Biomedical subjects

D G Monckton

Publications and source records attributed to D G Monckton.

At least 19 recordsLinked to original sources

PubMed2001

Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates.

The expansion of CAG.CTG trinucleotide repeats has been associated with an increasing number of human diseases. Once into the expanded disease-associated range, the repeats become dramatically unstable in the germline and also throughout the soma. Instability is expansion-biased, contributing towards the unusual genetics, and most likely the tissue-specificity and progressive nature of the symptoms. Such expansions constitute a unique form of dynamic mutation whose mechanism is poorly understood. It is generally assumed that repeat length changes arise via replication slippage, yet no direct evidence exists to support this hypothesis in a mammalian system. We have previously generated transgenic mouse models of unstable CAG.CTG repeats that reconstitute the dynamic nature of somatic mosaicism observed in humans. We have now used tissues from these mice to establish in vitro cell cultures. Monitoring of repeat stability in these cells has revealed the progressive accumulation of larger alleles as a result of repeat length changes in vitro, as confirmed by single cell cloning. We also observed the selection of cells carrying longer repeats during the first few passages of the cultures and frequent additional selective sweeps at later stages. The highest levels of instability were observed in cultured kidney cells, whereas the transgene remained relatively stable in eye cells and very stable in lung cells, paralleling the previous in vivo observations. No correlation between repeat instability and the cell proliferation rate was found, rejecting a simple association between length change mutations and cell division, and confirming a role for additional cell-type specific factors.

3' Untranslated Regions↗

PubMed2001

Frequency and stability of the myotonic dystrophy type 1 premutation.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is associated with the expansion of an unstable CTG repeat. Larger alleles are associated with a more severe form of the disease and almost always increase in length from one generation to the next, accounting for the clinical anticipation characteristic of DM1. As such, expanded alleles are rapidly lost from the population. However, the incidence of the disease appears to remain constant. It was the authors' aim to determine the frequency and germline stability of the DM1 premutation alleles that give rise to new DM1 families. METHODS: The authors measured the size of the DM1 CTG repeat in blood DNA derived from a large number of individuals in DM1 families, including distant and unaffected relatives. RESULTS: It was determined that DM1 premutation alleles can be identified both in distant relatives of DM1 probands and more rarely in unaffected spouses. These premutation alleles are not directly associated with a clinical phenotype in the carrier but are highly unstable and liable to expand in succeeding generations, particularly when transmitted by a man. In addition, the authors observed occasional expansion-biased instability of alleles within the high end of the normal size range. CONCLUSIONS: Individuals carrying premutation alleles are at high risk of having affected offspring within a limited number of generations. Such data indicate that premutation alleles cannot be the long-term source of new DM1 families, which must ultimately arise from mutations of alleles within the upper normal size range.

Adult↗

PubMed2001

Attempts to detect retrotransposition and de novo deletion of Alus and other dispersed repeats at specific loci in the human genome.

Dispersed repeat elements contribute to genome instability by de novo insertion and unequal recombination between repeats. To study the dynamics of these processes, we have developed single DNA molecule approaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci in human genomic DNA. Validation experiments showed these approaches could detect insertions and deletions at frequencies below 10(-6) per cell. However, bulk analysis of germline (sperm) and somatic DNA showed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of 2 x 10(-7) and 3 x 10(-7), respectively, in the individuals tested. Such re-arrangements at these loci therefore occur at a rate lower than that detectable by the most sensitive methods currently available.

Alu Elements↗

PubMed2000

Dramatic, expansion-biased, age-dependent, tissue-specific somatic mosaicism in a transgenic mouse model of triplet repeat instability.

Myotonic dystrophy type 1 (DM1) is one of a growing number of inherited human diseases whose molecular basis has been implicated as the expansion of a trinucleotide DNA repeat. Expanded disease-associated alleles of >50 CTG repeats are unstable in both the germline and soma. Expansion of the unstable alleles over time and variation of the level of mutation between the somatic tissues of an individual are thought to account at least partially for the tissue specificity and progressive nature of the symptoms. We previously generated a number of transgenic mouse lines containing a large expanded CTG repeat tract that replicated a number of the features of unstable DNA in humans, including frequent sex-specific changes in allele length during intergenerational transmission. Small length change mutations were apparent in the somatic tissues of young mice in all of the lines generated, but the gross instability observed in human DM1 patients was not replicated. We now show that in one of the lines, Dmt -D, spectacular, expansion-biased, tissue-specific instability is observed in older mice. The highest levels of instability were detected in kidney with gains of >500 repeats, representing a tripling of allele length, in some cells. Mosaicism accumulated in an age-dependent manner, but the tissue specificity did not obviously correlate with cell turnover. Such gross somatic mosaicism was not observed in three other lines examined, further emphasizing a role for flanking DNA in modulating repeat stability.

Age Factors↗

PubMed2000

Frequency of minisatellite repeat number changes at the MS205 locus in human sperm before and after cancer chemotherapy.

To determine whether the measurement of repeat number mutations at a minisatellite locus could detect human germline mutations induced by chemotherapy, we performed a longitudinal study of the mutation frequencies in sperm from 10 patients treated for Hodgkin's disease. Polymerase chain reaction on small pools of DNA equivalent to 100 sperm and Southern blotting were used to screen at least 7900 sperm in each sample to quantify the mutation frequency at the minisatellite MS205 locus. Pretreatment and posttreatment semen samples were obtained at least 2 months after completion of therapy from 4 patients treated with a regimen (Novantrone, Oncovin, vinblastine and prednisone [NOVP]) that lacks alkylating agents and from three patients treated with regimens (Cytoxan, vinblastine, procarbazine and prednisone/Adriamycin, bleomycin, dacarbazine, lomustine, and prednisone [CVPP/ABDIC] or mechlorethamine, Oncovin, procarbazine and prednisone [MOPP]) containing alkylating agents. There were no effects of NOVP or CVPP/ABDIC on the mutation frequencies. In the 1 patient treated with MOPP, the treatment with the highest dose of gonadotoxic alkylating agents, there was a statistically significant increase in mutation frequency from 0.79% pretreatment to 1.14% posttreatment, indicating induction of mutations in stem spermatogonia. During-treatment semen samples obtained from 2 patients treated with ABVD, which does not contain gonadotoxic alkylating agents, and 1 with NOVP also did not show any increases above the baseline mutation frequencies, indicating no increase in the minisatellite mutation frequency in spermatocytes. Thus, measurement of repeat number changes at minisatellite MS205 appears to be able to detect induced germline mutations in human sperm. However, most chemotherapy regimens do not significantly increase this class of mutations.

Adult↗

PubMed2000

Complex patterns of male germline instability and somatic mosaicism in myotonic dystrophy type 1.

The genetic basis of myotonic dystrophy type 1 (DM1) is the expansion of a CTG repeat in the 3' untranslated region of DM1PK. Once into the disease range, the repeat becomes highly unstable and is biased toward expansion in both somatic and germline tissues. Intergenerational differences usually reveal an increase in allele length, concordant with the clinical anticipation characteristic of DM1, but there have also been cases with intergenerational contractions of the repeat length, accompanied by apparent anticipation. In order to gain a better understanding of this intergenerational behaviour, we have obtained semen samples from six DM males and used single molecule analyses to compare the allele distributions present in their sperm and blood with those of their offspring. We have confirmed that the male germline mutational pathway is distinct from that of the soma, but the extent of variation is highly variable from one individual to another and not obviously correlated with progenitor allele length. Nonetheless, in all cases the alleles present in the father's sperm overlap with those observed in their offspring. These data also provide further indications that the interpretation of intergenerational transmissions by standard analyses is frequently compromised by the masking of germline differences by age-dependent somatic expansion in the parent.

Adolescent↗

PubMed1999

Very large (CAG)(n) DNA repeat expansions in the sperm of two spinocerebellar ataxia type 7 males.

Genetic anticipation, i.e. increasing disease severity and decreasing age of onset from one generation to the next, is observed in a number of diseases, including myotonic dystrophy type 1, Huntington's disease and several of the spinocerebellar ataxias. All of these disorders are associated with the expansion of a trinucleotide repeat and array length is positively correlated with disease severity and inversely correlated with the age of onset. The expanded repeat is highly unstable and continues to expand from one generation to the next, providing a molecular explanation for anticipation. Spinocerebellar ataxia type 7 (SCA7) is one of the latest additions to the list of triplet repeat diseases and is distinct from the other SCAs in that it is accompanied by retinal degeneration. Pedigree analyses have previously revealed that the SCA7 repeat is highly unstable and liable to expand, in particular when transmitted by a male. Surprisingly, though, an under-representation of male transmission has also been reported. We now demonstrate directly by single molecule analyses that the expanded repeat is extraordinarily unstable in the male germline and biased toward massive increases. Nearly all of the mutant sperm of two SCA7 males contain alleles that are so large that most of the affected offspring would at best have a severe infantile form of the disease. Indeed, the gross under-representation of such very large expanded alleles in patients suggests that a significant proportion of such alleles might be associated with embryonic lethality or dysfunctional sperm.

Alleles↗

PubMed1999

Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands.

An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with a phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels of instability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis -acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats.

Base Composition↗

PubMed1998

Progression of somatic CTG repeat length heterogeneity in the blood cells of myotonic dystrophy patients.

The genetic basis of myotonic dystrophy (DM) is the expansion of an unstable CTG repeat in the 34 UTR of the DM protein kinase gene on chromosome 19. One of the principal features of the DM mutation is an extraordinarily high level of somatic mosaicism, due to an extremely high degree of somatic instability both within and between different tissues. This instability appears to be biased towards further expansion and continuous throughout the life of an individual, features that could be associated with the progressive nature of the disease. Although increasing measured allele size between patients clearly correlates with an increased severity of symptoms and an earlier age of onset, this correlation is not precise and measured allele length cannot be used as an accurate predictor of age of onset. In order to further characterize the dynamics of DM CTG repeat somatic instability, we have studied repeat length changes over time in 111 myotonic dystrophy patients with varying clinical severity and CTG repeat size over time intervals of 1-7 years. We have found a direct progression of the size heterogeneity over time related to initial CTG repeat size and the time interval and always biased towards further expansion. Attempts to mathematically model the dynamics have proved only partially successful suggesting that individual specific genetic and/or environmental factors also play a role in somatic mosaicism.

Adult↗

PubMed1997

Hypermutable myotonic dystrophy CTG repeats in transgenic mice.

Myotonic dystrophy (DM) is one of a growing number of inherited human disorders associated with the expansion of triplet repeat DNA sequences. Expanded alleles are highly unstable in both the germline and soma, accounting in large part for the unusual genetics of this disorder, its phenotypic variability and probably, the progressive nature of the symptoms. However, the molecular mechanisms and the genetic factors modulating repeat stability in DM and the other human disorders associated with expanded repeats are not well understood. To provide a model system in which the turnover of triplet repeats could be studied throughout mammalian development, we have generated five transgenic mouse lines incorporating expanded CTG/CAG arrays derived from the human DM locus. Transgene analysis has revealed germline hypermutability, including expansions, deletions and parent-of-origin effects, somatic and early embryonic instability and segregation distortion. Mutational differences between lines and sexes demonstrate that stability, as in humans, is modulated by as yet unidentified cis and trans acting genetic elements.

Animals↗

PubMed1996

Instability of the expanded (CTG)n repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy.

The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG)n, in the 3'-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG)n repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10(6) cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG)n repeat region. Two types of mutations of the expanded (CTG)n repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG)n repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG)n repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the somatic heterogeneity of the (CTG)n repeat size observed in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG)n repeats in DM.

Alleles↗

PubMed1996

Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase.

Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features. Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A). The 167-amino-acid human PTP4A bears the signature active site of all PTPs, but does not show any other sequence homology to any of the previously described PTPs. Two cDNAs encoding PTP4A that differed in their noncoding regions were isolated. Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated. Northern analysis using a probe from a common 3'-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb. Both species of mRNA were seen in all human adult and fetal tissues tested. Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11. A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs. Our studies also suggest the existence of another processed pseudogene on chromosome 11.

Adult↗

PubMed1995

Unstable triplet repeat diseases.

Seven inherited human disorders are now associated with the intragenic expansion of triplet repeat DNA sequences. These repeats demonstrate extreme instability in both germline and somatic tissue, accounting for the unusual genetic inheritance patterns and symptom variability associated with these diseases.

Brain Diseases↗

PubMed1995

Myotonic dystrophy: an unstable CTG repeat in a protein kinase gene.

Myotonic dystrophy (DM) is caused by the amplification of CTG repeats in the 3' untranslated region of a gene encoding a protein homologous to serine/threonine protein kinases. In DM patients the CTG repeats are extremely unstable, varying in length from patient to patient and generally increasing in length in successive generations. There is a strong correlation between the size of the repeats and the age of onset and severity of the disease. The molecular basis of the effect of the CTG expansion on the development of the DM phenotype continues to be investigated. The first working hypothesis of the molecular mechanism of DM was a reduction in steady-state myotonin-protein kinase (Mt-PK) mRNA and protein levels. However, although the consensus finding is that the Mt PK mRNA and protein levels are decreased in DM patients, it is still not clear if this reduction leads directly to the DM phenotype. In this short review we discuss the molecular aspects of CTG instability and the expression of the myotonin-protein kinase gene in normal and DM populations.

DNA↗

PubMed1995

Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses.

In order to characterize the dynamics of CTG repeat instability in somatic and germline tissue from myotonic dystrophy (DM) males we have used small pool polymerase chain reaction (PCR) in a detailed quantitative analysis of repeat length variation. We demonstrate that the heterogeneous smear of CTG repeats observed in DM patients using standard analyses is comprised of multiple unresolved bands that may be dissected into discrete length alleles derived from single cells using single molecule PCR techniques. Analysis of somatic tissues demonstrates a bias toward increasing allele length and a lower boundary below which variant alleles are rare, consistent with a highly directional expansion pathway in the soma. Two sperm samples show extensive variation and a size increase bias, concordant with the phenomenon of anticipation. In addition, sperm analysis shows that large contractions, including reversions into the normal size range, are restricted to the germline. Detailed analysis of intergenerational 'reductions' paternally transmitted to two offspring suggests that some apparent reductions may be artifacts of somatic expansion in the parent. Our data indicate that in addition to germline variation, substantial somatic expansion can also contribute to the intergenerational differences usually observed in DM.

Adult↗

PubMed1995

Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent.

The most common form of adult muscular dystrophy, myotonic dystrophy (DM), is caused by the abnormal expansion of the CTG repeat, located in the 3' UTR of the DM gene. The expanded-CTG allele often presents as a diffused band on Southern blot analysis, suggesting somatic mosaicism. In order to study the somatic instability of the CTG repeat, we have investigated the dynamics of the size heterogeneity of the CTG expansion. Size heterogeneity is shown as a smear on Southern blot and is measured by the midpeak-width ratio of the expanded allele to the normal sized allele. The ratio is also corrected for compression in the higher-molecular-weight region. It is found that the size heterogeneity of the expanded-CTG repeats, of 173 DM patients, correlates well with the age of the patient (r = .81, P << .001). The older patients show larger size variation. This correlation is independent of the sex of either the patient or the transmitting parent. The size heterogeneity of the expansion, based on age groups, is also dependent on the size of the expanded trinucleotide repeat. However, obvious size heterogeneity is not observed in congenital cases, regardless of the size of expansion. Comparison of individual patient samples collected at two different times has confirmed that the degree of size heterogeneity increases with age and has revealed a subtle but definite upward shift in the size of the expanded-CTG allele. The progression of the CTG repeat toward larger expansion with age is further confirmed by small-pool PCR assay that resolved the heterogeneous fragments into discrete bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1994

Minisatellite isoalleles can be distinguished by single-stranded conformational polymorphism analysis in agarose gels.

Minisatellite isoallelism, i.e. the occurrence of minisatellite alleles with different internal sequence composition but indistinguishable length, is a common limitation of minisatellite allele length analysis. Internal sequence variation can be used to distinguish such isoalleles, provided that detailed sequence knowledge of its basis is available. We now show that minisatellite isoalleles can also be simply resolved by single-stranded conformational polymorphisms (SSCP) arising during agarose gel electrophoresis. SSCP on agarose gels can be used to distinguish minisatellite isoalleles either after PCR amplification, or by standard Southern blot analysis of genomic DNA.

Alleles↗

PubMed1994

Complex gene conversion events in germline mutation at human minisatellites.

Mutation at the human minisatellites MS32, MS205 and MS31A has been investigated by characterizing mutant alleles in pedigrees and in the case of MS32 by direct analysis of mutant molecules in single sperm. Most mutations at all three loci are polar, involving the preferential gain of a few repeat units at one end of the tandem repeat array. Incoming repeats can be derived from the same allele or the homologous chromosome, through they are frequently rearranged during mutation. Lack of exchange of flanking markers suggests the involvement of complex conversion-like events in the generation of mutant alleles. At MS32, high frequency mutation processes in sperm appear to be largely germline specific and to occur at a constant rate irrespective of allele size. Together with mutational polarity, this implies that germline instability is controlled by elements outside the tandem repeat array.

Alleles↗