Pathogenic mutations in APP
have previously been linked to familial early onset forms of dementia. A recent report looking at common variants in APP
in a large case-control sample consisting of 3,940 cases and 13,373 controls, found that common variants in these genes are unlikely to make strong contributions to susceptibility for LOAD 
. However, the impact of rare variants in these genes in late onset clinically diagnosed AD remains unclear. This is the first study to systematically screen for rare variants and pathogenic mutations in APP
in a large clinical series of well-characterized families densely affected by LOAD.
We found seven families carrying two known causative mutations in PSEN1
(A79V and G206A 
), and three families carrying two clearly causative mutations in GRN
(R110X, R493X 
. In some families these causative mutations (PSEN1
-A79V, and GRN
-R493X, ) did not completely segregate with disease, illustrating how phenocopies, potential presymptomatic individuals and reduced penetrance may complicate the interpretation of novel sequence variants in familial and sporadic LOAD. While we can identify the phenocopies in families with known mutations, as in the case of PSEN1
A79V and GRN
R110X (), it is more challenging in families with novel variants, because phenocopies might be interpreted as a failure of the variant to segregate with disease. Putative pathogenic variants in genes that cause late onset rather than early onset dementia could have a less severe effect on protein function due to other genetic or environmental modifiers that are not present when these same variants result in early onset disease. This would lead to the occurrence of reduced penetrance. Indeed, several cases of reduced penetrance have been reported in families with PSEN1
. Despite these problems in our segregation data, the absence of these variants in a large non-demented control series and the bioinformatics analyses suggest that the novel variants APP
c.1414-1G>T and MAPT
G201S are likely to be causative mutations or highly penetrant rare disease alleles.
In total 14 (3.2%) of the 439 sequenced samples from densely affected families carried a causative or likely-causative mutation. Although this percentage is low, the overall number of LOAD cases carrying mutations in these genes is likely to be higher than the number of early onset cases, because LOAD is much more frequent. Approximately 10–20% of all patients with LOAD have a family history of dementia. Of those, approximately 10% have a history of four or more affected family members. Based on our analysis, we estimate 3.2% of these densely affected families would have a causative mutation. Thus, 0.029–0.057% of all patients with LOAD may carry a pathogenic mutation in these genes, representing a minimum of 1,917–3,833 cases in the United States.
It is likely that we have underestimated the actual number of cases with causative mutations. First, the recurrence risk in family members peaks with an age-at-onset of 85 years in the proband 
. Several families with more than 10 affected individuals have no mutations in the genes studied here. Thus it is very likely that there are novel AD causative genes yet to be discovered. Second, some of the variants with unknown pathogenicity reported in this study, such as, APP G220E
and V340M, and MAPT
S427F, could be pathogenic, or risk factors. The scarcity of available DNA samples from other family members with LOAD made it difficult to determine whether or not these novel variants segregate with disease. However, we did not find these variants in additional controls and the bioinformatic analyses suggest that these variants may be pathogenic. To confirm this hypothesis more genetic and functional analyses will be necessary. Lastly, some of the variants we found in this study (APP
G201V and GRN
G515A) were also found in sporadic cases, but not in controls. These results indicate that novel mutations remain to be discovered and that mutations in these genes are also present in LOAD cases with no clear family history. Together these results suggest that any individual with a family history of dementia affecting a large number of relatives and with an onset in the mid-60s should be considered for mutation screening not only of the AD genes (APP
and 2), but also MAPT
. Our study also indicates that mutations in these genes can be present in 1 to 3% of the sporadic cases. These cases may be classified as sporadic because the family size is small, because of the lack of medical records for other family members or because the variant has low penetrance.
Another important finding is the observation of clinically diagnosed AD families carrying previously reported and likely novel pathogenic mutations in GRN
. Mutations in these genes are typically associated with frontotemporal lobar degeneration 
, but have been previously reported in clinically diagnosed AD cases 
. Our results show that mutations in GRN
in a clinical series of LOAD families are as common as mutations in the AD genes (APP
). Six families (1.37%) with pathogenic or likely pathogenic mutations in GRN
were found vs. eight families (1.82%) with mutations in APP
. None of the individuals with MAPT
mutations had autopsy confirmation of the clinical diagnosis. It is most likely that these individuals have been misdiagnosed and that neuropathological diagnosis for these individuals will be FTD and not AD.
Our study indicates that families carrying mutations in GRN
can present with disease that is clinically indistinguishable from probable AD even in specialist memory disorder clinics, which is analogous to the observations that “AD mutations” can present with frontotemporal lobar degeneration 
. Identification of families and individuals carrying mutations in genes associated with frontotemporal lobar degeneration will be important for clinical management of these patients, particularly as therapies are developed that target the specific pathophysiologic processes of these disorders.
Some of the rare variants discovered in this study appear to increase the risk for AD or modify the age at onset. We found a very significant association of PSEN2
R62H and R71W with age at onset. It is very likely that some of the variants classified as non-pathogenic also increase risk for AD: When the individuals with variants classified as pathogenic are removed (n
14), and the frequency of rare non-synonymous variants is compared with 1,000 genome project data (p
2.18, 95%CI: 1.31–3.62), the GSK dataset (p
1.89, 95%CI: 1.19–3.00) or the Exome Variant Server (p
1.06–2.06), there is still a significant excess of rare variants in these genes in the LOAD cases. In the three datasets (GSK 1,000 genome project, and Exome Variant Server) a large proportion of samples are of non-European origin, therefore some of the variants found in these datasets may represent population specific alleles, decreasing the power of our analyses. If we focus on the specific variants found in this study and compare their frequency in elderly non-demented individuals, we found that these variants may have a big impact on AD risk. The eight variants classified as “unknown pathogenicity” have a combined OR
1.65–11.57), and the variants classified as “non-pathogenic” have a combined OR
1.30–2.65). The common variants identified in the last GWAS for AD had ORs ranging from 1.26 to 1.11, with a combined OR of 2.23. Therefore an individual carrying a single rare variant in one of these genes has a higher likelihood of developing AD than individuals varying all of the risk alleles for the novel genes found in the recent GWAS studies. This is the first large study looking at the effect of rare variants in candidate genes in AD. Additional large studies are needed to replicate these findings.
This work provides some general guidelines to identify individuals and families that should be prioritized for genetic counseling and mutation screening. Although we did not find clear phenotypic differences between the families carrying missense, splice-site or nonsense variants compared with the families without novel variants, the number of reported affected individuals and the presence of at least one affected individual with an early age at onset were strong indicators of the presence of a pathogenic variant. Given the significant overlap in clinical presentation, individuals with a strong family history of AD should also be screened for mutations in MAPT and GRN, genes typically associated with frontotemporal lobar degeneration, when no autopsy is available. In families with a history of four or more affected individuals and with an onset in the mid-60s, genetic testing may be considered for PSEN1-2, APP, GRN and MAPT. Families with multiplex late onset dementia should be referred to genetic counsellors.
Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. Dividing AD into late onset and early onset is probably not useful from a mechanistic point of view because mutations in APP, PSEN1 and PSEN2 can be found in early onset and late onset AD. Similarly, APOE4 increases risk for AD in both early and late onset AD. Clearly factors other than the mutation can impact the age at onset and penetrance of at least some variants causing AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. Finding a mutation in these genes would change the clinical diagnosis in a demented individual. This will be particularly important when mechanism-based therapies become available because this would change the treatment of these individuals. Lastly, familial aggregation is more important than age at onset in determining the likelihood of an individual carrying a disease-causing variant.