Mutation of the pancreatic islet inward rectifier Kir6.2 also leads to familial persistent hyperinsulinemic hypoglycemia of infancy
Mutation of the pancreatic islet inward rectifier Kir6.2 also leads to familial persistent hyperinsulinemic hypoglycemia of infancyPamela Thomas*, Yuyang Ye and Elmer Lightner1
Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI 48109, USA and 1Department of Pediatrics, University Medical Center, Tucson, AZ 85724, USA
Received July 1, 1996;Revised and Accepted August 27, 1996
Closure of ATP-sensitive potassium channels in pancreatic islet [beta]-cells initiates a cascade of events that leads to insulin secretion. [beta]-Cell ATP-sensitive potassium currents can be reconstituted by coexpression of the inward rectifier Kir6.2 and the sulfonylurea receptor (SUR), a member of the ATP-binding cassette superfamily. Mutations in SUR have been identified in individuals affected with familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI), an autosomal recessive disorder of glucose metabolism which is linked to chromosome 11p15.1 and characterized by unregulated secretion of insulin and profound hypoglycemia. Because the Kir6.2 locus is within 5 kilobases (kb) of the SUR gene on chromosome 11p15.1 and it is a necessary member of the [beta]-cell KATP channel, we considered Kir6.2 as a candidate gene for PHHI. We identified a homozygous point mutation in Kir6.2 in the genomic DNA of a child, severely affected with PHHI, from a consanguineous family. This mutation is predicted to disrupt the conserved [alpha]-helical second transmembrane (M2) domain of the inward rectifier by substitution of a proline for a leucine residue (L147P). Mutation of Kir6.2, like SUR, appears to lead to the PHHI phenotype suggesting that Kir6.2 is necessary, although not sufficient, for normal regulation of insulin release.
Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is characterized by profound hypoglycemia due to unregulated secretion of insulin (1 ). Inheritance is in an autosomal recessive pattern, and linkage to chromosome 11p15.1 has been established (2 ,3 ). Subsequently, mutations in the first and second nucleotide binding folds of the sulfonylurea receptor (SUR) gene, a subunit of the pancreatic-islet [beta]-cell ATP-sensitive potassium (KATP) channel have been identified as causative for PHHI in some families (4 ,5 ).
The resting membrane potential of pancreatic islet [beta]-cells is set by KATP channels. As a result of glucose metabolism, alterations in the intracellular ATP/ADP ratio inhibit [beta]-cell ATP-sensitive potassium currents (IKATP) and ultimately result in an increase in exocytosis of insulin (6 ) thereby creating a link between the metabolic state of the cell and membrane electrical events. Reconstitution of IKATP requires the presence of the inward rectifier Kir6.2 in addition to the SUR (7 ). Kir6.2 has been mapped to chromosome 11p15.1, ~5 kb from SUR. We hypothesized that familial PHHI could also result from inactivation of Kir6.2.
Genomic DNA samples from members of 15 families affected with PHHI were evaluated. To assess whether mutation of Kir6.2 was present, screening was performed using the RNase cleavage assay (MisMatch Detecttm II, Ambion Inc., Austin, TX). Since Kir6.2 lacks introns within its coding region and this sequence is <1.5 kb in length (7 ), we were able to use genomic DNA as a template for polymerase chain reaction (PCR) amplification of a single product for screening the coding sequence.
The affected child of family 21 was of Mid-Eastern (Iranian) origin and the progeny of a first cousin mating. Profound hypoglycemia presented within hours of birth. The diagnosis of PHHI was made by one of us (E.L.) using established criteria (1 ), which included an insulin level of >30 [mu]U/ml with a simultaneous glucose determination of <30 mg/ dl, and glucose requirements of >15 mg/kg/min to maintain euglycemia. Hypoglycemia persisted despite medical therapy with diazoxide, glucagon and somatostatin. Subsequently, a sub-total pancreatectomy (95%) was performed. Histopathological examination of the operative specimen revealed diffuse nesidioblastosis.
Sequence analysis of 18 exons of SUR, including those coding for both nucleotide-binding folds, revealed wild-type sequence in this affected individual (data not shown), prompting the search for a mutation in Kir6.2.Analysis of samples from this affected individual for mutation of Kir6.2 revealed a cleavage pattern different from the wild-type pattern, and that Kir6.2 amplicon was cloned and sequenced. A homozygous T -> C point mutation was found at nucleic acid residue 649 of the genomic sequence (7 ) (Fig. 1 ), which would result in a proline substitution at residue 147 (L147P) of Kir6.2. This individual is the product of a consanguineous mating and, therefore, is expected to be homozygous by descent at the disease gene locus (8 ). The only other change found in the remainder of the Kir6.2 sequence in this individual was a T at nucleic acid position 652 instead of the G indicated in the published sequence (7 ) (Fig. 1 ). This change was also found in all 27 additional control and affected individuals evaluated (see below).
While searching for mutations in Kir6.2 we consistently found a T at nucleic acid position 652 instead of the G indicated in the published sequence (7 ) (Fig. 1 ). This substitution introduced an AlwI restriction endonuclease recognition site. Analysis of 13 normal controls and 14 affected individuals of diverse genetic background, by either direct sequence analysis or AlwI digestion of PCR amplified products, revealed that all were homozygous for the T residue, suggesting that the published sequence was in error. This T -> G substitution changes amino acid 148 from a serine to an isoleucine residue, which is in agreement with that found in the mouse Kir6.2 sequence at this position (7 ). Three additional sequence variants were found, including E23K in five affected families (frequency in 26 normal chromosomes 50%), A190t -> c in five affected families and L339V in three affected families. No correlation between genotype and phenotype could be established in those families exhibiting these variations.
We discovered a Kir6.2 gene mutation in an affected individual with severe familial PHHI demonstrating genetic heterogeneity for this disorder. No phenotypic difference is discernable between this family and those with mutations of the first or second nucleotide binding fold regions of SUR (P.M.Thomas, unpublished data). Interestingly, in rats Kir6.2 mRNA has been shown to be expressed in heart and brain (10 ), however, no discernable abnormalities were present in those systems in this patient.
The inward rectifier K+ channels are found in a variety of cell types where they allow K+ influx, but little K+ outflux, from the cell. They, therefore, have a significant role in the maintenance of the resting potential of the cell (11 ). This family of proteins characteristically contains two predicted [alpha]-helical transmembrane spanning domains (M1 and M2) (11 -13 ), which correspond to transmembrane regions S5 and S6 of voltage-gated K+ channels (14 ,15 ). The hydrophobic domain, comprised of M1, M2, and the intervening sequence H5 as well as the hydrophilic C-terminal domain, have been implicated as the major part of the ion channel pore (11 ,12 ,16 ,17 ). The presence of a proline substitution within the M2 region of Kir6.2 is predicted to result in disruption of this [alpha]-helical domain (18 ,19 ). Alignment of Kir6.2 with other members of the inward rectifier family revealed the presence of hydrophobic residues, and the absence of proline residues, in the M2 domain. Insertion of proline residues within transmembrane domains of other proteins, including the V2 vasopressin receptor, have been demonstrated to lead to loss of function (20 ).
IKATP functions in pancreatic islet [beta]-cells to link the metabolic state of the cell with membrane electrical events. When expressed individually neither Kir6.2 nor SUR produces IKATP (7 ,21 ). SUR has been shown to couple to several inward rectifier channels conferring sulfonylurea sensitivity upon them (21 ), and other inward rectifiers are expressed in the pancreas (22 ,23 ), raising the possibility that a combination different from SUR and Kir6.2 may be responsible for the [beta]-cell IKATP . However, demonstration that loss of function of either Kir6.2 or SUR leads to unregulated secretion of insulin and the PHHI phenotype, supports the model that these two subunits cooperate in the formation of IKATP. Detailed functional studies involving the L147P mutant form of Kir6.2, in combination with SUR, are required to confirm the effect of this mutation on IKATP .
Sulfonylurea drugs are used to elevate levels of insulin secretion, through inhibition of pancreatic islet [beta]-cell KATP channels, in the therapy of non-insulin dependent diabetes mellitua (24 ). Primary and secondary failures of sulfonylurea treatment frequently occur (25 ). The effects of sulfonylureas are not limited to the pancreas and adverse extrapancreatic effects have been described (24 ). The demonstration that mutation of Kir6.2 also leads to the PHHI phenotype may provide it as another target for control of insulin release. Whether additional mutations of Kir6.2 will be found to be correlated with PHHI or other more heterogeneous disorders of insulin secretion, including some forms of diabetes mellitus, remains to be determined.
Leukocyte DNA was obtained from peripheral blood samples according to the manufacturer's direction (Qiagen, Chatsworth, CA). Oligonucleotide primers were based on the published sequence (7 ). Kir6.2 coding sequence was amplified using primers 207 (5'-CTAGGCCACGTCCGAGG) and 208 (5'-ATGGTCCGTGTGTACA) in a 50 [mu]l PCR amplification reaction containing ~100-200 ng of genomic DNA, 250 [mu]M of each dNTP, 2.5 mM MgCl2, 1* PCR buffer (20 mM Tris-HCl, pH 8.4 and 50 mM KCl), 1 [mu]M concentrations of each primer and 1-5 U Taq DNA polymerase. Thermal cycler conditions were: 95oC for 5 min; 35 cycles of 94oC for 30 s, 56oC for 45 s, 72oC for 90 s; 72oC for 10 min.
Amplified products were either directly sequenced, as previously described (26 ), or cloned (TA Cloning Kit, Invitrogen, La Jolla, CA) and sequenced using a fluorescent sequencing protocol (Applied Biosystems). Sequencing primer 202 (5'-CTCATCGCCTTCGCCCAC) was used to detect the described mutation.
In order to test for the mutation, a PvuI restriction endonuclease digestion site (CGAT/CG) was introduced into the mutant sequence by PCR amplification of genomic DNA using PCR conditions as described above (with the exception that the PCR buffer was at pH 9.5), primer 202 and the modified downstream primer 210 (5'-TGTTCTGCACGATGACGATC). In the wild-type sequence, the nucleic acid at position 16 of primer 210, presented here in bold, is a G residue. One fifth of the PCR amplification reaction was digested with 5 U PvuI enzyme for 2 h and separated by electrophoresis on a 12% polyacrylamide gel.
To test the identity of base 652, either direct sequence analysis or restriction digestion with the endonuclease AlwI was performed on the 462 bp PCR amplicon obtained with primer 202 and 203 (5'-ACCCACGCCGTTCTCCAT), using the conditions and methods described above.
We thank A. Shenker and M. Egan for helpful discussions and review of the manuscript; G. Cote, E. Huang, and N. Wohllk for participation in the SUR sequence analysis of the affected individual; and the participating family members without whom this study would not have been possible. This work was supported by grants JDFI 195104, NICHD HD28820, and NIH DK02274 (to P.M.T.). Automated fluorescent sequencing was performed by the University of Michigan DNA core sequencing facility.
1 Aynsley-Green, A., Polak, J.M., Bloom, S.R., Gough, M.H., Keeling, J., Ashcroft, S.J.H., Turner, R.C. and Boum, J.D. (1981) Nesidioblastosis of the pancreas: definition of the syndrome and the management of the severe neonatal hyperinsulinaemic hypoglycaemia. Arch. Dis. Child.56, 496-508.MEDLINE Abstract
2 Glaser, B., Chiu, K.C., Anker, R., Nestoroqica, A., Landau, H., Ben-Bassat, H., Xhlomai, Z., Kaiser, N., Thornton, P.S., Stanley, C.A., Spielman, R.S., Gogolin-Ewens, K., Cerasi, E., Baker, L., Rice, J., Donis-Keller, H. and Permutt, M.A. (1994) Familial hyperinsulinism maps to chromosome 11p14-15.1, 30 cM centromeric to the insulin gene. Nature Genet.7, 185-188.MEDLINE Abstract
3 Thomas, P.M., Cote, G.J., Hallman, D.M. and Mathew, P.M. (1995) Homozygosity mapping, to chromosome 11p, of the gene for familial persistent hyperinsulinemic hypoglycemia of infancy. Am. J. Hum. Genet.56, 416-421.MEDLINE Abstract
4 Thomas, P.M., Cote, G.J., Wohllk, N., Haddad, B., Mathew, P.M., Rabl, W., Aguilar-Bryan, L., Gagel, R.F.and Bryan, J. (1995) Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science268, 426-429.MEDLINE Abstract
5 Thomas, P.M., Wohllk, N., Huang, E., Kuhnle, E., Rabl, W., Gagel R.F.and Cote, G.J. (1996) Inactivation of the first nucleotide binding fold of the sulfonylurea receptor and familial persistent hyperinsulinemic hypoglycemia of infancy. Am. J. Hum. Genet.. 59, 510-518.MEDLINE Abstract
6 Ashcroft, S.J.H. and Ashcroft, F.M. (1990) Properties and functions of ATP-sensitive K-channels. Cell. Signalling2, 197-214.
7 Ignaki, N., Gonoi, T., Clement J.P., Namba, N., Inazawa, J., Gonzalez, G., Atuilar-Bryan, L., Seino, S. and Bryan, J. (1995) Reconstitution of IKATP: An inward rectifier subunit plus the sulfonylurea receptor. Science270, 1166-1170.
8 Lander, E.S. and Botstein, D. (1987) Homozygosity mapping: A way to map human recessive traits with the DNA of inbred children. Science236, 1567-1570.MEDLINE Abstract
9 Glaser, B., Chiu K.C., Liu, L., Anker, R., Nestorowica, A., Cox, N.J., Landay, H., Kaiser, N., Thornton, P.S., Stanley, C.A., Cerasi, E., Baker, L., Donis-Keller, H. and Permutt, M.A. (1995) Recombinant mapping of the familial hyperinsulinism gene to an 0.8 cM region on chromosome 11p15.1 and demonstration of a founder effect in Ashkenazi Jews. Hum. Mol. Genet.4, 879-886.MEDLINE Abstract
10 Tokuyama, Y., Fan, Z., Furuta, H., Makielski, J.C., Polonsky, K.S., Bell, G.I. and Yano, H. (1996) Rat inwardly rectifying potassium channel Kir6.2: cloning electrophysiological characterization, and decreased expression in pancreatic islets of male Zucker diabetic fatty rats. Biochem. Biophys. Res. Commun. 220, 532-538.MEDLINE Abstract
11 Kubo, Y., Baldwin, T.J., Jan, Y.N. and Jan, L.Y. (1993) Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature362, 127-133.MEDLINE Abstract
12 Stanfield, P.R., Daview, N.W., Shelton, P.A., Sutcliffe, M.J., Khan, I.A., Brammar, W.J. and Conley, E.C. (1994) A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1. J. Physiol. (Lond) 478, 1-6.MEDLINE Abstract
13 Hong, K. and Driscoll, M. (1994) A transmembrane domain of the putative channel subunit MEC-4 influences mechanotransduction and neurodegeneration in C. elegans. Nature367, 470-473.MEDLINE Abstract
14 Jan, L.Y. and Jan, Y.N. (1994) Potassium channels and their evolving gates. Nature371, 119-122.MEDLINE Abstract
15 Durrel, S.R. and Guy, R.H. (1992) Atomic scale structure and functional models of voltage-gated potassium channels. Biophys. J.62, 238-250.
16 Wible, B.A., Tagilalatela, M., Ficker, E. and Brown, A.M. (1994) Gating of inwardly rectifying K+ channels localized to a single negatively charged residue. Nature371, 246-249.MEDLINE Abstract
17 Yang, J., Jan, N.Y. and Jan, L.Y. (1995) Control of rectification and permeation by residues in two distinct domains in an inward rectifier K+ channel. Neuron14, 1047-1054.MEDLINE Abstract
18 Creighton, T.E. (1993) In Proteins: Structure and Molecular Properties. W. H. Freeman and Company, New York, 2nd edition. p184
19 von Heijne, G. (1991) Proline kinks in transmembrane [alpha]-helices. J. Mol. Biol.218, 499-503.MEDLINE Abstract
20 Spiegel, A.M. (1995) Defects in G protein-coupled signal transduction in human disease. Annu. Rev. Physiol.58, 143-170.
21 Ammala, C., Moorhouse, A., Gribble, F., Ashfield, R., Proks, P., Smith, P.A., Sakura, H., Coles, B., Ashcroft, S.J.H. and Ashcroft, F.M. (1996) Promiscuous coupling between the sulphonylurea receptor and inwardly rectifying potassium channels. Nature379, 545-548.MEDLINE Abstract
22 Bond, C.T., Ammala, C., Ashfield, R., Blair, T.A., Gribble, F., Khan, R.N., Lee, K., Proks, P., Rowe, I.C.M., Sakura, H., Ashford, M.J., Adelman, J.P. and Ashcroft, F.M. (1995) Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic [beta]-cell and in the brain. FEBS Lett.367, 61-66.MEDLINE Abstract
23 Tanizawa, Y., Matsubara, A., Ueda, K., Katagiri, H., Kuwano, A., Ferrer, J., Permutt, M.A. and Oka, Y. (1996) A human pancreatic islet inwardly rectifying potassium channel: cDNA cloning, determination of the genomic structure and genetic variations in Japanese NIDDM patients. Diabetologia39, 447-452.MEDLINE Abstract
24 Panten, U., Schwanstecher, M. and Schwanstecher C. (1992) Pancreatic and extrapancreatic sulfonylurea receptors. Horm. Metab. Res.24, 549-554.MEDLINE Abstract
25 Bailey, C.J., Path, M.R.C. and Turner, R.C. (1996) Metformin. N. Engl. J. Med.334, 575-579.
26 Khorana, S., Gagel, R.F. and Cote, G.J. (1994) Direct sequencing of PCR products in agarose gel slices. Nucleic Acids Res.22, 3425-3426.MEDLINE Abstract
*To whom correspondence should be addressed
This page is maintained by OUP admin. Last updated Thu Oct 31 15:28:51 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
