A novel homozygous missense variant identified in the myosin VIIA motor domain of a Moroccan patient with usher syndrome.

BACKGROUND: Usher syndrome 1 (USH1) is the most severe subtype of Usher syndrome characterized by severe sensorineural hearing impairment, retinitis pigmentosa, and vestibular areflexia. USH1 is usually induced by variants in MYO7A, a gene that encodes the myosin-VIIa protein. Myosin-VIIA is effectively involved in intracellular molecular traffic essential for the proper function of the cochlea, the retinal photoreceptors, and the retinal pigmented epithelial cells. METHODS AND RESULTS: In this study, we report a new homozygous missense variant (NM_000260.4: c.1657 C > T p.(His553Tyr)) in MYO7A of a 28-year-old female with symptoms consistent with USH1. This variant, c.1657 C > T p.(His553Tyr) is positioned in the highly conserved myosin-VIIA motor domain. Previous studies showed that variants in this domain might disrupt the ability of the protein to bind to actin and thus cause the disorder. CONCLUSIONS: Our findings contribute to our understanding of the phenotypic and mutational spectrum of USH1 associated with autosomal recessive MYO7A variants and emphasize the important role of molecular testing in accurately diagnosing this syndrome. More advanced research is required to understand the functional effect of the identified variant and the genotype-phonotype correlations of MYO7A-related Usher syndrome 1.

RAS Mutations Predict Recurrence-Free Survival and Recurrence Patterns in Colon Cancer: A Unicenter Study in Morocco.

PURPOSE: To date, only a few studies have investigated the role of molecular alterations in cancer recurrence. This exploratory study aimed to evaluate the impact of molecular alterations on the time and site of recurrence in patients with stage I-IV CRC and to identify the risk factors predicting recurrence-free survival in colon cancer. METHODS: A total of 270 patients were retrospectively included. We assessed the full RAS status using Sanger and pyrosequencing. MSI status was determined by immunohistochemical analysis. Molecular alterations were correlated with recurrence timing (early or late), recurrence patterns, and recurrence-free survival. Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. RESULTS: Of the 270 patients, 85 (31%) experienced recurrence, among whom 53% had mutant full RAS status, 48% had KRAS mutations, and 31.4% had KRAS p. G12V mutation subtype. Compared with those with late recurrence, patients with early recurrence were significantly older (P = 0.02) and more likely to have poorly differentiated tumors, a higher rate of positive lymph nodes, KRAS mutations, and especially KRAS p. G12V mutation variant. RAS mutation status, KRAS mutations, and rare mutations are more common in patients with lung cancer recurrence. Multivariate logistic regression analysis revealed that differentiation, perineural invasion, full RAS mutation status, and KRAS codon 13 mutations were independent factors for recurrence-free survival in colon cancer. CONCLUSION: In this cohort, the timing and patterns of recurrence appeared to be associated with the patient's molecular profile. KRAS codon 12 mutations were the worst predictors of recurrence-free survival at all stages in our population.

Unraveling the Diversity of GJB2 Mutations in Nonsyndromic Hearing Loss: A Comprehensive Study in the Moroccan Population.

INTRODUCTION: Despite the high genetic heterogeneity of hearing loss, mutations in the GJB2 gene are a major cause of autosomal recessive nonsyndromic hearing loss (NSHL) worldwide. However, the mutation profile of GJB2 in NSHL is under-investigated in Morocco, especially among simplex cases. This study aimed to identify the spectrum and frequency of GJB2 mutations in the Moroccan population among simplex and multiplex families with NSHL. METHODS: Moroccan families with NSHL were selected according to well-defined criteria. Selected families were screened for GJB2 gene variants using direct sequencing of the entire coding region of GJB2. RESULTS: A total of 145 affected individuals from 115 families with NSHL were included in this study (49 simplex, 66 multiplex). Mutations in the GJB2 gene were noted in 28.69% of the families (33/115), of which 75.75% were multiplex families and 24.24% were simplex. In total, seven different mutations were detected: c.35delG(p.G12fs), c.551G>A(p.R184Q), c.139G>T(p.E47X), c.109G>A(p.V37I), c.167delT(p.L56fs), c.617A>G(p.N206S), c.94C>T(p.R32C). The last three mutations have not previously been reported in Morocco. The most common GJB2 mutation was c.35delG (21.73%), followed by p.V37I (2.60%) and p.E47X (1.73%). CONCLUSIONS: Our study confirms a high prevalence of GJB2 variants in the Moroccan population, particularly the c.35delG mutation. Additionally, we have identified previously unreported or rarely reported mutations, revealing a greater diversity of GJB2 mutations. These findings emphasize the importance of comprehensive screening beyond the 35delG mutation for patients with NSHL, regardless of their family history. Integrating this approach into clinical care will enhance diagnosis and management of hearing loss in the Moroccan population.

A homozygous missense variant in the PLCB4 gene causes severe phenotype of auriculocondylar syndrome type 2.

Auriculocondylar syndrome (ARCND) is a rare craniofacial birth defect characterized by malformations in the mandible and external ear (Question Mark Ear). Genetically, three distinct subtypes of ARCND (ARCND1, ARCND2, and ARCND3) have been identified. ARCND2 is linked to pathogenic variants in the PLCB4 gene (phospholipase C ß4). PLCB4 is a key effector of the EDN1-EDNRA pathway involved in craniofacial development via the induction, migration, and maintenance of neural crest cells. ARCND2 is typically inherited in an autosomal dominant pattern, with recessive inheritance pattern being rare. In this study, we report the first homozygous missense variant (NM_000933.4: c.2050G>A: p.(Gly684Arg)) in the PLCB4 gene causing ARCND in a 3-year-old patient with a severe clinical phenotype of the syndrome. The patient presented with typical craniofacial ARCND features, in addition to intestinal transit defect, macropenis, and hearing loss. These findings further delineate the phenotypic spectrum of ARCND associated with autosomal recessive PLCB4 loss of function variants. Notably, our results provide further evidence that these variants can result in a more severe and diverse manifestations of the syndrome. Clinicians should consider the rare features of this condition for better management of patients.

The Diagnosis and Genetic Mechanisms of Prader-Willi Syndrome: Findings From a Moroccan Population Study.

Background Prader-Willi syndrome (PWS) is a complex genetic disorder caused by a deficit in gene expression on the paternal inherited chromosome 15q11.2-q13. It affects various aspects of growth and development, including feeding, cognitive function, and behavior. Early diagnosis and management of PWS can significantly improve outcomes for patients and their families. Methods In this study, we analyzed a group of 29 clinically diagnosed patients suspected of PWS. All patients were referred to the medical genetics and onco-genetics service for genetic consultation and molecular analysis. We used DNA methylation analysis and fluorescence in situ hybridization (FISH) to confirm the diagnosis and identify the underlying genetic mechanisms. Results Our analysis showed that five out of seven patients (71.43%) with a positive methylation-specific PCR (MSP) had chromosomal deletion by FISH and presented major clinical signs summarized by morbid obesity in 65.21% of cases and neonatal hypotonia in 42.85% of cases. This finding indicates that paternal 15q11-q13 deletion is the most common genetic mechanism involved in PWS. Conclusion The results of this study highlight the importance of early diagnosis and molecular analysis in the management of Prader-Willi syndrome. Our findings contribute to a better understanding of the genotype-phenotype correlation in the Moroccan population and provide families with a rigorous molecular diagnosis, relevant genetic counseling, and multidisciplinary support. Further research is needed to explore the underlying mechanisms of PWS and develop effective interventions to improve outcomes for affected individuals.

Review of prostate cancer genomic studies in Africa.

Prostate cancer (PCa) is the second most commonly diagnosed in men worldwide and one of the most frequent cancers in men in Africa. The heterogeneity of this cancer fosters the need to identify potential genetic risk factors/biomarkers. Omics variations may significantly contribute to early diagnosis and personalized treatment. However, there are few genomic studies of this disease in African populations. This review sheds light on the status of genomics research on PCa in Africa and outlines the common variants identified thus far. The allele frequencies of the most significant SNPs in Afro-native, Afro-descendants, and European populations were compared. We advocate how these few but promising data will aid in understanding, better diagnosing, and precisely treating this cancer and the need for further collaborative research on the genomics of PCa in the African continent.

Molecular classification of soft tissue sarcomas for adequate diagnosis: A study on the northeast population of Morocco.

Background: Soft tissue sarcomas (STS) are a heterogeneous group of tumors. For adequate therapeutic management, an accurate diagnosis is necessary. In Morocco, the diagnosis is essentially based on the morphological and immunohistochemical study. Compared to other techniques, fluorescence in situ hybridization (FISH) is easier to develop and less expensive. This study aims to assess the feasibility and utility of implementing FISH technique to improve diagnostic accuracy and establish a good classification. Material and methods: This is a retrospective cohort study. 211 cases of mesenchymal tumors were included. Hematoxylin Eosin Safran (HES) staining was performed in all cases followed by immunohistochemistry (IHC). FISH was performed in all cases with suspected STS. The probes used were EWSR1, MDM2 and SS18. The performance of FISH and histopathological test were evaluated by the ROC curve method (receiver operating characteristic). We evaluated the concordance between FISH and real time PCR by Cohen test. Results: The real-time PCR technique showed good agreement with the FISH test by a Kappa coefficient of 60% (p = 0.035). FISH was able to confirm that it is more accurate (Youden's Index = 91%) than histological/immunohistochemical analysis (Youden's Index = 51%), as well as the positive predictive value was higher (100%) with an ROC curve finding a larger area under the curve of 0.953 (95% CI: 0.918-0.988), p = 0.000 which supports that FISH shows high performance to present an accurate final diagnosis. Conclusion: This is the first and the largest Moroccan series for the molecular diagnosis of STS by FISH. Our study shows that paraffin FISH is a sensitive and specific ancillary tool in the diagnosis of STS when used in the appropriate clinicopathological context.

Molecular and clinical assessment of maturity-onset diabetes of the young revealed low mutational rate in Moroccan families.

Background: Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance. To offer an adequate patient management and therapeutic treatment for MODY patients, in addition to an early efficient diagnosis of their asymptomatic relatives, it is crucial to set an accurate molecular diagnosis. Hence, our aim was to determine the frequency of HNF1A and GCK genes among Moroccan-suspected MODY patients. Methods: Twenty suspected MODY patients were screened for HNF1A and GCK mutations using Sanger sequencing and MLPA methods. Segregation analysis of identified mutations was performed among family members. The pathogenic nature of missense variants was predicted using bioinformatic tools. Results: A total of two mutations were revealed among all patients raising the diagnostic rate to 10%. We identified a large novel GCK deletion (c.209-?_1398+?del) by MLPA in one patient and a previously reported missense substitution (c.92G > A) in HNF1A gene. Conclusion: This is the first investigation to perform the molecular diagnosis of MODY suspected patients. Our findings constitute a primary contribution towards unraveling the genetic landscape involved in the pathogenesis of MODY disease in Morocco.

Detection of a new deleterious SGCE gene variant in Moroccan family with inherited myoclonus-dystonia.

Myoclonus-dystonia (M-D) is a pleiotropic neuropsychiatric disorder with autosomal dominant mode of inheritance with variable severity and incomplete penetrance. Pathogenic variants in ξ-sarcoglycan gene SGCE are the most frequently known genetic cause of M-D with maternal imprinting, and in most cases, a symptomatic individual inherits the pathogenic variant from his or her father. This work reported a missense mutation c.662G> T inherited in the M-D Moroccan family described for the first time, which is deleterious based on protein modeling analysis.

Dietary Fat Intake and KRAS Mutations in Colorectal Cancer in a Moroccan Population.

Epidemiologic data support an association between diet and mutations in the Kirsten-ras (KRAS) gene involved in colorectal cancer (CRC) development. This study aimed to explore the associations between fat intake and KRAS mutations in codons 12 and 13 in cases of CRC in the Moroccan population. A multicenter case-series study nested in a large-scale Moroccan CRC case-control study was conducted. Among all CRC cases recruited, 151 specimens were available for the DNA mutation analysis. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (Cis) for KRAS mutation status according to the fat intake variables. A KRAS mutation was detected in the CRC tumor of 34.4% of the patients among whom 65.4% had a single mutation at codon 12 and 34.6% had a single mutation at codon 13. Compared to low levels of consumption, a positive association was observed between high polyunsaturated fatty acids (PUFA) consumption (>16.9 g/day) and prevalence of KRAS mutations (OR = 2.15, 95% CI = 1.01-4.59). No statistically significant associations were observed for total fat, monounsaturated fatty acids, saturated fatty acids and KRAS mutations. The results of this study suggest that PUFA may be relevant in the etiology of CRC, possibly through the generation of G > A transitions at the KRAS oncogene. Further studies are needed to verify and explain this finding.

Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot.

PURPOSE: Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. METHODS: We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. RESULTS: Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). CONCLUSION: Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

Absence of GATA4 Mutations in Moroccan Patients with Atrial Septal Defect (ASD) Provides Further Evidence of Limited Involvement of GATA4 in Major Congenital Heart Defects.

OBJECTIVE: Atrial septal defect (ASD) is one of the most common types of congenital heart disease (CHD). It is mainly caused by mutations of NK2 homeobox 5, GATA binding protein 4 (GATA4), and myosin heavy chain 6 in non-syndromic cases. This study aims to carry out, for the first time, the GATA4 mutation screening in a Moroccan population affected by ASD and compare the obtained mutation rate across populations. MATERIALS AND METHODS: A total of 33 patients were enrolled in this study. DNAs were extracted from peripheral blood samples, and we performed PCR-sequencing for GATA4 coding regions. Sequences were analyzed by sequence alignment and functional impact prediction tools. Mutation rate comparisons were performed by R software using the appropriate statistical tests. RESULTS: We detected 7 variants, but no pathogenic mutation was revealed, except for Asn352= that was assessed by human splicing finder algorithms to have a potential impairing effect on the splicing mechanism. Until proven by in vitro functional studies, the current pathogenic mutation rate in our cohort seems to be 0%. Statistical comparison with previous studies from all over the world shows no significant difference. Seemingly, comparison of previous GATA4 mutation rates among tetralogy of Fallot (TOF) populations shows no significant difference. CONCLUSION: The low rates of GATA4 mutations observed throughout ASD and TOF international populations may suggest a limited causality of GATA4 mutations in the main CHDs, which further confirms the co-involvement of additional genetic and/or environmental factors in the manifestation of these phenotypes.

Associations between nutritional factors and KRAS mutations in colorectal cancer: a systematic review.

BACKGROUND: Between 30 and 50% of colon tumors have mutations in the Kirsten-ras (KRAS) gene, which have a large nutritional attributable risk. Despite its high frequency in colorectal cancer (CRC), data to support specific associations between KRAS mutations in CRC and diet are sparse. Here, we conducted a systematic review to summarize the current epidemiological evidence on the association between various dietary factors and KRAS mutations. METHODS: PubMed, Science Direct, and Cochrane databases were searched for relevant studies published until December 31, 2019, using inclusion and exclusion criteria in accordance with PRISMA guidelines. We analyzed the studies to find associations between nutritional factors and CRC tumors with KRAS mutations in humans. RESULTS: We identified 28 relevant studies to include in this systematic review. In-depth analyses showed unclear associations between nutritional factors and KRAS mutations in CRC. Most epidemiological studies in the same nutrient or food often reported conflicting and/or inconclusive findings, whereas for some dietary factors, the results were homogeneous. CONCLUSIONS: Further research using a more robust prospective cohort study is needed to lend more credence to the epidemiological associations found between KRAS mutations and dietary factors.

Cytogenetic and FISH analysis of 93 multiple myeloma Moroccan patients.

BACKGROUND: Multiple myeloma (MM) is a disease characterized by heterogeneous clinical presentations as well as complex genetic and molecular abnormalities. In MM, cytogenetic analysis is a challenge because of the low proliferation of malignant plasma cells. Thus, interphase fluorescence in situ hybridization (FISH), performed on sorted plasma cells detected abnormalities independently of a proliferative and infiltrative index. The purpose of this study was to explore, for the first time, the cytogenetic and molecular genetics features in Moroccan patients with multiple myeloma referred exclusively to National Reference Laboratory and to determine their risk stratification based on these features. METHODS: We performed cytogenetic analysis on 93 MM cases, all patients were subjected to FISH analysis, among which 45 patients have benefited from both FISH analysis and standard karyotype. RESULTS: Karyotype was normal in 78% (35/45) while, it was complex with varied structural and numerical abnormalities in 22% (10/45) of all patients, among which Hyperdiploid karyotype was found in 9% (n = 4 cases) and nonhyperdiploid in 13% (n = 6 cases). The most common numerical abnormalities were gains of chromosomes 3, 5, 9, 15, and 19. Whole chromosome losses were also frequent, affecting chromosomes X, 3, 14, 16 and 22. FISH analysis detected abnormalities in 50% of cases. The translocation t(4;14) and dup (1q) were the most frequent types of anomalies (14% and 13% respectively), followed by (17p) deletion and 14q32/IGH translocations with an undetermined origin (12% each) then the (1p) deletion (4%). For the normal karyotypes, FISH revealed chromosome abnormalities in 46%. CONCLUSION: This study compares the results of cytogenetic analysis of chromosomal abnormalities in the Moroccan population with other countries. ½ patient showed at least one type of molecular genetic abnormalities. Therefore, the introducing of the cytogenetic analysis is obligatory in the diagnosis of multiple myeloma.

Empowering newborn screening programs in African countries through establishment of an international collaborative effort.

In an effort to explore new knowledge and to develop meaningful collaborations for improving child health, the First Pan African Workshop on Newborn Screening was convened in June 2019 in Rabat, Morocco. Participants included an informal network of newborn screening stakeholders from across Africa and global experts in newborn screening and sickle cell disease. Over 150 attendees, representing 20 countries, were present including 11 African countries. The agenda focused on newborn screening rationale, techniques, system development, implementation barriers, ongoing research, and collaborations both globally and across Africa. We provide an overview of the workshop and a description of the newborn screening activities in the 11 African countries represented at the workshop, with a focus on sickle cell disease.

Analysis of Molecular Pretreated Tumor Profiles as Predictive Biomarkers of Therapeutic Response and Survival Outcomes after Neoadjuvant Therapy for Rectal Cancer in Moroccan Population.

Pathologic features depending on tumor response to preoperative chemoradiotherapy are important to determine the outcomes in patients with rectal cancer. Evaluating the potential predictive roles of biomarker expression and their prognostic impact is a promising challenge. We reported here the immunohistochemical staining of a panel marker of mismatch repair protein (MMR), Ki67, HER-2, and p53. Additionally, identification of somatic mutations of KRAS, NRAS, and BRAF genes were performed by direct sequencing and pyrosequencing in pretreated biopsy tissues from 57 patients diagnosed for rectal cancer. Clinical features and pathological criteria for postneoadjuvant treatment surgical resection specimen's data were collected. Immunohistochemical expression and mutational status were correlated with therapeutic response, overall survival, and disease progression. The mean age of patients was 56 years. Seven (12.3%) out of 57 patients had a complete therapeutic response. Our analysis showed that when using complete therapeutic response (Dworak 4) and incomplete therapeutic response (Dworak 3, 2, and 1) as grouping factor, high p53 expression at the pretreatment biopsy was significantly associated to an incomplete response (p = 0.002). For 20 and 2 out of 57, KRAS and NRAS mutations were detected, respectively. The majority of these mutations affected codon 12. KRAS mutations detected at codon 146 (A146T, A146V) was associated with the appearance of recurrence and distant metastasis (p = 0.019). A high expression of HER-2 corresponding to score 3+ was observed in 3 pretreatment biopsy specimens. This class was significantly associated with a short relapse-free survival (p = 0.002). Furthermore, the high expression of Ki67 was moderately correlated with an older age (p = 0.016, r = 0.319). In addition, this shows that high p53 expression in the pretreatment biopsy was associated with an incomplete response in surgical resection specimens after neoadjuvant treatment, and a HER-2 score 3+ can be a predictive factor of distant metastasis and local recurrence. Larger, prospective, and more studies are needed.

Cancer Omics in Africa: Present and Prospects.

During the last century, cancer biology has been arguably one of the most investigated research fields. To gain deeper insight into cancer mechanisms, scientists have been attempting to integrate multi omics data in cancer research. Cancer genomics, transcriptomics, metabolomics, proteomics, and metagenomics are the main multi omics strategies used currently in the diagnosis, prognosis, treatment, and biomarker discovery in cancer. In this review, we describe the use of different multi omics strategies in cancer research in the African continent and discuss the main challenges facing the implementation of these approaches in African countries such as the lack of training programs in bioinformatics in general and omics strategies in particular and suggest paths to address deficiencies. As a way forward, we advocate for the establishment of an "African Cancer Genomics Consortium" to promote intracontinental collaborative projects and enhance engagement in research activities that address indigenous aspects for cancer precision medicine.

Implication of Microsatellite Instability Pathway in Outcome of Colon Cancer in Moroccan Population.

BACKGROUND: Tumors with microsatellite instability (MSI tumors) have distinct clinicopathological features. However, the relation between these tumor subtypes and survival in colon cancer remains controversial. The aim of this study was to evaluate the overall survival (OS) in patients with MSI phenotype, in FES population. METHODS: The expression of MMR proteins was evaluated by immunohistochemistry for 330 patients. BRAF, KRAS, and NRAS mutations were examined by Sanger sequencing and pyrosequencing methods. The association of MSI status with a patient's survival was assessed by the Kaplan-Meier method and log-rank test. RESULTS: The mean age was 54.6 years (range of 19-90 years). The MSI status was found in 11.2% of our population. MSI tumors were significantly associated with male gender, younger patients, stage I-II, right localization, and a lower rate of lymph node and distant metastasis. The OS tends to be longer in MSI tumors than MSS tumors (109.71 versus 74.08), with a difference close to significance (P = 0.05). CONCLUSION: Our study demonstrates that MSI tumors have a particular clinicopathological features. The results of survival analysis indicate that the MSI status was not predictive of improved overall survival in our context with a lower statistical significance (P = 0.05) after multivariate analysis.

Molecular and presymptomatic analysis of a Moroccan Lynch syndrome family revealed a novel frameshift MLH1 germline mutation.

Lynch syndrome (LS) is an autosomal dominant disorder characterized by an increased risk of extracolonic cancers and early age of onset. It is associated with germline mutations in the DNA mismatch repair (MMR) genes. We report a case of a patient with colorectal cancer referred to our medical genetics department for molecular analysis and genetic counseling. The proband is a 64-year-old woman diagnosed with a tumor of the cecum. Histopathological examination showed a moderately differentiated mucinous adenocarcinoma categorized by pT3 N0. Analysis of her pedigree revealed three siblings who had colon cancer, as well as one relative with brain cancer. Based on these findings, molecular genetic investigation was found to be necessary in order to identify the disease-causing mutation. Immunohistochemistry staining of MMR proteins was performed on the tumor sample of the index proband. Mutational analysis of the MLH1/MSH2 genes was carried out. Analysis was extended to the family members and the general population. This led to the identification of a heterozygous frameshift duplication in the MLH1 gene at position 910 (c.910dupG). Three siblings had inherited the mutation from their mother, two of whom were asymptomatic at the time of diagnosis. To the best of our knowledge, this is a novel pathogenic duplication that has not been reported in the databases and literature. The outcome of the present case suggests that this mutation was the primary cause of LS in the family.