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The presenilin complex is composed of four core proteins (presenilin 1 or presenilin 2, APH1, nicastrin, and PEN2). Several endogenous proteins have been reported to selectively modulate the function of the presenilin complexes; these include transmembrane trafficking protein, 21-KD (TMP21), CD147 antigen (basigin), the γ-secretase-activating protein (gSAP), and the orphan G-protein-coupled receptor 3. Because the structure and assembly of these complexes underlies their activity, this review will discuss current work on the assembly of the complex and on presenilin-interacting proteins that regulate secretase activity.

Familial Alzheimer disease is a neurological disorder of adult onset. Three research centers have each contributed their families and genetic linkage data for combined analyses. The data from the Duke and Boston centers, comprising 73 pedigrees for whom numerous markers on chromosomes 19 and 21 were typed, are described

Familial Alzheimer's disease (FAD) has been shown to be genetically heterogeneous, with a very small proportion of early onset pedigrees being associated with mutations in the amyloid precursor protein (APP) gene on chromosome 21, and some late onset pedigrees showing associations with markers on chromosome 19. We now provide evidence for a major early onset FAD locus on the long arm of chromosome 14 near the markers D14S43 and D14S53 (multipoint lod score z = 23.4) and suggest that the inheritance of FAD may be more complex than had initially been suspected.

Probable Alzheimer disease (AD) is described in 79-year-old male twins with monozygosity confirmed by DNA examination. The first twin to be affected began to show signs of intellectual deterioration at age 70. In the other, onset was at age 72. Four of their living sibs (current age range = 75–92) are also suspected to have AD. The possible roles of genetic and environmental factors in the development of AD in this sibship are discussed. © 1992 Wiley-Liss, Inc.

Background: Frontotemporal dementia (FTD) in several 17q21-linked families was recently explained by truncating mutations in the progranulin gene ( GRN ). Objective: To determine the frequency of GRN mutations in a cohort of Caucasian patients with FTD without mutations in known FTD genes. Methods: GRN was sequenced in a series of 78 independent FTD patients including 23 familial subjects. A different Calabrian dataset (109 normal control subjects and 96 FTD patients) was used to establish the frequency of the GRN mutation. Results: A novel truncating GRN mutation (c.1145insA) was detected in a proband of an extended consanguineous Calabrian kindred. Segregation analysis of 70 family members revealed 19 heterozygous mutation carriers including 9 patients affected by FTD. The absence of homozygous carriers in a highly consanguineous kindred may indicate that the loss of both GRN alleles might lead to embryonic lethality. An extremely variable age at onset in the mutation carriers (more than five decades apart) is not explained by APOE genotypes or the H1/H2 MAPT haplotypes. Intriguingly, the mutation was excluded in four FTD patients belonging to branches with an autosomal dominant mode of inheritance of FTD, suggesting that another novel FTD gene accounts for the disease in the phenocopies. It is difficult to clinically distinguish phenocopies from GRN mutation carriers, except that language in mutation carriers was more severely compromised. Conclusion: The current results imply further genetic heterogeneity of frontotemporal dementia, as we detected only one GRN -linked family (about 1%). The value of discovering large kindred includes the possibility of a longitudinal study of GRN mutation carriers.

The metabolic pathways governing the turnover of presenilin 1 (PS1) have been incompletely worked out. The PS1 holoprotein has low abundance in many cells and appears to undergo endoproteolytic cleavage near residue 298. We provide evidence that one mechanism by which the PS1 holoprotein is degraded is through the action of the 26S proteasome. We also show that the proteasome does not participate in the endoproteolytic cleavage.

Some patients with familial Alzheimer's disease (FAD) have mutations in the presenilin-1 (PS-1) gene on chromosome 14. We report a Japanese family with AD and an Ala285Val substitution in exon 8 of the PS-1 gene. FAD in this family was characterized by relatively late onset (mean age, 50 years) and absence of myoclonus, seizures, or paratonia. Levels of tau were markedly elevated in CSF whereas CSF levels of amyloid beta protein were normal. MRI of the cranium showed marked linear signal abnormalities within white matter in the parieto-occipital lobes, consistent with cortical amyloid angiopathy of the type encountered in patients with the PS-1 gene mutation.

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts.

The possibility of an interaction of multiple genes has been speculated in pathogenesis of Alzheimer's disease (AD). Because we have recently cloned a novel gene S182 bearing five different missense mutations which segregate with early-onset familial AD, we sought the frequency of these mutations in familial and sporadic late-onset AD to clarify the incidence of these mutations in the disease. The current study showed lack of these mutations in 118 independent subjects affected with late-onset Alzheimer's disease.

The progressive cognitive dysfunction of Alzheimer’s disease (AD) is accompanied by a series of neuropathological changes, including deposition of amyloid β peptide in the parenchyma and blood vessels (Aβ) of the human brain. Many lines of evidence indicate that abnormal processing of Aβ (a proteolytic derivative of the β-amyloid precursor protein — βAPP) plays a central role in initiating the pathogenesis of AD (reviewed in Steineret al. 1999). There are also data indicating a positive correlation between β-amyloid plaques and cognitive impairment in AD patients (Cummings et al.1996; Haroutunian et al. 1998; Kanne et al.1998). However, there are gaps in our knowledge of the pathogenesis of even the “simple” genetic forms of AD. Thus there has been a need for an animal model that develops some or all aspects of this uniquely human disease in a reproducible fashion to decipher and stratify crucial pathogenic events. Such animal models would also, of course, be useful for testing therapies.

Nicastrin (Yu et al. 2000) and the presenilins (Rogaev et al. 1995; Sherrington et al. 1995) form high molecular weight complexes that are involved in the cleavage of the β-amyloid precursor protein (βAPP; Citron et al. 1997; De Strooper et al. 1998; Wolfe et al. 1999; Li et al. 2000; Yu et al. 2000) and Notch (Levitan and Greenwald 1995; De Strooper et al. 1999; Struhl and Greenwald 1999; Ye et al. 1999) within their transmembrane domains. The former process (termed γ-secretase cleavage) generates amyloid β-peptide (Aβ), which is involved in the pathogenesis of Alzheimer Disease (AD). The latter (termed S3-site cleavage) generates Notch intracellular domain NICD), which is involved in intercellular signaling during dorsal axis development in embryogenesis and during intercellular signaling in some post-natal tissues.

Background: Mutations in the presenilin-1 gene ( PS1 ) account for a majority of patients with early-onset familial AD. However, the clinical indications and algorithms for genetic testing in dementia are still evolving. Methods: The entire open reading frame of the PS1 gene was sequenced in a series of 414 consecutive patients referred for diagnostic testing, including 372 patients with AD and 42 asymptomatic persons with a strong family history of AD. Results: Forty-eight independent patients screened had a PS1 mutation including 21 novel mutations. In addition, 3% of subjects (11/413) had a known polymorphism, the Glu318Gly substitution. The majority of the mutations were missense substitutions but there were three insertions and Δexon 10 mutation. With six exceptions (codons 35, 178, 352, 354, 358, and 365) most of the mutations occurred at residues conserved in the homologous PS2 gene or in PS1 of other species. Conclusions: Eleven percent of a referral-based series of patients with AD can be explained by coding sequence mutations in the PS1 gene. The high frequency of PS1 mutations in this study indicates that screening for PS1 mutations in AD is likely to be successful, especially when directed at patients with a positive family history with onset before 60 years (90% of those with PS1 mutations were affected by age 60 years). This will also have significance for the secondary identification of at-risk relatives who might be candidates for future prophylactic therapies for AD.

Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments (NTF/CTF). Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of APP and Notch. We show that aspartate-mutant holoprotein presenilins are not incorporated into the high molecular weight, NTF/CTF-containing complexes. Aspartate-mutant presenilin holoproteins also preclude entry of endogenous wild-type PS1/PS2 into the high molecular weight complexes, but do not affect the incorporation of wild-type holoproteins into lower molecular weight holoprotein complexes. These data suggest that the loss-of-function aspartate-mutants cause altered PS complex maturation, and argue that the functional presenilin moieties are contained in the high molecular weight presenilin NTF/CTF-containing complexes.

At least four different genes have been identified that are associated with inherited susceptibiltiy to Alzheimer's disease. Some of the genes are highly penetrant (PS1, PS2, beta APP); the other, APOE, is a weaker susceptibility factor. Several additional genes are suspected to exist but have not yet been cloned.

The presenilin proteins are involved in the proteolytic processing of transmembrane proteins such as Notch/lin-12 and the beta-amyloid precursor protein (betaAPP). Mutation of a conserved cysteine (Cys60Ser) in the C. elegans presenilin sel-12 has a loss-of-function effect on Notch/lin-12 processing similar to that of null mutations in sel-12. In contrast, in mammalian cells, most missense mutations increase gamma-secretase cleavage of betaAPP. We report here that mutation of this conserved cysteine (Cys92Ser) in human presenilin 1 confers a loss-of-function effect in C. elegans, but causes increased A beta42 secretion in mammalian cells. These data suggest that the role of presenilins in Notch/lin-12 signalling and betaAPP processing are either separately regulated activities or independent activities of the presenilins.

Two children of an adult with early-onset, autopsy-confirmed Alzheimer disease (AD) developed dementia in their late 20s and were subsequently found to have novel mutations in codon 434 of the presenilin 1 (PS1) gene on chromosome 14, a G-to-T substitution at nucleotide 1548 and a C-to-G substitution at nucleotide 1549. The younger of the 2 children had AD confirmed at postmortem examination. The disease course in these 3 individuals was characterized by cognitive and behavioral problems accompanied by myoclonus, seizures, and aphasia within 5 years after onset. Two grandparents had clinically diagnosed AD with stroke beginning at ages 78 and 66 years, but neither had a PS1 mutation. No other living family member was demented, nor did any other family member have the PS1 mutation. We conclude that the affected parent of the proband was a likely recent founder for these novel mutations in PS1. The family demonstrates the clinical and genetic heterogeneity of AD. Arch Neurol. 2000;57:1454-1457

Alzheimer's disease (AD) is caused by multiple genetic and/or environmental etiologies. Because differences in the genetically determined pathogenesis may cause differences in the phenotype, we examined age at onset and age at death in 90 subjects with dominantly inherited AD due to different mutations (amyloid precursor protein, presenilin-1, and presenilin-2 genes). We found that among patients with dominantly inherited AD, genetic factors influence both age at onset and age at death.

Background: Mutations in the presenilin (PSEN) genes are responsible for the majority of early-onset Alzheimer disease (AD) cases. PSEN1 is a component of a high molecular weight, endoplasmic reticulum, membrane-bound protein complex, including beta-catenin. Pathogenic PSEN1 mutations were demonstrated to have an effect on beta-catenin and glycogen synthase kinase-3 beta(GSK-3 beta), two members of the wingless Wnt pathway. The nuclear translocation and the stability of beta-catenin, and the interaction between GSK3 beta and PSEN1 were influenced. Materials and Methods: Stably transfected human embryonic kidney (HEK) 293 cells overexpressing wild-type (wt) and mutant (mt) PSEN1, treated with and without LiCl, were used to isolate cytoplasmic and nuclear fractions. By Western blot analysis, endogenous beta-catenin levels were examined. By analyzing cytosolic fractions of PSEN1, transfected and nontransfected HEK 293 cells, and total brain extracts of AD patients and controls, we evaluated the effect of PSEN1 overexpression on beta-catenin stability. Finally, we analyzed the effect of pathogenic PSEN1 mutations on the interaction between PSEN1 and GSK3 beta by co-immunoprecipitation experiments. Results: We report reduced nuclear translocation of beta-catenin in cells stably expressing 1143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutation showed a similar pronounced effect on nuclear translocation of beta-catenin, as reported for processing of amyloid precursor protein (APP) into amyloid beta(A beta) Overexpression of PSEN1 and the presence of pathogenic mutations in PSEN1 had no significant effect on the stability of beta-catenin. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3 beta was observed when GSK3 beta was immunoprecipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3 beta. Conclusion: Our results further establish the nuclear translocation assay of beta-catenin as an adequate alternative for traditional A beta measurement to evaluate the effect of PSEN1 mutations on biochemical processes. We detected no significant effect of overexpressed wt or mt PSEN1 on the stabilty of beta-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3 beta was not observed in our experimental setup.

Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with familial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS1 as well as human wild-type (wtPS1) and the familial AD Met146Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PS1 antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PS1; (ii) significantly reduced levels of full-length human PS1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organelle-specific proteins revealed that PS1 was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PS1 showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PS1 distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to transfection systems. Our data also suggest that the molecular pathology associated with PS1 mutations results from subtle alterations in presenilin function, which can be further investigated using these transgenic neuronal cell culture models.

We previously described a splice donor site mutation in intron 4 of presenilin-1 (PSEN1) in two patients with autopsy-confirmed early-onset Alzheimer's disease (AD). Here we provide evidence that the intron 4 mutation is present in four additional unrelated early-onset AD cases, that the mutation segregates in an autosomal dominant manner and that all cases have one common ancestor. We demonstrate that the intron 4 mutation produces three different transcripts, two deletion transcripts (Delta4 and Delta4cryptic) and one insertion transcript (insTAC), by aberrant splicing. The deletion transcripts result in the formation of C-truncated (approximately 7 kDa) PSEN1 proteins while the insertion transcript produces a full-length PSEN1 with one extra amino acid (Thr) inserted between codons 113 and 114 (PSEN1 T113-114ins). The truncated proteins were not detectable in vivo in brain homogenates or lymphoblast lysates of mutation carriers. In vitro HEK-293 cells overexpressing Delta4, Delta4cryptic or insTACPSEN1 cDNAs showed increased Abeta42 secretion (approximately 3.4 times) only for the insertion cDNA construct. Increased Abeta42 production was also observed in brain homogenates. Our data indicate that in the case of intron 4 mutation, the AD pathophysiology results from the presence of the PSEN1 T113-114ins protein comparable with cases carrying dominant PSEN1 missense mutations.

Mutations in human CuZn superoxide dismutase (SOD) have been associated with familial amyotrophic lateral sclerosis (FALS). Although leading to many experimental advances, this finding has not yet led to a clear understanding of the biochemical mechanism by which mutations in SOD promote the degeneration of motorneurons that causes this incurable paralytic disease. To explore the biochemical mechanism of FALS SOD-mediated neuropathogenesis, we used transgenic methodology to target the expression of a human FALS SOD to motorneurons of Drosophila, an organism known for its phenotypic sensitivity to genetic manipulation of SOD. Earlier, we showed that targeted expression of human SOD in motorneurons of Drosophila causes a dramatic extension of adult lifespan (40%) and rescues most of the phenotypes of SOD-null mutants. Using the same genetic system, we now ask if targeted expression of a mutant allele of human SOD that is associated with FALS causes paralysis and premature death, or is otherwise injurious in Drosophila as it is in humans and transgenic mice. Here we report that high-level expression of a human FALS SOD in motorneurons is not detrimental and does not promote paralysis and premature death when expressed in motorneurons of Drosophila. In sharp contrast, the expression of FALS SOD in Drosophila actually extends lifespan, augments resistance to oxidative stress and partially rescues SOD-null mutants in a manner predicted by our earlier studies on the expression of wildtype human SOD in Drosophila motorneurons.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly and an increasingly significant health concern in our aging population. In the past 10 years, our understanding of this disease has increased dramatically. While the discovery of three rare genetic mutations that can cause AD has provided much information about the causes and progression of the disease, a great deal of attention has been focused on apolipoprotein (ApoE) because of its involvement in the more common, later onset form of AD. Due to the rapid pace of recent advances, it has not been easy for health care professionals, researchers and the general public to keep abreast of these developments. This paper reviews recent research in ApoE and late-onset AD, emphasizing molecular neuropathological, genetic and neuroimaging findings and highlighting current controversies that remain to be addressed.

The authors examined the association of Apolipoprotein-E (APO-E) genotype to symptoms of psychosis and depression in 501 patients diagnosed with probable (n=343) or possible (n=158) Alzheimer's disease (AD) according to NINCDS-ADRDA criteria. They observed the following APO-E genotypes: epsilon2/epsilon3 (n=19); epsilon2/epsilon4 (n=14); epsilon3/epsilon3 (n=228); epsilon3/epsilon4 (n=203); epsilon4/epsilon4 (n=37). In contrast to previous reports, the results did not indicate a relationship between either the epsilon4 allele or the epsilon2 allele and symptoms of mood disturbance in AD. However, an elevated risk for psychosis was shown, specifically, at the severe stage of cognitive impairment, among AD patients carrying the epsilon4 allele, after effects of age, gender, education, and level of cognitive impairment were controlled.

The prevalence of AD appears to vary widely in different ethnic groups. Certain risk factors for AD are well established for the general population, but there is little information regarding the relevance of these risk factors in specific ethnic groups.The authors examined the risk of AD associated with the APOE-epsilon4 allele, the APOE-epsilon2 allele, smoking, alcohol consumption, history of hypertension, low educational level, estrogen replacement therapy, and history of head trauma with loss of consciousness among samples of white non-Hispanics (WNH) (392 AD patients, 202 normal subjects) and white Hispanics (WHIS) (188 AD patients, 84 normal controls).This was a case-control study of patients evaluated at an outpatient memory disorders clinic and control subjects recruited from a free memory screening offered to the community.Increased risk for AD was associated with the APOE-epsilon4 allele after controlling for age, education, and gender among WNH (OR = 3.5; 95% CI = 2.3 to 5.5) and WHIS (OR = 3.1; 95% CI = 1.7 to 5.8). No protective effect was conferred by the APOE-epsilon2 allele, although this relationship approached significance among WNH (p = 0.02). Low levels of education increased the risk for AD among WNH (OR = 3.1; 95% CI = 1.8 to 5.9) but not WHIS. Alcohol use and hypertension approached significance as risk factors in WNH (p0.05) but not WHIS. Estrogen replacement treatment approached significance as a protective factor in both ethnic groups (p0.05).Although the APOE-epsilon4 allele is a risk factor for AD among WHIS and WNH, other risk factors such as low education and hypertension appear to be important only for WNH. Risk factors for AD reported or suggested previously that were not confirmed by this study include smoking and head trauma with loss of consciousness.

The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. We have used linear glycerol velocity and discontinuous sucrose gradient analysis to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF.

The K-variant of butyrylcholinesterase (BCHE-K) recently has been reported to be associated with Alzheimer disease (AD) in carriers of the epsilon4 allele of the apolipoprotein E (APOE) gene. We have re-examined the frequency of the BCHE-K allele in a large data set of both sporadic and familial cases of AD disease, and we have also examined the segregation of three genetic markers on chromosome 3 near BCHE . Our data neither support an association of BCHE-K with sporadic or familial AD, nor do they suggest the existence of another gene nearby on chromosome 3 as a common cause of familial AD.

We evaluated the influence of gender on penetrance of GTP-cyclohydrolase I (GCH) gene mutations in hereditary progressive dystonia/dopa-responsive dystonia (HPD/DRD) and determined whether some apparently sporadic HPD/DRD patients owe their disorder to a de novo mutation of the GCH gene. Previous clinical investigations of HPD/DRD have shown a predominance of affected women, with approximately half of HPD/DRD patients being sporadic. We conducted genomic DNA sequencing of the GCH gene in five HPD/DRD families having at least two generations of affected members and in four apparently sporadic cases and all of their parents. In the nine HPD/DRD pedigrees, we found independent mutations of the GCH gene (five deletions, one insertion, one nonsense mutation, and two point mutations at splice acceptor sites). The female-to-male ratio of the HPD/DRD patients was 4.3 with the penetrance of GCH gene mutations in women being 2.3 times higher than that in men (87% versus 38%, p = 0.026). There was no significant difference in the penetrance between maternally and paternally transmitted offspring. All of the four sporadic cases had de novo mutations because none of their parents were carriers. The results demonstrate gender-related incomplete penetrance of GCH gene mutations in HPD/DRD and suggest that this may not be due to genomic imprinting. Our data also suggest a relatively high spontaneous mutation rate of the GCH gene in this autosomal dominant disorder.

ALTHOUGH the clinical diagnosis of Alzheimer's disease (AD) in competent hands is quite accurate (approximately 85%) (15), the discovery that mutations in the β-amyloid precursor protein (βAPP)(16), presenilin I (PS1) (42), and presenilin 2 (PS2)(26,36) genes, and that polymorphisms in apolipoprotein E (APOE) gene (40) are associated with inherited susceptibility to AD has raised the possibility that these genes may be used in the diagnosis of patients with AD. This paper examines the conceptual arguments and limitations for use of genetic tests in AD. with emphasis on the use of PS1, PS2. and βAPP gene sequences in early-onset AD. Genetic testing for susceptibility to inherited disease, and in particular genetic testing for susceptibility to diseases that induce cognitive impairment, carry significant ethical and moral obligations on the part of those administering these tests. The ethical issues surrounding genetic testing in AD are described in depth elsewhere (1,34).

Molecular genetic studies in pedigrees with a reasonably unambiguous single gene autosomal dominant pattern of inheritance have led to the identification of the four different genes associated with inherited susceptibility to Alzheimer’s disease (AD) — namely β-amyloid precursor protein (βAPP; Goate etal. 1991; Murrell etal. 1991; Naruse etal. 1991; Hendricks etal. 1992; Karlinsky etal. 1992; Mullan et al. 1992), apolipoprotein E (APOE; Saunders et al. 1993), presenilin 1 (PS1; Sherrington etal. 1995), and presenilin 2 (PS2; Rogaev etal. 1995). There is also genetic evidence that some of these genes may function within the same biochemical pathway and have additive effects. Thus, carriers of (βAPPval717Ile who have one or more e4 alleles at APOE have an earlier onset than relatives with the same βAPP mutation and the e2 allele at APOE (St. George-Hyslop etal. 1994; Nacmias etal. 1995; Sorbi etal. 1995). However, because a significant number of pedigrees multiply affected by AD have been found which do not segregate mutations/polymorphisms in any of the four known AD susceptibility genes, it is suspected that at least one and possible several additional AD susceptibility genes must also exist but have not yet been identified (Sherrington et al. 1996).

In contrast to rare mutations in the amyloid presursor protein (APP) gene, missense mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes, on chromosomes 14 and 1 respectively, are the most common causes of early-onset familial Alzheimer’s disease (AD)(Sherrington et al. 1995)(Rogaev et al. 1995)(Levy-Lahad et al. 1995). Presenilin genes encode proteins with at least seven putative transmembrane domains and an extruded cytoplasmic “loop”, the latter with a preponderance of acidic amino acid residues: these proteins are expressed in a variety of cell types. While the physiologic function of these genes is unknown, their protein products have been demonstrated to accumulate in intracellular sites including the endoplasmic reticulum, and the Golgi apparatus. Similarly, the mechanism by which the 30 different point mutations have been identified in PS1 and PS2 to date cause the clinical and neuropathological hallmarks of Alzheimer disease is unknown. However, fibroblasts from heterozygous carriers of PS1 and PS2 mutations secrete increased levels of the amyloidogenic long-tailed amyloid β-peptides ending at residues 42 or 43 (Aβ42)(Martin 1995)(Scheuner et al. 1996). Increased levels of Aβ42 and other Aβ-peptides can also be measured in postmortem brain tissue from human patients dying with early-onset FAD associated with PS1 mutations(Lemere et al. 1996). To determine whether overproduction of Aβ peptides occurs in brain as an early biochemical event prior to the onset of neurodegeneration, we constructed transgenic mice with either mutant or wild-type human PS1 and mated them with another line of transgenic mice overexpressing wild-type human βAPP695 under the control of the same transcriptional regulatory element. These studies reveal that mutant PS1 transgenes but not wild-type PS1 transgenes act in a dominant fashion to programme over-production of long-tailed Aβ42 peptides in brain, and that this biochemical difference is present by at least 2–4 months of age and in the absence of any detectable neuropathologic lesions. These advances in our understanding of presenilin function are discussed in relation to the two schools of thought on AD pathogenesis, “tau-ist”and “saptist”, and also with regards the hypothesis that similarities between AD and prion diseases reflect the existence of shared pathogenic pathways.

Objective. —Primary caregivers should be aware of recent progress in the genetics of Alzheimer disease (AD) and of the clinical and ethical considerations raised regarding the introduction of genetic testing for purposes of disease prediction and susceptibility (risk) analysis in asymptomatic individuals and diagnosis in patients who present clinically with dementia. This statement addresses arguments for and against clinical genetic testing. Participants. —The 20 participants were selected by the investigators (S.G.P., T.H.M., A.B.Z., and P.J.W.) to achieve balance in the areas of genetics, counseling, ethics, and public policy, and to include leadership from related consensus projects. The consensus group met twice in closed meetings and carried on extensive correspondence over 2 years (1995-1997). The project was supported by the National Human Genome Research Institute of the National Institutes of Health. Evidence. —All 4 involved chromosomes were discussed in group meetings against a background of information from several focus group sessions with AD-affected families. The focus groups comprised volunteers identified by the Cleveland Area Chapter of the Alzheimer's Disease and Related Disorders Association and represented a variety of ethnic populations. Consensus Process. —The first draft was written in April 1996 by the principal investigator (S.G.P.) after the consensus group had met twice. The draft was mailed to all consensus group members 3 times over 6 months for extensive response and redrafting by the principal investigator until all members were satisfied. Conclusions. —Except for autosomal dominant early-onset families, genetic testing in asymptomatic individuals is unwarranted. Use ofAPOEgenetic testing as a diagnostic adjunct in patients already presenting with dementia may prove useful but it remains under investigation. The premature introduction of genetic testing and possible adverse consequences are to be avoided.

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.

Missense mutations in the presenilin 2 (PS-2) gene on chromosome 1 were sought by direct nucleotide sequence analysis of the open reading frame of 60 pedigrees with familial Alzheimer's disease (FAD). In the majority of these pedigrees, PS-1 and beta-amyloid precursor protein (beta APP) gene mutations had been excluded. While no additional PS-2 pathogenic mutations were detected, four silent nucleotide substitutions and alternative splicing of nucleotides 1338-1340 (Glu325) were observed. Analysis of additional members of a pedigree known to segregate a Met239Val mutation in PS-2 revealed that the age of onset of symptoms is highly variable (range 45-88 years). This variability is not attributable to differences in ApoE genotypes. These results suggest (i) that, in contrast to mutations in PS-1, mutations in PS-2 are a relatively rare cause of FAD; (ii) that other genetic or environmental factor modify the AD phenotype associated with PS-2 mutations; and (iii) that still other FAD susceptibility genes remain to be identified.

The subtype IV of Machado-Joseph disease (MJD), characterized by parkinsonism variably combined with ataxia, distal atrophy, and sensory loss, has been all but ignored in recent reports of MJD, including those describing the molecular biologic substrate of the disease. We have demonstrated expansion of the CAG trinucleotide repeat of the MJD1 gene located on chromosome 14q32.1 in 2 patients of Azorean descent who presented with levodopa-responsive atypical parkinsonism. Previous publications have documented the presence of this expanded repeat in the other more common MJD phenotypes (I-III). To our knowledge, this is the first molecular biologic confirmation of the presence of the MJD1 gene in the subtype IV phenotype. Patients presenting with parkinsonism and peripheral neuropathy should be screened for this genetic defect.

Allele frequencies for polymorphisms in the apolipoprotein E and the apolipoprotein CII genes were determined in subjects of Ashkenazi Jewish origin with late-onset Alzheimer's disease and in unaffected control subjects from the same ethnic group. A significant association was observed between late-onset Alzheimer's disease and the epsilon 4 (112Cys-->Arg) allele of apolipoprotein E; however, no association was detected with apolipoprotein CII. These results suggest that the association with epsilon 4 is probably not due to linkage disequilibrium.

A locus for Machado-Joseph disease (MJD) has recently been mapped to a 30-cM region of chromosome 14q in five pedigrees of Japanese descent. MJD is a clinically pleomorphic neurodegenerative disease that was originally described in subjects of Azorean descent. In light of the nonallelic heterogeneity in other inherited spinocere-bellar ataxias, we were interested to determine if the MJD phenotype in Japanese and Azorean pedigrees arose from mutations at the same locus. We provide evidence that MJD in five pedigrees of Azorean descent is also linked to chromosome 14q in an 18-cM region between the markers D14S67 and AACT (multipoint lod score +7.00 near D14S81). We also report molecular evidence for homozy-gosity at the MJD locus in an MJD-affected subject with severe, early-onset symptoms. These observations confirm the initial report of linkage of MJD to chromosome 14; suggest that MJD in Japanese and Azorean subjects may represent allelic or identical mutations at the same locus; and provide one possible explanation (MJD gene dosage) for the observed phenotypic heterogeneity in this disease.

Mapping of the genetic defect causing dominant palmoplantaris hyperkeratosis (PPHK) was continued based on the material of an extended Uzbek pedigree. No linkage between the PPHK gene and hypervariable DNA markers from 8p, 12p, 14q, and 22q were revealed. The study of PPHK gene linkage with DNA markers covering the entire length of 17th chromosome mapped the PPHK gene to 17q12-q24 and revealed close linkage with KRT10 and D17S800 loci (zero recombination frequency at a lod score7). The possible location of a PPHK mutation in one of the keratin genes mapped to the same region on the 17th chromosome is discussed.

In eukaryotic nuclei, the expression of genes is a complex series of events in which information encoded in the DNA molecule is transcribed into ribonucleic acid (RNA) and translated into proteins essential for normal cellular function. The first step in the gene expression pathway appears to be an initial “loosening up” of local chromatin structure within the nucleus to create a chromatin configuration that enables specific DNA sequences to be recognized by basal transcription factors and RNA polymerase systems.

Mutations in the Cu, Zn superoxide dismutase ( SOD1 ) gene have been reported in some pedigrees with Familial Amyotrophic Lateral Sclerosis (FALS). We have investigated the functional and structural effects of a Gly→Ser mutation at codon 41 of SOD1 in a pedigree with FALS and the topography of SOD 1 expression in the mammalian CNS. These analyses show that the 41 Gly→Ser mutation causes a 27% reduction in Cu, Zn SOD activity. SOD1 is transcribed at high levels in rat motoneurons and four other types of neurons homologous to upper motoneurons that degenerate in human ALS. However, SOD1 is transcribed at lower levels in other types of neurons, such as cerebellar Purkinje cells, which are not usually involved significantly in human ALS. On the other hand, immunocytochemical studies indicate that most types of rat neurons contain similar levels of Cu, Zn SOD immunoreactive protein. Nevertheless, these results suggest that the essential feature causing this subtype of ALS is either a reduction in Cu, Zn SOD activity in cell types that presumably critically require Cu, Zn SOD for protection against oxidative damage or the fact that the mutation in SOD1 associated with FALS results in a novel gain of function that is particularly deleterious to those cell types expressing SOD1 at high levels.

Alzheimer’s diseases (AD) is a major health problem which will continue to intensify in magnitude as the elderly in the population continue to increase in number. The age at which AD strikes is variable, ranging from the fourth to tenth decades, with the greatest proportion of cases occurring in the seventh and eighth decades. A genetic component of this disorder has been strongly indicated by family and survey studies, as well as life table analyses (reviewed in St George-Hyslop et al. 1989). Genetic linkage and association studies of kindreds displaying evidence for familial AD (FAD) have led to the localization of gene defects responsible for this genetically heterogeneous disorder on chromosomes 14, 19 and 21. In a small set of FAD kindreds, mutations have been found in the amyloid beta protein precursor (APP) gene. Yet, the available data indicate that the identity of the genes responsible for the majority of late-onset (> 65 years) as well as early-onset inherited AD remain unknown. Powerful and novel advances in the methodology available for performing genetic linkage analyses on genetically complex disorders have made it feasible to scan the entire human genome in a relatively fast and easy manner for the purpose of localizing the genes responsible for, or predisposing to, inherited AD. Here we describe progress on attempts to further localize and identify various FAD gene defects throughout the genome, with special emphasis on the major early-onset gene defect residing on the long arm of chromosome 14.

Wilson disease (WD), an autosomal recessive disorder of copper metabolism, has been previously mapped to chromosome 13q. Highly informative PCR-based polymorphic microsatellites closely linked to the WD locus (WND) at 13q14.3, as well as sequence-tagged sites for closely linked loci, are described. Two polymorphic microsatellite markers at D13S118 and D13S119 lie within 3 cM of WND. Two others (D13S227 and D13S228) were derived from a yeast artificial chromosome containing D13S31. These were placed on a genetic linkage map of chromosome 13 and were typed in 74 multiplex WD families from a variety of geographic origins (166 affected members). Multipoint analysis provides very high odds that the location of WND is between D13S31/D13S227/D13S228 and D13S59. Previous odds with RFLP-based markers were only 7:1 more likely than any other location. Current odds are 5,000:1. Preclinical testing of three cases of WD by using the highly informative polymorphic microsatellite markers is described. The markers described here ensure that 95% of predictive tests using DNA from both parents and from at least one affected sib will have an accuracy >99%.