Small Pool PCR Research Articles

Somatic CAG Repeat Stability in a Transgenic Sheep Model of Huntington's Disease.

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.

Masseter muscle volume as a disease marker in adult-onset myotonic dystrophy type 1

The advent of clinical trials in myotonic dystrophy type 1 (DM1) necessitates the identification of reliable outcome measures to quantify different disease manifestations using minimal number of assessments. In this study, clinical correlations of mean masseter volume (mMV) were explored to evaluate its potential as a marker of muscle involvement in adult-onset DM1 patients. We utilised data from a preceding study, pertaining to 39 DM1 patients and 20 age-matched control participants. In this study participants had undergone MRI of the brain, completed various clinical outcome measures and had CTG repeats measured by small-pool PCR. Manual segmentation of masseter muscles was performed by a single rater to estimate mMV. The masseter muscle was atrophied in DM1 patients when compared to controls (p<0.001). Significant correlations were found between mMV and estimated progenitor allele length (p = 0.001), modal allele length (p = 0.003), disease duration (p = 0.009) and and the Muscle Impairment Rating Scale (p = 0.008). After correction for lean body mass, mMV was also inversely correlated with self-reported myotonia (p = 0.014). This study demonstrates that changes in mMV are sensitive in reflecting the underlying disease process. Quantitative MRI methods demonstrate that data concerning both central and peripheral disease could be acquired from MR brain imaging studies in DM1 patients.

MYOTONIC DYSTROPHY: EP.238 Masseter muscle volume as a disease marker in adult-onset myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic condition characterized by multisystem involvement. Given the recent commencement of clinical trials in DM1, reliable outcome measures assessing different disease manifestations need to be investigated and validated, while keeping the total number of assessments to a minimum. In this study we used magnetic resonance (MR) images of the head of DM1 patients from a previous study to investigate the clinical correlations of masseter muscle volume and evaluate its potential use as a marker of muscle involvement. Data from 39 DM1 patients and 20 age-matched control participants who had previously undergone MR imaging of the head, completed a range of clinical outcome measures and had CTG repeat measured by small-pool PCR was used. Single rater performed manual segmentation of each masseter muscle in all participants to estimate its volume. The masseter muscle was atrophied in DM1 patients when compared to controls (p<0.001). Significant correlations were discovered between masseter volume and genetic determinants of disease severity, including estimated progenitor allele length (p=0.001) and modal allele length (p=0.003), as well as the disease duration (p=0.009). After correction for lean body mass, masseter volume was inversely correlated with self-reported myotonia (p=0.014), and with muscle function quantified using the muscle impairment rating scale (p=0.008). Masseter muscle atrophy is common in DM1 patients, and is easily quantified on MRI. The masseter volume correlates with genetic determinants of disease and measures of muscle involvement, suggesting these changes reflect the underlying disease process. Further studies evaluating masseter muscle through quantitative MRI methods may yield useful insights for outcome measure selection, and demonstrate that data regarding both central and peripheral disease may be gained from MRI brain in DM1 patients.

DNA methylation at the DMPK gene locus is associated with cognitive functions in myotonic dystrophy type 1.

Aim: Myotonic dystrophy type 1 (DM1) is caused by an unstable trinucleotide (CTG) expansion at the DMPK gene locus. Cognitive dysfunctions are often observed in the condition. We investigated the association between DMPK blood DNA methylation (DNAm) and cognitive functions in DM1, considering expansion length and variant repeats (VRs). Method: Data were obtained from 115 adult-onset DM1 patients. Molecular analyses consisted of pyrosequencing, small pool PCR and Southern blot hybridization. Cognitive functions were assessed by validated neuropsychological tests. Results: For patients without VRs (n=103), blood DNAm at baseline independently contributed to predict cognitive functions 9years later. Patients with VRs (n=12) had different DNAm and cognitive profiles. Conclusion: DNAm allows to better understand DM1-related cognitive dysfunction etiology.

Longitudinal increases in somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 are associated with variation in age-at-onset.

In myotonic dystrophy type 1 (DM1), somatic mosaicism of the (CTG)n repeat expansion is age-dependent, tissue-specific and expansion-biased. These features contribute toward variation in disease severity and confound genotype-to-phenotype analyses. To investigate how the (CTG)n repeat expansion changes over time, we collected three longitudinal blood DNA samples separated by 8-15years and used small pool and single-molecule PCR in 43 DM1 patients. We used the lower boundary of the allele length distribution as the best estimate for the inherited progenitor allele length (ePAL), which is itself the best predictor of disease severity. Although in most patients the lower boundary of the allele length distribution was conserved over time, in many this estimate also increased with age, suggesting samples for research studies and clinical trials should be obtained as early as possible. As expected, the modal allele length increased over time, driven primarily by ePAL, age-at-sampling and the time interval. As expected, small expansions <100 repeats did not expand as rapidly as larger alleles. However, the rate of expansion of very large alleles was not obviously proportionally higher. This may, at least in part, be a result of the allele length-dependent increase in large contractions that we also observed. We also determined that individual-specific variation in the increase of modal allele length over time not accounted for by ePAL, age-at-sampling and time was inversely associated with individual-specific variation in age-at-onset not accounted for by ePAL, further highlighting somatic expansion as a therapeutic target in DM1.

Brain Structural Features of Myotonic Dystrophy Type 1 and their Relationship with CTG Repeats.

BackgroundFew adequately-powered studies have systematically evaluated brain morphology in adult-onset myotonic dystrophy type 1 (DM1).ObjectiveThe goal of the present study was to determine structural brain differences between individuals with and without adult-onset DM1 in a multi-site, case-controlled cohort. We also explored correlations between brain structure and CTG repeat length.MethodsNeuroimaging data was acquired in 58 unaffected individuals (29 women) and 79 individuals with DM1 (50 women). CTG repeat length, expressed as estimated progenitor allele length (ePAL), was determined by small pool PCR. Statistical models were adjusted for age, sex, site, and intracranial volume (ICV).ResultsICV was reduced in DM1 subjects compared with controls. Accounting for the difference in ICV, the DM1 group exhibited smaller volume in frontal grey and white matter, parietal grey matter as well as smaller volume of the corpus callosum, thalamus, putamen, and accumbens. In contrast, volumes of the hippocampus and amygdala were significantly larger in DM1. Greater ePAL was associated with lower volumes of the putamen, occipital grey matter, and thalamus. A positive ePAL association was observed for amygdala volume and cerebellar white matter.ConclusionsSmaller ICV may be a marker of aberrant neurodevelopment in adult-onset DM1. Volumetric analysis revealed morphological differences, some associated with CTG repeat length, in structures with plausible links to key DM1 symptoms including cognitive deficits and excessive daytime somnolence. These data offer further insights into the basis of CNS disease in DM1, and highlight avenues for further work to identify therapeutic targets and imaging biomarkers.

DMPK gene DNA methylation levels are associated with muscular and respiratory profiles in DM1

ObjectiveTo assess the effects of dystrophia myotonica protein kinase (DMPK) DNA methylation (DNAme) epivariation on muscular and respiratory profiles in patients with myotonic dystrophy type 1 (DM1).MethodsPhenotypes were assessed with standardized measures. Pyrosequencing of bisulfite-treated DNA was used to quantify DNAme levels in blood from 90 patients with DM1 (adult form). Modal CTG repeat length was assessed using small-pool PCR. The presence of Acil-sensitive variant repeats was also tested.ResultsDNAme levels upstream of the CTG expansion (exon and intron 11) were correlated with modal CTG repeat length (rs = −0.224, p = 0.040; rs = −0.317, p = 0.003; and rs = −0.241, p = 0.027), whereas correlations were observed with epivariations downstream of the CTG repeats (rs = 0.227; p = 0.037). The presence of a variant repeat was associated with higher DNAme levels at multiple CpG sites (up to 10% higher; p = 0.001). Stepwise multiple linear regression modeling showed that DNAme contributed significantly and independently to explain phenotypic variability in ankle dorsiflexor (3 CpGs: p = 0.001, 0.013, and 0.001), grip (p = 0.089), and pinch (p = 0.028) strengths and in forced vital capacity (2 CpGs: p = 0.002 and 0.021) and maximal inspiratory pressure (p = 0.012).ConclusionsIn addition to the CTG repeat length, DMPK epivariations independently explain phenotypic variability in DM1 and could thus improve prognostic accuracy for patients.

The fragile X CGG repeat shows a marked level of instability in hereditary non-polyposis colorectal cancer patients.

The allelic variation of the FMR1 CGG repeat was investigated by small-pool PCR in nonneoplastic peripheral blood leukocytes from HNPCC patients and matched controls for similar CGG repeat lengths. The allelic variation for repeat lengths appears to be roughly twice as frequent in HNPCC patients as in controls, especially when patients are mutated in hMLH1. There are more expansions in HNPCC patients (42%) than in controls (20%) but this difference is statistically borderline. The mean length of expansions relative to the genuine size did not differ in HNPCC patients or controls (respectively 17% and 20% of the constitutional allelic length). The reported data suggest that instability within nonneoplastic cells of a subset of HNPCC patients might be one mechanism for transition from normal to the premutation range of the FMR1 CGG repeat.

Repeat Interruptions Modify Age at Onset in Myotonic Dystrophy Type 1 by Stabilizing DMPK Expansions in Somatic Cells.

CTG expansions in DMPK gene, causing myotonic dystrophy type 1 (DM1), are characterized by pronounced somatic instability. A large proportion of variability of somatic instability is explained by expansion size and patient’s age at sampling, while individual-specific differences are attributed to additional factors. The age at onset is extremely variable in DM1, and inversely correlates with the expansion size and individual-specific differences in somatic instability. Three to five percent of DM1 patients carry repeat interruptions and some appear with later age at onset than expected for corresponding expansion size. Herein, we characterized somatic instability of interrupted DMPK expansions and the effect on age at onset in our previously described patients. Repeat-primed PCR showed stable structures of different types and patterns of repeat interruptions in blood cells over time and buccal cells. Single-molecule small-pool PCR quantification of somatic instability and mathematical modeling showed that interrupted expansions were characterized by lower level of somatic instability accompanied by slower progression over time. Mathematical modeling demonstrated that individual-specific differences in somatic instability had greater influence on age at onset in patients with interrupted expansions. Therefore, repeat interruptions have clinical importance for disease course in DM1 patients due to stabilizing effect on DMPK expansions in somatic cells.

Outcome Measures for Central Nervous System Evaluation in Myotonic Dystrophy Type 1 May Be Confounded by Deficits in Motor Function or Insight.

Background: Central nervous system involvement in myotonic dystrophy type 1 (DM1) is associated with cognitive deficits, impaired social performance and excessive somnolence, which greatly impact quality of life. With the advent of clinical trials in DM1, there is a pressing need to identify outcome measures for quantification of central symptoms that are feasible and valid. In this context, we sought to evaluate neuropsychological and self-reported measures currently recommended by expert consensus, with particular reference to their specificity for central nervous system involvement in a moderate-sized DM1 cohort.Methods: Forty-five adults with DM1 and 20 controls completed neuropsychology assessments and symptom questionnaires. Those without contraindication also underwent MRI brain, from which global gray matter volume and white matter lesion volume were quantified. CTG repeat was measured by small pool PCR, and was screened for the presence of variant repeat sequences.Results: The neuropsychology test battery was well tolerated and detected impairment across various domains in the DM1 group vs. controls. Large effect sizes in the Stroop and Trail Making Tests were however attenuated by correction for basic speed, which could be influenced by dysarthria and upper limb weakness, respectively. Low mood was strongly associated with increased self-reporting of central symptoms, including cognitive impairment. Conversely, self-reported cognitive impairment did not generally predict poorer performance in neuropsychology assessments, and there was a trend toward greater self-reporting of low mood and cognitive problems in those with milder white matter change on MRI. Global gray matter volume correlated with performance in several neuropsychology assessments in a multivariate model with age and sex, while white matter lesion volume was associated with executive dysfunction reported by a proxy. Screening for variant repeats was positive in three individuals, who reported mild muscle symptoms.Conclusions: Identification of outcome measures with good specificity for brain involvement in DM1 is challenging, since complex cognitive assessments may be compromised by peripheral muscle weakness and self-reported questionnaires may be influenced by mood and insight. This highlights the need for further large, longitudinal studies to identify and validate objective measures, which may include imaging biomarkers and cognitive measures not influenced by motor speed.

De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3'-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3'-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

The flash-small-pool PCR: how to transform blotting and numerous hybridization steps into a simple denatured PCR.

Numerous human diseases are associated with abnormal expansion of unstable trinucleotide repeats (TNRs). TNR instability mechanisms are complex, and remain only partially understood. Small-pool-PCR (SP-PCR) is the reference method to assess TNR instability. SP-PCR amplifies a low number of DNA molecules and is followed by Southern blot. However, SP-PCR remains expensive and time consuming. Here, we describe an optimized SP-PCR that can be done in a day, which reduces cost and experimental biases: the flash-small-pool PCR (FSP-PCR). This method consists of a fluorescent PCR on a few DNA molecules, followed by an alkaline gel electrophoresis revealed with a near infra-red detector system. With reduced experimental steps, cost, and time consumption, microsatellite analysis will become more accessible due to FSP-PCR.

Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR (SP-PCR), such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We performed SP-PCR on myotonic-dystrophy-derived samples containing an expansion as large as 1000 repeats, demonstrating the applicability to clinically-relevant material. Thus, we expect the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders.

Comparison of Huntington's disease CAG Repeat Length Stability in Human Motor Cortex and Cingulate Gyrus.

Huntington's disease is caused by expansion of the CAG repeat in Huntingtin. This repeat has shown tissue-specific instability in mouse models and in a small number of post-mortem human samples. We used small-pool PCR to generate a modified instability index to quantify CAG instability within two brain regions from six human samples where cell loss has been associated with motor and mood symptoms: the motor cortex and cingulate gyrus. The expanded allele demonstrated instability in both regions, with minimal instability in the unexpanded allele. Region-specific differences were not observed, suggesting symptomatology may not be determined by repeat length instability.

Human embryonic stem cells show low-grade microsatellite instability.

It is well known that human embryonic stem cells (hESCs) frequently acquire recurrent chromosomal abnormalities, very reminiscent of those found in cancerous cells. Given the parallels between cancer and stem cell biology, we set out to investigate the occurrence of a common form of genome instability in tumors, namely microsatellite instability (MSI), in hESCs. MSI is caused by a deficiency in mismatch repair (MMR) genes, which leads to the accumulation of mutations during DNA replication. In this study, we analyzed up to 122 microsatellites in a total of 10 hESC lines, for 1-11 different passages, ranging from passage 7 to passage 334. In two lines, this revealed that two microsatellites had altered allelic patterns. Small-pool PCR for several microsatellites and testing of the Bethesda panel microsatellites (commonly used in cancer studies) revealed that, whilst MSI is common in all tested lines, it occurs at a very low and variable frequency, ranging from ∼1 to 20% of the total number of alleles. In cancerous cells, MSI leads to multiple large shifts in allele sizes within the majority of the cells, while hESCs show small changes in a minority of the cells. Since these genetic alterations do not consistently take over the culture, we assume that they are not concurrent with a selective advantage as it is in tumors. Finally, the MMR genes showed a very variable gene expression that could not be correlated with the variable (low) levels of MSI in the different hESC lines.

Non-Radioactive Detection of Trinucleotide Repeat Size Variability

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

CAG size-specific risk estimates for intermediate allele repeat instability in Huntington disease

IntroductionNew mutations for Huntington disease (HD) occur due to CAG repeat instability of intermediate alleles (IA). IAs have between 27 and 35 CAG repeats, a range just below the disease...

Determining microsatellite genotyping reliability and mutation detection ability: an approach using small-pool PCR from sperm DNA

Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 × 10(-2) to 2.2 × 10(-3) mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies.

Microsatellite instability in the peripheral blood leukocytes of HNPCC patients

Most hereditary nonpolyposis colorectal cancer (HNPCC) patients inherit a defective allele of a mismatch repair (MMR) gene, usually MLH1 or MSH2, resulting in high levels of microsatellite instability (MSI-H) in the tumors. Presence of MSI in the normal tissues of mutation carriers has been controversial. Here we directly compare MSI in the peripheral blood leukocyte (PBL) DNA of seven HNPCC patients carrying different types of pathogenic MMR mutations in MLH1 and MSH2 genes with the PBL DNA of normal age-matched controls and of patients with sporadic colorectal cancer (SCRC). Small pool PCR (SP-PCR) was used studying three microsatellite loci for at least 100 alleles each in most samples. The average frequencies of mutant microsatellite fragments in each HNPCC patient (0.04-0.24) were significantly higher (p<0.01) relative to their age-matched normal controls with mutant frequencies (MF) from 0.00 to 0.06, or SCRC patients (MF from 0.01-0.03). The data support the conclusions that higher MF in the PBL DNA of HNPCC patients is real and reproducible, may vary in extent according to the type of germline MMR mutation and the age of the individual, and provide a possible genetic explanation for anticipation in HNPCC families.

Somatic expansion of the Huntington's disease CAG repeat in the brain is associated with an earlier age of disease onset

The age of onset of Huntington's disease (HD) is determined primarily by the length of the HD CAG repeat mutation, but is also influenced by other modifying factors. Delineating these modifiers is a critical step towards developing validated therapeutic targets in HD patients. The HD CAG repeat is somatically unstable, undergoing progressive length increases over time, particularly in brain regions that are the targets of neurodegeneration. Here, we have explored the hypothesis that somatic instability of the HD CAG repeat is itself a modifier of disease. Using small-pool PCR, we quantified somatic instability in the cortex region of the brain from a cohort of HD individuals exhibiting phenotypic extremes of young and old disease onset as predicted by the length of their constitutive HD CAG repeat lengths. After accounting for constitutive repeat length, somatic instability was found to be a significant predictor of onset age, with larger repeat length gains associated with earlier disease onset. These data are consistent with the hypothesis that somatic HD CAG repeat length expansions in target tissues contribute to the HD pathogenic process, and support pursuing factors that modify somatic instability as viable therapeutic targets.