| 1. |
- Westin, Ida Maria, 1982-
(författare)
-
Genetics, epigenetics and functional mechanisms in inherited corneal and retinal dystrophies
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Inherited eye disorders (IED) are groups of genetically and clinically heterogenous conditions affecting different tissues in the eye. IED are most often progressive with reduced vision or legal blindness as outcome. This thesis is focused on investigating the underlying mechanisms in Fuchs’ endothelial corneal dystrophy (FECD) and two retinal dystrophies, Stargardt disease (STGD1) and autosomal recessive Retinitis pigmentosa (arRP, RP25).In FECD, we studied the association between FECD and the (CTG)n repeat expansion at the CTG18.1 locus in the TCF4 gene, in patients from northern Sweden. By using STR-PCR and TP-PCR, we found that 90% of FECD patients carry an expanded CTG18.1 allele, establishing the highest prevalence among FECD patients world-wide. With droplet digital PCR, we showed that transcripts spanning over the CTG18.1 have lower fractions in human corneal endothelium (CE) compared to skin, brain, muscle, and white blood cells. With Illumina Methylation arrays (850K), we detected a decreased global methylation in the CE at advanced age, that could possibly contribute to the late onset of FECD. We also found distinct differences in methylation between FECD patients and controls, that led us to two coagulation factors, found to be over-expressed in the CE from FECD patients.For the two retinal dystrophies, STGD1 and RP25, we investigated the functional effect of four genetic variants residing adjacent to or in splice consensus sequence of the ABCA4 gene (STDG1) and the EYS gene (RP25). With an in vitro mini-gene splicing assay we showed that all four genetic variants caused exon skipping in Retinal Pigment Epithelial cell line (ARPE-19) and Human Embryonic kidney cell line (HEK293T). Our results functionally proved these variants to be pathogenic and causative of STGD1 and RP25.In RP25, we also investigated the prevalence of pathogenic EYS variants in a cohort of patients from northern Sweden. DNA from 81 patients with a clinical diagnosis of RP were interrogated with a "cascade-targeted mutation analysis" approach, where NGS, MLPA and Sanger sequencing was used to find common EYS variants in this acknowledged genetically homogenous population. EYS mutations were present in at least 16% of all arRP patients and the most recurrent mutation in the study was an 8-bp deletion, previously found in the Finnish population.In conclusion, this thesis provides knowledge on disease causative mechanisms in IED and contributes with valuable information for future genetic counselling and genetic testing for affected families.
|
|
| 2. |
- Ljuslinder, Ingrid, 1968-
(författare)
-
Studies of LRIG1 and the ERBB receptor family in breast and colorectal cancer
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The LRIG1 gene (leucine-rich repeats and immunoglobulin like domains-1) at chromosome 3p14 is a proposed tumour suppressor gene whose gene product negatively regulates various receptor tyrosine kinases. This function has been the basis for classifying LRIG1 as a potential tumour suppressor gene (TSG). The ERBB receptor family is important in malignant cellular functions such as proliferation, survival, adhesion, migration and differentiation. In breast cancer, amplification of the ERBB2 proto-oncogene is an important negative prognostic factor. The epidermal growth factor receptor (EGFR/ERBB1), is expressed in colorectal cancer and has been correlated to a worse prognosis. Until recently, immunohistochemical analysis of EGFR expression was used to select patients suitable for treatment with EGFR targeted antibodies. This thesis characterizes LRIG1 in breast and colorectal cancer to gain further knowledge of the gene and its expression. Also, the EGFR expression in metastases and the invasive margin of colorectal cancers was investigated to correlate changes to clinical factors. Breast cancer samples and matched normal tissues were evaluated for LRIG1 and the ERBB receptors at gene, RNA and protein levels. An increase in copy number of the LRIG1 gene was evident. Also, increased LRIG1 copy number was associated with high levels of ERBB2 mRNA. Another set of breast cancer tumours were analysed for LRIG1 by FISH analysis. The results were coherent with the previous results. To further analyze the correlation to ERBB2, tumours with LRIG1 increased copy number were analysed for ERBB2. The data showed that 89% of tumours with increased LRIG1 copy number were either ERBB2 amplified or had an increased copy number of ERBB2. To investigate LRIG1 and the EGFR in colorectal cancer, the gene and protein expression was analysed by several methods in tumours and corresponding normal tissues. There were no significant changes at gene level found, but at the protein level, both over- and under expression were seen. No evident correlation between LRIG1 and EGFR expression was detected. The ERBB receptor family expression in colorectal cancer tumours and corresponding metastases was investigated to explore if the expression was altered in the metastatic lesion. The results showed that the EGFR expression was lost in the corresponding metastases in 33% of the tumours and that the same percentage of tumours gained expression in the metastases. Co-expression of the ERBB family members was also analysed; there was a significant increase of ERBB3/ERBB4 co-expression in late stage tumours. EGFR expression at the invasive margin of colorectal cancers was analysed to clarify whether expression correlated to the patient’s prognosis. Significant correlation to survival and the presence of budding was seen. In conclusion, 34% of the breast cancer tumours studied had an increased copy number of LRIG1 with a significant co-incidental increase in ERBB2 copy number. This raises the question of a functional correlation between LRIG1 and ERBB2, a finding that might be of clinical importance. The studies of EGFR and the ERBB receptors in colorectal cancer reflect the heterogeneity of EGFR expression in tumours. In addition, these findings suggest that survival of the patients correlates to an increasing EGFR expression at the invasive margin.
|
|
| 3. |
- Vikberg, Ann-Louise, 1979-
(författare)
-
Mitotic Kinesin-Like Protein 1 (MKLP1/KIF23) in hereditary congenital dyserythropoietic anemia type III and in cancer
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A hereditary form of autosomal dominant congenital dyserythropoietic anaemiatype III (CDA III) has been reported in four families from Sweden, Argentina, Cuba and USA. CDA III patients might experience signs of mild anaemia and some of them need occasional blood transfusions. Other clinical features seen in CDA III patients are retinal angioid streaks, monoclonal gammopathy of undetermined significance and multiple myeloma. Their bone marrow is characterised by presence of giant erythroblasts with up to 12 nuclei. Previously, CDA III was mapped to a region on chromosome 15q21-q25.In this study we aimed to identify the genetic cause of CDA III, investigate the reasons why erythroid lineage in the patients’ bone marrow is mostly affected, and seek the explanation of increased rate of cancer in the Swedish CDA III family.We identified the genetic cause of CDA III using targeted next generation sequencing. A novel missense mutation c.2747C>G, p.P916R in kinesin familymember 23 gene (KIF23) segregated with the disease in both the American and Swedish family, and was absent in databases of sequence variants from healthy individuals. Knock-down and rescue experiments in HeLa Kyoto cells showed that the P916R mutation caused cytokinesis failure which resulted in large cells with several nuclei. This was consistent with the CDA III phenotype.To reveal interaction partners of wild-type and mutant KIF23 proteins, pull-down experiments followed by mass spectrometry and Western blot analysis were performed. This identified Coatomer Protein Complex I (COPI), a vesicle forming complex responsible for intracellular transport, as a KIF23 interactor. By using immunofluorescence and fluorescence microscopy, we showed that COPI subunits COPα and COPβ localize to the midbody during cytokinesis. These findings indicate involvement of vesicle transport proteins in mitosis and cytokinesis, though the significance of COPI-KIF23 interaction in cell division remains to be uncovered.To address the question if other cells are affected by the KIF23 P916R mutation, we created a knock-in mouse model with Kif23 c.2726C>G, p.P909R, which corresponds to the human KIF23 c.2747C>G. However, the mice did not developany phenotype indicating CDA III. This result was consistent with the studies ofother CDA subtypes where mouse models failed, suggesting that CDA occur only in humans. Our study of human and mouse KIF23/Kif23 expression revealed novel, previously not annotated transcripts, one in human and two in mice. Expression analysis of total mRNA using droplet digital PCR demonstrated an extensive variation of KIF23 and Kif23 expression levels in all tissues. The shortest Kif23 transcript lacking exon 17 and 18 was prevalent in mice, while corresponding transcript in human was the least expressed.Considering the importance of KIF23 in cytokinesis and KIF23 association with cancer, we hypothesized that somatic KIF23 mutations might be overrepresented in cancer. For this purpose, we screened KIF23 and its promoter in non-small cell lung cancer samples that previously demonstrated KIF23 overexpression. No pathogenic driving KIF23 variants were detected by Sangersequencing; however, subsequent genome-wide genotyping (SNP-array) detected gain of chromosome 15 in most cases that could possibly explain KIF23 overexpression.
|
|
| 4. |
- Frida, Jonsson, 1979-
(författare)
-
Underlying genetic mechanisms of hereditary dystrophies in retina and cornea
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Inherited retinal and corneal dystrophies represent a group of disorders with great genetic heterogeneity. Over 250 genes are associated with retinal diseases and 16 genes are causative of corneal dystrophies. This thesis is focused on finding the genetic causes of corneal dystrophy, Leber congenital amaurosis (LCA), Stargardt disease and retinitis pigmentosa in families from northern Sweden. By whole exome sequencing a novel mutation, c.2816C>T, p.Thr939Ile, in Collagen Type XVII, Alpha 1 chain, COL17A1, gene was identified in several families with epithelial recurrent erosion dystrophy (ERED). We showed that the COL17A1 protein is expressed in the basement membrane of the cornea, explaining the mutation involvement in the corneal symptoms. We could link all the families in this study to a couple born in the late 1700s confirming a founder mutation in northern Sweden. Our finding highlights role of COL17A1 in ERED and suggests screening of this gene in patients with similar phenotype worldwide. Furthermore the genetic causes in several retinal degenerations were identified. In one family with two recessive disorders, LCA and Stargardt disease, a novel stop mutation, c.2557C>T, p.Gln853Stop, was detected in all LCA patients. In the Stargardt patients two intronic variants, the novel c.4773+3A>G and c.5461-10T>C, were detected in the ABCA4 gene. One individual was homozygous for the known variant c.5461-10T>C and the other one was compound heterozygote with both variants present. Both variants, c.4773+3A>G and c.5461-10T>C caused exon skipping in HEK293T cells demonstrated by in vitro splice assay, proving their pathogenicity in Stargardt disease. Finally, in recessive retinitis pigmentosa, Bothnia Dystrophy (BD), we identified a second mutation in the RLBP1 gene, c.677T>A, p.Met226Lys. Thus, BD is caused not only by common c.700C>T variant but also by homozygosity of c.677T>A or compound heterozygosity. Notably, known variant, c.40C>T, p.R14W in the CAIV gene associated with a dominant retinal dystrophy RP17 was detected in one of the compound BD heterozygote and his unaffected mother. This variant appears to be a benign variant in the population of northern Sweden.In conclusion, novel genetic causes of retinal dystrophies in northern Sweden were found demonstrating the heterogeneity and complexity of retinal diseases. Identification of the genetic defect in COL17A1 in the corneal dystrophy contributes to understanding ERED pathogenesis and encourages refinement of IC3D classification. Our results provide valuable information for future molecular testing and genetic counselling of the families.
|
|
