Two Proximally Close Priority Candidate Genes for diplopodia-1, an Autosomal Inherited Craniofacial-Limb Syndrome in the Chicken: MRE11 and GPR83.

Next-generation sequencing (NGS) and expression technologies were utilized to investigate the genes and sequence elements in a 586 kb region of chicken chromosome 1 associated with the autosomal recessive diplopodia-1 (dp-1) mutation. This mutation shows a syndromic phenotype similar to known human developmental abnormalities (e.g., cleft palate, polydactyly, omphalocele [exposed viscera]). Toward our goal to ascertain the variant responsible, the entire 586 kb region was sequenced following utilization of a specifically designed capture array and to confirm/validate fine-mapping results. Bioinformatic analyses identified a total of 6142 sequence variants, which included SNPs, indels, and gaps. Of these, 778 SNPs, 146 micro-indels, and 581 gaps were unique to the UCD-Dp-1.003 inbred congenic line; those found within exons and splice sites were studied for contribution to the mutant phenotype. Upon further validation with additional mutant samples, a smaller subset (of variants [51]) remains linked to the mutation. Additionally, utilization of specific samples in the NGS technology was advantageous in that fine-mapping methodologies eliminated an additional 326 kb of sequence information on chromosome 1. Predicted and confirmed protein-coding genes within the smaller 260 kb region were assessed for their developmental expression patterns over several stages of early embryogenesis in regions/tissues of interest (e.g., digits, craniofacial region). Based on these results and known function in other vertebrates, 2 genes within 5 kb of each other, MRE11 and GPR83, are proposed as high-priority candidates for the dp-1 mutation.

An APOO Pseudogene on Chromosome 5q Is Associated With Low-Density Lipoprotein Cholesterol Levels.

BACKGROUND: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease via its contribution to the development and progression of atherosclerotic lesions. Although the genetic basis of LDL-C has been studied extensively, currently known genetic variants account for only ≈20% of the variation in LDL-C levels. METHODS: Through an array-based association analysis in 1102 Amish subjects, we identified a variant strongly associated with LDL-C levels. Using a combination of genetic analyses, zebrafish models, and in vitro experiments, we sought to identify the causal gene driving this association. RESULTS: We identified a founder haplotype associated with a 15 mg/dL increase in LDL-C on chromosome 5. After recombination mapping, the associated region contained 8 candidate genes. Using a zebrafish model to evaluate the relevance of these genes to cholesterol metabolism, we found that expression of the transcribed pseudogene, APOOP1, increased LDL-C and vascular plaque formation. CONCLUSIONS: Based on these data, we propose that APOOP1 regulates levels of LDL-C in humans, thus identifying a novel mechanism of lipid homeostasis.

TM6SF2 rs58542926 impacts lipid processing in liver and small intestine.

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).

Assignment of Functional Relevance to Genes at Type 2 Diabetes-Associated Loci Through Investigation of ß-Cell Mass Deficits.

Type 2 diabetes (T2D) has been associated with a large number of genomic loci, many of which encompass multiple genes without a definitive causal gene. This complexity has hindered efforts to clearly identify functional candidate genes and interpret their role in mediating susceptibility to disease. Here we examined the relevance of individual genes found at T2D-associated loci by assessing their potential contribution to a phenotype relevant to the disease state: production and maintenance of ß-cell mass. Using transgenic zebrafish in which ß-cell mass could be rapidly visualized in vivo, we systematically suppressed the expression of orthologs of genes found at T2D-associated genomic loci. Overall, we tested 67 orthologs, many of which had no known relevance to ß-cell mass, at 62 human T2D-associated loci, including eight loci with multiple candidate genes. In total we identified 25 genes that were necessary for proper ß-cell mass, providing functional evidence for their role in a physiological phenotype directly related to T2D. Of these, 16 had not previously been implicated in the regulation of ß-cell mass. Strikingly, we identified single functional candidate genes at the majority of the loci for which multiple genes were analyzed. Further investigation into the contribution of the 25 genes to the adaptive capacity of ß-cells suggested that the majority of genes were not required for glucose-induced expansion of ß-cell mass but were significantly necessary for the regeneration of ß-cells. These findings suggest that genetically programmed deficiencies in ß-cell mass may be related to impaired maintenance. Finally, we investigated the relevance of our findings to human T2D onset in diabetic individuals from the Old Order Amish and found that risk alleles in ß-cell mass genes were associated with significantly younger age of onset and lower body mass index. Taken together, our study offers a functional approach to assign relevance to genes at T2D-associated loci and offers experimental evidence for the defining role of ß-cell mass maintenance in genetic susceptibility to T2D onset.

Differential effects on ß-cell mass by disruption of Bardet-Biedl syndrome or Alstrom syndrome genes.

Rare genetic syndromes characterized by early-onset type 2 diabetes have revealed the importance of pancreatic ß-cells in genetic susceptibility to diabetes. However, the role of genetic regulation of ß-cells in disorders that are also characterized by highly penetrant obesity, a major additional risk factor, is unclear. In this study, we investigated the contribution of genes associated with two obesity ciliopathies, Bardet-Biedl Syndrome and Alstrom Syndrome, to the production and maintenance of pancreatic ß-cells. Using zebrafish models of these syndromes, we identified opposing effects on production of ß-cells. Loss of the Alstrom gene, alms1, resulted in a significant decrease in ß-cell production whereas loss of BBS genes, bbs1 or bbs4, resulted in a significant increase. Examination of the regulatory program underlying ß-cell production suggested that these effects were specific to ß-cells. In addition to the initial production of ß-cells, we observed significant differences in their continued maintenance. Under prolonged exposure to high glucose conditions, alms1-deficient ß-cells were unable to continually expand as a result of decreased proliferation and increased cell death. Although bbs1-deficient ß-cells were similarly susceptible to apoptosis, the overall maintenance of ß-cell number in those animals was sustained likely due to increased proliferation. Taken together, these findings implicate discrepant production and maintenance of ß-cells in the differential susceptibility to diabetes found between these two genetic syndromes.

Disruption of ldlr causes increased LDL-c and vascular lipid accumulation in a zebrafish model of hypercholesterolemia.

Hyperlipidemia and arterial cholesterol accumulation are primary causes of cardiovascular events. Monogenic forms of hyperlipidemia and recent genome-wide association studies indicate that genetics plays an important role. Zebrafish are a useful model for studying the genetic susceptibility to hyperlipidemia owing to conservation of many components of lipoprotein metabolism, including those related to LDL, ease of genetic manipulation, and in vivo observation of lipid transport and vascular calcification. We sought to develop a genetic model for lipid metabolism in zebrafish, capitalizing on one well-understood player in LDL cholesterol (LDL-c) transport, the LDL receptor (ldlr), and an established in vivo model of hypercholesterolemia. We report that morpholinos targeted against the gene encoding ldlr effectively suppressed its expression in embryos during the first 8 days of development. The ldlr morphants exhibited increased LDL-c levels that were exacerbated by feeding a high cholesterol diet. Increased LDL-c was ameliorated in morphants upon treatment with atorvastatin. Furthermore, we observed significant vascular and liver lipid accumulation, vascular leakage, and plaque oxidation in ldlr-deficient embryos. Finally, upon transcript analysis of several cholesterol-regulating genes, we observed changes similar to those seen in mammalian systems, suggesting that cholesterol regulation may be conserved in zebrafish. Taken together, these observations indicate conservation of ldlr function in zebrafish and demonstrate the utility of transient gene knockdown in embryos as a genetic model for hyperlipidemia.

The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2.

talpid(2) is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid(3), talpid(2) has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the etiology of the craniofacial phenotype of this mutant. We confirmed that primary cilia were disrupted in talpid(2) mutants. Molecularly, we found disruptions in Hedgehog signaling. Post-translational processing of GLI2 and GLI3 was aberrant in the developing facial prominences. Although both GLI2 and GLI3 processing were disrupted in talpid(2) mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid(2) phenotype was linked to a 1.4 Mb region on GGA1q that contained the gene encoding the ciliary protein C2CD3. We cloned the avian ortholog of C2CD3 and found its expression was ubiquitous, but most robust in the developing limbs and facial prominences. Furthermore, we found that C2CD3 is localized proximal to the ciliary axoneme and is important for docking the mother centriole to the ciliary vesicle and cell membrane. Finally, we identified a 19 bp deletion in talpid(2) C2CD3 that produces a premature stop codon, and thus a truncated protein, as the likely causal allele for the phenotype. Together, these data provide insight into the cellular, molecular and genetic etiology of the talpid(2) phenotype. Our data suggest that, although the talpid(2) and talpid(3) mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.

Primary cilia in pancreatic development and disease.

Primary cilia and their anchoring basal bodies are important regulators of a growing list of signaling pathways. Consequently, dysfunction in proteins associated with these structures results in perturbation of the development and function of a spectrum of tissue and cell types. Here, we review the role of cilia in mediating the development and function of the pancreas. We focus on ciliary regulation of major pathways involved in pancreatic development, including Shh, Wnt, TGF-ß, Notch, and fibroblast growth factor. We also discuss pancreatic phenotypes associated with ciliary dysfunction, including pancreatic cysts and defects in glucose homeostasis, and explore the potential role of cilia in such defects.