Cinzia Gellera

Friedreich's Ataxia: Autosomal Recessive Disease Caused by an Intronic GAA Triplet Repeat Expansion

Friedreichs ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. The gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis

MATR3 is an RNA- and DNA-binding protein that interacts with TDP-43, a disease protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Using exome sequencing, we identified mutations in MATR3 in ALS kindreds. We also observed MATR3 pathology in ALS-affected spinal cords with and without MATR3 mutations. Our data provide more evidence supporting the role of aberrant RNA processing in motor neuron degeneration.

Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28

Autosomal dominant spinocerebellar ataxias (SCAs) are genetically heterogeneous neurological disorders characterized by cerebellar dysfunction mostly due to Purkinje cell degeneration. Here we show that AFG3L2 mutations cause SCA type 28. Along with paraplegin, which causes recessive spastic paraplegia, AFG3L2 is a component of the conserved m-AAA metalloprotease complex involved in the maintenance of the mitochondrial proteome. We identified heterozygous missense mutations in five unrelated SCA families and found that AFG3L2 is highly and selectively expressed in human cerebellar Purkinje cells. m-AAA–deficient yeast cells expressing human mutated AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment and deficiency of respiratory chain complex IV. Structure homology modeling indicates that the mutations may affect AFG3L2 substrate handling. This work identifies AFG3L2 as a novel cause of dominant neurodegenerative disease and indicates a previously unknown role for this component of the mitochondrial protein quality control machinery in protecting the human cerebellum against neurodegeneration.

The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/−FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/−FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/−FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10−8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.

Mutations of FUS Gene in Sporadic Amyotrophic Lateral Sclerosis

Background Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). Objective To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). Methods Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. Results Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. Conclusions The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.

Analysis of FUS gene mutation in familial amyotrophic lateral sclerosis within an Italian cohort.

Objective: Mutations in the FUS gene on chromosome 16 have been recently discovered as a cause of familial amyotrophic lateral sclerosis (FALS). This study determined the frequency and identities of FUS gene mutations in a cohort of Italian patients with FALS. Methods: We screened all 15 coding exons of FUS for mutations in 94 Italian patients with FALS. Results: We identified 4 distinct missense mutations in 5 patients; 2 were novel. The mutations were not present in 376 healthy Italian controls and thus are likely to be pathogenic. Conclusions: Our results demonstrate that FUS mutations cause ∼4% of familial amyotrophic lateral sclerosis cases in the Italian population.

Phenotypic manifestations associated with CAG-repeat expansion in the androgen receptor gene in male patients and heterozygous females: a clinical and molecular study of 30 families

Spinal and bulbar muscular atrophy (Kennedy disease) is an adult form of X-linked motor neuron disease caused by the expansion of a polymorphic CAG-repeat sequence in the first exon of the androgen receptor gene. We studied clinical and molecular features of 36 patients and 19 heterozygous females. Phenotypic manifestations and disease severity broadly varied among our spinal and bulbar muscular atrophy patients. The size of CAG expansion significantly influences the age of disease onset, but neither clinical features nor disease severity. The majority of carrier women presented signs of chronic denervation at neurophysiological examination and, in three cases, low-amplitude sensory action potentials were recorded. Notably, a few women developed mild signs of bulbar motor neuron impairment later in life. The identification of a large number of patients by the use of the molecular test further supports the hypothesis that Kennedy disease had been previously underdiagnosed, probably because of the great variability of clinical presentation. Although an early diagnosis may not be crucial for treatment, given the lack of effective therapy, the molecular testing can be of great relevance for disease prognosis and genetic counseling.

A majority of Huntington's disease patients may be treatable by individualized allele-specific RNA interference

Use of RNA interference to reduce huntingtin protein (htt) expression in affected brain regions may provide an effective treatment for Huntington disease (HD), but it remains uncertain whether suppression of both wild-type and mutant alleles in a heterozygous patient will provide more benefit than harm. Previous research has shown suppression of just the mutant allele is achievable using siRNA targeted to regions of HD mRNA containing single nucleotide polymorphisms (SNPs). To determine whether more than a minority of patients may be eligible for an allele-specific therapy, we genotyped DNA from 327 unrelated European Caucasian HD patients at 26 SNP sites in the HD gene. Over 86% of the patients were found to be heterozygous for at least one SNP among those tested. Because the sites are genetically linked, one cannot use the heterozygosity rates of the individual SNPs to predict how many sites (and corresponding allele-specific siRNA) would be needed to provide at least one treatment possibility for this percentage of patients. By computing all combinations, we found that a repertoire of allele-specific siRNA corresponding to seven sites can provide at least one allele-specific siRNA treatment option for 85.6% of our sample. Moreover, we provide evidence that allele-specific siRNA targeting these sites are readily identifiable using a high throughput screening method, and that allele-specific siRNA identified using this method indeed show selective suppression of endogenous mutant htt protein in fibroblast cells from HD patients. Therefore, allele-specific siRNA are not so rare as to be impractical to find and use therapeutically.

Identification of new ANG gene mutations in a large cohort of Italian patients with amyotrophic lateral sclerosis

Angiogenin (ANG) gene, coding for an angiogenic factor up-regulated by hypoxia and expressed in ventral horn motor neurons, is a novel candidate for the pathogenesis of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease characterized by the selective loss of cortical and spinal motor neurons. Missense mutations in ANG gene have been identified in two ALS populations from Northern Europe and North America, both in familial (FALS) and sporadic (SALS) patients, but they do not seem to be frequent in the Italian population. We performed a mutational screening in a large cohort of 737 Italian ALS patients, including 605 SALS and 132 FALS cases. We identified seven different mutations, five of which are novel, in nine patients (six SALS and three FALS), but not in 515 healthy controls. Three mutations are located in the signal peptide region, three in the coding sequence, and one in the 3′ untranslated region. In our ALS population, the observed mutational frequency of ANG gene accounts for about 1.2%, with an overrepresentation of FALS (2.3%) compared to SALS (1%) cases. We also found the previously described I46V substitution in six patients and four controls, suggesting that this mutation may represent a benign variant, at least in the Italian population. Our results provide further evidence of a tight link between angiogenesis and ALS pathogenesis and suggest that mutations in ANG gene are associated with an increased risk to develop ALS.

Visual system involvement in patients with Friedreich's ataxia

Optic neuropathy is common in mitochondrial disorders, but poorly characterized in Friedreichs ataxia (FRDA), a recessive condition caused by lack of the mitochondrial protein frataxin. We investigated 26 molecularly confirmed FRDA patients by studying both anterior and posterior sections of the visual pathway using a new, integrated approach. This included visual field testing and optical coherence tomography (OCT), pattern visual evoked potentials (P-VEPs) and diffusion-weighted imaging. The latter was used to study optic radiation by calculating water apparent diffusion coefficients (ADC). All patients suffered optic nerve involvement with their disorder. Different patterns of visual field defects were observed and a variably reduced retinal nerve fiber layer thickness was seen by OCT in all cases. P-VEPs were abnormal in approximately half of the patients. Decreased visual acuity and temporal optic disc pallor were present in advanced stages of the disease, but only five patients were symptomatic. Two of these patients suffered a sudden loss of central vision, mimicking Lebers hereditary optic neuropathy (LHON), and of the other three symptomatic patients two were noted to be compound heterozygotes. ADC values of optic radiations in patients were significantly higher than controls (P < 0.01). Retinal nerve fiber layer thickness at OCT and P-VEPs correlated with age at onset and ICARS total score. ADC values correlated with age at onset, disease duration, GAA triplet expansion size, ICARS total score and P-VEPs. Visual pathway involvement is found consistently in FRDA, being previously underestimated, and we here document that it also involves the optic radiations. Occasional LHON-like cases may occur. However, optic neuropathy in FRDA substantially differs from classic mitochondrial optic neuropathies implying a different pathophysiology of visual system degeneration in this mitochondrial disease.