Human Molecular Genetics in Two X-linked Diseases 
Stephen T. Warren, Ph.D. — Associate Investigator 
Dr. Warren is also Associate Professor of Biochemistry and of Pediatrics at Emory University School of 
Medicine. He received his Ph.D. degree in genetics from Michigan State University. Prior to joining the 
faculty at Emory, he did postdoctoral research with Richard Davidson at the University of Illinois School 
of Medicine in Chicago. 
MY laboratory is involved in human molecu- 
lar genetics and is especially interested in 
the identification of genes responsible for ge- 
netic disease. Our efforts currently focus on X- 
linked disease, particularly the fragile X syn- 
drome and Emery-Dreifuss muscular dystrophy. 
Fragile X Syndrome 
Fragile X syndrome is the commonest form of 
inherited mental retardation and one of the most 
prevalent genetic diseases known, affecting ap- 
proximately 1 per 1,000 persons worldwide. As 
the name implies, fragile X syndrome is asso- 
ciated with a fragile chromosomal site, which has 
been localized to band position Xq27.3. Fragile 
sites are heritable loci that form cytologically evi- 
dent gaps within chromosomes under specific 
biochemical induction. Although such sites are 
numerous throughout the human genome, the 
fragile X site is the only one associated with a 
disease. 
Fragile X syndrome is unusual among mamma- 
lian genetic disorders in that 20 percent of the 
males with a fragile X chromosome are not af- 
fected while approximately 30 percent of carrier 
females show some degree of mental impairment. 
The fragile X mutation is less frequently pene- 
trant (i.e., resulting in mental retardation) among 
the siblings of these normal carrier males (called 
transmitting males), and penetrance increases 
with each generation from a transmitting male 
until it reaches so-called Mendelian ratios, where 
half of the male children of a carrier female are 
affected, typical of an X-linked gene. One excep- 
tion is that daughters of transmitting males are 
never affected, but their children may be. This 
confusing hereditary pattern, unique among ge- 
netically studied organisms, has been referred to 
as the Sherman paradox, in reference to Steph- 
anie Sherman's description of it. 
Our work over the past year has not only identi- 
fied the gene responsible for fragile X syndrome 
but has also uncovered an unusual mutation 
whose behavior explains the Sherman paradox. 
Working with an international group of collabora- 
tors, including Thomas Caskey (HHMI, Baylor 
College of Medicine), David Nelson (Baylor), 
and Ben Oostra (Erasmus University, the Nether- 
lands) , we identified yeast artificial chromosome 
(YAC) clones, previously developed in my labora- 
tory, that mapped near chromosome breakpoints 
involving the fragile X site. Using cloned DNA 
derived from one of these YACs, we identified a 
cDNA encoded by a gene that the translocation 
breakpoints had interrupted. This gene, termed 
fragile X mental retardation 1 {FMR-1), pro- 
duces a 4.4-kb message expressed at high levels 
in the brain and testes. Male fragile X patients 
have macro-orchidism, or enlarged testes, in ad- 
dition to mental retardation. 
Within the FMR-1 mRNA is an unusual repeat 
of the trinucleotide CGG. In normal individuals, 
there are most frequently 29 repeats, though this 
can vary between 6 and 52. Among transmitting 
males and most normal carrier females, there are 
between 52 and 200 CGG repeats. Among men- 
tally retarded patients, the codon repeats up to 
1,300 times and is markedly unstable in mitotic 
cells. Importantly, when the repeat expands 
beyond 250, it spontaneously methylates the 
nearby DNA, turning off the FMR-1 gene. Re- 
moval of the gene product by hypermethylation 
in response to the massive augmentation of CGG 
repeats is believed to be the mechanism of fragile 
X syndrome. 
Work with our colleagues in Houston revealed 
an apparent relationship between the number of 
repeats in a normal carrier female and the proba- 
bility of having a mentally retarded son. In gen- 
eral, the smaller the abnormal repeat, the lower 
the risk of expansion to the full fragile X muta- 
tion in an offspring. Above a threshold of approxi- 
mately 200 repeats, the fragile X chromosome 
when passed down always undergoes expansion 
to the full mutation. Hence carrier mothers with 
200 repeats have a 50 percent risk of having a 
retarded son, while those with only 70 repeats 
have a 9 percent risk. This explains the paradox 
of penetrance in fragile X syndrome: the sequen- 
tial increase in the CGG repeat with each genera- 
tion imparts a concomitant increase in risk of 
having an affected child. 
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