Screening Alzheimer's Patients for Mutations in
Exons 16 & 17 of the ß-Amyloid Precursor Protein
Gene
|
Scott Crick |
|
With humans having longer life spans, and the baby boomer generation just entering the geriatric stage, Alzheimer's disease(AD) is a serious problem in our and many other developed countries. AD is the leading cause of dementia and the fourth leading cause of death in the elderly. Due to the very nature of the disease, regression to an infantile, totally dependent state, AD places an incredible stress on everyone associated with an affected person. The goal of my project is to screen AD families for genetic mutation on exons 16 and 17 of the ß-Amyloid Precursor Protein (APP) gene, which is located on chromosome 21.
Alzheimer's disease can be classified into two groups, Familial Alzheimer's Disease (FAD) and Multifactorial Alzheimer's disease. FAD shows autosomal dominant inheritance and is a single gene disorder. FAD accounts for only about 5% of all Alzheimer's cases and the age of onset is usually between 20 and 65 years. So far, three mutations have been identified that lead to FAD: Presenilin 1 (PS1) on chromosome14, Presenilin 2 (PS2) on chromosome 1, and APP on chromosome 21. These mutations lead, for the most part, to early-onset AD where the age of onset is less than 65 years. A PS2 mutation may lead to some late-onset AD. The other 95% of cases are multifactorial, which means that the AD is caused by many factors including genetics and yet unknown environmental factors. The only known genetic risk factor is an Apolipoprotein Epsilon (APOE) gene polymorphism on chromosome 19. A grey area occurs when the age of onset is between 50 and 70 years and the family exhibits an autosomal dominant inheritance pattern. No matter which type of AD is present, the clinical manifestations are always the same: gradual loss of memory, loss of speaking ability, and eventual total dependence.
The hallmark of an Alzheimer's patient is the brain contains an accumulation of aggregated senile plaques and neurofibrillary tangles that impede neuronal signaling and eventually lead to neuronal death. A post-mortem brain dissection is the only way to truly diagnosis an affected Alzheimer's patient. The plaques are comprised of ß-amyloid peptides.
ß-amyloid is a fragment of a protein that is snipped from another protein called
ß-amyloid precursor protein (BAPP). Three fragments can be formed by the proteolysis of BAPP, including a harmless p3 form when the protein is cleaved by alpha and gamma secretases. A 40 residue peptide is produced 90% of the time when beta and gamma secretases cleave the protein, but 10% of the time when the beta and gamma secretases cleave the protein a pathogenic form, 42 residues long, which forms fibrils and plaques more readily, is formed. The cause of production of this 42 residue peptide is still not fully understood.
A mutation at the genetic level could possibly cause this peptide to be produced. The reason for my focus on exons 16 and 17 of the APP gene, which is comprised of 19 exons, is these exons code for Amyloid ß (A ß). Mutations have already been found on both exons in several studies. All AD causing mutations in APP have been found on these two exons. Mutations at codon 717 occur in exon 17, which change valine to isoleucine, phenylalanine, or glycine, and a double mutation at codons 670-671 occurs in exon 16, which changes lysine to asparagine and methionine to leucine. The aim of my project was to search for mutations in APP exons 16 and 17 within several families from the Memory and Aging Project (MAP) from Washington University's Alzheimer's Disease Research Center (ADRC) that have apparent autosomal dominant inheritance and have an age of onset between 50 and 70 years.
I screened nine families for mutations in exon sixteen and four families for mutations in exon 17. The first step in this process was to obtain the DNA that had previously been extracted from a patient's blood. A Polymerase-Chain Reaction (PCR) was set up for exons sixteen and seventeen that would amplify only these exons. A check gel was run to verify if the correct product was amplified. The amplified PCR product was treated using a Qiagen column to clean up the primer front and remove extra DNTP's. The purified product was sequenced on an ABI 310, an automated DNA sequencer, using the D Rhodamine terminator chemistry. The sequences were analyzed and compared on the Sequence Navigator program. No mutation was identified in the nine families I screened for exon 16 or for four families screened for exon 17.
These families have already been screened for mutation in PS1 and PS2 with negative results. All but one of the affected individuals have been screened for the APOE e4 allele polymorphism. This being the case, a genetic mutation/risk factor must exist somewhere else that has yet to be found. My results allow the search for mutation to continue elsewhere in the genome.
|
|
|
Natural Sciences Learning Center
Washington University - Biology
All contents copyright © 2001
Email comments to nslc webmanager