A person who stutters severely has a hard time in life,socially and psychologically. His spasmodic repetition ofwords and syllables causes the stutterer intense anxiety orworse, and his listener, impatience or irritation.

But one whose DNA contains multiple repeats of acertain trio of bases may be damned with any of at leastnine neurodegenerative disorders, of which fragile-Xsyndrome is the widest spread, and Huntington's diseasethe best known, and most dreaded.

Fragile-X is the most common form of mental retardationin males. Its immediate cause is a DNA stutter in the Xchromosome, specifically, numerous repetitions of aCGG triplet codon. Huntington's disease (HD), far moredire than fragile-X, has repeats of a CAG triplet, onchromosome 4.

In the genetic code, C-A-G stands for the three nucleotidebases cytosine, adenine and guanine. CAG encodes theamino acid glutamine.

Besides HD, fragile-X and myotonic dystrophy (the mainform of muscular dystrophy), molecular neurogeneticistsstand watch over a clutch of rarer, congenital, triplet-driven disorders, whose very names suggest their sinistersymptomology:

* spino-bulbar muscular atrophy

* spino-cerebellar ataxia

* dentatorubral-pallidoluysian atrophy

* Haw River syndrome

* Machado-Joseph disease

The last disorder on this list afflicts mainly Portugueseinhabitants of the Azores islands. Haw River syndromestrikes an even narrower population, a single extendedfamily living along a valley in North Carolina (SeeBioWorld Today, Aug. 2, 1994, p. 1).

Venezuelans Model HD's Molecular Basis

Huntington's disease, of course, occurs widelythroughout the developed world, with an estimated30,000 patients in the U.S alone, plus 150,000 at risk.However, it too has a geographic concentration on theshores of Venezuela's Lake Maracaibo, with its owngenetic founder (See BioWorld Today, June 1, 1993, p.2). This population of 14,000 counts 500 HD sufferers,and more arising all the time. It has become a keyresource for geneticists working to lay bare the molecularbasis of HD.

Columbia University psychologist Nancy Wexler,president of the Hereditary Disease Foundation in SantaMonica, Calif., pioneered bringing the Venezuelan HDpopulation into mainstream HD research. She co-authored a paper in the September issue of the journalHuman Molecular Genetics, titled: "Single spermanalysis of the trinucleotide repeats in the Huntington'sdisease gene: quantitification of the mutation frequencyspectrum."

Another co-author is James Gusella of Boston'sMassachusetts General Hospital, who, with the HDCollaborative Research Group, discovered the HD geneon human chromosome 4 in 1993.

"In human semen," Wexler told BioWorld Today, "eachindividual sperm has a different number of CAG repeatsin it. In some men," she added, "that DNA stutter canexaggerate enormously in number. So if that man's ownrepeat length in all other tissues of his own bodynumbers, for example, 40, his sperm could have 100repeats."

This, Wexler explained, "has clinical as well as biologicalsignificance. Repeats of 60 or more can cause onset ofHD as young as age 20."

Molecular geneticist Norman Arnheim, of the Universityof Southern California, the paper's senior author, toldBioWorld Today: "Some people have very low repeatnumbers, some, very large numbers. We faced thequestion: What is the risk of a person at the high end ofthe CAG repeat normal spectrum having an offspringwith HD?"

He went on, "It's fairly well established that if you don'thave more than 33 or 34 repeats, you won't get HD. Butyou could have an offspring with 45; that child would.And you wouldn't necessarily know what the risk wouldbe of your having additional offspring with enoughrepeats to give them the disease."

Arnheim pointed out that "there are people with nofamily history at all who produce children with HD.These are spontaneous mutations, rather than inheritedones."

Catching A Single Sperm's DNA By The Tail

In an effort to define the risk and refine the prediction,Arnheim and his co-authors undertook a unique strategy:They zeroed in on the CAG repeats in the one paternallyinherited HD gene contained in a single sperm cell, ratherthan scrutinize the teeming swarm of spermatozoa in analiquot of seminal fluid, as others have done in the past.

Using fluorescence-activated cell-sorting, they isolated923 single sperm from three individuals with HD, andfrom two normal men. One of the latter, with a typicalnon-pathological 30 repeats, showed a moderate 11percent mutation frequency.

A 36-repeat sperm, intermediate between normal anddiseased, was mutating at a much faster rate, 53 percent.All of that person's gametes were expanding, whichtipped his scales into the HD range of 37 repeats or more.

Among the HD patients themselves, with 38 to 51repeats, the expansion rate reached 92 to 99 percent. Thisjibed with earlier ominous observations that the higherHD repeat numbers go, the higher still they are likely togo when inherited. (See BioWorld Today, Aug. 2, 1994,p. 1.)

"This paternal instability of those trinucleotide repeats,"Arnheim said, "proved significantly greater than maternalinstability. The repeats become more _ and very muchmore _ when they go through the male germline thanthrough the female.

"Our sperm-typing technique," he added, "allowed us toaccumulate the kind of data needed to evaluate the truenature of that instability at the molecular level."

Thus, he pointed out, "We know in principle that theindividual in our paper who carried one of those so-called`intermediate' alleles, was at an 8 percent risk of havingan offspring with HD."

Arnheim is not at all certain that such a predictivecapability can or should be applied to the population atlarge. "One in 100 people," he observed, "may carry oneof these large alleles."

Meanwile, he suggested, "Since the PCR protocol weused can amplify alleles with 100 triplet repeats fromsingle sperm cells, this method may be applied topreimplantation genetic diagnosis."

Wexler predicted, "Biotechnology research is going tofind the cure to Huntington's disease _ unquestionably.Somewhere out there is somebody who has a biotechanswer for fixing the DNA stutter, striking the protein,finding a treatment. We at the Hereditary DiseaseFoundation have grants and postdoctoral fellowshipsavailable. Phone 310-458-4183, or fax us at 310-458-3937." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.