Details regarding procedure and cost of DHPLC, observations regarding the dystrophin gene, and statistics about DMD carrier females.
The process for using DHPLC screening followed by sequencing

A technician performs the PCRs, in a 96 well plate or 200µl PCR tubes by standard methods. PCR amplicon from an unaffected individual is used as a control and the DHPLC system chromatogram for the unaffected individual is the reference to which the patient’s chromatogram is compared for variation in retention time or shape. In X-linked diseases such as DMD, PCR product from an unaffected male is also mixed 1:1 with PCR product from the male patient and the mixture is heated to 950C and allowed to cool to room temperature over 45 minutes in a thermocycler in order to create heteroduplexes and homoduplexes when variation exists. Mixing is not necessary for female patients since they have two X alleles. The samples are loaded into the DHPLC system and a sample spreadsheet is filled out. The DHPLC system automatically processes the samples at a rate of 6 to 8 minutes per sample. Chromatograms for samples are stored in the computer and those from patients are visually compared to those from an unaffected control to determine whether variations in shape or retention time are present. Any variant PCR fragments are prepared for sequencing on an automated fluorescent sequencer by standard methods. This level of convenience can be compared to other techniques.

Reagent cost analysis

The reagent cost of the existing multiplexed PCR diagnostic is estimated at $25.00 per patient. The reagent cost per patient for the process presented here is as follows. For large cohorts of patients to be screened, on average, likely disease-causative mutations would be found within approximately 43 fragments (1/2 the total number of fragments in the set) since in the dystrophin gene, point mutations tend to be fairly evenly distributed (see Leiden Muscular Dystrophy web pages ( and to the extent that there are hot spots, they can be screened first. The cost of a 50µl PCR is estimated at $0.50. The cost of the reagents to run a sample on the WAVE® system is estimated at $0.40 per injection. For male patients, an unaffected control fragment must be amplified and 5-10µl of it run on the DHPLC system in addition to a mixed sample of 5-10µl each of patient and unaffected control for each sample at each temperature. We would expect to run fragments an average of 2.5 temperatures (i.e. injections) per sample. Therefore, the cost of reagents to amplify and run all the fragment/temperature combinations required per patient is:

43 fragments x 2PCRs/fragment x $0.50/PCR + (86 x 2.5 injections) x $0.40/injection = $129

The number of fragments per patient exhibiting variation from the unaffected control will, of course, vary from patient to patient but should on average be no more than four. That is to say that before the likely disease-causative mutation is found in a patient, on average, three polymorphic variations will be found. The reagent costs to purify a single PCR reaction is estimated at $0.50. The reagent costs to sequence a single strand is estimated as $4.00, and to amplify and sequence four fragments in both directions would be:

4 PCRs x $0.50 + 4 PCR purifications x $0.50 + 8 x $4.00 = $36.00 per patient.

This results in a total reagent cost per patient for DHPLC screening followed by direct sequencing of $129 + $36 = $165

Combining $25.00/patient for 100% of patients with $165/patient for 35% of patients results in an average cost for reagents of:

($25 x 1) + ($165 x 0.35) = $82.75 per patient, or an increase over the current $25.00/patient of $57.75/patient. There are many costs other than reagent costs involved in performing diagnostic tests including consumables, technician time, instruments and capital equipment depreciation and overhead. It is beyond the scope of this article to estimate all of these costs, but we believe they will also constitute only a moderate increase in cost over the existing process.

Regarding the price of sequencing in a core facility, many core sequencing facilities are subsidized by grants that help to defray costs. The price per single strand sequence has been decreasing steadily over the past eight years due to technology improvements such that the price, typically $10.00 per single strand sequence currently, should continue to decline especially as 16- and 96-well capillary sequencers become more prevalent.

Two interesting facts about the dystrophin gene

It is interesting to note that base number 4441+2 (the donor splice site after exon 30) is a cytidine in all 8 patients and in fact in most wildtype/unaffected individuals. This occurs in 10 of 1799 splice sites (0.44%) looked at in the human genome while thymidine occurs in 1775 of 1799 (98.67%). This occurrence in the dystrophin gene of a common allele/wildtype GC instead of GT donor splice site represents a very rare phenomenon in the human genome[23]

Also of interest is the observation that all 1300 bp of the 5’-UTR and 3100 bp of the 3’-UTR are highly conserved in our population as they contained identical sequence for all eight of the patients. This indicates that these regions are potentially highly conserved across larger populations.

Additional statistics about carrier females

DMD, the most common form of dystrophinopathy, is a genetic disorder that manifests itself as follows. One of every 3500 live male births will be severely disabled boys[24] and an equivalent number of live female births will be carrier females, some manifesting and some non- manifesting. Sporadic mutations occur at approximately the same rate in females as in males[25]. Many of these carrier females, if aware of their carrier status, will have serious psychosocial dilemmas when facing procreation decisions. 5% to 10% of carrier females will be manifesting carriers with varying degrees of clinical involvement[5]. Approximately 10% of carrier females will have notable histological changes[5] and 8% (4 of 50 carriers) have right precordial lead electrocardiogram waves outside the normal range[26, 5]. Isolated non-familial cases of dystrophinopathy are found in approximately 10% of female neuromuscular disease patients[27]. Thus, DMD affects females as well as males, both in their experience of the disease state as well as in their role in procreation and care taking.