MOLECULAR BASIS OF X-LINKED DISORDERS 
JaneM. Gitschier, Ph.D., Assistant Investigator 
This laboratory is interested in a number of 
aspects of X-linked disorders, such as uncovering 
the genes responsible for some of these and finding 
the mutations that underlie them. In addition, re- 
search is aimed at improving genetic prediction and 
therapy for these diseases. Research in the past year 
has led to progress in a number of projects. 
Isolation of Candidate Disease 
Genes in Xq28 
Xq28 is the terminal band of the long arm of the 
human X chromosome. It is thought to be an ex- 
tremely gene-rich region, and at least 20 inherited 
diseases are known to map here. This laboratory is in 
the process of isolating genes in Xq28 and deter- 
mining whether mutations in any of them are asso- 
ciated with a particular inherited disorder. 
To date, six genes have been isolated, the most 
intriguing of which is the MPP-1 gene. MPP l was 
found to code for a membrane protein previously 
described in erythrocytes, but probably expressed 
in all cells. This protein, p55, is heavily palmitoyl- 
ated and contains domains homologous to SH3 (src- 
homology domain 3) and to the enzyme guanylate 
kinase. Overall, its sequence is reminiscent of the 
derived amino acid sequence of the Drosophila 
tumor-suppressor gene lethal discs large (dig) . Mu- 
tations in this gene lead to overgrowth of the epithe- 
lial cells in imaginal discs. 
It is tempting to speculate that mutations in 
MPP-1 could lead to a genetic disease, and several 
candidates come to mind. One is dyskeratosis con- 
genita, an Xq28-linked disorder characterized by 
leukoplakia of the oral mucosa, nail dystrophy, con- 
tinuous lacrimation, and a tendency for malignancy. 
Mutations in the p55 gene might also lead to one of 
the muscle disorders mapping to Xq28, since mus- 
cle cells may be particularly vulnerable to muta- 
tions that affect membrane integrity. 
The laboratory is in the process of screening pa- 
tient samples for mutations in the MPP-1 gene, as 
well as in other genes. Samples from more than 70 
patients with Xq28-linked disorders have been col- 
lected and immortalized. These are being assayed 
for abnormalities in DNA, RNA, and protein. 
Identification of Mutations 
in the V2 Vasopressin Receptor Gene 
Nephrogenic diabetes insipidus (NDI) is one of 
the inherited diseases that map to Xq28. In patients 
with this disorder, water retention by the kidney is 
not responsive to the antidiuretic hormone vaso- 
pressin. Recently isolation of the gene for the 
kidney-specific vasopressin receptor was reported. 
Because it was discovered to map to Xq27-28, it is a 
likely candidate for the NDI gene. Dr. Gitschier's 
laboratory made use of the published sequence to 
design primers for RT-PCR (reverse-transcriptase/ 
polymerase chain reaction) of RNA prepared from 
the immortalized NDI cell lines. Independent muta- 
tions were found in five of six unrelated patients 
tested. These mutations include one nonsense muta- 
tion, one frameshift, three missense mutations af- 
fecting conserved amino acids, and remarkably, in 
one patient, an additional 12 -bp deletion. These 
findings strongly support the hypothesis that the va- 
sopressin receptor is defective in NDI. 
Isolation of a Candidate Gene 
for Menkes Syndrome 
Menkes syndrome is an X-linked disorder of cop- 
per metabolism resulting in progressive neurologi- 
cal degeneration and death in early childhood. The 
underlying etiology is unknown, and there is no pre- 
dictably effective treatment. Genetic linkage studies 
point to Xql3 as the location of the Menkes gene. 
Although Menkes disease is almost exclusively 
found in males, a female patient has been identified 
with a balanced X:autosomal translocation, the 
breakpoint mapping to Xq 1 3 ■ It is hypothesized that 
the breakpoint of the translocated chromosome 
disrupts the Menkes gene. 
In collaboration with Dr. Seymour Packman (Uni- 
versity of California, San Francisco) , this laboratory 
has undertaken to isolate the gene responsible for 
Menkes disease. DNA probes from the Xql3 region 
were obtained and hybridized to blots of pulsed- 
field gels of normal and translocated DNA to local- 
ize the breakpoint. Yeast artificial chromosomes 
(YACs) that span the breakpoint were then isolated 
with the closest flanking probes, and the breakpoint 
position was refined to within 2 kb. These genomic 
sequences were used to isolate cDNA sequences by 
several approaches, including direct YAC screening 
and exon trapping. 
An excellent candidate cDNA has been obtained. 
The sequence for this large cDNA shows homology 
to a cadmium-transport protein from Staphylococ- 
cus aureus and to the class of cation-transporting 
ATPases. The occurrence of aberrations in both the 
GENETICS 1 99 
