Familial Alzheimer's disease associated with heterozygous NPC1 mutation.

INTRODUCTION: NPC1 mutations are responsible for Niemann-Pick disease type C (NPC), a rare autosomal recessive neurodegenerative disease. Patients harbouring heterozygous NPC1 mutations may rarely show parkinsonism or dementia. Here, we describe for the first time a large family with an apparently autosomal dominant late-onset Alzheimer's disease (AD) harbouring a novel heterozygous NPC1 mutation. METHODS: All the five living siblings belonging to the family were evaluated. We performed clinical evaluation, neuropsychological tests, assessment of cerebrospinal fluid markers of amyloid deposition, tau pathology and neurodegeneration (ATN), structural neuroimaging and brain amyloid-positron emission tomography. Oxysterol serum levels were also tested. A wide next-generation sequencing panel of genes associated with neurodegenerative diseases and a whole exome sequencing analysis were performed. RESULTS: We detected the novel heterozygous c.3034G>T (p.Gly1012Cys) mutation in NPC1, shared by all the siblings. No other point mutations or deletions in NPC1 or NPC2 were found. In four siblings, a diagnosis of late-onset AD was defined according to clinical characterisation and ATN biomarkers (A+, T+, N+) and serum oxysterol analysis showed increased 7-ketocholesterol and cholestane-3ß,5α,6ß-triol. DISCUSSION: We describe a novel NPC1 heterozygous mutation harboured by different members of a family with autosomal dominant late-onset amnesic AD without NPC-associated features. A missense mutation in homozygous state in the same aminoacidic position has been previously reported in a patient with NPC with severe phenotype. The alteration of serum oxysterols in our family corroborates the pathogenic role of our NPC1 mutation. Our work, illustrating clinical and biochemical disease hallmarks associated with NPC1 heterozygosity in patients affected by AD, provides relevant insights into the pathogenetic mechanisms underlying this possible novel association.

Novel POLG mutations and variable clinical phenotypes in 13 Italian patients.

POLG gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA (mtDNA) replication and repair. Mutations in POLG have been linked to a spectrum of clinical phenotypes, resulting in autosomal recessive or dominant mitochondrial diseases. These mutations have been associated with heterogeneous phenotypes, presenting with varying severity and at different ages of onset, ranging from the neonatal period to late adult life. We screened 13 patients for POLG mutations. All patients underwent a complete neurological examination, and in most of cases, muscle biopsy was performed. We detected 15 different variations in 13 unrelated Italian patients. Two mutations were novel and mapped in the pol domain (p.Thr989dup and p.Ala847Thr) of the enzyme. We also report new cases carrying controversial variations previously described as incompletely penetrant or a variant of unknown significance. Our study increases the range of clinical presentations associated with mutations in POLG gene, underlining some peculiar clinical features, such as PEO associated with corneal edema, and epilepsy, severe neuropathy with achalasia. The addition of two new substitutions, including the second report of an in-frame duplication, to the growing list of defects increases the value of POLG genetic diagnosis in a range of neurological presentations.

Evaluation of cholesterol metabolism in cerebrotendinous xanthomatosis.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is a treatable bile acid disorder caused by mutations of CYP27A1. The pathogenesis of neurological damage has not been completely explained. Oral chenodeoxycholic acid (CDCA) can lead to clinical stabilization, but in a subgroup of patients the disease progresses despite treatment. In the present study, we aimed at clarifying cholesterol metabolism abnormalities and their response to CDCA treatment, in order to identify reliable diagnostic and prognostic markers and understand if differences exist between stable patients and those with neurological progression. METHODS: We enrolled 19 untreated CTX patients and assessed serum profile of bile acids intermediates, oxysterols, cholesterol, lathosterol, and plant sterols. Then we performed a long-term follow up during CDCA therapy, and compared biochemical data with neurological outcome. RESULTS: We observed increase of cholestanol, 7α-hydroxy-4-cholesten-3-one (7αC4), lathosterol, and plant sterols, whereas 27-hydroxycholesterol (27-OHC) was extremely low or absent. CDCA treatment at a daily dose of 750 mg normalized all biochemical parameters except for 7αC4 which persisted slightly higher than normal in most patients, and 27-OHC which was not modified by therapy. Biochemical evaluation did not reveal significant differences between stable and worsening patients. DISCUSSION: Cholestanol and 7αC4 represent important markers for CTX diagnosis and monitoring of therapy. Treatment with CDCA should aim at normalizing serum 7αC4 as well as cholestanol, since 7αC4 better mirrors 7α-hydroxylation rate and is thought to be correlated with cholestanol accumulation in the brain. Assessment of serum 27-OHC is a very good tool for biochemical diagnosis at any stage of disease. Lathosterol and plant sterols should be considered as additional markers for diagnosis and monitoring of therapy. Further studies including long-term assessment of bile acid intermediates in cerebrospinal fluid are needed in patients who show clinical progression despite treatment.

Sporadic PEO caused by a novel POLG variation and a Twinkle mutation: digenic inheritance?

Progressive external ophthalmoplegia (PEO) with multiple deletions of mitochondrial DNA (mtDNA) is associated with several mutations in nuclear genes. They include POLG, POLG2, ANT1, C10orf2/Twinkle, and OPA1. However, digenic inheritance in mitochondrial disorders has been documented in a few cases over the years. Here we describe an 80-year-old man with sporadic PEO associated with mtDNA deletions. Sequencing of the POLG revealed a novel heterozygous mutation (c.2831A>G; p.Glu944Gly), predicted in silico as damaging, in the patient who also carried a heterozygous mutation in C10orf2/Twinkle (c.1142T>C; p.Leu381Pro). This case provides a second report of a PEO with different mutations in the POLG and C10orf2/Twinkle genes, supporting the hypothesis that the PEO phenotype can be determined by the co-existence of two abnormalities in separate genes, both involved in the maintenance and stability of mtDNA. Finally, this study expands the spectrum of POLG mutations and highlights the need to sequence the whole set of nuclear genes associated with PEO and multiple mtDNA deletions.

A suspicion index for early diagnosis and treatment of cerebrotendinous xanthomatosis.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid storage disorder characterized by a heterogeneous presentation and a broad spectrum of clinical manifestations. Since early diagnosis and replacement therapy with chenodeoxycholic acid can prevent clinical deterioration, our aim was to develop a diagnostic tool to identify and treat CTX patients at an initial stage of the disease. METHODS: We devised a suspicion index, composed of weighted scores assigned to indicators such as family history characteristics and common systemic and neurological features, on the basis of a pooled analysis of selected international CTX series. The indicators were classified as very strong (score 100), strong (50) or moderate (25). The suspicion index was then applied retrospectively to our CTX population. RESULTS: Early systemic signs such as cataract, diarrhea and neonatal cholestatic jaundice were considered strong indicators, together with neurological features such as intellectual impairment, psychiatric disturbances, ataxia, spastic paraparesis and dentate nuclei abnormalities at MRI. Tendon xanthomas were regarded as very strong indicators, as was an affected sibling. A total score ≥ 100 warranted serum cholestanol assessment. Elevated cholestanol or a total score ≥ 200, with one very strong or four strong indicators, warranted CYP27A1 gene analysis. In our patients, age at diagnosis was 35.5 ± 11.8 years (mean ± standard deviation), whereas with the diagnostic tool it became 10.6 ± 9.8 years (p < 0.01). CONCLUSIONS: Our suspicion index provides a simple and inexpensive diagnostic tool allowing diagnosis and treatment of CTX before neurological disability occurs.

Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD).

OBJECTIVE: To identify novel biomarkers as an alternative diagnostic tool for limb girdle muscular dystrophy (LGMD). BACKGROUND: LGMD encompasses a group of muscular dystrophies characterized by proximal muscles weakness, elevated CK levels and dystrophic findings on muscle biopsy. Heterozygous CAPN3 mutations are associated with autosomal dominant LGMD-4, while biallelic mutations can cause autosomal recessive LGMD-1. Diagnosis is currently often based on invasive methods requiring muscle biopsy or blood tests. In most cases Western blotting (WB) analysis from muscle biopsy is essential for a diagnosis, as muscle samples are currently the only known tissues to express the full-length CAPN3 isoform. METHODS: We analyzed CAPN3 in a cohort including 60 LGMD patients. Selected patients underwent a complete neurological examination, electromyography, muscle biopsy, and skin biopsies for primary fibroblasts isolation. The amount of CAPN3 was evaluated by WB analysis in muscle and skin tissues. The total RNA isolated from muscle, fibroblast and urine was processed, and cDNA was used for qualitative analysis. The expression of CAPN3 was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL. RESULTS: Among our patients, seven different CAPN3 mutations were detected, of which two were novel. After sequencing CAPN3 transcripts from fibroblast and urine, we detected different CAPN3 isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel CAPN3 mutation showed a 30% reduction in protein compared to controls from both tissues. CONCLUSIONS: Our findings showed for the first time the presence of the CAPN3 full-length transcript in urine and skin samples. Moreover, we demonstrated surprisingly comparable CAPN3 protein levels between muscle and skin samples, thus allowing us to hypothesize the use of skin biopsy and probably of urine samples as an alternative less invasive method to assess the amount of CAPN3 when molecular diagnosis turns out to be inconclusive.

Alu-element insertion in an OPA1 intron sequence associated with autosomal dominant optic atrophy.

PURPOSE: Autosomal dominant optic atrophy (ADOA) is the most common form of hereditary optic neuropathy caused by mutations in the optic atrophy 1 (OPA1) gene. It is characterized by insidious onset with a selective degeneration of retinal ganglion cells, variable loss of visual acuity, temporal optic nerve pallor, tritanopia, and development of central, paracentral, or cecocentral scotomas. Here we describe the clinical and molecular findings in a large Italian family with ADOA. METHODS: Routine ophthalmologic examination and direct sequencing of all coding regions of the OPA1 gene were performed. Further characterization of a new OPA1 gene insertion was performed by reverse transcription-PCR (RT-PCR) of RNA from patients and control subjects. RESULTS: We identified an Alu-element insertion located in intron 7 of OPA1 causing an in-frame deletion of exon 8 in 18 family members. CONCLUSIONS: The predicted consequence of this mutation is the loss of the guanosine triphosphatase (GTPase) activity of OPA1. Alu insertions have been reported in the literature as causing human genetic disease. However, this is the first report of a pathogenic OPA1 gene mutation resulting from an Alu insertion.

A second MNGIE patient without typical mitochondrial skeletal muscle involvement.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by mutations in the gene encoding thymidine phosphorylase (TYMP). Clinically, MNGIE is characterized by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. Most MNGIE patients have signs of mitochondrial dysfunction in skeletal muscle at morphological and enzyme level, as well as mitochondrial DNA depletion, multiple deletions and point mutations. A case without mitochondrial skeletal muscle involvement and with a TYMP splice-acceptor site mutation (c. 215-1 G>C) has been reported. Here, we describe an Italian patient with the same mutation and without mitochondrial skeletal muscle involvement, suggesting a possible genotype-phenotype correlation.

A novel heteroplasmic tRNA(Ser(UCN)) mtDNA point mutation associated with progressive external ophthalmoplegia and hearing loss.

We sequenced all mitochondrial tRNA genes from a patient with sporadic external ophthalmoplegia (PEO) and 5% COX-negative fibers in muscle biopsy, who had no detectable large mtDNA deletions. Direct sequencing showed a heteroplasmic mutation at nucleotide 7506 in the dihydrouridine stem of the tRNA(Ser(UCN)) gene. RFLP analysis confirmed that 30% of muscle and 20% of urinary epithelium mtDNA harbored the mutation, which was absent in other tissues of the proband as well as in mtDNA of his mother and 100 patients with various encephalomyopathies. Several point mutations on mitochondrial tRNA genes have been reported in PEO patients without large-scale rearrangements of mtDNA but no point mutations have hitherto been found in the gene coding for tRNA(Ser(UCN)).

Heterozygous mutations of HTRA1 gene in patients with familial cerebral small vessel disease.

AIMS: Cerebral small vessel disease (SVD) is the leading cause of vascular dementia. Although the most of cases are sporadic, familial monogenic causes have been identified in a growing minority of patients. CADASIL, due to mutations of NOTCH3 gene, is the most common genetic SVD, and CARASIL, linked to HTRA1 gene mutations, is a rare but well known autosomal recessive SVD. Recently, also heterozygous HTRA1 mutations have been described in patients with familial SVD. To detect a genetic cause of familial SVD, we performed mutational analysis of HTRA1 gene in a large cohort of Italian NOTCH3-negative patients. METHODS: We recruited 142 NOTCH3-negative patients and 160 healthy age-matched controls. Additional control data were obtained from five pathogenicity prediction software. RESULTS: Five different HTRA1 heterozygous mutations were detected in nine patients from five unrelated families. Clinical phenotype was typical of SVD, and the onset was presenile. Brain magnetic resonance imaging (MRI) showed a subcortical leukoencephalopathy, with involvement of the external and internal capsule, corpus callosum, and multiple lacunar infarcts. Cerebral microbleeds were also seen, while anterior temporal lobes involvement was not present. CONCLUSION: Our observation further supports the pathogenic role of the heterozygous HTRA1 mutations in familial SVD.

Analysis of opa1 isoforms expression and apoptosis regulation in autosomal dominant optic atrophy (ADOA) patients with mutations in the opa1 gene.

Autosomal dominant optic atrophy (ADOA) is a hereditary optic neuropathy characterized by bilateral symmetrical visual loss, decrease in retinal ganglion cells and a loss of myelin within the optic nerve. ADOA is associated to mutations in Optic atrophy 1 gene (OPA1), which encodes a mitochondrial protein involved in cristae remodeling, maintenance of mitochondrial membrane integrity, mitochondrial fusion and apoptosis regulation. We thus evaluated the rate of apoptosis and the expression levels of OPA1 isoforms in ADOA and control cells. Peripheral blood lymphocytes from eight patients with OPA1 mutation and age matched controls were cultivated both in basal conditions or with 2-deoxy-D-ribose, a reducing sugar that induces apoptosis through oxidative stress. Apoptosis was analyzed by flow cytometry, phosphatidylserine translocation, mitochondrial membrane depolarization and caspase 3 activation. We also analyzed the expression levels of OPA1 isoforms in ADOA and control cells cultured with and without 2-deoxy-D-ribose. We showed an increased percentage of apoptotic cells in ADOA patients compared to controls, both in basal culture conditions and after 2-deoxy-D-ribose treatment. This suggested a great susceptibility of ADOA cells to oxidative stress and a strong correlation between OPA1 protein dysfunctions and morphological-functional alterations to mitochondria. Moreover OPA1 protein expression was significantly decreased in lymphocytes from the ADOA patients after 2-deoxy-D-ribose treatment, implying a great sensitivity of the mutated protein to free radical damage. Concluding, we could confirm that oxidative stress-induced apoptosis may play a key role in the pathophysiological process bringing to retinal ganglion cells degeneration in ADOA.

First deep intronic mutation in the NOTCH3 gene in a family with late-onset CADASIL.

CADASIL is the most prominent inherited form of vascular dementia. The main clinical features include migraine with aura, stroke, mood disturbances, and cognitive decline, with a mid-life (30s-60s) adult onset. Genetic testing is the gold standard for the diagnosis. CADASIL is caused mostly by missense mutations in the NOTCH3 gene, invariably involving a cysteine residue. Only a couple of splice site mutations have been reported. In a few pathologically defined patients, genetic mutations remain unidentified. We report a family with late-onset CADASIL phenotype carrying a novel intronic deletion in the NOTCH3 gene (c.341-26_24delAAC). Transcript analysis revealed a splicing alteration, with the complete intron 3 retention. The insertion was in-frame and encoded an extra 25 amino acids, including 1 cysteine. This is the first report of an aberrant splicing event of the NOTCH3 gene associated with a mutation far away from the canonical splice site. Our finding suggests that the assays used to evaluate splicing should be mandatory in the diagnostic setting of genetically undefined CADASIL cases.

Mitochondria, oxidative stress and neurodegeneration.

Mitochondria are involved in ATP supply to cells through oxidative phosphorylation (OXPHOS), synthesis of key molecules and response to oxidative stress, as well as in apoptosis. They contain many redox enzymes and naturally occurring inefficiencies of oxidative phosphorylation generate reactive oxygen species (ROS). CNS functions depend heavily on efficient mitochondrial function, since brain tissue has a high energy demand. Mutations in mitochondrial DNA (mtDNA), generation and presence of ROS and environmental factors may contribute to energy failure and lead to neurodegenerative diseases. Many rare metabolic disorders have been associated with mitochondrial dysfunction. More than 300 pathogenic mtDNA mutations involve proteins that regulate OXPHOS and mitochondrial structural integrity, and have also been described in neurodegenerative diseases with autosomal inheritance. Mitochondria may have an important role in ageing-related neurodegenerative disorders like Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). In primary mitochondrial and neurodegenerative disorders, there is strong evidence that mitochondrial dysfunction occurs early and has a primary role in pathogenesis. In the present review, we discuss several mitochondrial diseases as models of neurodegeneration.

Sporadic myopathy, myoclonus, leukoencephalopathy, neurosensory deafness, hypertrophic cardiomyopathy and insulin resistance associated with the mitochondrial 8306 T>C MTTK mutation.

We report a new T8306C transition in the D-stem of the MTTK gene of a 67-year-old man who manifested severe adult onset myopathy, myoclonus, leukoencephalopathy, neurosensory hypoacusis, hypertrophic cardiomyopathy and insulin resistance. No other family member was affected, suggesting that our patient was a sporadic case. The T8306C mutation was heteroplasmic in several tissues of the proband, while it was absent from his asymptomatic siblings. Single fibre analysis confirmed the segregation of higher mutational load in cytochrome c oxidase-deficient fibres. The mutation T8306C is predicted to disrupt a highly conserved base pair and was not found in more than 120 controls. This finding broadens the phenotypic and molecular spectrum of mitochondrial tRNA(Lys) associated disorders.