Isabelle Coupry

Molecular analysis of the CBP gene in 60 patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RTS, MIM 180849) occurs in 1/125 000 births and is characterised by growth retardation and psychomotor developmental delay, broad and duplicated distal phalanges of the thumbs and halluces, typical facial dysmorphism, and an increased risk of neoplasia.1 RTS has been shown to be associated with chromosomal rearrangements in cytogenetic band 16p13.3,2–4 all involving the CREB binding protein gene, officially named CREBBP by the HUGO Nomenclature Committee, but generally referred to by its shorter acronym CBP .5 CBP is a transcriptional coactivator involved in different signal transduction pathways, thereby regulating the expression of many genes and playing an important role in the regulation of cell growth, cellular differentiation, and tumour suppression.6,7 To date, all studies concerning CBP in RTS have used FISH analysis with cosmids from the CBP region or the search for mutations at the molecular level using the protein truncation test.8,9 Taken together, these studies showed that translocations and inversions form the minority of CBP mutations in RTS, microdeletions account for only 10% of RTS cases, and PTT studies showed 10% truncating mutations. The structure of the CBP gene was recently described.8 CBP spans about 150 kb with 31 exons and its cDNA is 9 kb in length. We report here the use of different molecular techniques to analyse the CBP gene in a cohort of 60 RTS patients. These include cDNA probes to search for gross rearrangements by Southern blot analysis and to identify CBP mRNA of abnormal sizes on northern blots, intragenic microsatellite markers to look for intragenic deletions, as well as a complete series of primers to PCR amplify each of the 31 exons of the gene for mutation searching by direct sequencing. We have analysed 60 patients using these various techniques and identified 27 …

Thrombocytopenia resulting from mutations in filamin A can be expressed as an isolated syndrome

Filaminopathies A caused by mutations in the X-linked FLNA gene are responsible for a wide spectrum of rare diseases including 2 main phenotypes, the X-linked dominant form of periventricular nodular heterotopia (FLNA-PVNH) and the otopalatodigital syndrome spectrum of disorders. In platelets, filamin A (FLNa) tethers the principal receptors ensuring the platelet-vessel wall interaction, glycoprotein Ibα and integrin αIIbβ3, to the underlying cytoskeleton. Hemorrhage, coagulopathy, and thrombocytopenia are mentioned in several reports on patients with FLNA-PVNH. Abnormal platelet morphology in 2 patients with FLNA-PVNH prompted us to examine a third patient with similar platelet morphology previously diagnosed with immunologic thrombocytopenic purpura. Her enlarged platelets showed signs of FLNa degradation in Western blotting, and a heterozygous missense mutation in FLNA was detected. An irregular distribution of FLNa within the total platelet population was shown by confocal microscopy for all 3 patients. In vitro megakaryocyte cultures showed an abnormal differentiation, including an irregular distribution of FLNa with a frayed aspect, the presence of enlarged α-granules, and an abnormal fragmentation of the cytoplasm. Mutations in FLNA may represent an unrecognized cause of macrothrombocytopenia with an altered platelet production and a modified platelet-vessel wall interaction.

COL4A1 mutation in Axenfeld–Rieger anomaly with leukoencephalopathy and stroke

Several hereditary ischemic small‐vessel diseases of the brain have been reported during the last decade. Some of them have ophthalmological, mainly retinal, manifestations. Herein, we report on a family affected by vascular leukoencephalopathy and variable abnormalities of the anterior chamber of the eye.

REEP1 mutations in SPG31: Frequency, mutational spectrum, and potential association with mitochondrial morpho-functional dysfunction†

Hereditary spastic paraplegias (HSP) constitute a heterogeneous group of neurodegenerative disorders characterized at least by slowly progressive spasticity of the lower limbs. Mutations in REEP1 were recently associated with a pure dominant HSP, SPG31. We sequenced all exons of REEP1 and searched for rearrangements by multiplex ligation‐dependent probe amplification (MLPA) in a large panel of 175 unrelated HSP index patients from kindreds with dominant inheritance (AD‐HSP), with either pure (n = 102) or complicated (n = 73) forms of the disease, after exclusion of other known HSP genes. We identified 12 different heterozygous mutations, including two exon deletions, associated with either a pure or a complex phenotype. The overall mutation rate in our clinically heterogeneous sample was 4.5% in French families with AD‐HSP. The phenotype was restricted to pyramidal signs in the lower limbs in most patients but nine had a complex phenotype associating axonal peripheral neuropathy (= 5/11 patients) including a Silver‐like syndrome in one patient, and less frequently cerebellar ataxia, tremor, dementia. Interestingly, we evidenced abnormal mitochondrial network organization in fibroblasts of one patient in addition to defective mitochondrial energy production in both fibroblasts and muscle, but whether these anomalies are directly or indirectly related to the mutations remains uncertain. Hum Mutat 32:1118–1127, 2011. ©2011 Wiley‐Liss, Inc.

Ophthalmological Features Associated With COL4A1 Mutations

OBJECTIVE To investigate the wide variability of ocular manifestations associated with mutations in the COL4A1 gene that encodes collagen IValpha1. METHODS We clinically evaluated 7 patients from 2 unrelated families in whom ocular features segregated with COL4A1 mutations that were identified by direct sequencing. RESULTS The G2159A transition (c.2159G>A) that leads to the missense mutation p.Gly720Asp was identified in family A. An ocular phenotype of variable severity was observed in all affected relatives. The missense mutation c.2263G>A, p.Gly755Arg was identified in family B. One patient from family B also displayed notable ocular features. CONCLUSIONS The COL4A1 mutations may be associated with various ophthalmologic developmental anomalies of anterior segment dysgenesis type, which are reminiscent of Axenfeld-Rieger anomalies (ARA). Cerebrovascular disorders should be added to the list of signs potentially associated with ARA. CLINICAL RELEVANCE These data suggest that cerebral magnetic resonance imaging may be recommended in the clinical treatment of patients with apparently isolated ARA, even when neurological symptoms or signs are lacking.

A glial origin for periventricular nodular heterotopia caused by impaired expression of Filamin-A

Periventricular nodular heterotopia (PH) is a human brain malformation caused by defective neuronal migration that results in ectopic neuronal nodules lining the lateral ventricles beneath a normal appearing cortex. Most affected patients have seizures and their cognitive level varies from normal to severely impaired. Mutations in the Filamin-A (or FLNA) gene are the main cause of PH, but the underlying pathological mechanism remains unknown. Although two FlnA knockout mouse strains have been generated, none of them showed the presence of ectopic nodules. To recapitulate the loss of FlnA function in the developing rat brain, we used an in utero RNA interference-mediated knockdown approach and successfully reproduced a PH phenotype in rats comparable with that observed in human patients. In FlnA-knockdown rats, we report that PH results from a disruption of the polarized radial glial scaffold in the ventricular zone altering progression of neural progenitors through the cell cycle and impairing migration of neurons into the cortical plate. Similar alterations of radial glia are observed in human PH brains of a 35-week fetus and a 3-month-old child, harboring distinct FLNA mutations not previously reported. Finally, juvenile FlnA-knockdown rats are highly susceptible to seizures, confirming the reliability of this novel animal model of PH. Our findings suggest that the disorganization of radial glia is the leading cause of PH pathogenesis associated with FLNA mutations. Rattus norvegicus FlnA mRNA (GenBank accession number FJ416060).

Bilateral periventricular nodular heterotopia in France: frequency of mutations in FLNA, phenotypic heterogeneity and spectrum of mutations

Bilateral periventricular nodular heterotopia (BPNH) is the most common form of periventricular heterotopia. Mutations in FLNA, encoding filamin A, are responsible for the X linked dominant form of BPNH (FLNA-BPNH). Recently, atypical phenotypes including BPNH with Ehlers–Danlos syndrome (BPNH-EDS) have been recognised. A total of 44 FLNA mutations have so far been reported in this phenotype. Most of these mutations lead to a truncated protein, but few missense mutations have also been described. Here, the results of a mutation screening conducted in a series of 32 BPNH patients with the identification of 12 novel point mutations in 15 patients are reported. Nine mutations were truncating, while three were missense. Three additional patients with BPNH-EDS and a mutation in FLNA are described. No phenotype–genotype correlations could be established, but these clinical data sustain the importance of cardiovascular monitoring in FLNA-BPNH patients.

Spectrum of CREBBP gene dosage anomalies in Rubinstein-Taybi Syndrome patients

The Rubinstein–Taybi syndrome (RTS) is a rare autosomal-dominant disease associated with 10–15% of cases with 16p13.3 microdeletions involving the CREB-binding protein gene (CREBBP). We used array-comparative genomic hybridization and Quantitative multiplex fluorescent-PCR (QMF-PCR) to search for dosage anomalies in the 16p13.3 region and the CREBBP gene. We first constructed a microarray covering 2 Mb that carries seven BAC and 34 cosmid clones, as well as 26 low-molecular-weight probes (1000–1500 bp) that are spread along the CREBBP gene. To increase further the resolution inside the CREBBP gene, we used QMF-PCR assays providing a 7 kb resolution. The deletions characterized in this work extended between as little as 3.3 kb and 6.5 Mb. Some deletions were restricted to just a few exons of CREBBP, some deleted either the 5′ or the 3′ end of the gene plus adjacent genomic segments, others deleted the whole gene away. We also identified a duplication of exon 16. We showed that CREBBP dosage anomalies constitute a common cause of RTS. CREBBP high-resolution gene dosage search is therefore highly recommended for RTS diagnosis. No correlation was found between the type of deletion and the patients’ phenotype. All patients had typical RTS, and there was no particular severity associated with certain alterations.

Heterogeneity of Platelet Functional Alterations in Patients With Filamin A Mutations

Objective—We examined platelet functions in 4 unrelated patients with filaminopathy A caused by dominant mutations of the X-linked filamin A (FLNA) gene. Methods and Results—Patients P1, P2, and P4 exhibited periventricular nodular heterotopia, heterozygozity for truncating FLNA mutations, and thrombocytopenia (except P2). P3 exhibited isolated thrombocytopenia and heterozygozity for a p.Glu1803Lys FLNA mutation. Truncated FLNa was undetectable by Western blotting of P1, P2, and P4 platelets, but full-length FLNa was detected at 37%, 82%, and 57% of control, respectively. P3 FLNa (p.Glu1803Lys and full-length) was assessed at 79%. All patients exhibited a platelet subpopulation negative for FLNa. Platelet aggregation, secretion, glycoprotein VI signaling, and thrombus growth on collagen were decreased for P1, P3, and P4, but normal for P2. For the 2 patients analyzed (P1 and P4), spreading was enhanced and, more markedly, in FLNa-negative platelets, suggesting that FLNa negatively regulates cytoskeleton reorganization. Platelet adhesion to von Willebrand factor under flow correlated with platelet full-length FLNa content: markedly reduced for P1 and P4 and unchanged for P2. Interestingly, von Willebrand factor flow adhesion was increased for P3, consistent with a gain-of-function effect enhancing glycoprotein Ib-IX-V/von Willebrand factor interaction. These results are consistent with a positive role for FLNa in platelet adhesion under high shear. Conclusion—FLNA mutation heterogeneity correlates with different platelet functional impacts and points to opposite regulatory roles of FLNa in spreading and flow adhesion under shear.

New practical definitions for the diagnosis of autosomal recessive spastic ataxia of Charlevoix–Saguenay

Autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS) is caused by mutations in the SACS gene. SACS encodes sacsin, a protein whose function remains unknown, despite the description of numerous protein domains and the recent focus on its potential role in the regulation of mitochondrial physiology. This study aimed to identify new mutations in a large population of ataxic patients and to functionally analyze their cellular effects in the mitochondrial compartment.