Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG expansion on the huntingtin (HTT) gene and is characterized by progressive motor, cognitive, and neuropsychiatric decline. Recently, new genetic factors besides CAG repeats have been implicated in the disease pathogenesis. Most genetic modifiers are involved in DNA repair pathways and, as the cause of the loss of CAA interruption in the HTT gene, they exert their main influence through somatic expansion. However, this mechanism might not be the only driver of HD pathogenesis, and future studies are warranted in this field. The aim of the present review is to dissect the many faces of genetics in HD pathogenesis, from cis- and trans-acting genetic modifiers to RNA toxicity, mitochondrial DNA mutations, and epigenetics factors. Exploring genetic modifiers of HD onset and progression appears crucial to elucidate not only disease pathogenesis, but also to improve disease prediction and prevention, develop biomarkers of disease progression and response to therapies, and recognize new therapeutic opportunities. Since the same genetic mechanisms are also described in other repeat expansion diseases, their implications might encompass the whole spectrum of these disorders.

Expanded CAG repeats in coding regions of different genes are the most common cause of dominantly inherited spinocerebellar ataxias (SCAs). These repeats are unstable through the germline, and larger repeats lead to earlier onset. We measured somatic expansion in blood samples collected from 30 SCA1, 50 SCA2, 74 SCA3, and 30 SCA7 individuals over a mean interval of 8.5 years, along with postmortem tissues and fetal tissues from SCA1, SCA3, and SCA7 individuals to examine somatic expansion at different stages of life. We showed that somatic mosaicism in the blood increases over time. Expansion levels are significantly different among SCAs and correlate with CAG repeat lengths. The level of expansion is greater in individuals with SCA7 who manifest disease compared to that of those who do not yet display symptoms. Brain tissues from SCA individuals have larger expansions compared to the blood. The cerebellum has the lowest mosaicism among the studied brain regions, along with a high expression of ATXNs and DNA repair genes. This was the opposite in cortices, with the highest mosaicism and lower expression of ATXNs and DNA repair genes. Fetal cortices did not show repeat instability. This study shows that CAG repeats are increasingly unstable during life in the blood and the brain of SCA individuals, with gene- and tissue-specific patterns.

Somatic instability of the huntingtin (HTT) CAG repeat mutation modifies age-at-onset of Huntington\'s disease (HD). Understanding the mechanism and pathogenic consequences of instability may reveal therapeutic targets. Using small-pool PCR we analyzed CAG instability in the OVT73 sheep model which expresses a full-length human cDNA HTT transgene. Analyses of five- and ten-year old sheep revealed the transgene (CAG)69 repeat was remarkably stable in liver, striatum, and other brain tissues. As OVT73 sheep at ten years old have minimal cell death and behavioral changes, our findings support instability of the HTT expanded-CAG repeat as being required for the progression of HD.

Repeat expansion disorders (REDs) are monogenic diseases caused by a sequence of repetitive DNA expanding above a pathogenic threshold. A common feature of the REDs is a strong genotype-phenotype correlation in which a major determinant of age at onset (AAO) and disease progression is the length of the inherited repeat tract. Over a disease-gene carrier\'s life, the length of the repeat can expand in somatic cells, through the process of somatic expansion which is hypothesised to drive disease progression. Despite being monogenic, individual REDs are phenotypically variable, and exploring what genetic modifying factors drive this phenotypic variability has illuminated key pathogenic mechanisms that are common to this group of diseases. Disease phenotypes are affected by the cognate gene in which the expansion is found, the location of the repeat sequence in coding or non-coding regions and by the presence of repeat sequence interruptions. Human genetic data, mouse models and in vitro models have implicated the disease-modifying effect of DNA repair pathways via the mechanisms of somatic mutation of the repeat tract. As such, developing an understanding of these pathways in the context of expanded repeats could lead to future disease-modifying therapies for REDs.

Repeat expansion diseases are a large group of human genetic disorders caused by expansion of a specific short tandem repeat tract. Expansion in somatic cells affects age of onset and disease severity in some of these disorders. However, alleles in DNA derived from blood, a commonly used source of DNA, usually show much less expansion than disease-relevant cells in the central nervous system in both humans and mouse models. Here we examined the extent of expansion in different DNA sources from mouse models of the fragile X-related disorders, Huntington\'s disease, spinocerebellar ataxia type 1 and spinocerebellar ataxia type 2. We found that DNA isolated from stool is a much better indicator of somatic expansion than DNA from blood. As stool is a sensitive and noninvasive source of DNA, it can be useful for studies of factors affecting the risk of expansion, or the monitoring of treatments aimed at reducing expansion in preclinical trials, as it would allow expansions to be examined longitudinally in the same animal and allow significant changes in expansion to be observed much earlier than is possible with other DNA sources.

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington\'s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully confirmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantification was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for finding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantification of exogenous (CGG)n repeat instability in the transgenic cell lines\' genome.
Существует ряд наследственных заболеваний человека, причиной которых является экспансия тандемных повторов. К ним относятся миотоническая дистрофия первого типа, болезнь Хантингтона, заболевания, ассоциированные с ломкой Х-хромосомой. Синдром ломкой Х-хромосомы – наиболее распространенная причина наследственной умственной отсталости у человека. На сегодняшний день причины развития экспансии остаются неисследованными. Важная особенность протяженных повторов – их способность формировать альтернативные вторичные структуры ДНК. Существуют гипотезы, объясняющие природу нестабильности повторов, однако все они предполагают возникновение устойчивых вторичных структур ДНК на различных этапах клеточного цикла. Источником нестабильности считаются нарушения в различных процессах метаболизма ДНК (репликация, репарация и рекомбинация), вызванные образованием вторичных структур. Однако ни одна из гипотез до конца не подтверждена и, видимо, не является единственно верной. Вероятно, в различных типах клеток и на определенных стадиях клеточного цикла источником нестабильности выступает множество процессов. В настоящей работе мы предлагаем экспериментальную систему для изучения вклада транскрипции и ассоциированной с ней репарации в нестабильность повтора (CGG)n, поскольку это наименее изученный механизм возникновения нестабильности. Однако предложенные модели могут учитывать вклад и других процессов метаболизма ДНК, например репликации, что делает полученные системы универсальными и применимыми в разных исследованиях. Нами были созданы трансгенные клеточные линии, несущие повтор нормальной и премутантной длины под тетрациклин-индуцируемым промотором. Один тип линий содержит плазмиду с экзогенным повтором, интегрированным в геном посредством транспозона Sleeping Beauty, в другой клеточной линии вектор поддерживается в виде эписомы благодаря ориджину репликации SV40. Такие трансгенные клеточные линии могут служить экспериментальной системой для поиска причин нестабильности и создания терапевтических средств. Кроме того, был разработан критерий для оценки нестабильности экзогенного (CGG)n повтора в геноме трансгенных клеточных линий, расчет которого не зависит от эффективности синтеза протяженных повторов.

Recent work on Huntington disease (HD) suggests that somatic instability of CAG repeat tracts, which can expand into the hundreds in neurons, explains clinical outcomes better than the length of the inherited allele. Here, we measured somatic expansion in blood samples collected from the same 50 HD mutation carriers over a twenty-year period, along with post-mortem tissue from 15 adults and 7 fetal mutation carriers, to examine somatic expansions at different stages of life. Post-mortem brains, as previously reported, had the greatest expansions, but fetal cortex had virtually none. Somatic instability in blood increased with age, despite blood cells being short-lived compared to neurons, and was driven mostly by CAG repeat length, then by age at sampling and by interaction between these two variables. Expansion rates were higher in symptomatic subjects. These data lend support to a previously proposed computational model of somatic instability-driven disease.

Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual\'s lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich\'s ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well-particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich\'s ataxia pathogenesis.

Myotonic Dystrophy type 1 (DM1) is characterized by a high genetic and clinical variability. Determination of the genetic variability in DM1 might help to determine whether there is an association between CTG (Cytosine-Thymine-Guanine) expansion and the clinical manifestations of this condition. We studied the variability of the CTG expansion (progenitor, mode, and longest allele, respectively, and genetic instability) in three tissues (blood, muscle, and tissue) from eight patients with DM1. We also studied the association of genetic data with the patients\' clinical characteristics. Although genetic instability was confirmed in all the tissues that we studied, our results suggest that CTG expansion is larger in muscle and skin cells compared with peripheral blood leukocytes. While keeping in mind that more research is needed in larger cohorts, we have provided preliminary evidence suggesting that the estimated progenitor CTG size in muscle could be potentially used as an indicator of age of disease onset and muscle function impairment.