Abstract

Normal and pathological tandem repeats in human genomes

The human genome contains more than one million annotated tandem repeats (TRs).^1,2 Due to their repetitive nature, TRs have the highest mutational rate in the genome and are typically polymorphic and multiallelic, with the longest alleles being the most unstable. TRs are mainly scattered in non-coding regions of the genome although repetitions of triplets, usually limited in size, can also be found in coding parts of genes.¹ Coding TRs may occasionally become hotspots for harmful frameshift mutations.³

TRs are usually divided into microsatellites (1–9 bp repeats; or short tandem repeats, STRs) and minisatellites (10–99 bp repeats), which together form the variable number of tandem repeats (VNTRs), and satellites (≥100 bp repeats), which mainly constitute heterochromatin and centromeres. STRs have widely been used as markers in genetic linkage analyses and forensic studies, but they are still an enigmatic and understudied class of variation. STRs present in human genomes are recent in terms of evolution and are either human specific or conserved only in very close primate species.⁴ Most STRs have arisen from other repeated elements, such as long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE), including Alu elements.⁵ The evolutionary constraints applying to these dynamic, rapidly evolving variations and how TRs have contributed to shape human genomes are still largely unknown although recent evidence suggests that they could play an important role in the regulation of gene expression.^6,7

The expansion of TR length across generations is a well-characterized process that results in at least 50 known disorders (Tables 1, ​,2,2, and S1). The first expansions, both identified on chromosome (chr) X in 1991, were CGG repeats in the 5′ untranslated region (UTR) of FMR1 (MIM: 309550) and CAG repeats in the first exon of AR (MIM: 313700) as the underlying causes of fragile X syndrome^8,9 (FXS [MIM: 300624]) and spinal bulbar muscular atrophy (SBMA [MIM: 313200]),¹⁰ respectively (Figure 1). In the next 10 years, a wave of repeat expansion discovery followed, revealing the basis of more than 25 hereditary disorders, most of which primarily affect the central nervous system.

Open in a separate window

Figure 1

Timeline of repeat expansion discovery in human disorders

During the first twenty years, linkage analyses were the gold standard allowing to map the genomic region containing repeat expansions. The development of next-generation sequencing, and more particularly long-read sequencing, has marked a new milestone, making their identification at a genome-wide scale possible, and has allowed a new wave of repeat expansion discovery.

As illustrated by the initial discoveries, two main types of STR expansions exist: expansions affecting coding regions, mainly leading to abnormally long stretches of polyglutamine (polyQ, mostly encoded by CAG codons) or polyalanine (polyA, encoded by GCN codons) within proteins (Table 1), and expansions altering non-coding regions of genes (Figure 2).⁶¹ CAG triplet expansions, including those found in Huntington disease (HD [MIM: 143100]), spinocerebellar ataxias type 1 (SCA1 [MIM: 164400]), SCA2 (MIM: 183090), SCA3 (Machado-Joseph disease [MIM: 109150]), SCA6 (MIM: 183086), SCA7 (MIM: 164500), SCA17 (MIM: 607136), and dentatorubral-pallidoluysian atrophy (DRPLA [MIM: 125370]) are typically associated with neuronal intranuclear protein inclusions that contain the protein (or part of it) exhibiting the expanded polyQ stretch. These inclusions result from the propensity of these abnormal polyQ chains to form β sheet structures leading to intermolecular cross-beta oligomerization and to the formation of insoluble fibrillar aggregates, a mechanism usually described as a toxic gain-of-function at the protein level.⁶² PolyA peptides can also form β sheet structures, but polyA expansions, usually much more limited in size compared to their polyQ counterpart, have variable effects and can lead to either gain- and/or loss-of-function depending on the gene and protein where they occur.⁶³

Open in a separate window

Figure 2

Location of repeat expansions within genes

Repeat expansions may affect coding regions (mainly encoding polyglutamine or polyalanine tracts) or noncoding regions of genes. Noncoding repeat expansions are mainly located in promoters or 5′ untranslated regions (UTRs) or introns. Noncoding repeats in 5′ regions are often GC-rich and their expansion usually affects gene expression through methylation of other epigenetic processes. Intronic expansions and expansions affecting 3′ UTRs more often lead to RNA toxicity or polypeptide synthesis via repeat-associated non-AUG (RAN) translation and subsequent formation of cellular/nuclear aggregates. BCCD, brachydactyly and cleidocranial dysplasia; BSS, Baratela-Scott syndrome; CANVAS, cerebellar ataxia, neuropathy and vestibular areflexia syndrome; CCHS, congenital central hypoventilation syndrome; DM1, myotonic dystrophy type 1; DM2, myotonic dystrophy type 2; DRPLA, dentatorubral-pallidoluysian atrophy; EIEE1, early infantile epileptic encephalopathy type 1; EPM1, progressive myoclonus epilepsy type 1 (Unverricht-Lundborg disease); FAME, familial adult myoclonic epilepsy; FECD3, Fuchs endothelial corneal dystrophy type 3; FRAXE, fragile XE syndrome; FRDA, Friedreich ataxia; FTD/ALS, frontotemporal dementia / amyotrophic lateral sclerosis; FXS, fragile X syndrome; FXTAS, fragile X-associated tremor ataxia syndrome; GDPAG, global developmental delay, progressive ataxia, and elevated glutamine; HD, Huntington disease; HDL2, Huntington disease-like 2; HFGS, hand-foot-genital syndrome; HPE5, holoprosencephaly type 5; MRGH, mental retardation with isolated growth hormone deficiency; OPMD, oculopharyngeal muscular dystrophy; NIID, neuronal intranuclear inclusion disease; OPDM1, oculopharyngodistal myopathy type 1; OPDM2, oculopharyngeal muscular dystrophy type 2; OPML1, oculopharyngeal myopathy with leukoencephalopathy type 1; SBMA, spinal and bulbar muscular atrophy; SPD1, synpolydactyly type 1; SCA, spinocerebellar ataxia; SVA, SINE-VNTR-Alu retrotransposon; XDP, X-linked dystonia parkinsonism.

Non-coding repeat expansions are even more diverse and their impact strongly depends on the type, length, and location of the repeat motif within genes (Table 2).⁶¹ They can occur in either the 5′ UTRs, introns, or 3′ UTRs of genes (Figure 2). Repeat expansions located in the 5′ UTRs, promoter, or other regulatory regions are usually GC rich, like triplet (CGG) expansions in FMR1, dodecamer (CCCCGCCCCGCG) expansions in CSTB (MIM: 601145),³⁴ and hexanucleotide (GGGGCC) expansions in C9ORF72 (MIM: 614260).^43,44 Large, usually GC-rich expansions overlapping 5′ regulatory regions are frequently—but not always—associated with hypermethylation of the corresponding allele and gene silencing, as exemplified by full mutations (>200 repeats) causing FXS.⁶⁴ Expanded repeats falling into introns correspond to the majority of recently described expansion disorders and involve motifs of different lengths (mainly tri, tetra, penta, or hexa-nucleotides) and GC content. Only two repeat expansions in 3′ UTRs have been described to date, in myotonic dystrophy 1 (DM1 [MIM: 160900]; CTG expansion in the 3′ UTR of DMPK [MIM: 605377]) and SCA8 (MIM: 608768) (CTG/CAG expansion in the 3′ UTR of ATXN8OS [MIM: 603680]/ATXN8 [MIM: 613289]). Remarkably, non-coding, and more particularly intronic repeat expansions act through a variety of different mechanisms, most of which are associated with dominant inheritance although a few conditions are recessive, generally resulting from the loss-of-function of the gene as a result of the expansion.

Anticipation

A characteristic of dominant repeat expansion disorders is anticipation, a process by which the clinical manifestation appears at an earlier age and/or becomes more severe as the disorder is transmitted from one generation to the next. Strong correlations between the size of the expansion and the age at onset and/or the severity of the disorder have been established for many repeat expansion disorders, including all polyQ as well as many of the non-coding expansions. The larger the expansion is, the earlier the age at onset and the more severe the phenotype becomes. Moreover, expanded repeats, and especially trinucleotide repeats, tend to dynamically expand further during meiosis. Therefore, the offsprings of an affected carrier receiving the pathogenic allele are at higher risk of presenting a more severe or early form of the disorder. In extreme cases, including DM1, HD, and certain types of SCAs, the disorder can occur in infancy or childhood and be fatal in a few years whereas the transmitting parent has a milder adult-onset form of the same disorder.⁶⁵ The risk of expansion during meiosis generally increases with the number of repeats. Contractions of repeat numbers during meiosis have also been reported but are relatively unfrequent compared to expansions.⁶⁶ The instability of repeat expansions during meiosis and transmission of larger alleles to offprings is influenced by multiple factors including the size of the repeat, its structure, and the sex and age of the transmitting parent.^66,67 Interrupted repeats (for instance AGG interruptions in CGG repeats, or CAA interruptions in CAG repeats) are much more stable than pure ones. Contractions may be more frequent in the male germline, accounting for the exclusive maternal transmission of congenital DM1, or of full FMR1 expansions from premutated mothers, but not from premutated fathers. Males with FXS expansions have spermatozoa with premutation, suggesting that very long repeats may be subject to negative selection during spermatogenesis, by affecting completion of replication.⁶⁸ Conversely, CAG repeat expansions associated with juvenile HD forms arise only from paternal transmission, and somatic variability can be observed in sperm of HD expansion carriers.

Somatic instability

Repeat expansions are also mitotically unstable, each cell division being associated with a risk of error during DNA replication. This process has been initially attributed to the slippage of DNA polymerase while replicating repetitive DNA sequences and is further amplified by the ability of some repeats to form stable folded structures such as hairpins, stem-loops, cruciform, and/or tetraplexes that favor DNA polymerase pausing.^{68, 69, 70} However, the observations of expansions and contractions in non-dividing cells have indicated that other mechanisms are at play, and there is increasing evidence from mouse models, and very recently from affected individuals, that somatic stability or instability of repeat expansions largely depends on DNA mismatch repair processes and may impact progression of the disease.^68,69,71 Many repeat expansions are thus associated with somatic mosaicism, which degree is usually correlated with expansion size and also age, but can show large variation in different tissues, with a pattern that differs in the various expansion disorders.^{72, 73, 74, 75} Interruptions by alternative repeat motifs tend to stabilize expansions, making them less prone to alterations during DNA replication or repair.^{76, 77, 78} Moreover, interrupted expansions are associated with milder and/or different phenotypes compared to uninterrupted expansions, as shown for DM1, SCA2, or SCA10 (MIM: 603516).^79,80

Founder effects

Many repeat expansions, especially larger ones, are associated with founder effects, where the expanded allele is associated with a single or predominant haplotype, and result in geographical clustering of the corresponding disorder or highly variable prevalences depending on the population. For instance, DM1 and Friedreich ataxia (FRDA [MIM: 229300]) are, respectively, very rare in Africa and Japan.^81,82 This suggests that unexpanded alleles, contrary to their expanded counterparts, are relatively stable. Furthermore, risk haplotypes are in linkage disequilibrium with the large normal alleles at the cognate repeat loci, suggesting a multistep evolution process where an initial historical event is a mutation that generates a large normal allele (protomutation) that then acts as a reservoir for further gradual expansions that will finally result in pathological alleles.⁸² Another possibility for a rare founder event is the loss of stabilizing interruption by a variant motif in the target normal repeat (such as a loss of AGG interruptions in the CGG repeat at the fragile X locus).⁸³ Examples of repeat expansions for which founder effects have been demonstrated include DM1, FRDA, FXS, SCA3, SCA10, C9ORF72-related frontotemporal dementia and/or amyotrophic lateral sclerosis (FTD/ALS [MIM: 105550]), and myoclonic epilepsy of Unverricht and Lundborg (EPM1 [MIM: 254800]).^{84, 85, 86, 87}

Identification strategies: Remaining challenges

Although repeat expansions were identified as an important source of human disorders in the years 1991–2003, no additional expansion disorder was discovered in the next 5 years (Figure 1). In 2011, the identification of dominant hexanucleotide expansions in C9ORF72^43,44 at a locus mapped by linkage in multiple large families in 2006 revived some interest in the search for repeat expansion diseases. But attempts to identify repeat expansions remained limited by exome sequencing, which was the prevalent disease gene identification strategy.

Indeed, owing to their repetitive nature and their abundance in the human genome, TRs are very difficult to study at a genome-wide scale and have been for a long time masked by bioinformatic tools such as RepeatMasker. Short-read encompassing repeats typically map to multiple genomic regions, are clipped off, or are discarded. Furthermore, there is a computational challenge in accurately calling genotypes of polymorphic alleles composed of multiple copies of the same repeats. Detection of TRs is thus generally absent from standard NGS pipelines and requires specific tools in addition to standard SNP, indel, and structural/copy number variant pipelines. Bioinformatics tools specifically assessing repeat numbers from genome (or exome) data have been developed, including LobSTR,⁸⁸ HipSTR,⁸⁹ TREDPARSE,⁹⁰ ExpansionHunter⁹¹ STRetch,⁹² GangSTR,⁹³ and exSTRa.⁹⁴ However, until recently, most of these tools were only able to call genotypes at specific loci for a given motif. This has recently changed by the development of TRhist⁹⁵ and ExpansionHunter DeNovo,⁹⁶ which assess the presence of repeat expansions at a genome-wide scale. Nevertheless, the detection of expansions without linkage data or prior hypotheses on the expanded motif remains a challenge due to the huge number of possible motifs, the abundance of TRs in the human genome, and the difficulty in clearly distinguishing pathogenic expansions from normal polymorphic alleles based on short-read sequences.⁹⁰ TR expansions thus likely constitute an enormous unexplored reservoir of pathogenic variations. A recent study, which investigated tandem repeats in 17,231 individuals with autism spectrum disorders, suggested that repeat expansions at more than 2,500 loci may represent a collective contribution of 2.6% to autism risk, but the statistic analysis could not distinguish between potential highly penetrant expansions or those associated as polygenic low penetrance risk factors.⁹⁷

An alternative way to detect TR expansions more accurately is to use long-read technologies, including Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio) Single Molecule Real-Time (SMRT) sequencing.⁹⁸ These technologies allow researchers to obtain single read >10 kb in size, sometimes reaching up to 1–2 Mb, but specific DNA extractions from fresh blood samples or living cells are required to obtain good results. Repeat expansions are detected by pipeline calling structural variants although specific tools, like NanoSatellite⁹⁹ and tandem-genotypes,¹⁰⁰ have been developed to specifically study repeats present in long-read data. Remarkably, these technologies detect thousands of structural variants per individual that predominantly correspond to repeated regions. Because databases such as gnomAD are based on short-read sequencing data, most of the detected structural variants have not yet been described in large control population datasets and filtering of pathogenic variants amid this mass of information is not an easy task.

Overview of recently identified repeat expansion disorders

Despite the technical and computational difficulties, the last few years have clearly witnessed a second wave of repeat expansion discovery. While no expansions have been identified between 2003 and 2008, the genetic basis of more than 20 repeat expansion disorders have been unraveled since 2009, with 17 identified in the last 3 years (Figure 1 and Tables 1 and ​and2).2). In almost all studies, the identification of pathogenic expansions has taken many years and was only possible because of a large number of families that allowed reducing the candidate linkage intervals to regions containing only one or very few genes. This suggests that many more rarer expansion disorders remain to be identified. Comprehensive reviews have already covered many aspects of repeat expansion disorders identified before 2017.^61,101 We review in the following sections the clinical and genetic characteristics of novel disorders and recent advances made in the field since 2017.

Dominant expansions

Recessive expansions

X-linked expansion related to the insertion of a retrotransposon

X-linked dystonia-parkinsonism (XDP or “Lubag” syndrome [MIM: 314250]) is a neurodegenerative disorder characterized by severe and progressive torsion dystonia associated with extra-pyramidal signs including tremors, bradykinesia, and rigidity (parkinsonism) described in 1976 (see GeneReviews by Evidente in web resources). XDP mainly affects males and more rarely females, and is almost exclusively limited to individuals of Filipino descent originating from Panay Island, Philippines, where its prevalence is above 5 in 100,000. Age at onset varies from 12 to 79 years.¹²² Autopsy of XDP-affected brains has shown lesions in the striatum resembling those seen in individuals with HD.¹²³

Genome-wide linkage in XDP-affected families had identified the DYT3 locus on Xq13.1.¹²⁴ Using shotgun sequencing from BAC clones from a subject with XDP, Makino et al.¹²² identified a SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of TAF1 (MIM: 313650), encoding the largest component of the transcription factor IID complex. Gene expression analysis in a XDP postmortem brain showed a decrease of a neuron-specific TAF1 isoform associated with increased DNA methylation at the site of SVA insertion.¹²²

The inserted SVA is associated with an identical founder haplotype spanning 294 kb and including five single nucleotide substitutions and a 48-bp deletion.¹²² Further analysis of the SVA sequence revealed a polymorphic hexanucleotide (CCCTCT) tandem repeat located at the 5′ end of the SVA. Interestingly, the length of this repeat insertion is inversely correlated with the age at onset and its sequence is able to modulate the expression of a reporter gene, suggesting that this repeat expansion contributes to the pathogenesis of XDP.^47,125

Expansions of minisatellites

Pathophysiological mechanisms

Epigenetic gene silencing

Loss-of-function by gene silencing is one possible consequence of tandem repeat expansions (Figure 3A). Generally associated with recessive (or X-linked) disorders, these expansions are easily missed. Recessive expansions can be either bi-allelic or associated with a pathogenic point variant in trans and the phenotype associated with expansions and point variants is usually identical. Non-coding repeat expansions can modulate the expression of their recipient gene by directly or indirectly altering chromatin conformation and availability. GC-rich expansions may create new or strengthen existing CpG islands, leading to persistent DNA hypermethylation, as observed in FXS (FMR1)¹³⁵ and BSS (XYLT1).⁵⁰ However, some GC-rich expansions seem to lead to epigenetic gene silencing independently of DNA methylation, like expansions in CSTB (EPM1)¹³⁶ and more recently in GLS (GDPAG).⁵³ The recent example of GDPAG has shown that histone acetylation and methylation marks can be altered without any association with DNA hypermethylation. This example is reminiscent of the effect of GAA expansions in FXN (MIM: 606829) (causing FRDA), which alters transcription of Frataxin by creating secondary DNA/RNA structures (R-loops) that block RNA polymerase and are associated with repressive histone marks.^137,138 Of note, hypermethylation and inhibition of gene transcription can be only one of several consequences of the repeat expansions, as observed for C9ORF72-FTD/ALS, one of the few dominant disorders associated with hypermethylation.¹⁰¹ Although DNA methylation inversely correlates with repeat size and age at disease onset,¹³⁹ loss of function of C9ORF72 is likely insufficient to lead to FTD/ALS as no truncating mutations have been yet reported in this gene, but recent evidence suggests that it could contribute to disease pathogenesis by worsening the repeat-dependent gain-of-function mechanisms.¹⁴⁰

Open in a separate window

Figure 3

Main mechanisms associated with repeat expansions

(A) Epigenetic gene silencing. Large, usually GC-rich expansions located in promoters and/or 5′ UTRs are frequently associated with DNA hypermethylation at CpG sites. Methylated expanded alleles are locked in a chromatin configuration preventing gene expression, and therefore leading to a loss-of-function of the expanded allele. Examples of this mechanisms include full expansions in FMR1 (fragile X syndrome) and expansions in XYLT1 (Scott-Baratela syndrome).

(B) Sequestration of RNA-binding splicing factors. Specific expanded repeats within RNA molecules are able to form stable secondary structures such as hairpins, which can bind specific RNA-binding splicing factors (e.g., muscleblind-like proteins) with high affinity. These RNA molecules accumulate to form inclusions in the nucleus and sequester bound RNA-binding splicing factors, resulting in misplicing of a subset of transcripts requiring these factors. The illustration of this mechanism is provided by CTG/CAG expansions in DMPK.

(C) Protein misfolding and aggregation. Coding expansions, particularly those encoding polyQ stretches, lead to neuronal intranuclear protein inclusions. This mechanism results from the ability of abnormally long polyQ chains to form β sheet structures prone to form insoluble fibrillar aggregates.

(D) Repeat-associated non-AUG (RAN) translation. RAN translation is a non-canonical protein synthesis process in which peptide synthesis is initiated at the site of the expanded repeats in absence of an AUG codon. This process can theoretically occur in all reading frames on both sense and antisense DNA strands, resulting in up to six different polypeptides, although only some of these peptides are toxic and/or preferentially expressed. For example, intermediate CGG expansions (ranging from 55 to 200 repeats) associated with FXTAS mainly lead to synthesis of polyglycine peptides that are able to accumulate and form protein aggregates. The list of repeat expansion disorders associated with RAN occurs is rapidly increasing and so far includes DM2, FXTAS, FXPOI, HD, C9ORF72-FTD/ALS, SCA8, and SCA31.

Precision medicine in repeat expansions disorders

Precision medicine (previously referred to as personalized medicine), which consists of developing a treatment entirely adapted to an individual’s disease, taking the genome and environment into account, is increasingly becoming an accessible goal in genetic disorders.¹⁵² This concept has also been developed for several years for specific repeat expansion disorders, in particular HD. In this section of the review, we will focus on the recent progress in this field.

A possible way of developing personalized treatments is to understand the pathological consequences of the expansion at the cellular level and to find molecules that are able to either reverse or delay these consequences. For example, in polyQ disorders, the accumulation of polyQ protein aggregates leads to the dysfunction of several pathways, including altered transcription, reduced mitochondrial metabolism, excitotoxicity, authophagy, and apoptosis. Molecules targeting one or several of these deregulated pathways have therefore been suggested as possible treatments.¹⁵³ However, these approaches are usually limited as they are not able to fully restore the cellular dysfunctions. Yet, some of these strategies can prove useful on the long term in combination with other approaches. For expansion disorders associated with RAN translation, antibodies have been designed to target specifically the toxic peptides produced and block their effect in vivo. This strategy has recently proven successful in a mouse model of C9ORF72-FTD/ALS.¹⁵⁴

A promising way to treat dominant disorders associated with a gain of function of the expanded allele is to target and neutralize specifically this allele. This implies that one functional copy of the gene is sufficient and this strategy is not suitable for genes associated with haplointolerance. CRISPR-Cas9 technologies offer the possibility to edit individual genomes but so far they have limitations, including difficulties in delivering the proteins and RNA guide necessary to gene editing in the affected tissues, as well as possible off-target effects, which still restrict their use for clinical purposes.¹⁵⁵ Alternative approaches making use of antisense oligonucleotides (ASO) or siRNA delivered intrathecally have proven to be efficient in downregulating the expression of the expanded allele and have already reached phase III in clinical trials.¹⁵⁶ The designed oligonucleotides cannot directly target the repeats because of their repetitive nature and their presence in other genes and thus need to be complementary to a specific sequence located in cis of the expansion. In this setting, single-nucleotide polymorphisms (SNPs) are used to make the difference between the expanded and the normal alleles and target specifically the allele carrying the expansion.^157,158 This requires researchers to genotype and determine cis associations existing in each affected individual prior to designing the treatment, and this strategy can only be used for subjects with informative coding SNPs in the corresponding gene.

Another recent pioneering approach could become a game changer in HD and other CAG trinucleotide expansion disorders. A small molecule, naphthyridine-azaquinolone, was shown to specifically bind to expanded CAG repeats and induce their contraction in cells from affected individuals and the brains of mice with an expansion in Htt.¹⁵⁹ This approach could be used to reduce the number of CAG repeats in the brain of individuals with HD, hopefully leading to a delay in disease onset of several decades or even a lifetime protection.

Conclusion: Future studies and remaining challenges

The identification of tandem repeat expansions in human disorders is still in its infancy and many more disorders, monogenic or complex, likely remain to be uncovered. Furthermore, in the last few years we have witnessed an important widening of phenotypes related to known repeat expansions, as illustrated by the recent identification of HTT expansions in individuals with FTD and ALS.¹⁶⁰ Therefore, repeat expansions should be searched for in unsolved disorders of supposedly genetic origin, even in phenotypes not specifically evoking a known expansion.

The more recent studies have highlighted the extreme diversity in repeat motifs susceptible to expand and their location in genes. However, only specific repeat motifs appear to be pathogenic while others can be expanded in human genomes without any harmful consequences. Making the difference between these pathogenic and non-pathogenic motifs is of utmost importance as this information could be used to specifically chase the pathogenic ones among the many repeats contained in our genomes. Interestingly, most of the pentanucleotide repeat expansions recently discovered are composed of pathogenic motifs distinct from the one(s) present in reference and control genomes. Studying how these alternative pathogenic repeat insertions have arisen could help finding new repeat expansion disorders. In the case of SCA37 expansions, it has been hypothesized that the thymidine to cytosine transition has occurred after expansion of ATTTT repeats to ~200 copies, followed by expansion of the mutant ATTTC sequence itself.¹⁶¹ This mechanism is in agreement with previous studies showing the existence of susceptibility haplotypes predisposing to the disorders in specific populations.

The example of FMR1, in which CGG repeats in 5′ UTR have been associated with three different disorders depending on the repeat size and sex (due to the location of the gene on the X chromosome), has demonstrated that expansions may have different downstream effects depending on the expansion length and that intermediate expansion sizes should also be carefully studied as they could constitute susceptibility factors for more common disorders or be associated with other or late-onset phenotypes more difficult to recognize. So far, most expansion disorders affect the central nervous system and the muscles and there are only a few examples of expansion disorders limited to other tissues. This might be attributed to an increased susceptibility of some cells or tissues to mechanisms related to these expansions, for example somatic mosaicism of the repeats with higher repeat lengths in particular tissues,¹⁶² the sequestration of proteins that have a tissue-specific impact (e.g., muscle-blind proteins like), or an increased expression or specific function of the gene where the expansion occurs (e.g., DAB1).¹⁰² Neuronal and muscle cells, which are terminally differentiated non-cycling cells, might also be more susceptible to somatic repeat expansions or to toxic gain-of-function mechanisms. However, it is also likely that repeat expansions may affect other tissues, as observed for FXPOI.¹⁶³

A recent field of research is to understand the impact of normal and pathogenic tandem repeats on gene expression and chromatin conformation. This is now possible thanks to advanced techniques allowing researchers to study gene expression, chromatin conformation, or both at a high resolution, even from single cells. We have just started to fully acknowledge the role of intragenic microsatellites and minisatellites in modulation of gene expression and as a potential source of gene deregulation.^6,129 However, the study of minisatellites is associated with even greater challenges, including difficulties in determining sequence variation within repeats and integrating how both repeat number and sequence affect gene expression in a given cell type. This challenge is achievable only through novel long-read technologies. Furthermore, recent studies regarding minisatellites suggest that this category of repeated elements could act more frequently as susceptibility factors and modifiers rather than causing monogenic disorders, and establishing association with common or rare disorders might require large sample sizes. As for the role of satellites in human pathology, this is an almost untouched research topic. To date, only one disorder, facioscapulohumeral muscular dystrophy (FSHD1 [MIM: 158900]), has been linked to the heterozygous contraction of a satellite repeat array at the D4Z4 locus (encoding DUX4 [MIM: 606009]) in the subtelomeric region of chromosome 4q35. Diagnosing this disorder still represents a challenge as only specific techniques, such as Southern blotting, Fiber FISH, or optical genome mapping, can be used to specifically detect the contraction of these very large repeats (approx. 100 kb each) and distinguish them from non-pathogenic contractions of an almost identical repeat array at 10q26 (see GeneReviews by Preston et al. in web resources).

Finally, we have to keep in mind that repeat tracts can be wrongly annotated in the human reference genome. An accumulation of short reads in a region typically suggests that this region is either variable in copy number or wrongly annotated but this observation can be made for many regions. Moreover, there are still genomic regions whose sequence remains unknown because of their high complexity. Most of these regions, including centromeres and short arms of acrocentric chromosomes, actually correspond to repeats. The telomere-to-telomere (T2T) consortium has just started sequencing all the gaps remaining in the human genome reference, a tour de force that will undoubtedly result in clarifying many human disorders in the future. The first sequence, released in 2020, was a complete (i.e., gapless) telomere-to-telomere assembly of the single chr X present in the complete hydatidiform mole CHM13.¹⁶⁴ This achievement was possible thanks to a combination of high-coverage, ultra-long-read nanopore sequencing and alternative long-read technologies (SMRT sequencing and optical genome mapping) that improved the accuracy sequencing or facilitated the assembly. The T2T consortium has since announced the completion of the CHM13 genome sequence. The characterization of the variability of these previously unsequenceable regions and distinction between their normal and pathogenic states will require many years but this is obviously the start of a new genomic era where the systematic study of repeats will finally be possible.

Declaration of interests

The authors declare no competing interests.

Acknowledgments

Footnotes

References