Mutations in the sulfonylurea receptor gene are associated with familial hyperinsulinism in Ashkenazi Jews
Mutations in the sulfonylurea receptor gene are associated with familial hyperinsulinism in Ashkenazi JewsAnn Nestorowicz, Beth Anne Wilson, Kathleen P. Schoor, Hiroshi Inoue, Benjamin Glaser1, Heddy Landau2, Charles A. Stanley3, Paul S. Thornton3,+, John P. Clement IV4, Joseph Bryan4, Lydia Aguilar-Bryan4 and M. Alan Permutt*
Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, Box 8127, 660 South Euclid Ave., St Louis, MO 63110, USA, 1Department of Endocrinology and Metabolism, The Hebrew University, Hadassah Medical School, Jerusalem, Israel, 2Department of Pediatrics, The Hebrew University, Hadassah Medical School, Jerusalem, Israel, 3Division of Endocrinology/Diabetes, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA and 4Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, TX 77030, USA
Received July 1, 1996;Revised and Accepted August 29, 1996
Familial hyperinsulinism (HI) is a disorder of pancreatic [beta]-cell function characterized by persistent hyperinsulinism despite severe hypoglycemia. To define the molecular genetic basis of HI in Ashkenazi Jews, 25 probands were screened for mutations in the sulfonylurea receptor (SUR1) gene by single-strand conformation polymorphism (SSCP) analysis of genomic DNA and subsequent nucleotide sequence analyses. Two common mutations were identified: (i) a novel in-frame deletion of three nucleotides (nt) in exon 34, resulting in deletion of the codon for F1388 ([Delta]F1388) and (ii) a previously described g -> a transition at position -9 of the 3' splice site of intron 32 (designated 3992-9g -> a). Together, these mutations are associated with 88% of the HI chromosomes of the patients studied. 86Rb+ efflux measurements of COSm6 cells co-expressing Kir6.2 and either wild-type or [Delta]F1388 SUR1 revealed that the F1388 mutation abolished ATP-sensitive potassium channel (KATP) activity in intact cells. Extended haplotype analyses indicated that the [Delta]F1388 mutation was associated with a single specific haplotype whereas the 3992-9g -> a mutation was primarily associated with a single haplotype but also occurred in the context of several other different haplotypes. These data suggest that HI in Ashkenazi Jews is predominantly associated with mutations in the SUR1 gene and provide evidence for the existence of at least two founder HI chromosomes in this population.
Familial hyperinsulinism (HI; OMIM: 256450) is an autosomal recessive disorder of pancreatic [beta]-cell function and is characterized by inadequate suppression of insulin secretion in the presence of severe, recurrent, fasting hypoglycemia. Clinical manifestations of HI, which occurs predominantly in neonates and infants under 1 year of age, include seizures, coma and large birth weight for gestational age (1 -3 ). In the absence of treatment, HI may be lethal or result in irreversible neurologic sequelae. Estimates for the incidence of this disorder vary from 1/40 000 live births in northern Europe (4 ) to 1/2675 live births in Saudi Arabia, a country in which 51% of all births are from consanguineous marriages (5 ).
The HI locus was initially assigned to the region flanked by the microsatellite markers D11S926 and D11S899 on chromosome 11p14-15.1 by genetic linkage analyses and homozygosity mapping (6 ,7 ). Subsequent linkage and haplotype studies using additional genetic markers refined the position of the HI locus to the D11S419-D11S1310 interval (8 ). Recently, the gene for the sulfonylurea receptor (SUR1), a modulator of insulin secretion, was localized to chromosome 11p15.1 by fluorescence insitu hybridization (FISH) and two separate point mutations were identified in probands with HI by nucleotide sequence analyses of SUR1 cDNA and/or genomic DNA (9 ). Both mutations, a g -> a transition in a 5' splice site which results in exon skipping and a g -> a transition in intron 32, postulated to activate cryptic splice sites, are presumed to result in premature truncation of the SUR1 molecule (9 ).
To define the molecular basis of HI in Ashkenazi Jews, DNA samples from 25 probands were screened for mutations within all 39 exons of the SUR1 gene and also, within flanking intron-exon boundaries by SSCP analyses. Exons are numbered consecutively from the 5' end of the gene, with the two putative nucleotide-binding folds, NBF-1 and NBF-2, of the SUR1 protein encoded by exons 15-22 and 33-38, respectively (G. Gonzalez, L. Aguilar-Bryan and J. Bryan, GSDB accession #L78208) (10 ). Samples demonstrating electrophoretic mobility shifts were detected in some probands and/or control samples for exons 6, 12, 16, 18, 21, 27, 31, 33, 34, 35 and 39 (Figs 2 a and 3 a; data not shown). Mutations in the SUR1 gene in samples displaying such band shifts were identified by nucleotide sequence analyses of polymerase chain reaction (PCR) products amplified in independent reactions from genomic DNA samples. A total of 14 different mutations were identified, including 12 putative polymorphisms (see below).
The functional consequences of the [Delta]F1388 mutation upon KATP channel activity were examined by measurement of 86Rb+ efflux in intact COSm6 cells co-transfected with the murine homologue of the KATP channel subunit Kir6.2 (13 ) and either wild-type hamster SUR1 or SUR1 containing the [Delta]F1388 mutation (designated [Delta]F1388SUR1). 86Rb+ efflux was assayed in transfected cells following incubation with (i) metabolic inhibitors (2-deoxy- D-glucose and oligomycin) which reduce intracellular ATP levels and increase ADP levels, resulting in opening of KATP channels, or (ii) diazoxide, an activator of KATP channels in islet [beta]-cells or (iii) glibenclamide, an inhibitor of KATP channel activity. In cells expressing wild-type Kir6.2/SUR1 KATP channels, 86Rb+ efflux was stimulated above basal levels by the presence of either diazoxide or metabolic inhibitors (Fig. 4 ). The KATP channel activity elicited by metabolic inhibition in cells expressing wild-type Kir6.2/SUR1 KATP channels was sensitive to glibenclamide. Untransfected COSm6 cells or cells transfected with a control plasmid expressing [beta]-galactosidase displayed no stimulation of basal 86Rb+ efflux levels by diazoxide and inhibition of efflux by metabolic inhibitors (Fig. 4 ; data not shown). In contrast to cells expressing wild-type KATP channels, cells expressing Kir6.2/[Delta]F1388SUR1 channels displayed the same responses as untransfected control cells; no stimulation by diazoxide and reduced efflux in the presence of metabolic inhibitors. Photolabeling of the 140 kDa SUR1 receptor in transfected cells, using 125I-glibenclamide, indicated that similar levels of SUR1 were expressed in cells co-transfected with Kir6.2/[Delta]F1388SUR1 or wild-type Kir6.2/SUR1 KATP channels (data not shown). Together, these data suggest that [Delta]F1388SUR1 is expressed in transfected cells but is unable to form a functional KATP channel with Kir6.2.
To identify possible founder haplotypes associated with the [Delta]F1388 or 3992-9g -> a mutations, extended haplotypes were derived for HI and normal chromosomes in parents of the 25 Ashkenazi probands by typing all available individuals within each family for four microsatellite markers (D11S1397, D11S902, D11S921, D11S1890), which are linked to the HI locus (8 ,18 ,19 ). The relative order of these markers with respect to the SUR1 locus has been previously established as 11pter- D11S1397-(D11S902- SUR1- D11S921-D11S1890)-11pcen by genetic linkage (18 ,19 ), radiation hybrid (20 ) and physical (21 ) mapping. For 21 of these families, the D11S902-D11S921 haplotypes have been described previously (8 ). Complete haplotypes were constructed for 47 of 48 HI chromosomes and for 47 of 50 normal chromosomes. Subgroups were determined by combining haplotypes that differ by <= 2 bp at a single locus on the assumption that such differences are more likely to represent CA repeat slippage than different mutation events (22 ). No recombination was observed between the HI locus and any of the four microsatellite marker loci (data not shown). Table 1 shows the haplotypes present on Ashkenazi HI chromosomes and their corresponding frequencies on normal chromosomes. Mutations within SUR1 identified on HI chromosomes are also indicated for each haplotype. A total of 35 different haplotypes were detected, with 11 present on HI chromosomes (Table 1 ). A single haplotype, F, was predominant on HI chromosomes, accounting for 25 of 47 (53.2%) disease chromosomes and three of 47 (6.4%) normal chromosomes. The three most frequent F subgroups, F.I, F.II and F.IV, which together account for 48.9% HI chromosomes, were not detected on non-HI chromosomes. Two other frequent haplotypes, H and K, were detected on 10.6% and 19.1% of HI chromosomes, respectively, with corresponding frequencies on normal chromosomes of 2.1% and 10.6%. Together, haplotypes F, H and K account for 83.0% of the HI chromosomes and 19.1% normal chromosomes of the parents studied. All other haplotypes were rare, occurring on one of 47 (2.1%) HI chromosomes. Of the 34 HI chromosomes bearing the 3992-9g -> a mutation, the majority were represented by the F (73.5%) haplotype and less frequently, by haplotypes H (14.7%), G (2.9%), I (2.9%) and J (2.9%). In contrast, the [Delta]F1388 mutation was associated exclusively with haplotype K (sub- haplotypes K.I, K.II). Neither of these mutations were detected on normal chromosomes bearing the F, H, I, J or K haplotypes. These data are consistent with the presence of at least two founder chromosomes for HI in Ashkenazi Jews.
Table 1 Association between haplotypes on HI chromosomes and SUR1 mutations
In addition to the SSCP variants described above for exons 33 and 34, band shifts were also detected for some probands and/or control samples on SSCP for exons 6, 12, 16, 18, 21, 27, 31, 33, 34, 35 and 39 (data not shown). Nucleotide sequence analyses revealed a total of 12 different point mutations within the SUR1 gene which were classified as polymorphisms on the basis that they: (i) do not modify the amino acid sequence of the protein; or (ii) result in conservative amino acid substitutions in apparently non-highly conserved residues of the protein; or (iii) are present on normal chromosomes from the control Ashkenazi population (Table 2 ). To determine whether these polymorphisms created potential novel 5' or 3' splice sites, Shapiro/Senapathy consensus values (23 ) were calculated for positions -3 to +6 for possible 5' splice site sequences (containing GT) and for positions -14 to +1 for potential 3' splice site sequences (containing AG). Consensus values were calculated for all subsequences encompassing the putative polymorphism and varied from a minimum value of 0.214 (nt 4717g -> a) to a maximum of 0.731 (nt 1686t -> c) for possible 5' splice sites, and from 0.302 (nt 4123-25c -> t) to 0.829 (nt 3822g -> a) for 3' splice site consensus values. However, in each case the consensus values for the mutated sites were lower than values calculated for the normal site (data not shown), suggesting that novel 5' or 3' splice sites are unlikely to be created by the polymorphisms identified here. Nonetheless, the possibility that some of the identified polymorphisms either augment or ameliorate the deleterious effects of familial hyperinsulinism mutations upon KATP channel activity and insulin secretion cannot be excluded at present.
Table 2 Polymorphisms within the SUR1 geneaNucleotide and codon positions are according to the full-length human SUR1 cDNA sequence incorporating the alternative spliced form of exon 17 (GenBank accession no L78208; L78224).bTm designates putative transmembrane domain.cMutation was detected on HI and/or normal chromosomes.
The sulfonylurea receptor (SUR1) is involved in nucleotide regulation of KATP channel activity and is, therefore, a central component in the mechanism of glucose-regulated insulin secretion in pancreatic islet [beta]-cells (13 ,14 ). In this study, 25 HI patients of Ashkenazi Jewish descent were screened for mutations in the SUR1 gene and specific haplotypes associated with identified mutations were defined. Two common mutations associated with HI in this population were identified, a novel deletion of F1388 in NBF-2 and a previously described point mutation at position -9 of the 3' splice site of intron 32 (9 ). The 3992-9g -> a mutation, which is the most prevalent mutation in the SUR1 gene in Ashkenazis with HI, has also been previously detected in one proband of Saudi Arabian origin (9 ). In contrast, the [Delta]F1388 mutation, which accounted for 19% HI alleles, was not observed in RFLP analyses of 50 non-Ashkenazi probands of various ethnic origins and may, therefore, be associated predominantly with HI in Ashkenazi Jews (A. Nestorowicz, unpublished data). To date, only four mutations in the SUR1 gene have been reported. In addition to the 3992-9g -> a and [Delta]F1388 mutations described here, we previously decribed a missense mutation (G1479R) in exon 37 in a proband of Sephardic Jewish origin (17 ), and a g -> a transition at position -1 of the 5' splice site of intron 35 was reported for seven probands of Saudi Arabian origin and one proband of German descent (9 ). However, neither of these additional mutations were detected, either by RFLP or SSCP analyses of exons 35 and 37, in the 25 Ashkenazi Jewish families studied here (data not shown). The allelic homogeneity described here for HI in Ashkenazi Jews presumably reflects the relative isolation of this population (24 ) and has also been observed for several other autosomal recessive disorders in Ashkenazis, including Canavan disease (25 ,26 ), Gaucher disease (27 ), Fanconi anemia (28 ) and glycogenesis VII (29 ).
At present, it is unclear whether the 3992-9g -> a mutation has a causal role in the disease process or is a polymorphism highly associated with a disease causing mutation located elsewhere, either within the SUR1 gene or a nearby locus. The effect(s) of the 3992-9g -> a mutation upon RNA splicing have been examined by transfection of a human glioblastoma cell line (SNB19) with mini-gene constructs containing the exon 33 and flanking exons of SUR1 inserted downstream from a RSV promoter (9 ). Comparison of cDNA products from wild-type and mutant constructs revealed that activation of three separate 3' cryptic splice sites occurred in constructs containing the 3992-9g -> a mutation, resulting in a 7 bp addition or deletions of 20 or 30 bp. Consequent frameshifts associated with these additions/deletions are predicted to result in deletion of the entire NBF-2 domain (Fig. 1 ) (9 ). Although specific role(s) of NBF-2 remain to be elucidated, site-directed mutagenesis studies of NBF-2 in which G1479 was replaced with R have shown that MgADP stimulation of KATP channel activity occurs through binding in this domain (17 ). Deletion of NBF-2 in 3992-9g -> a alleles may, therefore, not only prevent binding of ATP to NBF-2 but may also abolish responsiveness of SUR1 to MgADP. Unresponsiveness to MgADP is predicted to result in a net reduction of channel activity at any ADP/ATP ratio, a hypothesis consistent with the loss of regulation of glucose-induced insulin secretion observed in the HI disease phenotype (17 ). Although cryptic spice site activation was demonstrated in transfected glioblastoma (SNB19) cells (9 ) it is unknown whether such aberrant RNA splicing of 3992-9g -> a mutant alleles occurs in islet [beta]-cells invivo in the HI patients studied here. Formal proof of cryptic splice site utilization will require isolation and nucleotide sequence analyses of SUR1 cDNA from affected probands possessing the 3992-9g -> a allele.
Intrafamilial variation in disease phenotype was observed for one family in which the proband, who was homozygous for the 3992-9g -> a mutation, had two unaffected haploidentical sibs (data not shown). However, both unaffected haploidentical sibs were large for gestational age (H. Landau and B. Glaser, unpublished data) and it is possible that these children exhibit(ed) subtle defects in regulation of glucose-induced insulin secretion. Such variation in phenotypic expression has also been described for a number of other autosomal recessive disorders including Gaucher disease (27 ). The mechanism(s) underlying this variable phenotypic expression of HI is unknown. Clinical heterogeneity may be due to the effects of modifier genes (genetic background), metabolic interferences and/or exogenous factors that modulate the expression of the HI phenotype. Alternately, if cryptic splice site utilization does occur in 3992-9g -> a mutant alleles in islet [beta]-cells invivo, it is feasible that some individuals may express variable proportions of wild-type transcript sufficient to maintain phenotypically normal regulation of insulin secretion.
A second common mutation identified in the Ashkenazi probands was deletion of the codon for F1388. 86Rb+ efflux measurements on intact cells expressing either Kir6.2/wild-type SUR1 or Kir6.2/[Delta]F1388SUR1 KATP channels revealed that channel activity was impaired by the [Delta]F1388 mutation (Fig. 4 ). Both metabolic inhibition and addition of diazoxide increased efflux in COSm6 cells expressing wild-type KATP channels whereas cells expressing Kir6.2/[Delta]F1388SUR1 channels were unresponsive to diazoxide and, similar to untransfected COSm6 control cells, showed reduced efflux in the presence of metabolic inhibitors. These data were obtained for intact cells and thus, are predicted to be representative of the activity of the mutant [Delta]F1388 KATP channel in pancreatic [beta]-cells. However, we have not yet resolved whether the [Delta]F1388 KATP channel is completely inactive or is unable to respond to changes in MgADP, as demonstrated for other mutations in NBF-2 (17 ). Amino acid alignments of SUR1 NBF-2 with other members of the ABC-superfamily indicates that residue 1388 is usually non-polar in nature but that the primary structure is not strictly conserved, with some members of this protein family containing L (cystic fibrosis transmembrane conductance regulator; CFTR), T (P-glycoprotein multidrug resistance protein family; MDR) or V(MDR) at the homologous position (30 ). Amino acid residue 1388 is located two residues downstream from the Walker A consensus sequence in NBF-2 and resides in a flexible cytoplasmic loop predicted to protrude from the core-nucleotide binding fold (Fig. 1 ) (10 -12 ,30 ). A conserved sequence (linker) motif located within this loop has been proposed for other members of the ABC protein family to couple, presumably via a conformational change(s), the energy of ATP hydrolysis to transport or activity of the protein (30 ). Three-dimensional modeling of a number of ABC proteins has shown that the core nucleotide-binding fold composed of Walker A and B motifs has a strict requirement for a particular conformation rather than primary structure to maintain binding and hydrolysis of ATP (11 ,31 ). It is possible that deletion of F1388 disrupts the requisite tertiary structure of the nucleotide-binding pocket, perhaps preventing binding of ATP or ADP and/or alters the conformation and function(s) associated with the conserved cytoplasmic linker motif in SUR1. Patch-clamp experiments on cells co-transfected with Kir6.2 and [Delta]F1388SUR1 cDNA constructs may provide further information regarding the effect(s) of the [Delta]F1388 mutation upon the properties of the KATP channel. Further studies are also needed to determine whether the [Delta]F1388 mutation disrupts post-translational processing of the SUR1 protein, as has been described for deletions of certain amino acids within the cytoplasmic loop of NBF-1 in CFTR (32 ,33 ).
Extended haplotype analyses revealed associations between the [Delta]F1388 and 3992-9g -> a mutations in SUR1 and specific haplotypes (Table 1 ). Although only a small number of probands with the [Delta]F1388 mutation were identified, the data suggest that this mutation may be associated exclusively with the group K haplotype, a finding consistent with a single origin of this mutation. In contrast to the [Delta]F1388 mutation, the 3992-9g -> a mutation was observed in the context of five (F, G, H, I, J) different (i.e. haplotypes that differ by >2 bp at one or more markers) haplotypes, with the majority of alleles carrying this mutation represented by haplotypes F and H. It is possible that these haplotypes represent independent origins of the 3992-9g -> a mutation and/or that this mutation is ancient, so that multiple recombination events with normal haplotypes may have resulted in variant haplotypes. As 12 polymorphisms were detected within the SUR1 gene, construction of haplotypes using these intragenic RFLP markers as well as intragenic microsatellite markers, may provide further insight into the origins of these chromosomes in Ashkenazi Jews. An additional five distinct haplotypes (A-E) were observed on single HI chromosomes for which no mutations were defined within the 39 exons of the SUR1 gene. These haplotypes may represent separate mutational events or rearrangements of more common haplotypes by an ancestral recombination event. Assuming that 2 bp variation are more likely to represent CA repeat slippage than different mutation events (22 ), we estimate that there may be in the order of five additional mutations in this Ashkenazi patient cohort that remain to be identified. These mutations may be present within the exon sequences or flanking intron-exon boundaries screened here but were undetected by SSCP analyses or may be located either within intronic sequences and/or the 5' or 3' untranslated regions of the SUR1 gene which have not yet been analyzed.
In summary, we have established that HI in Ashkenazi Jews is associated with two frequent mutations within the SUR1 gene. Identification of additional mutations in HI and characterization of their effects upon the structure and properties of the islet [beta]-cell KATP channel may provide further insight into the structure-function relationship of the SUR1 protein, the mechanism of insulin exocytosis in islet [beta]-cells and the molecular and cellular mechanisms underlying familial hyperinsulinism.
Table 3 . Oligonucleotide sequences, annealing temperatures and PCR product sizes for SSCP analysis of SUR1
A total of 25 Ashkenazi Jewish families were ascertained from Israel and the United States. Twenty-one of these families have been described previously (8 ) and an additional four families were recruited from Israel for this study. Consanguinity was present in three families on the panel in which the parents within each family were related to a common distant ancestor (8 ). Clinical diagnosis of HI was based on the following criteria (2 ,34 ,35 ): hypoglycemia with increased glucose utilization in infants that are large for gestational age and the combination of inappropriately elevated serum insulin levels, low plasma ketones and brisk response to glucagon injection. For this study, probands were classified as Ashkenazi Jewish when both parents were descended from eastern European ancestors (Germany, Czechoslovakia, Poland, Romania, Hungary, Ukraine, Russia or the Baltic States). Genomic DNA from probands and their families was isolated from Epstein-Barr virus (EBV)-transformed lymphoblasts or from peripheral blood lymphocytes using standard procedures. Genomic DNA from 89 unrelated, normal Ashkenazi Jewish individuals was kindly supplied by Dr D. Abliovitz (Department of Genetics, Hadassah Hospital, Jerusalem).
For SSCP analysis, individual exons and adjacent intron-exon boundaries were amplified by the polymerase chain reaction (PCR). Oligonucleotide sequences for SSCP were complementary to flanking intronic sequences (G. Gonzalez, L. Aguilar-Bryan and J. Bryan, GSDB accession nos L78208- L78222, L78224-L78243, L78254, L78255) and were designed to generate PCR products of 110-366 bp. Primer sequences and annealing temperatures for amplification of each exon are listed in Table 3 . Exons are numbered in consecutive order from the 5' end of the gene. For exon 25, the 366 bp PCR product was digested with the restriction endonuclease RsaI prior to electrophoresis through non-denaturing gels. Genomic DNA (50 ng) was amplified by PCR in a 10 [mu]l reaction containing: 10 pmol each primer, 0.2 mM each dNTP, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.0 U Taq DNA polymerase (Gibco) and 1 [mu]Ci [[alpha]-32P]-dCTP. For amplification of exon 1, the reaction mix included 5% formamide. Amplification conditions were an initial denaturation at 94oC (4 min); followed by 30 cycles of 94oC (1 min), optimal annealing temperature (1 min) and 72oC (1 min). Amplified samples were diluted two-fold with formamide buffer (95% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol), denatured at 94oC for 5 min and chilled on ice prior to electrophoresis. PCR products were analyzed on gels containing a 1/4 dilution of GeneAmp gel matrix (Perkin-Elmer) and 0.5*Tris-borate-EDTA (TBE), with or without 10% glycerol. Electrophoresis was done at 10-30 W for 5-12 h, at 4oC or room temperature (RT) and gels were exposed to Kodak X-OMAT film for autoradiography.
Nucleotide sequence analyses to identify mutations were done on patient samples exhibiting shifted bands relative to control samples on SSCP. Genomic DNA was amplified by PCR with primers used for SSCP analysis (Table 3 ). The resultant PCR products were excised from 2% low melting temperature (LMP) agarose gels, purified and directly sequenced by double-stranded DNA cycle sequencing (Perkin-Elmer) according to the manufacturer's instructions. Both strands of the PCR product were sequenced using PCR amplification primers end-labeled with 32P.
For mutations that created or abolished restriction endonuclease sites, the presence of the mutation was verified by restriction endonuclease digestion of PCR products with appropriate enzymes. To confirm the presence of the 3992-9g -> a and [Delta]F1388 mutations, genomic DNA was amplified using SSCP primers (Table 3 ) and the resultant PCR products were digested for 2 h with NciI or BseRI, respectively. Restriction digestion products were analyzed by electrophoresis on 3% NuSieve (3:1) (FMC BioProducts) agarose gels and were visualized by staining with ethidium bromide.
All available individuals within each family were genotyped with the four di- and tetranucleotide repeat polymorphic markers D11S1397, D11S902, D11S921 and D11S1890. D11S1397 is distal to the SUR1 locus but the relative order of D11S902- SUR1-D11S921-D11S1890 is unknown (18 -21 ). Haplotypes for 21 of these families at the D11S902 and D11S921 loci have been described previously (8 ). Oligonucleotide sequences and PCR amplification conditions for all microsatellite markers were obtained from Genome Data Base. Haplotypes were derived for parental chromosomes by inferring phase from their genotypes and those of their children: in 22 families there were at least two children, providing unequivocal haplotypes and in three families only one child was available for haplotype analyses. All haplotypes were constructed to minimize recombinants. For consanguineous marriages where the parental HI chromosomes were identical by descent (IBD), only one parental disease chromosome was included in the analyses. Control allele distributions were drawn from the non-carrier chromosomes within the same families.
Construction of the plasmids (i) pCMV-Kir6.2, expressing full-length murine Kir6.2, generously provided by Dr Susumu Seino (Chiba University, Japan) and (ii) pECE-SUR1 expressing full-length hamster SUR1 cDNA, have been described in detail elsewhere (10 ,13 ). F1388 was deleted from wild-type hamster SUR1 using an overlap PCR strategy. Briefly, complementary mutant oligonucleotides missing the phenylalanine codon (F) codon and two flanking primers were synthesized. The outside primers flanked unique restriction sites in the pECE-SUR1 plasmid. Two PCR products with a 20 bp overlap spanning the desired deletion were amplified using (i) the forward mutant primer (5'-AGTCCTCCTCCCTGGCCTTTTTCCG-3') with the downstream outside reverse primer (5'-CGTAGAGGATCCAGACATGA-3') and (ii) the forward outside primer (5'-GT- CATCTCCTATGTCACACC-3') and the reverse mutant primer (5'-AAAGGCCAGGGAGGAGGACTTCCCG-3'), respectively. Amplification was done with Taq DNA polymerase (Perkin-Elmer) for 30 cycles of 94oC for 1 min, 55oC for 1 min and 72oC for 1 min. The two resultant PCR products were purified by agarose gel electrophoresis, mixed and re-amplified using only the forward and reverse outside primers. The PCR product was digested with the restriction enzymes EcoRI and NotI, purified by agarose gel electrophoresis and subcloned using standard methodology into pECE-SUR1 (10 ) following digestion with EcoRI and NotI. The region amplified by PCR was sequenced to ensure no additional mutations were introduced during amplification. The resulting plasmid is designated pECE- [Delta]F1388SUR1.
COSm6 cells were maintained in Dulbecco's modified Eagle's medium with high glucose (DMEM-HG) and supplemented with 10% fetal calf serum (FCS). Cells were co-transfected with pCMV-Kir6.2 (5 [mu]g) and either pECE-SUR1 (5 [mu]g) or pECE-[Delta]F1388SUR1 (5 [mu]g) using DEAE-dextran as described previously (13 ). Co-transfected cells were incubated for 12-24 h with 86RbCl and assayed for efflux in the presence or absence of diazoxide, metabolic inhibitors (oligomycin and 2-deoxy-D-glucose) and glibenclamide as described in detail elsewhere (10 ,13 ). Co-transfected cells were photolabeled with 125I-glibenclamide as described previously (10 ,13 ).
We are grateful to the patients and their families for participating in this study. This work was supported in part by N.I.H. grants DK16746 (M.A.P.), RR-00240 (C.A.S.) and DK44311 (J.B.), by Grants #194164 (M.A.P.) and #195008 (J.B.) from the Juvenile Diabetes Foundation, by a Mentor-Based Fellowship Award from the American Diabetes Association (A.N., M.A.P.), Grant #93/00191/2 from the United States-Israel Binational Science Foundation (B.G., M.A.P.), Grant # 2677 from the Israel Ministry of Health (B.G.), and by grants from the Houston Endowment and American Diabetes Association (L.A.B.).
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*To whom correspondence should be addressed
+Present address: The Children's Hospital, Temple St, Dublin, Ireland
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