Expanding the genetic and phenotypic spectrum of TRAPPC9 and MID2-related neurodevelopmental disabilities: report of two novel mutations, 3D-modelling, and molecular docking studies.

Intellectual disabilities (ID) and autism spectrum disorders (ASD) have a variety of etiologies, including environmental and genetic factors. Our study reports a psychiatric clinical investigation and a molecular analysis using whole exome sequencing (WES) of two siblings with ID and ASD from a consanguineous family. Bioinformatic prediction and molecular docking analysis were also carried out. The two patients were diagnosed with profound intellectual disability, brain malformations such as cortical atrophy, acquired microcephaly, and autism level III. The neurological and neuropsychiatric examination revealed that P2 was more severely affected than P1, as he was unable to walk, presented with dysmorphic feature and exhibited self and hetero aggressive behaviors. The molecular investigations revealed a novel TRAPPC9 biallelic nonsense mutation (c.2920 C > T, p.R974X) in the two siblings. The more severely affected patient (P2) presented, along with the TRAPPC9 variant, a new missense mutation c.166 C > T (p.R56C) in the MID2 gene at hemizygous state, while his sister P1 was merely a carrier. The 3D modelling and molecular docking analysis revealed that c.166 C > T variant could affect the ability of MID2 binding to Astrin, leading to dysregulation of microtubule dynamics and causing morphological abnormalities in the brain. As our knowledge, the MID2 mutation (p.R56C) is the first one to be detected in Tunisia and causing phenotypic variability between the siblings. We extend the genetic and clinical spectrum of TRAPPC9 and MID2 mutations and highlights the possible concomitant presence of X-linked as well as autosomal recessive inheritance to causing ID, microcephaly, and autism.

Knowledge and attitudes toward epilepsy among people in Sfax region, Tunisia.

INTRODUCTION: Epilepsy is one of the most stigmatizing disorders. Stigma and negative attitudes associated with epilepsy are due to poor public awareness and knowledge. This study evaluated knowledge, awareness, and attitude toward epilepsy among Tunisian general population. METHODS: This was a cross-sectional study conducted between 2017 and 2019. On national epilepsy day on February and during awareness campaigns at Sfax Tunisia, we asked people who visited the epilepsy stand to anonymously answer a 31-item questionnaire on epilepsy. RESULTS: Five hundred and four participants have been included. About 43.6% of participants had personal or familial history of epilepsy. More than seventy percent of subjects thought that epilepsy is a neurological disease and 34.1% believed it is psychiatric. Majority (92.1%) of our population believed that epilepsy is non-contagious but 37.7% thought it is hereditary and 55.8% thought it causes intellectual deficiency. EEG was the most reported diagnostic method (61.7%). The two most popular therapeutic modalities reported in our population were drug treatment alone (85.3%) and associated with Quran (35.3%). Most (91.1%) of people thought that a person with epilepsy can get married. A person with epilepsy is able to study according to 92.7% of respondents, but 66.3% assumed that he/she suffers from difficulty concentrating. Subjects younger than 45 years were more aware of the ability of people with epilepsy to study and get married. We did not find any significant differences in knowledge and attitudes between subjects familiar with epilepsy and the rest of the population. CONCLUSION: The public knowledge and attitudes toward epilepsy were acceptable with regard to this study. However, negative attitudes and misunderstanding still exist.

A novel TBX1 missense mutation in patients with syndromic congenital heart defects.

Congenital heart defects represent a characteristic part of several genetic syndromes associated with chromosomal abnormalities such as 22q11.2 deletion syndrome; many genes located in this locus, mainly TBX1, are candidate genes for congenital heart defects. In our cohort of 27 subjects with congenital heart defect, both karyotype analysis and Fluorescence in situ hybridization (FISH) were performed. The TBX1 gene was sequenced in patients lacking chromosomal abnormalities. FISH analysis showed a de novo 22q11.2 deletion in two patients. The screening of TBX1 coding sequence identified a novel missense mutation c.569C > A (p.P190Q) in six unrelated patients and detected two associated known single nucleotide polymorphisms; the c.664C > T (rs2301558) in three patients and the c.420T > C (p.Phe140 Phe) (rs41298814) in one patient. Bioinformatic tools show that the novel missense mutation c.569C > A could modify the function and the stability of the TBX1 protein. The c.569C > A mutation was not found in 50 healthy controls. Ours results suggest a deleterious role of the c.569C > A mutation and strengthen the hypothesis that this mutation might be responsible for the same phenotype spectrum as the 22q11.2 deletion syndrome.

Frequent Infections, Hypotonia, and Anemia in a Breastfed Infant.

Vitamin B12 deficiency may be responsible of serious hematologic and neurodevelopmental abnormalities. We report the case of an infant who was hospitalized because of recurrent infections, failure to thrive, hypotonia, and weakness. He was 8 months old and had been exclusively breastfed. Blood cell count showed pancytopenia with megaloblastic bone marrow. The serum IgG concentration was low. Vitamin B12 level was very low and associated with increased urinary methylmalonic acid. Cobalamin deficiency was caused by mother's unrecognized pernicious anemia. Vitamin B12 supply led to rapid clinical and hematologic improvement.

Neonatal purulent meningitis in southern Tunisia: Epidemiology, bacteriology, risk factors and prognosis.

OBJECTIVES: To study the epidemiological, clinical and bacteriological aspects and outcome of purulent neonatal meningitis (PNM). METHODOLOGY: Retrospective analysis of 55 cases of PNM hospitalized in the pediatric ward of Hedi Chaker Hospital from 1990 to 2012. Infants less than 29 days of age were included. The diagnosis was made on either the presence of bacteria in the cerebrospinal fluid (CSF) or the combination of pleocytosis >30 cells/mm(3), protein level >1.3 g/l and glucose level <2.2 mmol/l or CSF/blood glucose ratio <0.4. RESULTS: The male:female sex ratio was 1.75. One or more maternal risk factors for infection were found in 24 cases. The main symptoms were fever and poor feeding. Soluble antigen was positive in four cases and cultures had isolated the bacteria in 28 cases. The mortality rate was 40%. The sequelae rate in the survivors was 16.4%. CONCLUSION: This study emphasizes the severity of PNM with high rates of mortality and neurological sequelae.

Founder effect confirmation of c.241A>G mutation in the L2HGDH gene and characterization of oxidative stress parameters in six Tunisian families with L-2-hydroxyglutaric aciduria.

L-2-hydroxyglutaric aciduria (L2HGA) is an autosomal recessive neurometabolic disorder characterized essentially by the presence of elevated levels of L-2-hydroxyglutaric acid (LGA) in plasma, cerebrospinal fluid and urine. L2HGA is caused by a deficiency in the L2-Hydroxyglutaric dehydrogenase (L2HGDH) enzyme involved in the oxidation of LGA to the alpha 2-ketoglutarate. LGA has been proposed as an endo- and exogenous cytotoxic organic acid that induces free radical formation and generation of reactive oxygen species (ROS). In this report, we analyzed 14 L2HGA patients belonging to six unrelated consanguineous families the south of Tunisia. The patients were diagnosed with L2HGA disease confirmed on the presence of high level of LGA in urine. We analyzed the L2HGDH gene in all probands and identified the same c.241A>G homozygous mutation, which was previously reported in Tunisia. We also used intragenic single nucleotide length polymorphisms (SNPs) and two extragenic microsatellites flanking the L2HGDH gene to confirm the founder effect of c.241A>G mutation in the 14 studied cases. In addition, we carried out the measurement of the oxidative stress parameters in the plasma of L2HGA patients which revealed a significant increase in the malondialdehyde levels (MDA), a biomarker of lipid peroxydation, and the reduced glutathione (GSH). A diminution of the antioxidant enzyme activities including superoxide dismutase (SOD), glutathione peroxidase (GPx), was also observed.

Hemolytic anemia and progressive neurologic impairment: think about triosephosphate isomerase deficiency.

We have reported the first Tunisian case of triosephosphate isomerase (TPI) deficiency in a 2-year-old girl. She was the first child of a nonconsanguineous couple. The disease included a neonatal onset of chronic hemolytic anemia, recurrent low-respiratory infections then progressive neurological involvement. The diagnosis was made after her death from the TPI values of her parents who exhibited intermediate enzyme deficiency. Molecular study of TPI genes showed that the father and the mother are heterozygous for Glu105Asp mutation. Pediatricians must be alert to the differential diagnosis in patients having hemolytic anemia and other concomitant manifestations.

[Bacterial meningitis in children: epidemiological data and outcome]. / La méningites purulentes de l'enfant dans le sud Tunisien: aspects épidémiologiques et évolutifs.

BACKGROUND: The bacterial meningitis in children remains a worrisome affection both by its frequency and by its gravity. AIM: describe the epidemiological, clinical and evolutionary data of bacterial meningitis in children aged between 3 months and 15 years. METHODS: we conducted a retrospective study of all cases of bacterial meningitis collected in the department of pediatrics of Hedi Chaker hospital, during a period of 6 years (2006-2011). We analyzed the files retrospectively and we studied the epidemiological data and the outcome. RESULTS: Thirty cases of bacterial meningitis were recorded. The most frequent species were Streptococcus pneumoniae and Haemophilus influenzae followed by Neisseria meningitidis with respectively 43%, 40%, and 6% of cases. The yearly distribution of these bacteria showed an increase of the rate of the bacterial meningitis during the last 2 years. The average age of our patients was 1 year 6months (3 months-13 years). All the patients were vaccinated according to the Tunisian vaccination calendar; and none of them had pneumococcal vaccine or the Hib vaccine. The main clinical manifestations were fever (100 %), seizures (14 %) and vomiting (44 %). Bulging fontanel was noted in 15cas (55 %), somnolence in 11 cases (40 %) and axial hypotonia in 12 cases (44 %). In the outcome, the neurological sequelae were showed in eight patients (26%). CONCLUSION: In our study, H. influenzae and S. pneumoniae were the main microorganisms responsible for bacterial meningitis. We strongly recommend the introduction pneumococcal vaccine in our vaccination calendar.

Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.

Metachromatic leukodystrophy (MLD) is a severe metabolic disorder caused by the deficient activity of arylsulfatase A due to ARSA gene mutations. According to the age of onset, MLD is classified into three forms: infantile, juvenile, and adult. In our study, we aimed to perform a genetic analysis for two siblings with juvenile MLD for a better characterization of the molecular mechanisms behind the disease. A consanguineous family including two MLD patients (PII.1 and PII.2) was enrolled in our study. The diagnosis was made based on the clinical and neuroimaging investigations. The sequencing of ARSA gene was performed followed by in silico analysis. Besides, the cis/trans distribution of the variants was verified through a PCR-RFLP. The ARSA gene sequencing revealed three known variants, two exonic c.1055A > G and c.1178C > G and an intronic one (c.1524 + 95A > G) in the 3'UTR region. All variants were present at heterozygous state in the two siblings and their mother. The assessment of the cis/trans distribution showed the presence of these variants in cis within the mother, while PII.2 and PII.2 present the c.1055A > G/c.1524 + 95A > G and the c.1178C > G in trans. Additionally, PII.1 harbored a de novo novel missense variant c.1119G > T, whose pathogenicity was supported by our predictive results. Our genetic findings, supported by a clinical examination, confirmed the affection of the mother by the adult MLD. Our results proved the implication of the variable distribution of the found variants in the age of MLD onset. Besides, we described a variable severity between the two siblings due to the de novo pathogenic variant. In conclusion, we identified a complex genotype of ARSA variants within two MLD siblings with a variable severity due to a de novo variant present in one of them. Our results allowed the establishment of an adult MLD diagnosis and highlighted the importance of an assessment of the trans/cis distribution in the cases of complex genotypes.

A novel mutation in FGD4/FRABIN causes Charcot Marie Tooth disease type 4H in patients from a consanguineous Tunisian family.

Charcot-Marie-Tooth (CMT) disease constitutes a clinically and genetically heterogeneous group of hereditary neuropathies characterized by progressive muscular and sensory loss in the distal extremities with chronic distal weakness, deformation of the feet, and loss of deep tendon reflexes. CMT4H is an autosomal recessive demyelinating subtype of CMT, due to mutations in FGD4/FRABIN, for which nine mutations are described to date. In this study, we describe three patients from a consanguineous Tunisian family, presenting with severe, early onset, slowly progressive, autosomal recessive demyelinating CMT, complicated by mild to severe kyphoscoliosis, consistent with CMT4H. In these patients, we report the identification of a novel homozygous frameshift mutation in FGD4: c.514_515insG; p.Ala172Glyfs*27. Our study reports the first mutation identified in FGD4 in Tunisian patients affected with CMT. It further confirms the important clinical heterogeneity observed in patients with mutations in FGD4 and the lack of phenotype/genotype correlations in CMT4H. Our results suggest that FGD4 should be screened in other early-onset CMT subtypes, regardless of the severity of the phenotype, and particularly in patients of consanguineous descent. In Tunisians, as in other populations with high consanguinity rates, screening of genes responsible for rare autosomal recessive CMT subtypes should be prioritized.

[Study of a Tunisian population of children with learning disorders]. / Etude d'une population tunisienne d'enfants en difficultés scolaires.

BACKGROUND: Learning disorders are increasingly a concern for Tunisians parents. These difficulties are divided into two groups: specific learning disabilities and non-specific learning disorders. AIM: Our work is part of a federated research project. Our aim is to determine the incidence, etiology and management of learning disorders in the region of Sfax. METHODS: We conducted a descriptive cross-sectional study on a population of 304 children assessed by their teachers as having academic difficulty. A multidisciplinary assessment including a neurological examination, an assessment of score of intelligence and language assessment has been performed for 209 children. RESULTS: Referring to our sample, learning disorders affect 21.3% of children in the region Sfax. The frequency of specific learning disorder is estimated at 10.3% (reading disorder 5.9%, dyscalculia 2.4%, reading disorder associated with dyscalculia 2%). Non-specific learning disorders were found in 11% of children. Etiologies in this group were dominated by mental retardation (2.1%), inappropriate education (2.3%). CONCLUSION: Our study revealed the high frequency of learning difficulties. It allows us to distinguish between specific learning disabilities and non specific learning disorders secondary to neurological or precarious socio-economic conditions. However, the profile and severity of specific learning disorders could not be studied due to the lack of standardized Arabic tests in Tunisia. In countries with a lack of professional and specialized unit care as in Tunisia, reading interventions in school should be proposed. Only children with remaining difficulties after this training will be sent to specialized professionals.

Juvenile Dermatomyositis Without Skin Lesions in an Antinuclear Matrix Protein 2 Antibody Seropositive Pediatric Case.

ABSTRACT: We report a 5-year-old boy who presented with progressive weakness in 4 limbs and gait disorders over 7 months. No skin rash was observed on admission. A symmetrical proximodistal weakness was found. The creatine kinase level was normal with a slightly elevated lactate dehydrogenase level. Biopsy specimens showed infiltration of mononuclear cells, few necrotic fibers, and perifascicular atrophy. Screening for myositis-specific antibodies was positive for the antinuclear matrix protein 2 antibody, which is mainly associated with dermatomyositis. Symptoms improved on receiving corticosteroids. Our findings suggest that in cases where inflammatory muscle disease is suspected, antinuclear matrix protein 2 antibody analyses should be considered for precise diagnosis, even with the absence of dermatological symptoms. The case suggests consideration of juvenile dermatomyositis in children with no associated skin manifestations or elevated creatine kinase levels and highlights the importance of screening for myositis-specific antibodies in helping with the diagnosis, given the possible heterogeneity of its clinical presentations.

Customized targeted massively parallel sequencing enables the identification of novel pathogenic variants in Tunisian patients with Developmental and Epileptic Encephalopathy.

OBJECTIVE: To develop a high throughput sequencing panel for the diagnosis of developmental and epileptic encephalopathy in Tunisia and to clarify the frequency of disease-causing genes in this region. METHODS: We developed a custom panel for next generation sequencing of the coding sequences of 116 genes in individuals with developmental and epileptic encephalopathy from the Tunisian population. Segregation analyses as well as in silico studies have been conducted to assess the identified variants' pathogenicity. RESULTS: We report 12 pathogenic variants in SCN1A, CHD2, CDKL5, SZT2, KCNT1, GNAO1, PCDH19, MECP2, GRIN2A, and SYNGAP1 in patients with developmental and epileptic encephalopathy. Five of these variants are novel: "c.149delA, p.(Asn50MetfsTer26)" in CDKL5; "c.3616C>T, p.(Arg1206Ter)" in SZT2; "c.111_113del, p.(Leu39del)" in GNAO1; "c.1435G>C , p.(Asp479His)" in PCDH19; as well as "c.2143delC, p. (Arg716GlyfsTer10)"in SYNGAP1. Additionally, for four of our patients, the genetic result facilitated the choice of the appropriate treatment. SIGNIFICANCE: This is the first report of a custom gene panel to identify genetic variants implicated in developmental and epileptic encephalopathy in the Tunisian population as well as the North African region (Tunisia, Egypt, Libya, Algeria, Morocco) with a diagnostic rate of 30%. This high-throughput sequencing panel has considerably improved the rate of positive diagnosis of developmental and epileptic encephalopathy in the Tunisian population, which was less than 15% using Sanger sequencing. The benefit of genetic testing in these patients was approved by both physicians and parents.

A Novel Mutation in the MAP7D3 Gene in Two Siblings with Severe Intellectual Disability and Autistic Traits: Concurrent Assessment of BDNF Functional Polymorphism, X-Inactivation and Oxidative Stress to Explain Disease Severity.

Intellectual disabilities (ID) and autism spectrum disorders (ASD) are characterized by extreme genetic and phenotypic heterogeneity. However, understanding this heterogeneity is difficult due to the intricate interplay among multiple interconnected genes, epigenetic factors, oxidative stress, and environmental factors. Employing next-generation sequencing (NGS), we revealed the genetic cause of ID and autistic traits in two patients from a consanguineous family followed by segregation analysis. Furthermore, in silico prediction methods and 3D modeling were conducted to predict the effect of the variants. To establish genotype-phenotype correlation, X-chromosome inactivation using Methylation-specific PCR and oxidative stress markers were also investigated. By analyzing the NGS data of the two patients, we identified a novel frameshift mutation c.2174_2177del (p.Thr725MetfsTer2) in the MAP7D3 gene inherited from their mother along with the functional BDNF Val66Met polymorphism inherited from their father. The 3D modeling demonstrated that the p.Thr725MetfsTer2 variant led to the loss of the C-terminal tail of the MAP7D3 protein. This change could destabilize its structure and impact kinesin-1's binding to microtubules via an allosteric effect. Moreover, the analysis of oxidative stress biomarkers revealed an elevated oxidative stress in the two patients compared to the controls. To the best of our knowledge, this is the first report describing severe ID and autistic traits in familial cases with novel frameshift mutation c.2174_2177del in the MAP7D3 gene co-occurring with the functional polymorphism Val66M in the BDNF gene. Besides, our study underlines the importance of investigating combined genetic variations, X-chromosome inactivation (XCI) patterns, and oxidative stress markers for a better understanding of ID and autism etiology.

Case report: Functional analysis of the p.Arg507Trp variant of the PIGT gene supporting the moderate epilepsy phenotype of mutations in the C-terminal region.

Pathogenic germline variants in the PIGT gene are associated with the "multiple congenital anomalies-hypotonia-seizures syndrome 3" (MCAHS3) phenotype. So far, fifty patients have been reported, most of whom suffer from intractable epilepsy. Recently, a comprehensive analysis of a cohort of 26 patients with PIGT variants has broadened the phenotypical spectrum and indicated that both p.Asn527Ser and p.Val528Met are associated with a milder epilepsy phenotype and less severe outcomes. Since all reported patients are of Caucasian/Polish origin and most harbor the same variant (p.Val528Met), the ability to draw definitive conclusions regarding the genotype-phenotype correlation remains limited. We report a new case with a homozygous variant p.Arg507Trp in the PIGT gene, detected on clinical exome sequencing. The North African patient in question displays a predominantly neurological phenotype with global developmental delay, hypotonia, brain abnormalities, and well-controlled epileptic seizures. Homozygous and heterozygous variants in codon 507 have been reported to cause PIGT deficiency without biochemical confirmation. In this study, FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the p.Arg507Trp variant leads to mildly reduced activity. Our result confirm the pathogenicity of this variant and strengthen recently reported evidence on the genotype-phenotype correlation of the PIGT variant.

Impact of single-nucleotide polymorphisms at the TP53-binding and responsive promoter region of BCL2 gene in modulating the phenotypic variability of LGMD2C patients.

Apoptosis of skeletal muscle fibers is a well-known event occurring in patients suffering from muscular dystrophies. In this study, we hypothesized that functional polymorphisms in genes involved in the mitochondrial apoptotic pathway might modulate the apoptotic capacity underlying the muscle loss and contributing to intrafamilial and interfamilial variable phenotypes in LGMD2C (Limb Girdle Muscular Dystrophy type 2C) patients sharing the same c.521delT mutation in SGCG gene. Detection of apoptosis was confirmed on muscle biopsies taken from LGMD2C patients using the TUNEL method. We genotyped then ten potentially functional SNPs in TP53, BCL-2 and BAX genes involved in the mitochondrial apoptotic pathway. Potential genotype-dependent Bcl-2 and p53 protein expressed in skeletal muscle was investigated using western blot and ELISA assays. The result showed that muscle cells carrying the TP53-R72R and TP53-16 bp del/del genotypes displayed an increased p53 level which could be more effective in inducing apoptosis by activation of the pro-apoptotic gene expression. In addition, the BCL2-938 AA genotype was associated with increased Bcl-2 protein expression in muscle from LGMD2C patients compared to -938CC genotype, while there was no evidence of significant difference in the BAX haplotype. Our findings suggest that increased Bcl-2 protein expression may counteract pro-apoptotic pathways and thus reduce the muscle loss. To the best of our knowledge, this is a pioneer study evaluating the role of apoptotic BCL-2 and TP53 genes in contributing to the phenotypic manifestation of c.521delT mutation in LGMD2C patients. Larger studies are needed to validate these findings.

Frontal motor seizure following non-convulsive status epilepticus in ring chromosome 20 syndrome.

The ring chromosome 20 syndrome is a rare syndrome characterized by intractable epilepsy with particular electro clinical features including episodes of prolonged confusional state and nocturnal frontal lobe seizures. We report a 17-year-old girl who had intractable epilepsy with frontal seizure and prolonged confusional state secondary to non-convulsive status epilepticus. The diagnosis of ring chromosome 20 was suspected and confirmed by karyotype. The cytogenetic study of CHRNA4 and KCNQ2 genes did not detect deletion in the ring chromosome 20. During video-EEG recording, this girl presented a non-convulsive status epilepticus that lasted more than 20 minutes followed by typical frontal lobe seizure. This association was not previously described, and was probably caused by chromosomal instability.

Combined in Silico Prediction Methods, Molecular Dynamic Simulation, and Molecular Docking of FOXG1 Missense Mutations: Effect on FoxG1 Structure and Its Interactions with DNA and Bmi-1 Protein.

FoxG1 encoded by FOXG1 gene is a transcriptional factor interacting with the DNA of targeted genes as well as with several proteins to regulate the forebrain development. Mutations in the FOXG1 gene have been shown to cause a wide spectrum of brain disorders, including the congenital variant of Rett syndrome. In this study, the direct sequencing of FOXG1 gene revealed a novel c.645C > A (F215L) variant in the patient P1 and a de novo known one c.755G > A (G252D) in the patient P2. To investigate the putative impact of FOXG1 missense variants, a computational pipeline by the application of in silico prediction methods, molecular dynamic simulation, and molecular docking approaches was used. Bioinformatics analysis and molecular dynamics simulation have demonstrated that F215L and G252D variants found in the DNA binding domain are highly deleterious mutations that may cause the protein structure destabilization. On the other hand, molecular docking revealed that F215L mutant is likely to have a great impact on destabilizing the protein structure and the disruption of the Bmi-1 binding site quite significantly. Regarding G252D mutation, it seems to abolish the ability of FoxG1 to bind DNA target, affecting the transcriptional regulation of targeted genes. Our study highlights the usefulness of combined computational approaches, molecular dynamic simulation, and molecular docking for a better understanding of the dysfunctional effects of FOXG1 missense mutations and their role in the etiopathogenesis as well as in the genotype-phenotype correlation.

Further insights into the spectrum phenotype of TRAPPC9 and CDK5RAP2 genes, segregating independently in a large Tunisian family with intellectual disability and microcephaly.

Intellectual disability (ID) often co-occurs with other neurologic phenotypes making molecular diagnosis more challenging particularly in consanguineous populations with the co-segregation of more than one ID-related gene in some cases. In this study, we investigated the phenotype of three patients from a large Tunisian family with significant ID phenotypic variability and microcephaly and performed a clinical exome sequencing in two cases. We identified, within the first branch, a homozygous variant in the TRAPPC9 gene (p.Arg472Ter) in two cases presenting severe ID, absent speech, congenital/secondary microcephaly in addition to autistic features, supporting the implication of TRAPPC9 in the "secondary" autism spectrum disorders and congenital microcephaly. In the second branch, we identified a homozygous variant (p.Lys189ArgfsTer15) in the CDK5RAP2 gene associated with an heterozygous TRAPPC9 variant (p.Arg472Ter) in one case harbouring primary hereditary microcephaly (MCPH) associated with an inter-hypothalamic adhesion, mixed hearing loss, selective thinning in the retinal nerve fiber layer and parafoveal ganglion cell complex, and short stature. Our findings expand the spectrum of the recently reported neurosensorial abnormalities and revealed the variable phenotype expressivity of CDK5RAP2 defect. Our study highlights the complexity of the genetic background of microcephaly/ID and the efficiency of the exome sequencing to provide an accurate diagnosis and to improve the management and follow-up of such patients.

Unusual double mutation in MECP2 and CDKL5 genes in Rett-like syndrome: Correlation with phenotype and genes expression.

INTRODUCTION: Rett syndrome (RTT) is a neuro-developmental disorder affecting almost exclusively females and it divided into classical and atypical forms of the disease. RTT-like syndrome was also described and presents an overlapping phenotype of RTT. RTT-like syndrome has been associated with several genes including MECP2 and CDKL5 having common biological pathways and regulatory interactions especially during neural maturation and synaptogenesis. METHODS: We report patient with Rett-like syndrome for whom clinical features and their progression guided toward the screening of two candidate genes MECP2 and CDKL5 by sequencing. Severity score was evaluated by "Rett Assessment Rating Scale" (R.A.R.S.). Predictions of pahogenicity and functional effects used several bioinformatic tools and qRT-PCR was conducted to evaluate gene expression. RESULTS: Mutational screening revealed two mutations c.1065 C > A (p.S355R) in MECP2 gene and c.616 G > A (p.D206N) mutation in CDKL5 gene in the patient with a high R.A.R.S. Bioinformatic investigations predicted a moderate effect of p.S355R in MECP2 gene but a more pathogenic one of p.D206N mutation in CDKL5. Effect of c.616 G > A mutation on structure and stability of CDKL5 mRNA was confirmed by qRT-PCR. Additionally, analysis of gene expression revealed a drastic effect of CDKL5 mutant on its MeCP2 and Dnmt1 substrates and also on its MYCN regulator. CONCLUSIONS: The co-existence of the two mutations in CDKL5 and MECP2 genes could explain the severe phenotype in our patient with RTT-Like and is consistent with the data related to the interactions of CDKL5 with MeCP2 and Dnmt1 proteins.