More than 200 different
ABCR mutations have been
associated with STGD1 (a listing is available at
http://www.uwcm.ac.uk/uwcm/mg/search/370748.html, provided by the
University of Wales College of Medicine, Cardiff, UK).
18 Among our subjects with STGD1 for whom mutational analysis has been
completed, none harbored two null
ABCR alleles, suggesting
that all subjects with STGD1 retain some ABCR
activity.
2 10 Three unrelated pedigrees segregated
ABCR mutations with recessive RP19.
3 4 5 In each
of these three families, the RP-associated mutations have been
truncating or splice mutations, consistent with the hypothesis that in
individuals with no ABCR activity, early-onset, severe, and rapidly
progressive retinal dystrophy develops, with a clinical phenotype
similar to classic RP and entirely distinct from STGD.
Pedigree AR682 manifests both STGD1 and RP. We performed extensive
sequence analyses of the
ABCR gene in one STGD1 proband and
one RP-affected individual from this pedigree. This search yielded
three different mutant
ABCR alleles
(Fig. 2) .
Pedigree AR682 segregated missense
ABCR mutations with both
STGD1 and RP. The 2588G→C alteration in STGD1 patient AR682-03 has
been observed previously in 26 unrelated patients with STGD1 and has
been classified as a mild mutant allele based on its association with
later onset disease and its pairing with presumed severe alleles in
patients with STGD1.
10 15 19 In addition, we observed the
polymorphism 2828G→A in
cis to the 2588G→C alteration,
consistent with linkage disequilibrium between these two alterations,
as reported previously.
15 19 The effects of the mutation
2588G→C have been studied by Sun et al.,
12 who report a
moderate reduction in expression of the G863A mutant protein and a
modest reduction in ATP-binding for the G863del variant of the
2588G→C mutation.
In individual AR682-03, the combination of the 2588G→C allele with
the complex allele [W1408R; R1640W] resulted in STGD1 with onset of
visual symptoms at age 15 years, consistent with classification of the[
W1408R; R1640W] allele as moderate to severe. RP-affected
individuals AR682-04 and -05 carry the complex allele [W1408R;
R1640W] in trans to the missense allele V767D. Both have
advanced RP at ages 71 and 73 years, each with a diagnosis of RP for
over five decades.
The three previously reported pedigrees with
ABCR-associated
RP had progressive retinal deterioration with onset of night blindness
before age 10, progressive rod and cone dystrophy including loss of
central vision, and pigment epithelial and choroidal
atrophy.
5 20 21 For each of the RP-affected individuals
presented in this study, symptomatic nyctalopia occurred in the late
first or early second decade and progressed to loss of functional
central acuity (e.g., loss of automotive license) by the fifth decade.
The retinal examinations in these adult subjects showed advanced RP.
ATP labeling and Western blot analysis of recombinant ABCR bearing the
mutations V767D or [W1408R; R1640W] showed that these mutant proteins
are not efficiently expressed in our transient transfection system,
despite expression of substantial amounts of mRNA
(Figs. 3 4) . Based
on our new observations, we predict that both RP-associated alleles
V767D and [W1408R; R1640W] represent severe mutations that result in
little or no ABCR activity. Either these mutations cause misfolding of
ABCR, or the nascent protein is unstable and quickly degraded, in that
little or no protein could be detected by Western blot analysis and ATP
labeling. In support of the misfolding hypothesis, the mutation V767D
is predicted to lie within a transmembrane region and may disrupt
proper folding of the nascent protein. However, the alterations W1408R
and R1640W are each predicted to affect the first intradiscal loop,
which has no known function.
22 Of interest, the mutation
V767D was reported in combination with the mutation 250delCAAA (a
frameshift mutation that is a presumed null allele) in a patient with
STGD1 with onset at age 8 years.
17 That this patient (now
24 years old) has an
ABCR genotype predicted to be equal to
or more severe than that of our patient with advanced RP suggests a
potential role for environmental or modifier gene effects.
The effects of the W1408R mutation on ABCR expression and ATP binding
are consistent with the classification of this as a mild to moderate
mutation. Our results confirm and refine those reported by Sun et
al.,
12 and show that this mutation has approximately 75%
of wild-type expression and ATP-binding activity. Analysis of the
R1640W mutation revealed a reduction in both expression (∼65%
wild-type) and ATP-binding capacity (∼60% wild-type). Furthermore,
our analysis of the mutations W1408R and R1640W alone and in
combination suggest that the null effects of the complex allele[
W1408R; R1640W] are due to a combination of the two alterations, and
are more severe than either mutation alone
(Fig. 4) . This represents an
important finding, because complex alleles have been reported in 7% of
our unselected cohort of 150 STGD-affected families,
10 as
well as in a large number of other reported patients with
STGD.
15 19 23
Our analysis and identification of missense ABCR mutations
in a family that segregates both STGD and RP is novel and interesting.
Based solely on the mutation data and model of ABCR activity and its
relation to pathogenesis, we hypothesized that the missense mutation
identified in patients with RP would have severe functional
consequences. Biochemical analysis of recombinant ABCR bearing these
mutations confirmed that the RP-associated missense mutations are null,
and further demonstrated that the effects of the complex allele W1408R;
R1640W are more severe than a simple additive effect of the two
constituent mutations. Our findings further support a model in which
disease severity is inversely correlated with ABCR activity and extend
the model to include missense mutations associated with RP.
The authors thank family AR682 and their attending physicians,
Robert D. Gourley, MD, Ronald M. Kingsley, MD, Alan L. Maberley, MD,
Stephen R. Martin, MD, Leslie W. Nesmith, MD, Raymond E. Townsend, MD,
and Mark J. Weiss, MD, for their willing and continued cooperation in
these studies; Jeremy Nathans, MD, PhD, and Robert S. Molday, PhD, for
reagents and advice; and Theodore Wensel, PhD, for abundant assistance
with the biochemical analysis of ABCR.