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.