The role of epigenetics in rare diseases.

Epigenetic control systems are based on chromatin modifications (DNA methylation, histone modifications and nucleosome positioning), which affect the local kinetics of gene expression. They play an important role in maintaining cell fate decisions, X inactivation and genomic imprinting. Aberrant chromatin states that are associated with a deleterious change in gene expression are called epimutations. An epimutation can be a primary epimutation that has occurred in the absence of any genetic change or a secondary epimutation that results from a mutation of a cis-acting regulatory element or trans-acting factor. Epimutations may play a causative role in disease, for example in imprinting disorders, or may be part of the pathogenetic mechanism as in the fragile X syndrome and in syndromes caused by a mutation affecting a chromatin modifier. For several diseases, DNA methylation testing is an important tool in the diagnostic work-up of patients.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. RESULTS: We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. CONCLUSION: Overall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

Early-set POMC methylation variability is accompanied by increased risk for obesity and is addressable by MC4R agonist treatment.

Increasing evidence points toward epigenetic variants as a risk factor for developing obesity. We analyzed DNA methylation of the POMC (pro-opiomelanocortin) gene, which is pivotal for satiety regulation. We identified sex-specific and nongenetically determined POMC hypermethylation associated with a 1.4-fold (confidence interval, 1.03 to 2.04) increased individual risk of developing obesity. To investigate the early embryonic establishment of POMC methylation states, we established a human embryonic stem cell (hESC) model. Here, hESCs (WA01) were transferred into a naïve state, which was associated with a reduction of DNA methylation. Naïve hESCs were differentiated via a formative state into POMC-expressing hypothalamic neurons, which was accompanied by re-establishment of DNA methylation patterning. We observed that reduced POMC gene expression was associated with increased POMC methylation in POMC-expressing neurons. On the basis of these findings, we treated POMC-hypermethylated obese individuals (n = 5) with an MC4R agonist and observed a body weight reduction of 4.66 ± 2.16% (means ± SD) over a mean treatment duration of 38.4 ± 26.0 weeks. In summary, we identified an epigenetic obesity risk variant at the POMC gene fulfilling the criteria for a metastable epiallele established in early embryonic development that may be addressable by MC4R agonist treatment to reduce body weight.

Loss of CpG island immunity to DNA methylation induced by mutation.

The inheritance of acquired traits in mammals is a highly controversial topic in biology. Recently, Takahashi et al. (Cell 186:715-731, 2023) have reported that insertion of CpG-free DNA into a CpG island (CGI) can induce DNA methylation of the CGI and that this aberrant methylation pattern can be transmitted across generations, even after removal of the foreign DNA. These results were interpreted as evidence for transgenerational inheritance of acquired DNA methylation patterns. Here, we discuss several interpretational issues raised by this study and consider alternative explanations.

Systematic analysis and prediction of genes associated with monogenic disorders on human chromosome X.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression, and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using machine learning classifiers trained to distinguish disease-associated from dispensable genes, we classify 247 genes, including 115 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

No evidence for intervention-associated DNA methylation changes in monocytes of patients with posttraumatic stress disorder.

DNA methylation patterns can be responsive to environmental influences. This observation has sparked interest in the potential for psychological interventions to influence epigenetic processes. Recent studies have observed correlations between DNA methylation changes and therapy outcome. However, most did not control for changes in cell composition. This study had two aims: first, we sought to replicate therapy-associated changes in DNA methylation of commonly assessed candidate genes in isolated monocytes from 60 female patients with post-traumatic stress disorder (PTSD). Our second, exploratory goal was to identify novel genomic regions with substantial pre-to-post intervention DNA methylation changes by performing whole-genome bisulfite sequencing (WGBS) in two patients with PTSD. Equivalence testing and Bayesian analyses provided evidence against physiologically meaningful intervention-associated DNA methylation changes in monocytes of PTSD patients in commonly investigated target genes (NR3C1, FKBP5, SLC6A4, OXTR). Furthermore, WGBS yielded only a limited set of candidate regions with suggestive evidence of differential DNA methylation pre- to post-therapy. These differential DNA methylation patterns did not prove replicable when investigated in the entire cohort. We conclude that there is no evidence for major, recurrent intervention-associated DNA methylation changes in the investigated genes in monocytes of patients with PTSD.

Evidence for correlations between BMI-associated SNPs and circRNAs.

Circular RNAs (circRNAs) are regulators of processes like adipogenesis. Their expression can be modulated by SNPs. We analysed links between BMI-associated SNPs and circRNAs. First, we detected an enrichment of BMI-associated SNPs on circRNA genomic loci in comparison to non-significant variants. Analysis of sex-stratified GWAS data revealed that circRNA genomic loci encompassed more genome-wide significant BMI-SNPs in females than in males. To explore whether the enrichment is restricted to BMI, we investigated nine additional GWAS studies. We showed an enrichment of trait-associated SNPs in circRNAs for four analysed phenotypes (body height, chronic kidney disease, anorexia nervosa and autism spectrum disorder). To analyse the influence of BMI-affecting SNPs on circRNA levels in vitro, we examined rs4752856 located on hsa_circ_0022025. The analysis of heterozygous individuals revealed an increased level of circRNA derived from the BMI-increasing SNP allele. We conclude that genetic variation may affect the BMI partly through circRNAs.

A critical appraisal of clinical epigenetics.

Modern epigenetics emerged about 40 years ago. Since then, the field has rapidly grown. Unfortunately, this development has been accompanied by certain misconceptions and methodological shortcomings. A profound misconception is that chromatin modifications are a distinct layer of gene regulation that is directly responsive to the environment and potentially heritable between generations. This view ignores the fact that environmental factors affect gene expression mainly through signaling cascades and the activation or repression of transcription factors, which recruit chromatin regulators. The epigenome is mainly shaped by the DNA sequence and by transcription. Methodological shortcomings include the insufficient consideration of genetic variation and cell mixture distribution. Mis- and overinterpretation of epigenetic data foster genetic denialism ("We can control our genes") and epigenetic determinism ("You are what your parents ate"). These erroneous beliefs can be overcome by using precise definitions, by raising the awareness about methodological pitfalls and by returning to the basic facts in molecular and cellular biology.

Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes.

BACKGROUND: Several studies have reported an association between male infertility and aberrant sperm DNA methylation patterns, in particular in imprinted genes. In a recent investigation based on whole methylome and deep bisulfite sequencing, we have not found any evidence for such an association, but have demonstrated that somatic DNA contamination and genetic variation confound methylation studies in sperm of severely oligozoospermic men. To find out whether testicular germ cells (TGCs) of such patients might carry aberrant DNA methylation, we compared the TGC methylomes of four men with cryptozoospermia (CZ) and four men with obstructive azoospermia, who had normal spermatogenesis and served as controls (CTR). RESULTS: There was no difference in DNA methylation at the whole genome level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 differentially methylated regions (DMRs), 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10-16). The DMRs were enriched for distal regulatory elements (p = 1.0 × 10-6) and associated with 132 genes, 61 of which are differentially expressed at various stages of spermatogenesis. Almost all of the 67 DMRs associated with the 61 genes (94%) are hypermethylated in CZ (63/67, p = 1.107 × 10-14). As judged by single-cell RNA sequencing, 13 DMR-associated genes, which are mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter is hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, eight of which downregulated in CZ, germ cell-specific enhancers may be affected. CONCLUSIONS: We found that impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome. We hypothesize that the described DNA methylation changes may reflect or contribute to premature abortion of spermatogenesis and therefore not appear in the mature, motile sperm.

The Diagnostic Journey of a Patient with Prader-Willi-Like Syndrome and a Unique Homozygous SNURF-SNRPN Variant; Bio-Molecular Analysis and Review of the Literature.

Prader-Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader-Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the 'Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations' (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype-phenotype relationship in PWS.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

GC-rich repeat expansions: associated disorders and mechanisms.

Noncoding repeat expansions are a well-known cause of genetic disorders mainly affecting the central nervous system. Missed by most standard technologies used in routine diagnosis, pathogenic noncoding repeat expansions have to be searched for using specific techniques such as repeat-primed PCR or specific bioinformatics tools applied to genome data, such as ExpansionHunter. In this review, we focus on GC-rich repeat expansions, which represent at least one third of all noncoding repeat expansions described so far. GC-rich expansions are mainly located in regulatory regions (promoter, 5' untranslated region, first intron) of genes and can lead to either a toxic gain-of-function mediated by RNA toxicity and/or repeat-associated non-AUG (RAN) translation, or a loss-of-function of the associated gene, depending on their size and their methylation status. We herein review the clinical and molecular characteristics of disorders associated with these difficult-to-detect expansions.

The adult phenotype of Schaaf-Yang syndrome.

BACKGROUND: MAGEL2-associated Schaaf-Yang syndrome (SHFYNG, OMIM #615547, ORPHA: 398069), which was identified in 2013, is a rare disorder caused by truncating variants of the paternal copy of MAGEL2, which is localized in the imprinted region on 15q11.2q13. The phenotype of SHFYNG in childhood partially overlaps with that of the well-established Prader-Willi syndrome (PWS, OMIM #176270). While larger numbers of younger individuals with SHFYNG have been recently published, the phenotype in adulthood is not well established. We recruited 7 adult individuals (aged 18 to 36) with molecularly confirmed SHFYNG and collected data regarding the clinical profile including eating habits, sleep, behavior, personal autonomy, psychiatric abnormalities and other medical conditions, as well as information about the respective phenotypes in childhood. RESULTS: Within our small cohort, we identified a range of common features, such as disturbed sleep, hypoactivity, social withdrawal and anxiety, but also noted considerable differences at the level of personal autonomy and skills. Behavioral problems were frequent, and a majority of individuals displayed weight gain and food-seeking behavior, along with mild intellectual disability or borderline intellectual function. Classical symptoms of SHFYNG in childhood were reported for most individuals. CONCLUSION: Our findings indicate a high variability of the functional abilities and social participation of adults with SHFYNG. A high prevalence of obesity within our cohort was notable, and uncontrollable food intake was a major concern for some caregivers. The phenotypes of PWS and SHFYNG in adulthood might be more difficult to discern than the phenotypes in childhood. Molecular genetic testing for SHFYNG should therefore be considered in adults with the suspected diagnosis of PWS, if testing for PWS has been negative.

A germ cell-specific ageing pattern in otherwise healthy men.

Life-long sperm production leads to the assumption that male fecundity remains unchanged throughout life. However, recently it was shown that paternal age has profound consequences for male fertility and offspring health. Paternal age effects are caused by an accumulation of germ cell mutations over time, causing severe congenital diseases. Apart from these well-described cases, molecular patterns of ageing in germ cells and their impact on DNA integrity have not been studied in detail. In this study, we aimed to assess the effects of 'pure' ageing on male reproductive health and germ cell quality. We assembled a cohort of 198 healthy men (18-84 years) for which end points such as semen and hormone profiles, sexual health and well-being, and sperm DNA parameters were evaluated. Sperm production and hormonal profiles were maintained at physiological levels over a period of six decades. In contrast, we identified a germ cell-specific ageing pattern characterized by a steady increase of telomere length in sperm and a sharp increase in sperm DNA instability, particularly after the sixth decade. Importantly, we found sperm DNA methylation changes in 236 regions, mostly nearby genes associated with neuronal development. By in silico analysis, we found that 10 of these regions are located in loci which can potentially escape the first wave of genome-wide demethylation after fertilization. In conclusion, human male germ cells present a unique germline-specific ageing process, which likely results in diminished fecundity in elderly men and poorer health prognosis for their offspring.

The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis.

BACKGROUND: In the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of oligozoospermic men, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown. RESULTS: Using deep bisulfite sequencing (DBS), we screened swim-up sperm samples from 40 normozoospermic and 93 patients diagnosed as oligoasthenoteratozoospermic, oligoteratozoospermic or oligozoospermic, which are termed OATs throughout the manuscript, for H19 and MEST methylation. Based on this screening, we defined three patient groups: normal controls (NC), abnormally methylated oligozoospermic (AMO; n = 7) and normally methylated oligozoospermic (NMO; n = 86). Whole-genome bisulfite sequencing (WGBS) of five NC and five AMO samples revealed abnormal methylation levels of all 50 imprinting control regions in each AMO sample. To investigate whether this finding reflected epigenetic germline mosaicism or the presence of residual somatic DNA, we made a genome-wide inventory of soma-germ cell-specific DNA methylation. We found that > 2000 germ cell-specific genes are promoter-methylated in blood and that AMO samples had abnormal methylation levels at these genes, consistent with the presence of somatic cell DNA. The comparison between the five NC and six NMO samples revealed 19 differentially methylated regions (DMRs), none of which could be validated in an independent cohort of 40 men. Previous studies reported a higher incidence of epimutations at single CpG sites in the CTCF-binding region 6 of H19 in infertile patients. DBS analysis of this locus, however, revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype. CONCLUSIONS: Our results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men. While we cannot exclude the existence of rare patients with slightly abnormal sperm methylation at non-recurrent CpG sites, the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated, which is reassuring for patients undergoing assisted reproduction.

wg-blimp: an end-to-end analysis pipeline for whole genome bisulfite sequencing data.

BACKGROUND: Analysing whole genome bisulfite sequencing datasets is a data-intensive task that requires comprehensive and reproducible workflows to generate valid results. While many algorithms have been developed for tasks such as alignment, comprehensive end-to-end pipelines are still sparse. Furthermore, previous pipelines lack features or show technical deficiencies, thus impeding analyses. RESULTS: We developed wg-blimp (whole genome bisulfite sequencing methylation analysis pipeline) as an end-to-end pipeline to ease whole genome bisulfite sequencing data analysis. It integrates established algorithms for alignment, quality control, methylation calling, detection of differentially methylated regions, and methylome segmentation, requiring only a reference genome and raw sequencing data as input. Comparing wg-blimp to previous end-to-end pipelines reveals similar setups for common sequence processing tasks, but shows differences for post-alignment analyses. We improve on previous pipelines by providing a more comprehensive analysis workflow as well as an interactive user interface. To demonstrate wg-blimp's ability to produce correct results we used it to call differentially methylated regions for two publicly available datasets. We were able to replicate 112 of 114 previously published regions, and found results to be consistent with previous findings. We further applied wg-blimp to a publicly available sample of embryonic stem cells to showcase methylome segmentation. As expected, unmethylated regions were in close proximity of transcription start sites. Segmentation results were consistent with previous analyses, despite different reference genomes and sequencing techniques. CONCLUSIONS: wg-blimp provides a comprehensive analysis pipeline for whole genome bisulfite sequencing data as well as a user interface for simplified result inspection. We demonstrated its applicability by analysing multiple publicly available datasets. Thus, wg-blimp is a relevant alternative to previous analysis pipelines and may facilitate future epigenetic research.

Common genetic variation in the Angelman syndrome imprinting centre affects the imprinting of chromosome 15.

Angelman syndrome (AS) is a rare neurogenetic imprinting disorder caused by the loss of function of UBE3A. In ~3-5% of AS patients, the disease is due to an imprinting defect (ID). These patients lack DNA methylation of the maternal SNRPN promotor so that a large SNRPN sense/UBE3A antisense transcript (SNHG14) is expressed, which silences UBE3A. In very rare cases, the ID is caused by a deletion of the AS imprinting centre (AS-IC). To search for sequence alterations, we sequenced this region in 168 patients without an AS-IC deletion, but did not detect any sequence alteration. However, the AS-IC harbours six common variants (five single nucleotide variants and one TATG insertion/deletion variant), which constitute five common haplotypes. To determine if any of these haplotypes is associated with an increased risk for an ID, we investigated 119 informative AS-ID trios with the transmission disequilibrium test, which is a family-based association test that measures the over-transmission of an allele or haplotype from heterozygous parents to affected offspring. By this we observed maternal over-transmission of haplotype H-AS3 (p = 0.0073). Interestingly, H-AS3 is the only haplotype that includes the TATG deletion allele. We conclude that this haplotype and possibly the TATG deletion, which removes a SOX2 binding site, increases the risk for a maternal ID and AS. Our data strengthen the notion that the AS-IC is important for establishing and/or maintaining DNA methylation at the SNRPN promotor and show that common genetic variation can affect genomic imprinting.

A human somatic cell culture system for modelling gene silencing by transcriptional interference.

Transcriptional interference and transcription through regulatory elements (transcriptional read-through) are implicated in gene silencing and the establishment of DNA methylation. Transcriptional read-through is needed to seed DNA methylation at imprinted genes in the germ line and can lead to aberrant gene silencing by DNA methylation in human disease. To enable the study of parameters and factors influencing transcriptional interference and transcriptional read-through at human promoters, we established a somatic cell culture system. At two promoters of imprinted genes (UBE3A and SNRPN) and two promoters shown to be silenced by aberrant transcriptional read-through in human disease (MSH2 and HBA2) we tested, if transcriptional read-through is sufficient for gene repression and the acquisition of DNA methylation. Induction of transcriptional read-through from the doxycycline-inducible CMV promoter resulted in consistent repression of all downstream promoters, independent of promoter type and orientation. Repression was dependent on ongoing transcription, since withdrawal of induction resulted in reactivation. DNA methylation was not acquired at any of the promoters. Overexpression of DNMT3A and DNMT3L, factors needed for DNA methylation establishment in oocytes, was still not sufficient for the induction of DNA methylation. This indicates that induction of DNA methylation has more complex requirements than transcriptional read-through and the presence of de novo DNA methyltransferases.

In vitro postovulatory oocyte aging affects H3K9 trimethylation in two-cell embryos after IVF.

BACKGROUND: The physiological time axis of oocyte maturation comprises highly sensitive processes. A prolonged time span between ovulation and fertilization may impair oocyte developmental competence and subsequent embryo development, possibly due to epigenetic modifications. Since post-translational histone modifications can modify chromatin activity, and trimethylation of H3K9 (H3K9me3) has been shown to increase in the murine oocyte during maturation, here the effect of postovulatory oocyte aging on H3K9me3 was analyzed. METHODS: The competence of murine oocytes which were aged for 2, 4, 6 and 8 h in vitro after oocyte retrieval to develop to the two-cell and blastocyst stage was determined. Degree of H3K9me3 was analyzed in the postovulatory aged oocytes as well as in the resulting two-cell embryos after IVF. RESULTS: The current study shows that postovulatory aging of oocytes for up to eight hours after oocyte retrieval exhibited no effect on two-cell embryo and blastocyst rate; however, changes in H3K9me3 in the resulting two-cell embryos were observed. CONCLUSION: Prolonged postovulatory oocyte aging leads to epigenetic modifications of H3K9. Such modifications may affect the developmental capacity of embryos at post-implantation developmental stages.

Unstable TTTTA/TTTCA expansions in MARCH6 are associated with Familial Adult Myoclonic Epilepsy type 3.

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements.