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1.  Modulation at Age of Onset in Tunisian Huntington Disease Patients: Implication of New Modifier Genes 
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. The causative mutation is an expansion of more than 36 CAG repeats in the first exon of IT15 gene. Many studies have shown that the IT15 interacts with several modifier genes to regulate the age at onset (AO) of HD. Our study aims to investigate the implication of CAG expansion and 9 modifiers in the age at onset variance of 15 HD Tunisian patients and to establish the correlation between these modifiers genes and the AO of this disease. Despite the small number of studied patients, this report consists of the first North African study in Huntington disease patients. Our results approve a specific effect of modifiers genes in each population.
doi:10.1155/2014/210418
PMCID: PMC4164136  PMID: 25254119
2.  A xenograft animal model of human arteriovenous malformations 
Background
Arteriovenous malformations (AVMs) are a type of high-flow vascular malformations that most commonly occurs in the head and neck. They are present at birth but are usually clinically asymptomatic until later in life. The pathogenesis of AVMs remains unclear and therapeutic approaches to AVMs are unsatisfied. In order to provide a tool for studying the pathogenesis and therapies of this disease, we established and studied a xenograft animal model of human AVMs.
Methods
Fresh human AVMs specimens harvested from 4 patients were sectioned (5x5x5 mm) and xenografted subcutaneously in 5 immunologically naïve nude mice (Athymic Nude-Foxn1nu). Each mouse had four pieces specimens in four quadrants along the back. The grafts were observed weekly for volume, color and texture. The grafts were harvested at every 30 days intervals for histologic examination. All grafts (n = 20) were sectioned and stained for hematoxylin and eosin (H&E). Comparative pathologic evaluation of the grafts and native AVMs were performed by two blinded pathologists. Immunohistochemical examination of human-specific nuclear antigen, vascular endothelial growth factor receptor-2 (VEGFR-2) and Ki-67 was performed.
Results
Clinical characteristics and pathologic diagnosis of native human derived AVMs were confirmed. 85% (n = 17) of AVM xenografts survived although the sizes decreased after implantation. Histological examination demonstrated numerous small and medium-size vessels and revealed structural characteristics matching the native AVMs tissue.76.5% (n = 13) of the surviving xenografts were positive for Ki-67 and human-specific nuclear antigen suggesting survival of the human derived tissue, 52.9% (n = 9) were positive for VEGFR-2.
Conclusions
This preliminary xenograft animal model suggests that AVMs can survive in the nude mouse. The presence of human-specific nuclear antigen, VEGFR-2, and Ki-67 demonstrates the stability of native tissue qualities within the xenografts.
doi:10.1186/1750-1172-8-199
PMCID: PMC3879430  PMID: 24377858
Arteriovenous malformations; Animal model; Nude mouse; In vivo
3.  Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma 
Nature genetics  2012;44(11):10.1038/ng.2444.
Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics1-3. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK (OMIM #148600), we report heterozygous loss-of-function mutations in AAGAB, encoding alpha- and gamma-adaptin binding protein p34, at a previously linked locus on 15q22. p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicative of a role in membrane traffic. Ultrastucturally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of p34 in keratinocytes led to increased cell division, which was linked to greatly increased epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and proliferation.
doi:10.1038/ng.2444
PMCID: PMC3836166  PMID: 23064416
4.  TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma 
Remke, Marc | Ramaswamy, Vijay | Peacock, John | Shih, David J. H. | Koelsche, Christian | Northcott, Paul A. | Hill, Nadia | Cavalli, Florence M. G. | Kool, Marcel | Wang, Xin | Mack, Stephen C. | Barszczyk, Mark | Morrissy, A. Sorana | Wu, Xiaochong | Agnihotri, Sameer | Luu, Betty | Jones, David T. W. | Garzia, Livia | Dubuc, Adrian M. | Zhukova, Nataliya | Vanner, Robert | Kros, Johan M. | French, Pim J. | Van Meir, Erwin G. | Vibhakar, Rajeev | Zitterbart, Karel | Chan, Jennifer A. | Bognár, László | Klekner, Almos | Lach, Boleslaw | Jung, Shin | Saad, Ali G. | Liau, Linda M. | Albrecht, Steffen | Zollo, Massimo | Cooper, Michael K. | Thompson, Reid C. | Delattre, Oliver O. | Bourdeaut, Franck | Doz, François F. | Garami, Miklós | Hauser, Peter | Carlotti, Carlos G. | Van Meter, Timothy E. | Massimi, Luca | Fults, Daniel | Pomeroy, Scott L. | Kumabe, Toshiro | Ra, Young Shin | Leonard, Jeffrey R. | Elbabaa, Samer K. | Mora, Jaume | Rubin, Joshua B. | Cho, Yoon-Jae | McLendon, Roger E. | Bigner, Darell D. | Eberhart, Charles G. | Fouladi, Maryam | Wechsler-Reya, Robert J. | Faria, Claudia C. | Croul, Sidney E. | Huang, Annie | Bouffet, Eric | Hawkins, Cynthia E. | Dirks, Peter B. | Weiss, William A. | Schüller, Ulrich | Pollack, Ian F. | Rutkowski, Stefan | Meyronet, David | Jouvet, Anne | Fèvre-Montange, Michelle | Jabado, Nada | Perek-Polnik, Marta | Grajkowska, Wieslawa A. | Kim, Seung-Ki | Rutka, James T. | Malkin, David | Tabori, Uri | Pfister, Stefan M. | Korshunov, Andrey | von Deimling, Andreas | Taylor, Michael D.
Acta Neuropathologica  2013;126(6):917-929.
Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1198-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-013-1198-2
PMCID: PMC3830749  PMID: 24174164
TERT promoter mutations; SHH pathway; Adult; Medulloblastoma
5.  Subgroup specific structural variation across 1,000 medulloblastoma genomes 
Northcott, Paul A | Shih, David JH | Peacock, John | Garzia, Livia | Morrissy, Sorana | Zichner, Thomas | Stütz, Adrian M | Korshunov, Andrey | Reimand, Juri | Schumacher, Steven E | Beroukhim, Rameen | Ellison, David W | Marshall, Christian R | Lionel, Anath C | Mack, Stephen | Dubuc, Adrian | Yao, Yuan | Ramaswamy, Vijay | Luu, Betty | Rolider, Adi | Cavalli, Florence | Wang, Xin | Remke, Marc | Wu, Xiaochong | Chiu, Readman YB | Chu, Andy | Chuah, Eric | Corbett, Richard D | Hoad, Gemma R | Jackman, Shaun D | Li, Yisu | Lo, Allan | Mungall, Karen L | Nip, Ka Ming | Qian, Jenny Q | Raymond, Anthony GJ | Thiessen, Nina | Varhol, Richard J | Birol, Inanc | Moore, Richard A | Mungall, Andrew J | Holt, Robert | Kawauchi, Daisuke | Roussel, Martine F | Kool, Marcel | Jones, David TW | Witt, Hendrick | Fernandez-L, Africa | Kenney, Anna M | Wechsler-Reya, Robert J | Dirks, Peter | Aviv, Tzvi | Grajkowska, Wieslawa A | Perek-Polnik, Marta | Haberler, Christine C | Delattre, Olivier | Reynaud, Stéphanie S | Doz, François F | Pernet-Fattet, Sarah S | Cho, Byung-Kyu | Kim, Seung-Ki | Wang, Kyu-Chang | Scheurlen, Wolfram | Eberhart, Charles G | Fèvre-Montange, Michelle | Jouvet, Anne | Pollack, Ian F | Fan, Xing | Muraszko, Karin M | Gillespie, G. Yancey | Di Rocco, Concezio | Massimi, Luca | Michiels, Erna MC | Kloosterhof, Nanne K | French, Pim J | Kros, Johan M | Olson, James M | Ellenbogen, Richard G | Zitterbart, Karel | Kren, Leos | Thompson, Reid C | Cooper, Michael K | Lach, Boleslaw | McLendon, Roger E | Bigner, Darell D | Fontebasso, Adam | Albrecht, Steffen | Jabado, Nada | Lindsey, Janet C | Bailey, Simon | Gupta, Nalin | Weiss, William A | Bognár, László | Klekner, Almos | Van Meter, Timothy E | Kumabe, Toshihiro | Tominaga, Teiji | Elbabaa, Samer K | Leonard, Jeffrey R | Rubin, Joshua B | Liau, Linda M | Van Meir, Erwin G | Fouladi, Maryam | Nakamura, Hideo | Cinalli, Giuseppe | Garami, Miklós | Hauser, Peter | Saad, Ali G | Iolascon, Achille | Jung, Shin | Carlotti, Carlos G | Vibhakar, Rajeev | Ra, Young Shin | Robinson, Shenandoah | Zollo, Massimo | Faria, Claudia C | Chan, Jennifer A | Levy, Michael L | Sorensen, Poul HB | Meyerson, Matthew | Pomeroy, Scott L | Cho, Yoon-Jae | Bader, Gary D | Tabori, Uri | Hawkins, Cynthia E | Bouffet, Eric | Scherer, Stephen W | Rutka, James T | Malkin, David | Clifford, Steven C | Jones, Steven JM | Korbel, Jan O | Pfister, Stefan M | Marra, Marco A | Taylor, Michael D
Nature  2012;488(7409):49-56.
Summary
Medulloblastoma, the most common malignant pediatric brain tumour, is currently treated with non-specific cytotoxic therapies including surgery, whole brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, prior attempts to identify targets for therapy have been underpowered due to small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup enriched. The most common region of focal copy number gain is a tandem duplication of the Parkinson’s disease gene SNCAIP, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1 that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGFβ signaling in Group 3, and NF-κB signaling in Group 4 suggest future avenues for rational, targeted therapy.
doi:10.1038/nature11327
PMCID: PMC3683624  PMID: 22832581
6.  A novel t(3;12)(q21;p13) translocation in a patient with accelerated chronic myeloid leukemia after imatinib and nilotinib therapy 
Cancer Biology & Medicine  2013;10(1):47-51.
The acquisition of secondary chromosomal aberrations in chronic myeloid leukemia (CML) patients with Philadelphia chromosome-positive (Ph+) karyotype signifies clonal evolution associated with the progression of the disease to its accelerated or blastic phase. Therefore, these aberrations have clinical and biological significance. T(3;12)(q26;p13), which is a recurrent chromosomal aberration observed in myeloid malignancies, is typically associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and extremely poor prognosis. We have identified a recurrent reciprocal translocation between chromosomes 3 and 12 with different breakpoint at bands 3q21 in the malignant cells from a 28-year-old man. The patient was initially diagnosed as having Ph+ CML in the chronic phase. The t(3;12)(q21;p13) translocation occurred 4 years after the patient was first diagnosed with CML while undergoing tyrosine kinase inhibitor therapy. We confirmed the t(3;12)(q21;p13) translocation via fluorescence in situ hybridization assay by using whole-chromosome paint probes for chromosomes 3 and 12. Our findings demonstrate that, similar to other recurrent translocations involving 3q26 such as t(3;3) and t(3;21), the t(3;12)(q21;p13) translocation is implicated not only in myelodysplastic syndrome and acute myeloid leukemia but also in the progression of CML. These findings extend the disease spectrum of this cytogenetic aberration.
doi:10.7497/j.issn.2095-3941.2013.01.008
PMCID: PMC3643689  PMID: 23691445
Philadelphia chromosome; t(3; 12)(q21; p13); chronic myeloid leukemia; accelerated phase; fluorescence in situ hybridization
7.  Detection of aneuploidy rate for chromosomes X, Y and 8 by fluorescence in-situ hybridization in spermatozoa from patients with severe non-obstructive oligozoospermia 
Purpose
To evaluate the frequency of sperm nuclei disomy for chromosomes 8, X, and Y in patients with severe non-obstructive oligozoospermia and to assess possible correlations between sperm nuclei aneuploidy and semen parameters or a particular clinical phenotype.
Materials and methods
The sperm aneuploidy rate for chromosomes X, Y, and 8 were assessed in 16 infertile men with severe non-obstructive oligozoospermia and 7 healthy men with normal semen parameters. The frequency of sperm aneuploidy was compared between several patients groups according to their clinical and biological factors.
Results
The total rate of chromosomally abnormal spermatozoa was significantly higher in patients with severe oligozoospermia compared to control group (P < 0.001). A significant relationship was found between the age of patients, sperm concentration, and morphology and the mean rate of sex chromosomes disomy. In addition to the low sperm count (<5 × 106/ml), an elevated FSH level and an exposed to an elevated temperature are two major predictive factors leading to the production of higher numbers of chromosomally abnormal gametes.
Conclusion
Patients with severe oligozoospermia, who are potential candidates for assisted reproduction technology, presented a high level of sex numerical chromosome abnormalities, and consequently are at high risk of chromosome abnormalities in their offspring.
doi:10.1007/s10815-011-9621-x
PMCID: PMC3220440  PMID: 21853383
Aneuploidy; FISH; Predictive factors; Severe oligozoospermia
8.  Chromosomal segregation in spermatozoa of five Robertsonian translocation carriers t(13;14) 
Purpose
To analyse the segregation of a Robertsonian translocation t(13;14) in five male carriers, and to verify a possible inter-chromosomal effect (ICE) of the Robertsonian translocation on chromosomes 18, X, and Y.
Methods
The spermatozoa of these patients (n = 5) and of 15 donors with normal semen parameters and 46,XY karyotype were analysed using triple colour FISH with locus specific probes for chromosomes 13, 14, and 21 and by triple colour FISH for chromosomes X, Y, and 18.
Results
The frequency of balanced spermatozoa resulting from alternate segregation varied between 62.16% and 81.70% with a mean of 71.5%. The rates of unbalanced spermatozoa resulting from adjacent segregation varied between 13.4% and 25.1% with a mean of 18.26%. Triple colour FISH X-Y-18 showed a significant increase in disomy frequencies of these chromosomes in comparison with controls, indicating an ICE.
Conclusion
In spite of the high number of normal/balanced frequencies, there remain many unbalanced spermatozoa resulting from adjacent mode of segregation. This raises the question of the unbalanced chromosomal risk for the offspring of 45,XY, t(13;14) males and the importance of the genetic counselling prior to ICSI or IVF treatment for couples where the male is a Robertsonian translocation carrier.
doi:10.1007/s10815-011-9560-6
PMCID: PMC3162055  PMID: 21448573
FISH; Meiotic segregation; Robertsonian translocation; Spermatozoa
9.  The effects of male aging on semen quality, sperm DNA fragmentation and chromosomal abnormalities in an infertile population 
Purpose
To investigate the effects of male aging on semen quality, DNA fragmentation and chromosomal abnormalities in the spermatozoa of infertile patients and fertile men.
Methods
Semen samples of 140 infertile patients (24–76 years) and 50 men with proven fertility (25–65 years) were analyzed according to WHO guidelines. DNA fragmentation was detected by TUNEL assay, while aneuploidy was assessed by FISH.
Results
In the patient group, semen volume and vitality of spermatozoa decreased significantly with age, while sperm concentration showed a statistically significant increase with age. DNA fragmentation as well as disomy of sex chromosomes and disomy 8 did not show a statistically significant change with age. However, the diploidy rate was significantly increased with patient’s age. In the control group, conventional semen parameters as well as DNA fragmentation and chromosomal abnormalities did not show a statistically significant with age.
Conclusion
Increased age in infertile men is associated with an increase in sperm concentration and diploidy, as well as a decline in semen volume and sperm vitality. However motility, morphology and DNA fragmentation are not affected by male age.
doi:10.1007/s10815-011-9537-5
PMCID: PMC3151353  PMID: 21287403
Aneuploidy; DNA fragmentation; Male infertility; Paternal age; Semen parameters
10.  Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men 
BMC Urology  2012;12:6.
Background
Growing evidence indicates that oxidative stress can be a primary cause of male infertility. Non-enzymatic antioxidants play an important protective role against oxidative damages and lipid peroxidation. Human seminal plasma is a natural reservoir of antioxidants. The aim of this study was to determine glutathione (GSH) concentrations, trace element levels (zinc and selenium) and the lipid peroxidation end product, malondialdehyde (MDA), in the seminal plasma of men with different fertility potentials.
Methods
Semen samples from 60 fertile men (normozoospermics) and 190 infertile patients (74 asthenozoospermics, 56 oligozoospermics, and 60 teratozoospermics) were analyzed for physical and biochemical parameters. Zinc (Zn) and selenium (Se) levels were estimated by atomic absorption spectrophotometry. Total GSH (GSHt), oxidized GSH (GSSG), reduced GSH (GSHr) and MDA concentrations were measured spectrophotometrically.
Results
Zn and Se concentrations in seminal plasma of normozoospermics were more elevated than the three abnormal groups. Nevertheless, only the Zn showed significant differences. On the other hand, Zn showed positive and significant correlations with sperm motility (P = 0.03, r = 0.29) and count (P < 0.01, r = 0.49); however Se was significantly correlated only with sperm motility (P < 0.01, r = 0.36). GSHt, GSSG and GSHr were significantly higher in normozoospermics than in abnormal groups. We noted a significant association between seminal GSHt and sperm motility (P = 0.03). GSSG was highly correlated to sperm motility (P < 0.001) and negatively associated to abnormal morphology (P < 0.001). GSHr was significantly associated to total sperm motility (P < 0.001) and sperm count (P = 0.01). MDA levels were significantly higher in the three abnormal groups than in normozoospermics. Rates of seminal MDA were negatively associated to sperm motility (P < 0.01; r = -0.24) and sperm concentration (P = 0.003; r = -0.35) Meanwhile, there is a positive correlation between seminal lipid peroxidation and the percentage of abnormal morphology (P = 0.008).
Conclusions
This report revealed that decreased seminal GSH and trace element deficiencies are implicated in low sperm quality and may be an important indirect biomarker of idiopathic male infertility. Our results sustain that the evaluation of seminal antioxidant status in infertile men is necessary and can be helpful in fertility assessment from early stages.
doi:10.1186/1471-2490-12-6
PMCID: PMC3349502  PMID: 22429816
Antioxidants; Idiopathic oligoasthenoteratozoospermia; Male infertility; Oxidative stress; Reactive oxygen species; Spermatozoa; Seminal plasma
11.  Detection of DNA fragmentation and meiotic segregation in human with isolated teratozoospermia 
Purpose
To evaluate levels of DNA fragmentation and chromosomal abnormalities in ejaculated sperm of males with isolated teratozoospermia and to determine if specific sperm morphological types occur simultaneously with these nuclear defects.
Methods
Sperm obtained from isolated teratozoospermic men (n = 70) and fertile men (n = 30) were analysed using fluorescence in situ hybridization and TUNEL assay.
Results
Teratozoospermic men, compared to fertile men, showed significantly higher rates of sex chromosomes disomy, and diploidy. Significant correlations were found between amorphous head, microcephalic head, short tail, and sex chromosomes disomy. Level of sperm DNA fragmentation was significantly higher in teratozoospermic men than in controls and positively correlated to the incidence of macrocephalic heads, amorphous heads, and short flagella.
Conclusions
Patients with isolated teratozoospermia have increased levels of DNA fragmentation and chromosomal aneuploidy. Some specific morphological abnormalities were shown to be predictive of chromosomal abnormalities and DNA alteration.
doi:10.1007/s10815-010-9482-8
PMCID: PMC3045490  PMID: 20872065
Aneuploidy; DNA fragmentation; Infertility; Teratozoospermia
12.  Altered Antioxidant Status and Increased Lipid Per-Oxidation in Seminal Plasma of Tunisian Infertile Men 
Human seminal plasma is a natural reservoir of antioxidants that protect spermatozoa from oxidative damages. There is evidence in literature supports the fact that impairments in seminal antioxidant and lipid per-oxidation status play important roles in the physiopathology of male infertility. Our present study forms the first one which was carried out in Tunisia. We evaluated the antioxidant status in the seminal plasma of 120 infertile men programmed to In Vitro Fertilization (IVF) for the first tentative. Patients were characterized by an idiopathic infertility. They were divided into three groups: normozoospermics who were considered as controls (n=40), asthenozoospermics (Astheno; n=45) and oligoasthenoteratozoospermics (OAT; n=35). Seminal activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) and the levels of glutathione (GSH), zinc (Zn) and malondialdehyde (MDA) were measured. With the significant increase of the seminal activities of SOD and GPX in normozoospermics group, there were positive correlations observed between this enzymes and sperm quality. Also, significant elevated rates of seminal zinc and GSH were observed in control group, but there was contradictory associations reflecting the effects of these antioxidants on semen parameters. However, we noted significant increase of MDA levels in groups with abnormal seminogram. We showed negative associations between this per-oxidative marker and sperm parameters. These results obviously suggested that impairment on seminal antioxidants is an important risk factor for low sperm quality associated to idiopathic infertility and as a result can lead to poor IVF outcome.
PMCID: PMC3248656  PMID: 22211112
Oxidative damage; Antioxidant enzymes; Semen quality; Male infertility; Sperm abnormalities; lipid per-oxidation.
13.  Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe 
Background
The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures.
Methods
Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue.
In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests.
Results
Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing.
After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r = 0.82, p < 0.05) or calcein AM/ethidium homodimer-1 staining (r = 0.76, p < 0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60 +/- 1.1% vs 52.2 +/- 7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26 ± 8.2% vs 38 ± 4.5%).
Conclusion
We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.
doi:10.1186/1477-7827-9-78
PMCID: PMC3128841  PMID: 21651765
14.  Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes 
Nature genetics  2010;42(7):619-625.
Joubert syndrome (JBTS), related disorders (JSRD) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and JBTS2 loci are allelic and due to mutations in TMEM216, encoding an uncharacterized tetraspan transmembrane protein. JBTS2 patients displayed frequent nephronophthisis and polydactytly, and two cases conformed to the Oro-Facio-Digital type VI phenotype, whereas skeletal dysplasia was common in MKS fetuses. A single p.R73L mutation was identified in all patients of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in patient fibroblasts or following siRNA knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 complexed with Meckelin, encoded by a gene also mutated in JSRD and MKS. Abrogation of tmem216 expression in zebrafish led to gastrulation defects that overlap with other ciliary morphants. The data implicate a new family of proteins in the ciliopathies, and further support allelism between ciliopathy disorders.
doi:10.1038/ng.594
PMCID: PMC2894012  PMID: 20512146
15.  Treatment of Acute Promyelocytic Leukemia with AIDA Based Regimen. Update of a Tunisian Single Center Study 
In Tunisia, the ATRA era began in 1998 with the use, consecutively, of two regimens combining ATRA and an anthracycline with cytarabine (APL93), and without cytarabine (LPA99). From 2004, 51 patients with confirmed APL either by t(15;17) or PML/RARA were treated according to the PETHEMA LPA 99 trial. Forty three patients achieved CR (86%). The remaining seven patients had early death (one died before treatment onset): four caused by differentiation syndrome (DS) and three died from central nervous system hemorrhage. Multivariate analysis revealed that female gender (P=0.045), baseline WBC> 10 G/L (P=0.041) and serum creatinine > 1.4mg/dl (P=0.021) were predictive of mortality during induction. DS was observed in 16 patients (32%) after a median onset time of 15 days from treatment onset (range, 2–29). Body mass index ≥ 30 (P=0.01) remained independent predictor of DS. Occurrence of hypertensive peaks significantly predicted occurrence of DS (P=0.011) and was significantly associated with high BMI (p=0.003). With a median follow-up of 50 months, 5 year cumulative incidence of relapse, event free and overall survival were 4.7%, 74% and 78%, respectively.
doi:10.4084/MJHID.2011.033
PMCID: PMC3212966  PMID: 22084648
16.  CC2D2A mutations in Meckel and Joubert syndromes indicate a genotype-phenotype correlation 
Human mutation  2009;30(11):1574-1582.
The Meckel syndrome (MKS) is a lethal fetal disorder characterized by diffuse renal cystic dysplasia, polydactyly, a brain malformation that is usually occipital encephalocele and/or vermian agenesis, with intrahepatic biliary duct proliferation. Joubert syndrome (JBS) is a viable neurological disorder with a characteristic “molar tooth sign” (MTS) on axial images reflecting cerebellar vermian hypoplasia/dysplasia. Both conditions are classified as ciliopathies with an autosomal recessive mode of inheritance. Allelism of MS and JBS has been reported for TMEM67/MKS3, CEP290/MKS4, and RPGRIP1L/MKS5. Recently, one homozygous splice mutation with a founder effect was reported in the CC2D2A gene in Finnish fetuses with MKS, defining the 6th locus for MKS. Shortly thereafter, CC2D2A mutations were reported in JBS also. The analysis of the CC2D2A gene in our series of MKS fetuses, identified 14 novel truncating mutations in 11 cases. These results confirm the involvement of CC2D2A in MKS and reveal a major contribution of CC2D2A to the disease. We also identified three missense CC2D2A mutations in two JBS cases. Therefore and in accordance with the data reported regarding RPGRIP1L, our results indicate phenotype-genotype correlations, as missense and presumably hypomorphic mutations lead to JBS while all null alleles lead to MKS.
doi:10.1002/humu.21116
PMCID: PMC2783384  PMID: 19777577
Meckel-Gruber syndrome; MKS; Joubert syndrome; JBS; CC2D2A; ciliopathy
17.  ETV6-RUNX1 Rearrangement in Tunisian Pediatric B-Lineage Acute Lymphoblastic Leukemia 
Advances in Hematology  2009;2009:924301.
In this study, Forty-one out of fifty-seven Tunisian children with B-lineage acute lymphoblastic leukemia (B-ALL), and without cytogenetically detectable recurrent abnormalities at the time of the diagnosis, were evaluated by fluorescence in situ hybridization (FISH) for the t(12;21). This translocation leads ETV6-RUNX1 (previously TEL-AML1) fusion gene. 16 patients (28%) had ETV6-RUNX1 rearrangement. In addition to this rearrangement, two cases showed a loss of the normal ETV6 allele, and three others showed an extra signal of the RUNX1 gene. Seven patients without ETV6-RUNX1 rearrangement showed extra signals of the RUNX1 gene. One out of the 7 patients was also associated with a t(3;12) identified by FISH. This is the first Tunisian study in which we report the incidence of t(12;21) among childhood B-lineage ALL and in which we have found multiple copies of RUNX1. Finally, our findings confirm that additional or secondary genetic changes are commonly encountered in pediatric B-lineage ALL with ETV6-RUNX1 gene fusion which is envisaged to play a pivotal role in disease progression.
doi:10.1155/2009/924301
PMCID: PMC2799269  PMID: 20049174
18.  Immunohistochemical Markers Associated with Brain Metastases in Patients with Non-Small Cell Lung Carcinoma 
Cancer  2008;113(8):2129-2138.
BACKGROUND
There are no reliable markers able to identify patients with non-small cell lung cancer (NSCLC) likely to metastasize to the brain. We investigated associations between immunohistochemical markers and development of brain metastases in patients with NSCLC.
METHODS
We performed a hospital-based, case-control study of patients with newly diagnosed NSCLC between 1989 and 2003 that developed brain metastases who had available pathology material from both primary NSCLC and brain metastases. The control patients had NSCLC and no evidence of brain metastases. We examined NSCLC for expression of Ki-67, caspase-3, VEGF-A, VEGF-C, E-cadherin and EGFR in 54 surgical pathology specimens using immunohistochemistry and evaluated associations with development of brain metastases.
RESULTS
Brain metastases developed after a median time of 12.5 months (range 1.7-89.4 months) from the diagnosis of NSCLC. A significantly increased risk of developing brain metastases was associated with patients who had high Ki-67 (adjusted odds ratio 12.2, 95% CI, 2.4 to 70.4, P<0.001), low caspase-3 (adjusted odds ratio 43.0, 95% CI, 5.3 to >100, P<0.001), high VEGF-C (adjusted odds ratio 14.6, 95% CI, 2.0 to >100, P<0.001), and low E-cadherin (adjusted odds ratio 3.6, 95% CI, 0.9 to 16.4, P=0.05), in the primary NSCLC. No significant risk was observed with VEGF-A and EGFR. A high Ki-67 was also associated with a shorter overall survival (P=0.04).
CONCLUSIONS
Patients with NSCLC and high Ki-67, low caspase-3, high VEGF-C, and low E-cadherin in their tumors may benefit from close surveillance since they may have an increased risk of developing brain metastases.
doi:10.1002/cncr.23826
PMCID: PMC2597625  PMID: 18720359
19.  Cathepsin S Deficiency Confers Protection from Neonatal Hyperoxia-induced Lung Injury 
Rationale: Bronchopulmonary dysplasia (BPD) is a chronic lung disease that adversely affects long-term pulmonary function as well as neurodevelopmental outcomes of preterm infants. Elastolytic proteases have been implicated in the pathogenesis of BPD. Cathepsin S (cat S) is a cysteine protease with potent elastolytic activity. Increased levels and activity of cat S have been detected in a baboon model of BPD.
Objectives: To investigate whether deficiency of cat S alters the course of hyperoxia-induced neonatal lung injury in mice.
Methods: Newborn wild-type and cat S–deficient mice were exposed to 80% oxygen for 14 days. Histologic and morphometric analysis were performed and bronchoalveolar lavage protein and cells were analyzed. Lung elastin was assessed by real-time polymerase chain reaction, in situ hybridization, desmosine analysis, and Hart's stain. Distribution of myofibroblasts was analyzed by immunofluorescence. Hydroxyproline content of lung tissues was measured.
Measurements and Main Results: Hyperoxia-exposed cat S–deficient mice were protected from growth restriction and had improved alveolarization, decreased septal wall thickness, lower number of macrophages, and lower protein concentration in bronchoalveolar lavage fluid. α-Smooth muscle actin–expressing myofibroblasts accounted for at least some of the increased interstitial cellularity in hyperoxia-exposed mouse lungs and were significantly less in cat S–deficient lungs. Lung hydroxyproline content was increased in hyperoxia-exposed wild-type, but not in cat S–deficient lungs. Desmosine content was significantly reduced in both genotypes with hyperoxia.
Conclusions: Cathepsin S deficiency improves alveolarization, and attenuates macrophage influx and fibroproliferative changes in hyperoxia-induced neonatal mouse lung injury.
doi:10.1164/rccm.200704-519OC
PMCID: PMC2020827  PMID: 17673697
cathepsin; bronchopulmonary dysplasia; hyperoxia; myofibroblast
20.  Orientation determination by wavelets matching for 3D reconstruction of very noisy electron microscopic virus images 
Background
In order to perform a 3D reconstruction of electron microscopic images of viruses, it is necessary to determine the orientation (Euler angels) of the 2D projections of the virus. The projections containing high resolution information are usually very noisy. This paper proposes a new method, based on weighted-projection matching in wavelet space for virus orientation determination. In order to speed the retrieval of the best match between projections from a model and real virus particle, a hierarchical correlation matching method is also proposed.
Results
A data set of 600 HSV-1 capsid particle images in different orientations was used to test the proposed method. An initial model of about 40 Å resolutions was used to generate projections of an HSV-1 capsid. Results show that a significant improvement, in terms of accuracy and speed, is obtained for the initial orientation estimates of noisy herpes virus images. For the bacteriophage (P22), the proposed method gave the correct reconstruction compared to the model, while the classical method failed to resolve the correct orientations of the smooth spherical P22 viruses.
Conclusion
This paper introduces a new method for orientation determination of low contrast images and highly noisy virus particles. This method is based on weighted projection matching in wavelet space, which increases the accuracy of the orientations. A hierarchical implementation of this method increases the speed of orientation determination. The estimated number of particles needed for a higher resolution reconstruction increased exponentially. For a 6 Å resolution reconstruction of the HSV virus, 50,000 particles are necessary. The results show that the proposed method reduces the amount of data needed in a reconstruction by at least 50 %. This may result in savings 2 to 3 man-years invested in acquiring images from the microscope and data processing. Furthermore, the proposed method is able to determine orientations for some difficult particles like P22 with accuracy and consistency. Recently a low PH sindbis capsid was determined with the proposed method, where other methods based on the common line fail.
doi:10.1186/1472-6807-5-5
PMCID: PMC555555  PMID: 15743519
21.  Wavelets filtering for classification of very noisy electron microscopic single particles images- application on structure determination of VP5-VP19C recombinant 
Background
Images of frozen hydrated [vitrified] virus particles were taken close-to-focus in an electron microscope containing structural signals at high spatial frequencies. These images had very low contrast due to the high levels of noise present in the image. The low contrast made particle selection, classification and orientation determination very difficult. The final purpose of the classification is to improve the signal-to-noise ratio of the particle representing the class, which is usually the average. In this paper, the proposed method is based on wavelet filtering and multi-resolution processing for the classification and reconstruction of this very noisy data. A multivariate statistical analysis (MSA) is used for this classification.
Results
The MSA classification method is noise dependant. A set of 2600 projections from a 3D map of a herpes simplex virus -to which noise was added- was classified by MSA. The classification shows the power of wavelet filtering in enhancing the quality of class averages (used in 3D reconstruction) compared to Fourier band pass filtering. A 3D reconstruction of a recombinant virus (VP5-VP19C) is presented as an application of multi-resolution processing for classification and reconstruction.
Conclusion
The wavelet filtering and multi-resolution processing method proposed in this paper offers a new way for processing very noisy images obtained from electron cryo-microscopes. The multi-resolution and filtering improves the speed and accuracy of classification, which is vital for the 3D reconstruction of biological objects. The VP5-VP19C recombinant virus reconstruction presented here is an example, which demonstrates the power of this method. Without this processing, it is not possible to get the correct 3D map of this virus.
doi:10.1186/1472-6807-3-9
PMCID: PMC317332  PMID: 14667245
22.  Roles of Triplex and Scaffolding Proteins in Herpes Simplex Virus Type 1 Capsid Formation Suggested by Structures of Recombinant Particles 
Journal of Virology  1999;73(8):6821-6830.
Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-Å diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-Å diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid.
PMCID: PMC112767  PMID: 10400780
23.  Genetics of low spinal muscular atrophy carrier frequency in sub‐Saharan Africa 
Annals of Neurology  2014;75(4):525-532.
Objective
Spinal muscular atrophy (SMA) is one of the most common severe hereditary diseases of infancy and early childhood in North America, Europe, and Asia. SMA is usually caused by deletions of the survival motor neuron 1 (SMN1) gene. A closely related gene, SMN2, modifies the disease severity. SMA carriers have only 1 copy of SMN1 and are relatively common (1 in 30–50) in populations of European and Asian descent. SMN copy numbers and SMA carrier frequencies have not been reliably estimated in Malians and other sub‐Saharan Africans.
Methods
We used a quantitative polymerase chain reaction assay to determine SMN1 and SMN2 copy numbers in 628 Malians, 120 Nigerians, and 120 Kenyans. We also explored possible mechanisms for SMN1 and SMN2 copy number differences in Malians, and investigated their effects on SMN mRNA and protein levels.
Results
The SMA carrier frequency in Malians is 1 in 209, lower than in Eurasians. Malians and other sub‐Saharan Africans are more likely to have ≥3 copies of SMN1 than Eurasians, and more likely to lack SMN2 than Europeans. There was no evidence of gene conversion, gene locus duplication, or natural selection from malaria resistance to account for the higher SMN1 copy numbers in Malians. High SMN1 copy numbers were not associated with increased SMN mRNA or protein levels in human cell lines.
Interpretation
SMA carrier frequencies are much lower in sub‐Saharan Africans than in Eurasians. This finding is important to consider in SMA genetic counseling in individuals with black African ancestry. Ann Neurol 2014;75:525–532
doi:10.1002/ana.24114
PMCID: PMC4112719  PMID: 24515897

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