QUARTERLY Genetic study of familial cases of Alzheimer’s disease

A small number (1-5%) of Alzheimer's disease (AD) cases associated with the early-onset form of the disease (EOAD) appears to be transmitted as a pure genetic, autosomal dominant trait. To date, three genes responsible for familial EOAD have been identified in the human genome: amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2). Mutations in these genes account for a significant fraction (18 to 50%) of familial cases of early onset AD. The mutations affect APP processing causing increased production of the toxic Abeta42 peptide. According to the "amyloid cascade hypothesis", aggregation of the Abeta42 peptide in brain is a primary event in AD pathogenesis. In our study of twenty AD patients with a positive family history of dementia, 15% (3 of 20) of the cases could be explained by coding sequence mutations in the PS1 gene. Although a frequency of PS1 mutations is less than 2% in the whole population of AD patients, their detection has a significant diagnostic value for both genetic counseling and treatment in families with AD.

cells.From genetic studies of families with autosomal dominant pattern of disease inheritance, three genes responsible for familial early onset AD (EOAD; first symptoms before 65 years of age) have been identified in the human genome: amyloid precursor protein (APP) on chromosome 21 at 21q21.1 (Tanzi et al., 1987), presenilin 1 (PS1) on chromosome 14 at 14q24.3 (Sherrington et al., 1995) and presenilin 2 (PS2) on chromosome 1 at 1q42.1 (Levy-Lahad et al., 1995).Moreover, the Apolipoprotein E (APOE) gene polymorphism was found as a strong susceptibility factor for AD (Saunders et al., 1993).Genetic analyses revealed that carriers of the APOE*4 allele are at a higher risk of the disease than APOE*4 non-carriers.The APP gene encodes a polypeptide of up to 770 amino acids which is probably involved in nuclear signaling (Selkoe, 1998).According to the "amyloid cascade hypothesis", abnormalities of APP metabolism with subsequent b-amyloid (Ab) generation play a central role in the pathogenesis of AD (Hardy & Higgins, 1992).APP is processed by three proteases, named a-, b-, and g-secretases.A series of endoproteolytic cleavages of APP leads to the formation of non-amyloidogenic (the Ab40 fragment generated by a-secretase) or amyloidogenic (the Ab42 peptide generated by band g-secretases) products.By contrast to Ab40, Ab42 has a greater tendency to form fibrillary b-amyloid deposits.The deposition of "seeding" Ab42 accelerates Ab40 accumulation and stimulates a cascade of processes leading to formation of plaques and neurofibrillary tangles (NFTs) with subsequent neuron death (Jarret & Lansbury, 1993).The presenilin genes (PS) encode multipass membrane proteins, named presenilins (PS) which have been found in the nuclear membrane, endoplasmic reticulum, and the Golgi (Kovacs et al., 1996).The most known topological model of PS suggests eight transmembrane domains.PS1 and PS2 display a high homology sharing 67% of aminoacid sequence.Their transmembrane domains are even more similar, with an identity of 84% (Levy-Lahad et al., 1995).The presenilins are involved in the Notch and Wnt/b-catenin signaling pathways (Levitan & Greenwald, 1995).The ps1 is necessary for normal neurogenesis and survival and localizes to synaptic membranes and neurite growth cones (Soriano et al., 2001).Moreover, presenilins have been suggested to regulate apoptosis and the unfolded protein stress response (Niwa et al., 1999).
Pathogenic mutations within the APP and PS genes account for up to 50% of familial EOAD cases.If one of the genes is mutated, the mutated protein leads to development of AD with a penetrance close to 100%.So far, 20 mutations in the APP gene, 124 mutations in the PS1 gene, and 8 mutations in the PS2 gene have been described worldwide.Most of the mutations are substitutions.Only a couple of deletions and insertions and two splicing defect mutations have been reported in the PS1 gene (Cruts et al., 1998).All APP mutations are clustered near the a-, b-, or g-secretase cleavage sites, demonstrating that they have a direct effect on APP processing and Ab formation.A majority of them affect the activity of secretases causing an increased production of Ab42 (Citron et al., 1992).Recent data show that PS mutations also affect APP processing causing overproduction of the amyloidogenic and toxic Ab42 peptide (Selkoe & Podlisny, 2002).However, the exact role of presenilins in the cleavage of APP is unclear, although multiple lines of evidence suggest that these proteins are essential for g-secretase activity (Kowalska & Wender, 1998;Kowalska, 2003).Presenilins probably constitute the active site of a large protein complex which is responsible for the g-secretase processing of the APP protein.PS mutations may disturb protein interactions in the complex through subtle conformational alterations (Esler & Wolfe, 2001).
To contribute to our knowledge of the genetic background of Alzheimer's disease in Poland, we performed a mutation analysis of the APP, PS1 and PS2 genes in patients with Alzheimer's disease from families in which dementia was transmitted as a genetic autosomal dominant trait.

MATERIAL AND METHODS
A sample of twenty patients with AD including 6 patients with EOAD (age of onset below 65 years), 6 patients with LOAD (late onset AD at or over 65 years) from families with autosomal dominant mode of dementia inheritance (at least three patients with dementia in at least two generations), and 8 patients with familial EOAD (at least two demented persons in patient's family) was screened for APP and PS mutations.The ages of the patients ranged from 30 to 94 years.The diagnosis, based on NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association) work group guidelines (McKhann et al., 1984), was made by clinical evaluations in the majority of cases including a CT scan and exclusion of other causes of dementia.We also examined 48 healthy control subjects from Poznañ region.The study was approved by the Medical Ethical Committee of the University of Medical Sciences in Poznań.
Blood samples were collected and genomic DNA was extracted with the QIAmp Blood Kit (Qiagen).Screening for mutations was carried out by the PCR-SSCP approach (Kowalska et al., 1997).Ten coding exons 3-12 of the PS1 and PS2 genes were amplified separately using primers designed to the flanking intronic sequences according to Kamimura et al. (1998).PCR conditions were 94°C for 10 min; 30 cycles of 94°C for 1 min, 54-69°C for 1 min, and 72°C for 2 min; and a final 10-min extension at 72°C.The reaction volume was 50 ml containing 500 ng of human DNA template, 50 pM primers, 200 mM dNTPs, 1.5 mM MgCl 2 , 10 mM Tris/HCl pH 8.2, 50 mM KCl, and 1-2 units of Taq poly-merase (TaKaRa).The PCR of exons 16 and 17 of the APP gene, encoding the b-amyloid fragment, was performed as described by Tanzi et al. (1992).A 5 ml aliquot of each PCR product was checked on 6% polyacrylamide gel.For SSCP, 2 ml a PCR product was applied to the GenePhor Electrophoresis system (Pharmacia).Gels were run for 100 min at 15 o C at running conditions: 600 V, 50 mA, 30 W. Bands were then visualized using the DNA silver staining kit (Pharmacia) in a Hoefer automated gel stainer.The exons presenting band shifts were subsequently subcloned into pAT vector (Invitrogen) and analyzed by DNA sequencing using the ABI PRISM Dye Terminator Cycle Sequencing Core Kit and the ABI 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.) according to the supplier's protocols.Apolipoprotein E genotyping was performed as described earlier (Wenham et al., 1991;Kowalska et al., 1998).

RESULTS
Screening for mutations in the APP, PS1, and PS2 genes was performed for 20 familial EOAD/LOAD cases.In addition, APOE genotyping was carried out.The results are summarized in Table 1.The following missense mutations in the PS1 gene: A246E in exon 7, P267L in exon 8, and L424R in exon 12 were found in patients from the families with autosomal dominant EOAD (ADEOAD) (Fig. 1).No mutations were found in 48 control individuals.The co-segregation of the mutations with AD was confirmed in the two ADEOAD pedigrees in which additional family members were available for genetic study (Fig. 1).Only one polymorphism, the A®C transversion, was found at position +16 in intron 8 of the PS1 gene, identical to that first reported by Wragg et al. (1996).No mutations were found in the PS2 gene.One polymorphism, the T®C transition, was observed in exon 4 at codon H87 of the PS2 gene.We did not detect any mutations in exons 16 and 17 of the APP gene.Therefore, we excluded both PS2 and APP mutations as the cause of AD in the analyzed cases.APOE genotyping revealed that patients carrying at least one APOE*4 allele constituted 40% (8 of 20) of the cohort.Among the 20 patients, four (20%) were homozygotes and another four heterozygotes for the APOE*4 allele.The two out of three patients with PS1 mutations were homozygotes for the APOE*3 allele (Table 1).

DISCUSSION
Most presenilin mutations reported so far were identified in or close to the highly conserved transmembrane (TM) regions of the proteins and in the large hydrophilic loop (HL VI) occurring after TM VI.There are two clusters of mutations in the PS1 gene.One of them is in the TM II domain encoded by exon 8 while the other extends from the TM VI domain (exons 7/8) through HL VI (exons 8/11) to the TM VII (exon 11).The mutations are predicted to interfere with the a-helical structure of TM II or the proteolytic processing of presenilins occurring in HL VI.The PS1 mutations found in this study were also located in functional domains of the protein: TM VI (A246E in exon 7), HL VI (P267L in exon 8), and TM VII (L424R in exon 12) (Fig. 2).The onset age in the PS1 mutation cases varied from 30 years (L424R) to 52 years (A246E) and 56 years (P267L).It was suggested that the age of AD onset could be determined by the nature of the mutation and its position in the gene (Cruts & Van Broeckhoven, 1998).Another possibility is that the onset age is modulated by additional genetic and/or environmental factors influencing expression of PS1 mutations, for example the APOE gene variability.However, there is no clear evidence for an effect of APOE genotype on the onset age in patients with PS1 mutations.Two out of three PS1 patients presented here had the APOE3/3 genotype, suggesting an absence of any correlation.The prevalence of Table 1.Analysis of PS1, PS2, APP and APOE genes in a cohort of familial EOAD/ LOAD cases ADEOAD, autosomal dominant early-onset Alzheimer's disease; ADLOAD, autosomal dominant late-onset Alzheimer's disease; FAD, familial Alzheimer's disease PS1 mutations among AD patients depends on criteria used to select the patients and varies from 18% (Cruts et al., 1998) to the over 50% (Campion et al., 1999) in presenile autosomal dominant Alzheimer's disease.Screening for mutations in a referral-based cohort of 414 patients with a high index of suspicion of familial AD revealed PS1 mutations in 11% of cases (Rogaeva et al., 2001).In the current study the frequency of PS1 mutations was 15% (3/20) in the whole sample of familial AD cases, or 50% (3/6) if the analysis was restricted to the EOAD families with a clear autosomal dominant mode of inheri- tance.The lack of APP and PS2 mutations in the analyzed subjects confirms their rare occurrence in patients with familial AD.The mutations are responsible for only a very small portion of familial AD.To gain better knowledge on the prevalence of the discussed mutations in the Polish population of AD patients, more extensive studies are required including a larger number of families with members from several generations.Besides, the vast majority of AD cases, over 90% of all patients, can be reffered to as sporadic AD with a negative family history of the disease and complex (multifactoral) inheritance.The genetic background of sporadic AD is still unknown and should be explained as soon as possible to allow the development of new genetic risk profiling strategies.

Figure 1 .
Figure 1.The PS1 gene mutations identified in three unrelated families with autosomal dominant early-onset Alzheimer's disease.

Figure 2 .
Figure 2. Putative structure of the Presenilin 1 gene product: a polytopic integral membrane protein with eight transmembrane domains.Black dots indicate positions of the three identified mutations.