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

Abstract

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.

Fig. 1.

A schematic of the INS gene showing the 10 mutations identified in 15 families. Positions of point mutations are indicated below the exons, while deletions are shown above the gene. The blue shaded regions are noncoding, the red text indicates a deletion, the blue text are noncoding mutations, and the green are coding mutations. The precise breakpoints of the multiexonic deletion are not known; the solid line represents the minimal deleted region. Mutation nomenclature is based on the coding sequence where nucleotide 1 represents translational start site.

Fig. 2.

Partial pedigrees of the 15 families with recessive INS mutations. (Del, deletion; n, Normal allele; M, mutation). Solid black-filled shapes represent patients with permanent neonatal diabetes, gray filled shapes represent patients with transient neonatal diabetes, and shapes filled with diagonal lines represent those patients diagnosed with diabetes after 6 months of age. Age at diagnosis and remission (where applicable) are shown below the symbols.

Fig. 3.

Functional evidence for the pathogenicity of recessive promoter INS mutations. (A) Schematic of the genomic sequence of the INS promoter structure with major cis regulatory elements, and the sequence context of mutated elements in several mammalian species that do not exhibit major divergence in these regions. Mutated bases are highlighted in red. The numbering of promoter landmarks is relative to the transcription start site (genomic numbering, where g.1 is equivalent to c.-238) consistent with the convention used in previous studies. Mutations are described according to Human Genome Variation Society guidelines (http://www.hgvs.org/mutnomen/) (cDNA numbering according to the translational start site where c.1 is equivalent to g.238), and distance to the conventional INS transcriptional start site is shown in parenthesis. (B) Evidence for loss-of-function of the c.-331(C > G, C > A) and c.-332C > G mutations. Firefly luciferase expression is compared in constructs containing the wild-type (WT) INS promoter sequence (INS WT), or c.-331 C > G, c.-331 C > A, c.-332 C > G, c.-339G > A mutations, after transfection in MIN6 β-cells. Data shown are means (+/−SE) from three independent constructs for each mutation (n = 3 replicates). c.-339G > A is a control mutation that does not impair INS transcription. Results are corrected for transfection efficiency using a vector that constitutively expresses Renilla luciferase, and are expressed relative to the INS WT results. The asterisks denote P < 0.0001 in ANOVA for the difference between INS WT and mutant constructs.

Fig. 4.

Functional evidence for the pathogenicity of recessive INS mutations affecting translation and mRNA stability (A) Homozygous mutations in the translation initiation codon of the INS gene result in reduced insulin content of transfected HeLa cells. The insulin content of HeLa cells was measured by RIA after transfection with wild-type insulin (INS WT) or either of two INS mutant constructs, as shown. Both nucleotide changes were identified in patients with permanent neonatal diabetes. Nonspecific values obtained with HeLa cells transfected with empty vector were subtracted from all samples and those data are presented as mean +/− SE (n = 3 replicates). (B) Allele-specific quantitative real-time PCR of c.*59A > G and normal transcripts. The graph shows the relative abundance of the wild-type and mutant RNA transcripts in mutant and normal cell lines. The rs3842753 A allele tags the c.*59A (wild type, shown in green); the c.*59G (mutant) was tagged by rs3842753 C allele (blue). The graph shows the level of transcripts in the control sample heterozygous only for rs3842753 and in the maternal sample (family DM1165), which is heterozygous for both rs3842753 and c.*59A/G. The level of the mutant transcript is reduced to less than 3 × 10⁻⁴ percent compared with the normal transcript in the heterozygous c.*59A > G cell line. Experimental error as calculated from the standard deviation of the replicate experiments is indicated. The standard deviation for the quantification of the c.*59G allele in the maternal sample is 3 × 10⁻⁶, and thus the experimental error is not visible in the figure.