ORIGINAL ARTICLE Mosaic Paternal Uniparental Isodisomy and an ABCC8 Mutation in a Patient With Permanent and Hemihypertrophy Julian P.H. Shield,1 Sarah E. Flanagan,2 Deborah J. Mackay,3,4 Lorna W. Harries,2 Peter Proks,5 Christophe Girard,5 Frances M. Ashcroft,5 I. Karen Temple,3,4 and Sian Ellard2

OBJECTIVE—Activating mutations in the KCNJ11 and ABCC8 developments have highlighted the importance of ion encoding the Kir6.2 and SUR1 subunits of the pancreatic channel mutations (“channelopathies”) in the etiology ATP-sensitive Kϩ channel are the most common cause of perma- of both permanent and transient forms of this condition nent neonatal diabetes. In contrast to KCNJ11, where only (3–5). The pancreatic ␤-cell ATP-sensitive Kϩ channel dominant heterozygous mutations have been identified, reces- (KATP channel) is composed of four inward rectifying sively acting ABCC8 mutations have recently been found in some Kϩ channel (Kir6.2) subunits, encoded by the gene patients with neonatal diabetes. These genes are co-located on 11p15.1, centromeric to the imprinted Beckwith- KCNJ11, and four (SUR1) sub- Wiedemann syndrome (BWS) locus at 11p15.5. We investigated a units encoded by ABCC8. Its role is to control insulin male with hemihypertrophy, a condition classically associated release in response to glucose metabolism within the with neonatal hyperinsulinemia and hypoglycemia, who devel- ␤-cell, through alterations in adenine nucleotide avail- oped neonatal diabetes at age 5 weeks. ability and binding (6). Inactivating mutations in either KCNJ11 or ABCC8 cause persistent hyperinsulinemic RESEARCH DESIGN AND METHODS—The KCNJ11 and ABCC8 genes and microsatellite markers on hypoglycemia of infancy (7,8). Recently, Gloyn and were analyzed in DNA samples from the patient and his parents. colleagues described activating mutations in KCNJ11 as the cause of many cases of permanent (9) and a few RESULTS—A paternally inherited activating mutation (N72S) in cases of transient (10) neonatal diabetes; this has been the ABCC8 gene was identified in the proband. The mutation was confirmed in a large follow-up series (5,11). The mutant present at 70% in the patient’s leukocytes and 50% in buccal cells. K channels display reduced sensitivity to ATP-in- Microsatellite analysis demonstrated mosaic segmental paternal ATP uniparental isodisomy (UPD) of 11pter-11p14 in the proband that duced closure, thus preventing both membrane depolar- encompassed the ABCC8 gene and the BWS locus. ization and insulin release in response to glucose. Clinically, it has been demonstrated that many, but not CONCLUSIONS—We report a patient with neonatal diabetes, all, children affected by mutations in KCNJ11 can hemihypertrophy, and relatively high birth weight resulting from replace insulin therapy with high-dose oral sulfonyl- telomeric segmental paternal UPD of chromosome 11, which ureas, usually glyburide (known in the U.K. as gliben- unmasks a recessively acting gain-of-function mutation in the ABCC8 gene and causes deregulation of imprinted genes at the clamide) (12). BWS locus on 11p15.5. Diabetes 57:255–258, 2008 Activating mutations in the ABCC8 gene have also been linked to neonatal diabetes (3–5,13–15). Mutations may result in either a transient or permanent form of the disease. Like the KCNJ11 mutations, they cause the KATP eonatal diabetes is a rare condition of hyper- channel to remain open even when ATP is elevated by glycemia usually presenting during infancy or glucose metabolism. Clinically, neonatal diabetes due to soon thereafter (1). Etiologies other than ABCC8 mutations can also be abrogated through the autoimmunity are far more prevalent if diabe- therapeutic use of sulfonylureas (3,4). N Hemihypertrophy describes asymmetric overgrowth of tes is diagnosed before the age of 6 months (2). Recent part or half of the body. It may be isolated or part of the Beckwith-Wiedemann syndrome (BWS), a condition char- From the 1Department of Endocrinology and Diabetes, Bristol Royal Hospital acterized by macroglossia, abdominal wall defects, neona- for Children and University of Bristol, Bristol, U.K.; the 2Institute of Biomed- tal hyperinsulinemia, and increased risk of embryonal ical and Clinical Science, Peninsula Medical School, Exeter, U.K.; 3Wessex Regional Genetics Laboratory and Clinical Genetics Service, Salisbury District tumors (16). Both isolated hemihypertrophy and BWS can Hospital, Salisbury, Wiltshire, U.K.; the 4Division of Human Genetics, Univer- be caused by mosaic paternal uniparental isodisomy of sity of Southampton, Southampton, U.K.; and the 5University Laboratory of chromosome 11p15 (17,18). Physiology, Oxford, U.K. Address correspondence and reprint requests to Professor Sian Ellard, Peninsula Medical School, Barrack Road, Exeter, EX2 5DW, U.K. E-mail: [email protected]. RESEARCH DESIGN AND METHODS Received for publication 19 July 2007 and accepted in revised form 11 October 2007. A male child weighing 3.87 kg at 40 weeks gestation (75th percentile) was born Published ahead of print at http://diabetes.diabetesjournals.org on 17 Octo- to nonconsanguineous, Somali parents. There had been two previous unevent- ber 2007. DOI: 10.2337/db07-0999. ful pregnancies. The proband was discharged home on the day of birth but J.P.H.S. and S.E.F. contributed equally to this work. returned to hospital aged 36 days, failing to thrive, severely dehydrated, and ϩ BWS, Beckwith-Wiedemann syndrome; KATP channel, ATP-sensitive K acidotic (pH 6.99 [normal range {NR} 7.3–7.4]). The initial blood glucose channel; UPD, uniparental isodisomy. measured was 50 mmol/l with a bicarbonate of 5 mmol/l (NR 21–34) and © 2008 by the American Diabetes Association. urinary ketones of Ͼ160 mg/dl. A diagnosis of neonatal diabetes was made, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance and subsequent to resuscitation the child was stabilized on twice-daily with 18 U.S.C. Section 1734 solely to indicate this fact. subcutaneous insulin. Both parents had normal fasting glucose levels (father

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no cardiomegaly, while serial abdominal ultrasounds identified increasing left renal hypertrophy compared with a normal right kidney. Investigations. The young age of the patient at presentation suggested a diagnosis of neonatal diabetes. Blood was collected for DNA analysis of chromosome 6 anomalies causing transient neonatal diabetes (19) and for KCNJ11 mutation analysis (which at this time was the only channelopathy described as causing permanent neonatal diabetes). These tests were both normal. GAD, insulin, and islet cell autoantibodies were all negative. The child’s karyotype was normal: 46 XY. However, microsatellite analysis of chromosome 11 revealed the child to be mosaic for paternal uniparental isodisomy (UPD) of 11pter-11p14 (Table 1 and Fig. 2A). As neonatal diabetes is not characteristic of either BWS or isolated hemihypertrophy, this prompted a review of potential candidate genes for diabetes that were encompassed within this region of UPD. The SUR1 gene (ABCC8), which lies within this region, was a logical candidate, as it coassembles with Kir6.2 (KCNJ11)to

form the KATP channel, and activating mutations in KCNJ11 are known to cause neonatal diabetes (9–11). The ABCC8 gene was sequenced as described previously (14). The father was identified as being heterozygous for a missense mutation, N72S (c.215AϾG), while the proband’s leukocyte DNA showed mosaicism for the N72S mutation (Fig. 2B). The N72S mutation was not found in 250 normal , and the affected residue was conserved across species. Quan- tification by real-time PCR (TaqMan assay) demonstrated that the N72S mutation was present at ϳ70% in leukocyte DNA and 50% in buccal cells. Briefly, genomic DNA from leukocytes and buccal cells was amplified using a mutation-specific TaqMan approach. Genomic DNA from the father, who is heterozygous for the N72S mutation, was used in serial dilution to produce standard curves to determine linear range and accuracy of quantification (primer and probe sequences are available on request). Reactions contained 5 ␮l TaqMan Fast Universal PCR Mastermix without Amperase, 0.5 ␮l Assays- by-Design (Applied Biosystems, Warrington, U.K.) probe and primer mix (corresponding to 36 ␮l of each primer and 8 ␮l of each probe), 2.5 ␮l water, and 2 ␮l DNA at a concentration of 100 ng/␮l. Amplification conditions consisted of a single cycle of 95°C for 20 s followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The relative level of mutant transcript relative to wild-type transcript was determined by the ⌬⌬CT method (20). Each test was carried out in triplicate. Functional studies in Xenopus oocytes (methods in 9,10) demonstrated that the N72S mutation results in a reduced sensitivity to inhibition by MgATP. FIG. 1. Photograph of proband showing hemihypertrophy of left leg. Half-maximal block of homozygous N72S channels was produced by 23 Ϯ 1 ␮mol/l ATP (n ϭ 10) compared with 15 Ϯ 1 ␮mol/l (n ϭ 6) for wild-type channels. Two-electrode voltage-clamp studies showed a concomitant small

5.2 mmol/l, age 36 years; mother 4.6 mmol/l, age 31 years) and no prior history increase in the whole-cell KATP channel resting current (14) (Fig. 3). Homozy- of diabetes. gous N72S channels were substantially blocked by the sulfonylurea tolbut- The child had left hemihypertrophy (Fig. 1) but no macroglossia, anterior amide in vitro (Fig. 3). abdominal wall defects, ear creases, or peri-auricular pits as seen in BWS. Identification of an ABCC8 mutation, together with the patient’s relatively Echocardiography of the heart revealed branch pulmonary artery stenosis but poor control on twice-daily insulin (GHb 8.3% NR 4.0–6.0%), the known

TABLE 1 Microsatellite analysis of proband revealing mosaic segmental paternal UPD of chromosome 11 D11S Mb from tel Proband blood Proband buccal Mother Father 2071 0.95 1 3 13 11 23 922 1.06 2 3 23 12 23 4177 1.44 3 4 34 13 24 4046 1.92 1 2 12 13 24 TH 2.15 1 2122212 1318 2.29 1 4142413 HBB 4.45 2 4 24 12 34 4149 8.99 1 3133312 1794 13.19 1 2122312 921 17.25 1 2 12 14 23 902 17.45 2 3231324 904 26.6 2 3232212 907 34.6 2 3233313 911 77.1 1 3 1 3 2 3 1 2 4143 79.1 1 3 1 3 1 3 2 3 1332 92 2 3 2 3 2 3 1 3 Microsatellite analysis was performed according to standard methods with the primer sets listed in the Table. After 28 cycles of PCR, amplimers were resolved on an ABI 3100 platform and maternal and paternal allele levels compared in DNA from blood and buccal samples of the proband. The bold figures indicate alleles present at unexpectedly high dosage. Microsatellites D11STH and D11S1318 lie within the BWS region, while D11S921 and D11S902 bracket KCNJ11 and ABCC8.

256 DIABETES, VOL. 57, JANUARY 2008 J.P.H. SHIELD AND ASSOCIATES

FIG. 3. Resting whole-cell KATP channel currents are slightly larger for homomeric N72S mutant channels and are blocked by sulfonylureas.

Mean steady-state whole-cell KATP channel currents for wild-type (WT), homomeric (hom) N72S, and heterozygous (het) N72S channels, ,as indicated evoked by a voltage step from ؊10 to ؊30 mV before (Ⅺ resting condition), after application of the metabolic inhibitor 3 mmol/l azide (o), and in the presence of 3 mmol/l azide plus 0.5 mmol/l tolbutamide (f). The number of oocytes is given below the bars.

DISCUSSION Uniparental isodisomy is a rare abnormality that provides insights into the genetic basis of human disease. Its identification in two cases of transient neonatal diabetes (22) led to identification of imprinting anomalies associ- ated with the transient form of this condition. UPD can also unmask genetic disorders with an autosomal reces- sive pattern of inheritance (23). In this case, the child’s hemihypertrophy and relatively high birth weight (75th centile) did not fit with a diagnosis of permanent neonatal diabetes as, if anything, these features would be expected to co-segregate with hypoglycemia secondary to hyperin- sulinemia (as seen in BWS). Since UPD can unmask recessive disorders, SUR1 (ABCC8) was an obvious can- didate, due to its position within the disomic region of interest, its importance in insulin secretion, and the fact that focal adenomatous hyperplasia of islet cells with congenital hyperinsulinemia has been described in which loss of maternal 11p15 is associated with homozygosity of FIG. 2. Results of microsatellite analysis and ABCC8 sequencing. A: an ABCC8 mutation inherited from a heterozygous father Analysis of microsatellite D11S1318 (2.29 Mb from pter, i.e., within the BWS locus). x-axis indicates product size (base pairs); y-axis indicates (24). product quantity (arbitrary units). Note the smaller peak for the Recent studies have identified activating mutations in maternal allele in the proband’s blood sample compared with the ABCC8 as causing both transient and permanent neonatal buccal sample. B: Pedigree showing mutation status and sequencing electropherograms. The solid symbol indicates the affected proband, diabetes (3–5). Several cases of permanent neonatal dia- and the father is shown as an unaffected carrier of the mutation (dot betes responded to glyburide at doses ranging from 0.22 to within an unfilled symbol). The mutated base is indicated by an arrow 0.59 mg ⅐ kgϪ1 ⅐ dayϪ1 (3), including one child with a on the electropherograms. dominant ABCC8 mutation (15) and at least four children with recessively inherited ABCC8 mutations (A.T. Hatter- efficacy of sulfonylureas in neonatal diabetes caused by KATP channel muta- tions (21), and the tolbutamide block of mutant N72S channels observed in sley, S.E.F., S.E., unpublished observations). Unfortu- functional studies, prompted a trial of the sulfonylurea glyburide. Over a nately, the child reported here was unresponsive to Ϫ Ϫ 2-week period, under intensive monitoring in the hospital, regular subcutane- glyburide at a dose of as much as 1 mg ⅐ kg 1 ⅐ day 1. The ous insulin was withdrawn and glyburide gradually increased from a starting Ϫ1 Ϫ1 Ϫ1 Ϫ1 complete absence of any clinical response in our infant dose of 0.1 mg ⅐ kg ⅐ day to a maximum dose of 1 mg ⅐ kg ⅐ day . prompted our withdrawal of glyburide after a 2-week trial, The therapeutic trial was terminated at 2 weeks due to the complete absence of a clinical response, with high blood glucose levels (up to 40 although a further trial when the child is older may be mmol/l) requiring the repeated administration of short-acting insulin to warranted given the in vitro response. The lack of a improve glycemia and prevent metabolic decompensation. The C-peptide level therapeutic response to sulfonyureas in our patient is before initiating glyburide was Ͻ94 pmol/l and remained at Ͻ94 pmol/l on 1 puzzling, as the mutant N72S channels were blocked by mg ⅐ kgϪ1 ⅐ dayϪ1 glyburide. The child is now over 18 months of age and is tolbutamide in in vitro studies. It is possible that other treated with a basal bolus insulin regimen of four injections daily (ϳ1 unit ⅐ Ϫ Ϫ imprinted genes involved in the paternal UPD might confer kg 1 ⅐ day 1) to maintain growth and health (recent A1C 6.6%). Although his neurological development at this stage seems normal, he remains under a reduced response. regular surveillance both for neurocognitive development and for increased In conclusion, we report a patient with UPD of chromo- tumor risk. some 11 who presented with neonatal diabetes as opposed

DIABETES, VOL. 57, JANUARY 2008 257 MOSAIC ABCC8 MUTATIONS AND NEONATAL DIABETES to the classical phenotype of hypoglycemia due to hyper- Howard N, Srinivasan S, Silva JM, Molnes J, Edghill EL, Frayling TM, insulinemia. Paternal UPD of chromosome 11pter-11p14, Temple IK, Mackay D, Shield JP, Sumnik Z, van Rhijn A, Wales JK, Clark encompassing the K channel genes and the BWS locus, P, Gorman S, Aisenberg J, Ellard S, Njolstad PR, Ashcroft FM, Hattersley ATP AT: Activating mutations in the gene encoding the ATP-sensitive potassi- unmasked a recessively acting gain-of-function ABCC8 um-channel subunit Kir6.2 and permanent neonatal diabetes. N Engl J Med gene mutation, thus causing diabetes in addition to fea- 350:1838–1849, 2004 tures of BWS. As has previously been described, the 10. Gloyn AL, Reimann F, Girard C, Edghill EL, Proks P, Pearson ER, Temple heterozygous father has no history of glucose intolerance IK, Mackay DJ, Shield JP, Freedenberg D, Noyes K, Ellard S, Ashcroft FM, or evidence of diabetes in adult life, as this is a recessively Gribble FM, Hattersley AT: Relapsing diabetes can result from moderately acting mutation (14). The level of mosaicism for the N72S activating mutations in KCNJ11. Hum Mol Genet 14:925–934, 2005 mutation varied between leukocytes (ϳ70% mutation) and 11. Flanagan SE, Edghill EL, Gloyn AL, Ellard S, Hattersley AT: Mutations in ϳ KCNJ11, which encodes Kir6.2, are a common cause of diabetes diagnosed buccal cells ( 50% mutation). As the child is alive and in the first 6 months of life, with the phenotype determined by genotype. well, the mutation load within the pancreas remains Diabetologia 49:1190–1197, 2006 unknown. Given the evidence that children with UPD11 12. Pearson ER, Flechtner I, Njolstad PR, Malecki MT, Flanagan SE, Larkin B, can have highly variable levels of mosaicism in different Ashcroft FM, Klimes I, Codner E, Iotova V, Slingerland AS, Shield J, Robert organs, we hypothesize that the pancreas must exhibit a JJ, Holst JJ, Clark PM, Ellard S, Sovik O, Polak M, Hattersley AT: Switching high level of paternal UPD cells to present with this from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 phenotype, the somatic recombination causing the pater- mutations. N Engl J Med 355:467–477, 2006 13. Proks P, Arnold AL, Bruining J, Girard C, Flanagan SE, Larkin B, Colclough nal UPD occurring very early during embryogenesis (25). K, Hattersley AT, Ashcroft FM, Ellard S: A heterozygous activating We have recently identified nine other patients with mutation in the sulphonylurea receptor SUR1 (ABCC8) causes neonatal recessively inherited ABCC8 mutations causing neonatal diabetes. Hum Mol Genet 15:1793–1800, 2006 diabetes (14). 14. Ellard S, Flanagan SE, Girard CA, Patch AM, Harries LW, Parrish A, Edghill EL, Mackay DJ, Proks P, Shimomura K, Haberland H, Carson DJ, Shield JP, ACKNOWLEDGMENTS Hattersley AT, Ashcroft FM: Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with We acknowledge funding from the Sir Graham Wilkins opposite functional effects. Am J Hum Genet 81:375–382, 2007 studentship to S.E.F. and support from the Research & 15. Masia R, De Leon DD, MacMullen C, McKnight H, Stanley CA, Nichols CG: Development Directorate at the Royal Devon & Exeter A mutation in the TMD0–L0 region of sulfonylurea receptor-1 (L225P) NHS Foundation Trust to S.E. causes permanent neonatal diabetes mellitus (PNDM). Diabetes 56:1357– 1362, 2007 16. Engstrom W, Lindham S, Schofield P: Wiedemann-Beckwith syndrome. REFERENCES Eur J Pediatr 147:450–457, 1988 1. Polak M, Shield J: Neonatal and very-early-onset diabetes mellitus. Semin 17. West PM, Love DR, Stapleton PM, Winship IM: Paternal uniparental disomy Neonatol 9:59–65, 2004 in monozygotic twins discordant for hemihypertrophy. J Med Genet 2. Edghill EL, Dix RJ, Flanagan SE, Bingley PJ, Hattersley AT, Ellard S, 40:223–226, 2003 Gillespie KM: HLA genotyping supports a nonautoimmune etiology in 18. Henry I, Puech A, Riesewijk A, Ahnine L, Mannens M, Beldjord C, Bitoun patients diagnosed with diabetes under the age of 6 months. Diabetes P, Tournade MF, Landrieu P, Junien C: Somatic mosaicism for partial 55:1895–1898, 2006 paternal isodisomy in Wiedemann-Beckwith syndrome: a post-fertilization 3. Babenko AP, Polak M, Cave H, Busiah K, Czernichow P, Scharfmann R, event. Eur J Hum Genet 1:19–29, 1993 Bryan J, Aguilar-Bryan L, Vaxillaire M, Froguel P: Activating mutations in 19. Temple IK, Shield JP: Transient neonatal diabetes, a disorder of imprinting. the ABCC8 gene in neonatal diabetes mellitus. N Engl J Med 355:456–466, J Med Genet 39:872–875, 2002 2006 20. Harries LW, Ellard S, Jones RW, Hattersley AT, Bingham C: Abnormal 4. Vaxillaire M, Dechaume A, Busiah K, Cave H, Pereira S, Scharfmann R, de splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes Nanclares GP, Castano L, Froguel P, Polak M: New ABCC8 mutations in syndrome. Diabetologia 47:937–942, 2004 relapsing neonatal diabetes and clinical features. Diabetes 56:1737–1741, 21. Lebovitz H, Melander A: Sulfonylureas: basic aspects and clinical uses. In 2007 International Textbook of Diabetes Mellitus. 2nd ed. Alberti KGMM, 5. Flanagan SE, Patch AM, Mackay DJ, Edghill EL, Gloyn AL, Robinson D, Zimmet P, DeFronzo RA, Eds. London, Wiley, 1997, p. 817–840 Shield JP, Temple K, Ellard S, Hattersley AT: Mutations in ATP-sensitive 22. Temple IK, James RS, Crolla JA, Sitch FL, Jacobs PA, Howell WM, Betts P, Kϩ channel genes cause transient neonatal diabetes and permanent Baum JD, Shield J: An imprinted gene(s) for diabetes? Nat Genet 9:110– diabetes in childhood or adulthood. Diabetes 56:1930–1937, 2007 112, 1995 6. Slingerland AS, Hattersley AT: Mutations in the Kir6.2 subunit of the KATP 23. Zlotogora J: Parents of children with autosomal recessive diseases are not channel and permanent neonatal diabetes: new insights and new treat- always carriers of the respective mutant alleles. Hum Genet 114:521–526, ment. Ann Med 37:186–195, 2005 2004 7. Thomas PM, Cote GJ, Wohilk N, Haddad B, Mathew PM, Rabel W, 24. Verkarre V, Fournet JC, de Lonlay P, Gross-Morand MS, Devillers M, Aquilar-Bryan L, Gagel RF, Byran J: Mutations in the sulphonylurea Rahier J, Brunelle F, Robert JJ, Nihoul-Fekete C, Saudubray JM, Junien C: receptor and familial persistent hyperinsulinemic hypoglycemia of infancy. Paternal mutation of the sulfonylurea receptor (SUR1) gene and maternal Science 268:426–429, 1995 loss of 11p15 imprinted genes lead to persistent hyperinsulinism in focal 8. Thomas PM, Yuyang Y, Lightner E: Mutation of the pancreatic islet inward adenomatous hyperplasia. J Clin Invest 102:1286–1291, 1998 rectifier, Kir6.2 also leads to familial persistent hyperinsulinemic hypogly- 25. Itoh N, Becroft DM, Reeve AE, Morison IM: Proportion of cells with cemia of infancy. Hum Mol Genet 5:1809–1812, 1996 paternal 11p15 uniparental disomy correlates with organ enlargement in 9. Gloyn AL, Pearson ER, Antcliff JF, Proks P, Bruining GJ, Slingerland AS, Wiedemann-beckwith syndrome. Am J Med Genet 92:111–116, 2000

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