First report of molecular diagnosis of Tunisian hemophiliacs A: identification of 8 novel causative mutations

ABSTRACT: INTRODUCTION: Hemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder. Aim In this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum. METHODS: We screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure. RESULTS: We identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles. CONCLUSION: The mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.     

Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A. French CMT Collaborative Research Group.

Charcot-Marie-Tooth type 1A (CMT1A) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are autosomal dominant neuropathies, associated, respectively, with duplications and deletions of the same 1.5-Mb region on 17p11.2-p12. These two rearrangements are the reciprocal products of an unequal meiotic crossover between the two chromosome 17 homologues, caused by the misalignment of the CMT1A repeat sequences (CMT1A-REPs), the homologous sequences flanking the 1.5-Mb CMT1A/HNPP monomer unit. In order to map recombination breakpoints within the CMT1A-REPs, a 12.9-kb restriction map was constructed from cloned EcoRI fragments of the proximal and distal CMT1A-REPs. Only 3 of the 17 tested restriction sites were present in the proximal CMT1A-REP but absent in the distal CMT1A-REP, indicating a high degree of homology between these sequences. The rearrangements were mapped in four regions of the CMT1A-REPs by analysis of 76 CMT1A index cases and 38 HNPP patients, who where unrelated. A hot spot of crossover breakpoints, located in a 3.2-kb region, accounted for three-quarters of the rearrangements, detected after EcoRI/SacI digestion, by the presence of 3.2-kb and 7.8-kb junction fragments in CMT1A and HNPP patients, respectively. These junction fragments, which can be detected on classical Southern blots, permit molecular diagnosis. Other rearrangements can also be detected by gene dosage on the same Southern blots.     

Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP)

Hereditary neuropathy with liability to pressure palsies (HNPP) is in most cases associated with an interstitial deletion of the same 1.5-Mb region at 17p11.2 that is duplicated in Charcot-Marie-Tooth type 1A (CMT1A) patients. Unequal crossing-over following misalignment at flanking repeat sequences (CMT1A-REP), either leads to tandem duplication in CMT1A patients or deletion in HNPP patients. With the use of polymorphic DNA markers located within the CMT1A/HNPP duplication/deletion region we detected the HNPP deletion in 16 unrelated HNPP patients, 11 of Belgian and 5 of French origin. In all cases, the 1.5-Mb size of the HNPP deletion was confirmed by EcoRI dosage analysis using a CMT1A-REP probe. In the 16 HNPP patients, the same 370/320-kb EagI deletion-junction fragments were detected with pulsed field gel electrophoresis (PFGE), while in CMT1A patients, a 150-kb EagI duplication-junction fragment was seen. Thus, PFGE analysis of EagI-digested DNA with a CMT1A-REP probe allows direct detection of the HNPP deletion or the CMT1A duplication for DNA diagnostic purposes.     

Impact of the overexpression of the StDREB1 transcription factor on growth parameters,yields, and chemical composition of tubers from greenhouse and field grown potato plants

Journal of Plant Research - Potato plants are often exposed to biotic and abiotic stresses that negatively impact their growth, development, and yield. Plants respond to different stresses by...     

Identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia

ABSTRACT: Inherited factor VII (FVII) deficiency is a rare disorder characterized by a bleeding phenotype varying from mild to severe. To date, more than 200 mutations have been described along the F7 gene encoding for FVII. The aim of this study was the identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia. Mutation detection was performed by sequencing the whole F7 gene coding region, exon-intron boundaries and about 400 bp of the promoter region. We identified 5 mutations in five unrelated families; the novel p.F328Y mutation and the reported mutations: p.R304Q, p.M298I, IVS1aG > A and nt1005C > T. For the remaining 5 patients we didn't identified any mutations using PCR/Sequencing protocol. In conclusion, this study represents the first comprehensive molecular series of FVII deficiency affected patients in Tunisia from the North. We will try in the future to continue the molecular study for Tunisian patients from Center and South provinces in order to have a complete idea about the FVII deficiency mutational profile in our country. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1288044089753085.