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.

Particular Mal de Meleda phenotypes in Tunisia and mutations founder effect in the Mediterranean region.

Mal de Meleda (MDM) is a rare, autosomal recessive form of palmoplantar keratoderma. It is characterized by erythema and hyperkeratosis of the palms and soles that progressively extend to the dorsal surface of the hands and feet. It is caused by mutations in SLURP-1 gene encoding for secreted mammalian Ly-6/uPAR-related protein 1 (SLURP-1). We performed mutational analysis by direct sequencing of SLURP-1 gene in order to identify the genetic defect in three unrelated families (families MDM-12, MDM-13, and MDM-14) variably affected with transgressive palmoplantar keratoderma. A spectrum of clinical presentations with variable features has been observed from the pronounced to the transparent hyperkeratosis. We identified the 82delT frame shift mutation in the SLURP-1 gene in both families MDM-12 and MDM-13 and the missense variation p.Cys99Tyr in family MDM-14. To date, the 82delT variation is the most frequent cause of MDM in the world which is in favour of a recurrent molecular defect. The p.Cys99Tyr variation is only described in Tunisian families making evidence of founder effect mutation of likely Tunisian origin. Our patients presented with very severe to relatively mild phenotypes, including multiple keratolytic pits observed for one patient in the hyperkeratotic area which was not previously reported. The phenotypic variability may reflect the influence of additional factors on disease characteristics. This report further expands the spectrum of clinical phenotypes associated with mutations in SLURP1 in the Mediterranean population.

Novel and recurrent mutations in the TAT gene in Tunisian families affected with Richner-Hanhart syndrome.

Tyrosinemia type II, also designated as oculocutaneous tyrosinemia or Richner-Hanhart syndrome (RHS), is a very rare autosomal recessive disorder. In the present study, we report clinical features and molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in two young patients, both born to consanguineous unions between first-degree cousins. These two unrelated families originated from Northern and Southern Tunisia. The clinical diagnosis was based on the observation of several complications related to Richner-Hanhart syndrome: recurrent eye redness, tearing and burning pain, photophobia, bilateral pseudodendritic keratitis, an erythematous and painful focal palmo-plantar hyperkeratosis and a mild delay of mental development. The diagnosis was confirmed by biochemical analysis. Sequencing of the TAT gene revealed the presence of a previously reported missense mutation (c.452G>A, p.Cys151Tyr) in a Tunisian family, and a novel G duplication (c.869dupG, p.Trp291Leufs 6). Early diagnosis of RHS and protein-restricted diet are crucial to reduce the risk and the severity of long-term complications of hypertyrosinemia such as intellectual disability.

Clinical and genetic investigation of pediatric cases of Wolff-Parkinson-White syndrome in Tunisian families.

BACKGROUND: Wolff-Parkinson-White (WPW) syndrome is an autosomal-dominant heart disease characterized by an accessory pathway that arises from an aberrant conduction from the atria to the ventricles. Several mutations within the PRKAG2 gene were shown to be responsible for WPW. This gene encodes the γ2 regulatory subunit of adenosine monophosphate (AMP)-activated protein kinase, which functions as a metabolic sensor in cells, responding to cellular energy demands. METHODS: This first study of WPW in a North African population comprises the clinical and genetic investigation of 3 Tunisian families, including 11 affected members. The involvement of the PRKAG2 and NKX2-5 genes was investigated. RESULTS: Mutation screening showed that with the exception of two already reported single-nucleotide polymorphisms, no mutations were detected within the coding region of PRKAG2 or in the NKX2-5 gene. CONCLUSIONS: This study provides further evidence of the genetic heterogeneity of WPW.

New mutations of Darier disease in Tunisian patients.

Darier's disease (DD, MIM 124200) also known as Darier-White disease and keratosis follicularis, is a rare autosomal dominant skin disorder characterized by warty papules and plaques in the seborrheic area (central trunk, flexures, scalp, and forehead). Pathogenic mutations in the ATP2A2 gene encoding the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) 2 gene underlie the disease. In the present study, we performed genetic investigation of three unrelated Tunisian families affected by DD. Mutation screening was performed by direct sequencing of the coding region and exon/intron boundaries of the ATP2A2 gene. Patients in the 3 studied families exhibited classical DD phenotype. DD was associated with neurological and cardiac disorders in one family. Two novel mutations were identified: a missense mutation (R559Q) and a frameshift mutation (1713-1714 del 2A). Both pathogenic mutations are located in exon 13 of the ATP2A2 gene and affected the ATP-binding site of the SERCA2 protein. In one family, no mutation was found within the coding region and exon/intron boundaries of the ATP2A2 gene. Our findings provide further evidence for the genetic heterogeneity of DD in Tunisia and that most mutations involved in this disease are family specific.

Clinical and mutational heterogeneity of Darier disease in Tunisian families.

OBJECTIVE: To study the mutation spectrum and phenotype-genotype correlation of Darier disease (DD) in Tunisian patients. DESIGN: Case series. SETTING: Referral center: Department of Dermatology (La Rabta Hospital), Tunis, Tunisia. PATIENTS: Eight large Tunisian families with DD, with a total of 23 patients and 9 unaffected family members. MAIN OUTCOME MEASURE: Patients were investigated at the clinical, histological, and genetic levels. Families were genotyped with 5 microsatellite markers spanning the ATP2A2 gene. Mutation screening was performed by direct sequencing of the coding region and exon/intron boundaries of the ATP2A2 gene. RESULTS: Typical clinical features of DD were constantly present. Phenotypic variation within and between the studied families was observed. Different neuropsychiatric disorders were seen in 5 families, and various cutaneous and extracutaneous original clinical associations were observed. The haplotype analysis led to the identification of different haplotypes cosegregating with the disease in the studied families. Mutation screening of the ATP2A2 gene revealed 3 recurrent mutations (119-120delAG, R677X, and D702N) and 4 novel variations: 2 missense mutations (G217A and L900R), one microinsertion (2772-2779 ins C), and one microdeletion (1747-1749 del 2T). CONCLUSIONS: Our findings provide evidence for clinical and mutational heterogeneity of Tunisian families with DD. No obvious phenotype-genotype correlation was established. To our knowledge, this is the first molecular investigation of DD in the North African population.

Immunohistological study of involucrin expression in Darier's disease skin.

BACKGROUND: Darier's disease (DD) is an autosomal dominant skin disorder characterized by acantholysis and abnormal keratinization. The gene responsible for DD, ATP2A2 encodes for the sarco/endoplasmic reticulum (ER) Ca2+-ATPase isoform 2 protein. Involucrin, considered as a marker of terminal epidermal differentiation, could be altered in some keratinization disorders including DD. PATIENTS AND METHODS: An immunohistochemical staining using anti-involucrin antibody was carried out on 16 DD patients epidermis. Involucrin staining was compared with biopsies from cutaneous lesions of three healthy individuals and of patients with Hailey-Hailey disease (five cases) and Mal de Meleda (four cases). A semi-quantitative analysis was performed in order to evaluate involucrin immunostaining on the basis of intensity, extension and epidermal distribution. The involucrin expression was examined afterward with confocal laser scanning microscopy. RESULTS: In contrast to normal skin, all DD cases showed premature expression of involucrin in the lower epidermal layers in four cases with a strong labeling in both keratinocytes cell membrane and cytoplasm. Other keratinization disorders share premature expression of involucrin but displayed differences in cytoplasm/cell membrane labeling. CONCLUSIONS: DD skin displayed a constant immunohistochemical involucrin pattern characterized by both premature expression and a particular cytoplasmic/cell membrane localization distribution.

Further evidence of the clinical and genetic heterogeneity of recessive transgressive PPK in the Mediterranean region.

Transgressive palmoplantar keratoderma (PPK) is the phenotypic hallmark of Mal de Meleda (MDM, MIM 24300). It is characterized by erythema and hyperkeratosis that extend to the dorsal face of the hands and feet. The disease is distributed worldwide and includes the Mediterranean population. The gene responsible for MDM, ARS (component B) mapped on chromosome 8qter, encodes for the SLURP-1 protein (Ly-6/uPAR related protein-1). A variety of mutations within the ARS gene have been shown to underlie MDM in different populations. Genetic heterogeneity of MDM is suspected. We have recently shown that three different homozygous mutations (82delT, C77R, C99Y) were responsible for MDM in 17 patients from Northern Tunisia belonging to eight unrelated consanguineous families. We report here a Tunisian family with three siblings presenting with recessive transgressive PPK closely resembling the MDM phenotype that excludes linkage to the ARS gene.