Immunohistochemical analysis of c-erbB-2, Bcl-2, p53, p21WAF1/Cip1, p63 and Ki-67 expression in hydatidiform moles.

Hydatidiform moles (HM) are characterized by an abnormal proliferating trophoblast with a potential for a malignant transformation. Similar to other human tumors, trophoblastic pathogenesis is likely a multistep process involving several molecular and genetic alterations. The study was performed to investigate the expression patterns of c-erbB-2 and Bcl-2 oncoproteins, p53, p21WAF1/CIP1 and p63 tumor suppressor proteins and Ki-67 cell proliferation marker in HM. We conducted a retrospective study of 220 gestational products, including 39 hydropic abortions (HA), 41 partial HM (PHM) and 140 complete HM (CHM). The expression of c-erbB-2, Bcl-2, p53, p21WAF1/CIP1, p63 and Ki-67 was investigated by immunohistochemistry on archival tissues. c-erbB-2 expression was observed in three PHM and 10 CHM. Bcl-2 immunostaining was significantly higher in PHM (61%) and CHM (70.7%) compared with HA (7.7%, p = 0.001 and p < 0.0001, respectively). p53 expression was stronger in CHM (73.6%) compared with PHM (24.4%, p < 0.0001) and HA (12.8%, p < 0.0001). p21WAF1/CIP1 staining was observed as well in molar and non-molar gestations (p > 0.05). p63 immunoexpression was significantly described in CHM (85.7%) and PHM (78%) compared with HA (10.2%, p < 0.0001 and p = 0.0001, respectively). Ki-67 was significantly expressed in CHM (72.1%) compared with HA (46.2%, p = 0.005). Altered expression of Bcl-2, p53, p63 and Ki-67 reflects the HM pathological development. Immunohistochemical analysis is beneficial to recognize the HM molecular and pathogenic mechanisms. Furthermore, it could serve as a useful adjunct to conventional methods for refining HM diagnosis.

Report of four new patients with protein-truncating mutations in C6orf221/KHDC3L and colocalization with NLRP7.

To date, two maternal-effect genes have been shown to have causative roles in recurrent hydatidiform moles (RHMs); NLRP7 that is mutated in 48-60% of patients with RHMs and C6orf221 (HUGO-approved nomenclature is now KHDC3L), a recently identified gene, that is mutated in 14% of patients with RHMs who are negative for NLRP7 mutations. We sequenced KHDC3L in 97 patients with RHMs and reproductive loss who are mostly negative for NLRP7 mutations. We identified three unrelated patients, each homozygous for one of the two protein-truncating mutations, a novel 4-bp deletion resulting in a frameshift, c.299_302delTCAA, p.Ile100Argfs*2, and a previously described 4-bp deletion, c.322_325delGACT, p.Asp108Ilefs*30, transmitted on a shared haplotype to three patients from different populations. We show that five HM tissues from one of these patients are diploid and biparental similar to HMs from patients with two defective NLRP7 mutations. Using immunofluorescence, we show that KHDC3L protein displays a juxta perinuclear signal and colocalizes with NLRP7 in lymphoblastoid cell lines from normal subjects. Using cell lines from patients, we demonstrate that the KHDC3L mutations do not change the subcellular localization of the protein in hematopoietic cells. Our data highlight the similarities between the two causative genes for RHMs, KHDC3L and NLRP7, in their subcellular localization, the parental contribution to the HM tissues caused by them, and the presence of several founder mutations and variants in both of them indicating positive selection and adaptation.

CITED2 mutations potentially cause idiopathic premature ovarian failure.

Anomalies in gonadal development in a mouse knockout model of Cited2 have been recently described. In Cited2(-/-) female gonads, an ectopic cell migration was observed and the female program of sex determination was transiently delayed. We hypothesize that, in humans, this temporary inhibition of genes should be sufficient to provoke a developmental impairment of the female gonads, conducive to premature ovarian failure (POF). To establish whether CITED2 mutations are a common cause of the disease, we performed a mutational analysis of this gene in a panel of patients with POF and in a group of control women with normal fertility. We amplified and directly sequenced the complete open reading frame of CITED2 in 139 patients with POF and 290 controls. This study revealed 5 synonymous and 3 nonsynonymous variants. Among these, 7 are novel. The nonsynonymous variant c.604C>A (p.Pro202Thr) was found uniquely in 1 woman from the POF group. In silico analysis of this mutation indicated a potential deleterious effect. We conclude that mutations in CITED2 may be involved in POF pathogenesis.

NLRP7 mutation analysis in sporadic hydatidiform moles in Tunisian patients: NLRP7 and sporadic mole.

CONTEXT: Hydatidiform mole, an aberrant human pregnancy, is commonly a nonrecurrent disease. Recently, a rare autosomal recessive form of familial and/or recurrent molar pregnancies was associated with mutations in the NLRP7 gene. OBJECTIVE: To investigate whether NLRP7 mutations exist in Tunisian women with sporadic hydatidiform moles. DESIGN: Genomic DNA from 38 unrelated Tunisian patients with sporadic hydatidiform moles were screened by sequencing all NLRP7 exons. A high-resolution melting curve analysis was performed on 170 DNA controls to analyze new sequence variants. RESULTS: More than 13% of these patients were heterozygous for NLRP7 mutations. We found 2 novel missense mutations in the heterozygous state, c.544G>A (p.Val182Met) in 1 patient and c.1480G>A (p.Ala494Thr) in 2 patients, and 2 already reported mutations, c.1532A>G (p.Lys511Arg) and c.2156C>T (p.Ala719Val), in 2 patients. None of these mutations were identified in 170 controls except for 1 woman who was heterozygous for p.Val182Met. CONCLUSION: As homozygous NLRP7 mutations are associated with recurrent hydatidiform mole or conception loss, the heterozygous state could represent a risk factor for nonrecurrent mole.

Cytogenetic and molecular aspects of absolute teratozoospermia: comparison between polymorphic and monomorphic forms.

OBJECTIVE: To compare the results of cytogenetic and molecular analysis between absolute polymorphic and monomorphic teratozoospermia. METHODS: The semen samples from patients with polymorphic teratozoospermia (n = 20), globozoospermia (n = 8), or macrocephalic sperm head syndrome (n = 12), and healthy fertile men (n = 20) were analyzed according to the World Health Organization criteria. The constitutional blood karyotype of the patients was performed on cultured lymphocytes, according to standard techniques. Microdeletion analysis of the Y chromosomes used a sequence tagged site-polymerase chain reaction technique. Triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18 were used to analyze the meiotic segregation. DNA fragmentation was detected using the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. RESULTS: Whatever the type of teratozoospermia, a normal karyotype and an absence of Y chromosome microdeletion were shown for all patients. A significant increase in the sperm aneuploidy rate and DNA fragmentation were shown, regardless of the type of teratozoospermia. Spermatozoa of the patients with globozoospermia carry an abnormal chromosomal constitution and DNA damage rate with the same frequency as that found in the sperm of patients with absolute polymorphic teratozoospermia. However, a greater sperm aneuploidy rate and DNA fragmentation were found in patients whose teratozoospermia was mainly characterized by increased rates of spermatozoa with macrocephalic head and multiple flagella. CONCLUSION: Our data have demonstrated that DNA fragmentation and sperm aneuploidy are critical tests in teratozoospermic men, because the results could negatively affect the intracytoplasmic sperm injection outcomes and might play an important role in the counseling of couples considering intracytoplasmic sperm injection.

Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte.

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.

Semen parameters and sperm DNA fragmentation as causes of recurrent pregnancy loss.

OBJECTIVES: To evaluate and compare standard sperm parameters, and sperm DNA fragmentation in seminal ejaculates from men whose partners had a history of recurrent pregnancy loss (RPL) and in a control group of men who had recently established their fertility. METHODS: Semen samples from 31 patients with a history of recurrent pregnancy loss and 20 men with proven fertility were analyzed according to World Health Organization guidelines. Sperm DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. RESULTS: When sperm quality of the control group was compared with that of the RPL group, a significant difference was observed in sperm motility, but not in other parameters. The mean number of sperm cells with fragmented DNA was significantly increased in the RPL group (32.22 ± 6.14%) compared with control donors (10.20 ± 2.1%). CONCLUSIONS: Our data indicate that sperm from men with a history of RPL have a higher incidence of DNA damage and poor motility than sperm from a control group, and this can explain in part the pregnancy loss in these patients.

The usefulness of p57(KIP2) immunohistochemical staining and genotyping test in the diagnosis of the hydatidiform mole.

Classification of molar gestations into complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM) and their differentiation from nonmolar hydropic abortions (HA) are traditionally accomplished by morphology alone. Sometimes, the process may be inaccurate or inconclusive especially in early diagnosed cases. With the availability of p57(KIP2) immunostaining (the product of a strongly paternally imprinted and maternally expressed gene), it may be possible to classify these lesions objectively. P57(KIP2) immunostaining is absent in CHM because it lacks a maternal genome, whereas PHM and HA show positive staining. The aims of this study were to evaluate the results of routine histopathological examination and p57(KIP2) immunoreactivity in a large series of molar and nonmolar HA in Tunisia, and to compare the accuracy of p57(KIP2) immunohistochemistry with that of nuclear DNA microsatellite polymorphism in identifying CHM. The immunohistochemical expression of p57(KIP2) protein was investigated in 220 specimens of first trimester hydropic abortuses, and it was compared with the original diagnosis based on morphology, including 132 CHM, 49 PHM, and 39 HA. Concordant results were obtained in 210 cases. In 9 of 10 cases with a discordant diagnosis (negative immunostaining in 8 cases morphologically diagnosed as PHM and one case diagnosed as HA), microsatellite DNA genotyping analysis agreed with the results of p57(KIP2) staining, confirming the diagnosis of CHM in these cases. Twenty cases of CHM with negative p57(KIP2) immunostaining were also analyzed by genotyping and indicated the absence of maternal contribution and the homozygosity for a single paternal allele in concordance with the androgenetic and monospermic origin of CHM in these cases. We confirm that for distinguishing CHM from its mimics, p57(KIP2) immunohistochemistry can be used as successfully as DNA microsatellite genotyping. However, molecular techniques are still required for the evaluation of some difficult cases with discordant positive p57(KIP2) staining.

Screening for NLRP7 mutations in familial and sporadic recurrent hydatidiform moles: report of 2 Tunisian families.

A familial or sporadic recurrent hydatidiform mole is a rare autosomal recessive condition that has been associated with biallelic mutations in the nucleotide-binding, leucine-rich repeat, pyrin domain 7 (NLRP7) gene (19q13.42). Cases from different ethnic origins have been reported earlier. Here we report the first Tunisian patients: 2 sisters with homozygous NLRP7 mutations (p.E570X) and 1 sporadic case with no mutation in NLRP7. Our results extend the number of familial recurrent reproductive wastages due to mutations in NLRP7. We suggest that mutations screening of NLRP7 could be proposed more systematically in women with recurrent pathologic pregnancy outcomes of unknown origin. The rare cases with a typical clinical picture, which were not related to NLRP7 mutation as in our sporadic case, should be investigated more to identify the causative gene.

Sequence analysis of the CDKN1B gene in patients with premature ovarian failure reveals a novel mutation potentially related to the phenotype.

Earlier reports demonstrated a key role of Cdkn1b during mouse ovarian development. In this study, the sequencing analysis of the complete coding region of this gene in a panel of premature ovarian failure patients and control subjects reveals a novel mutation potentially related to the phenotype.

Childhood cancer frequency in the center of Tunisia.

OBJECTIVE: In this paper, we analyzed the frequency of childhood cancer in the Center of Tunisia during 1993-2006. DESIGN: The different types of cancer were grouped according to the International Classification for Cancer in Children. The general and specific frequencies by age and by sex were analyzed. RESULTS: A total of 727 new cases of childhood cancer were registered, with a male to-female sex ratio of 1.7/1. Leukemias had the highest frequency (27%) and, of these, lymphoid leukemias were the most prevalent (73.5%). Thereafter, in descending order of frequency, were lymphomas (25.7%), tumors of the central nervous system (CNS, 9.2%), neuroblastomas (7.7%), sarcomas (6.9%), carcinomas (6.3%), bone tumors (5.8%), nephroblastomas (5.5%), and germinal cell tumors (2.6%). The highest frequency of cancer was found at age 10-14 years (34.9%). Leukemias were the most frequent in age groups 1-4 and 5-9 years, whereas, neuroblastomas and lymphomas were the most frequent at age under one year and 10-14 years, respectively. Of those cases of solid tumors, 55.8% were diagnosed as having advanced stages of the disease. CONCLUSION: Leukemias, lymphomas, and CNS tumors were the principal cancers in the Center of Tunisia. A childhood cancer registry with high-resolution data collection is advocated for in-depth analysis of pediatric malignancies.

Lung cancer in central Tunisia: epidemiology and clinicopathological features.

Lung cancer is the most common cancer worldwide but data from Tunisia are limited. The aim of this research was to describe the epidemiology, pathology and clinical features of lung cancer in Central Tunisia. All lung cancer cases diagnosed during a 15-year period were analyzed based on the data of the Cancer Registry of the Center of Tunisia. Five-year age-specific rates, world age-standardized rates (ASR), and annual percent change were calculated using annual data on population size and the estimated age structure. A total of 1,882 incident cases of lung cancer were registered (1,782 males, 100 females). The median age at diagnosis was 64 years for males and 61 years for females, with ASRs of 35.2 per 100,000 among males and 1.5 among females. Over time, there were significant decreasing trends by -6.5% (95% CI: -12.9%; -0.2%) for females and a stable incidence for males at an annual rate of +0.2% (95% CI: -1.6%; +1.8%). The predominant histological type was squamous cell carcinoma in males (36.9%) and adenocarcinoma in females (52%). During 2003-2007, adenocarcinoma became the most frequent (33.7%) followed by squamous cell carcinoma (30.3%) in males. The majority of tumor cases were diagnosed at advanced stages (79.9%). In conclusion, lung cancer has remained the most common cancer diagnosed at advanced stages among Tunisian men. Our findings justify the need to plan and develop effective programs aiming at the control and prevention of the spread of lung cancer in Tunisia.

Cervical adenocarcinoma and squamous cell carcinoma incidence trends among Tunisian women.

INTRODUCTION: Uterine cervix cancer is an important public health problem in Tunisia. In this study, we report trends in the incidence of adenocarcinoma and squamous cell carcinoma of the cervix uteri in the central region of Tunisia during 1993-2006. DESIGN: Data were obtained from the Cancer Registry of the Center of Tunisia which registers invasive cancer cases by active methods. Five-year age-specific rates, crude incidence rates (CR), world age-standardized rates (ASR), percent change (PC) and annual percent change (APC) were calculated using annual population data. RESULTS: Among all women cancers, cervix uteri cancer accounted for 5.9% and ranked the fourth during the study period with an ASR of 6.9 per 100,000. The ASRs decreased notably with an APC of -6.7% over the whole period. However, incidence rates of adenocarcinomas have increased during the last years (APC: +14.4%). CONCLUSION: The introduction of cytological screening programs has led to a marked decrease of the incidence rates of cervix uteri cancer among Tunisian women. The data underline the fact that the population-based cancer registry is an indispensable tool for providing data for planning and evaluation of programs for cancer control.

Pediatric rhabdomyosarcomas in Tunisia.

INTRODUCTION: Rhabdomyosarcoma is the most common soft tissue sarcoma in the first two decades of life. Since there is a paucity of reports on the pattern of its occurrence in Tunisia, we here analysed the epidemiological pattern, clinical features, and pathology. DESIGN: We retrospectively studied 30 consecutive cases of histologically proven rhabdomyosarcoma in children aged 0-15 years extracted from the database of the Cancer Registry of the Center of Tunisia for the period 1993-2007. RESULTS: Rhabdomyosarcomas represented 53.6% of soft tissue sarcomas and 3.8% of all children cancer cases registered during this period. The male/female ratio was 2.7 with a mean age at diagnosis of 5.9 years. The embryonal subtype was the most frequent (60%) and the two most common sites of disease were the head and neck (50%) and genito-urinary tract(23.3%). Chemotherapy was used in 90% of patients; 43.3% of patients had radical surgery and 26.7% of patients received radiation therapy. CONCLUSION: The epidemiology, pathology and clinical features of rhabdomyosarcoma in Tunisian children are close to those reported from other countries.

Type 1 diabetes mellitus, celiac disease, systemic lupus erythematosus and systemic scleroderma in a 15-year-old girl.

We report a case of an association of four autoimmune diseases in a 15-year-old girl and we discuss the etiopathogenic of this association and difficulties of treatment.

Assessment of the role of histopathology and DNA image analysis in the diagnosis of molar and non-molar abortion: a study of 89 cases in the center of Tunisia.

This study retrospectively evaluated the histopathological criteria commonly used in the literature on the diagnosis of hydatidiform mole, in correlation with the diagnosis rendered previously. The molar and non-molar cases seen in the first-trimester of pregnancy were separately reviewed by two pathologists. The correlation between the consensual histological diagnosis and the ploidy status was then evaluated. We retrospectively studied 89 specimens of abortus conception, including 35 complete hydatidiform moles (CHM), 12 partial hydatidiform moles (PHM), and 42 hydropic abortions (HA). The final histopathological diagnosis was compared with the results of DNA content detected by imaging analyzer (Samba 200), studying all cases of molar pregnancy and 4 cases of HA (initially diagnosed as molar pregnancies). In the consensus histological diagnosis, the cases were reclassified as follows: 30 CHM (initial diagnosis (ID): 27 CHM and 3 PHM), 12 PHM (ID: 6 PHM and 6 CHM), and one case with a persistent problem in differentiating PHM from HA and 46 HA (ID: 42 HA, 2 CHM, and 2 PHM). An agreement between the two pathologists was reached in 77 cases (K=0.72, 0.52, and 0.9, respectively, for CHM, PHM, and HA). The ploidy study demonstrated diploidy in 56.6% (17/30) of CHM and triploidy in 58.3% (7/12) of PHM. In the 4 cases of HA studied, 3 were diploid and 1 case was aneuploid. Our study demonstrated that several histopathological criteria could be used for the distinction between PHM, CHM, and HA. However, the study of DNA cannot be the technique of choice to distinguish between these entities. Some cases remain problematic since the morphological criteria are not easily reproducible. New sensitive techniques might resolve these dilemmas.

Severe osteomalacia due to undiagnosed coeliac disease: three case reports of Tunisian women.

We describe three cases of osteomalacia presenting in Tunisian women, all of whom had previously-undiagnosed coeliac disease (CD). Direct enquiry revealed an important weight loss and a history of diarrhoea in two patients, and a 15-year history of anaemia in one patient. Laboratory tests showed severe anaemia in the three cases. Reduced calcium was found in two cases, and corrected calcium was found in one case. Radiological examination showed fissure in two cases. The diagnosis of osteomalacia was made by clinical, biochemical and radiological features. Antigliadin, antireticulin, antiendomysial and anti-tissue transglutaminase antibodies were all positive in the three cases, and a small-bowel biopsy confirmed the diagnosis of CD. Treatment with gluten-free diet (GFD), supplemental calcium and vitamin D was initiated for the three patients, but only one patient complies strictly with the GFD; she showed a marked resolution of her symptoms.