The genetics of repeat disorders

Erdmann, Hannes (2023): The genetics of repeat disorders: development of novel long-read sequencing methods and phenotype-genotype studies. Dissertation, LMU München: Medizinische Fakultät

Abstract

The analysis of repetitive elements in the human genome remains a challenge in clinical genetics. As next-generation sequencing is limited in analyzing repeat disorders and complex regions within the human genome, specific diagnostic methods are required. This thesis describes (1) the implementation and validation of a long-read sequencing method for parallel repeat analysis of patients with adult-onset ataxia as well as (2) the analysis of the relevance of methylation profiles in the diagnosis and clinical evaluation of FSHD. For a comprehensive repeat analysis of patients with adult-onset ataxia, clinical nanopore Cas9-targeted sequencing (Clin-CATS) was designed to cover the ten repeat disorders most frequently causing adult-onset ataxia in Germany (status when publishing manuscript 1): spinocerebellar ataxias (SCA) 1–3, 6–8, 17, RFC1 spectrum disorder, Friedreich’s ataxia (FRDA) and fragile-X-associated tremor/ataxia syndrome (FXTAS). Associated repeat loci are enriched using CRIPSR/Cas9 and subsequently sequenced using Oxford Nanopore Technology long-read sequencing. Sequencing data are used to derive repeat length, repeat sequence to identify repeat interruptions and the repeat composition of the RFC1 repeat array, as well as FMR1 promoter methylation. Repeat lengths obtained by Clin-CATS show a high concordance to those determined by conventional PCR-based repeat analysis. Pathogenic repeat expansions were reliably detected and the comprehensive set of parameters determined improved diagnostic precision of Clin-CATS over conventional repeat testing. The analysis of 100 patients with an adult-onset ataxia phenotype by Clin-CATS revealed causative repeat expansions in 28 patients, including rare conditions such as a very-late onset FRDA or a high-function FXTAS male carrying a non-methylated FMR1 promotor despite a fully expanded FMR1 repeat array. Clin-CATs highlights the high polymorphism of the RFC1 repeat array and reveals RFC1 spectrum disorder to be a frequent cause of hereditary adult-onset ataxia in Germany. After verifying FSHD1 and FSHD2 patients as well as healthy individuals to significantly differ in the methylation patterns of their D4Z4 repeat arrays on chromosome 4q35, FSHD-MPA was established as a diagnostic method for diagnosing FSHD. Utilizing bisulfite conversion FSHD-MPA determines the methylation level of a region within the most distal D4Z4 repeat array of 4q35 alleles carrying the permissive haplotype (4qA or 4qAL, distal methylation) and the average methylation level of a second region present within each D4Z4 repeat unit of chromosome 4q35 (global methylation). Healthy individuals show global and distal hypermethylation, while FSHD1 patients show isolated distal hypomethylation and FSHD2 patients global and distal hypomethylation. Within a cohort of 148 patients with a clinical phenotype of FSHD or a positive family history of FSHD, methylation profiles are proven as precise diagnostic parameters for diagnosing FSHD by comparing the results from our epigenetic test with the results of Southern blotting and NGS sequencing of the epigenetic suppressor genes SMCHD1, DMNT3B and LRIF1 as well as the clinical phenotype. Furthermore FSHD1 and FSHD2 patients show an epigenetic overlap as some patients with global and distal hypomethylation have repeat contractions in the absence of pathogenic variants in known epigenetic suppressor genes. Methylation profiles allow to access the penetrance of genetic parameters indicating their potential in predictive testing. Distal methylation level and age-corrected clinical severity show high correlation level that are stronger than those of repeat length and age-corrected clinical severity in the cohort studied. As such distal methylation is a more precise and universal biomarker for disease severity in the present study accounting for FSHD1 as well as for FSHD2. Thus, the disease status of FSHD is better represented by epigenetic than by genetic parameters. Repeat contractions and pathogenic variants in epigenetic suppressor genes should be considered more as risk factors of the disease than as direct causes of the disease. Further refinements of FSHD diagnostics can be achieved by ONT long-read sequencing which yields all relevant diagnostic parameters within one analysis and specific for each allele including the methylation profile of the whole D4Z4 repeat locus.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: repeat disorders, FSHD, hereditary ataxia, long-read sequencing, clinical genetics, neurogenetics
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Deutsch
• Datum der mündlichen Prüfung: 19. Oktober 2023
• 1. Bericht­erstatter:in: Abicht, Angela