Positional cloning of "Lisch-Like", a candidate modifier of susceptibility to type 2 diabetes in mice

Abstract

In 404 Lep(ob/ob) F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lep(ob). The phenotypes of B6.DBA congenic mice include reduced beta-cell replication rates accompanied by reduced beta-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated "Lisch-like" (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646-amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes.

Figure 1. Genetic Map of Sub-Congenic Lines for Diabetes-Related Phenotypes in the Interval Chr 1 164–194 Mb.

Genetic map shows sub-congenic lines (1jc, 1jcdc, 1jcd, 1jcdt) in the interval Chr1:164–194 Mb that display hypoinsulinemic hyperglycemia in association with histological evidence of a relative reduction in β-cell mass in the first 21–28 days of life due to reduced β-cell proliferation. An expanded view of the Ll gene (chr1.1224.1) is shown at bottom. Above the map scale, in black type, are microsatellite markers that were used to genotype B6 and DBA alleles to establish general boundaries of these congenic intervals. D1mit110 is the peak of the F2/F3 QTL linkage map (see Mapping T2D-related Phenotypes in B6xDBA F2/F3 Progeny). Below map scale, RefSNP (rs) and D-markers in red type identify DBA sequence limits of the respective congenic lines. Markers in blue type identify the closest, confirmed non-DBA (B6) sequence. Sequences in intervals between markers in red and blue type are DBA vs. B6 invariant. Gray bars are DBA-derived sequences. Yellow box corresponds to a 3.2 Mb interval, conserved between DBA and B6. The red box identifies the N-scan predicted gene, chr1.1224.1, subsequently identified as Lisch-like (Ll), extending centromerically from line 1jcdt. In the expanded view of Ll, the B6 boundary (rs31968429) for lines 1jcdc, 1jcd, 1jcdt is 333 bp centromeric of exon 7; the DBA boundary, (rs33860076) is 2,700 bp telomeric of exon 7. 5330438I03Rik is an anti-sense transcript described in detail in the text. Marker positions are from the mouse genome annotation (NCBI Build 36, February 2006).

Figure 2. Fasting Blood Glucose and Glucose Tolerance Tests in Congenic Lines.

A) Blood glucose in Lep^ob/ob males congenic for the interval 1jcd (BB; homozygous for B6; DD; homozygous for DBA in the interval) and fed regular mouse chow diet (9% fat) ad libitum. Determinations made following a 4 h morning fast. 4–13 animals per genotype group. B) Blood glucose in Lep^+/+ males congenic for the interval 1jcd and fed high fat diet (60% of calories as fat) ad libitum for 13 wks, starting at 7 wks of age. Determinations made following a 4 h morning fast. C) ipGTT in 60-day old Lep^ob/ob males congenic for the interval 1jcdc. D) ipGTT in 200-day old Lep^ob/ob males congenic for the interval 1jcdc. E) ipGTT in 14-wk old male Lep^+/+ males congenic for the interval 1jc who had been fed the “Surwit” diet for 10 wks. In all panels, * indicates p<0.05 (2 tailed t-test for genotype effect of congenic interval; mean is +/−SEM.

Figure 3. Plasma Insulin/Glucose Ratios and Hyperglycemic Clamps in Age-Grouped 1jc Congenic Males.

A) Fasting plasma insulin/glucose ratios in 30- and 62-day old 1jc Lep^ob/ob B/B and D/D male mice, chow-fed since weaning. Raw data are shown in Table S1. Asterisk (*) indicates significant difference between B/B and D/D animals; p-value <0.05 for 2-tailed t-test. B) Hyperglycemic clamping in 100-day old 1jc males on Surwit Diet for 18 weeks. 1jc DD male mice fed a Surwit diet for 18 wks were clamped at a blood glucose concentration of 250 mg/dl for 1 hr and serum insulin concentrations measured at 1 hr. Asterisk (*) indicates p-value <0.05 for 2-tailed t-test.

Figure 4. Relationship between Islet Histology and Insulin Secretion.

A) Islet histology in 21-day old 1jcd Lep^ob/ob B/B and D/D male mice. 4 µm pancreatic sections from 21-day old 1jcd Lep^ob/ob B/B and D/D male mice were insulin-stained with anti-guinea pig IgG and visualized by light microscopy at 10× magnification. In D/D animals, islets were smaller and less numerous. By histomorphometry, the proportion of small islets (250–2000 µm²) in 21-day old Lep^ob/ob males was greater in D/D (1jc and 1jcd) mice (73%) than in B/B (60%); whereas the proportion of large islets (10,000–50,000 µm²) was lower (9% in D/D and 14% in B/B). B) In vitro glucose-stimulated insulin secretion in pancreatic islets in 28-day old 1jc Lep^ob/ob B/B and D/D males. Each congenic genotype group consisted of 3 male animals. Negative control consisted of 3 4-week old diabetes-prone Lepr^db/db KsJ male animals that are hypo-responsive to glucose stimulation ; positive control was 3 4-week old insulin-resistant animals segregating for a diabetes-susceptibility QTL on Chr5 at 78cM, characterized by hyperglycemia and hyperinsulinemia. B/B and D/D show dose response, but no B/B vs. D/D difference at any concentration of glucose. Response to 10 mM arginine in the same animals confirms that the β-cells of the B/B and D/D congenics are comparable with regard to insulin release to a non-glucose stimulus. The 0 mM arginine control in B/B is shown to establish baseline insulin levels.

Figure 5. β-Cell Mass and Replication Rates in 1jcd Lep^ob/ob Males.

A) Relative β-cell area in 20-, 60, and 150-day old Lep^ob/ob males segregating for B and D 1jcd congenic intervals. In 60 and 150-day old males segregating for the D/D sub-congenic interval, relative β-cell mass was approximately half that of B/B littermates; B/D animals were intermediate at 150 days. N = 10 for each of the 3 groups of animals. Mean+/−SEM. The asterisk (*) indicates that p<0.05 for D/D vs. B/B at 60 days, and D/D and B/D vs B/B at 150 days. These findings are consistent with in vivo data (see Figure 2C) showing onset of elevated blood glucose at rest and during ipGTT by 60 days. B) β-cell replication rates (Ki67) in 1- and 21-day old Lep^ob/ob B/B and D/D 1jcd males. To estimate the proportion of dividing cells, the number of Ki67-positive β-cells was normalized to the total number of insulin-positive cells. Replication of β-cells in 1-day old D/D males was ∼1/3 that of B/B littermates (p = 0.017). This difference, not present in 21-day old animals, was probably due to normally reduced β-cell replication by the time of weaning. Mean+/−SEM. The asterisk (*) indicates that p<0.05 for D/D vs. B/B in 1-day old animals.

Figure 6. Genes in the Minimal Congenic Interval on Chr1:168.1–170.3 Mb.

Gray background corresponds to the minimal DBA “variable” interval from 168.1 Mb–169.9 Mb, between markers rs33860076 and rs30708865. Yellow background corresponds to the centromeric end of the DBA vs. B6 “conserved” interval (i.e. nominally invariant). Genes in blue are from RefSeq; genes in black are predicted and locally confirmed as described in the text. The N-scan predicted gene chr1.1224.1 is designated here as “Lisch-like”. Amino acid variants are shown in red to the right of the corresponding gene. Nucleotide substitutions were confirmed by bidirectional sequencing in both C57BL/6J and DBA/2J DNA.

Figure 7. Expression Analysis of Candidate Genes and Liver Expression of Lisch-like.

A) Tissue-specific expression analysis of genes in the “variable” portion of the minimum congenic interval. Data for relative expression (B/B to D/D) from Table 1 for hypothalamus, islets, liver and EDL-muscle are displayed graphically and numerically below the graph. 21-day old DD and BB Lep^ob/ob 1jc congenic males were analyzed using Affymetrix #430A microarrays. B) Liver expression of Lisch-like in 1jc B/B and D/D males from 21–120 days. Samples from Lep^ob/ob 1jc males were analyzed by qPCR.

Figure 8. Predicted Structure of Ll Gene with Expanded Views of Critical Regions.

Lisch-like gene (middle of figure) is the full-length, 10-exon, splice variant (iso1) and includes 872 bp upstream of the transcriptional start site. Predicted domains are below exons. Exon 1 includes the 5′ UTR (narrow orange bar) and cleavable signal peptide (SP). Exons 2–4 are extra-cellular, within which exons 2–3 code for an Ig-like domain. Exon 5 includes the TMD with a very cysteine-rich cluster in the carboxyl half; exons 6–10 code for a serine- and proline-rich intracellular domain; exon 10 also includes a long 3′ UTR. The “xs” identify exons deleted in isoforms 2–4. A. 5′ upstream interval (expanded view); Black bars correspond to BLAT displays vs. the reference B6 genome. DBA variants are below the DBA bar. Annotations are composites of displays from the UCSC Genome Browser on Mouse February 2006 Assembly. Royal blue peaks correspond to sequences with predicted ESPERR regulatory potential (http://www.bx.psu.edu/projects/esperr). The darker blue blocks correspond to evolutionarily conserved sequences, from the UCSC “Conservation” track. Lighter blue blocks show positions of simple sequence motifs, with the consensus motif shown below. The CpG island track, provided by the UCSC Genome Browser, was generated using the unpublished cpglh program from Washington University (St. Louis) Genome Sequencing Center. B. Anti-sense interval corresponds to the sequences overlapping the Riken transcript 5339438I03Rik. The green triangle identifies a 37 nt unique sequence insertion in DBA. The two non-synonymous sequence variants in exon 9 sense transcript are shown. The SNP rs33860076 is the DBA marker at the centromeric end of congenic lines 1jcdc, 1jcd, and 1jcdt. C. 3′ UTR interval; vertical black bars represent positions of 52 B6 vs. DBA nucleotide sequence variants.

Figure 9. ClustalW Analysis of Lisch-like Homologs and the LSR Protein.

ClustalW analysis was performed on the EMBL-EBI server (www.ebi.ac.uk/clustalw/) using their default settings. We modified the display to emphasize exonic alignments. Positions of the two non-synonymous variants in exon 9 of Ll are identified by blue shading. The non-homologous extension of mouse Lsr exon 6 (pink background) is shown beneath exon 7. Mm_Ll; Mus musculus Lisch-like; Hs_C1orf32, Homo sapiens C1orf32; Dr_Ll, Danio rerio (zebra fish) Lisch-like ortholog; Mm_LSR, Mus musculus LSR-like ortholog. Pair-wise similarity scores by isoform and domain are shown in Table S2.

Figure 10. Expression Patterns and Morpholino Knockdown in Zebra Fish Embryos.

A) Developmental expression of zebra fish Lisch-like and Lsr-like orthologs. Lisch-like RNA was hybridized in situ to whole-mount zebra fish embryos at 48 hours post-fertilization (hpf), dorsal view with anterior towards the top; and 72 hpf, lateral view with anterior towards the top, ventral towards the right and yolk removed. Lsr-like RNA was hybridized at 48 hpf and 34 hpf. Ll panels show ventral views of embryos with yolks removed and anterior towards the top. Lsr-like panels show the same image captured in the focal plane of the anterior (ap) and posterior (pp) pancreatic buds, respectively. i, intestine; ph, pharynx; pn, pronephric ducts; l, liver; ap, anterior pancreatic bud; pp, posterior pancreatic bud; p, pancreas (after anterior and posterior bud fusion); b, brain; o, otic vesicle. B) Morpholino knockdown of Lisch-like and Lsr-like orthologs at 48 hpf. Two dimensional ventral views (anterior towards top) of confocal stacks of 48 hpf embryos, uninjected or injected with 15 ng morpholino: control, Lsr-like sp1, and Lisch-like ATG. Gut-GFP transgene expression (green); insulin immunolabelling (red).

Figure 11. Phenotypes of Mice Segregating for the W87* Allele of Lisch-like.

A) Western analysis of Lisch-like in hypothalamus of 1jc and homozygous W87* mice. The Western immunoblot shows differences in Ll expression in hypothalami of 1jc Lep^ob/ob B/B vs. D/D congenic males (left panel), and between wild-type C3HeB/FeJ and W87* C3HeB/FeJ males (right panel). The right panel immunoblot was incubated with rabbit anti-LL antiserum, prepared against a polypeptide corresponding to exons 7 and 8 of the ICD. The antiserum had been absorbed to fixed liver extracts from knock-out mice in order to block non-specific proteins from interacting with the antibody. The LL transcript isomers are visible as a 65 and 70 kD doublet in the B/B and C3HeB/FeJ wild-type lanes, but absent in the lanes of the 1jc-D/D congenic and C3HeB/FeJ W87* homozygous ENU mutants. B) Percent Replicating β-cells in 14-day old ENU-mutagenized mice. The percentage of Ki67-positive β-cells was estimated in 14-day old C3HeB/FeJ ENU-mutagenized mice, who were either homozygous wild-type (+/+), heterozygous (+/−), or homozygous for the W87* LL amber mutation (−/−). At 14 days there was a 2-fold difference in the % of Ki67⁺ β-cells in +/+ (3.75%) vs. −/− (1.75%) ENU W87* mice; +/− were intermediate (2.5%). Non-overlapping images of longitudinal pancreatic sections (200 µm apart) were acquired and analyzed using ImageJ software version 1.37 (NIH) to count insulin-positive and Ki67⁺ cells. Pancreatic weights of +/+ and −/− were not different. C) Fasting blood glucose (squares) and insulin/glucose ratios (diamonds) in W87* (−/−) and wild-type (+/+) littermates. P-value <0.05 for 2-tailed t-test at 63 days of age. Data points at other ages show trends. D) ipGTT on 50-day old Surwit-fed B6.CH3. N3F1 W87* males. Glucose intolerance is seen in W87* mice. Mice were fasted overnight prior to dextrose injection (50% dextrose solution, 0.5 g/kg, ip). Capillary tail bleeds were performed at the specified time points to determine circulating glucose levels by glucometer (FreeStyle Flash, Abbott). Blood glucose concentrations that are marked with an asterisk are significantly different (t-test; p<0.05; mean±SEM). Area under curve +/+ vs. −/− (p = 0.02).

Figure 12. LOD Scores for Markers along Chromosome 1.

LOD scores are shown for fasting blood glucose (black) and pancreatic grade (blue). Terminal phenotypes by genotype at D1Mit110 at 169.6 Mb are summarized in Table S5.