MOLECULAR GENETICS OF X-LINKED DISEASE 
Robert L. Nussbaum, M.D., Associate Investigator 
Dr. Nussbaum's laboratory has been engaged in 
positional cloning efforts directed toward identify- 
ing X-linked genes responsible for human genetic 
disease. Three disorders have been the targets of 
these efforts: fragile X syndrome, Lowe oculocere- 
brorenal syndrome, and choroideremia. As a result 
of the efforts of a number of laboratories around the 
world, including Dr. Nussbaum's, all three of these 
genes have been identified. During the past year Dr. 
Nussbaum's laboratory has shifted its efforts from 
positional cloning to a detailed genetic analysis of 
the biological basis of one of these diseases, the 
Lowe oculocerebrorenal syndrome (OCRL) , and the 
complex area of the metabolism that his research 
indicates is affected by this disease. 
OCRL is an X-linked disorder characterized by 
mental retardation, congenital cataracts, renal tubu- 
lar dysfunction in childhood, and progressive renal 
failure in adulthood. Dr. Nussbaum's laboratory 
used genomic cloning in the vicinity of two X chro- 
mosome-autosome translocations in two females 
with OCRL to identify the OCRL- J gene, which is 
interrupted by both translocations. Northern blot 
analysis revealed that the mRNA for this gene is ab- 
sent in both female patients and is missing or abnor- 
mal in size in 9 of 1 3 unrelated male patients with 
the disorder. This suggests that OCRL-1 is a reason- 
able candidate for the gene involved in the disease. 
(The preliminary work that formed the basis for the 
isolation of OCRL-1 was supported by a grant from 
the National Institute of Child Health and Develop- 
ment, National Institutes of Health.) 
The predicted amino acid sequence for OCRL- 1 is 
consistent with a 970-amino acid protein (theoreti- 
cal molecular weight 112 kDa) . Sequence analysis 
showed that OCRL- 1 is very similar but not identical 
to a previously described cDNA (HUMIN5P5), 
which encodes an enzyme, type III inositol poly- 
phosphate- 5 -phosphatase, that removes the phos- 
phate at the 5 position from inositol ! ,4, 5-trisphos- 
phate. Inositol is a six-carbon ring sugar alcohol 
with a complex cellular metabolism that has been 
the subject of intensive biochemical investigation. 
Inositol phosphate esterified to diacylglycerol 
(phosphatidylinositol) makes up 2-8% of cellular 
phospholipid. It undergoes a complex cycle of 
phosphorylation, dephosphorylation, phospholi- 
pase cleavage, and re-esterification. Inositol phos- 
phate is clearly involved in signal transduction and 
in anchoring membrane proteins by glypiation, but 
(as is the case with many biochemical investiga- 
tions) the physiological functions of most inositol 
phosphates, either free or esterified in phospho- 
lipids, are incompletely understood. The results in 
Dr. Nussbaum's laboratory suggest that OCRL may 
be the first human inborn error of inositol phos- 
phate metabolism and that at least one disease phe- 
notype can result from a defect in its metabolism. 
Strong evidence that OCRL- 1 is the genetic locus 
for OCRL has been obtained by demonstrating 
single-point mutations in the OCRL- 1 gene from se- 
verely affected OCRL patients. These mutations 
disrupt mRNA processing and translation and lead to 
premature translation termination and loss of the 
carboxyl-terminal 25-30 amino acids. This region 
of the protein is likely to be important for the func- 
tion of the enzyme, because it is highly conserved 
between the OCRL-1 protein and HUMIN5P5. 
The next goal is to define the mechanism by 
which this defect in inositol metabolism disrupts 
lens development and interferes with normal renal 
and neurological function. Because such studies 
cannot be undertaken in humans, the laboratory has 
begun to study the role of the OCRL- 1 gene product 
in frogs and mice. Embryonal stem cell technology 
is being used to create mouse models for OCRL. The 
mouse homologue for OCRL-1 has been isolated, 
and the exons encoding the terminal 100 amino 
acids of the protein have been isolated and se- 
quenced. DNA constructs have been made that will 
be used to recombine with the normal mouse gene 
and cause a deletion of exons encoding the carboxyl 
terminus of the protein, because human mutations 
that truncate the terminal 20-30 amino acids of the 
OCRL-1 protein cause severe disease. The Xenopus 
homologue for OCRL-1 has also been isolated. The 
timing and tissue distribution of expression of this 
gene during tadpole development is being studied, 
with emphasis on its role in the development of the 
amphibian lens. 
The OCRL- 1 gene product is only one of a myr- 
iad of enzymes implicated in inositol phosphate 
metabolism. Because it is difficult to assess the 
functional significance of all these enzymatic ac- 
tivities, the laboratory is beginning a systematic 
program to isolate and delete other genes in- 
volved in inositol metabolism in embryonal stem 
cells to create mice deficient in these enzymatic 
activities. In this way the functional significance 
of these metabolites and the physiological role of 
GENETICS 239 
