Christophe Philippe

The three stages of epilepsy in patients with CDKL5 mutations.

Mutations in the X‐linked cyclin‐dependent kinase‐like 5 (CDKL5) gene are responsible for a severe encephalopathy with early epilepsy. So far, the electroclinical phenotype remains largely unknown and no clear genotype–phenotype correlations have been established.

Mutation screening of the EXT1 and EXT2 genes in patients with hereditary multiple exostoses.

Hereditary multiple exostoses (HME), the most frequent of all skeletal dysplasias, is an autosomal dominant disorder characterized by the presence of multiple exostoses localized mainly at the end of long bones. HME is genetically heterogeneous, with at least three loci, on 8q24.1 (EXT1), 11p11-p13 (EXT2), and 19p (EXT3). Both the EXT1 and EXT2 genes have been cloned recently and define a new family of potential tumor suppressor genes. This is the first study in which mutation screening has been performed for both the EXT1 and EXT2 genes prior to any linkage analysis. We have screened 17 probands with the HME phenotype, for alterations in all translated exons and flanking intronic sequences, in the EXT1 and EXT2 genes, by conformation-sensitive gel electrophoresis. We found the disease-causing mutation in 12 families (70%), 7 (41%) of which have EXT1 mutations and 5 (29%) EXT2 mutations. Together with the previously described 1-bp deletion in exon 6, which is present in 2 of our families, we report five new mutations in EXT1. Two are missense mutations in exon 2 (G339D and R340C), and the other three alterations (a nonsense mutation, a frameshift, and a splicing mutation) are likely to result in truncated nonfunctional proteins. Four new mutations are described in EXT2. A missense mutation (D227N) was found in 2 different families; the other three alterations (two nonsense mutations and one frameshift mutation) lead directly or indirectly to premature stop codons. The missense mutations in EXT1 and EXT2 may pinpoint crucial domains in both proteins and therefore give clues for the understanding of the pathophysiology of this skeletal disorder.

Mutational spectrum of CDKL5 in early-onset encephalopathies: a study of a large collection of French patients and review of the literature.

The CDKL5 gene has been implicated in the molecular etiology of early‐onset intractable seizures with infantile spasms (IS), severe hypotonia and atypical Rett syndrome (RTT) features. So far, 48 deleterious alleles have been reported in the literature. We screened the CDKL5 gene in a cohort of 177 patients with early‐onset seizures, including 30 men and 10 girls with Aicardi syndrome. The screening was negative for all men as well as for women with Aicardi syndrome, excluding the CDKL5 gene as a candidate for this neurodevelopmental disorder. We report 11 additional de novo mutations in CDKL5 in female patients. For the first time, the MLPA approach allowed the identification of a partial deletion encompassing the promoter and the first two exons of CDKL5. The 10‐point mutations consist of five missenses (with recurrent amino acid changes at p.Ala40 and p.Arg178), four splicing variants and a 1‐base pair duplication. We present a review of all mutated alleles published in the literature. In our study, the overall frequency of mutations in CDKL5 in women with early‐onset seizures is around 8.6%, a result comparable with previous reports. Noteworthy, the CDKL5 mutation rate is high (28%) in women with early‐onset seizures and IS.

Mutations in SLC13A5 Cause Autosomal-Recessive Epileptic Encephalopathy with Seizure Onset in the First Days of Life

Epileptic encephalopathy (EE) refers to a clinically and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. We ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclinical seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clinical presentation in the two families, we hypothesized that the observed phenotype was due to mutations in the same gene, and we performed exome sequencing in three affected individuals. Analysis of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease association was confirmed by cosegregation analysis in additional family members. Screening of 68 additional unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one additional subject with compound heterozygous mutations of SLC13A5 and a similar clinical presentation as the index subjects. Mutations affected key residues for sodium binding, which is critical for citrate transport. These findings underline the value of careful clinical characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metabolism in epilepsy.

The DYRK1A gene is a cause of syndromic intellectual disability with severe microcephaly and epilepsy

Background DYRK1A plays different functions during development, with an important role in controlling brain growth through neuronal proliferation and neurogenesis. It is expressed in a gene dosage dependent manner since dyrk1a haploinsufficiency induces a reduced brain size in mice, and DYRK1A overexpression is the candidate gene for intellectual disability (ID) and microcephaly in Down syndrome. We have identified a 69 kb deletion including the 5′ region of the DYRK1A gene in a patient with growth retardation, primary microcephaly, facial dysmorphism, seizures, ataxic gait, absent speech and ID. Because four patients previously reported with intragenic DYRK1A rearrangements or 21q22 microdeletions including only DYRK1A presented with overlapping phenotypes, we hypothesised that DYRK1A mutations could be responsible for syndromic ID with severe microcephaly and epilepsy. Methods The DYRK1A gene was studied by direct sequencing and quantitative PCR in a cohort of 105 patients with ID and at least two symptoms from the Angelman syndrome spectrum (microcephaly < −2.5 SD, ataxic gait, seizures and speech delay). Results We identified a de novo frameshift mutation (c.290_291delCT; p.Ser97Cysfs*98) in a patient with growth retardation, primary severe microcephaly, delayed language, ID, and seizures. Conclusion The identification of a truncating mutation in a patient with ID, severe microcephaly, epilepsy, and growth retardation, combined with its dual function in regulating the neural proliferation/neuronal differentiation, adds DYRK1A to the list of genes responsible for such a phenotype. ID, microcephaly, epilepsy, and language delay are the more specific features associated with DYRK1A abnormalities. DYRK1A studies should be discussed in patients presenting such a phenotype.

Impairment of CDKL5 nuclear localisation as a cause for severe infantile encephalopathy

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome-like phenotype. To date, fewer than 20 different mutations have been reported. So far, no clear genotype–phenotype correlation has been established. We screened the entire coding region of CDKL5 in 151 affected girls with a clinically heterogeneous phenotype ranging from encephalopathy with epilepsy to atypical Rett syndrome by denaturing high liquid performance chromatography and direct sequencing, and we identified three novel missense mutations located in catalytic domain (p.Ala40Val, p.Arg65Gln, p.Leu220Pro). Segregation analysis showed that p.Arg65Gln was inherited from the healthy father, which rules out the involvement of CDKL5 in the aetiology of the phenotype in this patient. However, the de novo occurrence was shown for p.Ala40Val and p.Leu220Pro. The p.Ala40Val mutation was observed in two unrelated patients and represented the first recurrent mutation in the CDKL5 gene. For the two de novo mutations, we analysed the cellular localisation of the wild-type and CDKL5 mutants by transfection experiments. We showed that the two CDKL5 mutations cause mislocalisation of the mutant CDKL5 proteins in the cytoplasm. Interestingly these missense mutations that result in a mislocalisation of the CDKL5 protein are associated with severe developmental delay which was apparent within the first months of life characterised by early and generalised hypotonia, and autistic features, and as well as early infantile spasms.

BRAF, p53 and SOX2 in anaplastic thyroid carcinoma: evidence for multistep carcinogenesis

Aims: The aim of this study was to genotype a series of papillary thyroid carcinomas (PTCs) and anaplastic thyroid carcinomas (ATCs) for BRAF mutation, and to evaluate p53 and SOX2 expression as factors implicated in tumour progression. Methods: The study included 17 PTCs and 14 ATCs. Analysis of the exon 15 of BRAF was based on direct sequencing. Immunohistochemistry was used to evaluate p53 and SOX2 expression. Results: V600E (c.1799T>A) mutation was observed in 53% (9/17) of PTCs. Two cases of ATCs (2/14; 14%), both with PTC component, harboured BRAF mutation: the classical V600E mutation and an undocumented duplication of codon 599 (c.1795_1797dup; p.Thr599dup). These mutations were present in ATC as well as PTC tumour cells. Overexpression of p53 and SOX2 was depicted respectively in 64% (9/14) and 29% (4/14) of ATCs, and absent in PTCs. Conclusion: We confirm that V600E mutation is a frequent and specific event in PTC. BRAF-mutated ATCs are associated with a PTC component displaying the same mutation. We describe a new mutation of BRAF, T599dup, in a case of ATC with tall cell PTC component. Moreover, progression from PTC to ATC could be favoured by further TP53 mutation and SOX2 expression.

Neurologic Aspects of MECP2 Gene Duplication in Male Patients

Duplications in Xq28 involving the methyl CpG binding protein 2 gene (MECP2) have been described in male patients with severe mental disability, delayed milestones, absence of language, hypotonia replaced by spasticity and retractions, and recurrent and often severe infections. In a study involving five patients in two families, multiplex ligation-dependent probe amplification was used to screen the Xq28 region that includes MECP2, focusing on the presence of gene duplications. Some manifestations of the disease observed in these patients may occur less regularly than the classical abnormalities. Epilepsy with frequent seizures of the myoclonic-astatic type was observed in these patients and was associated with a slowing of the background electroencephalographic activity, rather than the generalized spike-waves or polyspike-waves usually observed in this type of seizure. In addition, cerebral abnormalities were observed with magnetic resonance imaging that were inconstant and nonspecific but that could nonetheless assist in diagnosis of this genetic pathology.

The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: experience of a national referral centre

Although the majority (65%) of boys with Duchenne muscular dystrophy (DMD) carry a deletion in the dystrophin gene, finding mutations in the remaining families is vital for counselling. We have provided a comprehensive mutation service as a national referral centre for France for over 10 years and we report here our experience. Mutation screening is on mRNA from a muscle biopsy. We have detected 79 mutations in 89 samples referred with a diagnosis of DMD, which is the most comprehensive survey to date of the full range of nondeletion mutations. Although some mutations were nonsense mutations, some frameshift mutations and some splicing mutations, all of them led to the generation of premature stop codons or a shortened product which could be detected using the Protein Truncation Test. We recommend a protocol which is robust and sensitive applied to the entire coding region reverse-transcribed from dystrophin transcripts from muscle biopsy.

Rapid detection of common autosomal aneuploidies by quantitative fluorescent PCR on uncultured amniocytes.

Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance for women with normal results can be performed by molecular analysis of uncultured foetal cells in less than 24 h. Today, all molecular techniques developed for a fast diagnosis of aneuploidies rely on the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. Our objective was to test a chromosome quantification method based on the analysis of fluorescent PCR products derived from non-polymorphic target genes. An easy to set up co-amplification of portions of DSCR1 (Down Syndrome Critical Region 1), DCC (Deleted in Colorectal Carcinoma), and RB1 (Retinoblastoma 1) allowed the molecular detection of aneuploidies for chromosomes 21, 18 and 13 respectively. Quantitative analysis was performed in a blind prospective study of 400 amniotic fluids. Four samples (1%) could not be analysed by PCR probably because of a low concentration of foetal DNA. Follow up karyotype analysis was done on all samples and molecular results were in agreement with the cytogenetic data with no false–positive or false–negative results. Our gene based fluorescent PCR approach is an alternative molecular method for a rapid and reliable detection of aneuploidies which can be helpful for the clinical management of high-risk pregnancies.