In this study, DNA samples of 140 unrelated patients with a familial history of dominant ataxia were screened for sequence abnormalities in exons 15 and 16 of the AFG3L2 gene using an SSCP approach. Furthermore, to check the SSCP results and to reveal intragenic DNA sequence variations, all 17 coding exons were sequenced for 20 index patients. Altogether, we found five different single-nucleotide exchanges in the coding sequence, as well as in the 3′-UTR and intronic regions. One of these variations leads to a change at the amino-acid level ().
Sequence variations within the AFG3L2 gene
SSCP screening of 140 unrelated ataxia patients showed a peculiarity in exon 16 for one sample. Sequencing of this exon revealed the heterozygous transition c.2098G>A in a 44-year-old female patient from Germany (III3). At the amino-acid level, this single-nucleotide substitution results in the missense exchange p.E700K.
Remarkably, the variation c.2098G>A was not found in DNA samples of 200 unrelated control individuals of German descent by allele-specific PCR. Furthermore, the amino-acid substitution p.E700K segregated with the disease in four additional affected members of the four-generation family (). In patient IV4, his mother III9 and in the twins IV1 and IV2, the first cerebellar signs of the disease developed within the first decade of life, whereas in five additional family members, symptoms were retrospectively noted within the third decade of life with an unusually slow progression and maintained free walking into the seventh decade of life. A more severe clinical presentation was only observed in both affected monozygotic twins, who in addition show a global developmental delay and currently attend a special school for children with cognitive impairment. However, given the normal cognitive function of the other affected relatives in this family and the few cases described so far in the literature, reported premature delivery and serious postnatal adaptation problems need to be considered as important cofactors for the more complex and severe clinical presentation of these monozygotic twins.
Figure 1 Pedigree of a four-generation German family with autosomal dominant spinocerebellar ataxia. Filled symbols indicate affected subjects. Open symbols specify unaffected spouses and presently asymptomatic family members. Deceased individuals are marked by (more ...)
In contrast, predictive testing of one not affected family member revealed the wild-type sequence. On the basis of these findings, a pathogenic impact of the missense mutation p.E700K on the ataxic phenotype is highly presumable. This assumption is supported by the strong conservation of glutamic acid (E) at position 700 in M. musculus
, R. norvegicus
, C. familiaris
, P. pygmaeus
, P. troglodytes
, E. caballus
and B. taurus
. Furthermore, the online programme ‘Mutation T@ster' (http://www.mutationtaster.org/
) classifies this amino-acid exchange as being presumably disease causing.
It is noteworthy that a pathogenic missense exchange E–K is already described in the literature.3
However, the authors do not specify the SCA28
gene name nor the exact position of this amino-acid substitution. Thus, it remains unclear whether both missense mutations are identical.
Subsequent to the SSCP screening, exons 15 and 16 were sequenced for 19 randomly selected individuals out of the 140 ataxia patients, as well as for the 44-year-old female patient III3 carrying the missense mutation p.E700K. No further DNA sequence abnormality was identified in any of the corresponding samples, indicating good reliability of the SSCP assay. Moreover, all remaining coding exons of the AFG3L2 gene were investigated by DNA sequence analysis for these 20 ataxia patients in order to reveal rare and underlying intragenic variations. This is due to the crucial necessity of gaining knowledge about the clinical impact of AFG3L2 variants to provide a firm basis for genetic testing in SCA28.
Single-nucleotide polymorphisms (SNPs) IVS7+6C>T and c.*28G>C were detected in intron 7 and in the 3′-UTR of the AFG3L2 gene, respectively. In addition to these noncoding SNPs, the two silent exchanges, p.L463 (exon 11) and p.E550 (exon 13), were found (). The intronic SNP and the two silent exchanges occur exclusively in the same individuals. Primarily, a pathogenic effect through impaired splicing would be possible for the IVS7+6C>T exchange because of its proximity to the exon/intron boundary. However, given the relatively high frequency of these four SNPs and the fact that no typical splice site is generated in all cases, a disease-causing impact through aberrant splicing is very unlikely for these variations.
Overall, only one ataxia-inducing mutation was found in 140 patients, illustrating that SCA28 is a relatively rare cause of disease in the German population. The neurological examination of the five affected family members further supports previous data regarding the clinical presentation of most SCA28 patients: an early-onset cerebellar ataxia with a slowly progressive phenotype. Notably, all AFG3L2 mutations known so far are located in a small interval between amino acids 654 and 700 in exons 15 and 16. Hence, for diagnostic purposes, investigating only these two exons should be taken into account. The clustering in the AFG3L2 peptidase M41 domain, as well as the missense nature of all described mutations, argues against haploinsufficiency as the pathogenetic mechanism. In fact, a dominant-negative effect through gain of function is more presumable.