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Human Molecular Genetics Pages 1119-1128  

Genetic heterogeneity in familial hyperinsulinism
Introduction
Results
   Identification of mutations in the SUR1 gene
   Splice-site mutations
   Nonsense mutation
   Deletions and insertions
   Missense mutations
   Additional nucleotide sequence changes within the SUR1 gene
Discussion
Materials And Methods
   Families
   Haplotype analysis
   SSCP analysis
   Identification of mutations
References

Genetic heterogeneity in familial hyperinsulinism

Genetic heterogeneity in familial hyperinsulinism

Ann Nestorowicz1,+, Benjamin Glaser2, Beth A. Wilson1,§, Show-Ling Shyng3, Colin G. Nichols3, Charles A. Stanley4, Paul S. Thornton4,, M. Alan Permutt1,*

1Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, St Louis, MO 63110, USA, 2Department of Endocrinology and Metabolism, The Hebrew University, Hadassah Medical School, Jerusalem, Israel, 3Department of Cell Biology, Washington University School of Medicine, St Louis, MO 63110, USA and 4Division of Endocrinology/Diabetes, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Received February 6, 1998; Revised and Accepted April 24, 1998

Familial hyperinsulinism (HI) is a disorder characterized by dysregulation of insulin secretion and profound hypoglycemia. Mutations in both the Kir6.2 and sulfonylurea receptor (SUR1) genes have been associated with the autosomal recessive form of this disorder. In this study, the spectrum and frequency of SUR1 mutations in HI and their significance to clinical manifestations of the disease were investigated by screening 45 HI probands of various ethnic origins for mutations in the SUR1 gene. Single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses of genomic DNA revealed a total of 17 novel and three previously described mutations in SUR1. The novel mutations comprised one nonsense and 10 missense mutations, two deletions, three mutations in consensus splice-site sequences and an in-frame insertion of six nucleotides. One mutation occurred in the first nucleotide binding domain (NBF-1) of the SUR1 molecule and another eight mutations were located in the second nucleotide binding domain (NBF-2), including two at highly conserved amino acid residues within the Walker A sequence motif. The majority of the remaining mutations was distributed throughout the three putative transmembrane domains of the SUR1 protein. With the exception of the 3993-9G->A mutation, which was detected on 4.5% (4/88) disease chromosomes, allelic frequencies for the identified mutations varied between 1.1 and 2.3% for HI chromosomes, indicating that each mutation was rare within the patient cohort. The clinical manifestations of HI in those patients homozygous for mutations in the SUR1 gene are described. In contrast with the allelic homogeneity of HI previously described in Ashkenazi Jewish patients, these findings suggest that a large degree of allelic heterogeneity at the SUR1 locus exists in non-Ashkenazi HI patients. These data have important implications for genetic counseling and prenatal diagnosis of HI, and also provide a basis to further elucidate the molecular mechanisms underlying the pathophysiology of this disease.

INTRODUCTION

Familial hyperinsulinism (HI; OMIM 256450) is a disorder characterized by inadequate suppression of insulin secretion in the presence of severe, recurrent, fasting hypoglycemia (1-5). Although both autosomal dominant and recessive inheritance of HI have been described (6-8), in the majority of familial cases this disorder is inherited as an autosomal recessive trait. Clinical manifestations of HI, which occur predominantly in neonates and infants under 1 year of age, are secondary to hypoglycemia and include seizures, coma and large birth weight for gestational age (1-5). For most HI patients, treatment involving inhibitors of insulin secretion and/or a sub-total pancreatectomy is required to prevent permanent neurologic sequelae.

A locus for autosomal recessive HI was assigned by linkage and extended haplotype analyses to human chromosome 11p14-15.1 (9-11). Mutations in both the sulfonylurea receptor (SUR1) and Kir6.2 genes, which are clustered on chromosome 11p15.1, have been implicated in HI (12-18). Evidence from electrophysiological and pharmacological studies of COSm6 cells co-expressing the SUR1 and Kir6.2 proteins indicates that both molecules form subunits of the pancreatic [beta]-cell ATP-sensitive potassium (KATP) channel (19,20). The Kir6.2 subunit is proposed to impart the gating and inward rectification properties as well as K+ ion selectivity of the KATP channel, whereas SUR1 confers pharmacological properties (19-23). Recent studies suggest that both Kir6.2 and SUR1 contribute to the ATP/ADP sensitivity of IKATP (23,24). In islet [beta]-cells, such channels function as regulators of glucose-induced insulin secretion by coupling the metabolic status of the cell to the membrane potential (20,25,26). The metabolic regulation of [beta]-cell KATP channel activity by intracellular nucleotides is complex (27). Elevations in blood glucose concentration lead to increased rates of glucose metabolism in [beta]-cells and consequent alterations in the intracellular ratio of ATP/ADP, resulting in KATP channel closure. The subsequent depolarization of the [beta]-cell plasma membrane activates voltage-sensitive Ca2+ channels and the ensuing influx of Ca2+ initiates insulin secretion. However, ADP in the absence of Mg2+ may cause channel inhibition as a weaker analog of ATP and MgADP may antagonize ATP-mediated inhibition of channel activity (15,22,24,27). KATP channel activity can also be modulated by pharmacological agents which act by directly binding to the SUR1 subunit (21-24). Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) and inhibit KATP channel activity, thereby stimulating insulin secretion. In contrast, diazoxide, which regulates channel activity by binding to SUR1 in normal pancreatic [beta]-cells, prevents closure of KATP channels, thus inhibiting membrane depolarization and insulin secretion. Diazoxide has been used successfully in the treatment of some cases of HI (5).

The SUR1 molecule is a member of the adenosine triphosphate binding cassette (ABC) protein superfamily (28). An initial model for the SUR1 protein based on homology analyses predicted four domains: two nucleotide binding folds (NBF-1, NBF-2) and two membrane-spanning domains, each composed of multiple transmembrane regions (28). More recently, an alternative model for the membrane topology of SUR1 was proposed in which two N-terminal hydrophobic membrane-spanning domains precede NBF-1 and a third transmembrane domain is located between NBF-1 and NBF-2 (29). Both the NBF-1 and NBF-2 domains of SUR1 contain two amino acid sequence motifs (Walker A and Walker B) characteristic of all nucleotide binding domains (30,31), as well as a third conserved sequence motif (C) which distinguishes the ABC family from other nucleotide binding proteins (31). Evidence that HI is associated with molecular defects in SUR1 and pancreatic [beta]-cell KATP channel function have been provided by the identification of eight different mutations in the SUR1 gene which co-segregate with the disease phenotype (12-15,18). Furthermore, two of these SUR1 mutations (G1479R and [Delta]F1388) were shown to impair KATP channel activity in electrophysiological studies of COSm6 cells expressing wild-type or mutant SUR1/Kir6.2 channels (13,15), a finding consistent with the HI phenotype. Recently, electrophysiological studies on pancreatic [beta]-cells isolated from HI patients demonstrated an absence of native [beta]-cell KATP channel activity, leading to spontaneous, persistent membrane hyperpolarization which is predicted to result in unregulated insulin secretion in vivo (32).

We have previously demonstrated allelic homogeneity in HI patients of Ashkenazi Jewish descent (13). Two frequent mutations in the SUR1 gene, a G->A transition at position -9 of the 3[prime] splice site of intron 32 (3993-9G->A) and deletion of the codon for Phe at position 1388 ([Delta]F1388), are associated with ~88% of HI chromosomes in the Ashkenazim (13). In this study, the molecular genetic basis of HI in an additional 45 unrelated HI probands of diverse racial and ethnic origins were investigated. A total of 20 different mutations in the SUR1 gene, including 17 novel mutations were identified. With one exception, each of these mutations was present on only ~1-2% disease chromosomes, suggesting that HI displays wide allelic and/or locus heterogeneity in this predominantly non-Ashkenazi Jewish patient cohort. These data have implications for genetic counseling and prenatal diagnosis of HI in non-Jewish patients and provide a basis for further definition of the structure-function relationship of the SUR1 protein.

RESULTS

Identification of mutations in the SUR1 gene


Figure 1. Schematic diagram of the locations of 20 mutations identified in the SUR1 gene in HI patients. All 39 exons of the SUR1 gene are depicted as closed boxes and are not drawn to scale. Splice-site mutations, insertions and deletions are shown below the gene and missense and nonsense mutations are indicated above the gene.

Probands from 44 unrelated HI families of diverse ethnic origins were screened for the presence of mutations in all 39 exons and flanking intron-exon boundaries of the SUR1 gene by single-strand conformation polymorphism (SSCP) analyses of genomic DNA. In addition, partial SSCP analysis was performed on DNA obtained from a single Ashkenazi Jewish proband. Mutations in DNA samples displaying electrophoretic mobility shifts, relative to control samples on SSCP, were identified by nucleotide sequence analyses of polymerase chain reaction (PCR) products re-amplified from genomic DNA samples. A total of 43 nucleotide sequence changes within the SUR1 gene were identified. Twenty of these sequence changes are classified as potential disease-causing mutations (Table 1 and Fig. 1) and the remainder as putative polymorphisms (Table 2 and see below). Seventeen of the putative causal mutations are novel. These include nonsense and missense mutations, frameshifts (due to small deletions), an insertion and splice-site mutations that are discussed in detail below. Representative examples of SSCP and nucleotide sequence analyses are given for each class of mutation (see below). In addition to SSCP and nucleotide sequence analyses of the SUR1 gene, DNA samples from all probands were analyzed by restriction fragment length polymorphism (RFLP) analyses for the presence of six previously described mutations in the SUR1 gene (12-15). Mutations in SUR1 were defined for both alleles in seven patients, 12 patients were presumed to be compound heterozygotes for a SUR1 mutation and an unknown mutation, and no SUR1 mutations were identified in the remaining 26 probands. Where DNA samples from first-degree relatives were available, mutations were examined for co-segregation with the HI phenotype by PCR amplification of the relevant exon and flanking intron-exon boundaries, followed by digestion of the resultant PCR products with restriction enzymes that allow mutant and wild-type alleles to be differentiated. In those cases where restriction endonuclease sites were neither created nor abolished by a specific mutation, allele-specific assays were developed (see below). Allele frequencies of each mutation for HI chromosomes were estimated by restriction enzyme digestion of PCR products amplified from genomic DNA of all 45 probands. Control allele distributions for mutations were estimated using genomic DNA obtained from 80-100 unrelated, unaffected normal individuals of Ashkenazi Jewish, Palestinian Arab or Caucasian populations.

A summary of the SUR1 mutations identified in the patient cohort, restriction endonucleases used to detect each mutation and allele frequencies on HI chromosomes are presented in Table 1.

Table 1. Mutations within the SUR1 gene
Patient Exon or intron Nucleotide changea Codonapredicted effect Domainb Restriction site change Frequency (%) HI chromosomes (n = 88) Segregation demonstrated Frequency 200 normal chromosomes
A1 exon 2 221G->A R74Q Tm PstI 1 (1.1%) NA 0
B2d exon3 375C->G H125Q Tm DdeIc 1 (1.1%) NA 0
C3e exon 4 563A->G N188S Tm TspRI 1 (1.1%) yes 0
D4 exon 6 949delC 317fs/ter Tm Bsp1286I 1 (1.1%) yes 0
E5 exon 8 1216A->G N406D Tm XcmI 1 (1.1%) NA 0
F6f intron 10 1630+1G->T aberrant splicing Tm BsrI 2 (2.3%) yes 0
G7 exon 12 1773C->G F591L Tm BsoF1 1 (1.1%) no 0
H8 exon 13 1893delT 631fs/ter Tm BstNI 1 (1.1%) yes 0
F6f intron 15 2117-1G->A aberrant splicing NBF-1 PstI 1 (1.1%) yes 0
I9 exon 24 2860C->T Q954X - BstNI 1 (1.1%) yes 0
J10g exon 28 3416C->Th T1139M Tm NlaIII 1 (1.1%) yes 0
K11 exon 29 3644G->A R1215Q Tm NciI 1 (1.1%) yes 0
J10g intron 32 3992-9G->Ai aberrant splicing NBF-2 NciI 4 (4.5%) yes 0
C3e intron 32 3992-3C->G aberrant splicing NBF-2 AvaI 1 (1.1%) yes 0
L12 exon 34 4135G->C G1379R NBF-2 EagI 1 (1.1%) yes 0
M13 exon 34 4144G->A G1382S NBF-2 BglI 1 (1.1%) yes 0
B2d exon 34 4162delTTCi,j delF 1388 NBF-2 BseRI 1 (1.1%) yes 0
J10g exon 34 4181G->Ah R1394H NBF-2 DraIII 1 (1.1%) yes 0
N14 exon 35 4310G->Ai aberrant splicing NBF-2 MspI 1 (1.1%) yes 0
O15 exon 37 4525insCGGCTT insertion of AlaSer after codon 1508 NBF-2 PvuIIk 1 (1.1%) yes 0
aNucleotide and codon positions are according to the full-length human SUR1 cDNA sequence incorporating the alternative spliced form of exon 17 (GenBank accession nos L78208 and L78216).
bTm and NBF refer to the transmembrane and nucleotide binding domains, respectively.
cRestriction enzyme site was created using mismatched primers as described in Materials and methods.
d-gProbands with more than one identified mutation in the SUR1 gene.
hMutation present on a complex allele.
iMutation described previously (12,13).
jNucleotide sequence analysis of mutant alleles do not indicate whether nucleotides 4162-4164 or 4163-4165 are deleted, but either mutation results in deletion of codon 1388 (13).
kMutation detected as described in Materials and methods.

Table 2. Novel polymorphisms identified within the SUR1 gene
Region Nucleotide changea Codona Amino acid substitution Domainb Reason for exclusion
Exon 2 207T->C 69 none Tm silent
Exon 3 330C->T 110 none Tm silent
Intron 3 412+18C->T - - Tm intronic
Exon 4 423G->A 141 none Tm silent
Intron 4 579+14C->T - - Tm intronic
Intron 4 579+29G->C - - Tm intronic
Intron 18 2294-36T->C - - NBF-1 normal chromosomes
Intron 18 2294-34C->T - - NBF-1 intronic
Intron 27 3402+13G->A - - Tm intronic
Intron 27 3402+45C->A - - Tm intronic
Exon 29 3615G->A 1115 none Tm silent
aNucleotide and codon positions are according to the full-length human SUR1 cDNA sequence incorporating the alternative spliced form of exon 17 (GenBank accession nos L78208 and L78216).
bTm and NBF refer to transmembrane and nucleotide binding domains, respectively.

Splice-site mutations

Three novel mutations in SUR1 that are predicted to result in aberrant splicing were identified in four patients by SSCP and nucleotide sequence analyses (Table 1). In patient C3 [who also had a missense mutation in exon 4 (563A->G)], SSCP and nucleotide sequence analyses of exon 33 revealed a heterozygous C->G transversion at position -3 of the 3[prime] splice site of intron 32 (designated 3992-3C->G; Figs 2a and b). This mutation is predicted to impair mRNA splicing since (i) it occurs at a position which is occupied by a pyrimidine in 97% of primate genes (33) and (ii) analogous mutations in other genes have been shown to result in splicing defects in vitro or in vivo (34,35). Since a recognition site for the restriction endonuclease AvaI is created by the3992-3C->G mutation, co-segregation of this mutation with the disease phenotype in the family of proband C3 was examined by PCR amplification of exon 33 from genomic DNA, followed by digestion of the resultant PCR products with AvaI.Both the father and proband were heterozygous for this mutation, possessing the 165 and 103 bp fragments derived from the mutant 3992-3C->G allele, as well as the wild-type allele product of 268bp (Fig. 2c).

Figure 2. Identification of a splice site mutation in the SUR1 gene. (a) SSCP analyses of exon 33 in seven unrelated individuals. Lane 1, control unaffected individual; lanes 2-7, six HI probands. Band shifts in a DNA sample (lane 4), possessing the 3992-3C->G mutation, are indicated by arrows. (b) Nucleotide sequence analyses of PCR products encompassing exon 33 amplified from genomic DNA samples shown in lanes 1 (normal) and 4 (patient) in (a). Exonic and intronic sequences are shown in upper and lower case, respectively. (c) Restriction fragment length polymorphism (RFLP) analyses of kindred C possessing the 3992-3C->G mutation. DNA fragments encompassing exon 33 were amplified by PCR from genomic DNA of available family members and digested with AvaI. Restriction digestion products were analyzed on a 3.5% NuSieve (3:1) agarose gel and visualized by staining with ethidium bromide. Lane 1, 123 bp ladder (Gibco); lane 2, undigested 268 bp PCR product; lane 3, paternal DNA, indicating the presence of the wild-type allele (268 bp) product and mutant allele products of 165 and 103 bp paternal DNA; lane 4, maternal DNA; lane 5, proband; lane 6, control without genomic DNA.


A novel G->T transition (1630+1G->T) at the conserved GT dinucleotide of the 5[prime] splice site of intron 10 was detected in two unrelated probands (F6 and P16) of Swedish descent (Table 1 and data not shown). BsrI restriction enzyme analyses of PCR products encompassing exon 9 amplified from the families of both probands indicated that the 1630+1G->T mutation was present on thematernaldisease chromosome of F6 and on the paternaldisease chromosome of P16 (data not shown).In proband F6, SSCP and nucleotide sequence analyses of exon 16 revealed a third novel splice site mutation, 2117-1G->A (Table 1 and data not shown). Although this mutation eliminates a PstI restriction endonuclease site, the same site is also absent in individuals possessing the adjacent common polymorphism 2117-3C->T (13). Therefore, to differentiate betweenthe 2117-3C->T polymorphism and 2117-1G->A mutation, an artificial PstI site was introduced into PCR products amplified from genomic DNA containing the 2117-3C->T polymorphism using a forward primer containing the wild-type C at position 2117-3, in combination with the exon 15 reverse primer (13). Thus, 2117-3C->T polymorphic and wild-type sequences may be distinguished from 2117-1G->A mutant alleles by the presence or absence of a PstI site, respectively. Analysis of kindred F using this allele-specific assay revealed that the 2117-1G->A mutation was present on the paternal HI chromosome (data not shown).

In addition to the three novel mutations described above, two previously characterized splice site mutations, a G->A transition at position -9 of the 3[prime] splice site of intron 32 (3993-9G->A) and a G->A transition at the last nucleotide of exon 35 (position -1 of the 5[prime] splice site of intron 35; 4310G->A), were also detected by SSCP and nucleotide sequence analyses of exons 33 and 35, respectively (Table 1 and data not shown) (12,13). Both mutations have been shown previously to activate cryptic splice sites in vitro and are predicted to result in frameshifts and premature termination of translation of SUR1 mRNA (12).

All five splice-site mutations were detected in the heterozygous state in the individual probands and each mutation was observed to co-segregate with the disease phenotype (Fig. 2c and data not shown). The 3993-9G->A mutation was the most common mutation detected in the HI probands in this study, occurring on 4.5% (4/88) HI chromosomes. Two of the HI chromosomes bearing this mutation were inherited from distant Ashkenazi Jewish ancestors, consistent with previous data indicating that the 3993-9G->A mutation is associated with ~69% HI chromo-somes in the Ashkenazim (13).Allelic frequencies for the 1630+1G->A, 2117-1G->A, 3992-3C->G and 4310G->A mutations were between 1.1 and 2.3% for HI chromosomes (Table 1). None of these mutations were detected on 200 chromosomes analyzed from 100 control individuals (data not shown).

Nonsense mutation

An electrophoretic mobility shift relative to control samples was observed in DNA from patient I9 by SSCP analysis of exon 24 (data not shown). Nucleotide sequence analysis of PCR products amplified from genomic DNA of proband I9 revealed a C->T transition at nucleotide (nt) 2860 (Fig. 3a and Table 1). This point mutation results in the introduction of a termination codon (TAG) for Gln (CAG) at position 954 and is designated Gln954Ter. The SUR1 protein product encoded by this mutant allele would be predicted to lack NBF-2. Since a BstNI restriction site in exon 24 is abolished by the Gln954Ter mutation, this property of the mutant allele was used to demonstrate co-segregation ofthe mutation with the HI phenotype in the family of proband I9 (Fig. 3b).BstN1 restriction enzyme analysis indicated that the proband and father were heterozygous for the Gln954Ter mutation, whereas the mother and an unaffected sibling were homozygous for the wild-type allele (Fig. 3b). The Gln954Ter mutation was detected on 1.1% (1/88) HI chromosomes but was absent in 100 control DNA samples (data not shown).

Figure 3. Identification of a nonsense (Gln954Ter) mutation in the SUR1 gene. (a)Nucleotide sequence analyses of PCR products encompassing exon 24 amplified from genomic DNA of the proband (I9) and an unaffected control individual (normal). (b) BstNI restriction enzyme analysis of PCR products, encompassing exon 24, amplified from genomic DNA of kindred I. PCR products amplified from mutant Gln954Ter alleles are digested into 147 and23 bp fragments whereas wild-type allele products are digested into 90, 57and 23 bp. The 23 bp fragment is not visible on the gel under the conditions used in this assay. Lane 1, 123 bp ladder (Gibco); lane 2, undigested 170 bp PCR product; lane 3, paternal DNA heterozygous for the Gln954Ter mutation; lane 4, maternal DNA homozygous for the wild-type allele; lane 5, proband; lane 6, unaffected sibling; lane 7, control without genomic DNA.

Deletions and insertions

Two novel 1 bp deletions were identified in patients D4 and H8 (Table 1). A 1893delT mutation was detected in exon 13 in proband H8, who is of Cajun descent (data not shown). This mutation is predicted to result in a frameshift at codon 631, resulting in the introduction of a premature termination codon at position 645and consequent deletion of both NBFs in the translated SUR1 product. Co-segregation of the 1893delT mutation with the disease phenotype was observed in the family of proband H8 (data not shown). PCR amplification of exon 13 from genomic DNA of available family members, followed by BstNI digestion of PCR products, indicated that both parents of the proband and five of six unaffected siblings were heterozygous for this mutation. In contrast, the proband was homozygous for the 1893delT mutation and one unaffected sibling inherited both maternal and paternal wild-type alleles. These data are consistent with the results of extended haplotype analyses which revealed identical paternal and maternal disease chromosome haplotypes for the region spanning D11S926-D11S1890, which encompasses the SUR1 locus (data not shown) (36,37). Although both parents of the proband were heterozygous for this rare mutation, a consanguineous relationship between these individuals was not evident from the available family history. It is possible, therefore, that the parents of proband H8 are either related to a common distant ancestor and/or that a founder chromosome bearing the 1893delT mutation exists within the Cajun population. Patient H8 had relatively mild disease that was diagnosed at 16 months of age and treated with diazoxide until age 5.

Proband D4, who is from a consanguineous Palestinian Arab family, was found to possess a homozygous 1 bp deletion in exon 6 (Table 1). Since this mutation occurs at a CC dinucleotide in the SUR1 cDNA sequence, it is not possible to determine whether nucleotide 949 or 950 is deleted in the mutant allele. In either case, this deletion is predicted to result in a frameshift at codon 317, resulting in the introduction of a termination codon at position 358 and synthesis of a truncated SUR1 molecule lacking both NBFs. Results of Bsp1286I restriction enzyme analyses of PCR products amplified from family members of proband D4 were consistent with co-segregation of the 949delC mutation with the HI phenotype (data not shown). In contrast with patient H8, all four affected members of the D family had severe disease that was clinically evident at birth. None of the affected siblings responded to diazoxide and each required a pancreatectomy. Persistent hypoglycemic symptoms were present in all affected children after surgery.

SSCP and nucleotide sequence analyses of PCR products encompassing exon 37 revealed a heterozygous in-frame insertion of 6 nt (CGGCTT) in patient O15 (Table 1). Since the inserted sequence is a direct duplication of nucleotides 4526-4531 in the wild-type cDNA sequence, it is unknown whether the insertion is located after nucleotide 4525 or 4531. The SUR1 molecule encoded by this mutant allele is predicted to contain an insertion of AlaSer after either codon 1508 or 1510.

In addition to the novel insertion and deletions described above, a previously described deletion of Phe at codon 1388 was detected by RFLP analysis in a proband (B2) of Ashkenazi descent (Table 1 and data not shown) (13). Each of the 949delC, 1893delT, [Delta]F1388 and 4525insCGGCTT mutations were detected on a single HI chromosome in the patient cohort (Table 1) and were absent in 100 control DNA samples (data not shown).

Missense mutations

Ten novel missense mutations were identified in nine unrelated families (Table 1). Representative SSCP (data not shown) and nucleotide sequence analysis for one of these mutations, an Arg to Gln substitution at codon 1215 (Arg1215Gln), is shown in Figure 4. With the exception of those mutations detected in patients A1, B2 and E5, for whom DNA samples from informative first-degree relatives were unavailable, co-segregation of each of the identified missense mutations with the HI phenotype was examined by PCR amplification of the relevant exon from genomic DNA and subsequent digestion with appropriate restriction enzymes (Fig. 4b and data not shown). For proband G7, BsoF1 restriction enzyme analysis indicated that although the proband possessed a Phe591Leu mutation in the heterozygous state, this mutation was neither present in the proband's parents nor in two unaffected siblings (data not shown). Nucleotide sequence analyses confirmed the absence of the Phe591Leu mutation in all first-degree relatives of proband G7. Since the results of extended haplotype analyses for kindred G were consistent with Mendelian inheritance for all offspring (data not shown), these data suggest that the Phe591Leu substitution occurred de novo in proband G7. With the exception of the Phe591Leu mutation, all other missense mutations examined co-segregated with the HI phenotype and displayed Mendelian inheritance (Fig. 4b and data not shown). For proband J10, restriction enzyme analyses also revealed the presence of a maternal complex allele harboring two missense mutations, Thr1139Met and Arg1394His (Table 1 and data not shown).

Figure 4. Identification of an Arg1215Gln mutation in the SUR1 gene. (a) Nucleotide sequence analyses of PCR products, encompassing exon 29, amplified from control (normal) and proband K11 (patient) DNA samples. The nucleotide sequence shown is of the reverse complementary DNA strand. (b) Restriction enzyme analysis of kindred K. PCR products encompassing exon 29 were amplified from genomic DNA of family members and digested with NciI. Lane 1, 123 bp ladder (Gibco); lane 2, undigested 305 bp PCR product; lane 3, paternal DNA; lane 4, maternal DNA, indicating the presence of the mutant uncut allele (305 bp) product and wild-type allele products of 177 and 128 bp; lane 5, unaffected sibling homozygous for the wild-type allele; lanes 6 and 7, affected siblings heterozygous for the Arg1215Gln mutation; lane 8, control without genomic DNA.

Each of the 10 missense mutations was rare, occurring on a single HI chromosome (Table 1). None of these mutations was detected in genomic DNA samples obtained from 100 unrelated, normal individuals.

Additional nucleotide sequence changes within the SUR1 gene

In addition to the 20 SUR1 mutations described above, another 23 point mutations in the SUR1 gene were identified that were classified as putative polymorphisms since they (i) are not predicted to alter the primary structure of the SUR1 molecule and/or (ii) are present on normal chromosomes. Furthermore, calculation of the Shapiro/Senepathy consensus values (33) for all sub-sequences encompassing each of the 23 polymorphisms suggested that new 5[prime] or 3[prime] splice sites were unlikely to be created by any of these nucleotide sequence changes (data not shown). Eleven of the putative polymorphisms were novel (Table 2) and the remaining 12 polymorphisms have been described previously (13). The reasons for exclusion of these variants as potential mutations is given in Table 2. As none resulted in codon changes or splice mutations, they were not screened in normal controls. Although the 579+14C->T and the 3402+13G->A mutations have been tentatively classified as polymorphisms on the basis that they are located in intronic sequences but not within consensus splice site or branch-point sequence motifs, the possibility that one or both of these sequence changes are causal mutations cannot be excluded at present. Both the 579+14C->T and 3402+13G->A sequence changes were detected in onlyone and two probands, respectively, for whom no other SUR1 mutations were identified, and neither mutation was observed in 100 control DNA samples (data not shown). Further studies are required to clarify the functional significance of the 579+14C->T and 3402+13G->A nucleotide sequence changes, as well as the other 21 identified polymorphisms, in the pathophysiology of HI.

DISCUSSION

KATP channels are involved in regulating glucose-dependent stimulus secretion coupling in pancreatic [beta]-cells (25,26). The SUR1 protein forms one subunit of the islet [beta]-cell KATP channel and is proposed to modulate channel activity via interactions with cytosolic nucleotides (19,20,24). To date, only eight SUR1 mutations have been reported in HI patients (12-15,18). In the present study, 16 novel mutations in the SUR1 gene in non-Ashkenazi Jewish probands and one novel mutation in a proband of Ashkenazi descent were identified (Table 1). Three previously described mutations, 3993-9G->A, 4310G->A and [Delta]F1388, were also detected (Table 1; 12,13). The cumulative data from this and other studies (12-15,18) indicate that mutations in SUR1 are distributed throughout the entire molecule but are predominantly located within NBF-2 and the transmembrane domains preceding NBF-1 (Table 1; 28,29). Since the total number of mutations defined is relatively small, analysis of additional HI patients is required to determine whether the observed clustering of mutations is biologically relevant and reflects intrinsic properties of the nucleotide sequence and/or critical contributions of the Tm and NBF-2 regions to SUR1 protein structure and function.

Although all 39 exons of the SUR1 gene were screened by SSCP analyses in 44 non-Ashkenazi probands, mutations associated with only 26.7% (23/86) of HI chromosomes were defined. The predominant mutation, 3993-9G->A, occurred on 4.5% HI alleles whereas all other mutations described were associated with 1.1-2.3% of HI chromosomes. Although these data suggest that SUR1 mutations identified in non-Ashkenazi probands are rare, it is conceivable that the relative frequencies of some of these mutations may vary amongst HI patients of different ethnic groups, as has been observed for the 3993-9G->A and [Delta]F1388 mutations (13; Table 1). The failure to detect mutations in SUR1 in the majority of HI alleles in the patients analyzed in this study may be due to several reasons. Firstly, mutations may be present in the untranslated regions of the SUR1 gene which were not analyzed by SSCP. Secondly, some cases of HI may be attributable to mechanisms affecting gene expression not apparent from the strategies used here for mutational analysis of SUR1. Finally, it is also possible that the disease phenotype is not linked to the SUR1 locus in all families ascertained for this study. Recent evidence suggests that a locus for an autosomal dominant form of HI is not linked to SUR1 (8) and that mutations in the Kir6.2 locus are also associated with the HI phenotype (16,17). However, SSCP analyses of the Kir6.2 gene in 44 of the probands failed to reveal any putative causal mutations (16), raising the possibility that mutations at loci other than SUR1 or Kir6.2 are responsible for the disease phenotype in a proportion of the families described here. Together, these data have implications for the early diagnosis and genetic counseling of affected children and their families. While standard mutation screening for the 3993-9G->A and [Delta]F1388 SUR1 mutations may identify ~88% HI alleles in the Ashkenazim (13), direct testing for specific mutations in non-Ashkenazi patients may be complicated by the existence of a large number of different mutant SUR1 alleles and, perhaps, differences in their geographic and ethnic distribution.

Splice-site, nonsense and frameshift mutations that are predicted to introduce premature termination signals for translation of SUR1 mRNA were identified (Table 1). Although the precise mechanism(s) by which these mutations cause loss of SUR1 and KATP channel activity await studies of mutant and wild-type mRNA transcripts in pancreatic tissue, premature termination codons in other genes have been shown to result in (i) reductions in steady-state mutant mRNA levels, (ii) aberrant splicing through exon skipping, intron retention or activation of cryptic splice sites, (iii) generation of an unstable mRNA and no detectable protein or (iv) production of an aberrant protein, containing deleted or novel sequences, which may undergo rapid degradation (for reviews see refs 34,38,39). Each of the identified mutations resulting in the introduction of a premature termination codon predicts the synthesis of a truncated SUR1 molecule lacking either one or both NBFs (Table 1). Since a functional SUR1 subunit in pancreatic cells is essential for KATP channel activity, mutations resulting in an absence of this protein in HI patients would lead to spontaneous [beta]-cell membrane depolarization and dysregulation of insulin secretion. Although little is known about the specific role(s) of NBF-1 and -2, evidence from site-directed mutagenesis studies of SUR1 suggests that Mg2+-independent ATP binding occurs at NBF-1 and that MgADP stimulation of KATP channel activity, via antagonism of ATP mediated channel inhibition, occurs through binding to NBF-2 (15,22-24). Deletion of the NBF-2 domain due to mutations in the SUR1 gene (Table 1), would therefore be predicted to result in unresponsiveness of SUR1 to MgADP, resulting in a net reduction of channel activity at any ATP/ADP ratio (15), a finding consistent with the HI phenotype. No correlations between genotype and phenotype were observed in patients (D4 and H8) who were homozygous for mutations predicted to result in deletion of both NBFs. Although patient H8 had relatively mild disease, which was responsive to diazoxide, all four HI patients in the family of proband D4 exhibited severe disease and required pancreatectomies. At present, the mechanism(s) responsible for such differences in the severity of clinical symptoms in HI patients possessing mutations resulting in deletion of both NBFs and formation of a presumed non-functional SUR1 subunit and KATP channel remain to be clarified.

The 10 novel missense mutations identified in SUR1 are distributed throughout the SUR1 molecule (Table 1). Two of these mutations, Gly1379Arg and Gly1382Ser, are located at glycine residues 1 and 2, respectively, of the Walker A consensus sequence motif (GXXGXGKS) in NBF-2 (28,30). Both Gly1 and Gly2 amino acid residues are strictly conserved between eukaryotic ABC proteins (31,40) and substantial evidence exists for the importance of these residues in the structure and function of other members of the ABC superfamily. Molecular modeling of the Walker A (P-loop) motif of ATP-binding proteins indicates that both Gly1 and Gly2 are required to maintain the correct conformation of the polypeptide for binding of ATP/ADP (41,42). Replacement of the corresponding Gly2 residue in F1-ATPase (42) and Gly1 residues in histidine permease (43)and F1-ATPase (42) results in either non-functional or dysfunctional molecules, indicating that both residues are critical for function of these ABC proteins. Furthermore, an analogous mutation (G716V) at Gly2 in the Walker A sequence motif in NBF-1 of the SUR1 protein has also been described in a HI patient (14). We have recently found that introduction of the Gly1382Ser mutation into SUR1 results in reduced KATP channel activity in vitro due to a decreased response to activation by MgADP (manuscript submitted). It is possible, therefore, that both the Gly1379Arg and Gly1382Ser mutations modify activity and/or regulation of the SUR1 protein by disrupting interactions of NBF-2 with cytosolic nucleotides, either by altering the conformation of the nucleotide binding pocket of NBF-2 or by occurring at residues that directly participate in nucleotide binding. A third missense mutation, Arg1394His, is located 8 amino acid residues downstream from the Walker A motif in NBF-2 and resides in a putative [alpha]-helix comprising residues of the adjacent Walker A sequence (28,30,31). Electrophysiological studies of R1394H-SUR1/Kir6.2 mutant channels indicates that KATP channel activity is abolished by the Arg1394His substitution (S.-L.Shyng, C. Nichols and M. Permutt, unpublished data). Since the Arg1394His mutation occurs at a position which is not highly conserved between ABC proteins, it is unknown whether this mutation impairs channel activity by causing defective protein folding and transport of the nascent SUR1 polypeptide or by altering the tertiary structure required for SUR1 function. Missense mutations have been shown to result in misfolding and subsequent degradation of the nascent polypeptide in a number of diseases (44), including cystic fibrosis (45-48) and X-linked adrenoleukodystrophy (49). It will be of interest to determine whether similar mechanisms are responsible for the observed defects in KATP channel activity arising from the Arg1394His and other missense mutations in HI.

A model for SUR1 (29) predicts that the remaining seven missense mutations (Arg74Gln, His125Gln, Asn188Ser, Asn406Asp, Phe591Leu, Thr1139Met, Arg1215Gln) are located either within transmembrane segments or are present on the extracellular or cytoplasmic loops connecting adjacent transmembrane helices. The identification of these missense mutations in HI patients provides a basis for further studies to elucidate the functions of these transmembrane domains in SUR1 and KATP channel activity. We have found that the His125Gln, Phe591Leu, Thr1139Met and Arg1215Gln mutations result in various reductions in the sensitivity of KATP channels to stimulation by MgADP in vitro (S.-L.Shyng et al., unpublished data). Furthermore, increased sensitivity of KATP channel activity to inhibition by ATP was observed in channels containing the Phe591Leu mutation. The functional significance of the Arg74Gln and Asn406Asp mutations upon SUR1 regulation of KATP channel activity are currently unknown. Expression of SUR1 and KATP channels containing these missense mutations and their electrophysiological characterization by single-channel recordings are required to provide further insight into the roles of these mutations in the pathophysiology of HI.

MATERIALS AND METHODS

Families

Forty-five kindreds of diverse ethnic origins were recruited via ascertainment of HI affected index cases. Five probands were of Danish descent, one Ashkenazi Jewish, two Mexican, one Swedish, 11 of mixed northern European origin, two Sephardic Jewish, two Arabic, three African/American, one African and one Cajun. Data on the ethnic origins of the remaining 16 families were unavailable. Forty-two families contained a single affected child and three families possessed multiple affected siblings. Consanguinity was known in two families on the panel in which the parents within each family were related to a common distant ancestor. Clinical diagnosis of HI was based upon the following criteria (50): 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. Genomic DNA was isolated from Epstein-Barr virus (EBV)-transformedlymphoblasts or from peripheral blood lymphocytes using standard procedures. Control DNA samples were obtained from 100 unrelated, normal Palestinian Arabs recruited in Eastern Jerusalem, 100 normal Caucasian individuals (kindly supplied by Dr B. Zehnbauer, Molecular Diagnostic Laboratory, Washington University, St Louis) and 89 unrelated, normal Ashkenazi Jews (supplied by Dr D. Abliovitz, Department of Genetics, Hadassah Hospital, Jerusalem). Patient records were reviewed and referring physicians asked to evaluate the clinical severity and responsiveness of each patient to diazoxide.

Haplotype analysis

All available individuals within each family were genotyped with the four di- or 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 has not been definitively established (36). Oligonucleotide sequences and PCR amplification conditions for all microsatellite markers were obtained from the Genome Data Base. In 42 of 45 families, haplotypes were derived for parental chromosomes by inferring phase from their genotypes and those of their children: in 30 families there were at least two children, providing unequivocal haplotypes, and in the remaining 15 families only one child was available for haplotype analysis. In three families, both parents were unavailable for genotyping. All haplotypes were constructed to minimize recombinants.

SSCP analysis

Individual exons and adjacent intron-exon boundaries of the SUR1 gene (GenBank accession nos L78208 and L78216) were amplified from genomic DNA by the polymerase chain reaction (PCR) and screened for the presence of mutations using SSCP as described in detail elsewhere (13). Oligonucleotide sequences for SSCP analyses were complementary to flanking intronic sequences and were designed to generate PCR products of 110-366 bp (13). For exon 25, the 366 bp PCR product was digested with the restriction endonuclease RsaI prior to electrophoresis through non-denaturing gels. Amplified samples were diluted 2-fold with formamide buffer (95% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol), denatured at 94°C for 5 min and chilled on ice prior to electrophoresis. PCR products were analyzed on gels containing a ¼ dilution of GeneAmp gel matrix (Perkin-Elmer) and 0.5× Tris-borate-EDTA (TBE) buffer, with or without 10% glycerol. Electrophoresis was performed at 10-30 W for 5-12 h at 4°C or room temperature (RT) and gels were exposed to Kodak X-OMAT film for autoradiography.

Identification of mutations

Mutations in DNA samples exhibiting altered electrophoretic mobilities, relative to control samples, on SSCP gels were identified by nucleotide sequence analysis. Genomic DNA was amplified by PCR with primers used for SSCP analysis (13). The resultant PCR products were isolated by electrophoresis on 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 genomic DNA samples from patients possessing the 1893delT and 4525insCGGCTT mutations (Table 1), the resultant PCR products were subcloned into the vector, pGEM-T (Promega, Wisconsin), prior to double-stranded cycle sequencing of recombinant plasmid DNA. For each mutation, both strands of the subcloned PCR fragment were sequenced in 10 separate recombinant plasmids.

The presence of mutations that either created or abolished restriction endonuclease sites were verified by digestion of PCR products amplified from genomic DNA with appropriate restriction enzymes (Table 1). To detect the His125Gln mutation, which did not alter a restriction enzyme site, PCR-directed site-specific mutagenesis was used to incorporate a DdeI restriction site into products derived from wild-type alleles. Genomic DNA was amplified by PCR using the mismatched forward primer: 5[prime]-CACCTCCGTGGTCTACTCTCA-3[prime], in combination with the reverse primer used for SSCP analysis of exon 3 (13), followed by digestion of the resultant PCR products with DdeI. Although the 2117-1G->A mutation eliminates a PstI site, this mutation cannot be differentiated from the common 2117-3C->T polymorphism (13) which eliminates the same PstI site. Therefore, a PstI site was created in PCR products derived from genomic DNA containing the 2117-3C->T polymorphism by using a forward oligonucleotide derived from the wild-type sequence: 5[prime]-ACTCACATCTGCCACCCTCCCTCCCTGCA-3[prime], together with the reverse oligonucleotide used for SSCP analysis of exon 16 (13). PCR products generatedfrom 2117-1G->A mutant alleles lack the PstI site, whereas wild-type and 2117-3C->T allele products possess the PstI site. Restriction digestion products were analyzed by electrophoresison 3.5% NuSieve (3:1) agarose (FMC BioProducts) gels and were visualized by staining with ethidium bromide. The 4525insCGGCTT was detected by PCR amplification of genomic DNA using SSCP primers for exon 37 (13) end-labeled with 32P, followed by PvuII digestion of the amplified fragments. Restriction digestion products were then electrophoresed on 6% polyacrylamide gels containing 7 M urea and 1× TBE buffer (pH 8.3), followed by exposure to Kodak X-OMAT film for autoradiography. Insertions in genomic DNA samples were detected as alterations in the electrophoretic mobilities of PCR fragments and were characterized by nucleotide sequence analyses as described above.

Genomic DNA samples were also analyzed for the presence ofsix previously described mutations (1671-20A->G, G716V, 2291-1G->A, 3993-9G->A, [Delta]F1388 and G1479R) by PCR amplification of relevant exons and flanking intron-exon boundaries, followed by restriction enzyme digestion as described in detail elsewhere (12-15).

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*To whom correspondence should be addressed at: Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, Box 8127, 660 South Euclid Avenue, St Louis, MO 63110, USA. Tel: +1 314 362 8680; Fax: +1 314 747 2692; Email: apermutt@imgate.wustl.edu
Present addresses: +Division of Endocrine Research, Eli Lilly and Company, Indianapolis, IN 46285, USA, §Department of Genetics, Washington University School of Medicine, St Louis, MO 63104, USA and [Dagger]The Children's Hospital, 1 Temple Street, Dublin, Ireland

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