Case report: Asp194Ala variant in MFN2 is associated with ALS-FTD in an Italian family.

Background: MFN2 gene encodes the protein Mitofusin 2, involved in essential mitochondrial functions such as fusion, trafficking, turnover, and cellular interactions. We describe a family carrying a novel MFN2 mutation associated with ALS-frontotemporal dementia (FTD) clinical phenotype in the mother and Charcot-Marie-Tooth disease type 2A (CMT2A) in her son. Case presentation: The mother, a 67-year-old woman, referred to us for a three year-history of mood disturbance and gait impairment, and a more recent hypophonia, dysarthria, dysphagia, and diffuse muscle wasting. Family history was positive for psychiatric disorders and gait disturbances. Brain 18F-FDG PET showed severe hypometabolism in the fronto-temporal brain cortex bilaterally. Electrodiagnostic studies (EDX) showed severe motor axonopathy in the bulbar, cervical and lumbosacral districts. Her 41-year-old son had a history of mood depression and sensory disturbances in the limbs, along with mild muscle wasting, weakness, and reduced reflexes. Nerve conduction studies revealed a moderate sensory-motor polyneuropathy, while brain MRI was normal. Whole exome sequencing of the patients' DNA identified the novel MFN2 (NM_014874.4) variant c.581A>C p.(Asp194Ala). Conclusion: Our findings provide evidence of heterogenous clinical manifestations in family members sharing the same MFN2 molecular defect. Additionally, we present the first documented case of ASL-FTD associated with an MFN2 mutation, thereby expanding the range of MFN-related disorders. Further research involving larger cohorts of patients will be needed to better understand the role of MFN2 as a contributing gene in the development of ALS-FTD.

Peak width of skeletonized mean diffusivity (PSMD) and cognitive functions in relapsing-remitting multiple sclerosis.

Peak width of skeletonized mean diffusivity (PSMD) is a new MRI marker, which has shown clinical relevance in some neurological conditions and, in preliminary data, in multiple sclerosis (MS). We aimed here to investigate, in a group of relapsing-remitting MS (RRMS) patients, the relationship between PSMD and cognitive performances, in comparison with other MRI measures. RRMS patients (n = 60) and normal controls (n = 15) underwent a 3 T MRI examination. MRI-based white matter (WM) lesion volume, microstructural integrity (assessed with Tract-Based Spatial Statistics of diffusion tensor imaging [DTI] images) and brain volumes (i.e., total brain, grey matter [GM] and WM) were computed. In addition, PSMD was calculated through "skeletonization" of WM tracts and diffusion histograms. Cognition was evaluated with Rao's Brief Repeatable Battery (BRB), which incorporated tests of verbal and visual memory, attention, concentration, information processing speed and verbal fluency. PSMD closely correlated with symbol digit modalities test (SDMT) (r = -0.70, p < 0.001) and, to a lesser extent, with verbal and visual memory tests. Multiple regression analysis showed that PSMD explained SDMT variance (R2 = 0.54, p < 0.001) more than other MRI measures. Results point out the relevance of microstructural damage, as assessed by PSMD, as a reliable marker of cognition in MS, especially in explaining dysfunction in information processing speed.

Peak width of skeletonized mean diffusivity (PSMD) as marker of widespread white matter tissue damage in multiple sclerosis.

BACKGROUND: Peak width of skeletonized mean diffusivity (PSMD) is a novel and fully automated, MRI biomarker, which has shown clinical relevance in cerebral small vessel diseases (SVD). We aimed here to assess PSMD levels across the brain of patients with multiple sclerosis (MS), in comparison to normal controls (NC) and patients with CADASIL, a genetically defined form of severe SVD. METHODS: We assessed PSMD in relapsing-remitting (RR) MS patients (n = 47) in comparison to age-matched CADASIL patients (n = 25) and NC (n = 28). Diffusion Tensor Imaging data were acquired on 1.5T MR clinical scanner to automatically compute PSMD through "skeletonization" of WM tracts and diffusion histograms. RESULTS: RRMS had lower WM lesion volume (LV) than CADASIL (8.6 ±â€¯8.2 vs 24.4 ±â€¯17.4 cm3, p < 0.001). After correction for LV, PSMD values in MS were higher than in CADASIL patients (adjusted mean values: 4.5 vs 3.9 × 10-4 mm2/s, p = 0.03) and in both patient groups were higher than in NC (2.8 ±â€¯0.3 × 10-4 mm2/s, p < 0.001). PSMD values correlated with LV in both patient groups (r = 0.8, p < 0.001 in MS; r = 0.6, p = 0.002 in CADASIL). CONCLUSIONS: In both patient groups, PSMD was higher than in NC and closely correlated with LV, suggesting sensitivity in assessing brain tissue damage in these disorders. In MS patients, PSMD levels were higher than in CADASIL patients, despite the lower LV. This might be related to more severe normal-appearing WM abnormalities occurring in the MS brains. This novel, fully automated, MRI metric may represent a useful marker for a robust quantification of the diffuse WM tissue damage in MS.

Recurrent F8 and F9 gene variants result from a founder effect in two large French haemophilia cohorts.

INTRODUCTION: Haemophilia A (HA) and haemophilia B (HB) are X-linked recessive diseases, caused by a large number of pathogenic variants in the F8 and F9 genes. With the exception of introns 22 and 1 inversions which are frequent in severe HA cases, about 2000 unique variants in F8 and 1000 in F9 have been described in databases and their recurrence remains limited. AIM AND METHODS: During routine analysis, we identified two recurrent missense variants, the F8 gene c.1244C>T, p.Ala415Val variant in 27 HA patients and the F9 gene c.835G>A, p.Ala279Thr variant in 34 HB patients, in two groups of haemophiliac patients from two different regions of France. We aimed to identify whether these variants result from a founder effect. We performed haplotype reconstruction after analysis of extragenic and intragenic polymorphic markers. The ESTIAGE programme was used to estimate the age of the variant. RESULTS: We identified a common ancestral haplotype HA1, in all the HA patients sharing the p.Ala415Val variant, and HB1 for 22 of 34 HB patients sharing the p.Ala279Thr variant. The estimated time of occurrence of the founder variant was between the 13th and 17th century (95% CI: 16 to 29 generations) for the F8 variant and between the 3rd and the 11th century for the F9 variant (95% CI: 44 to 72 generations). CONCLUSION: This study supports a founder effect for these two variants in the two largest reported cohorts of haemophilia patients with an identical variant. These pathogenic variants are among the three most early reported variants in haemophilia.

Molecular cytogenetic characterization of five F8 complex rearrangements: utility for haemophilia A genetic counselling.

BACKGROUND: Genomic inversions are usually balanced, but unusual patterns have been described in haemophilia A (HA) patients for intron 22 (Inv22) and intron 1 (Inv1) inversions leading to the hypothesis of more complex rearrangements involving deletions or duplications. AIM: To characterize five abnormal patterns either in Southern blot and long-range PCR for Inv22 or in PCR for Inv1. MATERIALS AND METHODS: All patients were studied using cytogenetic microarray analysis (CMA). RESULTS: In all cases, CMA analysis found that each inversion was associated with complex Xq28 rearrangement. In three patients, CMA analysis showed large duplication ranging from 230 to 1302 kb and encompassing a various number of contiguous genes among which RAB39B. RAB39B duplication is a strong candidate gene for X-linked intellectual disability (XLID). Surprisingly, none of the severe HA patients with RAB39B duplication reported in this study or in the literature exhibited XLID. We hypothesise that F8 complex rearrangement down regulated RAB39B expression. In the two remaining patients, CMA analysis found Xq28 large deletion (from 285 to 522 kb). Moyamoya syndrome was strongly suspected in one of them who carried BRCC3 deletion. CONCLUSION: Because several F8 neighbouring genes are associated with other pathologies such as XLID and cardiovascular disease, all HA patients where complex Xq28 rearrangement was suspected should be referred to a geneticist for possible utility of a pangenomic study. Such investigation should be carefully considered in genetic counselling in female carriers to assess the risk of transmitting severe HA with a "contiguous gene syndrome".

Study of six patients with complete F9 deletion characterized by cytogenetic microarray: role of the SOX3 gene in intellectual disability.

Essentials Some hemophilia B (HB) patients with complete F9 deletion present with intellectual disability (ID). We delineate six F9 complete deletions and investigate genotype/phenotype correlation. We identify SOX3 as a candidate gene for ID, acting through haploinsufficiency, in HB patients. All complete F9 deletions in ID patients should be explored with cytogenetic microarrays. SUMMARY: Background Large deletions encompassing both the complete F9 gene and contiguous genes have been detected in patients with severe hemophilia B (HB). Some of these patients present other clinical features, such as intellectual disability (ID). Objectives/Methods In this study, we characterized six unrelated large deletions encompassing F9, by cytogenetic microarray analysis (CMA), to investigate genotype/phenotype correlation. Results Five of the six patients included in this study presented with ID associated with HB. CMA showed that the six large deletions, ranging in size from approximately 933 kb to 9.19 Mb, were located within the Xq26.3 to Xq28 bands. In all cases, the complete deletion of F9 was associated with the loss of various neighboring genes (5-28 other genes). The smallest region of overlap for ID was a 1.26-Mb region encompassing seven OMIM genes (LOC389895, SOX3, LINC00632, CDR1, SPANXF1, LDOC1, SPANXC). SOX3, our candidate gene for ID, encodes an early transcription factor involved in pituitary development. All of the patients studied who had both HB and ID had deletion of the SOX3 gene. Conclusions All HB patients with an atypical phenotype, especially if complete deletion of F9 is suspected, should be referred to a geneticist for possible pangenomic assessment, because haploinsufficiency of genes flanking F9, such as SOX3 in particular, may result in a broader phenotype, including ID. Such assessment would be of particular value for the genetic counseling of female carriers with F9 deletions, as it would facilitate analysis of the risk of transmitting HB associated with ID.

[Identification of knowledge deficits of pharmacy students at the beginning of the fifth year of pharmacy practice experience: Proposals to change the content of academic programs]. / Identification des lacunes de connaissances des étudiants en filière officine débutant le stage de 5(e) année : propositions pour modifier le contenu de l'enseignement.

INTRODUCTION: In France, community pharmacy students performed a hospital pharmacy practice experience during the 5th year of the university curriculum. The purpose of a part of the content of the academic teaching program delivered before this practice experience is to prepare the students for their future hospital activities. It should enable them for the practical use of knowledge in order to improve pharmacotherapy, laboratory diagnosis and monitoring of patients' care. The aim of this study was to show if there are gaps in this program. METHODS: Fourteen students performing their clerkship in a teaching hospital were invited to highlight these gaps when they were gradually immersed in the pharmaceutical care. They did so under the careful observation of hospital pharmacist preceptors. These practitioners referred to professional guidelines, documentary tools used in daily clinical practice and publications supporting their pharmaceutical care practices. RESULTS: Shortcomings and gaps identified were: how to communicate with other healthcare professionals and the content of verbal exchanges, how to conduct a patient-centered consultation, documentation tools required for relevant pharmacist' interventions, codification of pharmacist's interventions, risks related to drug packaging and benefit risk assessment of health information technologies. DISCUSSION: These gaps represent a handicap by delaying the process that led to move from student to healthcare professional. Hospital pharmacist preceptors have to fill in these gaps before engaging students in pharmaceutical care. CONCLUSION: These results invite to revise partly the content of the academic teaching program delivered before the 5th year hospital pharmacy practice experience.

Characterization of five associations of F8 missense mutations containing FVIII B domain mutations.

INTRODUCTION: Haemophilia A (HA) is a bleeding disorder due to an absence or a reduced activity of coagulation factor VIII (FVIII) caused by mutations in F8 gene. Missense mutations represent approximately 45% of the reported molecular defects in HA. However, only few missense mutations in FVIII B domain have been described. AIM: The aim of this study was to characterize five genetic variations (three novels and two previously reported) localized in the FVIII B domain. In all cases, an additional missense variation located outside the FVIII B domain was found. We investigated each of these variations separately and in combination too for their contribution to HA phenotype. METHODS: F8 variants were transiently expressed in COS-1 cells. Media and cell lysates were collected after 72 h. Then, FVIII activity, secretion and thermostability were analysed and compared to FVIII wild-type. RESULTS: The 5 FVIII B domain variants showed normal FVIII: C (98.5-128.5%) and FVIII: Ag (97.7-154%). No synergistic effect was observed between the B domain variant and their associated mutations. In contrast, the variants located outside the B domain, p.V682L, p.S714L, p.V592D and p.C573F revealed significantly decrease of FVIII: C with values in the range 3.5-44.5% (p < 0.05). However, the p.G224R variant showed FVIII: C and FVIII: Ag values no significantly different from FVIII-WT. CONCLUSION: The FVIII B domain variants, p.D963N, p.S806T, p.G873D, p.H998Q and p.Q1225R may be considered as polymorphism or non-pathologic mutations. In five patients, clinical phenotype could be explained by the additional causative missense mutation. For the p.G224T variant further splicing studies are necessary to determine its pathogenicity.

Usefulness of an in vitro cellular expression model for haemophilia A carrier diagnosis: illustration with five novel mutations in the F8 gene in women with isolated factor VIII:C deficiency.

This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low FVIII: C levels (<0.50 IU mL(-1) ), F8 gene variations and without family history of HA. Except p.Ser577Tyr, F8 gene variations that we reported have never been described (p.Leu107His, p.Pro521Leu, p.Val682Leu, p.Leu2032Pro, p.Ala315dup). Prediction of their potential causal impact was studied by two strategies: bioinformatics approaches and site-directed mutagenesis followed by FVIII cellular expression into COS-1 cell. FVIII clotting assay ( FVIII: C) and antigen ( FVIII: Ag) were assayed in vitro. In silico analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The in vitro variant expression model showed abnormal intra and/or extracellular FVIII: C and FVIII: Ag levels for five mutations, which suggest their causality in HA and provide informations about the involved mechanism. We suspect a defect in synthesis and secretion for p.Leu107His, p.Ala315dup and p.Pro521Leu. The mutation p.Val682Leu only affects the FVIII function while p.Ser577Tyr alters function and synthesis. The variant p.Leu2032Pro is probably a polymorphism because no alteration of the FVIII protein expression was observed in vitro. In vitro results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this in vitro cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel F8 gene variation and to provide new criteria for the genetic counselling.

Characterization of four novel molecular changes in the promoter region of the factor VIII gene.

Haemophilia A (HA) is an X-linked recessive bleeding disorder, caused by a wide variety of mutations in the factor VIII (F8) gene, leading to deficiency in the activity of coagulation FVIII. These mutations can affect all the F8 exons from the initiation codon to the termination codon, however, only few molecular changes in the promoter region of the F8 gene were reported so far. Here, we describe six nucleotide variations (c.-51G>A, c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A and c.-664G>A) detected in the F8 promoter and their correlation with clinical phenotype of the patients. Potential role of these mutations in HA was also assessed. Causality was demonstrated with transient transfection experiments using luciferase reporter gene plasmids and computational analysis. Two molecular changes (c.-51G>A and c.-664G>A) did not seem to affect the promoter function of the F8 gene whereas c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A mutations had an impact on the F8 promoter function and were responsible for HA. Furthermore, these mutations were associated with resistance to 1-deamino-8-D-argininevasopressin (desmopressin) therapy when they were causative. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to desmopressin and this clinical implication should be considered by clinicians.

[Student assessment of pharmacy practice experiences in France: a national survey]. / Activités et ressenti des étudiants concernant les stages des études de pharmacie en France.

Pharmacy practice experience (PPE) aims to help trainees to become independent professional practitioners and is a prerequisite for developing skills and competence. In France, it does not exist any study that describes student satisfaction related to preregistration trainings. The aim of this study was to assess student perceptions related to the four PPE types that are planned along the course of study. A questionnaire was sent to each student. Concerning the first and second year introductory PPE, among the 8491 responses, 73% of the students stated that the length was too long regarding their actual knowledge and the objectives were not always achieved. Four thousand and eleven responses regarding third and fourth year PPE were analyzed. Sixty-two percent of the students did not deliver drugs related to the practice's subject and 57% declared doing similar task assigned during the first year PPE especially storage one's. One thousand six hundred and seven questionnaires regarding hospital practices were received. Forty-one percent of the trainees were not asked to perform task that were related to their actual knowledge. Additional comments focused on overmuch shadowing. Among the 853 responses related to the 6th year PPE, 88% of the students considered that they were overall satisfied of the training. Although 30% felt they acquired insufficient skills and professionalism and declared they were not ready to integrate the work world. Those results should prompt decision makers to modify in depth PPE programs in France in order to improve trainees' professionalism and skills acquisition. Promoting and developing researches in the field of PPE is urgently needed.

Synthesis of GPIb beta with novel transmembrane and cytoplasmic sequences in a Bernard-Soulier patient resulting in GPIb-defective signaling in CHO cells.

The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.

Two novel mutations in EGF-like domains of human factor IX dramatically impair intracellular processing and secretion.

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

Description of 10 new mutations in platelet glycoprotein IIb (alphaIIb) and glycoprotein IIIa (beta3) genes.

In this study we have used denaturing gradient gel electrophoresis (DGGE) for identifying sequence alterations in glycoprotein (GP) IIb and IIIa genes from 20 patients affected by Glanzmann's thrombasthenia. These patients were from 16 different families. Using computer modelling, we divided the promoters, coding sequences and flanking splicing regions, in 31 segments for the GPIIb gene and 19 domains for the GPIIIa gene. We were able to find a mutation potentially affecting GPIIb-IIIa expression or function in 16 patients out of 20. In six patients from three families, the gypsy mutation modifying the splice donor site of intron 15 of the GPIIb gene was detected. In the other patients, 10 novel mutations were characterised, which were located either in the GPIIb gene (nine cases) or in the GPIIIa gene (one case). The type of mutation was nonsense mutation (one case), missense mutation (five cases), small insertion of 1 bp (one case) and splicing modifications (three cases). Among these genetic events, three were directly responsible for Glanzmann's thrombasthenia, four were localised in regions known to be involved in GPIIb-IIIa complex expression and three mutations were potentially responsible for Glanzmann's thrombasthenia.