Effect of genetic variation in a Drosophila model of diabetes-associated misfolded human proinsulin

Abstract

The identification and validation of gene-gene interactions is a major challenge in human studies. Here, we explore an approach for studying epistasis in humans using a Drosophila melanogaster model of neonatal diabetes mellitus. Expression of the mutant preproinsulin (hINS(C96Y)) in the eye imaginal disc mimics the human disease: it activates conserved stress-response pathways and leads to cell death (reduction in eye area). Dominant-acting variants in wild-derived inbred lines from the Drosophila Genetics Reference Panel produce a continuous, highly heritable distribution of eye-degeneration phenotypes in a hINS(C96Y) background. A genome-wide association study (GWAS) in 154 sequenced lines identified a sharp peak on chromosome 3L, which mapped to a 400-bp linkage block within an intron of the gene sulfateless (sfl). RNAi knockdown of sfl enhanced the eye-degeneration phenotype in a mutant-hINS-dependent manner. RNAi against two additional genes in the heparan sulfate (HS) biosynthetic pathway (ttv and botv), in which sfl acts, also modified the eye phenotype in a hINS(C96Y)-dependent manner, strongly suggesting a novel link between HS-modified proteins and cellular responses to misfolded proteins. Finally, we evaluated allele-specific expression difference between the two major sfl-intronic haplotypes in heterozygtes. The results showed significant heterogeneity in marker-associated gene expression, thereby leaving the causal mutation(s) and its mechanism unidentified. In conclusion, the ability to create a model of human genetic disease, map a QTL by GWAS to a specific gene, and validate its contribution to disease with available genetic resources and the potential to experimentally link the variant to a molecular mechanism demonstrate the many advantages Drosophila holds in determining the genetic underpinnings of human disease.

Keywords: Drosophila; genome-wide association study; heparan sulfate proteoglycan; mutant insulin; sulfateless.

Figure 1

Distribution of eye area in hINS^C96Y × DGRP crosses. Mean ±1 SD, sorted by the mean, is shown for crosses between the transgenic {GMR >> hINS^C96Y} line to 178 DGRP lines, and two randomly chosen DGRP inbred lines (red). Representative photographs of eyes from across the range of the distribution are shown. The rightmost image is of a nontransgenic wild-type fly eye.

Figure 2

Genome-wide scan identifies candidate locus associated with the hINS^C96Y-induced phenotype. Quantile–quantile (Q–Q) plot reveals an excess of small P-values on autosomes (A) but not on the X chromosome (B), which is not variable in the mapping population due to cross design. (C) Manhattan plot shows a strong peak (green) on chromosome 3L. The blue and red horizontal lines indicate raw P < 10⁻⁵ and Bonferroni corrected P < 0.05, respectively. (D) UCSC browser view of the sfl locus containing the association peak. The intron containing the peak also contains a nested gene CG32396.

Figure 3

RNAi knockdown confirms sfl and excludes CG32396 as the causal gene. The effect of knocking down either CG32396 or sfl was tested in the absence ({UAS–RNAi} × {GMR–Gal4}) or presence ({UAS–RNAi} × {GMR–Gal4, UAS–hINS^C96Y}) of hINS^C96Y. Compared to the control crosses (first and third columns in both sexes), significant difference in mean eye area was observed only with RNAi against sfl and only in the presence of hINS^C96Y (n = 15, asterisks above a box plot indicate significant differences at 0.05 level determined by a student’s t-test, with Bonferroni correction for multiple testing). In box plots, the median (black dot), interquartile (box), and 1.5 times the interquartile range (whiskers) are indicated; data points outside the range are represented by circles.

Figure 4

RNAi and mutant analysis for heparin sulfate biosynthesis pathway genes. The experimental design is the same as in Figure 3. Left: the effect of RNAi or mutant alleles in the absence of hINS^C96Y expression. Right: the effect when hINS^C96Y is expressed in the eye imaginal disc. Mutants were tested in heterozygous states for a dominant interaction with hINS^C96Y. Fifteen male flies are measured for each group. The statistical significance of differences from the control cross (gray, w1118) was determined by a two-sided student’s t-test. Those that are significant at 0.05 level after Bonferroni correction are marked with a red arrowhead.

Figure 5

Sequencing of a 3-kb region in sfl and the LD patterns in the region. (A) Alignment of 19 DGRP sequences ordered by their eye-degeneration phenotype (mean, most severe on the bottom). The hINS^C96Y transgenic line (asterisk) was also sequenced. Red ticks and white spaces indicate SNPs and deletions relative to the reference sequence. No insertions relative to the reference were found. The purple track shows the −log₁₀ of GWAS P-values. The bottom track shows the linkage blocks as determined by Haploview (4.02) using the solid spine method with default settings (D’ > 0.8). (B) Detailed haplotype block structures. Each numbered column represents a polymorphic site, with the alleles colored as blue or red; each row represents a haplotype with frequency >0.01. An arrowhead marks the 18-/4-bp indel polymorphism (see text; 18 bp, blue; 4 bp, red). Finally, the number between any two blocks represents the multiallelic D’, which quantifies the associations between adjacent blocks.

Figure 6

Pyro-sequencing measure of sfl allele-specific transcript ratio in 18-/4-bp heterozygotes. (A) Schematic diagram of the pyrosequencing approach. Colored lines represent transcripts (mRNA) associated with either the 18 or the 4bp allele, expressed at different levels. Common primers were used to amplify both transcripts of the gene of interest from the cDNA library made from eye imaginal disc tissues. Pyrosequencing was carried out on the amplified products. (B) A pyrogram of a heterozygote with the polymorphic site (G/C) that is diagnostic for the 18-/4-bp indel highlighted. The ratio of the two peaks (light intensity, y-axis) are used to calculate the relative ratio of the two alleles. (E, enzyme; S, substrate; A/C/G/T, nucleotides). (C) Log2-transformed ratio of 18-/4-bp allele expression in 15 crosses between randomly paired 18- and 4-bp lines. Estimates of the ratio and 95% confidence intervals are plotted. The dotted line corresponds to equal expression from the two alternative alleles.