Emma L. Edghill

Insulin gene mutations as a cause of permanent neonatal diabetes

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with β cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially β cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of β cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

Mutations in PTF1A cause pancreatic and cerebellar agenesis.

Individuals with permanent neonatal diabetes mellitus usually present within the first three months of life and require insulin treatment. We recently identified a locus on chromosome 10p13–p12.1 involved in permanent neonatal diabetes mellitus associated with pancreatic and cerebellar agenesis in a genome-wide linkage search of a consanguineous Pakistani family. Here we report the further linkage analysis of this family and a second family of Northern European descent segregating an identical phenotype. Positional cloning identified the mutations 705insG and C886T in the gene PTF1A, encoding pancreas transcription factor 1α, as disease-causing sequence changes. Both mutations cause truncation of the expressed PTF1A protein C-terminal to the basic-helix-loop-helix domain. Reporter-gene studies using a minimal PTF1A deletion mutant indicate that the deleted region defines a new domain that is crucial for the function of this protein. PTF1A is known to have a role in mammalian pancreatic development, and the clinical phenotype of the affected individuals implicated the protein as a key regulator of cerebellar neurogenesis. The essential role of PTF1A in normal cerebellar development was confirmed by detailed neuropathological analysis of Ptf1a−/− mice.

Insulin Mutation Screening in 1,044 Patients With Diabetes: Mutations in the INS Gene Are a Common Cause of Neonatal Diabetes but a Rare Cause of Diabetes Diagnosed in Childhood or Adulthood

OBJECTIVE— Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood. RESEARCH DESIGN AND METHODS— The INS gene was sequenced in 285 patients with diabetes diagnosed before 2 years of age, 296 probands with maturity-onset diabetes of the young (MODY), and 463 patients with young-onset type 2 diabetes (nonobese, diagnosed <45 years). None had a molecular genetic diagnosis of monogenic diabetes. RESULTS— We identified heterozygous INS mutations in 33 of 141 probands diagnosed at <6 months, 2 of 86 between 6 and 12 months, and none of 58 between 12 and 24 months of age. Three known mutations (A24D, F48C, and R89C) account for 46% of cases. There were six novel mutations: H29D, L35P, G84R, C96S, S101C, and Y103C. INS mutation carriers were all insulin treated from diagnosis and were diagnosed later than ATP-sensitive K+ channel mutation carriers (11 vs. 8 weeks, P < 0.01). In 279 patients with PND, the frequency of KCNJ11, ABCC8, and INS gene mutations was 31, 10, and 12%, respectively. A heterozygous R6C mutation cosegregated with diabetes in a MODY family and is probably pathogenic, but the L68M substitution identified in a patient with young-onset type 2 diabetes may be a rare nonfunctional variant. CONCLUSIONS— We conclude that INS mutations are the second most common cause of PND and a rare cause of MODY. Insulin gene mutation screening is recommended for all diabetic patients diagnosed before 1 year of age.

Mutations in ATP-Sensitive K+ Channel Genes Cause Transient Neonatal Diabetes and Permanent Diabetes in Childhood or Adulthood

Transient neonatal diabetes mellitus (TNDM) is diagnosed in the first 6 months of life, with remission in infancy or early childhood. For ∼50% of patients, their diabetes will relapse in later life. The majority of cases result from anomalies of the imprinted region on chromosome 6q24, and 14 patients with ATP-sensitive K+ channel (KATP channel) gene mutations have been reported. We determined the 6q24 status in 97 patients with TNDM. In patients in whom no abnormality was identified, the KCNJ11 gene and/or ABCC8 gene, which encode the Kir6.2 and SUR1 subunits of the pancreatic β-cell KATP channel, were sequenced. KATP channel mutations were found in 25 of 97 (26%) TNDM probands (12 KCNJ11 and 13 ABCC8), while 69 of 97 (71%) had chromosome 6q24 abnormalities. The phenotype associated with KCNJ11 and ABCC8 mutations was similar but markedly different from 6q24 patients who had a lower birth weight and who were diagnosed and remitted earlier (all P < 0.001). KATP channel mutations were identified in 26 additional family members, 17 of whom had diabetes. Of 42 diabetic patients, 91% diagnosed before 6 months remitted, but those diagnosed after 6 months had permanent diabetes (P < 0.0001). KATP channel mutations account for 89% of patients with non-6q24 TNDM and result in a discrete clinical subtype that includes biphasic diabetes that can be treated with sulfonylureas. Remitting neonatal diabetes was observed in two of three mutation carriers, and permanent diabetes occurred after 6 months of age in subjects without an initial diagnosis of neonatal diabetes.

Mutations in hepatocyte nuclear factor-1beta and their related phenotypes.

Background: Hepatocyte nuclear factor-1 beta (HNF-1β) is a widely distributed transcription factor which plays a critical role in embryonic development of the kidney, pancreas, liver, and Mullerian duct. Thirty HNF-1β mutations have been reported in patients with renal cysts and other renal developmental disorders, young-onset diabetes, pancreatic atrophy, abnormal liver function tests, and genital tract abnormalities. Methods: We sequenced the HNF-1β gene in 160 unrelated subjects with renal disease, 40% of whom had a personal/family history of diabetes. Results: Twenty three different heterozygous HNF-1β mutations were identified in 23/160 subjects (14%), including 10 novel mutations (V61G, V110G, S148L, K156E, Q176X, R276Q, S281fsinsC, R295P, H324fsdelCA, Q470X). Seven (30%) cases were proven to be due to de novo mutations. Renal cysts were found in 19/23 (83%) patients (four with glomerulocystic kidney disease, GCKD) and diabetes in 11/23 (48%, while three other families had a family history of diabetes. Only 26% of families met diagnostic criteria for maturity-onset diabetes of the young (MODY) but 39% had renal cysts and diabetes (RCAD). We found no clear genotype/phenotype relationships. Conclusion: We report the largest series to date of HNF-1β mutations and confirm HNF-1β mutations as an important cause of renal disease. Despite the original description of HNF-1β as a MODY gene, a personal/family history of diabetes is often absent and the most common clinical manifestation is renal cysts. Molecular genetic testing for HNF-1β mutations should be considered in patients with unexplained renal cysts (including GCKD), especially when associated with diabetes, early-onset gout, or uterine abnormalities.

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

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.

HNF1B Mutations Associate with Hypomagnesemia and Renal Magnesium Wasting

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (<1.58 mg/dl) compared with 2% (one of 48) of those without mutations (P < 0.0001). The median plasma magnesium was significantly lower among mutation carriers than those without mutations (1.68 versus 2.02 mg/dl; P < 0.0001). Because hypermagnesuria and hypocalciuria accompanied the hypomagnesemia, we analyzed genes associated with hypermagnesuria and detected highly conserved HNF1 recognition sites in FXYD2, a gene that can cause autosomal dominant hypomagnesemia and hypocalciuria when mutated. Using a luciferase reporter assay, we demonstrated HNF1B-mediated transactivation of FXYD2. These results extend the phenotype of HNF1B mutations to include hypomagnesemia. HNF1B regulates transcription of FXYD2, which participates in the tubular handling of Mg(2+), thus describing a role for HNF1B not only in nephrogenesis but also in the maintenance of tubular function.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

HLA Genotyping Supports a Nonautoimmune Etiology in Patients Diagnosed With Diabetes Under the Age of 6 Months

Children with permanent diabetes are usually assumed to have type 1 diabetes. It has recently been shown that there are genetic subgroups of diabetes that are often diagnosed during the neonatal period but may present later. A recent Italian study proposed that type 1 diabetes is rare before 6 months of age. We aimed to examine genetic susceptibility to type 1 diabetes in patients diagnosed with diabetes before the age of 2 years. We analyzed HLA class II genotypes, markers of autoimmune diabetes, in 187 children with permanent diabetes diagnosed at <2 years of age. Of the 79 subjects diagnosed at <6 months of age, 41% (95% CI 0.30–0.51) had type 1 diabetes–associated high-risk genotypes, a proportion similar to that in healthy population control subjects (44%, P = 0.56). This group included 32 patients with mutations in the KCNJ11 gene, which encodes Kir6.2 (44% high-risk HLA class II genotypes), and 47 in whom the etiology of diabetes was unknown (38% high-risk HLA class II genotypes). Of 108 patients diagnosed between 6 and 24 months of age, 93% (0.86–0.99) had high-risk HLA class II genotypes compared with 44% of the population control subjects (P < 0.0001). We conclude that infants diagnosed with diabetes before 6 months of age are unlikely to have autoimmune type 1 diabetes and are most likely to have a monogenic etiology.

KCNJ11 activating mutations are associated with developmental delay, epilepsy and neonatal diabetes syndrome and other neurological features

Heterozygous activating mutations in the gene encoding for the ATP-sensitive potassium channel subunit Kir6.2 (KCNJ11) have recently been shown to be a common cause of permanent neonatal diabetes. Kir6.2 is expressed in muscle, neuron and brain as well as the pancreatic beta-cell, so patients with KCNJ11 mutations could have a neurological phenotype in addition to their diabetes. It is proposed that some patients with KCNJ11 mutations have neurological features that are part of a discrete neurological syndrome termed developmental Delay, Epilepsy and Neonatal Diabetes (DEND), but there are also neurological consequences of chronic or acute diabetes. We identified KCNJ11 mutations in four of 10 probands with permanent neonatal diabetes and one affected parent; this included the novel C166F mutation and the previously described V59M and R201H. Four of the five patients with mutations had neurological features: the patient with the C166F mutation had marked developmental delay, severe generalised epilepsy, hypotonia and muscle weakness; mild developmental delay was present in the patient with the V59M mutation; one patient with the R201H mutation had acute and chronic neurological consequences of cerebral oedema and another had diabetic neuropathy from chronic hyperglycaemia. In conclusion, the clinical features in these patients support the existence of a discrete neurological syndrome with KCNJ11 mutations. The severe DEND syndrome was seen with the novel C166F mutation and mild developmental delay with the V59M mutation. These features differ markedly from the neurological consequences of acute or chronic diabetes.