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• W2009059663 abstract "Congenital afibrinogenemia (CAF, OMIM #202400) is a rare, recessively inherited, clotting disorder characterized by the severe deficiency of fibrinogen [1Lak M. Keihani M. Elahi F. Peyvandi F. Mannucci PM. Bleeding and thrombosis in 55 patients with inherited afibrinogemia.Br J Haematol. 1999; 107: 204-6Crossref PubMed Scopus (210) Google Scholar, 2Mannucci P.M. Duga S. Peyvandi F. Recessively inherited coagulation disorders.Blood. 2004; 104: 1243-52Crossref PubMed Scopus (384) Google Scholar], a plasma glycoprotein representing the precursor of fibrin monomers involved in the haemostatic plug formation. Fibrinogen is synthesized in hepatocytes as a 340-kDa hexamer composed of two Aα, two Bβ, and two γ chains [3Mosesson MW. Fibrinogen structure and fibrin clot assembly.Semin Thromb Hemost. 1998; 24: 169-74Crossref PubMed Scopus (48) Google Scholar], encoded by the FGA, FGB, and FGG gene, respectively. The three genes are clustered in a 50-Kb region at 4q31.3 [4UCSC Genome Browser. Available at http://genome.ucsc.edu/. (accessed 12 July 2005).Google Scholar]. Congenital afibrinogenemia is a highly heterogeneous genetic disease with no prevalent mutations and a total of 38 different genetic defects so far reported, all located within the fibrinogen gene cluster [5The Fibrinogen Database. Available at http://www.geht.org/databaseang/fibrinogen/ (accessed 12 July 2005).Google Scholar]. The molecular diagnosis of diseases with high allelic/locus heterogeneity (such as CAF) normally implies the direct sequencing of all the genes known to be involved. This tedious, costly, and time-consuming process is required to assess the different genetic defect in each patient. Pooling DNA samples from different individuals is a short-cut approach to high-throughput genetic variation screening [6Sham P. Bader J.S. Craig I. O'Donovan M. Owen M. DNA pooling: a tool for large-scale association studies.Nat Rev Genet. 2002; 3: 862-71Crossref PubMed Scopus (469) Google Scholar]. While several genotyping methods have been successfully applied to DNA pools, mutational screening by direct sequencing of PCR-amplified DNA pools was seldom reported. This was mainly due to the requirement for a relatively high concentration of the minor allele in the pool. This issue is less limiting in case of homozygous mutations, as the signal of the mutant (i.e. the minor) allele is doubled, allowing the pooling of twice the number of DNAs without any sensitivity loss. Homozygous patients are frequent in rare inherited recessive diseases, such as CAF, as a consequence of the high level of consanguinity among affected families [7Peyvandi F. Mannucci PM. Rare coagulation disorders.Thromb Haemost. 1999; 82: 1207-14Crossref PubMed Scopus (176) Google Scholar]. In this study, the feasibility of the DNA pooling for the molecular diagnosis of CAF was investigated, by mutational screening of the fibrinogen gene cluster in three unrelated CAF patients (R76, R79, and R99), whose clinical details are summarized in Fig. 1A. A sensitivity assessment of the DNA-pooling approach to direct sequencing was carried out prior to its application to the molecular diagnosis of CAF. In this sensitivity assessment, equimolar quantities of DNA from one, two, or three wild-type (WT) healthy controls were pooled with the DNA from an individual previously characterized as being heterozygote at a specific locus. Therefore the obtained wild-type to mutant allele ratios, in these trial DNA pools, were 3:1, 5:1, or 7:1, respectively. Direct sequencing of PCR-amplified pooled DNA allowed the detection of the heterozygote allele at each tested concentration (Fig. 1B). The area ratios of overlapping double peaks in sequence electropherograms were strikingly similar with the allele ratios in the DNA pools (data not shown). Thus, having assessed its sensitivity and reliability, this approach was applied to the mutational screening of three CAF patients. Firstly, the homozygosity state for the fibrinogen gene cluster in the three patients was tested by polymorphism genotyping, as described [9Menegatti M. Asselta R. Duga S. Malcovati M. Bucciarelli P. Mannucci P.M. Tenchini ML. Identification of four novel polymorphisms in the Aalpha and gamma fibrinogen genes and analysis of association with plasma levels of the protein.Thromb Res. 2001; 103: 299-307Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar]. The patients were homozygous for each of the analyzed markers (data not shown), suggesting the probable homozygosity of the whole cluster. After an accurate quantitation step, equal amounts of genomic DNA from the three patients were mixed together, PCR amplified and then simultaneously sequenced throughout the fibrinogen cluster (as described, [8Duga S. Asselta R. Santagostino E. Zeinali S. Simonic T. Malcovati M. Mannucci P.M. Tenchini ML. Missense mutations in the human beta fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion.Blood. 2000; 95: 1336-41Crossref PubMed Google Scholar]). An internal homozygous positive control (B1), previously demonstrated to carry the FGA-Arg110stop mutation [10Asselta R. Duga S. Spena S. Santagostino E. Peyvandi F. Piseddu G. Targhetta R. Malcovati M. Mannucci P.M. Tenchini ML. Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen A alpha-chain gene are not associated with the decay of the mutant mRNAs.Blood. 2001; 98: 3685-92Crossref PubMed Scopus (72) Google Scholar], was also included in the pool. Sequence variations among pooled samples were highlighted by the presence of overlapping peaks in sequence electropherograms. Known polymorphisms were excluded from further investigation, while each of the newly found sequence variations was considered as candidate mutation. Single DNA sequencing of the relevant PCR fragments allowed the assignment of each mutation to a patient (Fig. 1C). All newly identified mutations were found in FGA at the homozygous state. In particular, patient R99 showed a novel G > A transition occurring at the first nucleotide of intron 1 (IVS1 + 1G > A). Patient R76 showed a novel insertion of a thymine in exon 3 between nucleotides 1754 and 1755 (1754_1755insT); this mutation results in a frameshift (FS56stop) that, starting from residue Phe47, introduces nine aberrant amino acids followed by a premature termination codon (PTC) at position 56. Patient R79 showed a novel in-frame insertion of a trinucleotide (TGA) in exon 5 between positions 3884 and 3885 (3884_3885insTGA); this insertion introduces a PTC at position 185 (Leu185stop). The mutation of the internal control, B1, was also confirmed [10Asselta R. Duga S. Spena S. Santagostino E. Peyvandi F. Piseddu G. Targhetta R. Malcovati M. Mannucci P.M. Tenchini ML. Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen A alpha-chain gene are not associated with the decay of the mutant mRNAs.Blood. 2001; 98: 3685-92Crossref PubMed Scopus (72) Google Scholar]. The IVS1 + 1G > A mutation involves the highly conserved [11Cartegni L. Chew S.L. Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing.Nat Rev Genet. 2002; 3: 285-98Crossref PubMed Scopus (1752) Google Scholar] first nucleotide of intron 1; it is supposed to abolish a donor splice site and therefore generate a PTC in consequence of a frameshift, as demonstrated for a similar CAF mutation [12Asselta R. Duga S. Simonic T. Malcovati M. Santagostino E. Giangrande P.L. Mannucci P.M. Tenchini ML. Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation.Blood. 2000; 96: 2496-500Crossref PubMed Google Scholar]. The remaining two mutations, 1754_1755insT and 3884_3885insTGA, are also predicted to cause the synthesis of severely truncated Aα chains, lacking 91.2% and 70.6% of the mature WT protein, respectively. Truncated Aα chains have been demonstrated to dramatically interfere with fibrinogen assembly causing its complete absence in the plasma of homozygous patients [10Asselta R. Duga S. Spena S. Santagostino E. Peyvandi F. Piseddu G. Targhetta R. Malcovati M. Mannucci P.M. Tenchini ML. Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen A alpha-chain gene are not associated with the decay of the mutant mRNAs.Blood. 2001; 98: 3685-92Crossref PubMed Scopus (72) Google Scholar]. The DNA-pooling approach was thus successfully applied to the mutational screening of a pool of four homozygotes (three patients and one positive control), demonstrating its reliability and effectiveness in a 4:1 wild-type to mutant allele ratio. The molecular diagnosis of other highly heterogeneous inherited diseases could benefit from the optimization allowed by this protocol. The larger is the locus to be screened, the more effective is the DNA-pooling strategy in reducing the number of DNA sequence analyses to be performed. In fact, in a standard single-patient sequencing approach, the total number of requested sequences is T = N × F where N is the number of patients and F the number of fragments to be amplified and sequenced. DNA pooling reduces T (by minimizing unnecessary sequencing of WT fragments) to a value comprised between N2 + F and 2N2 + F, depending on the number of heterozygotes in the pool (e.g. if all individuals are homozygous, T = N2 + F). In the case of four homozygotes, DNA pooling is advantageous for any F bigger than five (e.g. when F = 100, a 70% reduction of T is achieved). Obviously, this strategy is recommended only when the chance of large deletions and major chromosomal rearrangements is low. In conclusion, this study widened the mutational spectrum of CAF, confirming the heterogeneity of this rare disease. The description of three novel mutations will benefit prenatal diagnosis of CAF, as recently demonstrated in a Palestinian [13Neerman-Arbez M. Vu D. Abu-Libdeh B. Bouchardy I. Morris MA. Prenatal diagnosis for congenital afibrinogenemia caused by a novel nonsense mutation in the FGB gene in a Palestinian family.Blood. 2003; 101: 3492-4Crossref PubMed Scopus (42) Google Scholar] and in two Italian families (S. Duga, unpublished data). We thank Prof. F. Peyvandi (Angelo Bianchi Bonomi, Fondazione Luigi Villa, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Fondazione IRCCS, University of Milan, Milan, Italy), whose name has not been included among the authors only to meet the guidelines concerning the number of authors, for the recruitment of patients and for the measurement of fibrinogen plasma levels. We also wish to thank all family members for their participation in this study. The financial support of Telethon, Italy (grant no. GGP030261) is gratefully acknowledged. This work was partially funded by IRCCS Maggiore Hospital, Milan, Italy and by a grant of Fondazione Italo Monzino to F. Feyrand." @default.
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• W2009059663 title "The DNA-pooling technique allowed for the identification of three novel mutations responsible for afibrinogenemia" @default.
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