Tiny Chip Will Speed Up Genetic Diagnosis of Huntington’s Disease

Researchers have developed a miniature device that can measure the length of disease-related DNA regions in less than five minutes. The technology was tested using DNA samples from myotonic dystrophy type 1 and Huntington’s disease patients.

The study, “μLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes,” was published in Scientific Reports.

Several genetic diseases that directly affect the nervous system, including Huntington’s disease, are caused by a triplet repeat expansion, i.e. when the number of trinucleotide repeats in a mutated gene is greater than the number found in the normal version of that gene. In Huntington’s disease there are excessive CAG repeats within the gene that codes for the huntingtin protein.

Of note, a CAG trinucleotide repeat is a segment made up of a series of three DNA building blocks (cytosine, adenine, and guanine) that appear multiple times in a row.

Because of the highly repetitive nature of this kind of mutation, reliable quantification of expansion length with existing molecular technology is difficult and time-consuming. However, determining triplet repeat size is important to predict disease progression and establish clinical management, as longer repeats are associated with more severe disease.

Therefore, “there is a need for novel methods to determine repeat sizes that are sensitive, that provide information about repeat size variation, and that reduce the time and effort required to obtain the results,” researchers said.

Now, researchers from the Université de Toulouse and the University of Lausanne tested the potential of a lab-on-a-chip device called μLAS (μLAboratory for DNA Separation, Picometrics Technologies) for measuring expanded trinucleotide repeats associated with Huntington’s disease and myotonic dystrophy type 1.

A lab-on-a-chip system handles extremely small fluid volumes while integrating several laboratory functions on a single miniature chip.

In a laboratory setting, scientists can make many copies of a specific DNA region (called target DNA) by using a technique known as polymerase chain reaction (PCR). In a PCR, the target DNA is mixed up with specific enzymes, small strands of DNA and other organic molecules, and submitted to a series of cycled temperature changes that will allow many copies of the target region to be produced. After that, the amplified portion of DNA can be quantified (i.e. DNA sequence length can be determined) or used for other purposes.

In μLAS, voltage and pressure are applied to two identical, side-by-side, funnel-shaped channels less than a millimeter wide, forcing the separation of previously amplified, negatively charged DNA fragments according to their size. Of note, smaller fragments are pushed down the funnel, in contrast with larger ones.

By using a reference molecular-weight size marker (also known as DNA ladder) and fluorescent dye, scientists can then easily detect DNA fragments’ position under a microscope and determine their length.

Five Huntington’s disease and three myotonic dystrophy type 1 DNA samples, with various triplet repeat sizes, were quantified using the new system.

μLAS was able to detect expanded triplets, containing up to 750 CAG/CTG repeats, in all tested samples and in 5 minutes or less, thus covering the entire range of expansions seen in Huntington’s disease and much of those seen in myotonic dystrophy type 1, in which repeat length varies from 50 to 5,000 CTG.

Tests also revealed μLAS was highly sensitive, meaning samples do not need to be highly amplified before running them through the chip. As a result, scientists decreased the number of amplification cycles (i.e. used shorter PCR programs), reducing the number of amplification artifacts without compromising the accurate sizing of the expanded repeats.

PCR artifacts often occur depending on the concentrations of each reagent used in the reaction, including the DNA template. Amplification artifacts can be shorter or longer than the original target DNA, and depending on the amount of artifacts produced, reliable sizing of DNA fragment could be endangered.

“These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders,” researchers concluded.