No association found between Alzheimer's disease and a mitochondrial tRNA glutamine gene variant

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Neuroscience Letters 201 (1995) 107-110

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No association found between Alzheimer's Disease and a mitochondrial tRNA glutamine gene variant M i c h e l l e A. W r a g g a,*, C h r i s t o p h e r J. T a l b o t a, J o h n C. M o r r i s b, C o r i n n e L. L e n d o n a, A l i s o n M . G o a t e a aDepartment of Psychiatry, Washington University Medical School, St. Louis, MO 63110, USA bDepartment of Neurology, Washington University Medical School, St. Louis, MO 63110, USA Received 27 September 1995; accepted 20 October 1995

Abstract

We have screened a large sample of patients with sporadic late-onset dementia of the Alzheimer type (DAT) and age-matched controis for a mitochondrial tRNA Gin variant previously reported to be associated with increased risk of developing Alzheimer's disease (AD). The frequency of an Ava H site gain was determined by restriction analysis of a PCR-amplified mitochondrial DNA product. One of 155 DAT cases and four of 105 age-matched controls carded the variant. Both the affected and control frequencies are statistically different from those previously reported. The mitochondrial lineage of those individuals harboring the variant was determined by sequencing a short region of the hypervariable mitochondrial D-loop. The affected individual and three of the four controls carrying the Ava H variant belong to the same mitochondrial lineage previously reported to be associated with AD. Keywords: Alzheimer's disease; Mitochondrial DNA; tRNA GIngene; Ava H variant; Mitochondrial lineage; Oxidative phosphorylation

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, in which clinical symptoms appear in middle to late life. AD is clinically, pathologically and genetically heterogeneous. Recent years have seen many advances in the identification of genetic loci implicated in the etiology of AD. Missense mutations within the amyloid precursor protein (APP) gene (chromosome 21) segregate with the disease in a small number of early-onset AD pedigrees [3,9]. The majority of familial early-onset AD cases are thought to be caused by mutations in the PS-1 gene (chromosome 14) [18]. A third locus on chromosome 1 (PS-2) carries a mutation in Volga German AD families [12]. The e4 and the e2 alleles of the apolipoprotein E (ApoE) gene (chromosome 19) have been associated with both familial and sporadic early- and late-onset AD [4,5, 21]. The presence of the ApoE e4 allele increases the likelihood of developing AD. The existence of healthy elderly people who are ApoE e4 homozygotes indicates that there must be other genetic and/or environmental risk factors that modify e4 risk. The existence of late-onset * Corresponding author. Tel.: +1 314 3628668; fax: +1 314 3628649; e-mail: wragg @icarus.wustl.edu.

AD cases with no e4 alleles indicates that other genetic and/or environmental risk factors increase the risk for AD in the absence of e4 alleles. Disturbances in mitochondrial metabolism due to a variety of mutations in mitochondrial DNA (mtDNA) or nuclear DNA have been associated with degenerative diseases and the normal aging process [6,8,13,16,20,23]. Both mitochondrial encoded polypeptides and nuclear encoded polypeptides are necessary components of the five multisubunit enzyme complexes involved in oxidative phosphorylation [22]. As mutations accumulate in the mtDNA, oxidative phosphorylation declines and may fall below the threshold necessary for cellular metabolism [23]. It is hypothesized that when this occurs clinical symptoms may manifest, the severity of which is dependent on the tissue or organ involved. Organs with a high energy consumption, such as the brain and the heart, are likely to suffer severely when this occurs [ 14]. It has been proposed that the high rate of neuronal cell death observed in AD may be in part caused by defects in oxidative phosphorylation [7]. AD patients have a substantial reduction in cytochrome oxidase (oxidative phosphorylation complex IV) activity in the cerebral cortex, which may result in reduced ATP production and subsequently

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Table 1 Proportion of individuals carrying the tRNAGln variant in DAT and controlpopulations AD/DAT(%) Shoffneret al. 1993 ([19]) 2/62 (3.2) Hutchin and Cortopassi 1995 ([10]) 6/72(8.3) Wragg et al. 1995 1/155 (0.6)

Controls(%) 12/1691 (0.7) 1/296 (0.3 4/105 (3.8)

neuronal cell death [11,17]. Treatment of normal human fibroblasts with an oxidative phosphorylation uncoupler results in an elevated level of immunoreactivity of the cells with the Alz50 monoclonal antibody [2]. This suggests that a defect in oxidative phosphorylation could lead to NFT formation and may be involved in AD. Shoffner et al. [19] reported an elevated frequency of a homoplasmic mitochondrial tRNA Gin gene variant in a small sample of autopsy-confirmed AD cases. An A-to-G transition at nucleotide pair (np) 4336, which alters a moderately conserved nucleotide and creates an Ava H site, was observed in two of 62 (3.2%) AD patients (see Table 1). The frequency of the Ava H site in Caucasian controls was estimated to be 0.7%, from 125 laboratory controls (1/125) and 1566 (11/1566) literature controls [19]. Frequencies of 8% (6/72) in AD patients and 0.3% (1/296) in controls, for the Ava H site, were recently reported by Hutchin and Cortopassi [10]. Individuals carrying the variant were shown to belong to a single mitochondrial lineage by phylogenetic analysis. We have performed a follow-up study on a large Caucasian sample of late-onset, sporadic DAT patients and age-matched controis. The mitochondria~ lineage of those people harboring the variant was determined by sequencing a region of the mitochondrial D-loop. Participants were Caucasian and were recruited from the Greater St. Louis metropolitan community through the Washington University Alzheimer's Disease Research Center. Recruitment was not based upon a prior family history of Alzheimer's Disease; however, approximately 50% of participants have a first degree relative with a diagnosis dementia of the Alzheimer type (DAT). Diagnostic criteria for dementia of the Alzheimer type (DAT) are equivalent to but more stringent than those for probable and possible AD by the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association (NINCDS/ADRDA) Workshop [15]. Clinical diagnostic accuracy for histological AD in our subjects is 96% [1]. Control subjects meet all exclusionary criteria for DAT and are not demented. Blood samples were collected from 155 DAT patients (mean age 76.6 years, SD 8.5 years) and 105 age-matched controls (mean age 78.2 years, SD 9.5 years). The mean age of onset of dementia in the DAT patients was 70 years (SD 8.5 years).

Genomic DNA was extracted by a standard method from blood samples. A 286 bp DNA fragment of the mitochondrial tRNA gene was amplified by PCR using the following cocktail: 0.4/~M forward (5'-cgctacgaccaactcataca-3') and reverse (5'-gatagcttatttagctgacc-3') primers, 200#M dNTPs, 1.5 mM MgCI 2, 0.5 U Amplitaq (Perkin Elmer Cetus) and standard Cetus buffer. Amplification was performed on a Hybaid Omnigene using the following conditions: 94°C 4 min, 1 cycle; 94°C 30 s, 50°C 30s, 72°C 30 s, 35 cycles; 72°C 5 min. Amplified products were digested with 5 U Ava H for 4 h and electrophoresed through a 2.5% agarose gel at 4.5 V/cm for 45 min. A 300 bp fragment of the mitochondrial D-loop was amplified by PCR using the following cocktail: 0.4/~M forward (5'-cttgaccacctgtagtacat-3') and reverse (5'-cctgaagtaggaaccagatg-3") (biotinylated) primers, 200/~M dNTPs, 1.5 mM MgCI 2, 0.5 U Amplitaq (Perkin Elmer Cetus) and standard Cetus buffer. Amplification was performed on a Hybaid Omnigene using the following conditions: 94°C 4 min, 1 cycle; 94°C 30 s, 50°C 30 s, 72°C 30 s, 35 cycles; 72°C 5 min. Single-stranded DNA was collected with M-280 streptavidin Dynabeads (Dynal) and dideoxy sequencing was performed using standard protocols. One of 155 (0.6%) DAT patients harbored the Ava H variant. While a statistically significant difference from the frequency of Shoffner et al. [19] could not be obtained, due to the sample size, the frequency of the tRNA GIn variant in our DAT sample is statistically different from that of Hutchin and Cortopassi [10] at the P < 0.01 level (Yates Z2 = 7.4). The frequency of the variant in our control sample was 4/105 (3.8%), which is statistically different from the reported frequency of 0.7% in the Caucasian population at the P < 0.01 level (Yates Z2 = 7.5). Sequencing of a region of the mtDNA D-loop determined that the DAT individual and three of the controis harboring the Ava H variant had an Rsa I site loss at np 16304. These individuals belong to the same mitochondrial lineage as the AD patients reported by Shoffner et al. [19] and Hutchin and Cortopassi [10]. The fourth control carrying the Ava H variant had retained the Rsa 1 site and had a T-to-C transition at np 16356, and thus was not part of the same lineage. This suggests that either the Ava H variant arose before the Rsa 1 variant, or that the Ava H variant has arisen at least twice. Of the four individuals we have identified, three were control subjects, aged 73, 80 and 89 years. These individuals could still develop AD. A sampling bias in the selection of control subjects could have allowed us to identify a larger proportion (3/105) of this lineage than would normally be expected. This may be due to an elevated level of a particular ethnic origin in our control sample compared to the DAT group. However, this seems unlikely as all three studies have used North American Caucasian control groups of presumably similar ethnic composition. The three controls we have identified in this lineage may carry

M.A, Wragg et al. / Neuroscience Letters 201 (1995) 107-110

genetic elements that protect against AD. One of the individuals (aged 80) carries an A p o E e2 allele, which has been shown to play a protective role in A D [4]. The D A T individual (aged 65) belonging to the 4336 + 16304 lineage carries an A p o E e4 allele, which may account for the dementia in this individual [5]. One of the controls (aged 73) also carries an A p o E e4 allele, and thus is at risk for developing AD. The third control (aged 89) is homozygous for the A p o E e3 allele. This individual has a family history of AD, with three affected first degree relatives (average age of onset, 81.3 years; affected relatives died before the age of 89). The A p o E genotype of the individuals reported in [19] and [10] is not known. Both the 4336 and 16304 polymorphisms have ancient origins. It is possible that an additional mutation occurred elsewhere in the mitochondrial genome at a later time, thereby creating another lineage. Such a mutation could either predispose to or protect against AD. Although a definite diagnosis of A D relies on histopathological examination of the brain, the diagnostic criteria used in our D A T sample has historically been 96% accurate in the diagnosis of AD. Thus, it is unlikely that our D A T sample contains patients suffering from a dementia other than AD, which may have caused a lack of association o f the tRNA ~ln variant with the disease. Both the D A T and the control samples have been typed for ApoE. Both populations have the predicted allelic frequencies, suggesting that they are not atypical in other respects. The discrepancies in the frequency of the A v a H variant in both the A D groups and the age-matched controls may be due to an unknown sampling bias, or small sample sizes, which may either enhance or disguise an effect. Hutchin and Cortopassi [10] reported, as a note in proof, screening of a further 122 A D cases for the A v a H variant. Of these, only two individuals carried the variant. This greatly reduced frequency of the variant in this A D group could be due to either of the above explanations. The combination and analysis of all the data thus far reported should give a better indication of whether people belonging to this lineage are at increased risk for developing AD. W e have calculated the Odds Ratio (OR)for the total number of reported A D cases (11/411) and controls (5/ 526) who belong to this lineage and found no statistical difference between the two groups (OR = 2.87; 95% CI 0.99-8.2). A total of 11 A D / D A T patients and five controis belonging to the 16304 + 4336 lineage have thus far been reported. These numbers are too small to permit statistical analysis for an increased risk for A D in this lineage. Further elderly members of this lineage need to be identified and clinically assessed to determine whether there is an increased risk for A D associated with this lineage. W e thank the physicians and staff of the Memory and Aging Project of the Washington University Alzheimer's

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Disease Research Center for performing the clinical evaluations, and Dr. Nick Craddock for helpful discussions. This work was funded by Metropolitan Life Foundation award (AG) and NIH Career Development award (AG00634-01). Thanks to Elizabeth A. Grant for data management.

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