Manuele Mine

The SR Protein SC35 Is Responsible for Aberrant Splicing of the E1α Pyruvate Dehydrogenase mRNA in a Case of Mental Retardation with Lactic Acidosis

ABSTRACT Pyruvate dehydrogenase (PDH) complex deficiency is a major cause of lactic acidosis and Leighs encephalomyelopathies in infancy and childhood, resulting in early death in the majority of patients. Most of the molecular defects have been localized in the coding regions of the E1α PDH gene. Recently, we identified a novel mutation of the E1α PDH gene in a patient with an encephalopathy and lactic acidosis. This mutation, located downstream of exon 7, activates a cryptic splice donor and leads to the retention of intronic sequences. Here, we demonstrate that the mutation results in an increased binding of the SR protein SC35. Consistently, ectopic overexpression of this splicing factor enhanced the use of the cryptic splice site, whereas small interfering RNA-mediated reduction of the SC35 protein levels in primary fibroblasts from the patient resulted in the almost complete disappearance of the aberrantly spliced E1α PDH mRNA. Our findings open the exciting prospect for a novel therapy of an inherited disease by altering the level of a specific splicing factor.

Splicing Error in E1α Pyruvate Dehydrogenase mRNA Caused by Novel Intronic Mutation Responsible for Lactic Acidosis and Mental Retardation

An intronic point mutation was identified in theE1α PDH gene from a boy with delayed development and lactic acidosis, an X-linked disorder associated with a partial defect in pyruvate dehydrogenase (PDH) activity. Protein analysis demonstrated a corresponding decrease in immunoreactivity of the α and β subunits of the PDH complex. In addition to the normal spliced mRNA product of the E1α PDH gene, patient samples contained significant levels of an aberrantly spliced mRNA with the first 45 nucleotides of intron 7 inserted in-frame between exons 7 and 8. The genomic DNA analysis found no mutation in the coding regions but revealed a hemizygous intronic G to A substitution 26 nucleotides downstream from the normal exon 7 5′-splice site. Splicing experiments in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which involves a switch from the use of the normal 5′-splice site (intron 7 position 1) to the cryptic 5′-splice site downstream of the mutation (intron 7 position 45). The intronic mutation is unusual in that it generates a consensus binding motif for the splicing factor, SC35, which normally binds to exonic enhancer elements resulting in increased exon inclusion. Thus, the aberrant splicing phenotype is most likely explained by the generation of a de novo splicing enhancer motif, which activates the downstream cryptic 5′-splice site. The mutation documented here is a novel case of intron retention responsible for a human genetic disease.

Molecular characterization of 82 patients with pyruvate dehydrogenase complex deficiency. Structural implications of novel amino acid substitutions in E1 protein

BACKGROUND Pyruvate dehydrogenase complex (PDHc) deficiencies are an important cause of primary lactic acidosis. Most cases result from mutations in the X-linked gene for the pyruvate dehydrogenase E1α subunit (PDHA1) while a few cases result from mutations in genes for E1β (PDHB), E2 (DLAT), E3 (DLD) and E3BP (PDHX) subunits or PDH-phosphatase (PDP1). AIM To report molecular characterization of 82 PDHc-deficient patients and analyze structural effects of novel missense mutations in PDHA1. METHODS PDHA1 variations were investigated first, by exon sequencing using a long range PCR product, gene dosage assay and cDNA analysis. Mutation scanning in PDHX, PDHB, DLAT and DLD cDNAs was further performed in unsolved cases. Novel missense mutations in PDHA1 were located on the tridimensional model of human E1 protein to predict their possible functional consequences. RESULTS PDHA1 mutations were found in 30 girls and 35 boys. Three large rearrangements, including two contiguous gene deletion syndrome were identified. Novel missense, frameshift and splicing mutations were also delineated and a nonsense mutation in a mosaic male. Mutations p.Glu75Ala, p.Arg88Ser, p.Arg119Trp, p.Gly144Asp, p.Pro217Arg, p.Arg235Gly, p.Tyr243Cys, p.Tyr243Ser, p.Arg245Gly, p.Pro250Leu, p.Gly278Arg, p.Met282Val, p.Gly298Glu in PDHA1 were predicted to impair active site channel conformation or subunit interactions. Six out of the seven patients with PDHB mutations displayed the recurrent p.Met101Val mutation; 9 patients harbored PDHX mutations and one patient DLD mutations. CONCLUSION We provide an efficient stepwise strategy for mutation screening in PDHc genes and expand the growing list of PDHA1 mutations analyzed at the structural level.

Activating NOTCH3 Mutation in a Patient with Small-vessel-disease of the Brain

The most common causative diagnosis of hereditary small‐vessel‐disease of the brain, CADASIL, is due to highly stereotyped mutations in the NOTCH3 receptor. NOTCH3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor‐like Repeats encoded by exons 2–24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal NOTCH3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of NOTCH3 in a patient with cerebral small‐vessel‐disease but lacking GOM deposits and NOTCH3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of NOTCH3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical NOTCH3 signaling, although in a ligand‐independent fashion. Biochemical analysis suggests that the mutation renders NOTCH3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of NOTCH3 mutations and that NOTCH3 activating mutations should be further considered for molecular analysis of patients with cerebral small‐vessel‐disease.

Leigh's disease due to a new mutation in the PDHX gene

To describe the clinical course, neuroradiological presentation, biochemical and molecular studies of a new patient with pyruvate dehydrogenase complex (PDHc) deficiency. To compare this case with the data on other published cases.

A new case of pyruvate dehydrogenase deficiency due to a novel mutation in the pdx1 gene

We report a case of neonatal congenital lactic acidosis associated with pyruvate dehydrogenase E3‐binding protein deficiency in a newborn girl. She had a severe encephalopathy, and magnetic resonance imaging of the brain showed large subependymal cysts and no basal ganglia lesions. She died 35 days after birth. We detected a novel homozygous deletion (620delC) in the PDX1 gene, which encodes for the E3BP subunit of the pyruvate dehydrogenase complex.

Acute urinary retention due to a novel collagen COL4A1 mutation.

The gene COL4A1 encodes the α1 chain of type IV collagen, a basement-membrane protein implying vascular parietal strength. Mutations in the gene COL4A1 have been described in families with diffuse small-vessel disease of the brain, resulting in perinatal stroke, congenital porencephaly, extensive leukoencephalopathy, intracerebral hemorrhage, and retinal arteriolar tortuosity.1–4 Our observation extends the clinical and magnetic resonance (MR) phenotype of COL4A1 mutation. ### Case reports. A 21-year-old man without neurologic medical history presented with acute urinary retention. He had a right amblyopia due to a congenital strabismus surgically treated in childhood. Clinical examination showed brisk lower limb reflexes and a left Babinski sign. Blood pressure was normal. The Mini-Mental State Examination score was strictly normal. The patient declined further neuropsychological testing. Brain CT showed diffuse leukoencephalopathy and multiple supratentorial microcalcifications (figure, A). Brain MRI revealed widespread white matter hyperintensities on T2 and fluid-attenuated inversion recovery sequences, dilated perivascular spaces, and lacunar infarctions. Leukoencephalopathy involved the cerebral periventricular and cerebellar deep white matter (figure, B). Multiple microbleeds were seen on gradient echo sequences, involving brainstem and supratentorial white matter (figure, C). MR angiography of cervical and intracranial arteries was normal, as was spinal …

COL4A1 mutation revealed by an isolated brain hemorrhage.

Discussion The spectrum of COL4A1-related disorders includes perinatal cerebral hemorrhage and porencephaly [2] , cerebral small vessel disease with retinal arteriolar tortuosity and leukoencephalopathy [3] , HANAC syndrome (Hereditary Angiopathy, Nephropathy, Aneurysms, and Muscle Cramps) and other eye abnormalities, including the Axenfeld-Rieger anomaly and cataract. A frequent white matter involvement is reported [3, 4] . Interestingly, some HANAC patients may have a normal brain MRI. However, except for one patient who suffered from migraine, these patients were free from neurologic symptoms [5, 6] . Recently, Weng et al. [7] reported COL4A1 variants in 2/96 patients with sporadic ICH. Their phenotype was not described in Introduction Ten to fifteen percent of strokes are related to an intracerebral hemorrhage (ICH). Etiologies are mainly arterial hypertension, amyloid deposition, and vascular malformations. In cases of a young age without vascular risk factors and vascular malformations, a genetic cause has to be suspected, especially a mutation in the gene coding for type IV collagen alpha 1 (COL4A1) [1] . We report a COL4A1 gene mutation in a 45-year-old woman with an isolated ICH in the absence of any other MRI abnormality, which further extends the clinical spectrum of COL4A1 mutations.

A novel hereditary extensive vascular leukoencephalopathy mapping to chromosome 20q13

ABSTRACT Objective: The detection of a leukoencephalopathy is a frequent situation in neurologic practice. In a number of cases, the etiology remains obscure despite extensive investigations. We characterized the clinical, pathologic, and genetic features of a novel hereditary vascular leukoencephalopathy. Methods: After the observation of a similar leukoencephalopathy in 2 sisters, clinical, neuroimaging, and molecular genetics investigations were conducted in 21 of their consenting relatives. Pathologic data were obtained in one patient. Results: Fourteen members presented with significant white matter lesions at MRI examination, among whom only 5 individuals were symptomatic. The main clinical manifestations included gait disturbances, transient movement disorders, stroke, and cognitive dysfunction. The 9 remaining members aged from 26 to 60 years were asymptomatic. The MRI pattern was highly stereotyped with symmetric white matter hyperintensities worsening with patients age. We mapped the gene involved in this condition on chromosome 20q13. Neuropathologic examination suggested that this leukoencephalopathy is underlaid by a cerebral arteriolopathy affecting small preterminal arterioles, clearly distinct from amyloid angiopathy and hypertension-related small-vessel disease. Conclusions: These data establish that this family is affected by a novel autosomal dominant vascular leukoencephalopathy mapping to chromosome 20q13. This disease is characterized by a progressive and age-related hemispheric and brainstem leukoencephalopathy contrasting with the paucity and late onset of clinical symptoms in most of the cases.

Intracerebral hemorrhage and COL4A1 mutations, from preterm infants to adult patients.

Intracerebral hemorrhage (ICH) accounts for 10% of stroke and is a particularly severe form of stroke. Therapeutic tools are still limited, and prevention remains the most important way to reduce morbidity and mortality. However, for effective prevention, we need to understand the factors underlying the occurrence of hemorrhagic stroke and to identify individuals at greatest risk. Hypertension is a leading factor for ICH in adult patients, and cerebrovascular malformations are the most common causes of ICH in children. However, ICH is a heterogeneous condition, and in a substantial proportion of ICH patients, particularly in the younger ones, no cause can be identified despite extensive investigations. Recently, mutations of COL4A1 have been shown to cause ICH and porencephaly both in mouse and human. 8 COL4A1 encodes the 1 chain of type IV collagen, a major component of basement membranes, including vascular basement membrane. Mutated mice and human patients show an increased fragility of brain vessels that renders them highly sensitive to environmental factors such as birth trauma or antithrombotic agents. Gould and colleagues previously showed that surgical delivery of mouse pups dramatically reduces the risk for cerebral bleeding in the perinatal period and suggested that caesarean delivery may decrease the risk for ICH in human neonates. In this issue, de Vries and coworkers report on two siblings born preterm, at 33 weeks, and at 31 weeks of gestation, with a very low birth weight and in whom perinatal cranial routine ultrasound and magnetic resonance imaging (in the second infant) detected resolving hemorrhages and porencephalic cavities. Both siblings carry a deleterious mutation affecting a glycine residue located in the second C-terminal tandem repeated domain of COL4A1 1 chain. This mutation was inherited from their asymptomatic mother. The presence of these lesions in the first days of life strongly suggests that these hemorrhagic events occurred antenatally and not during vaginal delivery. If these two preterm infants would have been delivered at term, one would assume that they might have been diagnosed later on as having congenital hemiplegia. The first obvious and important implication of this observation is that caesarean delivery would not be sufficient to completely prevent the occurrence of ICH in mutated infants. The second one is that prevention and genetic counseling are important in this condition. This observation raises a number of questions of interest not only for neuropediatricians, obstetricians, and geneticists but also for neurologists taking care of adult patients. One major question is when should we suspect a COL4A1 mutation and conduct a genetic testing in a preterm or at-term infant showing an ICH. This question is not only important for medical care and genetic counseling but has also medicolegal implications. Currently, in cases of a familial porencephaly (more than one affected case), genetic screening of COL4A1 should be considered mandatory and will be of great help for genetic counseling. However, there are many situations in which the answer is so far unclear. Presumed antenatal or perinatal onset of a unilateral intraventricular/ICH leading to a porencephalic cyst is observed in 5 to 8% of very-low-birth-weight infants. Should all these infants be tested for COL4A1 mutations in the absence of any affected relative and any other known causative factor? What should be done when intracranial bleeding is detected in utero by ultrasound screening? Another major question is when should we suspect a COL4A1 mutation in an adult patient, who would then be at risk for ICH and at risk to have an affected child? COL4A1 mutations lead to a number of distinct phenotypes both in mouse and human, depending on the genetic background or environmental factors, or both. Relatives of familial porencephaly children have been shown to have leukoencephalopathy and an increased risk for ICH and/or cerebral microbleeds and/or ischemic lacunar infarcts. Another autosomal dominant condition characterized by the association of arteriolar retinal tortuousities, infantile hemiplegia, and leukoencephalopathy has been associated with a COL4A1 mutation. A distinct phenotype associating anterior eye chamber developmental anomalies including congenital cataract, leukoencephalopathy, ischemic lacunar infarcts, and microbleeds has been reported in a COL4A1-mutated family. At last, a novel phenotype associated with COL4A1 mutations has recently been reported under the acronym HANAC (hereditary angiopathy nephropathy aneurysms and muscle cramps). Screening of COL4A1 is clearly indicated when a patient with hemorrhagic stroke and leukoencephalopathy has suggestive associated symptoms such as retinal arteriolar tortuosity and/or hematuria, or has a history of infantile hemiplegia with porencephaly and/or anterior eye malformation, regardless of whether the proband has affected relatives. However, associated symptoms are often missed, particularly in relatives. COL4A1 mutaEDITORIALS