# Repeat Expansions with Small TTTCA Insertions in MARCHF6 Cause Familial Myoclonus without Epilepsy

Theresa Kühnel, Elsa Leitão, Renate Lunzer, Fabian Kilpert, Sabine Kaya, Claudia del Gamba, Kelly Astudillo, Steven Frucht, Marion Simonetta-moreau, Eric Bieth, et al.

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► To cite this version:

Theresa Kühnel, Elsa Leitão, Renate Lunzer, Fabian Kilpert, Sabine Kaya, et al. Repeat Expansions with Small TTTCA Insertions in MARCHF6 Cause Familial Myoclonus without Epilepsy. Movement Disorders, 2025, 40 (7), pp.1401-1408. 10.1002/mds.30192. hal-05525467

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RESEARCH ARTICLE

# Repeat Expansions with Small TTTCA Insertions in MARCHF6 Cause Familial Myoclonus without Epilepsy

Theresa Kühnel, PhD,<fnref n="1" /> Ⓓ Elsa Leitão, PhD,<fnref n="1" /> Ⓓ Renate Lunzer, MD, PhD,² Fabian Kilpert, PhD,<fnref n="1" /> Ⓓ Sabine Kaya, MS,<fnref n="1" />

Claudia Del Gamba, MD,³,⁴ Ⓓ Kelly Astudillo, MD,³ Ⓓ Steven Frucht, MD, MD,³ Ⓓ

Marion Simonetta-Moreau, MD, PhD,⁵ Ⓓ Eric Bieth, MD, PhD,⁶ Ⓓ Iris Unterberger, MD,⁷ Ⓓ

Giulietta Maria Riboldi, MD, PhD,³ Ⓓ and Christel Depienne, PhD<fnref n="1" />* Ⓓ

**ABSTRACT:** **Background:** Familial adult myoclonus epilepsy (FAME) is a rare autosomal dominant disorder caused by the same intronic TTTTA/TTTCA repeat expansion in seven distinct genes. TTTTA-only expansions are benign, whereas those containing TTTCA insertions are pathogenic.

**Objective:** We investigated the genetic basis of dominant cortical myoclonus without seizures in two unrelated families.

**Methods:** Repeat-primed polymerase chain reaction (PCR), long-range PCR, and nanopore sequencing were used to detect and characterize expansions at known FAME loci.

**Results:** We identified a novel repeat expansion in MARCHF6, comprising 388 to 454 elongated TTTTA repeats and 5 to 11 TTTCA repeats at the 3'-terminus, segregating with cortical myoclonus in 8 affected individuals. This

configuration shows meiotic stability but low-level somatic variability in blood. We observed an inverse correlation between the number of TTTCA repeats and the age at myoclonus onset.

**Conclusions:** These findings indicate that as little as five TTTCA repeats combined with expanded TTTTA repeats can cause cortical myoclonus without epilepsy, highlighting the potential mechanisms underlying FAME pathology. © 2025 The Author(s). *Movement Disorders* published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

**Key Words:** myoclonus; repeat expansion; epilepsy; MARCHF6; familial adult myoclonus epilepsy (FAME)

Familial adult myoclonus epilepsy (FAME), also known as familial adult myoclonic epilepsy, benign adult familial myoclonic epilepsy (BAFME), autosomal dominant cortical myoclonus and epilepsy (ADCME), or familial cortical myoclonic tremor with epilepsy (FCMTE),<fnref n="1" />⁻³ is a rare autosomal dominant disorder characterized by cortical myoclonus and epilepsy. Patients present with brief, irregular, involuntary movements, mainly in the upper limbs and occasionally in the lower limbs. Cortical tremor, the rhythmic variant of cortical myoclonus, may be present in some patients.

Generalized tonic-clonic or myoclonic seizures are present in 15% to 100% of cases.⁴ Symptoms typically start in adulthood about the third decade, but the age of onset may vary from 10 to >60 years. The progression of the disorder also varies among individuals, with some experiencing mild symptoms that remain stable over time, whereas others may develop more severe symptoms that worsen with age.<fnref n="1" />⁻³

FAME is caused by the same pentanucleotide repeat expansion in introns of seven different genes: SAMD12 (FAME1/BAFME1), STARD7 (FAME2/

<footnote n="1">¹Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, Essen, Germany; ²Institut für Humangenetik, Medizinische Universität Innsbruck, Innsbruck, Austria; ³Department of Neurology, The Marlene and Paolo Fresco Institute for Parkinson's and Movement Disorders, NYU Langone Health, New York, New York, USA; ⁴Department of Neurology, ASL Toscana Centro, Prato, Italy; ⁵Department of Neurosciences, University Hospital of Toulouse France, Neuro-Imaging Center, INSERM, UMR1214 Toulouse France, Paul Sabatier Toulouse 3 University, Toulouse, France; ⁶Génétique Médicale, Centre Hospitalier Universitaire de Toulouse, Toulouse, France; ⁷Department of Neurology, Innsbruck Medical University, Innsbruck, Austria</footnote>

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>*Correspondence to: Prof. Christel Depienne, Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstraße 55, 45147 Essen, Germany; E-mail: christel.depienne@uk-essen.de</footnote>

<footnote>Theresa Kühnel and Elsa Leitão contributed equally to this article.</footnote>

<footnote>**Relevant conflicts of interest/financial disclosures:** None.</footnote>

<footnote>**Funding agency:** Deutsche Forschungsgemeinschaft (DFG), project number 455314768.</footnote>

<footnote>**Received:** 9 September 2024; **Revised:** 11 March 2025; **Accepted:** 21 March 2025</footnote>

<footnote>Published online 9 April 2025 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.30192</footnote>
FCMTE2/ADCME), MARCHF6 (FAME3/FCMTE3), YEATS2 (FAME4/BAFME4), TNRC6A (FAME6/BAFME6), RAPGEF2 (FAME7/BAFME7), and RAI1 (FAME8/BAFME8).⁵ Pathogenic expansions consist of elongated TTTTA repeats always containing a TTTCA insertion absent in healthy populations. These dynamic expansions, ranging from 2.2 to 18.4 kb, exhibit somatic variability. The typical structure is 5'-(TTTTA)exp (TTTCA)exp-3', but TTTCA repeats can also insert within TTTTA repeats⁶,⁷ or combine with other pentanucleotide motifs.⁷,⁸ TTTTA-only expansions, existing in some populations (eg, 6% of Japanese in SAMD12), are benign.⁶

FAME repeat expansions exhibit geographic clustering and population-specific prevalence due to founder effects. FAME1 (SAMD12) predominantly occurs in Asian populations (Japan, China, India, and Sri Lanka),⁹ whereas FAME2 (STARD7) and FAME3 (MARCHF6) are mainly found in European patients, with FAME2 prevalent near Naples, Italy.¹⁰⁻¹² Rare subtypes include FAME4 (Thailand), FAME6 and FAME7 (Japan), and FAME8 (Mali), potentially linked to specific geographic backgrounds.⁶,¹³,¹⁴ FAME subtypes may exhibit slightly distinct clinical characteristics. For instance, more than 70% of FAME3 patients exhibit seizures, sometimes as the first symptom. FAME2 and FAME3 cases occasionally present with focal rather than generalized seizures.¹¹,¹⁵⁻¹⁷

In this study, we investigated two unrelated families with cortical myoclonus but no seizures in which we identified a new repeat expansion configuration at the MARCHF6 FAME locus.

# Patients and Methods

## Patients

Two unrelated families of European descent, with multiple individuals affected with autosomal dominant cortical myoclonus (Fig. 1A), were referred for genetic testing of FAME expansions in a research context. Family 5 (E22-0392) is of Austrian origin and includes 9 individuals with cortical myoclonus, 6 of whom were sampled. Family members were evaluated at the Institute of Human Genetics and the outpatient seizure clinic of the Department of Neurology, Innsbruck Medical University. Family 6 (E23-0117) is of French origin and includes 4 affected members, 2 of whom were available for genetic analysis. Family members were evaluated at the Fresco Institute for Parkinson's Disease, NYU Langone Health NY, USA, and the University Hospital Toulouse, France. Genetic testing was conducted at the Institute of Human Genetics, University Hospital Essen. Informed consent was obtained for all family members. The study was approved by ethics boards at NYU Langone Health (s21-00207) and the University Hospital Essen (18-8176-BO, 21-10155-BO).

# Expansion Detection and Characterization

Repeat-primed polymerase chain reaction (RP-PCR) was performed as previously described.¹¹ Repeat expansions at the MARCHF6 locus were amplified using long-range PCR (LR-PCR) from genomic DNA extracted from blood. Samples were barcoded, multiplexed, and sequenced using nanopore sequencing on MinION Mk1B R9.4.1 flow cells (Data S1). BaseCALLing and analysis of nanopore data were performed using Guppy,¹⁸ pycoQC,¹⁹ NanoPlot²⁰ and BBMap²¹. Flanking sequences were trimmed off using Cutadapt.²² This in-house bioinformatics pipeline using Snakemake²³ is available on Github (https://github.com/kilpert/FAME3_analyses.git).

# Results

Clinical data were available for 4 affected members of family 5. At neurological examination, symptoms included cortical myoclonus in the lower more than the upper limbs, mild dysdiadochokinesia, ataxia, or action tremor (Table 1). These symptoms remained relatively stable over time. Routine electroencephalography images showed interictal generalized spike-and-wave discharges, but none had epilepsy. Two family members without myoclonus experienced seizures: one due to hyponatremia and another due to juvenile myoclonic epilepsy.

The proband of family 6 (6-II-1) was a 63-year-old man with an 8-year history of action-induced cortical myoclonus of the upper limbs triggered by stress, associated with anxiety and obsessive-compulsive features. His father (deceased) and sister presented with similar but more severe symptoms (both myoclonus and anxiety) with progression over time. The sister presented generalized cortical myoclonus that worsened during sleep. There was no personal or family history of seizures.

Across the two identified families, the age at myoclonus onset ranged from 27 to 55 years. Treatment with antiseizure medications, including valproic acid, topiramate, and levetiracetam, was beneficial in all of the 5 treated subjects but did not entirely suppress myoclonus.

In addition to cortical myoclonus, 5 affected family members (4 in family 5 and the sister in family 6) reported brief paroxysmal episodes of ataxia and sudden leg weakness, triggered by motor activity or prolonged exercise such as walking on uneven ground or steep terrain, going up and down the stairs, or dancing. These episodes were not observed in consultation, and their nature could not be precisely determined, but their description may correspond to negative myoclonus.

We tested the presence of repeat expansions at the FAME2 (STARD7) and FAME3 (MARCHF6) loci
using RP-PCR. This analysis was negative for FAME2 but revealed a sawtooth pattern that stopped abruptly after a few peaks in contrast to the positive control,

displaying the classical continuous pattern associated with a long TTTCA expansion at the FAME3 locus (Fig. 1B). We then amplified the alleles at this

<figure><img src="image_3.png" /><figcaption>FIG. 1. Legend on next page.</figcaption></figure>
locus using LR-PCR in all available members of both families. A PCR product between 2 and 3 kb was identified in the 2 available affected individuals of family 6 (E23-0117) and all individuals with myoclonus in family 5 (E22-0392), whereas the 15 family members without myoclonus did not have the expansion (Fig. 1C; Fig. S1). To investigate the repeat number, we sequenced the LR-PCR amplicons of the 8 individuals with an expanded allele using nanopore sequencing. This analysis revealed a comparable expansion structure in the two families, with an expanded TTTTA fragment ranging from 385 to 453 repeats, followed by a short TTTCA stretch composed of 5 to 11 repeats ending with three TTTTA repeats in family 5 and one TTTTA repeat in family 6 (Fig. 1D,E). Interestingly, nanopore sequencing, which was done on a new flow cell and multiplexing only the samples included in this study, also detected reads with a higher number of TTTCA repeats in all individuals although this phenomenon was limited (Fig. 1F), as well as a somatic variability in TTTTA repeat numbers (Fig. 1G).

Finally, we investigated whether there was any correlation between the age at onset (AAO) and the number of repeats. We observed significant inverse correlations between the AAO and the number of both TTTTA and TTTCA repeats as well as the size of the expansion (Fig. 1H,I). In addition, we observe that the number of TTTCA repeats was positively correlated to the number of TTTTA repeats (Fig. 1K).

Discussion

FAME represents a new paradigm of noncoding repeat expansion disorders in which the same intronic expansion is pathogenic regardless of the function of the gene harboring the expansion. The seven genes where FAME expansions occur encode proteins with completely different functions and subcellular localizations, and expansions do not appear to alter gene expression or splicing.²⁴ The pathophysiological mechanisms leading to FAME remain largely unknown,²⁴,²⁵

but currently favored hypotheses include the accumulation of expansion-carrying RNA molecules in RNA foci followed by sequestration of RNA-binding proteins as in myotonic dystrophy,²⁶⁻²⁸ or the expression of toxic polypeptides as a result of repeat-associated non-AUG (RAN) translation.²⁹

Here, we describe two families with a new expansion in MARCHF6, associated with a milder phenotype characterized by different degrees of cortical myoclonus but no seizures. In addition, some family members reported action-induced paroxysmal episodes. As no neurophysiology or videos were available, the nature of these movement disorders remains unclear. The expansion, consisting of expanded TTTTA repeats associated with 5-11 TTTCA repeats at its 3'-extremity, segregates with cortical myoclonus in both families. The existence of short TTTCA stretches within SAMD12 expansion has been reported in two previous studies: 1 isolated patient had an expansion containing 14 TTTCA repeats,⁷ whereas a small family with 2 affected individuals had a 4.5-kb expansion containing 10 TTTCA repeats interrupted by 7 TTTTA repeats.³⁰ In both cases, patients had late-onset seizures. Our findings provide definite evidence of segregation of these atypical expansions with cortical myoclonus and further show that as little as five TTTCA repeats next to TTTTA expansions are sufficient to lead to cortical myoclonus. Although we observed low levels of somatic mosaicism with longer TTTCA stretches in nanopore data, these expansions remained relatively stable during transmission, consistent with the absence of anticipation in these families. Nevertheless, we observed an inverse correlation between the age at myoclonus onset and repeat number, which applies to both TTTTA and TTTCA repeats, as they correlate with each other. This suggests that introducing a “C” into an AT-rich sequence may be selectively favored, potentially turning nonpathogenic TTTTA expansions into pathogenic repeats.

The observation that myoclonus occurs in the presence of a few TTTCA repeats, whereas seizures tend to occur with larger TTTCA expansions, suggests that

**FIG. 1.** (A) Pedigrees of families 5 and 6. Half-black symbols indicate affected individuals with neurological examination. Squares indicate males, circles indicate females, and diamonds indicate both sexes. Half-gray symbols indicate affected subjects with a history but no examination. Half-green symbol: juvenile myoclonic epilepsy; Half-blue symbols acute symptomatic seizure due to hyponatremia. The numbers in the symbols indicate the number of siblings of the same sex. The numbers below the symbols correspond to the individual ID within the generation (left side of the pedigree). Numbers in red indicate an expansion, and numbers in black indicate its absence. (B) Identification of the expansion at the MARCHF6 locus using repeat-primed polymerase chain reaction (RP-PCR). RP-PCR profiles using TTTCA primer for a control individual (top left, negative control), a patient with a typical expansion in MARCHF6 (top right, positive control), and the 8 affected individuals from families 5 and 6. (C) Gel electrophoresis of long-range PCR (LR-PCR) amplicons spanning the MARCHF6 expansion in individuals with myoclonus from families 5 and 6. (D–G) Characterization of repeat expansion sequence and variability using nanopore sequencing. (D) Waterfall plots showing selected nanopore reads after separation of alleles based on the presence of at least two consecutive TTTCA repeats (Methods). Three hundred randomly chosen reads are shown in each graph. Yellow: TTTTA, red: TTTCA, cyan: TTTTG, blue: TTATG, black: other. (E) Median number of TTTTA and TTTCA repeats calculated from nanopore reads for each of the 8 affected individuals from families 5 and 6. (F) Box plots showing the distribution of the number of TTTCA repeats in each affected individual. (G) Box plots showing the distribution of the number of TTTTA repeats in each affected individual. (H–J) Genotype–phenotype correlations. Pearson’s correlations of the age at onset (AAO) and the median number of (H) TTTCA repeats, (I) TTTTA repeats, and (J) median size of the expansion including both motifs. (K) Pearson’s correlation between the median number of TTTCA and TTTTA repeats.
<table><caption>TABLE 1 Continued</caption><thead><tr><th>Treatment</th><th>VPA-beneficial</th><th>VPA-beneficial</th><th>VPA-beneficial</th><th>LEV-beneficial</th><th>TPM, LEV-beneficial</th><th>None</th><th>LEV-beneficial</th></tr></thead><tbody><tr><td>Seizures (Y/N)</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>Cognitive abilities</td><td>Normal</td><td>Normal</td><td>Normal</td><td>Normal</td><td>N/A</td><td>Normal</td><td>Normal</td></tr><tr><td>Anxiety</td><td>Y</td><td>N/A</td><td>N/A</td><td>N/A</td><td>Y</td><td>Y</td><td>Y</td></tr><tr><td>OCD</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N</td><td>Y</td><td>N/A</td></tr><tr><td>Other</td><td>N/A</td><td>Insomnia</td><td>Insomnia</td><td>Syncope</td><td>None</td><td>None</td><td>N/A</td></tr><tr><td>EEG</td><td>Generalized SPW</td><td>Generalized SPW</td><td>Generalized SPW</td><td>Generalized SPW</td><td>Generalized SPW</td><td>N/A</td><td>Normal</td></tr><tr><td>Cerebral MRI</td><td>Nonlesional</td><td>Nonlesional</td><td>Nonlesional</td><td>Nonlesional</td><td>Nonspecific cortical atrophy</td><td>Nonlesional</td><td>Olfactory meningioma treated by radiosurgery</td></tr><tr><td>Neurological exam</td><td>Resting tremor, action tremor, bilateral dysdiadochokinesis, ataxia</td><td>Action tremor, bilateral dysdiadochokinesis, ataxia</td><td>Action tremor</td><td>Mild bilateral dysdiadochokinesis</td><td>Upper- and lower-limb myoclonus</td><td>Mild and occasional myoclonic jerks on the left more than the right upper limb, especially with fine motor skills (i.e., writing and drawing a spiral)</td><td>No tremor. No ataxia. Hand clumsiness in fine motor tasks possibly related to post radiotherapy corpus callosum lesion</td></tr></tbody></table>

*Reported by the son.

Abbreviations: AAO, age at onset; N/A, not assessed or not available; Y, yes; N, no; VPA, valproic acid; LEV, levitiracetam; TPM, topiramate; OCD, obsessive-compulsive disorder; EEG, electroencephalography; SPW, spike and waves; MRI, magnetic resonance imaging.
these clinical features may result from different degrees
of cortical hyperexcitability and/or distinct effects of
the expansion in different brain regions (e.g., cerebel-
lum and cortex). This aligns with our previous finding
that the number of TTTCA repeats correlates with the
age of seizure onset but not the onset of myoclonus.¹¹
Moreover, due to the benign nature of expansions
exclusively consisting of TTTTA repeats, the fact that a
few TTTCA repeats alongside TTTTA expansion is suf-
ficient to confer pathogenicity raises questions about
current hypotheses. Although this does not entirely rule
out the ability of the corresponding RNA to aggregate
into RNA foci and sequester RNA-binding proteins or
to express toxic polypeptide by RAN translation, it is
more likely that the introduction of TTTCA repeats
may modify the ability of the DNA or the RNA to fold
or to bind protein partners. TTTCA repeats adjacent to
TTTTA are rare in the human reference genome, with
one occurrence in the *MED12L* gene. The reason why
expression of TTTCA adjacent to TTTTA repeats is
nonpathogenic in the context of *MED12L* is of interest,
but it is worth observing that the expression of this
gene remains low in the brain (as well as in other
tissues).

In conclusion, our results confirm the pathogenic-
ity of expansions containing small TTTCA stretches
and further contribute to a better understanding of
FAME conditions. It also implies that FAME loci
should be tested in patients who have cortical myoc-
lonus even in the absence of a familial history of
epilepsy.

**Author Roles:** 1. Research project: A. Conception, B. Organization, C. Execution; 2. Statistical analysis: A. Design, B. Execution, C. Review and critique; 3. Manuscript: A. Writing of the first draft, B. Review and critique.
T.K.:1A, 1B, 1C, 3B.
E.L.:1C, 2A, 2B, 3A, 3B.
R.L.:1B, 1C, 3B.
F.K.:1C, 2C, 3B.
S.K.:1B, 1C, 3B.
C.D.G.:1C, 3B.
K.A.:1C, 3B.
S.F.:1C, 3B.
M.S.M.:1C, 3B.
E.B.:1C, 3B.
I.U.:1C, 3B.
G.M.R.:1B, 1C, 3B.
C.D.:1A, 1B, 1C, 3A, 3B.

**Acknowledgments:** We thank all patients and families who partici-
pated in this study. We are grateful to Dr. Isabel Silveira (i3s Institute,
Porto, Portugal) for sharing conditions allowing LR-PCR amplification of
SCA37 expansions that helped us establish the LR-PCR assay used for
FAME3 in this study. Open Access funding enabled and organized by
Projekt DEAL.

**Full financial disclosures of all authors for the previous 12 months:** This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants (project numbers 455314768 and 458099954) to C.D. G.M.R. was supported by grants from Michael J Fox Foundation, Parkinson's Foundation, Department of Defense (PD210038), NIH (R01 NS116006; R01 NS133742; 1OT2OD038130). M.S.M. has received Honoraria from IPSEN BIOTECH for training courses and ABBVIE for lectures. All remaining authors had no financial disclosures or conflict of interest to report.

Data Availability Statement

Nanopore reads have been deposited in the European
Genome-Phenome Archive (EGA, http://www.ebi.ac.uk/
ega) (https://ega-archive.org/studies/EGAS50000000570).
All other data supporting the findings described in this
manuscript are available in the article or can be requested
from the corresponding author.

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## Supporting Data

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