Osteoprotegerin gene polymorphisms and otosclerosis: an additional genetic association study, multilocus interaction and meta-analysis.

BACKGROUND: Otosclerosis (OTSC) is among the most common causes of a late-onset hearing loss in adults and is characterized by an abnormal bone growth in the otic capsule. Alteration in the osteoprotegerin (OPG) expression has been suggested in the implication of OTSC pathogenesis. METHODS: A case-control association study of rs2228568, rs7844539, rs3102734 and rs2073618 single nucleotide polymorphisms (SNPs) in the OPG gene was performed in a Tunisian-North African population composed of 183 unrelated OTSC patients and 177 healthy subjects. In addition, a multilocus association and a meta-analysis of existing studies were conducted. RESULTS: Rs3102734 (p = 0.013) and rs2073618 (p = 0.007) were significantly associated with OTSC, which were predominantly detected in females after multiple corrections. Among the OPG studied SNPs, the haplotypes A-A-C-G (p = 0.0001) and A-A-C-C (p = 0.0004) were significantly associated with OTSC in females. Multilocus association revealed that the SNPs: rs2073618 in OPG, rs1800472 in TGFß1, rs39335, rs39350 and rs39374 in RELN, and rs494252 in chromosome 11 showed significant OTSC-associated alleles in Tunisian individuals. In addition, meta-analysis of the rs2073618 SNP in Tunisian, Indian and Italian populations revealed evidence of an association with OTSC (OR of 0.826, 95% CI [0.691-0.987], p = 0.035). CONCLUSIONS: Our findings suggest that rs3102734 and rs2073618 variants are associated with OTSC in North African ethnic Tunisian population. Meta-analysis of the rs2073618 in three different ethnic population groups indicated an association with OTSC.

Fine needle non-aspiration cytology for the diagnosis of cervical lymph node tuberculosis: a single center experience / Punção não aspirativa com agulha fina para o diagnóstico de tuberculose linfonodal cervical: experiência de centro único

Abstract Introduction: The fine-needle cytology is being used as a first line of investigation in the diagnosis of head and neck swellings, as it is simple, cost effective and less invasive as compared to biopsy. Objective: The aims of this study were to evaluate the results of the fine-needle non-aspiration cytology of cervical lymphadenopathy and to study the factors influencing the rate of non-diagnosis results. Methods: This retrospective study was conducted on selected patients with cervical lymphadenopathy that had undergone a fine-needle non-aspiration cytology followed by a histological biopsy. The sensitivity, specificity, positive predictive value and negative predictive value of fine-needle non-aspiration cytology for diagnosing tuberculosis were estimated. The risk factors of non-diagnosis results were evaluated. Results: The sensitivity, specificity, positive predictive value rates of fine-needle non-aspiration cytology for tuberculosis were 83.3%, 83.3%, 78.9% and 86.9% respectively. In total, 47 out of the 131 samples (35.8%) were considered non-diagnosis. Of the non-diagnosis samples, 84.2% (38 out of 47) were benign mostly due to tuberculosis (30 cases). Among the studied factors, only tuberculosis (confirmed by histopathological examination) was significantly associated with non-diagnosis cytology (p = 0.02, Odds-Ratio = 2.35). Conclusion: Tuberculosis is currently the commonest cause of cervical lymphadenopathy in North Africa. Fine-needle non-aspiration cytology is safe and accurate in the diagnosis of cervical tuberculous lymph node that is associated with the risk of non-diagnosis cytology.

Resumo Introdução: A punção não aspirativa com agulha fina tem sido utilizada como primeira linha de investigação no diagnóstico de tumores de cabeça e pescoço, por ser uma técnica simples, custo-efetiva e menos invasiva quando comparada à biópsia. Objetivo: Os objetivos deste estudo foram avaliar os resultados de citologia por punção não-aspirativa com agulha fina de linfadenopatias cervicais e estudar os fatores que influenciam a taxa de falha diagnóstica. Método: Este estudo retrospectivo foi realizado em pacientes selecionados com linfadenopatia cervical submetidos a punção não aspirativa com agulha fina, seguida por biópsia histológica. Foram estimadas a sensibilidade, especificidade, o valor preditivo positivo e valor preditivo negativo da punção não aspirativa com agulha fina para o diagnóstico de tuberculose. Os fatores de risco dos resultados com falha diagnóstica foram avaliados. Resultados: As taxas de sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da punção não aspirativa com agulha fina para tuberculose foram de 83,3%, 83,3%, 78,9% e 86,9%, respectivamente. Das 131 amostras, 47 (35,8%) foram consideradas como falha diagnóstica. Das amostras não diagnosticadas, 84,2% (38 de 47) eram benignas, principalmente devido à tuberculose (30 casos). Entre os fatores estudados, apenas a tuberculose (confirmada pelo exame histopatológico) estava significativamente associada à citologia com falha diagnóstica (p = 0,02, odds ratio = 2,35). Conclusão: A tuberculose é atualmente a causa mais comum de linfadenopatia cervical no norte da África. A punção não aspirativa com agulha fina é uma técnica segura e precisa no diagnóstico de linfonodos cervicais associados ao risco de citologia com falha diagnóstica.

Fine needle non-aspiration cytology for the diagnosis of cervical lymph node tuberculosis: a single center experience.

INTRODUCTION: The fine-needle cytology is being used as a first line of investigation in the diagnosis of head and neck swellings, as it is simple, cost effective and less invasive as compared to biopsy. OBJECTIVE: The aims of this study were to evaluate the results of the fine-needle non-aspiration cytology of cervical lymphadenopathy and to study the factors influencing the rate of non-diagnosis results. METHODS: This retrospective study was conducted on selected patients with cervical lymphadenopathy that had undergone a fine-needle non-aspiration cytology followed by a histological biopsy. The sensitivity, specificity, positive predictive value and negative predictive value of fine-needle non-aspiration cytology for diagnosing tuberculosis were estimated. The risk factors of non-diagnosis results were evaluated. RESULTS: The sensitivity, specificity, positive predictive value rates of fine-needle non-aspiration cytology for tuberculosis were 83.3%, 83.3%, 78.9% and 86.9% respectively. In total, 47 out of the 131 samples (35.8%) were considered non-diagnosis. Of the non-diagnosis samples, 84.2% (38 out of 47) were benign mostly due to tuberculosis (30 cases). Among the studied factors, only tuberculosis (confirmed by histopathological examination) was significantly associated with non-diagnosis cytology (p=0.02, Odds-Ratio=2.35). CONCLUSION: Tuberculosis is currently the commonest cause of cervical lymphadenopathy in North Africa. Fine-needle non-aspiration cytology is safe and accurate in the diagnosis of cervical tuberculous lymph node that is associated with the risk of non-diagnosis cytology.

CDH23 Methylation Status and Presbycusis Risk in Elderly Women.

Introduction: Presbycusis, an age-related hearing impairment (ARHI) disease, is the most common cause for HI in adults worldwide. One of the best candidate genes for ARHI susceptibility is Cadherin 23 (CDH23) which encodes stereocilia tip-links of the inner ear sensory hair cell. Although alterations in the methylation status of CpG dinucleotides across various genes were reported to be associated with HI, methylation changes in CDH23 gene have not been reported previously. Objectives: This study aimed at investigating whether DNA methylation level of CDH23 gene at intragenic CpG island overlapping an exonic-intronic region at position chr10:73565570-73565827 (GRCh37/hg19) could be risk factor associated with ARHI. Materials and Methods: We screened for methylation changes in this particular position for CDH23 gene in 50 blood samples of elderly women affected with presbycusis and healthy control cohort. Methylation of CpG sites were assessed using Quantitative methylation-specific PCR (qMSP) following sodium bisulfite DNA conversion chemistry. Methylation levels were normalized against TSH2B reference gene. Results: DNA methylation analysis for the common CpG islands in CDH23 gene revealed 3.27-folds significant increase (p < 0.0001) in methylation profile for ARHI women as compared to healthy controls with an elevated risk odds ratio (OR) of 2.219 [95% CI 1.071-4.597]. Conclusion: Our study is the first of its kind to prove that higher CpG site methylation levels in CDH23 gene are likely to be associated with ARHI.

[Neoadjuvant chemotherapy with concurrent chemoradiotherapy in the treatment of nasopharyngeal cancer: Southern Tunisian experience]. / La chimiothérapie néoadjuvante associée à la chimioradiothérapie concomitante dans le traitement du cancer du nasopharynx : expérience du Sud-Tunisien.

PURPOSE: A retrospective study to evaluate the efficacy and safety of the addition of neoadjuvant chemotherapy to concurrent chemoradiotherapy in the treatment of nasopharyngeal carcinoma. PATIENTS AND METHODS: Data from 62 patients treated for non-metastatic nasopharyngeal carcinoma were analyzed by comparing two groups of patients: a first group of 32 patients treated with 3 cycles of neoadjuvant chemotherapy based on docetaxel, cisplatin and 5-fluoro-uracil every 21 days followed by concurrent chemoradiotherapy (weekly cisplatin 40mg/m2 with radiotherapy 70Gy, 2Gy per session, 5 sessions per week) and a second group of 30 patients treated with the same concurrent chemoradiotherapy. RESULTS: After a median follow-up of 53.5 months, neoadjuvant chemotherapy showed a significant reduction in the rate of a distant metastatic relapses (3.3% vs. 10%, P=0.03). No significant difference in disease-free survival at 5 years (65.6% vs. 68.8%, P=0.46) or overall survival at 5 years (68.8% vs. 73.3%, P=0.46) was noted between the two groups. Induction chemotherapy was associated with febrile neutropenia of 15.6%. During concurrent chemoradiotherapy, hematological complications were greater in the first chemotherapy group (53% vs. 33%). CONCLUSION: Induction chemotherapy by docetaxel, cisplatin and 5-fluoro-uracil is a safe and effective option in the treatment of nasopharyngeal carcinoma. A better definition of high risk of relapse group would optimize the indications of this chemotherapy in the therapeutic arsenal.

Bupivacaine wound infiltration reduces postoperative pain and analgesic requirement after thyroid surgery.

BACKGROUND AND OBJECTIVE: We conducted a prospective double-blind randomized study assessing bupivacaine end-of-surgery wound infiltration for pain relief in thyroid surgery. METHODS: Patients were randomly divided into two groups: Group S, local wound infiltration with saline solution; Group B, bupivacaine 0.5% was administered. Pain perception was measured using visual analogue scale (VAS) during post-anaesthetic care unit (PACU) stay every 10 min and during the 24 postoperative hours admission at 2, 4, 6, 12, and 24 h after surgery. The total consumption of analgesics (morphine and nefopam) was recorded. RESULTS: Sixty patients were studied. The VAS scores were significantly lower in the bupivacaine administered group in the post-anaesthetic care unit (PACU) at 0, 10, 20, 30, 40, 50 and 60 min, and during the hospital stay at hours 6, 12, 18 and 24. The number of patients who required postoperative opioid rescue was significantly lower in group B. No patient in group B developed neurological or cardiological complications after infiltration. CONCLUSION: Bupivacaine application is effective in decreasing postoperative pain and analgesic requirement during the hospital stay for patients with thyroidectomy.

Down-expression of P2RX2, KCNQ5, ERBB3 and SOCS3 through DNA hypermethylation in elderly women with presbycusis.

CONTEXT: Presbycusis, an age-related hearing impairment (ARHI), represents the most common sensory disability in adults. Today, the molecular mechanisms underlying presbycusis remain unclear. This is in particular due to the fact that ARHI is a multifactorial complex disorder resulting from several genomic factors interacting with lifelong cumulative effects of: disease, diet, and environment. OBJECTIVE: Identification of novel biomarkers for presbycusis. MATERIALS AND METHODS: We selectively ascertained 18 elderly unrelated women lacking environmental and metabolic risk factors. Subsequently, we screened for methylation map changes in blood samples of women with presbycusis as compared to controls, using reduced representation bisulfite sequencing. We focused on hypermethylated cytosine bases located in gene promoters and the first two exons. To elucidate the related gene expression changes, we performed transcriptomic study using gene expression microarray. RESULTS: Twenty-seven genes, known to be expressed in adult human cochlea, were found in the blood cells to be differentially hypermethylated with significant (p < 0.01) methylation differences (>30%) and down-expressed with fold change >1.2 (FDR <0.05). Functional annotation and qRT-PCR further identified P2RX2, KCNQ5, ERBB3 and SOCS3 to be associated with the progression of ARHI. DISCUSSION AND CONCLUSION: Down-expressed genes associated with DNA hypermethylation could be used as biomarkers for understanding complex pathogenic mechanisms underlying presbycusis.

Proteins oxidation and autoantibodies' reactivity against hydrogen peroxide and malondialdehyde -oxidized thyroid antigens in patients' plasmas with Graves' disease and Hashimoto Thyroiditis.

The aim of this study was to evaluate proteins oxidation in plasmas of two autoimmune thyroid diseases (AITD): Graves' disease (GD) and Hashimoto Thyroiditis (HT), and to determine whether oxidative modification of thyroid antigens (T.Ag) enhanced the reactivity of autoantibodies in plasmas of AITD patients compared with the reactivity towards native T.Ag. Carbonyl and thiol groups and MDA-protein adducts were assessed spectrophotometric methods in plasmas of 74 AITD patients and 65 healthy controls. The reactivities immunoglobulin (Ig)G autoantibodies towards malondialdéhyde (MDA)-modified T.Ag, hydrogen peroxide (H2O2)-modified T.Ag, native T.Ag and native derm were checked by enzyme-linked immunosorbent assay (ELISA). Evaluation of oxidized proteins exhibited high levels of MDA bound to proteins and carbonyl groups, as well as reduced thiol level in plasmas of AITD patients by comparison to healthy controls (p < 0.05). The ELISA test showed that AITD patients' plasmas' reactivity to native T.Ag was significantly increased to the reactivity towards native derm, whereas, no differences were found in the reactivity to native T.Ag and derm in controls plasmas. In addition, treatment of T.Ag by oxidants revealed enhanced reactivity of IgG circulating autoantibodies against H2O2-oxidized T.Ag compared to native ones (p < 0.001) in plasmas of both AITD. Also, reactivity's to MDA-oxidized T.Ag in GD plasmas decreased compared to native ones (p < 0.05) and no changes were noted for HT. Pearson correlation study resulted in positive correlation between reactivity's to H2O2-oxidized T.Ag and free triodotyronine level in GD patients (r = 0.42, p < 0.05) in one hand and thyroid stimulating hormone level in HT patients in the other (r = 0.65, p < 0.001). The data suggest that high production of H2O2 probably occurred during hormone synthesis could contribute to protein oxidation in AITD and to create neoepitopes responsible for autoantibody reactivity's to H2O2-oxidized T.Ag enhancement. These results provide support to the involvement of oxidative stress in AITD development and/or exacerbation.

Long-term clinical follow-up and molecular testing for diagnosis of the first Tunisian family with Alström syndrome.

Alström syndrome is a clinically complex disorder characterized by progressive degeneration of sensory functions, resulting in visual and audiological impairment as well as metabolic disturbances. It is caused by recessively inherited mutations in the ALMS1 gene, which codes for a centrosomal/basal body protein. The purpose of this study was to investigate the genetic and clinical features of two Tunisian affected siblings with Alström syndrome. Detailed clinical examinations were performed including complete ophthalmic examination, serial audiograms and several biochemical and hormonal blood tests. For the molecular study, first genomic DNA was isolated using a standard protocol. Then, linkage analysis with microsatellite markers was performed and DNA array was used to detect known mutations. Subsequently, all ALMS1 exons were simultaneously sequenced for one affected patient with the TaGSCAN targeted sequencing panel. Finally, segregation of the causal variant was performed by Sanger sequencing. Both affected siblings had cone rod dystrophy with impaired visual acuity, sensorineural hearing loss and truncal obesity. One affected individual showed insulin resistance without diabetes mellitus. Other clinical features including cardiac and pulmonary dysfunction, hypothyroidism, hyperlipidemia, acanthosis nigricans, renal and hepatic dysfunction were absent. Genetic analysis showed the presence of a homozygous splice site mutation (c.10388-2A > G) in both affected siblings. Although Alström syndrome is relatively well characterized disease, this syndrome is probably misdiagnosed in Tunisia. Here, we describe the first report of Tunisian patients affected by this syndrome and carrying a homozygous ALMS1 mutation. The diagnosis was suspected after long-term clinical follow-up and confirmed by genetic testing.

Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis.

PURPOSE: Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, cadherin-23, protocadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively. METHODS: In the present study, we recruited four Tunisian families with a diagnosis of USH1, together with healthy unrelated controls. Affected members underwent detailed audiologic and ocular examinations. We used the North African Deafness (NADf) chip to search for known North African mutations associated with USH. Then, we selected microsatellite markers covering USH1 known loci to genotype the DNA samples. Finally, we performed DNA sequencing of three known USH1 genes: MYO7A, PCDH15, and USH1C. RESULTS: Four biallelic mutations, all single base changes, were found in the MYO7A, USH1C, and PCDH15 genes. These mutations consist of a previously reported splicing defect c.470+1G>A in MYO7A, three novel variants, including two nonsense (p.Arg3X and p.Arg134X) in USH1C and PCDH15, respectively, and one frameshift (p.Lys615Asnfs*6) in MYO7A. CONCLUSIONS: We found a remarkable genetic heterogeneity in the studied families with USH1 with a variety of mutations, among which three were novel. These novel mutations will be included in the NADf mutation screening chip that will allow a higher diagnosis efficiency of this extremely genetically heterogeneous disease. Ultimately, efficient molecular diagnosis of USH in a patient's early childhood is of utmost importance, allowing better educational and therapeutic management.

A mutation in SLC22A4 encoding an organic cation transporter expressed in the cochlea strial endothelium causes human recessive non-syndromic hearing loss DFNB60.

The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.

Over-expression of miR-10b in NPC patients: correlation with LMP1 and Twist1.

Aberrant expression of miR-10b has been described in many cancers but remains unexplored in nasopharyngeal carcinoma (NPC). Therefore, we aimed to study the miR-10b expression level in 43 NPC biopsies collected from Tunisian patients and three NPC xenografts. Then, we investigated the correlation between miR-10b expression and its upstream regulators LMP1/Twist1 as well as its adjacent gene HoxD4. We showed that miR-10b was significantly up-regulated in NPC biopsies compared to non-tumor nasopharyngeal tissues (fold change 153; p = 0.004) and associated with advanced clinical stage and young age at diagnosis (p = 0.005 and p = 0.011, respectively). In addition, over-expression of miR-10b was positively associated with the transcription factor Twist1 as well as the EBV oncoprotein LMP1 (fold change 6.32; p = 0.014, fold change 6.58; p = 0.01 respectively). Furthermore, higher level of miR-10b was observed in tumors with simultaneous expression of LMP1 and Twist1, compared to those expressing only Twist1 (fold change 2.49; p = 0.033). Meanwhile, the analysis of the link between miR-10b and its neighbor gene HoxD4 did not show any significant correlation (Fisher test p = 0.205; Mann-Whitney test p = 0.676). This study reports the first evidence of miR-10b over-expression in NPC patients. Furthermore, our findings can support hsa-miR-10b gene regulation through LMP1/Twist1 in NPC malignancy.