MOLECULAR GENETICS OF HUMAN DISEASE 
Francis S. Coluns, M.D., Ph.D., Associate Investigator 
Dr. CoUins's laboratory is involved in four major 
projects that involve the molecular basis of human 
disease. Three of these projects involve the identifi- 
cation of genes that cause disease but whose pro- 
tein products have not been possible to character- 
ize directly. With the new technique of reverse 
genetics, much progress has been achieved. 
L Cystic Fibrosis. 
Intense efforts over the last four years culminated 
in the successful cloning of the cystic fibrosis (CF) 
gene in the late summer of 1989, in collaboration 
with Drs. Lap-Chee Tsui and Jack Riordan (Hospital 
for Sick Children, Toronto). A region of —500,000 
bp was cloned, using a combination of chromo- 
some jumping and chromosome walking. Genetic 
analysis of a large number of CF families indicated 
the most likely position of the CF mutation; a sur- 
vey of that region for conserved and/or transcribed 
sequences yielded a genomic fragment of DNA that 
proved to be the 5' end of the CF gene. The com- 
plete cDNA sequence has been obtained. This se- 
quence encodes a protein of ~168 kDa, which has 
homologies to the multidrug resistance gene and a 
variety of other genes for transporter pumps of 
lower organisms. Specifically, the protein product 
contains several hydrophobic domains that pre- 
sumably anchor it to the cell membrane. It also 
contains a region that is probably an ATP-binding 
site, which indicates that the protein may be an 
ATP-activated pump. The proof that this represents 
the CF gene is the identification of a 3 bp deletion 
in exon 10 that occurs in 70% of CF chromosomes 
but is never found in normal chromosomes. This 
deletion results in the loss of a single phenylala- 
nine residue in the protein product, which is lo- 
cated in the ATP-binding domain. 
This achievement represents the first time that a 
gene has been cloned by reverse genetics without 
the availability of gross chromosomal rearrange- 
ments. The door is now open to a large number of 
experiments that may lead to an understanding of 
the basic defect in CF. Current priorities include the 
identification of the other mutations responsible 
for CF, which undoubtedly will affect the same 
gene, and the development of a gene transfer sys- 
tem to show that expression of the normal version 
of this gene is capable of correcting the defect in CF 
cells. Dr. Collins and his colleagues have named the 
gene CFTR (cystic fibrosis transmembrane regula- 
tor). (See Science 245:1058-1065, 1066-1073, and 
1073-1080 for details of the cloning.) 
II. Neurofibromatosis. 
Neurofibromatosis (NFl) is a common autosomal 
dominant human disease characterized by the de- 
velopment of multiple benign tumors and an in- 
creased risk of cancer. Considerable progress has 
been made in identifying the NFl gene, which had 
been previously mapped to chromosome 17 by 
linkage analysis. DNA samples from two patients 
who have NFl in association with balanced translo- 
cations involving 17qll.2 have been used to nar- 
row the location of the NFl gene to a region of 
—500 kb. This achievement was made possible by 
cloning a large number of Not\ linking clones from 
chromosome 17 and testing them against pulsed- 
field gel electrophoresis (PFGE) blots from these 
two patients. A probe was eventually identified, de- 
noted 17L1, which lies <400 kb away from both 
translocation breakpoints. This region is being in- 
vestigated for candidate genes, in collaboration 
with Dr. Raymond L. White (HHMI, University of 
Utah). 
III. Huntington Disease. 
The Collins laboratory is an active participant in 
the collaborative effort to identify the Huntington 
disease gene on the short arm of chromosome 4. 
This genetic analysis, primarily carried out by Dr. 
James Gusella, has led to conflicting information 
about the precise location of the gene. Current ef- 
forts are focused on applying the chromosome 
jumping technique to fill in the gaps in the physical 
map of the terminal 6 million bp of 4p, as well as 
constructing high-quality cDNA libraries from vari- 
ous regions of the human brain to search for candi- 
date transcripts. 
IV Hemoglobin Switching. 
The switch from production of fetal hemoglobin 
to adult hemoglobin at about the time of birth in 
the human provides an interesting model system 
for understanding the developmental regulation of 
gene expression and has the additional importance 
of potential clinical utility: if this switch could be 
Continued 
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