HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Correspondence to:

ARTICLES: V. Bonifati, C. F. Rohé, G. J. Breedveld, E. Fabrizio, M. De Mari, C. Tassorelli, A. Tavella, R. Marconi, D. J. Nicholl, H. F. Chien, E. Fincati, G. Abbruzzese, P. Marini, A. De Gaetano, M. W. Horstink, J. A. Maat-Kievit, C. Sampaio, A. Antonini, F. Stocchi, P. Montagna, V. Toni, M. Guidi, A. Dalla Libera, M. Tinazzi, F. De Pandis, G. Fabbrini, S. Goldwurm, A. de Klein, E. Barbosa, L. Lopiano, E. Martignoni, P. Lamberti, N. Vanacore, G. Meco, B. A. Oostra, and The Italian Parkinson Genetics Network Early-onset parkinsonism associated with PINK1 mutations: Frequency, genotypes, and phenotypes Neurology 2005; 65: 87-95 [Abstract] [Full text] [PDF]

Correspondence: Submit a response to this article

Correspondence published:

Biologic effect of a PINK1 mutation: mRNA levels of the c.1366C>T (Gln456Stop) change

Christine Klein, MD, Anne Grünewald (Department of Neurology), Katja Hedrich (Department of Neurology and Human Genetics, University of Lübeck   (8 November 2005)

Reply to authors

Vincenzo Bonifati, Christan F. Rohé, Guido J. Breedveld, and Ben A. Oostra.   (8 November 2005)

Biologic effect of a PINK1 mutation: mRNA levels of the c.1366C>T (Gln456Stop) change 8 November 2005
Christine Klein, MD, University of Luebeck Neurology Ratzeburger Allee 160, 23538 Lübeck, Anne Grünewald (Department of Neurology), Katja Hedrich (Department of Neurology and Human Genetics, University of Lübeck Send Correspondence to journal: Re: Biologic effect of a PINK1 mutation: mRNA levels of the c.1366C>T (Gln456Stop) change christine.klein{at}neuro.uni-luebeck.de Christine Klein, MD, et al.	We read with great interest the recent report by Bonifati et al demonstrating that the transcript of an allele bearing the c.1366C>T (Gln456Stop) mutation in the PINK1 gene associated with early-onset Parkinson’s disease (PD) is not detectable by PCR in two unrelated families. [1] The authors explained this finding by either lack of expression of the mutant allele or by instability due to the mutation or another change in linkage disequilibrium with the c.1366T alteration. They conclude that this variant is an example of a mutant allele that exerts its major pathogenic effect in PD at the mRNA rather than at the protein level. [1] We identified a large German PD family with four affected siblings who were all homozygous for the c.1366C>T mutation. We also studied eleven of their asymptomatic heterozygous children and five mutation- negative family members. To address the question of an effect of this mutation on the mRNA level, we collected fresh blood from all 20 family members and extracted RNA that was reverse transcribed. We first carried out an RT-PCR spanning Exons 4-7 including the mutation (Primers Ex4F: CCAAGAGAGGTCCCAAGC; Ex7R: CCTCACCAACTGTCTCACG). We observed no difference in product intensity even in the homozygous mutation carriers, suggesting that the mutated allele is expressed at a similar level and has a similar stability as the wild type allele (Figure 1A). To separately investigate the mRNA level of the wild type and mutated allele, we designed mutation-specific primers and performed another RT-PCR (Ex7FWT: TCAATCCCTTCTACGGCC, Ex7FMUT: TCAATCCCTTCTACGGCT, Ex8R: CTCCTCAGTCCAGCCTCAT). We demonstrated the specificity of the primers and the expression of both alleles in all heterozygous carriers (Figure 1B). We showed that the pathogenic effect of the recurrent c.1366C>T mutation in our family is not related to lack of expression or instability. Our results rather support the hypothesis that the lack of mutated transcripts in the families described by Bonifati et al may be caused by another change in linkage disequilibrium with the mutation. Interestingly, the c.1366C>T mutation likely has arisen twice independently in our family or bases on an ancient founder since the haplotypes are partly different (Figure 1C). It remains unclear which haplotypes the reported unrelated Italian families carry. [1] Our heterozygous mutation carriers were asymptomatic, whereas Bonifati’s patients had full-blown PD. It remains to be determined whether RNA expression levels of the mutated allele may be correlated with disease status. References 1. Bonifati V, Rohe CF, Breedveld GJ, et al. Early-onset parkinsonism associated with PINK1 mutations: frequency, genotypes, and phenotypes. Neurology 2005; 65: 87-95. Figure Figure legend mRNA level and haplotypes in a large German family with the c.1366C>T mutation in PINK1. A) An RT-PCR product of PINK1 is shown for three homozygous mutation carriers (MUT/MUT) and for four mutation- negative (WT/WT) family members. The intensity of the product is comparable, suggesting similar mRNA levels. B) With mutation specific primers we selectively amplified either the wild type allele (top panel) or the mutated allele (bottom panel) of PINK1 (643 bp product). As expected, the wild type primer did not lead to a PCR product in the homozygous mutation carriers, whereas the mutation-specific primer fails to amplify the wild type allele in the mutation-negative family members as shown for three examples each. In the heterozygous mutation carriers both alleles are expressed which is demonstrated here for three of the eleven carriers. The co-amplification of beta-globin as a housekeeping gene (313 bp product) demonstrates RNA loading in all samples. C) The PINK1 haplotype at six microsatellite markers surrounding the mutation is shown for the four affected homozygous siblings (black symbols). A crossing over event occurred in the male patient. In the other three siblings only the markers flanking the mutation were homozygous indicating either an ancient founder or independently arisen mutations. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (CK, KH), the Bundesministerium für Bildung und Forschung (01GI0201, CK), and a Lichtenberg Grant from the Volkswagen Foundation (CK).
Reply to authors 8 November 2005
Vincenzo Bonifati, Department of Clinical Genetics, Erasmus MC Rotterdam P.O. Box 1738, 3000 DR Rotterdam, The Netherlands, Christan F. Rohé, Guido J. Breedveld, and Ben A. Oostra. Send Correspondence to journal: Re: Reply to authors v.bonifati{at}erasmusmc.nl Vincenzo Bonifati, et al.	We thank Klein et al for their interest in our paper. [1] Their identification of a fourth family with autosomal recessive early-onset PD (AREP) associated with the PINK1 c.1366C>T (Gln456Stop) mutation confirms our findings of a recurrent pathogenic mutation. We cannot exclude that the c.1366C>T mutation (in heterozygous or homozygous state) has different biological effects in different patients, because of a variation in cis or many other factors. However, we believe that the conclusions by Klein et al concerning the biologic effect of this mutation are not supported by their findings. First, they present data about amplification of wild type and mutant PINK1 transcripts from cDNA material obtained from heterozygous and homozygous carriers of this mutation. However, none of the experiments shown by Klein et al are quantitative, and therefore, their results do not allow any conclusion to be made about levels or stability of mRNA. These conclusions can only be drawn from quantitative PCR analysis. Second, regarding the haplotype analysis, Klein et al. should explore whether their patients are compound heterozygous for c.1366C>T and a PINK1 genomic deletion. Since the time of the publication of our paper [1], we repeated the experiments shown there using new cDNA material prepared from blood, and novel sets of primers. The results (not shown) replicate those of figure 2 of our paper [1], again suggesting a major pathogenic effect of the c.1366C>T mutation at the mRNA level, most likely an example of nonsense-mediated mRNA decay. [2] Similar to the results of Klein et al, our agarose-gel analysis (not shown) using non-quantitative allele-specific PCR yielded evidence of robust amplification of both wild type and c.1366C>T PINK1 transcripts, a finding compatible with the presence of even minimal residual amounts of mutant c.1366C>T transcript. We then performed different quantitative real-time PCR assays for the total PINK1 transcript (protocols and results available on request); the results show consistently a ~50% decrease in levels of PINK1 mRNA in heterozygous carriers of c.1366C>T compared with non-carriers. Unfortunately, mRNA was not available from our homozygous mutation carrier. In our view, the data presented by Klein et al confirm that the PINK1 c.1366C>T mutation is associated with AREP. Further quantitative PCR assays are necessary to assess the biological effects of this mutation on the levels and stability of mRNA. References 1. Bonifati V, Rohe CF, Breedveld GJ, et al. Early-onset parkinsonism associated with PINK1 mutations: frequency, genotypes, and phenotypes. Neurology 2005; 65: 87-95. 2. Holbrook JA, Neu-Yilik G, Hentze MW, Kulozik AE. Nonsense-mediated decay approaches the clinic. Nat Genet 2004; 36: 801-8.