Current phenotypic and genetic spectrum of syndromic deafness in Tunisia: paving the way for precision auditory health.

Hearing impairment (HI) is a prevalent neurosensory condition globally, impacting 5% of the population, with over 50% of congenital cases attributed to genetic etiologies. In Tunisia, HI underdiagnosis prevails, primarily due to limited access to comprehensive clinical tools, particularly for syndromic deafness (SD), characterized by clinical and genetic heterogeneity. This study aimed to uncover the SD spectrum through a 14-year investigation of a Tunisian cohort encompassing over 700 patients from four referral centers (2007-2021). Employing Sanger sequencing, Targeted Panel Gene Sequencing, and Whole Exome Sequencing, genetic analysis in 30 SD patients identified diagnoses such as Usher syndrome, Waardenburg syndrome, cranio-facial-hand-deafness syndrome, and H syndrome. This latter is a rare genodermatosis characterized by HI, hyperpigmentation, hypertrichosis, and systemic manifestations. A meta-analysis integrating our findings with existing data revealed that nearly 50% of Tunisian SD cases corresponded to rare inherited metabolic disorders. Distinguishing between non-syndromic and syndromic HI poses a challenge, where the age of onset and progression of features significantly impact accurate diagnoses. Despite advancements in local genetic characterization capabilities, certain ultra-rare forms of SD remain underdiagnosed. This research contributes critical insights to inform molecular diagnosis approaches for SD in Tunisia and the broader North-African region, thereby facilitating informed decision-making in clinical practice.

A regionally based precision medicine implementation initiative in North Africa:The PerMediNA consortium.

Precision Medicine is being increasingly used in the developed world to improve health care. While several Precision Medicine (PM) initiatives have been launched worldwide, their implementations have proven to be more challenging particularly in low- and middle-income countries. To address this issue, the "Personalized Medicine in North Africa" initiative (PerMediNA) was launched in three North African countries namely Tunisia, Algeria and Morocco. PerMediNA is coordinated by Institut Pasteur de Tunis together with the French Ministry for Europe and Foreign Affairs, with the support of Institut Pasteur in France. The project is carried out along with Institut Pasteur d'Algérie and Institut Pasteur du Maroc in collaboration with national and international leading institutions in the field of PM including Institut Gustave Roussy in Paris. PerMediNA aims to assess the readiness level of PM implementation in North Africa, to strengthen PM infrastructure, to provide workforce training, to generate genomic data on North African populations, to implement cost effective, affordable and sustainable genetic testing for cancer patients and to inform policy makers on how to translate research knowledge into health products and services. Gender equity and involvement of young scientists in this implementation process are other key goals of the PerMediNA project. In this paper, we are describing PerMediNA as the first PM implementation initiative in North Africa. Such initiatives contribute significantly in shortening existing health disparities and inequities between developed and developing countries and accelerate access to innovative treatments for global health.

Genetic testing for hereditary cancer syndromes in Tunisian patients: Impact on health system.

INTRODUCTION: Cancer management in Africa faces diverse challenges due to limited resources, health system challenges, and other matters. Identifying hereditary cancer syndromic cases is crucial to improve clinical management and preventive care in these settings. This study aims to explore the clinicopathological features and genetic factors associated with hereditary cancer in Tunisia, a North African country with a rising cancer burden MATERIALS AND METHODS: Clinicopathological features and personal/family history of cancer were explored in 521 patients. Genetic analysis using Sanger and next-generation sequencing was performed for a set of patients RESULTS: Hereditary breast and ovarian cancer syndrome was the most frequent cluster in which 36 BRCA mutations were identified. We described a subgroup of patients with likely ''breast cancer-only syndrome'' among this cluster. Two cases of Li-Fraumeni syndrome with distinct TP53 mutations namely c.638G>A and c.733G>A have been identified. Genetic investigation also allowed the identification of a new BLM homozygous mutation (c.3254dupT) in one patient with multiple primary cancers. Phenotype-genotype correlation suggests the diagnosis of Bloom syndrome. A recurrent MUTYH mutation (c.1143_1144dup) was identified in three patients with different phenotypes CONCLUSION: Our study calls for comprehensive genetic education and the implementation of genetic screening in Tunisia and other African countries health systems, to reduce the burden of hereditary diseases and improve cancer outcomes in resource-stratified settings.

Uncovering the clinical relevance of unclassified variants in DNA repair genes: a focus on BRCA negative Tunisian cancer families.

Introduction: Recent advances in sequencing technologies have significantly increased our capability to acquire large amounts of genetic data. However, the clinical relevance of the generated data continues to be challenging particularly with the identification of Variants of Uncertain Significance (VUSs) whose pathogenicity remains unclear. In the current report, we aim to evaluate the clinical relevance and the pathogenicity of VUSs in DNA repair genes among Tunisian breast cancer families. Methods: A total of 67 unsolved breast cancer cases have been investigated. The pathogenicity of VUSs identified within 26 DNA repair genes was assessed using different in silico prediction tools including SIFT, PolyPhen2, Align-GVGD and VarSEAK. Effects on the 3D structure were evaluated using the stability predictor DynaMut and molecular dynamics simulation with NAMD. Family segregation analysis was also performed. Results: Among a total of 37 VUSs identified, 11 variants are likely deleterious affecting ATM, BLM, CHEK2, ERCC3, FANCC, FANCG, MSH2, PMS2 and RAD50 genes. The BLM variant, c.3254dupT, is novel and seems to be associated with increased risk of breast, endometrial and colon cancer. Moreover, c.6115G>A in ATM and c.592+3A>T in CHEK2 were of keen interest identified in families with multiple breast cancer cases and their familial cosegregation with disease has been also confirmed. In addition, functional in silico analyses revealed that the ATM variant may lead to protein immobilization and rigidification thus decreasing its activity. We have also shown that FANCC and FANCG variants may lead to protein destabilization and alteration of the structure compactness which may affect FANCC and FANCG protein activity. Conclusion: Our findings revealed that VUSs in DNA repair genes might be associated with increased cancer risk and highlight the need for variant reclassification for better disease management. This will help to improve the genetic diagnosis and therapeutic strategies of cancer patients not only in Tunisia but also in neighboring countries.

Association between epidemiological and clinico-pathological features of breast cancer with prognosis, family history, Ki-67 proliferation index and survival in Tunisian breast cancer patients.

Breast cancer has different epidemio-clinical characteristics in Middle East and North-African populations compared to those reported in the Western countries. The aim of this study is to analyze the epidemiological and clinico-pathological features of breast cancer in Tunisia and to determine prognostic factors with special interest to family history, Ki-67 proliferation index and comorbidity. We retrospectively reviewed epidemiological and clinico-pathological data from patients' medical records, treated in the Medical Oncology Department at Abderrahmane Mami Hospital, in the period 2011-2015. Data has been collected on 602 breast cancer patients and analyzed using SPSS software V.23.0. Our study showed high fractions of young breast cancer patients and cases with dense breasts. The most prevalent comorbidities observed in the studied cohort were cardiovascular diseases and diabetes. Familial breast cancer was found in 23.3% of cases and was associated with younger age at diagnosis (p<0.001) and advanced stage (p = 0.015). Ki-67 index >20% was significantly associated with early age at diagnosis, lymph node involvement (p = 0.002), advanced tumor grade (p<0.001) and high risk of relapse (p = 0.007). Ki-67 cut-off 30% predicted survival in luminal cases. Survival was worse in patients with triple negative breast cancer compared to non-triple negative breast cancer, inflammatory breast cancer compared to non-inflammatory breast cancer, moderately to poorly differentiated tumors compared to well-differentiated tumors and with positive lymph nodes compared to pN0 (p<0.05). Our study showed new insights into epidemiological and clinico-pathological characteristics of breast cancer that are not well explored in Tunisian population. Considering our findings along with the implementation of electronic health record system may improve patient health care quality and disease management.

Health influenced by genetics: A first comprehensive analysis of breast cancer high and moderate penetrance susceptibility genes in the Tunisian population.

Significant advances have been made to understand the genetic basis of breast cancer. High, moderate and low penetrance variants have been identified with inter-ethnic variability in mutation frequency and spectrum. Genome wide association studies (GWAS) are widely used to identify disease-associated SNPs. Understanding the functional impact of these risk-SNPs will help the translation of GWAS findings into clinical interventions. Here we aim to characterize the genetic patterns of high and moderate penetrance breast cancer susceptibility genes and to assess the functional impact of non-coding SNPs. We analyzed BRCA1/2, PTEN, STK11, TP53, ATM, BRIP1, CHEK2 and PALB2 genotype data obtained from 135 healthy participants genotyped using Affymetrix Genome-Wide Human SNP-Array 6.0. Haplotype analysis was performed using Haploview.V4.2 and PHASE.V2.1. Population structure and genetic differentiation were assessed using principal component analysis (PCA) and fixation index (FST). Functional annotation was performed using In Silico web-based tools including RegulomeDB and VARAdb. Haplotype analysis showed distinct LD patterns with high levels of recombination and haplotype blocks of moderate to small size. Our findings revealed also that the Tunisian population tends to have a mixed origin with European, South Asian and Mexican footprints. Functional annotation allowed the selection of 28 putative regulatory variants. Of special interest were BRCA1_ rs8176318 predicted to alter the binding sites of a tumor suppressor miRNA hsa-miR-149 and PALB2_ rs120963 located in tumorigenesis-associated enhancer and predicted to strongly affect the binding of P53. Significant differences in allele frequencies were observed with populations of African and European ancestries for rs8176318 and rs120963 respectively. Our findings will help to better understand the genetic basis of breast cancer by guiding upcoming genome wide studies in the Tunisian population. Putative functional SNPs may be used to develop an efficient polygenic risk score to predict breast cancer risk leading to better disease prevention and management.

African Genomic Medicine Portal: A Web Portal for Biomedical Applications.

Genomics data are currently being produced at unprecedented rates, resulting in increased knowledge discovery and submission to public data repositories. Despite these advances, genomic information on African-ancestry populations remains significantly low compared with European- and Asian-ancestry populations. This information is typically segmented across several different biomedical data repositories, which often lack sufficient fine-grained structure and annotation to account for the diversity of African populations, leading to many challenges related to the retrieval, representation and findability of such information. To overcome these challenges, we developed the African Genomic Medicine Portal (AGMP), a database that contains metadata on genomic medicine studies conducted on African-ancestry populations. The metadata is curated from two public databases related to genomic medicine, PharmGKB and DisGeNET. The metadata retrieved from these source databases were limited to genomic variants that were associated with disease aetiology or treatment in the context of African-ancestry populations. Over 2000 variants relevant to populations of African ancestry were retrieved. Subsequently, domain experts curated and annotated additional information associated with the studies that reported the variants, including geographical origin, ethnolinguistic group, level of association significance and other relevant study information, such as study design and sample size, where available. The AGMP functions as a dedicated resource through which to access African-specific information on genomics as applied to health research, through querying variants, genes, diseases and drugs. The portal and its corresponding technical documentation, implementation code and content are publicly available.

Identification of Eleven Novel BRCA Mutations in Tunisia: Impact on the Clinical Management of BRCA Related Cancers.

BACKGROUND: Breast cancer is the world's most common cancer among women. It is becoming an increasingly urgent problem in low- and middle-income countries (LMICs) where a large fraction of women is diagnosed with advanced-stage disease and have no access to treatment or basic palliative care. About 5-10% of all breast cancers can be attributed to hereditary genetic components and up to 25% of familial cases are due to mutations in BRCA1/2 genes. Since their discovery in 1994 and 1995, as few as 18 mutations have been identified in BRCA genes in the Tunisian population. The aim of this study is to identify additional BRCA mutations, to estimate their contribution to the hereditary breast and ovarian cancers in Tunisia and to investigate the clinicopathological signatures associated with BRCA mutations. METHODS: A total of 354 patients diagnosed with breast and ovarian cancers, including 5 male breast cancer cases, have been investigated for BRCA1/2 mutations using traditional and/or next generation sequencing technologies. Clinicopathological signatures associated with BRCA mutations have also been investigated. RESULTS: In the current study, 16 distinct mutations were detected: 10 in BRCA1 and 6 in BRCA2, of which 11 are described for the first time in Tunisia including 3 variations that have not been reported previously in public databases namely BRCA1_c.915T>A; BRCA2_c.-227-?_7805+? and BRCA2_c.249delG. Early age at onset, family history of ovarian cancer and high tumor grade were significantly associated with BRCA status. BRCA1 carriers were more likely to be triple negative breast cancer compared to BRCA2 carriers. A relatively high frequency of contralateral breast cancer and ovarian cancer occurrence was observed among BRCA carriers and was more frequent in patients carrying BRCA1 mutations. CONCLUSION: Our study provides new insights into breast and ovarian cancer genetic landscape in the under-represented North African populations. The prevalence assessment of novel and recurrent BRCA1/2 pathogenic mutations will enhance the use of personalized treatment and precise screening strategies by both affected and unaffected North African cancer cases.

Identification of BRCA2 Cis Double Heterozygous Breast Cancer Cases Using Whole Exome Sequencing: Phenotypic Expression and Impact on Personalized Oncology.

BRCA1 and BRCA2 are the most commonly mutated breast cancer susceptibility genes that convey a high risk of breast and ovarian cancer. Most BRCA1 or BRCA2 mutation carriers have inherited a single heterozygous mutation. In recent years, very rare cases with biallelic or trans double heterozygous mutations on BRCA1 and or BRCA2 have been identified and seem to be associated with distinctive phenotypes. Given that this genotype-phenotype correlation in cancer predisposing hereditary conditions is of relevance for oncological prevention and genetic testing, it is important to investigate these rare BRCA genotypes for better clinical management of BRCA mutation carriers. Here we present the first report on Cis double heterozygosity (Cis DH) on BRCA2 gene identified using Whole exome sequencing (WES) in a Tunisian family with two BRCA2 mutations namely: c.632-1G>A and c.1310_1313DelAAGA that are both reported as pathogenic in ClinVar database. Subsequent analysis in 300 high-risk Tunisian breast cancer families detected this Cis double heterozygous genotype in 8 additional individuals belonging to 5 families from the same geographic origin suggesting a founder effect. Moreover, the observed Cis DH seems to be associated with an early age of onset (mean age = 35.33 years) and severe phenotype of the disease with high breast cancer grade and multiple cancer cases in the family. The identification of unusual BRCA genotypes in this Tunisian cohort highlights the importance of performing genetic studies in under-investigated populations. This will also potentially help avoiding erroneous classifications of genetic variants in African population and therefore avoiding clinical misdiagnosis of BRCA related cancers. Our findings will also have an impact on the genetic testing and the clinical management of North African breast cancer patients as well as patients from different other ethnic groups in regard to several emerging target therapies such as PARP inhibitors.

Germline copy number variations in BRCA1/2 negative families: Role in the molecular etiology of hereditary breast cancer in Tunisia.

Hereditary breast cancer accounts for 5-10% of all breast cancer cases. So far, known genetic risk factors account for only 50% of the breast cancer genetic component and almost a quarter of hereditary cases are carriers of pathogenic mutations in BRCA1/2 genes. Hence, the genetic basis for a significant fraction of familial cases remains unsolved. This missing heritability may be explained in part by Copy Number Variations (CNVs). We herein aimed to evaluate the contribution of CNVs to hereditary breast cancer in Tunisia. Whole exome sequencing was performed for 9 BRCA negative cases with a strong family history of breast cancer and 10 matched controls. CNVs were called using the ExomeDepth R-package and investigated by pathway analysis and web-based bioinformatic tools. Overall, 483 CNVs have been identified in breast cancer patients. Rare CNVs affecting cancer genes were detected, of special interest were those disrupting APC2, POU5F1, DOCK8, KANSL1, TMTC3 and the mismatch repair gene PMS2. In addition, common CNVs known to be associated with breast cancer risk have also been identified including CNVs on APOBECA/B, UGT2B17 and GSTT1 genes. Whereas those disrupting SULT1A1 and UGT2B15 seem to correlate with good clinical response to tamoxifen. Our study revealed new insights regarding CNVs and breast cancer risk in the Tunisian population. These findings suggest that rare and common CNVs may contribute to disease susceptibility. Those affecting mismatch repair genes are of interest and require additional attention since it may help to select candidates for immunotherapy leading to better outcomes.

Identification of Novel BRCA1 and RAD50 Mutations Associated With Breast Cancer Predisposition in Tunisian Patients.

BACKGROUND: Deleterious mutations on BRCA1/2 genes are known to confer high risk of developing breast and ovarian cancers. The identification of these mutations not only helped in selecting high risk individuals that need appropriate prevention approaches but also led to the development of the PARP-inhibitors targeted therapy. This study aims to assess the prevalence of the most frequent BRCA1 mutation in Tunisia, c.211dupA, and provide evidence of its common origin as well as its clinicopathological characteristics. We also aimed to identify additional actionable variants using classical and next generation sequencing technologies (NGS) which would allow to implement cost-effective genetic testing in limited resource countries. PATIENTS AND METHODS: Using sanger sequencing, 112 breast cancer families were screened for c.211dupA. A set of patients that do not carry this mutation were investigated using NGS. Haplotype analysis was performed to assess the founder effect and to estimate the age of this mutation. Correlations between genetic and clinical data were also performed. RESULTS: The c.211dupA mutation was identified in 8 carriers and a novel private BRCA1 mutation, c.2418dupA, was identified in one carrier. Both mutations are likely specific to North-Eastern Tunisia. Haplotype analysis supported the founder effect of c.211dupA and showed its recent origin. Phenotype-genotype correlation showed that both BRCA1 mutations seem to be associated with a severe phenotype. Whole Exome Sequencing (WES) analysis of a BRCA negative family revealed a Variant of Unknown Significance, c.3647C > G on RAD50. Molecular modeling showed that this variant could be classified as deleterious as it is responsible for destabilizing the RAD50 protein structure. Variant prioritization and pathway analysis of the WES data showed additional interesting candidate genes including MITF and ANKS6. CONCLUSION: We recommend the prioritization of BRCA1-c.211dupA screening in high risk breast cancer families originating from the North-East of Tunisia. We also highlighted the importance of NGS in detecting novel mutations, such as RAD50-c.3647C > G. In addition, we strongly recommend using data from different ethnic groups to review the pathogenicity of this variant and reconsider its classification in ClinVar.

Family specific genetic predisposition to breast cancer: results from Tunisian whole exome sequenced breast cancer cases.

BACKGROUND: A family history of breast cancer has long been thought to indicate the presence of inherited genetic events that predispose to this disease. In North Africa, many specific epidemio-genetic characteristics have been observed in breast cancer families when compared to Western populations. Despite these specificities, the majority of breast cancer genetics studies performed in North Africa remain restricted to the investigation of the BRCA1 and BRCA2 genes. Thus, comprehensive data at a whole exome or whole genome level from local patients are lacking. METHODS: A whole exome sequencing (WES) of seven breast cancer Tunisian families have been performed using a family-based approach. We focused our analysis on BC-TN-F001 family that included two affected members that have been sequenced using WES. Relevant variants identified in BC-TN-F001 have been confirmed using Sanger sequencing. Then, we conducted an integrative analysis by combining our results with those from other WES studies in order to figure out the genetic transmission model of the newly identified genes. Biological network construction and protein-protein interactions analyses have been performed to decipher the molecular mechanisms likely accounting for the role of these genes in breast cancer risk. RESULTS: Sequencing, filtering strategies, and validation analysis have been achieved. For BC-TN-F001, no deleterious mutations have been identified on known breast cancer genes. However, 373 heterozygous, exonic and rare variants have been identified on other candidate genes. After applying several filters, 12 relevant high-risk variants have been selected. Our results showed that these variants seem to be inherited in a family specific model. This hypothesis has been confirmed following a thorough analysis of the reported WES studies. Enriched biological process and protein-protein interaction networks resulted in the identification of four novel breast cancer candidate genes namely MMS19, DNAH3, POLK and KATB6. CONCLUSIONS: In this first WES application on Tunisian breast cancer patients, we highlighted the impact of next generation sequencing technologies in the identification of novel breast cancer candidate genes which may bring new insights into the biological mechanisms of breast carcinogenesis. Our findings showed that the breast cancer predisposition in non-BRCA families may be ethnic and/or family specific.