Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects

Abstract

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.

Figure  1.

ABCC8 and KCNJ11 gene mutations in patients with PNDM. Pedigrees are shown for recessively inherited ABCC8 mutations. Solid symbols denote patients with neonatal diabetes and unaffected heterozygous mutation carriers are indicated by a dot. Asterisks denote those mutations selected for functional analysis.

Figure  2.

Quantification of normal and c.536delATGG mutant transcripts in a heterozygous lymphoblastoid cell line. The relative levels of normal (gray bar) and mutant mRNA transcripts were quantified in a lymphoblastoid cell line derived from the proband’s mother, who was heterozygous for the c.536delATGG mutation. Error bars show the upper and lower limits of quantification based on three independent measurements. Cells were incubated in the presence (diagonal stripes) or absence (black) of cycloheximide (CHX) before RNA extraction.

Figure  3.

Schematic representation of K_ATP channels in family ISPAD 78. The unaffected parents are heterozygous for the c.536del4 frameshift or P207S missense mutation. Lymphoblastoid cells showed a decrease in c.536del4 mRNA (see fig. 2), and the majority of K_ATP channels in the proband are predicted to be homomeric for the P207S mutation.

Figure  4.

Schematic of the transmembrane topology of SUR1 showing the missense mutations identified. Recessive mutations are shown in black and dominant mutations in green. NBD, nucleotide binding domain.

Figure  5.

A, Mean (± SEM) concentration of ATP required to half-maximally inhibit the K_ATP current (IC₅₀) for the wild-type K_ATPchannel (WT), channels carrying the indicated SUR1 mutations which cause neonatal diabetes (grey bars) and channels carrying the SUR1 mutations found in the unaffected parents (hatched bars). Numbers above the bars indicate the number of patches (oocytes) tested. To simulate the patient’s genotype, we coinjected Kir6.2 mRNA with hetF132L (1:1 mix of WT and F132L SUR1 mRNAs); homA1185E or P207S (mutant SUR1 only); or V1523L+T229I (1:1 mix of V1523L and T229I SUR1). B, Mean (±SEM) K_ATP current remaining in the presence of 3mM MgATP in the inside-out patch for the wild-type K_ATP channel (WT; white bars) channels carrying the indicated SUR1 mutations which cause neonatal diabetes (grey bars) and channels carrying the SUR1 mutations found in the unaffected parents (hatched bars). Numbers above the bars indicate the number of patches (oocytes) tested.

Figure  6.

ATP sensitivity of K_ATP channels corresponding to those found in children with neonatal diabetes and their unaffected parents. Mean relationship between [ATP] and the macroscopic K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_C) for Kir6.2/SUR1 channels (WT, dashed lines), probands (dotted lines) or unaffected parents (solid lines). The smooth curves are the best fit of the Hill equation to the mean data. For wild-type channels, IC₅₀=15 μM, h=1.11, n=6. For hetSUR1-F132L (a), IC₅₀=105 μM, h=0.72, and n=5. For homSUR1-P207S (b), IC₅₀=65 μM, h=0.87, and n=6. For SUR1-T229I+SUR1V1523L (c), IC₅₀=157 μM, h=0.91, and n=8. For homSUR1-A1185E (d), IC₅₀=35 μM, h=0.69, and n=6. For hetA1185E channels (d), IC₅₀=32 μM, h=1.0, and n=5. For hetT229I channels (e), IC₅₀=17 μM, h=1.11, and n=9. For hetV1523L channels (f), IC₅₀=45 μM, h=0.7, and n=6.