HID1 as a novel disease-causing gene in early onset neurological disorders: molecular, functional and phenotypic studiesDoctoral thesis
Date of Examination:2022-08-25
Date of issue:2022-09-07
Advisor:Prof. Dr. Jutta Gärtner
Referee:Prof. Dr. Jutta Gärtner
Referee:Prof. Dr. Bernd Wollnik
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EnglishRare neurological diseases with genetic causes are gaining relevance for child morbidity and mortality due to improvements in health care, prevention and treatment of conventional life-limiting diseases in infants. Emerging and improving high-throughput sequencing technologies constitute a key tool in determining disease-causing mutations and allow the identification of potential new disease-related genes. This way, high-temperature induced dauer 1 (HID1), a known player in large dense core vesicle (LDCV)-based peptidergic signaling in C. elegans and rodents, was identified as a novel potential disease-related gene in two patients presenting with a unique neurologic and neuroendocrine phenotype. The aim of this project was to molecularly link the detected homozygous mutation in HID1 to the presented clinical defects in patients. First, this study investigated HID1 and a CRISPR/Cas9-introduced patient-derived mutation in a human neuroendocrine cell line, which constitutes a widely accepted model system for neurological disease. Basic research on HID1 revealed that it is a Golgi-associated protein but also localizes to the cytosol, possibly to LDCV in human neuroendocrine cells. It could be proven that HID1 expression is exceptionally high in the pituitary gland and increases with neuronal differentiation, linking HID1 to neurodevelopment. Co-immunoprecipitation followed by mass spectrometry-based identification of interacting protein candidates could substantially broaden the spectrum of potential HID1 functions, linking HID1 especially to the biological processes of intracellular localization establishment, intracellular and transmembrane transport and cytoskeletal organization. Considering allocation of interactors to cellular components, HID1 appears to be prominently associated with the Golgi apparatus and related vesicles, which corresponds to the results of the localization study. Patient mutation-carrying cells and HID1 knockout cells were used to investigate molecular genotype-phenotype relations in suspected HID1-related disease. It could be demonstrated that the patient-derived HID1 mutation leads to the absence of detectable protein, resulting from degradation of the corresponding mutant HID1 mRNA via nonsense-mediated mRNA decay (NMD). In order to investigate the functional effect of the patient-derived mutation, LDCV cargo secretion was stimulated and secretory output was determined. Cargo secretion is significantly reduced in disease-modeling cells, which is likely caused by impaired LDCV biogenesis since defective HID1 protein leads to diminished protein expression of the LDCV cargo protein and biogenesis driver secretogranin II (SGII), probably due to downregulation of SCG2 gene expression. Furthermore, highly diminished cytosolic signal of LDCV marker protein chromogranin A indicates accumulation of LDCV cargo at the trans-Golgi network in the absence of HID1 protein, which may be caused by defective vesicle formation. Along with the significantly reduced secretion of LDCV cargo, known to have neuromodulating functions, disease-modeling cells exhibit aberrant ability to differentiate into mature neurons. Cells show morphological differences as significantly reduced neurite outgrowth and clustering of cell soma. Also, pronounced cell death and diverging expression of differentiation markers and LDCV cargo proteins was observed. In conclusion, this study could show that HID1 is involved in LDCV biogenesis by affecting cargo protein biosynthesis and vesicle formation. Furthermore, the data point towards the involvement of HID1 in LDCV trafficking and neurodevelopment. Experiments with the patient-derived HID1 mutation indicate that the mutation is a loss-of-function mutation that negatively influences the secretion of hormones and neuromodulators. These defects are especially reflected in pituitary insufficiency and severe statomotor development delay in patients, substantiating the assumption that detected HID1 mutation is pathogenic and thus is the driver of the neurological disease in the described patients.
Keywords: HID1; LDCV; Secretion; Novel disease-causing gene; Large dense core vesicles; Genotype-phenotype correlation; Patient-related research; Early onset neurological disorder; Neurodifferentiation