Paced by Huntington's chorea a decade ago, and cystic fibrosis afew years back, the gene-hunters keep turning up the inherited rootsof more and more maladies caused by defects in one isolated gene.With such single-gene genetic diseases, aberrant DNA sequences canbe mapped by pinpointing a few tens of thousands of polymorphisms_ deviant stretches of nucleic acid along the genome _ that are foundmore often in patients than in health individuals.But most of the medically important illnesses on which the moleculargeneticists are training their sights _diabetes, hypertension, asthma,epilepsy, to name a few _ are multi-gene disorders. Pinning down theinherited genomic errors that predispose to such multifactorialills involves analyzing hundreds of thousands, if not millions ofmarker sequences _ hundreds of thousands, if not millions.Hence, the search for such genetic first causes of complex diseases isslow, costly, often downright frustrating.Using radioisotopes and today's gene-probing technologies, includinga PCR automatic cycler and multichannel pipetting devices, onetrained technician, using short tandem repeat polymorphisms, cangenerate some 1,500 to 2,000 genotypes a week, at a cost of about $1.50 each.This estimate comes from a world leader in pioneering DNA diseaselinkage markers. He is James Weber, who heads the Molecular GeneticsLaboratory at the Marshfield Medical Research Foundation inMarshfield, Wis.
Know Thy Genome"Know thy genome," an editorial by Weber, appears in the July issue ofNature Genetics, out today. "Current bottlenecks in the typing processfor these markers," he wrote, "are polyacrylamide gel electrophoresisand scoring." To overcome them, Weber wrote, "Numbers of gels can bereduced by loading multiple markers in each gel lane that overlaps insize but which are labeled by different fluorescent dyes. Scoring markerscan be improved by capturing images of the electrophoresis patternin computers and then computationally converting these imagesinto genotypes."He added, "This is the approach now described by Todd and colleagues."They do so in the same issue of Nature Genetics, in a paper titled,"Chromosome-specific microsatellite sets for fluorescence-based,semi-automated genome mapping."Molecular geneticist John Todd, its principal author, is at theUniversity of Oxford, England. He told BioWorld Today that hisgroup's approach to large-scale genetic mapping of the human genome"could get 750 genotypes in four hours, a tenfold increase inefficiency over conventional methods." Weber, in an interview,observed, "My guess is that that number has not quite been achievedyet, but the tenfold factor, I think, is there."The Oxford team has developed 254 dinucleotide repeat marker lociarranged into 39 sets, covering all 22 human somatic chromosomes,plus the sex-linked X one. Four-fifths of these markers came fromFrance's Genethon research center; others from the Genome Data Basein Baltimore.Genethon, a $10.6 million automated laboratory facility in Evry, issupported by the French Muscular Dystrophy Assn. and France's NationalCenter for Human Polymorphism Research. His system's main feature,Todd said, "is that it's semi-automated, and the data is capturedelectronically, not manually." He elaborated: "The `Genotyper' softwarenewly developed by Applied Biosystems, Inc. of Foster City, Calif.,which correlates the fluorescent intensity of labeled DNA fragmentswith marker loci information, is tested here by us for the first time."Todd's test run screened 96 sibling pairs and their parents (384individuals) from families with more than two affected members forsusceptibility loci of insulin-dependent diabetes mellitus. Thescreen generated over 87,000 genotypes, and results matched thoseobtained independently by Genethon. The 254 genetic markers nowavailable, Todd said, will increase to "400 or 500 in evenlyspaced sets across all the chromosomes within a year." He added,"These sets allow analyzing hundreds of genotypes, or hundredsof individuals, in one gel run."A "small" benefit of Todd's system, Weber mentioned, "is the eliminationof isotopes. That makes the work in the lab go faster, moreefficiently, and probably a little bit cheaper."
Automation Is The AdvantageHe added that "the big advantage in moving from radioactively labeledsamples to fluorescently labeled ones is that the analysis can becarried out automatically. It has the potential of allowing one toload the samples on the gel, then walk away, and get the resultsprinted out on a computer."Funded by Britain's Medical Research Council, the U.K. Human GenomeMapping Project, through its Resource Center in Harrow, U.K., isoffering the primer-pair sets, free of charge, to countries in theEuropean Community that have a national genome program. These,according to a recent bulletin, include laboratories in Denmark,France, Germany, Italy, Netherlands, Russia and the U.K. itself.The bulletin stated that "a charge at commercial rates will bemade to non-European groups and to the private sector."Among companies using Resource Center facilities are Glaxo,SmithKline Beecham, British Biotechnology, Yamanouchi ResearchInstitute, Zeneca Pharmaceuticals and the Wellcome Foundation.The Center's director, Ramnath Elaswarapu, told BioWorld Todaythat he is "organizing a meeting of fluorescent primer recipients,in association with Applied Biosystems, on Tuesday, Aug. 16, 1994,near Oxford." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.