ABCMdb

PMID: 11687513

Shroyer NF, Lewis RA, Yatsenko AN, Lupski JR
Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa.
Invest Ophthalmol Vis Sci. 2001 Nov;42(12):2757-61., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg STGD-affected individual AR682-03 was compound heterozygous for the mutation 2588G3C and a complex allele, [W1408R; R1640W]. Login to comment 8 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg RP-affected individuals AR682-04 and-05 were compound heterozygous for the complex allele [W1408R; R1640W] and the missense mutation V767D. Login to comment 9 ABCA4 p.Val767Asp Functional analysis of the mutation V767D by Western blot and ATP binding revealed a severe reduction in protein expression. Login to comment 10 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg In vitro analysis of ABCR protein with the mutations W1408R and R1640W showed a moderate effect of these individual mutations on expression and ATP-binding; the complex allele [W1408R; R1640W] caused a severe reduction in protein expression. Login to comment 14 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg These studies of the complex allele W1408R; R1640W suggest a synergistic effect of the individual mutations. Login to comment 45 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg Oligos corresponding to each mutation were: V767D, GTC TGG CAG CAG CCT GTA GTG GTG ACA TCT ATT TCA C; W1408R, GAC CCT TCA CCC CCG GAT ATA TGG GCA G; R1640W, CAA CGC CAT CTT ATG GGC CAG CCT GCC (mutant nucleotide positions are in bold type). Login to comment 46 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg Oligos corresponding to each mutation were: V767D, GTC TGG CAG CAG CCT GTA GTG GTG ACA TCT ATT TCA C; W1408R, GAC CCT TCA CCC CCG GAT ATA TGG GCA G; R1640W, CAA CGC CAT CTT ATG GGC CAG CCT GCC (mutant nucleotide positions are in bold type). Login to comment 75 ABCA4 p.Gly863Ala ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg Sequencing of STGD1-affected proband AR682-03 revealed three ABCR mutations: the transition 4222T3C that encodes the missense substitution W1408R, the transition 4918C3T that results in the missense substitution R1640W, and the transversion 2588G3C that gives rise to equal amounts of proteins with either a deletion of glycine at residue 863 or the missense substitution G863A (Fig. 2, Table 1).10,15,16 Sequencing of her RP-affected paternal grandmother, AR682-04, also revealed three ABCR mutations: the missense substitutions W1408R and R1640W and a transversion 2300T3A that encodes the missense substitution V767D (Fig. 2, Table 1).17 Direct DNA sequencing of all members of pedigree AR682 for the exons corresponding to these mutations revealed segregation of the mutation 2588G3C from the maternal lineage and the complex allele [W1408R; R1640W] from the paternal lineage; the mutation V767D was identified only in the two RP-affected individuals (Fig. 2). Login to comment 76 ABCA4 p.Gly863Ala ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg Sequencing of STGD1-affected proband AR682-03 revealed three ABCR mutations: the transition 4222T3C that encodes the missense substitution W1408R, the transition 4918C3T that results in the missense substitution R1640W, and the transversion 2588G3C that gives rise to equal amounts of proteins with either a deletion of glycine at residue 863 or the missense substitution G863A (Fig. 2, Table 1).10,15,16 Sequencing of her RP-affected paternal grandmother, AR682-04, also revealed three ABCR mutations: the missense substitutions W1408R and R1640W and a transversion 2300T3A that encodes the missense substitution V767D (Fig. 2, Table 1).17 Direct DNA sequencing of all members of pedigree AR682 for the exons corresponding to these mutations revealed segregation of the mutation 2588G3C from the maternal lineage and the complex allele [W1408R; R1640W] from the paternal lineage; the mutation V767D was identified only in the two RP-affected individuals (Fig. 2). Login to comment 79 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg Biochemical Analysis of Recombinant ABCR Plasmid constructs encoding recombinant ABCR proteins bearing the missense mutations V767D, W1408R, and R1640W, and the complex allele [W1408R; R1640W] were transiently transfected into HEK 293T cells. Login to comment 80 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg Biochemical Analysis of Recombinant ABCR Plasmid constructs encoding recombinant ABCR proteins bearing the missense mutations V767D, W1408R, and R1640W, and the complex allele [W1408R; R1640W] were transiently transfected into HEK 293T cells. Login to comment 87 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg Western blot analysis of proteins from cells transfected with the V767D or [W1408R; R1640W] mutation-bearing constructs showed very little, if any, protein (Fig. 4). Login to comment 88 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg Western blot analysis of proteins from cells transfected with the V767D or [W1408R; R1640W] mutation-bearing constructs showed very little, if any, protein (Fig. 4). Login to comment 89 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg In contrast, proteins bearing the W1408R or R1640W mutations appeared to have mild or moderate defects in expression or stability (Fig. 4). Login to comment 90 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg Recombinant ABCR bearing the V767D or [W1408R; R1640W] mutations showed no labeling with azido-ATP, even when fivefold total protein was used in the assay (Fig. 4 and data not shown). Login to comment 91 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg Recombinant ABCR bearing the V767D or [W1408R; R1640W] mutations showed no labeling with azido-ATP, even when fivefold total protein was used in the assay (Fig. 4 and data not shown). Login to comment 92 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg The recombinant protein bearing the R1640W mutation had a moderate effect on ATP labeling, with approximately 55% of wild-type activity. Login to comment 93 ABCA4 p.Trp1408Arg As reported by Sun et al.,12 the mutation W1408R had a moderate effect on ATP labeling. Login to comment 99 ABCA4 p.Gly863Ala For brevity, the mutation 2588G3C was denoted G863A. Login to comment 100 ABCA4 p.Gly863Ala For brevity, the mutation 2588G3C was denoted G863A. Login to comment 101 ABCA4 p.Gly863Ala ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Asn1868Ile ABCA4 p.Trp1408Arg ABCA4 p.Arg943Gln ABCA4 p.His423Arg ABCR Alterations in Patients with Stargardt Disease and Retinitis Pigmentosa Exon Nucleotide Amino Acid AR682-03 AR682-04 3 302af9;26 A/A A/G 10 1268G 3 A H423R A/A A/G 1356af9;11delG 6G/6G 6G/7G 15 2300T 3 A V767D T/T T/A 17 2588G 3 C G863A G/C G/G 19 2828G 3 A R943Q G/A G/G 24 3523-30 A/T A/T 28 4203C 3 A P1401P C/A C/C 4222T 3 C W1408R C/T C/T 33 4667af9;48 C/T T/T 35 4918C 3 T R1640W C/T C/T 40 5585-70 C/T T/T 5603A 3 T N1868I A/T A/A 5682G 3 C L1894L G/C G/G Mutations are indicated in bold. Login to comment 102 ABCA4 p.Gly863Ala ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Asn1868Ile ABCA4 p.Trp1408Arg ABCA4 p.Arg943Gln ABCA4 p.His423Arg ABCR Alterations in Patients with Stargardt Disease and Retinitis Pigmentosa Exon Nucleotide Amino Acid AR682-03 AR682-04 3 302ϩ26 A/A A/G 10 1268G 3 A H423R A/A A/G 1356ϩ11delG 6G/6G 6G/7G 15 2300T 3 A V767D T/T T/A 17 2588G 3 C G863A G/C G/G 19 2828G 3 A R943Q G/A G/G 24 3523-30 A/T A/T 28 4203C 3 A P1401P C/A C/C 4222T 3 C W1408R C/T C/T 33 4667ϩ48 C/T T/T 35 4918C 3 T R1640W C/T C/T 40 5585-70 C/T T/T 5603A 3 T N1868I A/T A/A 5682G 3 C L1894L G/C G/G Mutations are indicated in bold. Login to comment 114 ABCA4 p.Gly863Ala The 2588G3C 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 2828G3A in cis to the 2588G3C alteration, consistent with linkage disequilibrium between these two alterations, as reported previously.15,19 The effects of the mutation 2588G3C 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 2588G3C mutation. Login to comment 115 ABCA4 p.Gly863Ala ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg The 2588G3C 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 2828G3A in cis to the 2588G3C alteration, consistent with linkage disequilibrium between these two alterations, as reported previously.15,19 The effects of the mutation 2588G3C 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 2588G3C mutation. Login to comment 116 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg In individual AR682-03, the combination of the 2588G3C 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. Login to comment 117 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg RP-affected individuals AR682-04 and -05 carry the complex allele [W1408R; R1640W] in trans to the missense allele V767D. Login to comment 120 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg 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). Login to comment 121 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg 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. Login to comment 123 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg 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. Login to comment 124 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg 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. Login to comment 125 ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg 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. Login to comment 126 ABCA4 p.Arg1640Trp ABCA4 p.Val767Asp ABCA4 p.Trp1408Arg 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. Login to comment 127 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg 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. Login to comment 128 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg 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). Login to comment 129 ABCA4 p.Arg1640Trp Analysis of the R1640W mutation revealed a reduction in both expression (ϳ65% wild-type) and ATP-binding capacity (ϳ60% wild-type). Login to comment 130 ABCA4 p.Arg1640Trp ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg ABCA4 p.Trp1408Arg 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). Login to comment 136 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg 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. Login to comment 139 ABCA4 p.Arg1640Trp ABCA4 p.Trp1408Arg 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. Login to comment