[Creation and report of the Tunisian Fanconi Anemia Registry (TFAR)]. / Création et rapport du registre tunisien de l'anémie de Fanconi (TFAR).

INTRODUCTION: Fanconi anemia (FA) is a genetically and phenotypically heterogeneous inherited disease. Many groups have established FA registries. In Tunisia, in collaboration with the Tunisian Fanconi Anemia Study Group (TFASG), we set up the Tunisian Fanconi Anemia Registry (TFAR). PATIENTS AND METHODS: We contacted all hematology and pediatrics departments to include their FA patients diagnosed between January 1983 and December 2008. The registry is available on the TFASG web site (www.fanconi-tunisie.net). RESULTS: Sorting the files brought out 142 patients belonging to 118 families. The mean age at diagnosis was 11 years. There was consanguinity in 86%, malformative syndrome in 91%, and pancytopenia at diagnosis in 69%. Of 28 patients, 95% belonged to the FANCA group. Androgen treatment was given in 109 cases and genoidentical bone marrow transplantation (BMT) in 27 patients. The diagnosis of a myelodysplastic syndrome was retained in 4%, acute leukemia in 6%, and a solid tumor in 2%. The median overall survival time in all patients is 17 years 5 months; it is significantly better in patients having received allografts (p=0.01). CONCLUSION: FA seems frequent in Tunisia, which is in part explained by the high consanguinity and endogamy in this country. Hematologic impairment is still the most frequent revealing circumstance of the disease. It is often severe or moderate and requires androgen treatment or bone marrow transplantation. BMT should be proposed to all patients with an HLA-compatible donor.

Study of aneuploidy rate and sperm DNA fragmentation in large-headed, multiple-tailed spermatozoa.

The aim of this study was to analyse the meiotic segregation and DNA fragmentation rates in ejaculated spermatozoa of Tunisian men who presented the macrocephalic sperm head syndrome and to compare the results with those from 20 fertile men with normal semen profiles. Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay. Fluorescence in situ hybridisation for chromosomes X, Y and 18 was performed for the study of meiotic segregation. Despite a normal blood karyotype, patients with large-headed spermatozoa showed a significantly higher incidence of sperm chromosomal abnormalities compared with the control group. For all the patients, tetraploidy, triploidy and diploidy were the most observed abnormalities. A very high level of DNA fragmentation was shown for these patients. In conclusion, our results demonstrated that patients with large-headed, multiple-tailed spermatozoa had significantly higher incidence of sperm chromosomal abnormalities and very high level of DNA fragmentation. So intracytoplasmic sperm injection should not be recommended to these patients, not only because of its low chances of success rate but also because of its high genetic risk.

Aneuploidy rate in spermatozoa of selected men with severe teratozoospermia.

The aim of this study was to evaluate the incidence of spermatic aneuploidies in men with severe teratozoospermia and to determine an eventual relation between aneuploidies and a specific morphology of spermatozoa. Fluorescence in situ hybridisation (FISH) using a probe cocktail containing the alpha satellite for the centromeric region of chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of thirty patients with severe teratozoospermia (abnormal forms >80%) and 15 fertile men with normal semen profiles. The mean frequency of teratozoospermia in patients was 91 ± 6.99%. There was statistically a significantly increased frequency of 1818, XY, XX and YY disomies in sperm with severe teratozoospermia compared with normal sperm (1.24% versus 0.08%, 1.42% versus 0.31%, 1.13% versus 0.19% and 1.11% versus 0.24%, respectively, P < 0.001 in all comparisons). The rate of total diploidy was significantly increased in patients compared with controls (1.46% versus 0.16%, P < 0.001). There was a correlation between macrocephalic spermatozoa and diploidy (r = 0.37, P < 0.05). Our data add further evidence that patients with severe teratozoospermia have an increased sperm aneuploidy rate and that this is particularly high in macrocephalic spermatozoa; FISH analysis on sperm could help to improve risk assessment and reproductive counselling in these individuals who are frequently candidates for intracytoplasmic sperm injection (ICSI) as a treatment of their infertility, as the use of ICSI has created consequential debate concerning the genetic risk for the offspring.

Clinical and molecular characterization of a combined 17p13.3 microdeletion with partial monosomy 21q21.3 in a 26-year-old man.

We led a clinical and molecular characterization of a patient with mild mental delay and dysmorphic features initially referred for cytogenetic exploration of an azoospermia. We employed FISH and array CGH techniques for a better definition and refinement of a double chromosome aberration associating a 17p microdeletion with partial monosomy 21q due to 1:3 meiotic segregation of a maternal reciprocal translocation t(17;21)(p13.3;q21.2) revealed after banding analysis. Brain MRI depicted partial callosal and mild diffuse cerebral atrophies, but without expected signs of lissencephaly. The patient's karyotype formula was: 45,XY,der(17)t(17;21)(p13.3;q21.2)mat,-21. FISH study confirmed these rearrangements and array CGH analysis estimated the loss sizes to at least 635 kb on chromosome 17 and to 15.6 Mb on chromosome 21. The absence of lissencephaly and major brain malformations often associated with 17p terminal deletions could be attributed to the retention of PAFAH1B1, YWHAE and CRK genes. Dysmorphic features, moderate mental impairment and minor brain malformations could result from the 21q monosomy and particularly the partial deletion of the APP-SOD1 region. Azoospermia should result from gamete apoptosis induced by a control mechanism triggered in response to chromosome imbalances. Our study provides an additional case for better understanding and delineating both 17p and 21q deletions.

Semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity.

The objective of this study was to determine the effects of density gradient centrifugation on sperm cell DNA integrity and to correlate any detected DNA damage with semen analysis parameter. A total of 40 semen samples were collected from nonazoospermic men presenting for infertility evaluation at our department. Individual samples were divided into two parts: one part of the semen was washed and the remainder was prepared using the PureSperm density gradient centrifugation. Sperm DNA fragmentation as evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay, was monitored in the initially washed sample and in the different layers of the density gradient centrifugation. No significant correlations were observed between sperm DNA fragmentation, age of patient, concentration and motility. However, a significant correlation existed with strict spermatic morphology. Following density gradient centrifugation, the proportion of spermatozoa with DNA fragmentation decreased significantly when compared with whole semen. In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of DNA damage when compared with those remaining in the 70% and 50% layers. These results demonstrate that semen processing by the PureSperm gradient is useful in selecting sperm with higher double-strand DNA integrity.

[Place of pelvic ultrasonography using transabdominal technique in the investigation of premature ovarian failure]. / Place de l'échographie pelvienne réalisée par voie sus-pubienne dans l'exploration de l'insuffisance ovarienne prématurée.

OBJECTIVES: The aim of the present study was to characterize women with premature ovarian failure (POF) by their ovarian ultrasonographic appearances using transabdominal technique to establish the relationship to clinical, hormonal status, and genetic analysis. PATIENTS AND METHODS: We studied a cohort of 80 patients suffering from POF. The surface of the ovary was calculated and we identified the detection or not of follicles. RESULTS: The detection of the two ovaries by ultrasound was positive in 33 patients; only one ovary was identified in seven patients; none was noted in 40 patients. The surface of the ovaries ranged between 0.74 et 5.92 cm(2) (2.2+/-1.13 cm(2)). Ultrasonography identified follicles in 23 patients (28.75%). The presence of follicles suggested at ultrasonography was detected in 14 cases (70%) in normal-sized ovaries (> or =2 cm(2)) and in nine cases (45%) in small-sized ovaries (p=0.1). No significative statistical difference was found between the ultrasonographic appearances and the type of amenorrhea, pubertal development, hormonal status (estradiol, testosterone and delta-4-androstendione) and the chromosomal analysis. CONCLUSION: The clinical and hormonal status and the genetic analysis can't predict the presence or not of follicles in the ovaries of patients with POF.

[Postnatal diagnosis and prognosis of 2 cases of triploidy]. / Diagnostic postnatal et pronostic de 2 cas de triploïdie.

Triploidy is one of the most common chromosomal aberrations in spontaneous abortions characterized by a 69-chromosome karyotype. This chromosome abnormality is rare in live-born children. Prevalence is lower than 1/50,000. We report on two premature newborns, male and female, born at 35 and 37 weeks of gestation, who presented with severe intrauterine growth retardation, facial dysmorphy, myelomeningocele, and syndactyly. They died during the first hours of life due to respiratory distress syndrome. Analysis of the karyotype showed a homogeneous triploidy on all mitoses: 69 XXY and 69 XXX. The parental origin of the triploidy can have specific effects in the fetal phenotype and the development of the placenta.

Cytogenetic analyses of premature ovarian failure using karyotyping and interphase fluorescence in situ hybridization (FISH) in a group of 1000 patients.

To evaluate the implication of chromosome abnormalities in the etiology of premature ovarian failure (POF), 1000 patients with POF recruited at the Department of Cytogenetics of Farhat Hached Hospital (Sousse, Tunisia) between January 1996 and December 2008. Chromosome analyses were performed by using karyotyping and interphase fluorescent in situ hybridisation (FISH) using a centromeric probe of the chromosome X to look for low-level mosaicism of X-chromosome monosomy. Hundred and eight chromosomal abnormalities (10.8%) were found using karyotype analysis. Anomalies were detected in 61 cases out of 432 primary amenorrhea patients (14.12%) and 47 cases out of 568 secondary amenorrhea patients (8.27%). In 23 POF patients among 200 (11.5%) with 46,XX normal karyotype and explored using interphase FISH analysis, the percentage of cells with X-chromosome monosomy was significantly higher as compared with controls in the same age. The cytogenetic study of POF patients showed a high prevalence of chromosome anomalies either in primary or in secondary amenorrhoea. Mosaic X-chromosome s aneuploïdy was the most frequent abnormality and some patients with POF may be attributable to low-level 45,X/46,XX mosaicism detectable using FISH analysis.

Prenatal diagnosis and prognosis of triple X syndrome: 47, XXX.

Triple X syndrome is a relatively common sex chromosomal abnormality occurring in 0,1% of live-born female infants. Most of these infants have a normal phenotype and only a few cases with 47, XXX karyotype have congenital malformations. We report three cases of triple X syndrome that were diagnosed prenatally by genetic amniocentesis for advanced maternal age and have been observed from birth to age of 3 to 12 years. A description of their growth and development is presented. The birth weight was normal in all patients and one of them had facial dysmorphism with right microphtalmia and auricular septal defect. During the first 2 years of life, the neuromotor development of these infants was not distinguishable from chromosomally normal children. By 3 years of age, two patients have a moderate developmental delay in speech and language. One girl 12-year-old had normal schooling. The diagnosis of the triple X syndrome can be never made because clinical demonstrations are not rather important to arouse the demand of a karyotype. Prenatal diagnosis is often made in front of the advanced maternal age. Expectant parents must be counseled as to the significance of this 47, XXX karyotype and prognostic information must be given.

Functional evidence implicating FOXL2 in non-syndromic premature ovarian failure and in the regulation of the transcription factor OSR2.

BACKGROUND: FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), involving craniofacial/palpebral abnormalities often associated with premature ovarian failure (POF). RESULTS: We describe a FOXL2 variant (p.Gly187Asp) in a case of POF without BPES. The subcellular localisation of FOXL2-G187D was normal but its transactivation capacity tested on two reporter promoters, one of which should be relevant to the ovary, was significantly lower than that of normal FOXL2. However, FOXL2-G187D was able to activate strongly a reporter construct driven by the promoter of Osr2 (odd-skipped related 2 transcription factor), which we have suggested to be a crucial target of FOXL2 in the craniofacial region. This is compatible with the absence of BPES in our patient. CONCLUSIONS: Our data provide evidence in favour of the implication of FOXL2 variants in non-syndromic POF and confirm the regulatory interaction between FOXL2 and OSR2 whose perturbation might contribute to the palpebral abnormalities observed in BPES patients.

[Genetic analysis of premature ovarian failure: role of forkhead and TGF-beta genes]. / Analyse génétique des insuffisances ovariennes prématurées: implication des gènes forkhead et TGF-beta.

Premature ovarian failure is a common pathology affecting 1% of women. Although multiple etiologies have been described the majority of cases are idiopathic. Forkhead transcription factors as FOXL2 and FOXO3A are of particular interest in the research of genetic factors related with the pathology as they are present in diverse developmental pathways and ovarian physiology. Similarly, some TGF-beta factors (i.e. BMP 15 and GDF-9) have been demonstrated to play a key role in the regulation, at ovarian level, of female reproduction. In recent years numerous studies have been performed in order to elucidate the implication of these factors in the ovarian physiopathology. The aim of this manuscript is to describe some of these advances in the context of premature ovarian failure.

Placental mesenchymal dysplasia with beckwith-wiedemann syndrome fetus in the context of biparental and androgenic cell lines.

Placental mesenchymal dysplasia (PMD) is a distinct placental disorder that may coexist with a normal fetus. In one-third of cases, the fetus exhibits Beckwith-Wiedemann Syndrome (BWS). In the present study, we report a case of PMD changes associated with an unusual genetic constitution. Pathological examination showed an enlarged placenta with a mixture of normal but also numerous clusters of grape-like fluid-filled vesicles confined to the stem villi without trophoblast proliferation. Some stem villi contained many large vessels filled by partially organized thrombi consistent with PMD. The fetus presented an enlarged liver and cytomegaly in the adrenal glands, hyperplastic islets of Langerhans in the pancreas, and some microcysts with cuboidal epithelium in the kidneys. These findings suggest the Beckwith-Wiedemann syndrome phenotype. DNA genetic markers showed three alleles for three independent markers and two alleles for the 12 others. Fluorescent in situ hybridization (FISH) demonstrated that villous trophoblast and fetal tissues are diploid. The haploid paternal complement found in the androgenetic cells was different from that found in biparental cells, suggesting a double fertilization event. Preferential distribution of the androgenetic cells into the placenta explains the predominance of molar villi with an apparently normal fetus. This represents a well-documented case of androgenic and biparental mixture of cell types in both fetal and placental tissues.

[Nosologic discussion between Fanconi disease and congenital dyskeratosis: 1 case of congenital bone marrow aplasia]. / Discussion nosologique entre dyskératose congénitale et maladie de Fanconi: à propos de 1 cas d'aplasie médullaire constitutionnelle.

UNLABELLED: Based on a case report of aplastic anemia associated with malformation, we discuss the diagnostic criteria and the nosologic problem between the 2 principal aplastic anemia accompanied with malformation: Fanconi disease and dyskeratosis congenita. CASE REPORT: A 19-year-old girl, issued from a third degree consanguineous marriage, was admitted because of anemic and hemorrhagic syndrome. Physical examination showed several malformations: microphtalmia, brownish spots, generalized hyperpigmentation and ungueal dystrophy without mucosal leucoplasia. Statural and ponderal retardation were noted. On the hemogram there was a pancytopenia and on biopsy, the bone marrow was desertic. The caryotype performed on peripheral blood lymphocytes after sensibilisation with mitomycin C revealed chromosomal instability aspects. Based on these clinical and biological features, the diagnosis of hereditary aplastic anaemia was retained. The patient was given norethandrolone. She died 3 months later by septic shock. DISCUSSION: Coexistence of aplastic anemia with a malformative syndrome suggests most probably an hereditary form of aplastic anemia. Fanconi anemia is the most frequent. It associates characteristic anomalies of the face, with microphtalmia, brownish spots, statural and ponderal retardation, and thumb anomalies. Ungueal dystrophy, mucosal leucoplasia are almost pathognomonic of congenital dyskeratosis. When the malformative syndrome is not characteristic, the cytogenetic study may also fail to make the differential diagnosis, as was the situation in our case.

Prenatal diagnosis and characterization of an analphoid marker chromosome 16.

We report on a fetus with intrauterine growth retardation and multiple malformations diagnosed on ultrasound at 32 weeks. Examination of amniotic fluid cells in culture showed a 47,XY, i(16)(q10), +mar karyotype. Chromosome analysis of both parents was normal. Using spectral karyotyping, we identified the marker chromosome as a mitotically stable acentric marker chromosome derived from chromosome 16. Further studies using subtelomeric fluorescent probes confirmed the presence of an isochromosome for the long arm of chromosome 16 and showed that the acentric marker chromosome derived from the short arm of chromosome 16 leading to a trisomy for the long arm of chromosome 16. After genetic counseling, the parents decided to terminate the pregnancy. Fetal autopsy showed a male fetus with ambiguous external genitalia, cardiac malformation, megacystis and limbs anomalies as observed in other cases of trisomy for the long arm of chromosome 16. In addition, fetal brain examination showed vermian and olfactory bulb hypoplasia.

[Spectral karyotyping (SKY) principle, avantages and limitations]. / Le caryotype spectral (SKY): principe, avantages et limites en cytogénétique constitutionnelle et tumorale.

Banding karyotype is a routine technique, which allows the identification of numerous aneusomy and/or aneuploïdy in congenital diseases and cancers. However, this analysis fails to detect small or complex chromosome rearrangements. Molecular cytogenetic techniques like fluorescence in situ hybridization (FISH) analysis can overlap these limitations. Particularly, multicolor karyotyping by spectral karyotyping (SKY) may rectify or precise the conventional karyotype results. With two examples, we present here, the principle, the indications and the limits of this technique for constitutional and cancer chromosomal abnormalities characterization. Moreover, we present an easy way to build efficient sky probes with a best sensitivity than the probes classically used.

Reassessment of childhood B-lineage lymphoblastic leukemia karyotypes using spectral analysis.

We studied a stratified cohort of 51 childhood B-lineage acute lymphoblastic leukemias (B-ALLs) to evaluate the efficiency of spectral karyotyping (SKY) in the detection of chromosome aberrations previously diagnosed using chromosome banding and/or reverse transcriptase polymerase chain reaction. Despite the small number of cases analyzed, several important features emerge from the study: (a) The result of banding analysis was revised in two-thirds of the cases. Eighty-three chromosome anomalies previously undetected or not characterized using chromosome banding were identified by spectral karyotyping, even in patients with apparently normal karyotypes. (b) All hyperdiploidy cases showed one or more extra copies of chromosomes X, 14, and 21. (c) Two hidden rearrangements, a t(7;12)(?p12;p13), and a new translocation, a t(9;12)(q31;p13), both involving the TEL gene, were characterized. (d) Some cryptic rearrangements, such as the der(21) t(12;21) translocation, remained undetected. (e) No new recurrent chromosome anomalies were discovered with this technique. In conclusion, the present study confirms the efficiency of the SKY technique in resolving and characterizing many complex chromosome anomalies seen in childhood B-ALLs, but it raises questions about the ability of this technique to detect cryptic rearrangements, such as the t(12;21) translocation.