Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility.

BACKGROUND: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). METHODS: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. RESULTS: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators. CONCLUSIONS: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

Generation and characterisation of four multiple sclerosis iPSC lines from a single family.

Multiple sclerosis (MS) is a complex neuroinflammatory/degenerative disease of the central nervous system (CNS) that results in the formation of demyelinated lesions and axon degeneration. MS aetiology is complex, with genetics estimated to account for ∼48% of MS risk (International Multiple Sclerosis Genetics Consortium, 2019). Despite this, families with a high incidence of MS are rare. We have generated four induced pluripotent stem cell (iPSC) lines from individuals with relapsing-remitting and secondary progressive MS within a single family. The generation of disease-specific iPSC lines from multiple members of a single family will facilitate MS genetic and functional studies.

Using MS induced pluripotent stem cells to investigate MS aetiology.

Multiple sclerosis (MS) is a complex disease, and its pathophysiology impacts the function of immune and central nervous system cell types. Despite extensive investigation into the aetiology of MS, the underlying cause/s remain elusive and consequently, faithful in vitro or in vivo preclinical models of MS do not exist. Advances in human stem cell technologies have enabled the generation of induced pluripotent stem cells (iPSCs) from people with MS. This review summarises the discoveries made using iPSCs derived from people with MS and explores their current and potential application/s in MS research.

Multi-phenotype genome-wide association studies of the Norfolk Island isolate implicate pleiotropic loci involved in chronic kidney disease.

Chronic kidney disease (CKD) is a persistent impairment of kidney function. Genome-wide association studies (GWAS) have revealed multiple genetic loci associated with CKD susceptibility but the complete genetic basis is not yet clear. Since CKD shares risk factors with cardiovascular diseases and diabetes, there may be pleiotropic loci at play but may go undetected when using single phenotype GWAS. Here, we used multi-phenotype GWAS in the Norfolk Island isolate (n = 380) to identify new loci associated with CKD. We performed a principal components analysis on different combinations of 29 quantitative traits to extract principal components (PCs) representative of multiple correlated phenotypes. GWAS of a PC derived from glomerular filtration rate, serum creatinine, and serum urea identified a suggestive peak (pmin = 1.67 × 10-7) that mapped to KCNIP4. Inclusion of other secondary CKD measurements with these three kidney function traits identified the KCNIP4 locus with GWAS significance (pmin = 1.59 × 10-9). Finally, we identified a group of two SNPs with increased minor allele frequencies as potential functional variants. With the use of genetic isolate and the PCA-based multi-phenotype GWAS approach, we have revealed a potential pleotropic effect locus for CKD. Further studies are required to assess functional relevance of this locus.

Identifying the Lipidomic Effects of a Rare Loss-of-Function Deletion in ANGPTL3.

BACKGROUND: The identification and understanding of therapeutic targets for atherosclerotic cardiovascular disease is of fundamental importance given its global health and economic burden. Inhibition of ANGPTL3 (angiopoietin-like 3) has demonstrated a cardioprotective effect, showing promise for atherosclerotic cardiovascular disease treatment, and is currently the focus of ongoing clinical trials. Here, we assessed the genetic basis of variation in ANGPTL3 levels in the San Antonio Family Heart Study. METHODS: We assayed ANGPTL3 protein levels in ≈1000 Mexican Americans from extended pedigrees. By drawing upon existing plasma lipidome profiles and genomic data we conducted analyses to understand the genetic basis to variation in ANGPTL3 protein levels, and accordingly the correlation with the plasma lipidome. RESULTS: In a variance components framework, we identified that variation in ANGPTL3 was significantly heritable (h2=0.33, P=1.31×10-16). To explore the genetic basis of this heritability, we conducted a genome-wide linkage scan and identified significant linkage (logarithm of odds =6.18) to a locus on chromosome 1 at 90 centimorgans, corresponding to the ANGPTL3 gene location. In the genomes of 23 individuals from a single pedigree, we identified a loss-of-function variant, rs398122988 (N121Kfs*2), in ANGPTL3, that was significantly associated with lower ANGPTL3 levels (ß=-1.69 SD units, P=3.367×10-13), and accounted for the linkage signal at this locus. Given the known role of ANGPTL3 as an inhibitor of endothelial and lipoprotein lipase, we explored the association of ANGPTL3 protein levels and rs398122988 with the plasma lipidome and related phenotypes, identifying novel associations with phosphatidylinositols. CONCLUSIONS: Variation in ANGPTL3 protein levels is heritable and under significant genetic control. Both ANGPTL3 levels and loss-of-function variants in ANGPTL3 have significant associations with the plasma lipidome. These findings further our understanding of ANGPTL3 as a therapeutic target for atherosclerotic cardiovascular disease.

Mesenchymal plasticity of devil facial tumour cells during in vivo vaccine and immunotherapy trials.

Immune evasion is critical to the growth and survival of cancer cells. This is especially pertinent to transmissible cancers, which evade immune detection across genetically diverse hosts. The Tasmanian devil (Sarcophilus harrisii) is threatened by the emergence of Devil Facial Tumour Disease (DFTD), comprising two transmissible cancers (DFT1 and DFT2). The development of effective prophylactic vaccines and therapies against DFTD has been restricted by an incomplete understanding of how allogeneic DFT1 and DFT2 cells maintain immune evasion upon activation of tumour-specific immune responses. In this study, we used RNA sequencing to examine tumours from three experimental DFT1 cases. Two devils received a vaccine prior to inoculation with live DFT1 cells, providing an opportunity to explore changes to DFT1 cancers under immune pressure. Analysis of DFT1 in the non-immunised devil revealed a 'myelinating Schwann cell' phenotype, reflecting both natural DFT1 cancers and the DFT1 cell line used for the experimental challenge. Comparatively, immunised devils exhibited a 'dedifferentiated mesenchymal' DFT1 phenotype. A third 'immune-enriched' phenotype, characterised by increased PDL1 and CTLA-4 expression, was detected in a DFT1 tumour that arose after immunotherapy. In response to immune pressure, mesenchymal plasticity and upregulation of immune checkpoint molecules are used by human cancers to evade immune responses. Similar mechanisms are associated with immune evasion by DFTD cancers, providing novel insights that will inform modification of DFTD vaccines. As DFT1 and DFT2 are clonal cancers transmitted across genetically distinct hosts, the Tasmanian devil provides a 'natural' disease model for more broadly exploring these immune evasion mechanisms in cancer.

1q21.1 distal copy number variants are associated with cerebral and cognitive alterations in humans.

Low-frequency 1q21.1 distal deletion and duplication copy number variant (CNV) carriers are predisposed to multiple neurodevelopmental disorders, including schizophrenia, autism and intellectual disability. Human carriers display a high prevalence of micro- and macrocephaly in deletion and duplication carriers, respectively. The underlying brain structural diversity remains largely unknown. We systematically called CNVs in 38 cohorts from the large-scale ENIGMA-CNV collaboration and the UK Biobank and identified 28 1q21.1 distal deletion and 22 duplication carriers and 37,088 non-carriers (48% male) derived from 15 distinct magnetic resonance imaging scanner sites. With standardized methods, we compared subcortical and cortical brain measures (all) and cognitive performance (UK Biobank only) between carrier groups also testing for mediation of brain structure on cognition. We identified positive dosage effects of copy number on intracranial volume (ICV) and total cortical surface area, with the largest effects in frontal and cingulate cortices, and negative dosage effects on caudate and hippocampal volumes. The carriers displayed distinct cognitive deficit profiles in cognitive tasks from the UK Biobank with intermediate decreases in duplication carriers and somewhat larger in deletion carriers-the latter potentially mediated by ICV or cortical surface area. These results shed light on pathobiological mechanisms of neurodevelopmental disorders, by demonstrating gene dose effect on specific brain structures and effect on cognitive function.

Transcriptomic Profiling of Fibropapillomatosis in Green Sea Turtles (Chelonia mydas) From South Texas.

Sea turtle fibropapillomatosis (FP) is a tumor promoting disease that is one of several threats globally to endangered sea turtle populations. The prevalence of FP is highest in green sea turtle (Chelonia mydas) populations, and historically has shown considerable temporal growth. FP tumors can significantly affect the ability of turtles to forage for food and avoid predation and can grow to debilitating sizes. In the current study, based in South Texas, we have applied transcriptome sequencing to FP tumors and healthy control tissue to study the gene expression profiles of FP. By identifying differentially expressed turtle genes in FP, and matching these genes to their closest human ortholog we draw on the wealth of human based knowledge, specifically human cancer, to identify new insights into the biology of sea turtle FP. We show that several genes aberrantly expressed in FP tumors have known tumor promoting biology in humans, including CTHRC1 and NLRC5, and provide support that disruption of the Wnt signaling pathway is a feature of FP. Further, we profiled the expression of current targets of immune checkpoint inhibitors from human oncology in FP tumors and identified potential candidates for future studies.

Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors.

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.

Genotype phasing in pedigrees using whole-genome sequence data.

Phasing is the process of inferring haplotypes from genotype data. Efficient algorithms and associated software for accurate phasing in pedigrees are needed, especially for populations lacking reference panels of sequenced individuals. We present a novel method for phasing genotypes from whole-genome sequence data in pedigrees, called PULSAR (Phasing Using Lineage Specific Alleles/Rare variants). The method is based on the property that alleles specific to a single founding chromosome within a pedigree are highly informative for identifying haplotypes that are shared identical by descent. Simulation studies are used to assess the performance of PULSAR with various pedigree sizes and structures, and the effect of genotyping errors and the presence of nonsequenced individuals is investigated. In pedigrees with complete sequencing and realistic genotyping error rates, PULSAR correctly phases >99.9% of heterozygous genotypes, excluding sites at which all individuals are heterozygous, and does so with a switch error rate frequently below 10-4. PULSAR is highly accurate, capable of genotype error correction and imputation, and computationally competitive with alternative phasing software applicable to pedigrees. Our method has the significant advantage of not requiring reference panels that are essential for other population-based phasing algorithms. A software implementation of PULSAR is freely available.

Association of Copy Number Variation of the 15q11.2 BP1-BP2 Region With Cortical and Subcortical Morphology and Cognition.

Importance: Recurrent microdeletions and duplications in the genomic region 15q11.2 between breakpoints 1 (BP1) and 2 (BP2) are associated with neurodevelopmental disorders. These structural variants are present in 0.5% to 1.0% of the population, making 15q11.2 BP1-BP2 the site of the most prevalent known pathogenic copy number variation (CNV). It is unknown to what extent this CNV influences brain structure and affects cognitive abilities. Objective: To determine the association of the 15q11.2 BP1-BP2 deletion and duplication CNVs with cortical and subcortical brain morphology and cognitive task performance. Design, Setting, and Participants: In this genetic association study, T1-weighted brain magnetic resonance imaging were combined with genetic data from the ENIGMA-CNV consortium and the UK Biobank, with a replication cohort from Iceland. In total, 203 deletion carriers, 45 247 noncarriers, and 306 duplication carriers were included. Data were collected from August 2015 to April 2019, and data were analyzed from September 2018 to September 2019. Main Outcomes and Measures: The associations of the CNV with global and regional measures of surface area and cortical thickness as well as subcortical volumes were investigated, correcting for age, age2, sex, scanner, and intracranial volume. Additionally, measures of cognitive ability were analyzed in the full UK Biobank cohort. Results: Of 45 756 included individuals, the mean (SD) age was 55.8 (18.3) years, and 23 754 (51.9%) were female. Compared with noncarriers, deletion carriers had a lower surface area (Cohen d = -0.41; SE, 0.08; P = 4.9 × 10-8), thicker cortex (Cohen d = 0.36; SE, 0.07; P = 1.3 × 10-7), and a smaller nucleus accumbens (Cohen d = -0.27; SE, 0.07; P = 7.3 × 10-5). There was also a significant negative dose response on cortical thickness (ß = -0.24; SE, 0.05; P = 6.8 × 10-7). Regional cortical analyses showed a localization of the effects to the frontal, cingulate, and parietal lobes. Further, cognitive ability was lower for deletion carriers compared with noncarriers on 5 of 7 tasks. Conclusions and Relevance: These findings, from the largest CNV neuroimaging study to date, provide evidence that 15q11.2 BP1-BP2 structural variation is associated with brain morphology and cognition, with deletion carriers being particularly affected. The pattern of results fits with known molecular functions of genes in the 15q11.2 BP1-BP2 region and suggests involvement of these genes in neuronal plasticity. These neurobiological effects likely contribute to the association of this CNV with neurodevelopmental disorders.

Association of CREBRF variants with obesity and diabetes in Pacific Islanders from Guam and Saipan.

AIMS/HYPOTHESIS: Variants in CREBRF (rs12513649 and rs373863828) have been strongly associated with increased BMI and decreased risk of type 2 diabetes in Polynesian populations; the A allele at rs373863828 is common in Polynesians but rare in most other global populations. The aim of the present study was to assess the association of CREBRF variants with obesity and diabetes in Pacific Islander (largely Marianas and Micronesian) populations from Guam and Saipan. METHODS: CREBRF rs12513649 and rs373863828 were genotyped in 2022 participants in a community-based cross-sectional study designed to identify determinants of diabetes and end-stage renal disease (ESRD). Associations were analysed with adjustment for age, sex, ESRD and the first four genetic principal components from a genome-wide association study (to account for population stratification); a genomic control procedure was used to account for residual stratification. RESULTS: The G allele at rs12513649 had an overall frequency of 7.7%, which varied from 2.2% to 20.7% across different Marianas and Micronesian populations; overall frequency of the A allele at rs373863828 was 4.2% (range: 1.1-5.4%). The G allele at rs12513649 was associated with higher BMI (ß = 1.55 kg/m2 per copy; p = 0.0026) as was the A allele at rs373863828 (ß = 1.48 kg/m2, p = 0.033). The same alleles were associated with lower risk of diabetes (OR per copy: 0.63 [p = 0.0063] and 0.49 [p = 0.0022], respectively). Meta-analyses combining the current results with previous results in Polynesians showed a strong association between the A allele at rs373863828 and BMI (ß = 1.38 kg/m2; p = 2.5 × 10-29) and diabetes (OR 0.65, p = 1.5 × 10-13). CONCLUSIONS/INTERPRETATION: These results confirm the associations of CREBRF variants with higher BMI and lower risk of diabetes and, importantly, they suggest that these variants contribute to the risk of obesity and diabetes in Oceanic populations.

Rare DEGS1 variant significantly alters de novo ceramide synthesis pathway.

The de novo ceramide synthesis pathway is essential to human biology and health, but genetic influences remain unexplored. The core function of this pathway is the generation of biologically active ceramide from its precursor, dihydroceramide. Dihydroceramides have diverse, often protective, biological roles; conversely, increased ceramide levels are biomarkers of complex disease. To explore the genetics of the ceramide synthesis pathway, we searched for deleterious nonsynonymous variants in the genomes of 1,020 Mexican Americans from extended pedigrees. We identified a Hispanic ancestry-specific rare functional variant, L175Q, in delta 4-desaturase, sphingolipid 1 (DEGS1), a key enzyme in the pathway that converts dihydroceramide to ceramide. This amino acid change was significantly associated with large increases in plasma dihydroceramides. Indexes of DEGS1 enzymatic activity were dramatically reduced in heterozygotes. CRISPR/Cas9 genome editing of HepG2 cells confirmed that the L175Q variant results in a partial loss of function for the DEGS1 enzyme. Understanding the biological role of DEGS1 variants, such as L175Q, in ceramide synthesis may improve the understanding of metabolic-related disorders and spur ongoing research of drug targets along this pathway.

Glycated Serum Protein Genetics and Pleiotropy with Cardiometabolic Risk Factors.

Measurements of fasting glucose (FG) or glycated hemoglobin A1c (HbA1c) are two clinically approved approaches commonly used to determine glycemia, both of which are influenced by genetic factors. Obtaining accurate measurements of FG or HbA1c is not without its challenges, though. Measuring glycated serum protein (GSP) offers an alternative approach for assessing glycemia. The aim of this study was to estimate the heritability of GSP and GSP expressed as a percentage of total serum albumin (%GA) using a variance component approach and localize genomic regions (QTLs) that harbor genes likely to influence GSP and %GA trait variation in a large extended multigenerational pedigree from Jiri, Nepal (n = 1,800). We also performed quantitative bivariate analyses to assess the relationship between GSP or %GA and several cardiometabolic traits. Additive genetic effects significantly influence variation in GSP and %GA levels (p values: 1.15 × 10-5 and 3.39 × 10-5, respectively). We localized a significant (LOD score = 3.18) and novel GSP QTL on chromosome 11q, which has been previously linked to type 2 diabetes. Two common (MAF > 0.4) SNPs within the chromosome 11 QTL were associated with GSP (adjusted pvalue < 5.87 × 10-5): an intronic variant (rs10790184) in the DSCAML1 gene and a 3'UTR variant (rs8258) in the CEP164 gene. Significant positive correlations were observed between GSP or %GA and blood pressure, and lipid traits (p values: 0.0062 to 1.78 × 10-9). A significant negative correlation was observed between %GA and HDL cholesterol (p = 1.12 × 10-5). GSP is influenced by genetic factors and can be used to assess glycemia and diabetes risk. Thus, GSP measurements can facilitate glycemic studies when accurate FG and/or HbA1c measurements are difficult to obtain. GSP can also be measured from frozen blood (serum) samples, which allows the prospect of retrospective glycemic studies using archived samples.

Rediscovering the value of families for psychiatric genetics research.

As it is likely that both common and rare genetic variation are important for complex disease risk, studies that examine the full range of the allelic frequency distribution should be utilized to dissect the genetic influences on mental illness. The rate limiting factor for inferring an association between a variant and a phenotype is inevitably the total number of copies of the minor allele captured in the studied sample. For rare variation, with minor allele frequencies of 0.5% or less, very large samples of unrelated individuals are necessary to unambiguously associate a locus with an illness. Unfortunately, such large samples are often cost prohibitive. However, by using alternative analytic strategies and studying related individuals, particularly those from large multiplex families, it is possible to reduce the required sample size while maintaining statistical power. We contend that using whole genome sequence (WGS) in extended pedigrees provides a cost-effective strategy for psychiatric gene mapping that complements common variant approaches and WGS in unrelated individuals. This was our impetus for forming the "Pedigree-Based Whole Genome Sequencing of Affective and Psychotic Disorders" consortium. In this review, we provide a rationale for the use of WGS with pedigrees in modern psychiatric genetics research. We begin with a focused review of the current literature, followed by a short history of family-based research in psychiatry. Next, we describe several advantages of pedigrees for WGS research, including power estimates, methods for studying the environment, and endophenotypes. We conclude with a brief description of our consortium and its goals.

Reliability of genomic predictions of complex human phenotypes.

Genome-wide association studies have helped us identify a wealth of genetic variants associated with complex human phenotypes. Because most variants explain a small portion of the total phenotypic variation, however, marker-based studies remain limited in their ability to predict such phenotypes. Here, we show how modern statistical genetic techniques borrowed from animal breeding can be employed to increase the accuracy of genomic prediction of complex phenotypes and the power of genetic mapping studies. Specifically, using the triglyceride data of the GAW20 data set, we apply genomic-best linear unbiased prediction (G-BLUP) methods to obtain empirical genetic values (EGVs) for each triglyceride phenotype and each individual. We then study 2 different factors that influence the prediction accuracy of G-BLUP for the analysis of human data: (a) the choice of kinship matrix, and (b) the overall level of relatedness. The resulting genetic values represent the total genetic component for the phenotype of interest and can be used to represent a trait without its environmental component. Finally, using empirical data, we demonstrate how this method can be used to increase the power of genetic mapping studies. In sum, our results show that dense genome-wide data can be used in a wider scope than previously anticipated.

Genome-wide linkage scan for loci influencing plasma triglyceride levels.

We conducted a genome-wide linkage scan to detect loci that influence the levels of fasting triglycerides in plasma. Fasting triglyceride levels were available at 4 time points (visits), 2 pre- and 2 post-fenofibrate intervention. Multipoint identity-by-descent (MIBD) matrices were derived from genotypes using IBDLD. Variance-component linkage analyses were then conducted using SOLAR (Sequential Oligogenic Linkage Analysis Routines). We found evidence of linkage (logarithm of odds [LOD] ≥3) at 5 chromosomal regions with triglyceride levels in plasma. The highest LOD scores were observed for linkage to the estimated genetic value (additive genetic component) of the log-normalized triglyceride levels in plasma. Our results suggest that a chromosome 10 locus at 37 cM (LODpre = 3.01, LODpost = 3.72) influences fasting triglyceride levels in plasma regardless of the fenofibrate intervention, and that loci in chromosomes 1 at 170 cM and 4 at 24 cM ceases to affect the triglyceride levels when fenofibrate is present, while the regions in chromosomes 6 at 136 to 162 cM and 11 at 39 to 40 cM appear to influence triglyceride levels in response to fenofibrate.

Heritability and genetic associations of triglyceride and HDL-C levels using pedigree-based and empirical kinships.

The heritability of a phenotype is an estimation of the percent of variance in that phenotype that is attributable to additive genetic factors. Heritability is optimally estimated in family-based sample populations. Traditionally, this involves use of a pedigree-based kinship coefficient generated from the collected genealogical relationships between family members. An alternative, when dense genotype data are available, is to directly measure the empirical kinship between samples. This study compares the use of pedigree and empirical kinships in the GAW20 data set. Two phenotypes were assessed: triglyceride levels and high-density lipoprotein cholesterol (HDL-C) levels pre- and postintervention with the cholesterol-reducing drug fenofibrate. Using SOLAR (Sequential Oligogenic Linkage Analysis Routines), pedigree-based kinships and empirically calculated kinships (using IBDLD and LDAK) were used to calculate phenotype heritability. In addition, a genome-wide association study was conducted using each kinship model for each phenotype to identify genetic variants significantly associated with phenotypic variation. The variant rs247617 was significantly associated with HDL-C levels both pre- and post-fenofibrate intervention. Overall, the phenotype heritabilities calculated using pedigree based kinships or either of the empirical kinships generated using IBDLD or LDAK were comparable. Phenotype heritabilities estimated from empirical kinships generated using IBDLD were closest to the pedigree-based estimations. Given that there was not an appreciable amount of unknown relatedness between the pedigrees in this data set, a large increase in heritability in using empirical kinship was not expected, and our calculations support this. Importantly, these results demonstrate that when sufficient genotypic data are available, empirical kinship estimation is a practical alternative to using pedigree-based kinships.

Rare, potentially pathogenic variants in 21 keratoconus candidate genes are not enriched in cases in a large Australian cohort of European descent.

Many genes have been suggested as candidate genes for keratoconus based on their function, their proximity to associated polymorphisms or due to the identification of putative causative variants within the gene. However, very few of these genes have been assessed for rare variation in keratoconus more broadly. In contrast, VSX1 and SOD1 have been widely assessed, however, the vast majority of studies have been small and the findings conflicting. In a cohort of Australians of European descent, consisting of 385 keratoconus cases and 396 controls, we screened 21 keratoconus candidate genes: BANP, CAST, COL4A3, COL4A4, COL5A1, FOXO1, FNDC3B, HGF, IL1A, IL1B, ILRN, IMMP2L, MPDZ, NFIB, RAB3GAP1, RAD51, RXRA, SLC4A11, SOD1, TF and VSX1. The candidate genes were sequenced in these individuals by either whole exome sequencing or targeted gene sequencing. Variants were filtered to identify rare (minor allele frequency <1%), potentially pathogenic variants. A total of 164 such variants were identified across the two groups with no variants fulfilling these criteria in cases in IL1RN, BANP, IL1B, RAD51 or SOD1. The frequency of variants was compared between cases and controls using chi-square or Fishers' Exact tests for each gene with at least one rare potentially pathogenic variant identified in the case cohort. The number of rare potentially pathogenic variants per gene ranged from three (RXRA) to 102 (MPDZ), however for all genes, there was no difference in the frequency between the cases and controls. We conclude that rare potentially pathogenic variation in the 21 candidate genes assessed do not play a major role in keratoconus susceptibility and pathogenesis.