Insulin mutations and diabetes
Insulin gene mutations as a cause of diabetes were identified independently by ourselves (Støy et al. 2007) and by Colombo et al. (2008) [13
]. We carried out a linkage analysis in a three-generation family with PNDM of unknown etiology. The results suggested that the putative diabetes gene was located on either chromosome 2, 3, 6 or 11. Cataloguing the genes under the linkage peaks of each of these chromosomes revealed two candidate genes—NEUROD1
. There were no mutations in the coding region of NEUROD1
. Sequencing of INS
revealed the missense mutation G32S in affected family members suggesting that an INS
mutation was responsible for PNDM in this family. The association of INS
mutations with PNDM was confirmed by the identification of mutations in 15 additional probands. Interestingly, the majority of the mutations were not inherited but were de novo
in origin. All were in the heterozygous state. Colombo et al. [17
] used a candidate gene approach based on the fact that a mutation in the Ins2
gene (the mouse homolog of the insulin gene in human) was the cause of diabetes (described as maturity-onset diabetes of the young) in the Akita mouse model of diabetes [18
]. They identified seven different INS
mutations in 9 probands with PNDM.
Mutations (in the heterozygous state) in INS
have now been reported in 66 probands with a variety of diagnoses including PNDM, infancy-onset diabetes (i.e. diabetes diagnosed before 12 months of age), type 1b diabetes (i.e. non-autoimmune type 1 diabetes (T1DM)), maturity-onset diabetes of the young (MODY), and early-onset type 2 diabetes (T2DM) (Figs. – and Table ) [13
]. There are multiple affected family members in 23/66 families. Of the 107 carriers in these families, 104 have diabetes and three have normal glucose tolerance.
Fig. 2 Summary of human insulin gene mutations and disease phenotype. The numbers in brackets indicate the number of probands with that specific mutation. PNDM, permanent neonatal diabetes mellitus; TNDM, transient neonatal diabetes mellitus; MODY, maturity-onset (more ...)
Insulin gene mutations and diabetes
In the large Exeter cohort of patients with PNDM diagnosed before 6 months of age, 14% of patients born to non-consanguineous parents were found to carry a heterozygous INS mutation. The incidence of INS mutations in patients diagnosed with diabetes outside the neonatal period is less well defined, but is estimated to be less than 2%. The mutations in 18/66 (27%) of the probands were inherited from an affected parent whereas in the remaining 48 probands (73%) the mutation was de novo in origin. In one family, two affected children with the same mutation were born to unaffected parents, neither of them carriers, suggesting that one of the parents was a germline mosaic.
The diabetes-associated mutations lead to the synthesis of a structurally abnormal preproinsulin or proinsulin protein. The mutations are located in the signal peptide, the B-chain and A-chain regions and the pairs of basic residues that flank the C-peptide. Those located in the signal peptide impair the function of this domain of preproinsulin including absent or altered cleavage of the signal peptide. The mutations in proinsulin affect formation of the disulfide bonds linking the B-chain and A-chain as well as the intra-A-chain disulfide bond thereby leading to a misfolded proinsulin molecule that is retained in the ER impairing normal beta-cell function and resulting in cell death [17
Garin et al. [32
] recently reported recessive mutations in INS
resulting in neonatal diabetes. These mutations affect proinsulin biosynthesis per se
and function as null mutations, doing so by several different mechanisms. One mutation results in deletion of exons 1 and 2 of the insulin gene. There are several mutations located in the promoter region of the gene that result in decreased transcription. A mutation in the region encoding the 3′-untranslated region of the mRNA affects polyadenylation and mRNA stability. Other mutations affect translation of insulin mRNA by mutation of the translation initiation codon, or lead to synthesis of a truncated proinsulin molecule (Figs. and ).
Fig. 3 A schematic of the human insulin gene showing the locations of mutations in the homozygous or compound heterozygous state in probands with PNDM and TNDM. Mutations involving coding regions are shown above the gene while those involving non-coding regions (more ...)
Homozygous mutations in INS
have been reported in 15 families/21 patients. These patients were diagnosed with PNDM (67% of patients), type 1b diabetes (9% of patients) and, in contrast to heterozygous INS
mutation carriers, with transient neonatal diabetes mellitus (TNDM) (24% of patients). Homozygous INS
mutations are the most common cause of PNDM without extra-pancreatic features in patients born to consanguineous parents (~32%), but are a rare cause of neonatal diabetes in patients born to non-consanguineous parents (~1%) [32
Characteristics of mutations
All the mutations in the preproinsulin protein reported to date are shown in Fig. . In addition to the mutations associated with diabetes, hyperinsulinemia or hyperproinsulinemia, there are four mutations that are unlikely to be pathogenic: A23S, A23T, L68M and G84R [14
] (Figs. and ). The mutation A23S has been reported to be associated with type 1b diabetes. However, this site is not highly conserved amongst preproinsulin sequences and is Ser in the chimpanzee sequence strongly suggesting that it does not lead to diabetes (Fig. ). Functional studies indicate that G84R does not affect proinsulin biosynthesis suggesting that the putative diabetes-associated mutation is not pathogenic [30
The dominantly-acting diabetes-associated mutations involve 19/110 amino acids of preproinsulin and include 23 missense mutation, one nonsense mutation and one insertion/deletion mutation. They are located in all regions of the preproinsulin molecule except the C-peptide: signal peptide (3 mutations), B-chain (12 mutations), A-chain (8 mutations) and pairs of basic amino acids that flank the C-peptide (2 mutations). The latter mutations are found at the enzymatic cleavage site between the B-chain and C-peptide (R55C) and the C-peptide and A-chain (R89C). Other mutations of Arg89 have been described in patients with hyperproinsulinemia. Thirteen mutations were identified in more than one proband (Fig. , Table ) and mutations at five codons (A24D, G32S/R, F48C, R89C, and C96Y/S) account for 56% (37/66) of all mutations identified to date. Six residues are sites for different amino acid substitutions: R6C/H, A23S/T, L30M/P/V, G32S/R, C96Y/S and Y108C/X. Five mutations were identified in families where all affected family members were diagnosed with diabetes outside the neonatal period or infancy (R6C, R6H, L30M, R46Q and R55C) highlighting the different effects of these various mutations on pancreatic beta-cell function. As noted above, A23S/T, L68M, and G84R are most likely rare variants that are not pathogenic and patients carrying these variant are not included in the genotype/phenotype analyses described below.
Ten different homozygous INS
mutations have been reported to date [32
]. Five homozygous mutations have been found in regions of INS
affecting transcription or mRNA processing: three single base substitutions and a 24-base pair deletion are located in the promoter and one single base substitution is in the 3´-untranslated region (3′-UTR). One patient was a compound heterozygote for two promoter mutations. Four homozygous mutations have been identified that affect the coding regions of INS
: two different mutations that result in mutation of the translational initiation site (M1I), a large deletion of exons 1 and 2 (M1_Q62del) and a nonsense mutation in the C-peptide region (Q62X). The site in the promoter at c.-331 seems to be a mutational hotspot, with seven probands identified with mutations at this site (c.-331C>G and c.-331C>A).
Clinical characteristics of patients with PNDM due to heterozygous (dominant-negative) insulin mutations
The majority of patients with heterozygous mutations in INS
were diagnosed with diabetes before 6 months of age (85% in the Exeter cohort). However, in contrast to the majority of patients with mutations in KCNJ11
mutations have also been found in patients diagnosed from 6–12 months of age. Ninety-six percent of patients with a heterozygous INS
mutation in the Exeter cohort were diagnosed with diabetes in the first year of life. The age-at-diagnosis of diabetes (excluding patients with the mutations R6C/H, L30M, R46Q and R55C) varies from 0–1,560 weeks with a median of 11 weeks in the Exeter cohort and 26 weeks in the remaining patients. In contrast to the Akita mouse where there appears to be a difference in age-at-onset of diabetes between male and female mice (with males having an earlier onset with more severe hyperglycemia, at least on a C57BL/6 background) [18
], there is no difference in age-at-diagnosis between male and female human carriers of a heterozygous INS
gene mutation. Both male and female human carriers present with severe beta-cell failure at diagnosis including, in some instances, diabetic ketoacidosis or severe symptomatic hyperglycemia, a sign of almost complete insulin deficiency. The majority of patients are treated with insulin in full replacement doses [14
Mutation carriers are born small for gestational age with median birth weights from 2,700–3,087 g (6th to 27th percentile) most likely reflecting in utero insulin deficiency. Interestingly, male carriers have been reported to have more severe growth retardation than female carriers [14
More than half of patients with diabetes due to a heterozygous INS
mutation have been screened for the presence of beta-cell auto-antibodies (anti-GAD65, islet cell antibodies (ICA) and insulin antibodies). There were no detectable auto-antibodies except for anti-insulin antibodies in some patients, most likely secondary to longstanding insulin therapy. The majority of patients had no residual beta-cell function as evidenced by low or undetectable basal or stimulated C-peptide levels. These measurements were in most cases performed months or years after the diagnosis of diabetes. However in one study, the C-peptide levels were determined at onset of diabetes and in most patients at one or two other times. These data suggest a gradual and progressive decline in beta-cell function over time. Six of 11 patients had normal, and in some instances, high C-peptide level at diagnosis of diabetes, declining levels at the repeat measurements and finally undetectable C-peptide months or years after the onset of diabetes [17
Neurological dysfunction is a key feature of the phenotype of some patients with PNDM due to mutations in KCNJ11
]. Patients with INS
mutations do not have other associated extra-pancreatic features. Some patients have complications secondary to longstanding diabetes such as neuropathy and retinopathy. Signs of insulin resistance (e.g. the presence of acanthosis nigricans) have been noted in some overweight patients [13
Clinical characteristics of patients with MODY
A subgroup of INS
mutations is found exclusively in patients diagnosed with diabetes outside infancy and early childhood: R6H, R6C, L30M, R46Q and R55C. These patients fulfill traditional MODY criteria: non-obese, diagnosis generally <25 years of age and a family history of diabetes consistent with autosomal dominant inheritance. The diabetes is non-ketotic and the patients are treated with diet, oral hypoglycemic agents (OHA) or insulin. The majority of patients have residual beta-cell function as evidenced by detectable C-peptide levels. The extent of beta-cell failure could also be progressive, as some patients have declining C-peptide levels over time. One exception to the generally mild phenotype of this group of patients is a Norwegian family with the R55C mutation. The proband and her mother were diagnosed with diabetes under the dramatic setting of diabetic ketoacidosis and they now both require insulin in full replacement doses. A French family with the same mutation has mild, non-ketotic diabetes. This variation is presumably due to differences in genetic background and its effect on diabetes phenotype. Carriers of the R6H, R6C, L30M, R46Q and R55C mutations appear to have a normal birth weight (median, 4,000 g). However, birth weight in this group may be influenced by the presence of diabetes in the mothers and more studies are required to clarify the effect of these mutations on birth weight [14
The relationship between genotype (i.e. specific INS mutation) and phenotype is beginning to emerge from the studies to date. Some mutations (R6C/H, L30M, R46Q and R55C) are associated with a later age-at-diagnosis and a milder clinical course with patients maintaining adequate glycemic control on diet or oral hypoglycemic agents alone. However, the majority of INS mutations results in diabetes in the neonatal period and requires treatment with replacement doses of insulin. There can be, however, differences in the age-at-diagnosis of diabetes between carriers with the same mutation even within the same family indicating the role of other factors (genetic and nongenetic) in modifying the effect of the mutant protein on beta-cell function.
Clinical characteristics of patients with neonatal diabetes due to homozygous or compound heterozygous mutations
The phenotype of patients with diabetes due to a homozygous (or compound heterozygous) mutation in INS is characterized by severe intrauterine growth retardation (birth weight, <1 percentile) and diabetes most likely reflecting severe insulin deficiency in the pre- and postnatal life, respectively. The majority of patients are diagnosed with diabetes in the first days or weeks of life. Again, there are no extra-pancreatic manifestations.
The diabetes in this group of patients can be permanent or transient. The patients with PNDM appear to have lower birth weights (median SD scores for birth weight −3.9 vs. −1.8, P
0.03) and diabetes diagnosed earlier in life (2 days vs. 24 days, P
0.04) when compared to patients with TNDM. The mutations c. −366_−343del, c.3G>A (p.0?), c.3G>T (p.0?), c.184C>T (p.Q62X), c.−370−?186+?del) and c.*59A>G) appear to be associated with PNDM whereas the mutations at c.−218 and c.−331 have been identified in patients with both PNDM (n
6) and TNDM (n
5) as well as type 1b diabetes (n
Interestingly, the carrier parents (i.e. heterozygous for the mutation) of patients who are homozygous or compound heterozygous for an INS mutation have normal glucose tolerance indicating that a single insulin allele is sufficient to provide the insulin required to maintain normal glycemia.