactory thresholds. The optimised method is a suitable technique for the quantitative analysis of short-chain fatty acids from the aliphatic series in real samples of white, rose and red wines.

A new method for the determination of carbonyl compounds in wines by headspace solid-phase microextraction coupled to gas chromatography-ion trap mass spectrometry.

A new analytical method for the determination of 18 carbonyl compounds [2,3-pentadione, hexanal, (E)-2-hexen-1-al, octanal, acetoin, (E)-2-octenal, furfural, decanal, (E)-2-nonenal, benzaldehyde, 5-methylfurfural, (E,E)-2-cis-6-nonadienal, ß-damascenone, phenylacetaldehyde, acetophenone, (E,E)-2,4-decadienal, benzophenone, and vanillin] in wines using automated headspace solid-phase microextraction (HS/SPME) coupled to gas chromatography-ion trap mass spectrometry (GC-ITMS) was developed. Five fibers with different polarities were tested, and a study of the influence of various factors such as time and extraction temperature, desorption time and temperature, pH, and ionic strength and content in tannins, anthocyans, sucrose, SO(2), and alcoholic degree was conducted. These factors were optimized using a synthetic wine doped with the different analytes. The proposed method affords wide ranges of linearity, good linearity (r(2) > 0.998), values of repeatability and reproducibility lower than 5.5% of RSD, and detection limits ranging from 0.62 µg/L for ß-damascenone to 129.2 µg/L for acetoin. Therefore, the optimized method was applied to the quantitative analysis of the aforementioned analytes in real samples of wines.

Metallic content of wines from the Canary Islands (Spain). Application of artificial neural networks to the data analysis.

Eleven elements, K, Na, Ca, Mg, Fe, Cu, Zn, Mn, Sr, Li and Rb, were determined in dry and sweet wines bearing the denominations of origin of El Hierro, La Palma and Lanzarote islands (Canary Islands, Spain). Analyses were performed by flame atomic absorption spectrophotometry, with the exceptions of Li and Rb for which flame atomic emission spectrophotometry was used. The content in copper and iron did not present risks of cases. All samples presented a copper and zinc content below the maximum amount recommended by the Office International de la Vigne et du Vin (OIV) for these elements. Significant differences in the metallic content were found among the different islands. Thus, Lanzarote presented the highest mean content in sodium and lithium and the lowest mean content in rubidium, and La Palma presented the highest mean content in strontium and rubidium. Sweet wines from La Palma, elaborated as naturally sweet with over-ripe grapes, presented mean contents significantly higher with regard to dry wines from the same island in the majority of the analysed elements. Cluster analysis and Kohonen self-organising maps showed differences in wines according to the island of origin and the ripening state of the grapes. Back-propagation artificial neural networks showed better prediction ability than stepwise linear discriminant analysis.

Classification of commercial wines from the Canary Islands (Spain) by chemometric techniques using metallic contents.

Eleven elements, K, Na, Ca, Mg, Fe, Cu, Zn, Mn, Sr, Li and Rb, were determined in dry and sweet wines bearing the denominations of origin of El Hierro, La Palma and Lanzarote islands (Canary Islands, Spain). Analyses were performed by flame atomic absorption spectrophotometry, with the exceptions of lithium and rubidium for which flame atomic emission spectrophotometry was used. Sweet wines from La Palma were elaborated as naturally sweet with over-ripe grapes and significant differences were found in all the analysed elements with the exceptions of sodium, iron and rubidium with regard to dry wines from the same island. Contrarily, sweet wines from Lanzarote elaborated with grapes in a similar ripening state to dry wines did not present significant differences between them with the exception of strontium, the content of which was greater in dry wines. Among the three islands, significant differences in mean content were found with the exceptions of iron and copper. Cluster analysis and principal component analysis show differences in wines according to the island of origin and the ripening state of the grapes. Linear discriminant analysis using rubidium, sodium, manganese and strontium, the four most discriminant elements, gave 100% recognition ability and 95.6% prediction ability. The sensitivity and specificity obtained using soft independent modelling of class analogy (SIMCA) as a modelling multivariate technique were both 100% for El Hierro and Lanzarote, and 100 and 95%, respectively, for La Palma. The modelling and discriminant capacities of the different metals were also studied.