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Mutations in the DNA mismatch repair (MMR) genes are associated with the inheritance of hereditary non-polyposis colorectal cancer, also known as Lynch syndrome, a cancer syndrome with an average age at onset of 44. Individuals presenting with colorectal cancer are diagnosed with Lynch I, whereas individuals who present with extra-colonic tumors (such as endometrial, stomach, etc.) are identified as patients with Lynch syndrome II. Recently, 30 families have been reported with inheritance of biallelic mutations in the MMR genes. Here we summarize the phenotype of individuals with inheritance of homozygous or compound heterozygous mutations in the MMR genes that result in a complete lack of protein or greatly compromised protein function. In contrast to individuals with Lynch syndrome I and II, individuals with no MMR function present with childhood onset of hematological and brain malignancies, whereas residual MMR function can also result in gastrointestinal cancers and an age of onset in the second to fourth decade. Individuals with biallelic MMR mutations often present with café-au-lait spots, regardless of the level of MMR function remaining. Thus, the inheritance of two MMR gene mutations is a separate entity from Lynch I or II or the subtypes Turcot and Muir-Torre.

DNA mismatch repair (MMR) deficiency is associated with an increased mutational burden and predisposition to certain malignancies. Relatively little is known, however, about gene-specific mutation frequencies within MMR-deficient primary tumors. Thymic lymphomas from Msh2 −/− mice were thus analyzed by using a lacI -based transgenic shuttle-phage mutation detection system. All tumors exhibited greatly elevated lacI gene mutation frequencies, ranging from 3.2- to 17.4-fold above the ≈15-fold elevations present within normal Msh2 −/− thymi. In addition, lacI genes harboring multiple changes, including clusters of mutations, were found in thymic tumor DNA. The results suggest that an additional mutator activity, such as an error-prone DNA polymerase, leads to increased genomic instability in these MMR-deficient tumors.

The mutational response of mismatch repair-deficient animals to the alkylating agent N -methyl- N -nitrosourea was evaluated by using a transgenic lacI reporter system. Although the mutations detected in MSH2 heterozygotes were similar to those of controls, MSH2 −/− animals demonstrated striking increases in mutation frequency in response to this agent. G:C to A:T transitions at GpG sites, as opposed to CpG sites, dominated the mutational spectrum of both MSH2 +/+ and MSH2 −/− N -methyl- N -nitrosourea -treated animals. Extrapolating to humans with hereditary non-polyposis colorectal cancer, the results suggest that MSH2 heterozygotes are unlikely to be at increased risk of mutation, even when exposed to potent DNA methylating agents. In contrast, mismatch repair-deficient cells spontaneously arising within individuals with hereditary non-polyposis colorectal cancer would likely exhibit hypermutability in response to such mutagens, an outcome predicted to accelerate the pace of tumorigenesis.

Tumors derived from individuals with hereditary non-polyposis colorectal cancer syndrome frequently demonstrate mutations in both alleles of hMSH2, a key gene in DNA mismatch repair (MMR). Sporadic tumors also frequently exhibit MMR deficiency. In keeping with the role of MMR in the maintenance of genome integrity, mice deficient in MSH2 via gene targeting demonstrate a high incidence of thymic lymphomas and small intestinal adenocarcinomas. To investigate the effects of MSH2 deficiency in normal tissues, mice containing a retrievable transgenic lacI reporter gene for mutation detection were crossed with MSH2−/− mice. Mice homozygous for MSH2 deficiency revealed 4.8, 11.0 and 15.2-fold elevations in spontaneous mutation frequency in DNA obtained from brain, small intestine, and thymus, respectively, as compared to heterozygous or wild-type mice. Mutations most frequently recovered from MSH2−/− mice were single base substitutions (77%), particularly base transitions (64%). Frameshifts occurred less frequently (19%) and fell within very short (3 – 5 bp) mononucleotide runs. Thus the number of key growth control genes potentially impacted by MMR deficiency extends beyond those containing repetitive sequences. These results highlight the capacity for MSH2 deficiency to serve as a potent driving force during the multi-step evolution of tumors.

The gene for Huntington disease (HD) has been localized close to the telomere on the short arm of chromosome 4. However, refined mapping using recombinant HD chromosomes has resulted in conflicting findings and mutually exclusive candidate regions. Previously reported significant nonrandom allelic association between D4S95 and HD provided support for a more proximal location for the defective gene. In this paper, we have analyzed 17 markers, spanning approximately 6 Mb of DNA distal to locus D4S62, for nonrandom association to HD. We confirm the previous findings of nonrandom allelic association between D4S95 and HD. In addition, we provide new data showing significant nonrandom association between HD and 3 markers at D4S133 and D4S228, which are approximately 3 Mb telomeric to D4S95.

Hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch syndrome types I and II, and the related subtypes Turcot and Muir-Torre syndrome, have all been associated with inheritance of germ line mutations in the DNA mismatch repair (MMR) genes. Fifty individuals have recently been identified with an early onset of a different spectrum of cancers associated with inheritance of two MMR mutations - resulting either in a constitutive loss of MMR function, or greatly impaired MMR function. In contrast to Lynch I and II individuals, individuals with inheritance of homozygous or compound heterozygous mutations in the MMR genes that result in a complete lack of protein, present with hematological and brain malignancies in the first decade of life. Biallelic mutations with compromised but residual protein function present with a broader spectrum of cancers (brain, hematological or gastrointestinal) in the second to fourth decades of life. We propose that inheritance of two MMR mutations in an individual and the unique tumor spectrum that occurs with an early onset should be defined separately from Lynch syndrome I and II, or the subtypes Turcot and Muir-Torre. We suggest Lynch III as an appropriate name for identifying individuals with constitutively compromised MMR associated with biallelic mutations.

At least 12 disorders including Huntington disease (HD) are associated with expansion of a trinucleotide repeat (TNR). Factors contributing to the risk of expansion of TNRs and the mechanism of expansion have not been elucidated. Data from Saccharomyces cerevisiae suggest that the flap endonuclease FEN1 plays a role in expansion of repetitive DNA tracts. It has been hypothesized that insufficiency of FEN1 or a mutant FEN1 might contribute to the occurrence of expansion events of long repetitive DNA tracts after polymerase slippage events during lagging strand synthesis. The expression pattern of FEN1 was determined, and ubiquitous tissue expression, including germ cells, suggested that FEN1 has the potential to be involved in HD. Fifteen HD parent/child pairs that demonstrated intergenerational increases in CAG length of greater than 10 repeats were examined for possible mutations or polymorphisms within the FEN1 gene that could underlie the saltatory repeat expansions seen in these individuals. No alterations were observed compared to 50 controls, excluding FEN1 as a trans-acting factor underlying TNR expansion. The identification of a candidate gene(s) in HD or other CAG-expansion disorders implicated in TNR instability will elucidate the mechanism of expansion for this growing family of neurological disorders.

DNA mismatch repair (MMR) deficiency leads to an increased mutation frequency and a predisposition to neoplasia. 'Knockout' mice deficient in the MMR proteins Msh2 and Pms2 crossed with mutation detection reporter (supF, lacI and cII) transgenic mice have been used to facilitate a comparison of the changes in mutation frequency and spectra. We find that the mutation frequency was consistently higher in Msh2-deficient mice than Pms2-deficient mice. The lacI target gene, which is highly sensitive to point mutations, demonstrated that both Msh2- and Pms2-deficient mice accumulate transition mutations as the predominant mutation. However, when compared with Msh2(-/-) mice, lacI and cII mutants from Pms2-deficient mice revealed an increased proportion of +/-1 bp frameshift mutations and a corresponding decrease in transversion mutations. The supF target gene, which is sensitive to frameshift mutations, and the cII target gene revealed a strong tendency for -1 bp deletions over +1 bp insertions in Msh2(-/-) compared with Pms2(-/-) mice. These data indicate that Msh2 and Pms2 deficiency have subtle but differing effects on mutation avoidance which may contribute to the differences in tumor spectra observed in the two 'knockout' mouse models. These variances in mutation accumulation may also play a role, in part, in the differences seen in prevalence of MSH2 and PMS2 germline mutations in hereditary non-polyposis colorectal cancer patients.

We reported previously that thymic lymphomas arising in mice lacking the DNA mismatch repair (MMR) gene, Msh2(-/-), exhibited striking elevations in the mutation frequency of a transgenic lacI reporter gene when compared with normal Msh2(-/-) tissues. To investigate whether hypermutation was a feature of all tumors arising in MMR-deficient mice, lacI transgene mutation frequencies were obtained from several different mouse tumors deficient for PMS2 and/or MSH2. While lacI gene hypermutation was again clearly evident in Msh2 +/- ms2(-/-) and Msh2(-/-)Pms2(-/-) thymic lymphomas, three non-thymic MSH2-deficient tumors failed to show lacI gene mutation frequency elevations when compared with a normal tissue of MMR-deficient mice. The elevated mutation frequencies in the lymphoid tumors, and the finding of multiple clustered mutations in lacI genes rescued from these tumors, suggest that they are possibly generated by a lymphoma-specific hypermutational mechanism.

INTRODUCTION Disorders caused by triplet expansions present unique challengesfor understanding and correlating the genotype with the clinicalphenotype. Currently, there is some confusion over ranges fornormal and disease alleles and regarding understanding ofpenetrance and intermediate alleles. Various centres have adopteddifferent definitions, resulting in varying interpretations of allelesizes and their relationship to phenotype by a particular age.Furthermore, the observation in some trinucleotide repeat (TNR)diseases of new mutations arising from ‘intermediate’ sizedalleles has resulted in different interpretations over what is a‘normal’ and what is a ‘disease’ allele. This has also beenaccompanied by an emerging recognition that many factors,including genetic changes outside of the causative gene andpotentially even environmental features, might influence theseclassical ‘single gene’ disorders. We have collected data from >80peer-reviewed manuscripts on the seven disorders associated withexpansion of a CAG repeat, as well as consulted with experts inthe field on unpublished data, in order to define the normal anddisease range of alleles, determine the zone of reduced pene-trance, if any, and define the size range for intermediate sizedalleles proven molecularly to expand to a CAG size in the diseaserange. Here we present an approach for the interpretation of allelesizes and their relationship to a phenotype for this class ofdisorders.CAG SIZE RANGES ASSOCIATED WITH DISEASEThe ranges of allele sizes in the seven disorders is based on the totalnumber of individuals, normal and affected, that have been assessed.This in turn is influenced by the frequency of the disorder as well as,to a lesser extent, the length of time since the gene has been cloned(Table 1) (1–88). The affected range is defined as the range of CAGsdetected in persons with the clinical phenotype of a particulardisorder caused by mutations in the gene in question. The dataavailable to date are shown in Figure 1 (1–88). For spinocerebellarataxia type 1 and 2 (SCA1, SCA2), spinal and bulbar muscularatrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA) andespecially spinocerebellar ataxia type 6 (SCA6), the small numbersof affected chromosomes examined means that the ranges will likelychange at their margins, as more individuals are assessed (Table 1).Except for SCA6, the similarity between the ranges of disease(>40 CAGs) and normal size alleles at each of the seven TNRdiseases suggests a common mechanism of expansion. However,the interval between normal and disease range can also be usedto divide these seven disorders into two groups. For example,SBMA, SCA6, DRPLA and SCA3 clearly have mutuallyexclusive normal and disease range CAG alleles. On the otherhand, there is no obvious gap between normal and disease rangesfor Huntington’s disease (HD), SCA1 and SCA2 (Fig. 1).PENETRANCE, INDIVIDUALITY AND DISEASEDespite our extensive genetic variability and exposure to differentenvironments, it is remarkable that, for all these disorders, thatCAG size is associated so strongly with age of onset—a criteraitself subject to significant differences in interpretation. A diseasegene has reduced penetrance when a fraction of patients do notmanifest the disorder despite inheriting a known disease-causingmutation (89). However, for many individuals with TNRdiseases, the onset of illness is often late and highly variable. Thuspenetrance is age-dependent. In these instances it is appropriateto define reduced penetrance as occurring if persons haveinherited a mutation in the disease range but lived beyond theexpected lifespan without manifesting any signs of the disease.Reduced penetrance is the one end of the spectrum ofexpression of a disease-causing mutation and is clear evidence ofhow unique individuals accommodate to a particular mutation inthe context of the whole genome, environment and experience.The delineation of the molecular basis of these diseases hasallowed new and definitive approaches to assessment ofpenetrance in this class of disorders. For all of the CAG TNRdiseases combined, the reported instances of non-penetrance arefew, given the large number of individuals molecularly assessed(8,30,44,90–92).

This study was planned to determine the number of origins of the mutation underlying Huntington's disease (HD) in Sweden. Haplotypes were constructed for 23 different HD families, using six different polymorphisms [(CCG)n, GT70, 674, BS1, E2 and 4.2], including two within the gene. In addition, extensive genealogical investigations were performed, and the geographical origin of the haplotypes was studied. Ten different haplotypes were observed suggesting multiple origins for the HD mutation in Sweden. Analysis of the two polymorphic markers within the HD gene (the CCG repeat and GT70) indicates that there are at least three origins for the HD mutation in Sweden. One of these haplotypes (7/A) accounts for 89% of the families, suggesting that the majority of the Swedish HD families are related through a single HD mutation of ancient origin. Furthermore, three of the families that were previously considered to be unrelated could be traced to a common ancestor in the 15th century, a finding that is consistent with this hypothesis.

Huntington disease (HD) has been shown to be associated with an expanded CAG repeat within a novel gene on 4p16.3 (IT15). A total of 30 of 1,022 affected persons (2.9% of our cohort) did not have an expanded CAG in the disease range. The reasons for not observing expansion in affected individuals are important for determining the sensitivity of using repeat length both for diagnosis of affected patients and for predictive testing programs and may have biological relevance for the understanding of the molecular mechanism underlying HD. Here we show that the majority (18) of the individuals with normal sized alleles represent misdiagnosis, sample mix-up, or clerical error. The remaining 12 patients represent possible phenocopies for HD. In at least four cases, family studies of these phenocopies excluded 4p16.3 as the region responsible for the phenotype. Mutations in the HD gene that are other than CAG expansion have not been excluded for the remaining eight cases; however, in as many as seven of these persons, retrospective review of these patients' clinical features identified characteristics not typical for HD. This study shows that on rare occasions mutations in other, as-yet-undefined genes can present with a clinical phenotype very similar to that of HD.

Huntington's disease (HD) is associated with expansion of a CAG repeat in a new gene. We have recently defined a premutation in a paternal allele of 30 to 38 CAG repeats in the HD gene which is greater than that seen in the general population (< 30 repeats) but below the range seen in patients with HD (> 38). These intermediate alleles are unstable during transmission through the germline and in sporadic cases expand to the full mutation associated with the clinical phenotype of HD. Here we have analysed three new mutation families where, in each, the proband and at least one sib have CAG sizes in the HD range. In one of these families, two sibs with expanded CAG repeats are both clinically affected with HD, thus presenting a pseudorecessive pattern of inheritance. In all three families the parental intermediate allele has expanded in more than one offspring, thus showing a previously unrecognised risk of inheriting HD to sibs of sporadic cases of HD.

Alu repetitive elements represent the most common short interspersed elements (SINEs) found in primates, with an estimated 500,000 members in the haploid human genome. Considerable evidence has accumulated that these elements have dispersed in the genome by active transcription followed by retroposition, and that this process is ongoing. Sequence variation between the individual elements has lead to the hierarchical classification of Alu repeats into families and subfamilies. Young subfamilies that are still being actively transposed are of considerable interest, and the identification of one such subfamily (designated 'PV') has lead to the hypothesis that the most recent retroposition events are due to a single master Alu source gene. In the course of our search for the gene causing Huntington disease, we have detected an Alu retroposition event in two families. Sequence analysis demonstrates that this Alu element is not a member of the PV subfamily, but is similar to 5 other Alu elements in the GenBank database. Together, these Alu elements, all of which contain a 7 base-pair internal duplication, define a distinct subfamily, designated as the Sb2 subfamily, providing evidence for a second actively retroposing Alu source gene. These data provide support for multiple source genes for Alu retroposition in the human genome.

As part of the search for the Huntington disease (HD) gene we have cloned and sequenced 34 kb of genomic DNA containing the full-length gene for the beta-subunit of the human cGMP phosphodiesterase (beta-cGMP PDE). This gene is localized to 4p16.3 about 700 kb proximal to the 4p telomere and represents the most telomeric gene characterized on 4p to date. We show that this gene is comprised of 22 exons spanning approximately 43 kb of genomic DNA. We also provide 400 bp immediately 5' to the putative initiator methionine and 700 bp of 3' flanking sequences. Northern blot analysis of several human tissues revealed a highly abundant 3.5 kb transcript and a minor signal of 4.5 kb in retinal tissue. Alignment of the deduced amino acid sequence to the previously identified beta-subunits of the cGMP PDEs of mouse and cow demonstrates highly significant similarities and, therefore, confirms the identity of the cloned gene. A defect in the beta-subunit of the cGMP PDE gene has been shown recently to be the cause for the retinal degeneration in the rd mouse. The cloning of the human homolog and the knowledge of its genomic organization with exon/intron boundaries will allow rapid assessment of the role of this gene in the causation of human retinopathies.

Late onset Huntington's disease is characterised by onset of symptoms after the age of 50 and is usually associated with a milder course. We have analysed the CAG trinucleotide repeat within the HD gene in 133 late onset patients from 107 extended families. The median upper allele size for the CAG repeat was 42 with a range of 38 to 48 repeats. A significant negative correlation (r = -0.29, p = 0.001) was found between the length of repeat and age of onset for the total cohort. However, for persons with age of onset greater than 60, no significant correlation was found. In addition, no significant correlation was found between age of onset and size of the lower allele and the sex of the affected parent or grandparent. There was no preponderance of maternal descent for late onset cases in this series. This study shows that variation in repeat length only accounts for approximately 7% of the variation in age of onset for persons beyond the age of 50 and clearly shows how with increasing onset age the effect of the repeat length on this onset age seems to diminish.