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Heterozygosity for a hypomorphic Polβ mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

Lokanga RA, Senejani AG, Sweasy JB, Usdin K - PLoS Genet. (2015)

Bottom Line: The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene.Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals.This may have interesting implications for the mechanism by which BER generates expansions.

View Article: PubMed Central - PubMed

Affiliation: Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

ABSTRACT
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

No MeSH data available.


Related in: MedlinePlus

The effect of heterozygosity for the PolBC mutation on the number of expansions, contractions and unchanged alleles seen in the gametes of 11-month-old male mice.Small pool PCR was carried out on sperm DNA isolated from three 11-month-old PolB+/+ and three 11-month-old PolB+/C male mice as described in the Materials and Methods. These animals all had ~140 repeats. The error bars represent the 95% confidence interval. The difference between the number of expansions, contractions and unchanged alleles in the two groups of animals was evaluated by Fisher’s exact test but no significant differences were found.
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pgen.1005181.g003: The effect of heterozygosity for the PolBC mutation on the number of expansions, contractions and unchanged alleles seen in the gametes of 11-month-old male mice.Small pool PCR was carried out on sperm DNA isolated from three 11-month-old PolB+/+ and three 11-month-old PolB+/C male mice as described in the Materials and Methods. These animals all had ~140 repeats. The error bars represent the 95% confidence interval. The difference between the number of expansions, contractions and unchanged alleles in the two groups of animals was evaluated by Fisher’s exact test but no significant differences were found.

Mentions: When small pool PCR was carried out on the sperm of 11-month-old animals, no significant difference in the number of expansions was seen in the gametes of PolB+/+ and PolB+/C mice (Fig 3). This was not unexpected since we had previously shown that the number of expansions in the germ line increases with paternal age [36]. Since the number of gametes that had sustained at least one expansion in PolB+/C mice was 42% at 3 months of age, it was not surprising that this number had risen to ~90% by 11 months of age. However, when we examined the distribution of residual expansions in these animals, we were surprised to see a relative enrichment for larger alleles (Fig 4). In WT animals the average number of repeats added had increased from an average of ~1 repeat in 3 month old mice to an average of ~7 repeats in the older animals consistent with our previous reports [36]. In contrast, in PolB+/C mice, a similar deficit of smaller alleles was seen in older mice as was seen in younger ones. In addition, the number of larger alleles had increased such that 39% of gametes that had expanded had gained more than 15 repeats compared to 9% in PolB+/+ animals. This data would be consistent with the idea that while the expansion frequency is lower in PolB+/C mice than it is in PolB+/+ animals, when expansions do occur, they tend to be larger. Thus repeated rounds of expansion in the germ line of PolB+/+ males would, for the most part, lead to an incremental increase in repeat number with time, while in PolB+/C mice, each expansion event, while less frequent, would add a larger number of repeats. The distribution of allele sizes in the gametes of 11-month-old PolB+/C mice showed a series of local maxima corresponding to gametes with 10, 16, 21 and 27 added repeats (indicated by the gray arrowheads in Fig 4). Taken together with the data for 3 month old fathers, the pattern would be consistent with many gametes in PolB+/C mice having undergone multiple rounds of expansion each involving the addition of ~5–6 repeats, whereas in PolB+/C mice most expansions only add ~1 repeat.


Heterozygosity for a hypomorphic Polβ mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

Lokanga RA, Senejani AG, Sweasy JB, Usdin K - PLoS Genet. (2015)

The effect of heterozygosity for the PolBC mutation on the number of expansions, contractions and unchanged alleles seen in the gametes of 11-month-old male mice.Small pool PCR was carried out on sperm DNA isolated from three 11-month-old PolB+/+ and three 11-month-old PolB+/C male mice as described in the Materials and Methods. These animals all had ~140 repeats. The error bars represent the 95% confidence interval. The difference between the number of expansions, contractions and unchanged alleles in the two groups of animals was evaluated by Fisher’s exact test but no significant differences were found.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401650&req=5

pgen.1005181.g003: The effect of heterozygosity for the PolBC mutation on the number of expansions, contractions and unchanged alleles seen in the gametes of 11-month-old male mice.Small pool PCR was carried out on sperm DNA isolated from three 11-month-old PolB+/+ and three 11-month-old PolB+/C male mice as described in the Materials and Methods. These animals all had ~140 repeats. The error bars represent the 95% confidence interval. The difference between the number of expansions, contractions and unchanged alleles in the two groups of animals was evaluated by Fisher’s exact test but no significant differences were found.
Mentions: When small pool PCR was carried out on the sperm of 11-month-old animals, no significant difference in the number of expansions was seen in the gametes of PolB+/+ and PolB+/C mice (Fig 3). This was not unexpected since we had previously shown that the number of expansions in the germ line increases with paternal age [36]. Since the number of gametes that had sustained at least one expansion in PolB+/C mice was 42% at 3 months of age, it was not surprising that this number had risen to ~90% by 11 months of age. However, when we examined the distribution of residual expansions in these animals, we were surprised to see a relative enrichment for larger alleles (Fig 4). In WT animals the average number of repeats added had increased from an average of ~1 repeat in 3 month old mice to an average of ~7 repeats in the older animals consistent with our previous reports [36]. In contrast, in PolB+/C mice, a similar deficit of smaller alleles was seen in older mice as was seen in younger ones. In addition, the number of larger alleles had increased such that 39% of gametes that had expanded had gained more than 15 repeats compared to 9% in PolB+/+ animals. This data would be consistent with the idea that while the expansion frequency is lower in PolB+/C mice than it is in PolB+/+ animals, when expansions do occur, they tend to be larger. Thus repeated rounds of expansion in the germ line of PolB+/+ males would, for the most part, lead to an incremental increase in repeat number with time, while in PolB+/C mice, each expansion event, while less frequent, would add a larger number of repeats. The distribution of allele sizes in the gametes of 11-month-old PolB+/C mice showed a series of local maxima corresponding to gametes with 10, 16, 21 and 27 added repeats (indicated by the gray arrowheads in Fig 4). Taken together with the data for 3 month old fathers, the pattern would be consistent with many gametes in PolB+/C mice having undergone multiple rounds of expansion each involving the addition of ~5–6 repeats, whereas in PolB+/C mice most expansions only add ~1 repeat.

Bottom Line: The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene.Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals.This may have interesting implications for the mechanism by which BER generates expansions.

View Article: PubMed Central - PubMed

Affiliation: Section on Gene Structure and Disease, Laboratory of Cell and molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

ABSTRACT
The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

No MeSH data available.


Related in: MedlinePlus