Mutational spectrum of BRCA1/2 genes in Moroccan patients with hereditary breast and/or ovarian cancer, and review of BRCA mutations in the MENA region.

PURPOSE: Breast cancer (BC) is the most common form of female cancer around the world. BC is mostly sporadic, and rarely hereditary. These hereditary forms are mostly BRCA1- and BRCA2-associated hereditary breast and ovarian cancer syndrome. BRCA1 and BRCA2 genes are large and had some recurrent mutations specific to some populations. Through this work we analyze the most recurrent mutations in Moroccan population and compared them to a large review of other BRCA1/2 spectrum mutations in the MENA region. METHODS: We report in this work a series of 163 unrelated patients (the largest series of Moroccan patients) with familial breast and/or ovarian cancer, selected among patients referred to our oncogenetic outpatient clinic, from 2006 to 2021. To identify genetic variants in these two genes, different genetic analysis strategies have been carried out, using Sanger Sequencing DNA or Target Panel Sequencing. RESULTS: Pathogenic variants were identified in 27.6% of patients. The most frequent mutation identified in our patients was the c.1310_1313delAAGA, BRCA2 (33%), and three other mutations seem more frequent in the Moroccan population (33%) of all reported patients: c.798_799delTT, BRCA1; and c.3279delC, BRCA1; and c.7234_7235insG in BRCA2 gene. CONCLUSION: Through this work, we emphasize the importance of screening for BRCA1 and BRCA2 recurrent mutations in Moroccan patients. Other MENA (MENA: English-language acronym referring to the Middle East and North Africa region) countries had also some recurrent BRCA mutations, which will allow a fast and unexpensive first line genetic analysis and a precise molecular diagnosis. This will allow an adapted follow-up of the patients and a pre-symptomatic diagnosis of their relatives.

Novel copy number variation of COLQ gene in a Moroccan patient with congenital myasthenic syndrome: a case report and review of the literature.

BACKGROUND: Congenital myasthenic syndromes (CMSs) are rare genetic diseases due to abnormalities of the neuromuscular junction leading to permanent or transient muscle fatigability and weakness. To date, 32 genes were found to be involved in CMSs with autosomal dominant and/or recessive inheritance patterns. CMS with acetylcholinesterase deficiency, in particular, was determined to be due to biallelic mutations of COLQ gene with early-onset clinical signs. Here, we report clinical features and novel molecular findings of COLQ-related CMS in a Moroccan patient with a review of the literature for this rare form. CASE PRESENTATION: In this study, we report the case of a 28-month-old Moroccan female patient with hypotonia, associated to axial muscle weakness, global motor delay, bilateral ptosis, unilateral partial visual field deficiency with normal ocular motility, and fatigable muscle weakness. Clinical exome sequencing revealed a novel homozygous deletion of exon 13 in COLQ gene, NM_005677.4(COLQ):c.(814+1_815-1)_(954+1_955-1) del p.(Gly272Aspfs*11). This finding was subsequently confirmed by quantitative real-time PCR (qPCR) in the proband and her parents. In silico analysis of protein-protein interaction network by STRING tool revealed that 12 proteins are highly associated to COLQ with an elevated confidence score. Treatment with Salbutamol resulted in clear benefits and recovery. CONCLUSIONS: This clinical observation illustrates the important place of next-generation sequencing in the precise molecular diagnosis of heterogeneous forms of CMS, the appropriate management and targeted treatment, and genetic counseling of families, with a better characterization of the mutational profile of this rare disease in the Moroccan population.

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.

GNB5 Mutations Cause an Autosomal-Recessive Multisystem Syndrome with Sinus Bradycardia and Cognitive Disability.

GNB5 encodes the G protein ß subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision.

Heimler Syndrome Is Caused by Hypomorphic Mutations in the Peroxisome-Biogenesis Genes PEX1 and PEX6.

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. We ascertained eight families affected by HS and, by using a whole-exome sequencing approach, identified biallelic mutations in PEX1 or PEX6 in six of them. Loss-of-function mutations in both genes are known causes of a spectrum of autosomal-recessive peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome. PBDs are characterized by leukodystrophy, hypotonia, SNHL, retinopathy, and skeletal, craniofacial, and liver abnormalities. We demonstrate that each HS-affected family has at least one hypomorphic allele that results in extremely mild peroxisomal dysfunction. Although individuals with HS share some subtle clinical features found in PBDs, the diagnosis was not suggested by routine blood and skin fibroblast analyses used to detect PBDs. In conclusion, our findings define HS as a mild PBD, expanding the pleiotropy of mutations in PEX1 and PEX6.

Developments in FINDbase worldwide database for clinically relevant genomic variation allele frequencies.

FINDbase (http://www.findbase.org) aims to document frequencies of clinically relevant genomic variations, namely causative mutations and pharmacogenomic markers, worldwide. Each database record includes the population, ethnic group or geographical region, the disorder name and the related gene, accompanied by links to any related databases and the genetic variation together with its frequency in that population. Here, we report, in addition to the regular data content updates, significant developments in FINDbase, related to data visualization and querying, data submission, interrelation with other resources and a new module for genetic disease summaries. In particular, (i) we have developed new data visualization tools that facilitate data querying and comparison among different populations, (ii) we have generated a new FINDbase module, built around Microsoft's PivotViewer (http://www.getpivot.com) software, based on Microsoft Silverlight technology (http://www.silverlight.net), that includes 259 genetic disease summaries from five populations, systematically collected from the literature representing the documented genetic makeup of these populations and (iii) the implementation of a generic data submission tool for every module currently available in FINDbase.

Distribution of allelic and genotypic frequencies of NAT2 and CYP2E1 variants in Moroccan population.

BACKGROUND: Several pathogenesis and genetic factors influence predisposition to antituberculosis drug-induced hepatotoxicity (ATDH) especially for isoniazid (INH). However, the major susceptibility genes for ATDH are N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1). NAT2 gene determines the individual's acetylator status (fast, intermediate or slow) to metabolize drugs and xenobiotics, while CYP2E1 c1/c1 genotype carriers had an increased risk of ATDH. Polymorphisms of the NAT2 and CYP2E1 genes vary remarkably among the populations of different ethnic origins. The aim of this study was to determine, for the first time, the frequency of slow acetylators in Moroccan population by genotyping of NAT2 gene variants and determining the genotype c1/c1 for CYP2E1 gene, in order to predict adverse effects of Tuberculosis treatment, particularly hepatotoxicity. RESULTS: The frequencies of specific NAT2 alleles were 53%, 25%, 2% and 4% for NAT2*5, NAT2*6, NAT2*7 and NAT2*14 respectively among 163 Moroccan studied group. Genotyping of CYP2E1 gene, by real-time polymerase chain reaction using TaqMan probes, revealed frequencies of 98.5% for c1/c1 and 1.5% for c1/c2 among 130 Moroccan studied group. CONCLUSION: The most prevalent genotypes of NAT2 gene in Moroccans are those which encode slow acetylation phenotype (72.39%), leading to a high risk of ATDH. Most Moroccans are homozygous for c1 allele of CYP2E1 gene which aggravates hepatotoxicity in slow acetylators. This genetic background should be taken into account in determining the minimum dose of INH needed to treat Moroccan TB patients, in order to decrease adverse effects.

Exome sequencing reveals a novel Moroccan founder mutation in SLC19A3 as a new cause of early-childhood fatal Leigh syndrome.

Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes have been reported to cause Leigh syndrome, indicating an extreme genetic heterogeneity for this disorder, but still only explaining part of the cases. The possibility of whole exome sequencing enables not only mutation detection in known candidate genes, but also the identification of new genes associated with Leigh syndrome in small families and isolated cases. Exome sequencing was combined with homozygosity mapping to identify the genetic defect in a Moroccan family with fatal Leigh syndrome in early childhood and specific magnetic resonance imaging abnormalities in the brain. We detected a homozygous nonsense mutation (c.20C>A; p.Ser7Ter) in the thiamine transporter SLC19A3. In vivo overexpression of wild-type SLC19A3 showed an increased thiamine uptake, whereas overexpression of mutant SLC19A3 did not, confirming that the mutation results in an absent or non-functional protein. Seventeen additional patients with Leigh syndrome were screened for mutations in SLC19A3 using conventional Sanger sequencing. Two unrelated patients, both from Moroccan origin and one from consanguineous parents, were homozygous for the same p.Ser7Ter mutation. One of these patients showed the same MRI abnormalities as the patients from the first family. Strikingly, patients receiving thiamine had an improved life-expectancy. One patient in the third family deteriorated upon interruption of the thiamine treatment and recovered after reinitiating. Although unrelated, all patients came from the province Al Hoceima in Northern Morocco. Based on the recombination events the mutation was estimated to have occurred 1250-1750 years ago. Our data shows that SLC19A3 is a new candidate for mutation screening in patients with Leigh syndrome, who might benefit from high doses of thiamine and/or biotin. Especially, Moroccan patients with Leigh syndrome should be tested for the c.20C>A founder mutation in SLC19A3.

High frequency of hotspot mutation in PTPN11 gene among Moroccan patients with Noonan syndrome.

Noonan syndrome (NS; OMIM 163950) is an autosomal dominant RASopathy with variable clinical expression and genetic heterogeneity. Clinical manifestations include characteristic facial features, short stature, and cardiac anomalies. Variants in protein-tyrosine phosphatase, non-receptor-type 11 (PTPN11), encoding SHP-2, account for about half of NS patients, SOS1 in approximately 13%, RAF1 in 10%, and RIT1 each in 9%. Other genes have been reported to cause NS in less than 5% of cases including SHOC2, RASA2, LZTR1, SPRED2, SOS2, CBL, KRAS, NRAS, MRAS, PRAS, BRAF, PPP1CB, A2ML1, MAP2K1, and CDC42. Several additional genes associated with a Noonan syndrome-like phenotype have been identified. Clinical presentation and variants in patients with Noonan syndrome are this study's objectives. We performed Sanger sequencing of PTPN11 hotspot (exons 3, 8, and 13). We report molecular analysis of 61 patients with NS phenotype belonging to 58 families. We screened for hotspot variants (exons 3, 8, and 13) in PTPN11 gene by Sanger sequencing. Twenty-seven patients were carrying heterozygous pathogenic variants of PTPN11 gene with a similar frequency (41.4%) compared to the literature. Our findings expand the variant spectrum of Moroccan patients with NS phenotype in whom the analysis of hotspot variants showed a high frequency of exons 3 and 8. This screening test allowed us to establish a molecular diagnosis in almost half of the patients with a good benefit-cost ratio, with appropriate management and genetic counseling.

A maternally derived complex small supernumerary marker chromosome involving chromosomes 8 and 14: case report and review of the literature.

Introduction: The majority of small supernumerary marker chromosomes (sSMCs) are derived from one single chromosome. Complex sSMCs, on the other hand, consist of genetic material derived from more than one, normally two chromosomes. Complex sSMCs involving chromosomes 8 and 14 are rarely encountered. Case presentation: We present here a 14-month-old boy born from an unrelated couple. At birth, the baby was hypotonic and had a cleft lip and palate, as well as ocular involvement. Throughout the course of development, the baby experienced feeding difficulties, stunted growth, and delayed psychomotor development. Banding together with molecular cytogenetics revealed a balanced maternal translocation t(8;14)(p22.3;q21)mat, leading due to meiotic 3:1 segregation to a partial trisomy of chromosomes 8 and 14 in the affected boy. Discussion/Conclusion: This report highlights the importance of cytogenetics in diagnosis of rare genetic disorders, with impact on genetic counselling of patients and their families. There are three comparable cases in the literature involving both chromosomes 8 and 14, but with different breakpoints; the complex sSMC derived from chromosomes 8 and 14 in this case, characterized as der(14)t(8;14) (p22.3;q21)mat.

Currarino Syndrome in Two Moroccan Siblings with Inherited 7q36 Deletion due to Maternal t(7;21)(q36;p11)mat: A Case Report.

Introduction: Currarino syndrome is a rare syndrome with multiple congenital anomalies including sacral agenesis, anorectal malformation, and presence of a presacral mass. Currarino syndrome is considered to be an autosomal dominant inherited disorder, with low penetrance and variable expressivity, but sporadic cases have also been reported. Mutations in MNX1 gene, mapped to 7q36, are the main causes of this syndrome. To the best of our knowledge, less than 400 cases of this syndrome have been mentioned in the literature. Currarino syndrome is often seen in children and considered to be rare in adults; it is mostly found as incidental finding and suspected to be underdiagnosed. Case Presentation: Recognizing the rarity of this syndrome, we present here two siblings with incomplete form of Currarino syndrome combined with microcephaly and intellectual disability. Banding and molecular cytogenetics were used to characterize the origin of this disorder. Banding cytogenetics together with molecular cytogenetics revealed an unbalanced translocation t(7;21)(q36.2;p11.3)mat, leading to a deletion of the 7q36 region in both affected children. Conclusion: This report highlights the importance of cytogenetics in diagnosis of rare genetic syndromes, with impact on genetic counseling of patients and their families. To the best of our knowledge, this is the first Moroccan Currarino syndrome case due to an unbalanced translocation leading to a der(7)t(7;21)(q36.2;p11.3). Also, this is the first Currarino syndrome case associated with a deletion in 7q36 to be reported in Morocco.

Genome and Epigenome Disorders and Male Infertility: Feedback from 15 Years of Clinical and Research Experience.

Infertility affects around 20% of couples of reproductive age; however, in some societies, as many as one-third of couples are unable to conceive. Different factors contribute to the decline of male fertility, such us environmental and professional exposure to endocrine disruptors, oxidative stress, and life habits with the risk of de novo epigenetics dysregulation. Since the fantastic development of new "omes and omics" technologies, the contribution of inherited or de novo genomes and epigenome disorders to male infertility have been further elucidated. Many other techniques have become available to andrology laboratories for the investigation of genome and epigenome integrity and the maturation and the competency of spermatozoa. All these new methods of assessment are highlighting the importance of genetics and epigenetics investigation for assisted reproduction pathology and for supporting professionals in counselling patients and proposing different management strategies for male infertility. This aims to improve clinical outcomes while minimizing the risk of genetics or health problems at birth.

Phenotypic spectrum of Simpson-Golabi-Behmel syndrome in a series of 42 cases with a mutation in GPC3 and review of the literature.

Simpson-Golabi-Behmel syndrome (SGBS) is a rare X-linked multiple congenital abnormality/intellectual disability syndrome characterized by pre- and post-natal overgrowth, distinctive craniofacial features, macrocephaly, variable congenital malformations, organomegaly, increased risk of tumor and mild/moderate intellectual deficiency. In 1996, Glypican 3 (GPC3) was identified as the major gene causing SGBS but the mutation detection rate was only 28-70%, suggesting either genetic heterogeneity or that some patients could have alternative diagnoses. This was particularly suggested by some reports of atypical cases with more severe prognoses. In the family reported by Golabi and Rosen, a duplication of GPC4 was recently identified, suggesting that GPC4 could be the second gene for SGBS but no point mutations within GPC4 have yet been reported. In the genetics laboratory in Tours Hospital, GPC3 molecular testing over more than a decade has detected pathogenic mutations in only 8.7% of individuals with SGBS. In addition, GPC4 mutations have not been identified thus raising the question of frequent misdiagnosis. In order to better delineate the phenotypic spectrum of SGBS caused by GPC3 mutations, and to try to define specific clinical criteria for GPC3 molecular testing, we reviewed the clinical features of all male cases with a GPC3 mutation identified in the two molecular laboratories providing this test in France (Tours and Paris). We present here the results of the analysis of 42 patients belonging to 31 families and including five fetuses and three deceased neonates.

Complex translocation leading to13q interstitial deletion in a Moroccan child with retinoblastoma and intellectual disability.

BACKGROUND: Retinoblastoma (RB) is the most common malignant intraocular tumor in children; it affects their eyes often even prenatally. RB may be sporadic or familial, due to germinal mutation in RB1 gene or by abnormal chromosomal abnormalities involving RB1 gene, located in 13q14. Monosomy of subband 13q14 as a partial deletion can also be responsible for RB with additional symptoms. The latter may be RB associated with psychomotor retardation, macrocephaly, broad forehead, thick earlobes, and bulbous nose. MATERIALS AND METHODS: We present here the case of a boy from a consanguineous marriage with bilateral retinoblastoma, intellectual disability and facial dysmorphic features. Classical and molecular cytogenetics were used to recognize genotype-phenotype association. RESULTS: The karyotype showed a three way translocation involving chromosomes 5, 12 and 13. Further molecular cytogenetics analysis revealed a deletion of 13q14 involving the tumor suppressor gene RB1. CONCLUSION: This case highlights the impact of classical and molecular cytogenetics in diagnosis of rare genetic syndromes and for the genetic counselling of patients and their families. Pure molecular karyotyping analyses would miss the underlying chromosomal mechanism leading to the rearrangement.

Identification of a novel LAMA2 c.2217G > A, p.(Trp739*) mutation in a Moroccan patient with congenital muscular dystrophy: a case report.

BACKGROUND: Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a rare autosomal recessive genetic condition caused by deleterious mutations in the LAMA2 gene encoding the laminin-α2 chain. It is the most frequent subtype of congenital muscular dystrophies (CMDs) characterized by total laminin-α2 deficiency with muscle weakness at birth or in the first six months of life. To the best of our knowledge, this study reports the first molecular diagnosis and genetic defect of this heterogeneous form of CMD performed in a Moroccan medical genetic center using next-generation sequencing (NGS). It allows us to expand the mutational spectrum of the LAMA2 gene. CASE PRESENTATION: We report the case of a female Moroccan child with clinical and paraclinical features in favor of a CMD. She has global congenital hypotonia with generalized muscle weakness, psychomotor retardation, increased serum creatine kinase, and normal brain scan at the age of six months. Targeted NGS leads to the identification of a novel homozygous nonsense mutation c.2217G > A, p.(Trp739*) in the exon 16 of LAMA2. Sanger sequencing confirmed this mutation in the affected patient and showed that her parents are heterozygous carriers. CONCLUSIONS: A modern genetic analysis by NGS improves the genetic diagnosis pathway for adequate genetic counseling of affected families more precisely. An accession number from the National Center for Biotechnology Information (NCBI) ClinVar database was retrieved for this novel LAMA2 mutation.

Homozygous nonsense mutation of WNT10B gene in a Moroccan family with split-hand foot malformation identified by exome sequencing: a case report.

Split-hand foot malformation (SHFM) is a clinically heterogeneous congenital limb defect affecting predominantly the central rays of hands and/or feet. The clinical expression varies in severity between patients as well between the limbs in the same individual. SHFM might be non-syndromic with limb-confined manifestations or syndromic with extra-limb manifestations. Isolated SHFM is a rare condition with an incidence of about 1 per 18,000 live born infants and accounts for 8-17 % of all limb malformations. To date, many chromosomal loci and genes have been described as associated with isolated SHFM, i.e., SHFM1 to 6. SHFM6 is one of the rarest forms of SHFM, and is caused by mutations in WNT10B gene. Less than ten pathogenic variants have been described. We have investigated a large consanguineous Moroccan family with three affected members showing feet malformations with or without split hand malformation phenotypes. Using an exome sequencing approach, we identified a homozygous nonsense variant p.Arg115* of WNT10B gene retaining thereby the diagnosis of SHFM6. This homozygous nonsense mutation identified by exome sequencing in a large family of split hand foot malformation highlights the importance of exome sequencing in genetically heterogeneous entities.

Clinical description and mutational profile of a Moroccan series of patients with Rubinstein Taybi syndrome.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS; OMIM 180849) is a rare autosomal dominant developmental disorder with an estimated prevalence of one case per 125,000 live births. RSTS is characterized by typical face, broad thumbs and halluces, short stature, and intellectual disability. Facial dysmorphy is characteristic with microcephaly, low frontal hairline, arched eyebrows, long eyelashes, convex profile of nose, narrow palate, and micrognathia. RSTS is mainly due to mutations or microdeletions of the CREBBP gene (about 60%) and more rarely of the EP300 gene (8%). OBJECTIVE: Clinical description and identification of mutations of patients with Rubinstein Taybi syndrome. METHODS: PCR and direct sequencing of CREBBP gene. RESULTS: We report here, the clinical and molecular data of a series of six Moroccan patients with a phenotype of RSTS. The molecular study of the major gene CREBBP (by Sanger Sequencing followed by CGH array, if sequence normal) revealed point mutations in five patients. For the sixth patient, CGH array revealed a microdeletion carrying the CREBBP gene. Through this work, we emphasize the importance of clinical expertise in the diagnosis, management and genetic counseling in Rubinstein Taybi syndrome.

Germinal mosaicism in Noonan syndrome: A family with two affected siblings of normal parents.

Noonan syndrome (NS; OMIM 163950) is an autosomal dominant disorder with variable clinical expression and genetic heterogeneity. Clinical manifestations include characteristic facial features, short stature, and cardiac anomalies. Mutations in protein-tyrosine phosphatase, non-receptor-type 11 (PTPN11), encoding SHP-2, account for about half of NS patients. We report on a Moroccan family with two children with NS and apparently unaffected parents. The molecular studies showed the heterozygous mutation c.922A>G of PTPN11 gene in the two affected sibs. Neither the parents, nor the oldest brother carries this mutation in hematologic cells. The mutation was also absent in buccal epithelial cells and fingernails of both parents. We believe this is the first report of germ cell mosaicism in NS and suggest an empirical risk for recurrence of that is less than 1%.