Identification of novel and de novo variant in the SCN1A gene confirms Dravet syndrome in Moroccan child: a case report.

Dravet syndrome is a severe form of epilepsy characterised by recurrent seizures and cognitive impairment. It is mainly caused by variant in the SCN1A gene in 90% of cases, which codes for the α subunit of the voltage-gated sodium channel. In this study, we present one suspected case of Dravet syndrome in Moroccan child that underwent exome analysis and were confirmed by Sanger sequencing. The variant was identified in the SCN1A gene, and is a new variant that has never been described in the literature. The variant was found de nova in our case, indicating that it was not inherited from the parents. The variant, SCN1A c.965-2A>G p.(?), is located at the splice site and results in an unknown modification of the protein. This variant is considered pathogenic on the basis of previous studies. These results contribute to our knowledge of the SCN1A gene mutations associated with Dravet syndrome and underline the importance of genetic analysis in the diagnosis and confirmation of this disorder. Further studies are needed to better understand the functional consequences of this variant and its implications for therapeutic strategies in Dravet syndrome.

Clinical Exome Sequencing Reveals Novel Mutations in SPTB Gene Associated with Hereditary Spherocytosis in Patients with Suspected Congenital Hemolytic Anemia.

Congenital hemolytic anemia (CHA) is defined as the premature destruction of red blood cells (RBC) due to congenital or acquired defects. The hereditary form of hemolytic anemia can be divided into hemoglobinopathies, membranopathies, and enzymopathies. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to congenital hemolytic anemia. To date; five genes have been associated with HS coding for cytoskeleton and transmembrane proteins, those genes are SPTB, SLC4A1, EPB42, ANK1, and SPTA1. Due to genetic heterogeneity, clinical exome sequencing (CES) was performed on four unrelated Moroccan patients referred for CHA investigation. Sanger sequencing and qPCR were performed to confirm CES results and to study the de novo character of identified variants. The molecular analysis revealed 3 novel mutations and one previously reported pathogenic variant of the SPTB gene confirming the diagnosis of HS in the four patients. Hereditary spherocytosis anemia is a genetically heterogenous disease which could be misdiagnosed clinically. The introduction of novel sequencing technologies can facilitate accurate genetic diagnosis, allowing an adapted care of the patient and his family.

Myeloperoxidase Modulates Inflammation in Generalized Pustular Psoriasis and Additional Rare Pustular Skin Diseases.

Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19%-41% of affected individuals harbor bi-allelic mutations in IL36RN, the genetic cause is not known in most cases. To identify and characterize new pathways involved in the pathogenesis of GPP, we performed whole-exome sequencing in 31 individuals with GPP and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO). We discovered eight MPO mutations resulting in MPO -deficiency in neutrophils and monocytes. MPO mutations, primarily those resulting in complete MPO deficiency, cumulatively associated with GPP (p = 1.85E-08; OR = 6.47). The number of mutant MPO alleles significantly differed between 82 affected individuals and >4,900 control subjects (p = 1.04E-09); this effect was stronger when including IL36RN mutations (1.48E-13) and correlated with a younger age of onset (p = 0.0018). The activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO deficiency. Phorbol-myristate-acetate-induced formation of neutrophil extracellular traps (NETs) was reduced in affected cells (p = 0.015), and phagocytosis assays in MPO-deficient mice and human cells revealed altered neutrophil function and impaired clearance of neutrophils by monocytes (efferocytosis) allowing prolonged neutrophil persistence in inflammatory skin. MPO mutations contribute significantly to GPP's pathogenesis. We implicate MPO as an inflammatory modulator in humans that regulates protease activity and NET formation and modifies efferocytosis. Our findings indicate possible implications for the application of MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases.

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.

A novel non sense mutation in WDR62 causes autosomal recessive primary microcephaly: a case report.

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a rare genetically heterogeneous disorder of neurogenic brain development characterized by a reduced head circumference at birth with no remarkable anomalies of brain architecture and variable degrees of intellectual impairment. Clinical and genetic heterogeneity in genetic disorders represent a major diagnostic challenge. CASE PRESENTATION: Two patients, 11 and 9 years old, born from consanguineous parents, were referred to the department of medical genetics at the National Institute of Health in Rabat. The diagnosis of MCPH was made, based on reduced head circumference without brain architecture abnormalities. The two patients were subject to the whole-exome sequencing, which allowed to diagnose a novel homozygous mutation c.1027C > T; p.Gln343* in exon 8 of WDR62, a gene already known to be related to MCPH. Sanger sequencing confirmed the segregation of the mutation in the family. CONCLUSION: Our data expends the spectrum of mutations in WDR62 gene, proves the efficiency and cost-effectiveness of whole exome sequencing for the molecular diagnosis of genetically heterogeneous disorders such MCPH. Exome sequencing led to the rapid and cost-effective identification of a novel homozygous mutation in WDR62 gene, thereby facilitating genetic counseling.

Exome sequencing reveals a novel PLP1 mutation in a Moroccan family with connatal Pelizaeus-Merzbacher disease: a case report.

BACKGROUND: Epilepsy regroups a common and diverse set of chronic neurological disorders that are characterized by spontaneous, unprovoked, and recurrent epileptic seizures. Epilepsies have a highly heterogeneous background with a strong genetic contribution and various mode of inheritance. X-linked epilepsy usually manifests as part of a syndrome or epileptic encephalopathy. The variability of clinical manifestations of X-linked epilepsy may be attributed to several factors including the causal genetic mutation, making diagnosis, genetic counseling and treatment decisions difficult. We report the description of a Moroccan family referred to our genetic department with X-linked epileptic seizures as the only initial diagnosis. CASE PRESENTATION: Knowing the new contribution of Next-Generation Sequencing (NGS) for clinical investigation, and given the heterogeneity of this group of disorders we performed a Whole-Exome Sequencing (WES) analysis and co-segregation study in several members of this large family. We detected a novel pathogenic PLP1 missense mutation c.251C > A (p.Ala84Asp) allowing us to make a diagnosis of Pelizaeus-Merzbacher Disease for this family. CONCLUSION: This report extends the spectrum of PLP1 mutations and highlights the diagnostic utility of NGS to investigate this group of heterogeneous disorders.

Correction to: Exome sequencing reveals a novel PLP1 mutation in a Moroccan family with connatal Pelizaeus-Merzbacher disease: a case report.

After publication of the original article [1] it was brought to our attention that author Bouchra Ouled Amar Bencheikh was incorrectly included as Bouchra Oulad Amar Bencheikh.

Novel DDR2 mutation identified by whole exome sequencing in a Moroccan patient with spondylo-meta-epiphyseal dysplasia, short limb-abnormal calcification type.

Spondylo-meta-epiphyseal dysplasia (SMED), short limb-abnormal calcification type (SMED, SL-AC), is a very rare autosomal recessive disorder with various skeletal changes characterized by premature calcification leading to severe disproportionate short stature. Twenty-two patients have been reported until now, but only five mutations (four missense and one splice-site) in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene has been identified. We report here a novel DDR2 missense mutation, c.370C > T (p.Arg124Trp) in a Moroccan girl with SMED, SL-AC, identified by whole exome sequencing. Our study has expanded the mutational spectrum of this rare disease and it has shown that exome sequencing is a powerful and cost-effective tool for the diagnosis of clinically heterogeneous disorders such as SMED.

Further evidence for causal FAM20A mutations and first case of amelogenesis imperfecta and gingival hyperplasia syndrome in Morocco: a case report.

BACKGROUND: Amelogenesis imperfecta represents a group of developmental conditions, clinically and genetically heterogeneous, that affect the structure and clinical appearance of enamel. Amelogenesis imperfecta occurred as an isolated trait or as part of a genetic syndrome. Recently, disease-causing mutations in the FAM20A gene were identified, in families with an autosomal recessive syndrome associating amelogenesis imperfecta and gingival fibromatosis. CASE PRESENTATION: We report, the first description of a Moroccan patient with amelogenesis imperfecta and gingival fibromatosis, in whom we performed Sanger sequencing of the entire coding sequence of FAM20A and identified a homozygous mutation in the FAM20A gene (c.34_35delCT), already reported in a family with this syndrome. CONCLUSION: Our finding confirms that the mutations of FAM20A gene are causative for amelogenesis imperfecta and gingival fibromatosis and underlines the recurrent character of the c.34_35delCT in two different ethnic groups.

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.

Isolation and characterization of ovine mesenchymal stem cells derived from peripheral blood.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells with capacity to differentiate into several mesenchymal lineages. This quality makes MSCs good candidates for use in cell therapy. MSCs can be isolated from a variety of tissues including bone marrow and adipose tissue, which are the most common sources of these cells. However, MSCs can also be isolated from peripheral blood. Sheep has been proposed as an ideal model for biomedical studies including those of orthopaedics and transmissible spongiform encephalopathies (TSEs). The aim of this work was to advance these studies by investigating the possibility of MSC isolation from ovine peripheral blood (oPB-MSCs) and by subsequently characterizing there in vitro properties. RESULTS: Plastic-adherent fibroblast-like cells were obtained from the mononuclear fraction of blood samples. These cells were analysed for their proliferative and differentiation potential into adipocytes, osteoblasts and chondrocytes, as well as for the gene expression of cell surface markers. The isolated cells expressed transcripts for markers CD29, CD73 and CD90, but failed to express the haematopoietic marker CD45 and expressed only low levels of CD105. The expression of CD34 was variable. The differentiation potential of this cell population was evaluated using specific differentiation media. Although the ability of the cultures derived from different animals to differentiate into adipocytes, osteoblasts and chondrocytes was heterogeneous, we confirmed this feature using specific staining and analysing the gene expression of differentiation markers. Finally, we tested the ability of oPB-MSCs to transdifferentiate into neuronal-like cells. Morphological changes were observed after 24-hour culture in neurogenic media, and the transcript levels of the neurogenic markers increased during the prolonged induction period. Moreover, oPB-MSCs expressed the cellular prion protein gene (PRNP), which was up-regulated during neurogenesis. CONCLUSIONS: This study describes for the first time the isolation and characterization of oPB-MSCs. Albeit some variability was observed between animals, these cells retained their capacity to differentiate into mesenchymal lineages and to transdifferentiate into neuron-like cells in vitro. Therefore, oPB-MSCs could serve as a valuable tool for biomedical research in fields including orthopaedics or prion diseases.

Changes in HSP gene and protein expression in natural scrapie with brain damage.

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

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.

Novel mutation in the TGFBI gene in a Moroccan family with atypical corneal dystrophy: a case report.

BACKGROUND: Corneal dystrophies (CDs) are a heterogeneous group of bilateral, genetically determined, noninflammatory bilateral corneal diseases that are usually limited to the cornea. CD is characterized by a large variability in the age of onset, evolution and visual impact and the accumulation of insoluble deposits at different depths in the cornea. Clinical symptoms revealed bilateral multiple superficial, epithelial, and stromal anterior granular opacities in different stages of severity among three patients of this family. A total of 99 genes are involved in CDs. The aim of this study was to identify pathogenic variants causing atypical corneal dystrophy in a large Moroccan family and to describe the clinical phenotype with severely different stages of evolution. CASE PRESENTATION: In this study, we report a large Moroccan family with CD. Whole-exome sequencing (WES) was performed in the three affected members who shared a phenotype of corneal dystrophy in different stages of severity. Variant validation and familial segregation were performed by Sanger sequencing in affected sisters and mothers and in two unaffected brothers. Whole-exome sequencing showed a novel heterozygous mutation (c.1772C > A; p.Ser591Tyr) in the TGFBI gene. Clinical examinations demonstrated bilaterally multiple superficial, epithelial and stromal anterior granular opacities in different stages of severity among three patients in this family. CONCLUSIONS: This report describes a novel mutation in the TGFBI gene found in three family members affected by different phenotypic aspects. This mutation is associated with Thiel-Behnke corneal dystrophy; therefore, it could be considered a novel phenotype genotype correlation, which will help in genetic counselling for this family.

Clinical, cytogenetic and molecular findings in nine Moroccan patients with Fanconi anemia.

INTRODUCTION: Fanconi anemia (FA) is a rare inherited hematological disease due to a defect in the DNA repair pathway resulting in congenital abnormalities and high susceptibility to develop cancers. The cytogenetic analysis using alkylating agents is still a reference test to establish the diagnosis. Despite the genetic heterogeneity, the identification of the causal mutation is actually performed especially after the development of next generation sequencing (NGS). METHODS: we report here nine Moroccan patients referred to the department of Medical Genetics for suspicion of FA. We realized a genetic consultation to establish a clinical record with biological data before carrying out the genetic analysis. Karyotyping with mitomycin was performed for all the probands before elaborating molecular study. We used massively parallel sequencing to analyse the three most frequent mutated genes FANCA, FANCC, and FANCG, representing 84% of all genes involved in FA. RESULTS: all the patients showed hematological signs associated with at least one extra-hematological congenital anomaly. The chromosomal breaks were significantly higher for the nine patients, compared to the controls. The molecular diagnosis was confirmed in 8 of the 9 families tested (88.8%) with 4 novel mutations. The next generation based sequencing identified 9 variations: 6 in the FANCA gene (66.6%), 3 in the FANCG gene (33.3%) and no FANCC variation was found. Of those, 7 were homozygous and 2 were compounds heterozygous. CONCLUSION: to the best of our knowledge, this is the first molecular report of Moroccan patients with FA suggesting the predominance of two genes without any recurrent mutation. The molecular analysis of FANCA and FANCG genes should be offered first for all patients in Morocco.

Molecular diagnosis of dystrophinopathies in Morocco and report of six novel mutations.

Dystrophinopathies are the most common genetic neuromuscular disorders during childhood, with an X-linked recessive inheritance pattern. Because of clinical and genetic heterogeneity of dystrophinopathies, genetic testing of dystrophin gene at Xp21.2 is constantly evolving. Multiplex Polymerase Chain Reaction (MPCR) is used in the first line to detect common exon deletions of dystrophin gene (accounting for 65% of mutations), followed by the Multiplex Ligation-dependent Probe Amplification (MLPA) technique to reveal deletions of exons outside the usual hotspot and duplications in male and female carriers. (MLPA adds another 10-15% positive cases to MPCR). Recently, Next Generation Sequencing allows to screen for rare large and point mutations. We report here, molecular analysis results of dystrophin gene during 27 years in a large Moroccan cohort of 356 patients, using the multiplex polymerase chain reaction (MPCR) to screen for hot-spot exon deletions. First applications of whole dystrophin gene sequencing in our lab lead to the identification of six novel mutations.

Clinical exome sequencing identifies two novel mutations of the SCN1A and SCN2A genes in Moroccan patients with epilepsy: a case series.

BACKGROUND: Epilepsy is the most common neurological disorder that causes spontaneous, unprovoked, and recurrent seizures. Epilepsy is clinically and genetically heterogeneous with various modes of inheritance. The complexity of epilepsy presents a challenge and identification of the causal genetic mutation allows diagnosis, genetic counseling, predicting prognosis, and, in some cases, treatment decisions. Clinical exome sequencing is actually becoming a powerful approach for molecular diagnosis of heterogeneous neurological disorders in clinical practice. CASE PRESENTATION: We report our observations of three unrelated Moroccan patients referred to our genetics department for molecular diagnosis of epilepsy: a 4-year-old Moroccan boy, a 3-year-old Moroccan girl, and a 7-year-old Moroccan boy. Due to the heterogeneity and complexity of epilepsy, we performed clinical exome sequencing followed by targeted analysis of 936 epilepsy genes. A total of three mutations were identified in known epilepsy genes (SCN1A, SCN2A). By clinical exome sequencing, we identified two novel mutations: c.4973C>A (p.Thr1658Lys) in SCN1A gene and c.1283A>G (p.Tyr428Cys) in the SCN2A gene, whereas the third mutation c.3295G>T (p.Glu1099*) was already described in patients with Dravet syndrome. CONCLUSION: This study demonstrates that clinical exome sequencing is an effective diagnosis tool to investigate this group of diseases with huge diversity and defends its use in clinical routine.

Differential expression and protein distribution of Bax in natural scrapie.

Bax is a pro-apoptotic member of the Bcl-2 family that plays an important role in neuronal apoptosis. However, the results are controversial, especially regarding its function in the apoptosis involved in prion diseases. This work analyzes the gene expression and protein distribution of Bax in the central nervous systems of sheep naturally infected with scrapie. Gene expression profiling, obtained by means of real-time RT-PCR analysis, has shown a significant over-expression of this pro-apoptotic factor in medulla oblongata and diencephalon, whereas its expression was stable in cerebellum and prefrontal cortex. Immunohistochemistry confirmed the expression results and extended the investigation to 13 different regions. A high degree of variability was found in Bax immunoreactivity, mainly in the scrapie group, which also corresponded to the degree of PrP(Sc) deposition. Despite this variability, qualitative differences were found between scrapie and control groups. Intraneuronal reactivity for Bax was mainly observed in the spinal cord, brain stem, hypothalamus, and colicullus of scrapie animals, whereas controls displayed immunoreactivity almost exclusively in the neuropile. Moreover, a significant positive correlation was observed between Bax and prion deposition. Despite Bax over-expression, the activated form of caspase-3 was never observed in neurons showing apoptotic-like morphology. In contrast, activated caspase-3 staining appeared as cytoplasmic granules in apparently healthy neurons. We conclude that apoptosis either occurs in an extremely low number of neurons or neuroprotective mechanisms arrest the mitochondrial pathway after Bax induction.

Novel splice site mutation in CNNM4 gene in a family with Jalili syndrome.

Jalili syndrome is a rare autosomal recessive genetic disease characterized by the association of amelogenesis imperfecta and cone-rod retinal dystrophy. This syndrome is caused by mutations in the CNNM4 gene. Different types of CNNM4 mutations have been reported; missense, nonsense, large deletions, single base insertion, and duplication. We used Sanger sequencing to analyze a large consanguineous family with three siblings affected with Jalili syndrome, suspected clinically after dental and ophthalmological examination. These patients are carrying a novel homozygous mutation in the splice site acceptor of intron 3 (c.1682-1G > C) in the CNNM4 gene. We compare the findings of the present family to those from literature, in order to further delineate Jalili syndrome.