The molecular basis of human retinal and vitreoretinal diseases

During the last two to three decades, a large body of work has revealed the molecular basis of many human disorders, including retinal and vitreoretinal degenerations and dysfunctions. Although belonging to the group of orphan diseases, they affect probably more than two million people worldwide. Most excitingly, treatment of a particular form of congenital retinal degeneration is now possible. A major advantage for treatment is the unique structure and accessibility of the eye and its different components, including the vitreous and retina. Knowledge of the many different eye diseases affecting retinal structure and function (night and color blindness, retinitis pigmentosa, cone and cone rod dystrophies, photoreceptor dysfunctions, as well as vitreoretinal traits) is critical for future therapeutic development. We have attempted to present a comprehensive picture of these disorders, including clinical, genetic and molecular information. The structural organization of the review leads the reader through non-syndromic and syndromic forms of (i) rod dominated diseases, (ii) cone dominated diseases, (iii) generalized retinal degenerations and (iv) vitreoretinal disorders, caused by mutations in more than 165 genes. Clinical variability and genetic heterogeneity have an important impact on genetic testing and counselling of affected families. As phenotypes do not always correlate with the respective genotypes, it is of utmost importance that clinicians, geneticists, counsellors, diagnostic laboratories and basic researchers understand the relationships between phenotypic manifestations and specific genes, as well as mutations and pathophysiologic mechanisms. We discuss future perspectives

Genomic Approaches to Understanding Variable Expressivity in Alagille Syndrome and Genetic Susceptibility to Biliary Atresia

The biliary system facilitates transport of bile from the liver, where it is produced, to the gall bladder, where it is stored and later released to aid digestion. Obstruction and defects in the biliary system are a primary indication of liver transplantation in children. Alagille syndrome (ALGS) and biliary atresia (BA) are two cholangiopathies with different etiopathology, but both affect bile flow and can lead to end-stage liver disease. ALGS is a dominant, multisystemic disease caused by mutations in JAG1 or NOTCH2 characterized by intrahepatic ductopenia and variable expressivity. BA is a multifactorial disease characterized by necroinflammatory obliteration of the extrahepatic biliary tree with unknown etiology. I used genomic tools including genome-wide gene expression analysis, genome-wide association (GWA) studies of single nucleotide polymorphisms (SNPs) and copy number variation (CNVs), and exome sequence analysis to uncover the genetic component to variable expressivity in ALGS and susceptibility to BA. In the first part of this thesis, I searched for genetic modifiers of the ALGS phenotype by investigating the effects of the non-mutated JAG1 allele, characterizing downstream gene expression of Notch signaling, and performing a SNP and CNV GWA studies of liver disease severity. This work revealed that cardiac defects are correlated with liver disease severity and the presence of butterfly vertebrae. I uncovered an enhancer element upstream of THBS2 that may affect the progression of liver disease in ALGS patients. In the second part of this thesis, I looked for markers of genetic susceptibility to BA, using an association study, exome sequencing of unrelated patients, and exome sequencing and CNV analysis of familial recurrence of BA-associated phenotypes. This work suggested several BA candidate genes, including FOXA2, which we propose as a BA susceptibility gene. Additionally, fine mapping of a previously reported susceptibility locus demonstrated an intronic risk allele in ADD3. Exome sequencing of candidate genes and a pathway-enriched analysis revealed alterations in several other genes that support the hypothesis that BA is a genetically heterogeneous disorder. This work identifies multiple areas for future research to better understand the genetics of these two biliary disorders

Genetics in Familial Intrahepatic Cholestasis: Clinical Patterns and Development of Liver and Biliary Cancers: A Review of the Literature

The family of inherited intrahepatic cholestasis includes autosomal recessive cholestatic rare diseases of childhood involved in bile acids secretion or bile transport defects. Specific genetic pathways potentially cause many otherwise unexplained cholestasis or hepatobiliary tumours in a healthy liver. Lately, next-generation sequencing and whole-exome sequencing have improved the diagnostic procedures of familial intrahepatic cholestasis (FIC), as well as the discovery of several genes responsible for FIC. Moreover, mutations in these genes, even in the heterozygous status, may be responsible for cryptogenic cholestasis in both young and adults. Mutations in FIC genes can influence serum and hepatic levels of bile acids. Experimental studies on the NR1H4 gene have shown that high bile acids concentrations cause excessive production of inflammatory cytokines, resistance to apoptosis, and increased cell regeneration, all risk conditions for developing hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). NR1H4 gene encodes farnesoid X-activated receptor having a pivotal role in bile salts synthesis. Moreover, HCC and CCA can emerge in patients with several FIC genes such as ABCB11, ABCB4 and TJP2. Herein, we reviewed the available data on FIC-related hepatobiliary cancers, reporting on genetics to the pathophysiology, the risk factors and the clinical presentation

Notch signaling requiem : orchestral role of notch signaling in cancer and developmental disease

Notch signaling is an evolutionary conserved contact-dependent cell-cell communication pathway. This “contact” spans from hydra to fruit flies to human; orchestrating development, homeostasis and cancer, thus the Requiem, a song of life and death. Upon the “contact” of Notch receptor and ligand, the intracellular domain NICD is released and translocates to the nucleus. NICD, together with the DNA binding protein CSL and other co-activators, activate downstream targets. In this thesis, I have investigated the role of Notch signaling in multiple contexts with a modular approach. This includes: the non-canonical role of CSL in breast cancer, crosstalk of Notch signaling with hypoxia signaling in cancer, canonical Notch signaling in blood development, a novel mouse model for Alagille syndrome, and the hyperactivated Notch during mammary development and tumourigenesis. Here I phrase them in five sections of a requiem (Mozart’s Requiem, 1791): Introitus: In Paper I, we found that ablation of CSL unleashed a hypoxic response in normoxic conditions and enhanced tumour growth in breast cancer. A large part of the deregulated genes in the CSL null cell line is Notch independent. We demonstrated a non-canonical role of CSL and the possible implication of loss of CSL in breast cancer. Kyrie: In Paper II, we established that Notch signaling can modulate hypoxia signaling in multiple cancer cell types. By siRNA knocked down of HIF2α, we found that Notch signaling requires HIF2α for regulating a subset of Notch targets in medulloblastoma cells. Differences in the effect of N1ICD and N2ICD were also shown in the medulloblastoma cells. Lastly, we presented evidence of Notch signaling contributing to the HIF1α-to-HIF2α switch. Dies Irae: In Paper III, we revealed that canonical Notch signaling is dispensable in adult steady-state and stress myelo-erythropoiesis by ablating CSL in the myeloid lineage. Some of the Notch targets were derepressed in some of the progenitor stages, indicating CSL could act as a repressor in some contexts. Rex tremendae: In Paper IV, we established and characterized a mouse model for Alagille syndrome in human, recapitulating defects in multiple organ-systems. We showed a mutation in Jag1 caused delay differentiation and structural abnormalities in the bile ducts. From transcriptomics of mice and patients samples, we also found some commonly affected genes across species. Lastly, we discovered that the mutated Jag1 failed to bind to Notch1 and reduced the extent of Notch2 and Notch3 activation. Lacrymosa: In Paper V, we observed that hyperactive Notch in the luminal lineage during lactation cause defect in ductal development and led to mammary tumour development. Furthermore, we showed that this lineage can contribute to a large part of the mammary tumour

Genetic screening in patients with kidney failure from hypertension or unknown cause

Hypertensiv nyresygdom og nyresygdom af ukendt årsag er, næst efter diabetes, de hyppigst rapporterede årsager til nyresvigt, hvilket vanskeliggør implementering af præcisionsmedicin indenfor nefrologi. Genetisk screening udgør et diagnostisk værktøj, der muliggør identifikation af sygdomsgivende genvarianter uafhængigt af nyresygdommens stadium. Den optimale strategi for genetisk screening af nyrepatienter er dog forsat uafklaret. En anden udfordring er, at der ofte påvises genvarianter af ukendt klinisk betydning (VUS). Der er derfor behov for nye metoder til at vurdere genvarianters patogenicitet.Det primære formål med ph.d.-projektet var at undersøge forekomsten af uopdagede genetiske nyresygdomme hos patienter, der udviklede nyresvigt ≤50 år som følge af hypertension eller af ukendt årsag, for at optimere en strategi for genetisk screening. I den første del rekrutterede vi en kohorte bestående af 124 probander med uforklaret nyresvigt og udførte et omfattende genetisk screeningsprogram, herunder single-nukleotid-polymorfi (SNP)-array, MUC1-genanalyse og helgenomsekventering (whole-genome sequencing, WGS). WGS-analysen blev foretaget i tre trin: 1) proband-baseret analyse, 2) familie-baseret analyse, og 3) kohorte-baseret re-analyse. Vi identificerede en genetisk årsag hos 31% af probanderne (38/124). Varianter blev hovedsageligt påvist i COL4A3/A4/A5, NPHP1 og MUC1. Alle diagnostiske varianter kunne påvises med WGS, undtagen varianterne i et komplekst område i MUC1-genet, som blev påvist hos 6% af probanderne (7/124). En familiehistorik med nyresvigt var den eneste signifikante prædiktor for et positivt genetisk fund. Vi fandt ingen forskel i diagnostisk udbytte ved sammenligning af patienter med og uden hypertension ved diagnosetidspunktet af nyresygdom. Projektets andet formål var at udvikle metoder til funktionel karakterisering af gener associeret med nyresygdom ved brug af biologisk materiale fra patienter. Vi udviklede en filteringsbaseret, centrifugeringsfri metode til oprensning af ekstracellulære vesikler i urinen (uEV’er) og demonstrerede dens anvendelighed ved at påvise tab af SLC3A1 protein hos patienter med SLC3A1-cystinuri via af western blotting.  Under et forskningsophold på Charité Universitätsmedizin karakteriserede vi effekten af en loss-of-function variant i BICC1-genet i fibroblaster fra en patient. Vi fandt mislokalisation af BICC1 proteinet og samtidig dysregulering af downstream proteinet β-catenin.Sammenfattende fandt vi således et højt diagnostisk udbytte ved genetisk screening af patienter med nyresvigt ≤50 år som følge af hypertension eller af ukendt årsag, samt en overraskende høj forekomst af MUC1-associeret nyresygdom. Den stigende anvendelse af genetisk screening øger behovet for implementering af nye metoder til funktionel karakterisering af genvarianter af ukendt klinisk betydning og nye gener associeret med nyresygdom. Isolering af uEV’er viser lovende potentiale som en enkel, ikke-invassiv metode til at undersøge funktionelle effekter i nyrerne.Next to diabetic kidney disease, hypertension and unknown cause are the leading etiologies of incident kidney failure (KF), posing a hindrance for precision nephrology strategies. Genetic screening has become a diagnostic resource that enables detection of causative genetic variants regardless of the kidney disease stage. However, the optimal strategy for genetic screening in kidney disease is still being explored. Moreover, identification of variants of unknown significance (VUS) poses a current bottleneck in genetic screening, which may be remedied by novel approaches to determine variant pathogenicity. This PhD project primarily aimed to investigate the prevalence of undiscovered genetic kidney disease in patients with KF ≤50 years, attributed to hypertension or an unknown cause. The goal was to improve genetic screening strategies for this patient group. In the first part of the project, we recruited a cohort of 124 probands with unexplained KF and conducted multifaceted genetic screening comprising single-nucleotide-polymorphism (SNP)-array, MUC1 gene analysis, and whole-genome sequencing (WGS). The WGS analysis was performed in three steps: 1) proband-based analysis, 2) family-based analysis, and 3) cohortbased re-analysis. We identified a genetic cause of KF in 31% of the probands (38/124), primarily involving the genes COL4A3/A4/A5, NPHP1, and MUC1. Notably, all diagnostic variants were detectable by WGS except those in the variable number tandem repeat region of MUC1, which accounted for 6% of diagnoses (7/124). A family history of KF was the only significant predictor of positive genetic screening. Importantly, we found no difference in diagnostic yield between patients with or without hypertension at kidney disease presentation. The second part of the project aimed to explore methods for functionally characterizing kidney disease genes using patient-derived materials. We developed a filtering-based, centrifugation-free enrichment method for urine-derived extracellular vesicles (uEVs) and demonstrated its utility by detecting the loss of SLC3A1 protein on western blots from patients with SLC3A1-cystinuria. Additionally, during a collaborative research stay at Charité Universitätsmedizin, we characterized a loss-of-function variant in BICC1 using patient-derived fibroblasts, revealing cellular BICC1 mislocalization and concordant dysregulation of the downstream target β-catenin.In conclusion, we found a high diagnostic yield from genetic screening in patients with KF ≤50 years from hypertension or unknown cause, with a surprisingly high prevalence of MUC1-associated autosomal dominant tubulointerstitial kidney disease. The increased use of genetic screening demands novel approaches to characterize VUS findings and novel kidney disease genes, and isolated uEVs show promising potential as a straightforward and non-invasive approach to study functional effects on the kidneys

A genome-wide association study identifies a susceptibility locus for biliary atresia on 2p16.1 within the gene EFEMP1

Biliary atresia (BA) is a rare pediatric cholangiopathy characterized by fibrosclerosing obliteration of the extrahepatic bile ducts, leading to cholestasis, fibrosis, cirrhosis, and eventual liver failure. The etiology of BA remains unknown, although environmental, inflammatory, infectious, and genetic risk factors have been proposed. We performed a genome-wide association study (GWAS) in a European-American cohort of 343 isolated BA patients and 1716 controls to identify genetic loci associated with BA. A second GWAS was performed in an independent European-American cohort of 156 patients with BA and other extrahepatic anomalies and 212 controls to confirm the identified candidate BA-associated SNPs. Meta-analysis revealed three genome-wide significant BA-associated SNPs on 2p16.1 (rs10865291, rs6761893, and rs727878; P < 5 ×10-8), located within the fifth intron of the EFEMP1 gene, which encodes a secreted extracellular protein implicated in extracellular matrix remodeling, cell proliferation, and organogenesis. RNA expression analysis showed an increase in EFEMP1 transcripts from human liver specimens isolated from patients with either BA or other cholestatic diseases when compared to normal control liver samples. Immunohistochemistry demonstrated that EFEMP1 is expressed in cholangiocytes and vascular smooth muscle cells in liver specimens from patients with BA and other cholestatic diseases, but it is absent from cholangiocytes in normal control liver samples. Efemp1 transcripts had higher expression in cholangiocytes and portal fibroblasts as compared with other cell types in normal rat liver. The identification of a novel BA-associated locus, and implication of EFEMP1 as a new BA candidate susceptibility gene, could provide new insights to understanding the mechanisms underlying this severe pediatric disorder

Sequencing of a Chinese tetralogy of Fallot cohort reveals clustering mutations in myogenic heart progenitors

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations