Die Etablierung einer neuen NGS-basierten Panelanalyse zur Identifikation der genetischen Ursachen isolierter und syndromaler Formen der Mikrophthalmie
by Leonie Naumann née Thiele
Date of Examination:2025-03-27
Date of issue:2025-02-25
Advisor:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Michael Schittkowski
Persistent Address:
http://dx.doi.org/10.53846/goediss-11090
Files in this item
Name:Die Etablierung einer neuen NGS-basierten Pa...pdf
Size:4.03Mb
Format:PDF
This file will be freely accessible after 2025-04-24.
Abstract
English
Microphthalmia and anophthalmie are part of the spectrum of rare diseases. Microphthalmia is defined as a developmental disorder of the eye, in which the globe of the eye is more than two standard deviations below the population mean, whereas anophthalmia is defined as the complete absence of visual tissue with, in some cases, some residual neuroectodermal tissue still being detectable. Both, microphthalmia as well as anophthalmia, can occur as isolated findings or they can be associated with additional phenotypic features as part of a syndromic disorder. In case of microphthalmia, clinical manifestation can be highly variable ranging from restricted view up to complete amaurosis. High clinical variability often complicates the identification of underlying causes in affected patient. Still, this is of vital importance for not only resolving the diagnostic odyssey of affected patients, but it also plays a role in the management of treatment options in these patients and underlines the necessity of a multidisciplinary team with ophthalmologists, human geneticist, ocularists and pediatricians in treatment of these patients. The major aim of my thesis was the genetic analysis and characterization of patients with microphthalmia/anophthalmia using next generation sequencing technologies. In the first part of my thesis, a NGS-based multigene-panel was designed, which in total encompasses 53 genes that are associated with isolated and syndromic forms of microphthalmia/anophthalmia. I established and validated this multigene-panel for routine diagnostic testing and used it for identification of the underlying genetic cause in a patient cohort with an ocular phenotype. Secondly, selected cases of this patient cohort that could not be solved by multigene-panel analysis, were subjected to whole-exome sequencing to detect the underlying genetic cause and possibly identify new genes that are involved in the pathogenesis of microphthalmia/anophthalmia. Detected variants were analyzed regarding their functional effect, classified in terms of pathogenicity and specific variants were tested for cosegregation in the respective families. Based on this strategy, I was able to identify pathogenic and likely pathogenic in four patients that were subjected to exome sequencing. Based on this strategy, I was able to identify a pathogenic hemizygous variant in MED12 (c.886C>T) in a patient with a complex ocular malformation and additional phenotypical features. Using bioinformatic and molecular genetic approaches, I was able to provide evidence for causality of this variant, and thereby I could expand the spectrum of pathogenic variants in MED12. As a result, MED12 was included in the multigene-panel for microphthalmia/anophthalmia that was designed in course of this thesis. In a second case, I was able to identify a homozygous variant in NCAPH (c.391C>T) by using exome sequencing and subsequent bioinformatic analysis of homozygous variants. So far, only a single case with ocular malformations has been described in the literature carrying a pathogenic variant in this gene (recessive inheritance, homozygous variant on position c.728C>T in NCAPH). The identification of pathogenic variants in NCAPH in a second patient with an overlapping phenotype supports the results of the previously reported case and introduced NCAPH as a causative gene for a Mendelian disorder in humans. In summary, the data presented in this thesis established new diagnostic tools for the genetic examination of patients with isolated and syndromic forms of microphthalmia/ anophthalmia. Additionally, using exome sequencing, I was able to identify the underlying genetic cause in patients with ocular malformations. These results will support reliable identification of causative variants in affected patients, thereby directly having an impact on patients and their families affected by undiagnosed disorders and providing new perspective to therapeutic approaches.
Keywords: microphthalmia; anophthalmia; rare diseases; multigene-panel; Necxt Generation Sequencing; NCAPH; MED12
