Autosomal Dominant MPAN: Mosaicism Expands the Clinical Spectrum to Atypical Late-Onset Phenotypes.

BACKGROUND: Mitochondrial membrane protein-associated neurodegeneration (MPAN) is caused by mutations in the C19orf12 gene. MPAN typically appears in the first two decades of life and presents with progressive dystonia-parkinsonism, lower motor neuron signs, optic atrophy, and abnormal iron deposits predominantly in the basal ganglia. MPAN, initially considered as a strictly autosomal recessive disease (AR), turned out to be also dominantly inherited (AD). OBJECTIVES: Our aim was to better characterize the clinical, molecular, and functional spectra associated with such dominant pathogenic heterozygous C19orf12 variants. METHODS: We collected clinical, imaging, and molecular information of eight individuals from four AD-MPAN families and obtained brain neuropathology results for one. Functional studies, focused on energy and iron metabolism, were conducted on fibroblasts from AD-MPAN patients, AR-MPAN patients, and controls. RESULTS: We identified four heterozygous C19orf12 variants in eight AD-MPAN patients. Two of them carrying the familial variant in mosaic displayed an atypical late-onset phenotype. Fibroblasts from AD-MPAN showed more severe alterations of iron storage metabolism and autophagy compared to AR-MPAN cells. CONCLUSION: Our data add strong evidence of the realness of AD-MPAN with identification of novel monoallelic C19orf12 variants, including at the mosaic state. This has implications in diagnosis procedures. We also expand the phenotypic spectrum of MPAN to late onset atypical presentations. Finally, we demonstrate for the first time more drastic abnormalities of iron metabolism and autophagy in AD-MPAN than in AR-MPAN. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Whole-exome sequencing confirms implication of VPS13D as a potential cause of progressive spastic ataxia.

BACKGROUND: VPS13D is a large ubiquitin-binding protein playing an essential role in mitophagy by regulating mitochondrial fission. Recently, VPS13D biallelic pathogenic variants have been reported in patients displaying variable neurological phenotypes, with an autosomic recessive inheritance. The objectives of the study were to determine the genetic etiology of a patient with early onset sporadic progressive spastic ataxia, and to investigate the pathogenicity of VPS13D variants through functional studies on patient's skin fibroblasts. CASE PRESENTATION: We report the case of a 51-year-old patient with spastic ataxia, with an acute onset of the disease at age 7. Walking difficulties slowly worsened over time, with the use of a wheelchair since age 26. We have used trio-based whole-exome sequencing (WES) to identify genes associated with spastic ataxia. The impact of the identified variants on mitochondrial function was assessed in patient's fibroblasts by imaging mitochondrial network and measuring level of individual OXPHOS complex subunits. Compound heterozygous variants were identified in VPS13D: c.946C > T, p.Arg316* and c.12416C > T, p.(Ala4139Val). Primary fibroblasts obtained from this patient revealed an altered mitochondrial morphology, and a decrease in levels of proteins from complex I, III and IV. CONCLUSIONS: Our findings confirmed implication of VPS13D in spastic ataxia and provided further support for mitochondrial defects in patient's skin fibroblasts with VPS13D variants. This report of long-term follow up showed a slowly progressive course of the spastic paraplegia with cerebellar features. Furthermore, the performed functional studies could be used as biomarker helping diagnosis of VPS13D-related neurological disorders when molecular results are uneasy to interpret.

Plasma oxysterols: biomarkers for diagnosis and treatment in spastic paraplegia type 5.

The hereditary spastic paraplegias are an expanding and heterogeneous group of disorders characterized by spasticity in the lower limbs. Plasma biomarkers are needed to guide the genetic testing of spastic paraplegia. Spastic paraplegia type 5 (SPG5) is an autosomal recessive spastic paraplegia due to mutations in CYP7B1, which encodes a cytochrome P450 7α-hydroxylase implicated in cholesterol and bile acids metabolism. We developed a method based on ultra-performance liquid chromatography electrospray tandem mass spectrometry to validate two plasma 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC) as diagnostic biomarkers in a cohort of 21 patients with SPG5. For 14 patients, SPG5 was initially suspected on the basis of genetic analysis, and then confirmed by increased plasma 25-OHC, 27-OHC and their ratio to total cholesterol. For seven patients, the diagnosis was initially based on elevated plasma oxysterol levels and confirmed by the identification of two causal CYP7B1 mutations. The receiver operating characteristic curves analysis showed that 25-OHC, 27-OHC and their ratio to total cholesterol discriminated between SPG5 patients and healthy controls with 100% sensitivity and specificity. Taking advantage of the robustness of these plasma oxysterols, we then conducted a phase II therapeutic trial in 12 patients and tested whether candidate molecules (atorvastatin, chenodeoxycholic acid and resveratrol) can lower plasma oxysterols and improve bile acids profile. The trial consisted of a three-period, three-treatment crossover study and the six different sequences of three treatments were randomized. Using a linear mixed effect regression model with a random intercept, we observed that atorvastatin decreased moderately plasma 27-OHC (∼30%, P < 0.001) but did not change 27-OHC to total cholesterol ratio or 25-OHC levels. We also found an abnormal bile acids profile in SPG5 patients, with significantly decreased total serum bile acids associated with a relative decrease of ursodeoxycholic and lithocholic acids compared to deoxycholic acid. Treatment with chenodeoxycholic acid restored bile acids profile in SPG5 patients. Therefore, the combination of atorvastatin and chenodeoxycholic acid may be worth considering for the treatment of SPG5 patients but the neurological benefit of these metabolic interventions remains to be evaluated in phase III therapeutic trials using clinical, imaging and/or electrophysiological outcome measures with sufficient effect sizes. Overall, our study indicates that plasma 25-OHC and 27-OHC are robust diagnostic biomarkers of SPG5 and shall be used as first-line investigations in any patient with unexplained spastic paraplegia.

CYP2U1 activity is altered by missense mutations in hereditary spastic paraplegia 56.

Hereditary spastic paraplegia (HSP) is an inherited disorder of the central nervous system mainly characterized by gradual spasticity and weakness of the lower limbs. SPG56 is a rare autosomal recessive early onset complicated form of HSP caused by mutations in CYP2U1. The CYP2U1 enzyme was shown to catalyze the hydroxylation of arachidonic acid. Here, we report two further SPG56 families carrying three novel CYP2U1 missense variants and the development of an in vitro biochemical assay to determine the pathogenicity of missense variants of uncertain clinical significance. We compared spectroscopic, enzymatic, and structural (from a 3D model) characteristics of the over expressed wild-type or mutated CYP2U1 in HEK293T cells. Our findings demonstrated that most of the tested missense variants in CYP2U1 were functionally inactive because of a loss of proper heme binding or destabilization of the protein structure. We also showed that functional data do not necessarily correlate with in silico predictions of variants pathogenicity, using different bioinformatic phenotype prediction tools. Our results therefore highlight the importance to use biological tools, such as the enzymatic test set up in this study, to evaluate the effects of newly identified variants in clinical settings.

Gain-of-Function Mutation in Filamin A Potentiates Platelet Integrin αIIbß3 Activation.

OBJECTIVE: Dominant mutations of the X-linked filamin A (FLNA) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. APPROACH AND RESULTS: We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100-amino acid-long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in αIIbß3 integrin activation and a parallel increase in talin recruitment to ß3, contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of αIIbß3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by αIIbß3. CONCLUSIONS: Altogether, our results are consistent with a less binding of mutant FLNa to ß3 and the facilitated recruitment of talin by ß3 on platelet stimulation, explaining the increased αIIbß3 activation and the ensuing gain-of-platelet functions.

Mitochondrial morphology and cellular distribution are altered in SPG31 patients and are linked to DRP1 hyperphosphorylation.

Hereditary spastic paraplegia, SPG31, is a rare neurological disorder caused by mutations in REEP1 gene encoding the microtubule-interacting protein, REEP1. The mechanism by which REEP1-dependent processes are linked with the disease is unclear. REEP1 regulates the morphology and trafficking of various organelles via interaction with the microtubules. In this study, we collected primary fibroblasts from SPG31 patients to investigate their mitochondrial morphology. We observed that the mitochondrial morphology in patient cells was highly tubular compared with control cells. We provide evidence that these morphological alterations are caused by the inhibition of mitochondrial fission protein, DRP1, due to the hyperphosphorylation of its serine 637 residue. This hyperphosphorylation is caused by impaired interactions between REEP1 and mitochondrial phosphatase PGAM5. Genetically or pharmacologically induced decrease of DRP1-S637 phosphorylation restores mitochondrial morphology in patient cells. Furthermore, ectopic expression of REEP1 carrying pathological mutations in primary neuronal culture targets REEP1 to the mitochondria. Mutated REEP1 proteins sequester mitochondria to the perinuclear region of the neurons and therefore, hamper mitochondrial transport along the axon. Considering the established role of mitochondrial distribution and morphology in neuronal health, our results support the involvement of a mitochondrial dysfunction in SPG31 pathology.

Functional validation of ABHD12 mutations in the neurodegenerative disease PHARC.

ABHD12 mutations have been linked to neurodegenerative PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract), a rare, progressive, autosomal, recessive disease. Although ABHD12 is suspected to play a role in the lysophosphatidylserine and/or endocannabinoid pathways, its precise functional role(s) leading to PHARC disease had not previously been characterized. Cell and zebrafish models were designed to demonstrate the causal link between an identified new missense mutation p.T253R, characterized in ABHD12 from a young patient, the previously characterized p.T202I and p.R352* mutations, and the associated PHARC. Measuring ABHD12 monoacylglycerol lipase activity in transfected HEK293 cells demonstrated inhibition with mutated isoforms. Both the expression pattern of zebrafish abhd12 and the phenotype of specific antisense morpholino oligonucleotide gene knockdown morphants were consistent with human PHARC hallmarks. High abhd12 transcript levels were found in the optic tectum and tract, colocalized with myelin basic protein, and in the spinal cord. Morphants have myelination defects and concomitant functional deficits, characterized by progressive ataxia and motor skill impairment. A disruption of retina architecture and retinotectal projections was observed, together with an inhibition of lens clarification and a low number of mechanosensory hair cells in the inner ear and lateral line system. The severe phenotypes in abhd12 knockdown morphants were rescued by introducing wild-type human ABHD12 mRNA, but not by mutation-harboring mRNAs. Zebrafish may provide a suitable vertebrate model for ABHD12 insufficiency and the study of functional impairment and potential therapeutic rescue of this rare, neurodegenerative disease.

Otopalatodigital spectrum disorders: refinement of the phenotypic and mutational spectrum.

Otopalatodigital spectrum disorders (OPDSD) constitute a group of dominant X-linked osteochondrodysplasias including four syndromes: otopalatodigital syndromes type 1 and type 2 (OPD1 and OPD2), frontometaphyseal dysplasia, and Melnick-Needles syndrome. These syndromes variably associate specific facial and extremities features, hearing loss, cleft palate, skeletal dysplasia and several malformations, and show important clinical overlap over the different entities. FLNA gain-of-function mutations were identified in these conditions. FLNA encodes filamin A, a scaffolding actin-binding protein. Here, we report phenotypic descriptions and molecular results of FLNA analysis in a large series of 27 probands hypothesized to be affected by OPDSD. We identified 11 different missense mutations in 15 unrelated probands (n=15/27, 56%), of which seven were novel, including one of unknown significance. Segregation analyses within families made possible investigating 20 additional relatives carrying a mutation. This series allows refining the phenotypic and mutational spectrum of FLNA mutations causing OPDSD, and providing suggestions to avoid the overdiagnosis of OPD1.

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

OBJECTIVE: 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. METHODS: A total of 321 index patients with spastic ataxia selected from the SPATAX network were analyzed by direct sequencing of the SACS gene, and 156 patients from the ATAXIC project presenting with congenital ataxia were investigated either by targeted or whole exome sequencing. For functional analyses, primary cultures of fibroblasts were obtained from 11 patients carrying either mono- or biallelic variants, including 1 case harboring a large deletion encompassing the entire SACS gene. RESULTS: We identified biallelic SACS variants in 33 patients from SPATAX, and in 5 nonprogressive ataxia patients from ATAXIC. Moreover, a drastic and recurrent alteration of the mitochondrial network was observed in 10 of the 11 patients tested. INTERPRETATION: Our results permit extension of the clinical and mutational spectrum of ARSACS patients. Moreover, we suggest that the observed mitochondrial network anomalies could be used as a trait biomarker for the diagnosis of ARSACS when SACS molecular results are difficult to interpret (ie, missense variants and heterozygous truncating variant). Based on our findings, we propose new diagnostic definitions for ARSACS using clinical, genetic, and cellular criteria.

Liver disease in infancy caused by oxysterol 7 α-hydroxylase deficiency: successful treatment with chenodeoxycholic acid.

A child of consanguineous parents of Pakistani origin developed jaundice at 5 weeks and then, at 3 months, irritability, a prolonged prothrombin time, a low albumin, and episodes of hypoglycaemia. Investigation showed an elevated alanine aminotransferase with a normal γ-glutamyl-transpeptidase. Analysis of urine by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that the major peaks were m/z 480 (taurine-conjugated 3ß-hydroxy-5-cholenoic acid) and m/z 453 (sulphated 3ß-hydroxy-5-cholenoic acid). Analysis of plasma by gas chromatography-mass spectrometry (GC-MS) showed increased concentrations of 3ß-hydroxy-5-cholenoic acid, 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol, indicating oxysterol 7 α-hydroxylase deficiency. The patient was homozygous for a mutation (c.1249C>T) in CYP7B1 that alters a highly conserved residue in oxysterol 7 α-hydroxylase (p.R417C) - previously reported in a family with hereditary spastic paraplegia type 5. On treatment with ursodeoxycholic acid (UDCA), his condition was worsening, but on chenodeoxycholic acid (CDCA), 15 mg/kg/d, he improved rapidly. A biopsy (after 2 weeks on CDCA), showed a giant cell hepatitis, an evolving micronodular cirrhosis, and steatosis. The improvement in liver function on CDCA was associated with a drop in the plasma concentrations and urinary excretions of the 3ß-hydroxy-Δ5 bile acids which are considered hepatotoxic. At age 5 years (on CDCA, 6 mg/kg/d), he was thriving with normal liver function. Neurological development was normal apart from a tendency to trip. Examination revealed pes cavus but no upper motor neuron signs. The findings in this case suggest that CDCA can reduce the activity of cholesterol 27-hydroxylase - the first step in the acidic pathway for bile acid synthesis.

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. CONCLUSIONS: FLNA mutation heterogeneity correlates with different platelet functional impacts and points to opposite regulatory roles of FLNA in spreading and flow adhesion under shear.

Atypical male and female presentations of FLNA-related periventricular nodular heterotopia.

Periventricular nodular heterotopia, the most common form of cortical malformation in adulthood, is characterized by nodules of neurons ectopically placed along the lateral ventricles. Classically, ectopic nodules are bilateral and symmetric defining bilateral periventricular nodular heterotopia (BPNH). BPNH can lead to epilepsy and intellectual disability of variable severity. The X-linked dominant form of BPNH, related to mutations in FLNA encoding filamin A, is the major cause of BPNH, causing prenatal and neonatal lethality in males that explain the excess of affected women. However, few living males have been described with this condition. In addition, mutations in FLNA have been also exceptionally associated with unilateral nodular heterotopia. We describe here three new patients, all carrying a novel missense mutation in FLNA. Two of the patients were adult males with BPNH; both had normal cognitive development and one did not manifest any seizure until he died at age 57. The last patient was a female adult with epilepsy and focal nodules essentially located along the right ventricle. We compare the clinical and imaging data of our patients with those of previously described similar cases. The type and location of FLNA mutations leading to such atypical presentations are discussed.

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).

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 αIIbß3, 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.

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.

Lung disease associated with periventricular nodular heterotopia and an FLNA mutation.

X-linked periventricular nodular heterotopia (PH) is a neuronal migration disorder caused by mutations in the gene encoding filamin A (FLNA). High phenotypic diversity, ranging from PH to otopalatodigital syndrome and frontometaphyseal dysplasia has been described in association with FLNA mutations. Extra-neurological features including cardiovascular abnormalities, coagulopathy, skeletal dysplasia and joint hypermobility have sometimes been described in patients with PH. Respiratory manifestations have not been associated with FLNA disorders with the exception of tracheal stenosis and pulmonary hypoplasia associated with frontometaphyseal dysplasia and Melnick-Needles syndrome. Here, we report on a male patient aged 6 years presenting with a mosaic nonsense mutation c.994delG within the FLNA gene, PH and severe congenital lung disease comprising bilateral atelectasis, lung cysts, tracheobronchomalacia, pulmonary arterial hypertension and long-term oxygen dependence; histology of resected lung showed panpulmonary emphysema with marked reduction of bronchial cartilage. Rare male patients with PH and FLNA mutations have already been reported, usually with early lethality. These observations suggest the possibility of a link between FLNA mutations and congenital lung disease. A prospective study of patients with PH and FLNA mutations would be helpful in order to test this hypothesis.

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 mutation in the 3'-UTR of the HDAC6 gene abolishing the post-transcriptional regulation mediated by hsa-miR-433 is linked to a new form of dominant X-linked chondrodysplasia.

A family with dominant X-linked chondrodysplasia was previously described. The disease locus was ascribed to a 24 Mb interval in Xp11.3-q13.1. We have identified a variant (c.*281A>T) in the 3' untranslated region (UTR) of the HDAC6 gene that totally segregates with the disease. The variant is located in the seed sequence of hsa-miR-433. Our data showed that, in MG63 osteosarcoma cells, hsa-miR-433 (miR433) down-regulated both the expression of endogenous HDAC6 and that of an enhanced green fluorescent protein-reporter mRNA bearing the wild-type 3'-UTR of HDAC6. This effect was totally abrogated when the reporter mRNA bore the mutated HDAC6 3'-UTR. The HDAC6 protein was found to be over-expressed in thymus from an affected male fetus. Concomitantly, the level of total alpha-tubulin, a target of HDAC6, was found to be increased in the affected fetal thymus, whereas the level of acetylated alpha-tubulin was found to be profoundly decreased. Skin biopsies were obtained from a female patient who presented a striking body asymmetry with hypotrophy of the left limbs. The mutated HDAC6 allele was expressed in 31% of left arm-derived fibroblasts, whereas it was not expressed in the right arm. Overexpression of HDAC6 was observed in left arm-derived fibroblasts. Altogether these results strongly suggest that this HDAC6 3'-UTR variant suppressed hsa-miR-433-mediated post-transcriptional regulation causing the overexpression of HDAC6. This variant is likely to constitute the molecular cause of this new form of X-linked chondrodysplasia. This represents to our knowledge the first example of a skeletal disease caused by the loss of a miRNA-mediated post-transcriptional regulation on its target mRNA.

Detection of an intragenic deletion expands the spectrum of CTSC mutations in Papillon-Lefèvre syndrome.

The Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder. The gene responsible for the disease, cathepsin C (CTSC), is localized in 11q14.1-q14.21. We performed mutational and functional analyses of CTSC in two patients affected by this condition. Three previously unreported CTSC mutations were identified. The first patient had a compound heterozygous status with a p.G386R missense mutation and an intragenic deletion spanning exons 3-7. Second patient carried a homozygous splice site mutation, p.A253SfsX30. CTSC activity was undetectable in both patients, thus demonstrating the pathological effect of these mutations. We describe early evidence of an original intragenic deletion reported in PLS. Since this mutational mechanism could not be detected by direct sequencing, intragenic deletion has to be specifically investigated using gene dosage analysis techniques such as quantitative multiplex fluorescent polymerase chain reaction. We consider that this technique should be performed in patients with apparently homozygous CTSC mutations when one parent does not carry the expected mutation or is not available for analysis.

COL4A1 mutation in Axenfeld-Rieger anomaly with leukoencephalopathy and stroke.

OBJECTIVE: 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. METHODS: After the occurrence of a small, deep infarct associated with white matter lesions in a patient with a medical history of congenital cataract and amblyopia, we conducted clinical and neuroradiological investigations in 10 of her relatives. RESULTS: Diffuse leukoencephalopathy associated with ocular malformations of the Axenfeld-Rieger type was observed in five individuals. Familial genetic analyses led to the identification of a novel missense mutation in the COL4A1 gene, p.G720D, which cosegregates with the disease. INTERPRETATION: Our data corroborate previous observations demonstrating the role of COL4A1 in cerebral microangiopathy and expand the phenotypic spectrum associated with mutations in this gene. We delineate a novel association between the Axenfeld-Rieger anomaly and leukoencephalopathy and stroke. Ann Neurol 2007.