Molecular Basis of Genetic Diseases and Chromosome Mapping 
an obligate heterozygote. The amplified DNA du- 
plexes are screened for the presence of base dif- 
ferences by denaturing gradient gel electrophore- 
sis (DGGE) or single-strand conformational 
polymorphism (SSCP) studies. Amplified exons 
that appear to have base differences, identified by 
altered melting behavior or conformational prop- 
erties, are sequenced to reveal the precise base 
differences. 
In a rare form of inherited dv^^arfism, the Laron 
syndrome, individuals are unresponsive to 
growth hormone, lack the activity of a specific 
growth hormone-binding protein in serum, and 
have low levels of insulin-like growth factor I. 
The growth hormone receptor is a likely candi- 
date gene for mutations leading to this disorder. 
The receptor gene consists of 10 exons that are 
amplified individually from flanking primers. In 
a study of 38 Laron syndrome individuals from a 
highly inbred population in the mountains of 
southern Ecuador, we identified a base substitu- 
tion in the extracellular domain of the receptor 
that does not alter the encoded amino acid but 
creates a new donor splice site. The resulting mu- 
tant mRNA is deleted for 24 bases, which predicts 
a protein lacking eight amino acids in a highly 
conserved region of the molecule. The location 
of the deleted eight amino acids does not involve 
the hormone-binding or receptor dimerization 
sites. The most likely effect of this mutation is a 
poorly folded, unstable protein that might de- 
grade rapidly. Further studies are under way to 
test this prediction. 
The same strategy is now being pursued to find 
mutations in the growth hormone receptor gene 
in other patients with Laron syndrome who are 
from different ethnic and racial populations. The 
Ecuadorean growth hormone receptor mutation 
represents the first instance of a disease-causing 
base substitution that acts by creating a new do- 
nor splice site and leads to its exclusive use while 
the original site is unaltered. 
The gonadotropin-releasing hormone (GnRH) 
is produced in specialized cells in the hypothala- 
mus, travels to the pituitary gland, and causes the 
release of the gonadotropic follicle-stimulating 
hormone (FSH) and luteinizing hormone (LH). 
We previously mapped the gene to the short arm 
of human chromosome 8. In the mouse, partial 
deletion of this gene causes a recessive pheno- 
type, termed hypogonadal, leading to lack of sex- 
ual development. In human families with in- 
herited incomplete sexual development due to 
low levels of gonadotropin hormones, the GnRH 
gene is a candidate for involvement, as in the 
mouse model. We have studied a family with 
three affected siblings that show a sequence 
polymorphism in the signal peptide of the GnRH 
gene, but have found that this polymorphism 
does not cosegregate with the disease phenotype, 
thus excluding a GnRH gene defect in this partic- 
ular family. 
The Marfan syndrome is an autosomal domi- 
nant condition that involves abnormalities in 
elastic tissue of the aorta and the fibrils that sus- 
pend the optic lens and in connective tissue of 
many organs, including bones, tendons, and lung. 
The estimated incidence is 1 in 10,000, and clin- 
ical severity is highly variable. Dissection and 
rupture of the aorta is a life-threatening compli- 
cation. During the past year, linkage studies in 
other laboratories have pinpointed the site of the 
Marfan gene on human chromosome 15, and co- 
incidentally the gene encoding fibrillin, the ma- 
jor microfibrillar protein, was also mapped to the 
same location. Subsequently, a mutation in the 
fibrillin gene was documented in two unrelated 
Marfan patients. 
Stanford University is a center for Marfan diag- 
nosis, treatment, and research. Over the last three 
years, we have established a broad-based research 
program that involves a detailed clinical and ge- 
nealogical database and the collection of blood 
and tissue samples from affected persons and fam- 
ily members. We hypothesize that a mutation in 
the fibrillin gene causes the Marfan phenotype by 
producing an abnormal protein that when in- 
corporated into the microfibrils causes their 
instability. 
We are now screening the mRNA produced by 
skin or aortic fibroblasts from patients with clas- 
sical Marfan syndrome and related connective tis- 
sue disorders. After reverse transcription of the 
10,000-bp fibrillin mRNA, small overlapping 
sections are amplified and screened for point mu- 
tations by DGGE and SSCP studies. Our goal is not 
only to develop a molecular diagnostic test for a 
disorder that is clinically often difficult to diag- 
nose, but also to gain insight into the relation- 
ships between particular types of mutations and 
the phenotypes they produce. 
Since there are fibrillin-related genes else- 
where in the genome and many connective tissue 
disorders that overlap the Marfan phenotype to 
some degree, this will be a large fruitful field of 
investigation. 
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