ABCMdb

PMID: 17466004

Muzyamba M, Farzaneh T, Behe P, Thomas A, Christesen HB, Brusgaard K, Hussain K, Tinker A
Complex ABCC8 DNA variations in congenital hyperinsulinism: lessons from functional studies.
Clin Endocrinol (Oxf). 2007 Jul;67(1):115-24. Epub 2007 Apr 27., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC8 p.Arg1436Gln ABCC8 p.Asp1193Val Results The first case had diffuse disease and was homozygous for the mutations D1193V and R1436Q in SUR1. Login to comment 4 ABCC8 p.Arg1436Gln ABCC8 p.Asp1193Val Channel complexes containing the D1193V mutant were delivered to the plasma membrane and were functional and those containing R1436Q were also present at the plasma membrane but were nonfunctional. Login to comment 5 ABCC8 p.Arg1436Gln Combining the two mutations (SUR1D1193V/R1436Q) led to intracellular retention of the channel complex. Login to comment 6 ABCC8 p.Asp1471Asn ABCC8 p.Gly228Asp ABCC8 p.Val1572Ile In a second family, the patient had histologically focal disease and was heterozygous for two mutations from his father (G228D and D1471N) and one from his mother (V1572I). Login to comment 7 ABCC8 p.Asp1471Asn ABCC8 p.Gly228Asp SUR1 G228D and D1471N singly or in combination led to intracellular retention of the channel complex and loss of function. Login to comment 8 ABCC8 p.Val1572Ile By contrast,V1572I is trafficked appropriately and is functional,consistent with a mechanism of reduction to hemizygosity of paternal ABCC8 in focal disease.V1572I is likely to be a benign DNA variant. Login to comment 23 ABCC8 p.Asn188Ser Whereas compound mutations occur in approximately 13% of CHI patients,39 the simultaneous presence of two homozygous CHI mutations has to our knowledge only been reported in one patient, in which one of the mutations (N188S) was thought to be nonfunctional.40 In this report,we describe two patients with more than one suspected mutation in diffuse and focal disease and delineate the consequences these DNA variations have on KATP channel function. Login to comment 30 ABCC8 p.Arg1436Gln ABCC8 p.Arg1436Gln ABCC8 p.Asp1193Val ABCC8 p.Asp1471Asn ABCC8 p.Gly228Asp There is also a notation based on a potential splice variant of 1582 amino acids in length.19 The SUR1 mutations,D1193V and R1436Q (referred to as SUR1D1193V/R1436Q), and G228D and D1471 (referred to as SUR1G228D/D1471N),were made in the same SUR1 cDNA construct to mimic the fact that the patients had mutations on the same chromosome.Mouse Kir6·2 was used and expressed as described previously.41 Mouse Kir6·2-GFP (Kir6·2 fused in frame with the enhanced variant of the green fluorescent protein) and Kir6·2-HA (Kir6·2 engineered to contain an extracellular antigenic haemagglutinin epitope tag between the first transmembrane domain and the H5 segment) were kind gifts of Drs Ribalet and Jan, respectively. Login to comment 64 ABCC8 p.Arg1436Gln ABCC8 p.Asp1193Val He was homozygous for two mutations in SUR1, namely D1193V and R1436Q, with both mutations occurring on each allele. Login to comment 76 ABCC8 p.Asp1471Asn ABCC8 p.Gly228Asp 4411 g > a, leading to the amino acid change G228D and D1471N. Login to comment 86 ABCC8 p.Val1572Ile It is pertinent to note that V1572I has previously been reported to be a polymorphism, on the basis that it is a conservative amino acid substitution, in an Askenazi Jewish population but has not been studied functionally.19 Trafficking and expression studies We first examined the potential subcellular distribution of the Kir6·2/ SUR1 mutant channel complexes. Login to comment 93 ABCC8 p.Arg1436Gln In particular, SUR1D1193V/R1436Q was less efficiently transfected. Login to comment 95 ABCC8 p.Arg1436Gln In addition, in HEK293 cells we have not established a clear correlation between glycosylation pattern and trafficking status.48 We next examined the behaviour of SUR1D1193V, SUR1R1436Q and SUR1D1193V/R1436Q. Login to comment 97 ABCC8 p.Asp1471Asn By contrast, SUR1G228D, SUR1D1471N and SUR1G228D/D1471N all failed to translocate Kir6·2-GFP to the plasma membrane. Login to comment 98 ABCC8 p.Asp1471Asn SURV1572I translocated Kir6·2-GFP to the plasma membrane (Fig. 2b).We confirmed that Kir6·2-GFP was located in the ER when expressed with one of the SUR1 mutants (SUR1G228D/D1471N) that could not deliver a complex to the membrane by colocalizing with DsRed2-ER (Fig. 2c). Login to comment 99 ABCC8 p.Asp1471Asn In addition, we performed quantitative colocalization analysis as described previously, measuring the fraction of green pixels (Kir6·2-GFP) that also contain red (DsRed2-ER).45 In some representative images Kir6·2-GFP = 89 ± 5% (n = 7), SUR1 + Kir6·2-GFP = 31 ± 6% (n = 5) and SUR1G228D/D1471N + Kir6·2-GFP = 82 ± 2% (n = 6, P < 0·001 vs. SUR1 + Kir6·2-GFP and not significant vs. Kir6·2-GFP). Login to comment 103 ABCC8 p.Arg1436Gln We next examined the behaviour of SUR1D1193V, SUR1R1436Q and SUR1D1193V/R1436Q. Login to comment 105 ABCC8 p.Asp1471Asn By contrast, SUR1G228D, SUR1D1471N and SUR1G228D/D1471N all failed to translocate Kir6·2-HA to the surface of the cell (Fig. 3c). Login to comment 114 ABCC8 p.Arg1436Gln ABCC8 p.Asp1471Asn SUR1D1193V and SUR1V1572I were functional in Rb86 flux assays whereas SUR1R1436Q, SUR1D1193V/R1436Q,SUR1G228D,SUR1D1471NandSUR1G228D/ D1471N were not (Fig. 4). Login to comment 120 ABCC8 p.Arg1436Gln ABCC8 p.Asp1471Asn SUR1D1193V, SUR1V1572I and SUR1D1471N were functional in patch clamp assays whereas SUR1R1436Q, SUR1D1193V/R1436Q, SUR1G228D and SUR1G228D/D1471N were not.In the knowledge of these results we repeated the flux assays and trafficking assays with Kir6·2-GFP with SUR1D1471N. Login to comment 123 ABCC8 p.Asp1471Asn Finally, we mimicked the complex genotype in the patient in the second family by coexpressing SUR1V1572I and SUR1G228D/ D1471N together with Kir6·2-GFP (Fig. 6a) or Kir6·2 (Fig. 6b). Login to comment 125 ABCC8 p.Asp1471Asn In addition, the expression of SUR1V1572I with SUR1G228D/D1471N and Kir6·2 led to a recovery of function as indicated by substantial Rb+ fluxes (Fig. 6b). Login to comment 128 ABCC8 p.Arg1436Gln Patient 1 had severe diffuse disease and was homozygous for two ABCC8 mutations in the cis configuration (SUR1D1193V/ R1436Q). Login to comment 129 ABCC8 p.Asp1193Val The mutation D1193V introduced alone into SUR1 led Fig. 2 The trafficking and expression of SUR1 mutants. Login to comment 137 ABCC8 p.Asp1471Asn (c) Representative confocal images of HEK293 cells transiently transfected with Kir6·2-GFP, DsRed2-ER and SUR1G228D/D1471N. Login to comment 141 ABCC8 p.Arg1436Gln The R1436Q mutation led to channel complexes present at the plasma membrane; however,under our assay conditions we were not able to demonstrate significant channel activity. Login to comment 145 ABCC8 p.Asp1471Asn In contrast to the first family, mutations expressed singly or in combination (SUR1G228D,SUR1D1471N and SUR1G228D/D1471N) led to a degree of loss of function due to the failure of the channel complex to traffic. Login to comment 146 ABCC8 p.Asp1471Asn The mutations SUR1G228D and SUR1G228D/ D1471N were unambiguous in that in all the assays used, the mutant channel complexes failed to traffic and were nonfunctional. Login to comment 155 ABCC8 p.Val1572Ile The SUR1G228D and SUR1D1471N mutations are paternally inherited; however,the situation is further complicated by the potential inheritance of a maternal DNA variation (SUR1V1572I).V1573I (alternative notation) has been reported to be a polymorphism on the basis that it is a conservative amino acid and found in controls with a low frequency.19,40 V1572I/V1573I has,however,not been studied functionally and a potential impact was not excluded a priori. Login to comment 158 ABCC8 p.Asp1471Asn The disease in patient 2 arose because of paternal inheritance of SUR1G228D/D1471N and a focal somatic event,and in contrast to patient 1,both mutations singly or in combination are deficient in trafficking and function. Login to comment