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.

Several diagnostic strategies have been applied to the detection of FMR1 gene repeat expansions in fragile X syndrome. Here, we report a novel polymerase chain reaction-based strategy using the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany) and the osmolyte betaine. Repeat expansions up to ϳ330 CGGs in males and up to at least ϳ160 CGGs in carrier women could be easily visualized on ethidium bromide agarose gels. We also demonstrated that fluorescence analysis of polymerase chain reaction products was a reliable tool to verify the presence of premutation and full mutation alleles both in males and in females. This technique, primarily designed to detect premutation alleles, can be used as a routine first screen for expanded FMR1 alleles. (J Mol Diagn 2005, 7:605-612)Fragile X syndrome is the most common inherited form of mental retardation. This syndrome is caused by the expansion of CGG repeats in the 5Ј-untranslated region of the fragile X mental retardation 1 (FMR1) gene and hypermethylation of its 5Ј upstream CpG island.1 The CGG repeat element is polymorphic, varying from 6 to 44 repeats in the normal range, from 45 to 54 repeats in the gray zone, and from 55 to 200 repeats in the premutation range.2 For alleles below the gray zone, the CGG repeat is generally stable in parent-to-offspring transmissions. However, CGG elements in the premutation range become increasingly unstable with increasing repeat number, and alleles exceeding ϳ59 CGG repeats can expand to a full mutation in a single generation, almost exclusively by transmission from mother to son. The FMR1 premutation is typically associated with specific clinical manifestations unique to the premutation range: premature ovarian failure has been observed in ϳ20% of women, 3-6 whereas the fragile X-associated tremor/ ataxia syndrome has been found in at least one-third of carrier males more than 50 years old.7-9 Individuals affected with fragile X syndrome have FMR1 alleles with a CGG repeat number greater than 200.At present, DNA analysis of the CGG expansion is primarily performed using Southern blot analysis, which is able to detect alleles spanning the range from normal to large full mutation alleles; however, this method lacks the resolution to accurately size alleles. An alternative approach, using polymerase chain reaction (PCR) amplification of the region spanning the CGG repeat, provides much greater resolution, although it suffers from the difficulty of amplifying CGG repeats greater than ϳ100 to 150 repeats, because of the high GC content of the sequence being amplified.Radioactive or chemiluminescent probing, or fluorescence PCR, can overcome most problems, at least in the premutation range. Several studies have already described a number of PCR techniques, which use diverse combinations of DNA polymerase, 7-deaza-dGTP, and co-solvents such as dimethyl sulfoxide (DMSO) and betaine. 10 -15 However, the largest allele that has been amplified to date is 250 CGG repeats, 13 and PCR results with alleles of greater than ϳ...

We describe a four-generation Italian family with a novel form of juvenile-onset, slowly progressive, autosomal dominant cerebellar ataxia. Eleven affected family members have been evaluated. The mean age at onset was 19.5 years with no evidence of anticipation. The first symptoms were invariably unbalanced standing and mild gait incoordination. Gaze-evoked nystagmus was prominent at onset, while patients with longer disease duration developed slow saccades, ophthalmoparesis and, often, ptosis. Deep tendon reflexes in lower limbs were increased in 80% of the cases. Genetic analysis excluded the presence of pathological repeat expansions in spinocerebellar ataxia (SCA) types 1-3, 6-8, 10, 12 and 17, and DRPLA genes. Linkage exclusion tests showed no evidence of association with other known SCA loci. A genome-wide screen analysis identified linkage with chromosome 18 markers. A maximum two-point limit of determination score of 4.20 was found for marker D18S53. Haplotype analysis refined a critical region of 7.9 Mb between markers D18S1418 and D18S1104. This new SCA locus on 18p11.22-q11.2 has been designated SCA28. Candidate genes within the critical interval are currently screened for mutations.

Spinocerebellar ataxia type 28 is an autosomal dominant form of cerebellar ataxia (ADCA) caused by mutations in AFG3L2, a gene that encodes a subunit of the mitochondrial m-AAA protease. We screened 366 primarily Caucasian ADCA families, negative for the most common triplet expansions, for point mutations in AFG3L2 using DHPLC. Whole-gene deletions were excluded in 300 of the patients, and duplications were excluded in 129 patients. We found six missense mutations in nine unrelated index cases (9/366, 2.6%): c.1961C>T (p.Thr654Ile) in exon 15, c.1996A>G (p.Met666Val), c.1997T>G (p.Met666Arg), c.1997T>C (p.Met666Thr), c.2011G>A (p.Gly671Arg), and c.2012G>A (p.Gly671Glu) in exon 16. All mutated amino acids were located in the C-terminal proteolytic domain. In available cases, we demonstrated the mutations segregated with the disease. Mutated amino acids are highly conserved, and bioinformatic analysis indicates the substitutions are likely deleterious. This investigation demonstrates that SCA28 accounts for ∼3% of ADCA Caucasian cases negative for triplet expansions and, in extenso, to ∼1.5% of all ADCA. We further confirm both the involvement of AFG3L2 gene in SCA28 and the presence of a mutational hotspot in exons 15-16. Screening for SCA28, is warranted in patients who test negative for more common SCAs and present with a slowly progressive cerebellar ataxia accompanied by oculomotor signs.

LMNB1 gene duplication appears characteristic of a subset of adult-onset autosomal dominant leucoencephalopathies, sharing autonomic dysfunction at onset, diffuse T2-hyperintensity of supra- and infratentorial white matter, sparing of U-fibres and optic radiations. The variable phenotypes in the remaining cases lacking LMNB1 defects (five with autosomal dominant transmission) suggest that adult-onset leucoencephalopathies are genetically heterogeneous.

This observation suggests that a mutation in an LMNB1 regulatory sequence underlies the variant ADLD phenotype. Thus, adult forms of ADLD linked to 5q23 appear to be more heterogeneous clinically and genetically than previously thought.

We have recently mapped the spinocerebellar ataxia type 28 (SCA28) locus on chromosome 18p11.22 in a four-generation Italian family. The clinical phenotype in affected individuals of this family was characterized by juvenile onset, slowly progressive gait and limb ataxia, dysarthria, hyperreflexia at lower limbs, nystagmus, and ophthalmoparesis. The mean age at onset was 19.5 years, and no evidence of anticipation between generations was observed. The disease locus on chromosome 18p11.22-q11.2 was found to span a region of 7.9 Mb of genomic DNA. Direct sequencing of candidate genes within the critical interval led to the identification of a heterozygous point mutation in one of them. The mutation was located in a highly conserved domain with proposed functional properties in the protein product of the SCA28 gene, and segregated with the disease phenotype in all affected members of this family. Thereafter we have screened 105 patients with autosomal dominant spinocerebellar ataxia who had resulted negative for mutations in known SCA genes. Genetic screening allowed the identification in a second Italian family of a distinct missense mutation located in the very same functional domain of the protein. The affected members of this second family exhibited a neurological phenotype similar to that of the original family. Both mutations, not found in more than 500 chromosomes, are associated with amino acid changes (Glu-->Lys and Ser-->Leu, respectively) in evolutionarily conserved residues of the alleged SCA28 gene. Our data point to a putative pathogenic role of these mutations, and indicate SCA28 as the sixth recognized SCA genotype caused by point mutations.

Spinocerebellar ataxia type15 (SCA15) is a pure ataxia characterized by very slow progression. Only seven families have been identified worldwide, in which partial deletions and a missense mutation of the inositol triphosphate receptor type I gene (ITPR1) have been reported. We examined a four-generation Italian family segregating an autosomal dominant cerebellar ataxia, in which linkage analysis was positive for the SCA15 locus. We performed a genomic real-time polymerase chain reaction to search for ITPR1 gene deletions in this family and in 60 SCA index cases negative for mutations in the SCA1-3, 6-8, 10, 12,and dentatorubral-pallidoluysian atrophy genes. The deleted segments were characterized using a custom array comparative genomic hybridization analysis. We have identified two families with an ITPR1 gene deletion: in one, the deletion involved ITPR1 only, while in the other both sulfatase-modifying factor 1 and ITPR1. Clinical data of ten patients and brain MRI (available for six) showed that the phenotype substantially overlapped known SCA15 cases,but we also noted buccolingual dyskinesias, facial myokymias,and pyramidal signs never reported in SCA15. ITPR1 expression analysis of two deleted cases showed a half dose. Our results further support ITPR1 gene as causative of SCA15. The families reported show that SCA15 is present in Italy and has a greater variability in the age at onset and clinical features than previously reported. We propose that the search for ITPR1 deletions is mandatory in the clinical hypothesis of SCA15 and that ITPR1-reduced expression in blood may be a useful marker to identify SCA15 patients harboring genomic deletions and possibly point mutations causing reduction of mRNA level.