Download Generating Genetic Biomarkers in Clinical and Research

GENERATING GENETIC BIOMARKERS IN CLINICAL AND
RESEARCH APPLICATIONS BY THE DENATURING HIGHPERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC)
TECHNOLOGY
Dani Bercovich
Human Molecular Genetics & Pharmacogenetics,
Migal – Galilee Technology Center
Genetic diagnosis depends heavily on the availability of efficient and sensitive methods for detecting
DNA mutations and sequence variations. With the expansion in our understanding of the human
genome sequence and of the relevance of gene mutations to human disease, there is a compelling
need for improved methods of mutation detection having high sensitivity and allowing for high
throughput using partial or complete automation. A relatively new addition to DNA scanning
methods utilizes denaturing high-performance liquid chromatography (DHPLC). Scanning for DNA
mutations and variants using DHPLC involves subjecting PCR products to chromatography using an
ion-pair reverse-phase cartridge. PCR products are denatured and allowed to reanneal. Under
conditions of partial denaturation with a linear acetonitrile gradient, heteroduplexes from PCR
samples having an internal sequence variation display a reduced column retention time relative to
their homoduplex counterparts. The elution profile for heterozygous samples is typically quite
distinct from that of either homozygous sequence, making the identification of heterozygous
mutations relatively straightforward.
We used the DHPLC to diagnose DNA alterations in the UBE3A gene, which is known to cause
Angelman syndrome. We have also used this method to diagnose DNA alterations in the MACP2
gene. The MACP2 gene is known to cause Rett syndrome. Using the DHPLC, we were able to detect
all mutations in Familial hypercholesterolemia (FH) patients at the gene encoding for the receptor
that transports low-density lipoproteins (LDL) into cells. Using the DHPLC method reviled genetic
haplotypes (by identifying 38 SNP’s from two different genes of about 80 patients) that have a strong
association with the differences in response to Statin treatment in those patients. We also used the
DHPLC for the first time to genotype haploid organisms, using DNA from Meningococcal bacteria
Outbreaks in Israel.
Through collaborations, the DHPLC technology was used to identify some common mutations in
the Israeli population such as the Bloom syndrome, Fanconi syndrome, BRCA1 & BRCA2, and the
APC gene in FAP patients with colon cancer. Screening Ashkenazi protest cancer patient reviled a
novel mutation. With the DHPLC we found mutations in Exon 2 of GATA1 gene in Megakaryocytic
Malignancies that was associated with trisomy 21. Other uses of the DHPLC method were applied
for SNP scanning along the human nAChRs Beta-4 and Alpha-3. From our experience, DHPLC
detected 100% of DNA alterations and in some cases found mutations that were missed by
sequencing. This data indicates that using the DHPLC method is advantageous over direct
sequencing for finding DNA alterations.