X inactivation plays a major role in the gender bias in somatic expansion in a mouse model of the fragile X-related disorders: implications for the mechanism of repeat expansion.

The Fragile X-related disorders are X-linked disorders resulting from the inheritance of FMR1 alleles with >54 CGG/CCG repeats in their 5' UTR. The repeats expand both somatically and on intergenerational transmission and increased repeat numbers are associated with increased risk of disease and increased risk of further expansion. The mechanism responsible for expansion is unknown. Here, we show in a knockin mouse model of these disorders that somatic expansion is much less common in females than in males. We show that this is due in large part to the fact that expansions occur only when the repeat is on the active X chromosome. However, even when this is taken into account, expansions in females are still less common than expected. This additional gender effect is not due to a protective effect of estrogen, a deleterious effect of testosterone or to differences in the expression of the Fmr1 gene or a variety of X-linked and autosomal DNA repair genes. However, our data do suggest that a higher level of expression of genes that protect against oxidative damage in females may contribute to their lower levels of expansion. Whatever the basis, our data suggest that the risk for somatic expansion may be lower in women than it is in men. This could help explain the reduced penetrance of some aspects of disease pathology in women. The fact that expansion only occurs when the Fmr1 allele is on the active X chromosome has important implications for the mechanism of repeat expansion.

Somatic expansion in mouse and human carriers of fragile X premutation alleles.

Repeat expansion diseases result from expansion of a specific tandem repeat. The three fragile X-related disorders (FXDs) arise from germline expansions of a CGG•CCG repeat tract in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. We show here that in addition to germline expansion, expansion also occurs in the somatic cells of both mice and humans carriers of premutation alleles. Expansion in mice primarily affects brain, testis, and liver with very little expansion in heart or blood. Our data would be consistent with a simple two-factor model for the organ specificity. Somatic expansion in humans may contribute to the mosaicism often seen in individuals with one of the FXDs. Because expansion risk and disease severity are related to repeat number, somatic expansion may exacerbate disease severity and contribute to the age-related increased risk of expansion seen on paternal transmission in humans. As little somatic expansion occurs in murine lymphocytes, our data also raise the possibility that there may be discordance in humans between repeat numbers measured in blood and that present in brain. This could explain, at least in part, the variable penetrance seen in some of these disorders.

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.

A mouse model of the fragile X premutation: effects on behavior, dendrite morphology, and regional rates of cerebral protein synthesis.

Carriers of FMR1 premutation alleles have 55-200 CGG repeats in the 5' untranslated region of the gene. These individuals are at risk for fragile X associated primary ovarian insufficiency (females) and, in late life, fragile X associated tremor and ataxia syndrome (males, and to a lesser extent, females). Premutation carrier status can also be associated with autism spectrum disorder, attention deficit hyperactivity disorder, and some cognitive deficits. In premutation carriers, FMR1 mRNA levels are often higher than those with normal sized alleles. In contrast, in subjects with full mutation alleles, (>200 repeats) the FMR1 gene is silenced and FMR1 mRNA and its product, FMRP, are absent. We have studied a male knock-in (KI) mouse model of the fragile X premutation (120-140 repeats) during young adulthood. In comparison to wild type, KI mice were hyperactive, exhibited less anxiety in both the open field and the elevated zero maze, were impaired on the passive avoidance test, and showed some subtle deficits on a test of social interaction. Motor learning as assessed by the rotarod test was normal. Dendritic arbors were less complex and spine densities and lengths increased in medial prefrontal cortex, basal lateral amygdala, and hippocampus compared with wild type. Regional rates of cerebral protein synthesis measured in vivo in KI mice were increased. KI mice also had elevated levels of Fmr1 mRNA and decreased levels of FMRP. Our results highlight similarities in phenotype between KI and Fmr1 knockout mice and suggest that the decreased concentration of FMRP contributes to the phenotype in young adult KI mice.

ATM and ATR protect the genome against two different types of tandem repeat instability in Fragile X premutation mice.

Expansion of a tandem repeat tract is responsible for the Repeat Expansion diseases, a group of more than 20 human genetic disorders that includes those like Fragile X (FX) syndrome that result from repeat expansion in the FMR1 gene. We have previously shown that the ATM and Rad3-related (ATR) checkpoint kinase protects the genome against one type of repeat expansion in a FX premutation mouse model. By crossing the FX premutation mice to Ataxia Telangiectasia-Mutated (Atm) mutant mice, we show here that ATM also prevents repeat expansion. However, our data suggest that the ATM-sensitive mechanism is different from the ATR-sensitive one. Specifically, the effect of the ATM deficiency is more marked when the premutation allele is paternally transmitted and expansions occur more frequently in male offspring regardless of the Atm genotype of the offspring. The gender effect is most consistent with a repair event occurring in the early embryo that is more efficient in females, perhaps as a result of the action of an X-linked DNA repair gene. Our data thus support the hypothesis that two different mechanisms of FX repeat expansion exist, an ATR-sensitive mechanism seen on maternal transmission and an ATM-sensitive mechanism that shows a male expansion bias.

Potassium bromate, a potent DNA oxidizing agent, exacerbates germline repeat expansion in a fragile X premutation mouse model.

Tandem repeat expansion is responsible for the Repeat Expansion Diseases, a group of human genetic disorders that includes Fragile X syndrome (FXS). FXS results from expansion of a premutation (PM) allele having 55-200 CGG.CCG-repeats in the 5' UTR of the FMR1 gene. The mechanism of expansion is unknown. We have treated FX PM mice with potassium bromate (KBrO(3)), a potent DNA oxidizing agent. We then monitored the germline and somatic expansion frequency in the progeny of these animals. We show here that KBrO(3) increased both the level of 8-oxoG in the oocytes of treated animals and the germline expansion frequency. Our data thus suggest that oxidative damage may be a factor that could affect expansion risk in humans.

ATR protects the genome against CGG.CCG-repeat expansion in Fragile X premutation mice.

Fragile X mental retardation syndrome is a repeat expansion disease caused by expansion of a CGG.CCG-repeat tract in the 5' UTR of the FMR1 gene. In humans, small expansions occur more frequently on paternal transmission while large expansions are exclusively maternal in origin. It has been suggested that expansion is the result of aberrant DNA replication, repair or recombination. To distinguish amongst these possibilities we crossed mice containing 120 CGG.CCG-repeats in the 5' UTR of the mouse Fmr1 gene to mice with mutations in ATR, a protein important in the cellular response to stalled replication forks and bulky DNA lesions. We show here that ATR heterozygosity results in increased expansion rates of maternally, but not paternally, transmitted alleles. In addition, age-related somatic expansions occurred in mice of both genders that were not seen in ATR wild-type animals. Some ATR-sensitive expansion occurs in postmitotic cells including haploid gametes suggesting that aberrant DNA repair is responsible. Our data suggest that two mechanisms of repeat expansion exist that may explain the small and large expansions seen in humans. In addition, our data provide an explanation for the maternal bias of large expansions in humans and the lower incidence of these expansions in mice.

Clinical utility of multigene analysis in over 25,000 patients with neuromuscular disorders.

OBJECTIVE: Molecular genetic testing for hereditary neuromuscular disorders is increasingly used to identify disease subtypes, determine prevalence, and inform management and prognosis, and although many small disease-specific studies have demonstrated the utility of genetic testing, comprehensive data sets are better positioned to assess the complexity of genetic analysis. METHODS: Using high depth-of-coverage next-generation sequencing (NGS) with simultaneous detection of sequence variants and copy number variants (CNVs), we tested 25,356 unrelated individuals for subsets of 266 genes. RESULTS: A definitive molecular diagnosis was obtained in 20% of this cohort, with yields ranging from 4% among individuals with congenital myasthenic syndrome to 33% among those with a muscular dystrophy. CNVs accounted for as much as 39% of all clinically significant variants, with 10% of them occurring as rare, private pathogenic variants. Multigene testing successfully addressed differential diagnoses in at least 6% of individuals with positive results. Even for classic disorders like Duchenne muscular dystrophy, at least 49% of clinically significant results were identified through gene panels intended for differential diagnoses rather than through single-gene analysis. Variants of uncertain significance (VUS) were observed in 53% of individuals. Only 0.7% of these variants were later reclassified as clinically significant, most commonly in RYR1, GDAP1, SPAST, and MFN2, providing insight into the types of evidence that support VUS resolution and informing expectations of reclassification rates. CONCLUSIONS: These data provide guidance for clinicians using genetic testing to diagnose neuromuscular disorders and represent one of the largest studies demonstrating the utility of NGS-based testing for these disorders.

Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia.

Friedreich ataxia (FRDA), the most common hereditary ataxia, is caused by mutations in the frataxin (FXN) gene. The vast majority of FRDA mutations involve expansion of a GAA*TTC-repeat tract in intron 1, which leads to an FXN mRNA deficit. Bisulfite mapping demonstrates that the region adjacent to the repeat was methylated in both unaffected and affected individuals. However, methylation was more extensive in patients. Additionally, three residues were almost completely methylation-free in unaffected individuals but almost always methylated in those with FRDA. One of these residues is located within an E-box whose deletion caused a significant drop in promoter activity in reporter assays. Elevated levels of histone H3 dimethylated on lysine 9 were seen in FRDA cells consistent with a more repressive chromatin organization. Such chromatin is known to reduce transcription elongation. This may be one way in which the expanded repeats contribute to the frataxin deficit in FRDA. Our data also suggest that repeat-mediated chromatin changes may also affect transcription initiation by blocking binding of factors that increase frataxin promoter activity. Our results also raise the possibility that the repeat-mediated increases in DNA methylation in the FXN gene in FRDA patients are secondary to the chromatin changes.

Possible precision medicine implications from genetic testing using combined detection of sequence and intragenic copy number variants in a large cohort with childhood epilepsy.

OBJECTIVE: Molecular genetic etiologies in epilepsy have become better understood in recent years, creating important opportunities for precision medicine. Building on these advances, detailed studies of the complexities and outcomes of genetic testing for epilepsy can provide useful insights that inform and refine diagnostic approaches and illuminate the potential for precision medicine in epilepsy. METHODS: We used a multi-gene next-generation sequencing (NGS) panel with simultaneous sequence and exonic copy number variant detection to investigate up to 183 epilepsy-related genes in 9769 individuals. Clinical variant interpretation was performed using a semi-quantitative scoring system based on existing professional practice guidelines. RESULTS: Molecular genetic testing provided a diagnosis in 14.9%-24.4% of individuals with epilepsy, depending on the NGS panel used. More than half of these diagnoses were in children younger than 5 years. Notably, the testing had possible precision medicine implications in 33% of individuals who received definitive diagnostic results. Only 30 genes provided 80% of molecular diagnoses. While most clinically significant findings were single-nucleotide variants, ~15% were other types that are often challenging to detect with traditional methods. In addition to clinically significant variants, there were many others that initially had uncertain significance; reclassification of 1612 such variants with parental testing or other evidence contributed to 18.5% of diagnostic results overall and 6.1% of results with precision medicine implications. SIGNIFICANCE: Using an NGS gene panel with key high-yield genes and robust analytic sensitivity as a first-tier test early in the diagnostic process, especially for children younger than 5 years, can possibly enable precision medicine approaches in a significant number of individuals with epilepsy.

Regional FMRP deficits and large repeat expansions into the full mutation range in a new Fragile X premutation mouse model.

Carriers of FMR1 alleles with 55-200 repeats in the 5' UTR are at risk for Fragile X associated tremor and ataxia syndrome. The cause of the neuropathology is unknown but is thought to be RNA-mediated. Maternally transmitted premutation alleles are also at risk of expansion of the repeat tract into the "full mutation" range (>200 repeats). The mechanism responsible for expansion is unknown. Full mutation alleles produce reduced amounts of the FMR1 gene product, FMRP, which leads to Fragile X mental retardation syndrome. We have developed a murine model for Fragile X premutation carriers that recapitulates key features seen in humans including a direct relationship between repeat number and Fmr1 mRNA levels, an inverse relationship with FMRP levels and Purkinje cell dropout that have not been seen in a previously described knock-in mouse model. In addition, these mice also show a differential deficit of FMRP in different parts of the brain that might account for symptoms of the full mutation that are seen in premutation carriers. As in humans, repeat instability is high with expansions predominating and, for the first time in a mouse model, large expansions into the full mutation range are seen that occur within a single generation. Thus, contrary to what was previously thought, mice may be good models not only for the symptoms seen in human carriers of FMR1 premutation alleles but also for understanding the mechanism responsible for repeat expansion, a phenomenon that is responsible for a number of neurological and neurodevelopmental disorders.

Ovarian abnormalities in a mouse model of fragile X primary ovarian insufficiency.

FMR1 premutation (PM) alleles have 55-200 CGG·CCG-repeats in their 5' UTR. PM carriers are at risk of fragile X-associated tremor and ataxia syndrome (FXTAS). Females are also at risk for FX primary ovarian insufficiency (FXPOI). PM pathology is generally attributed to deleterious properties of transcripts with long CGG-tracts. For FXPOI, hormone changes suggest a reduced residual follicle pool. Whether this is due to a smaller than normal original follicle pool or an increased rate of follicle depletion is unclear. A FX-PM mouse the authors generated with 130 CGG·CCG-repeats in the endogenous Fmr1 gene recapitulates features of FXTAS. Here the authors demonstrate that the gross development of the ovary and the establishment of the primordial follicle pool is normal in these mice. However, these animals show a faster loss of follicles of all follicle classes, suggesting that the problem is intrinsic to the ovary. In addition, many oocytes show aberrant nuclear accumulation of FMRP and elevated levels of ubiquitination. Furthermore, PM follicles are smaller and have fewer granulosa cells (GCs) than normal. Thus, these animals have ovarian abnormalities involving both the oocytes and GCs that may shed light on the molecular basis of FXPOI in humans.

Fitness cost of LINE-1 (L1) activity in humans.

The self-replicating LINE-1 (L1) retrotransposon family is the dominant retrotransposon family in mammals and has generated 30-40% of their genomes. Active L1 families are present in modern mammals but the important question of whether these currently active families affect the genetic fitness of their hosts has not been addressed. This issue is of particular relevance to humans as Homo sapiens contains the active L1 Ta1 subfamily of the human specific Ta (L1Pa1) L1 family. Although DNA insertions generated by the Ta1 subfamily can cause genetic defects in current humans, these are relatively rare, and it is not known whether Ta1-generated inserts or any other property of Ta1 elements have been sufficiently deleterious to reduce the fitness of humans. Here we show that full-length (FL) Ta1 elements, but not the truncated Ta1 elements or SINE (Alu) insertions generated by Ta1 activity, were subject to negative selection. Thus, one or more properties unique to FL L1 elements constitute a genetic burden for modern humans. We also found that the FL Ta1 elements became more deleterious as the expansion of Ta1 has proceeded. Because this expansion is ongoing, the Ta1 subfamily almost certainly continues to decrease the fitness of modern humans.

Ancient repeated DNA elements and the regulation of the human frataxin promoter.

Friedreich ataxia results from frataxin insufficiency caused by repeat expansion in intron 1 of the frataxin gene. Since the coding sequence is unchanged, the potential exists to ameliorate symptoms by increasing frataxin promoter activity. We therefore defined the minimal frataxin promoter in humans. Despite the fact that frataxin is an essential gene, its promoter is not well conserved in mammals, in part because it has been the frequent target of retroelement insertions. Most of the activity of the human frataxin promoter can be attributed to these retroelements, illustrating how these elements, considered parasitic by some, have been co-opted to drive critical genes. Individuals with the milder French Acadian form and those with the classic form of the disease have no biologically relevant sequence differences in the promoter or 3' UTR, suggesting that some other region of the gene, perhaps the repeat itself, is responsible for the difference in disease severity.

The insertional history of an active family of L1 retrotransposons in humans.

As humans contain a currently active L1 (LINE-1) non-LTR retrotransposon family (Ta-1), the human genome database likely provides only a partial picture of Ta-1-generated diversity. We used a non-biased method to clone Ta-1 retrotransposon-containing loci from representatives of four ethnic populations. We obtained 277 distinct Ta-1 loci and identified an additional 67 loci in the human genome database. This collection represents approximately 90% of the Ta-1 population in the individuals examined and is thus more representative of the insertional history of Ta-1 than the human genome database, which lacked approximately 40% of our cloned Ta-1 elements. As both polymorphic and fixed Ta-1 elements are as abundant in the GC-poor genomic regions as in ancestral L1 elements, the enrichment of L1 elements in GC-poor areas is likely due to insertional bias rather than selection. Although the chromosomal distribution of Ta-1 inserts is generally a function of chromosomal length and gene density, chromosome 4 significantly deviates from this pattern and has been much more hospitable to Ta-1 insertions than any other chromosome. Also, the intra-chromosomal distribution of Ta-1 elements is not uniform. Ta-1 elements tend to cluster, and the maximal gaps between Ta-1 inserts are larger than would be expected from a model of uniform random insertion.