Genetics

3.1. REPEAT INSTABILITY Each of the above-described disorders is characterized by the presence of a trinucleotide repeat within the gene responsible for that disorder. For fragile X syndrome and SCA12, the trinucleotide repeat is localized to the 5' untranslated region and for DM I and SCA8 it is present in the 3' untranslated region. For SBMA, HD, SCA1, SCA2, SCA3/MJD, SCA6, SCA7, and DRPLA, the repeat is within the coding region. In 96% of patients with Friedreich ataxia, the repeat is located within an intron with approx 4% of the remaining affected patients, presenting as compound heterozygotes for the intronic GAA expansion on one allele and a point mutation within the Frataxin (FRDA) gene on the other allele. Common to each of these disorders is that the trinucleotide repeat is polymorphic within the normal population, with alle-les inherited stably from one generation to another (10). Also, in each of these disorders, expansion of the repeat beyond the normal range results in either abnormal gene function or abnormal levels of gene product and, ultimately, disease.

In the disease state, the trinucleotide repeat for each of these disorders demonstrates instability when transmitted from parents to offspring. Expansion of unstable trinucleotide repeats during transmission is most often the case, although contractions have also been documented. In general, instability of trin-ucleotide repeats is directly related to their size (i.e., the longer the repeat, the more likely it is to undergo expansion) (10-12). This is particularly well documented for FRAXA and DM I. Stability of repeats also appears to be related to the primary sequence. In FRAXA and SCA1, for example, the trinucleotide repeat sequences are not perfect sequences, but are interrupted: interspersed AGG (instead of CGG) for Fragile X syndrome and one to three CAT repeats within the CAG repeat for SCA1 (13,14). Loss of these interruption sequences appears to render the resulting repeat susceptible to greater instability with minimal expansion. For SCA1, the age of onset is determined by the number of uninterrupted CAG repeats. The presence of one or more CAT interruptions within an expanded allele have been associated with a milder phenotypic presentation as well as a later age of onset. However, when a repeat reaches a critical size threshold, the repeat becomes very unstable regardless of interruptions. For example, a repeat size of 100 or greater in FRAXA almost always leads to a full mutation (>200) in subsequent generations (15). Recent studies indicate that the loss of AGG interruptions leading to an expansion of premutations to full mutations occurs as one of the final events in the expansion process that is observed in Fragile X families (16).

3.2. GENOTYPE AND PHENOTYPE CORRELATION For each of the trinucleotide repeat disorders, there is a correlation between increasing repeat size and disease severity (17,18). Anticipation (worsening of disease severity and decreasing of age of onset over successive generations) is well documented and correlates with increasing expansion size (10,11,17-19). For example, both congenital DM patients and the most severely retarded FRAXA syndrome patients nearly always have dramatic repeat expansions, whereas those with smaller expansions typically have milder disease (10,11,20). Similarly, in individuals with HD, larger repeats are associated with prominent atrophy at the head of the caudate, a region of brain responsible for movement integration (21). In case of the CAG repeat disorders, increasing repeat length correlates with earlier disease onset (17-19,22,23). This correlation is strongest for SCA1, where approx 70% of the variability in age of disease onset is accounted for by repeat length (22). In Friedreich ataxia, the presence of two expanded alleles as a result of the autosomal recessive nature of this disease precludes the occurrence of anticipation as seen in other dominantly inherited autosomal or X-linked trinucleotide repeat disorders. However, the age of onset and severity of disease correlate with the size of the smaller of the two repeats (24). Although strong correlations exist in all triplet repeat diseases, other factors apparently also influence the severity and age onset of the diseases (17,20,23). For example, in SBMA and HD, it has been reported that affected siblings with very similar repeat lengths have had onset of symptoms at very different ages (17,23). Similarly, several patients with Friedreich ataxia show a later age of onset or retained reflexes despite a similar distribution of repeat sizes (25,26). Thus, generally, a larger triplet repeat size correlates with more severe disease, although significant but unidentified genetic/environmental modifiers might also play a role in severity.

Fragile X syndrome, FRDA, and DM I also have repeat sizes considered to be in a "premutation" range. The premutation, which is intermediate in size between the normal repeat and "full mutation" size ranges, usually causes minimal (if any) phenotypic abnormalities. However, the premutation is unstable and often leads to further expansion and full phenotypic expression in the subsequent generation (11). In addition, there is evidence for the existence of a "gray zone" in which normal and abnormal repeat sizes might overlap. The stability of repeats in this range differs between families and, thus, requires evaluation of multiple generations before repeat stability can be determined. Furthermore, stability of gray zone repeats can be affected by interruption of the trinucleotide repeat sequence, as discussed in Section 3.1.

3.3. BIAS OF PARENTAL TRANSMISSION Interestingly, parental bias has been observed with respect to trinucleotide repeat expansion in subsequent generations. For several disorders (SBMA, HD, SCA1, SCA2, SCA3/MJD, SCA7, and DRPLA), paternal transmission of an abnormal allele often produces expansions that are relatively large, whereas maternal transmission might result in expansions of only a few repeats (2,19). Large expansions of the CAG repeat in a paternal transmission, from 43 to over 200 and from 49 to over 200, have been observed in the case of infantile onset SCA2 and SCA7, respectively (27,28). These observations might reflect the potential for high repeat instability during spermatogenesis. Affected males are therefore more likely to transmit a greatly expanded repeat that could cause juvenile-onset disease. On the other hand, the untranslated CTG and CGG repeats of DM I, FRAXA, and SCA8 tend to have maternal bias of transmission (29,30). Almost all cases of congenital myotonic dystrophy are maternally transmitted, and in FRAXA, the expansion from premutation to full mutation occurs through a oogenesis. In Friedrich ataxia, recent findings suggest that the expanded

GAA alleles are likely to expand further during maternal transmission (31). A uniform mechanism responsible for transmission bias of trinucleotide repeat diseases has not yet been elucidated. Prezygotic selection, at the level of DNA replication, against full mutation carrying sperm from premutation males has been proposed as leading to the observed maternal bias in transmission in case of Fragile X syndrome (32).

3.4. MOLECULAR MECHANISMS The eight CAG repeat neurodegenerative disorders (SBMA, HD, SCA1, SCA2, SCA3/MJD, SCA6, SCA7, and DRPLA) are caused by modest expansions of CAG repeats that are subsequently translated into enlarged polyglutamine tracts (2,4). It has been proposed that these mutations result in an abnormal protein that is directly responsible for the observed neuronal toxicity. This "gain of function" hypothesis is supported by the finding that patients with mutations other than (CAG) repeat expansions within the androgen receptor gene results in phenotypes (i.e., testicular feminization and androgen insensitivity syndrome but no weakness and neurodegeneration) distinct from the SBMA phenotype (33). Additionally, the "gain of function" hypothesis is consistent with the dominant pattern of inheritance observed for these diseases.

Altered protein function is unlikely to be the underlying mechanism for those trinucleotide repeat diseases in which the repeat is not translated (FRAXA, DM I, and Friedreich ataxia). Rather, (CGG)n repeat expansion in the FMR-1 gene is associated with decreased mRNA and protein levels. Thus, a CGG repeat expansion greater than 200 repeats in length is associated with increased DNA methylation of an adjacent CpG island in the FMR-1 promotor region. Additionally, deacetyla-tion of histones by histone deacetylases recruited in the vicinity of the hypermethylated FMR-1 promotor region has been shown to result in chromatin condensation and transcriptional silencing of the FMR1 gene leading to decreased protein levels (34). Somatic mosaicism for FMR-1 methylation in leukocytes with fully expanded CGG repeats has been reported in high functioning Fragile X males with borderline or no mental retardation. However, because of the lack of available information on the methylation status of FMR-1 locus and FMRP expression level in the brain of these individuals, it has not been possible to extrapolate the findings of methylation mosaicism in leukocyte DNA to the severity of cognitive deficit. The appearance of a fully methylated Fragile X mutation, greater than 200 repeats, is always preceded by transmission of an unmethylated premutation, usually between 55 and 200 repeats. Premutation carriers have minimal (if any) phenotypic abnormalities. Therefore, it is likely that the decreased transcription of the FMR-1 gene in cases of fully methylated Fragile X repeat expansions is what leads to complete phenotypic expression of Fragile X syndrome. In Friedreich ataxia, the homozygous presence of expanded GAA repeats (range from 66 to greater than 1700 triplets) result in a "loss of function" because of suppression of expression of Frataxin, the FRDA gene product (35). However, in contrast to Fragile X syndrome, this is not linked to an abnormal methylation of a CpG island and the mechanism(s) of transcriptional repression has not been identified (35).

In DM I, it is generally believed that repeat expansion results in a reduction of steady-state myotonin protein kinase

Fig. 1. Example of Southern blot and PCR analysis of DNA from individuals of a Fragile X family. Left: Southern blot of DNA digested with EcoRI and Nrul and hybridized with the DNA probe StB12.3; Right: PCR amplification of the CGGrepeat followed by denaturing gel electrophoresis. Lane numbers in the right panel correspond to those in the left. Numbers below the pedigree correspond to the CGG repeat number. The great grandmother in lane 5 has two normal alleles (23,32). Her daughter (lane 4) has a normal allele of 23 and an expanded allele of 57. In subsequent transmissions, the abnormal allele of 57 expanded to 65 in one generation (lane 3) and then to 110 and >200 in the next generation (lanes 2 and 1, respectively).

Fig. 1. Example of Southern blot and PCR analysis of DNA from individuals of a Fragile X family. Left: Southern blot of DNA digested with EcoRI and Nrul and hybridized with the DNA probe StB12.3; Right: PCR amplification of the CGGrepeat followed by denaturing gel electrophoresis. Lane numbers in the right panel correspond to those in the left. Numbers below the pedigree correspond to the CGG repeat number. The great grandmother in lane 5 has two normal alleles (23,32). Her daughter (lane 4) has a normal allele of 23 and an expanded allele of 57. In subsequent transmissions, the abnormal allele of 57 expanded to 65 in one generation (lane 3) and then to 110 and >200 in the next generation (lanes 2 and 1, respectively).

(Mt-PK) mRNA and protein (36). It has been proposed that the "toxic gain of function" by abnormal RNA transcripts bearing the 3'-CUG repeats, retained within the nucleus, are directly responsible for the observed neuronal toxicity through inappropriate association with other DNA/RNA or sequestration of nuclear transcription factors required for development and maturation (37). An alternate model proposes chromatin restructuring caused by CTG repeats in the 3' UTR, to result in a decreased expression of Mt-PK as well as other nearby genes (37). One of the genes identified as playing a key role in the pathophysiology of myotonic dystrophy is SIX5, a homeobox domain gene immediately adjacent to the DMPK gene on chromosome 19q. The CTG repeat expansion in the 3' UTR region of DMPK extends into the promotor region of SIX5, thereby influencing the expression of its gene product (38).

3.5. MOLECULAR DIAGNOSIS The understanding of trinucleotide repeat diseases at the molecular level has had a major impact on the laboratory diagnosis of these diseases. Molecular testing has greatly improved the accuracy of the diagnosis, has allowed for presymptomatic testing of at risk family members, and has provided a means for the differential diagnosis of those diseases with overlapping clinical features. Laboratory diagnosis of the trinucleotide repeat diseases generally involves two approaches: polymerase chain reaction (PCR) and Southern blot analysis.

Primers flanking the region of DNA that contains the trinucleotide repeat are used to amplify that region by PCR. The PCR product can be analyzed by a number of techniques, including gel electrophoresis. The size of the product is determined by comparison with a standardized sizing ladder. Utilizing this approach, one can determine repeat sizes up to 200 (Fig. 1). This includes both normal and abnormal alleles of CAG repeat disorders (SCA1, SCA2, MJD/SCA3, SCA6, SCA7, SBMA, HD, and DRPLA), and normal alleles and pre-mutations in FRAXA and Friedreich ataxia. Because the efficiency of conventional PCR is inversely correlated with the number of repeats in each allele, alleles with more than 200 repeats are more difficult to amplify and might yield no PCR product. PCR analysis, however, is simple, inexpensive, and capable of providing accurate sizing of most alleles. One of the ways to overcome this limitation in efficiency of conventional PCR is with the use of long-range PCR. This technique has been used succesfully to amplify expanded alleles (up to 1.5 kb + 3n, where n is the number of GAA repeat triplets) in Friedreich ataxia (39). Many conventional PCR-based methodologies used in the detection of trinucleotide repeat expansions are transitioning into platforms that use automated fluorescence detections. These applications, when coupled with the use of chemicals such as betaine to reduce DNA secondary structure around the trinucleotide repeat expansions and pfu DNA

polymerase to correct for errors in replication during PCR, are expected to have a major impact in the automation of detection of trinucleotide repeat disorders in the clinical laboratory (40).

In contrast to PCR, Southern blot analysis allows detection of full mutations in those diseases with large repeat expansions (FRAXA, DM I, and Friedreich ataxia) (Fig. 1). Additionally, for FRAXA, Southern blot analysis provides information concerning the methylation status of an abnormal allele. The methylation status might be of diagnostic importance when the number of repeats is near the upper end of the premutation range (41). Southern blot analysis is more labor-intensive than PCR and requires larger quantities of genomic DNA. Southern blot can detect alleles in most size ranges, but in contrast to PCR analysis, it does not allow for precise sizing of trinucleotide repeats in the normal and premutation range. Full mutation alleles appear as a smear on Southern blots, because of a mosaicism in the length of the repeat in somatic cells. Radiolabeling of probes used in Southern blot analysis remains common, although nonisotopic detection methods are available as alternatives for clinical laboratories.

For diseases with small repeat expansions (SBMA, SCA1, SCA2, HD, SCA3/MJD, SCA6, SCA7, and DRPLA), PCR analysis alone is generally adequate for diagnosis. However, for those disorders with larger repeat expansions (FRAXA, DM I, and Friedreich ataxia) combined Southern blot and PCR analysis are most often used. Follow-up analysis for SCA2 and SCA7, to rule out the possibility of extreme CAG repeat expansions that have been associated with infantile or juvenile-onset ataxia (27,28), is performed on samples from infants and juvenile patients identified as homozygous by PCR for a CAG repeat allele in the normal range. This is performed as a PCR-Southern blot, followed by hybridization with a (CAG)n oligonucleotide. For some of the trinucleotide repeat disorders, additional studies might be recommended. For example, the stability of an SCA1 allele with CAG repeats at the upper end of the normal range can be assessed by Snfll restriction endonuclease digestion, which detects the presence of CAT interruptions. In SCA1, the normal-sized (CAG) and stable gray zone alleles are nearly always interrupted by one to three CAT trinucleotide repeats, whereas the expanded or unstable gray zone SCA1 repeat is an uninterrupted sequence of CAG repeats (14).

Routine cytogenetic analysis is recommended as part of a comprehensive genetic evaluation of patients referred for Fragile X syndrome testing. This testing strategy enables detection of constitutional chromosome abnormalities that might have overlapping phenotypic features of Fragile X syndrome. Finally, it should be realized that disease-causing mutations other than trinucleotide repeat expansion might occur within the aforementioned genes. Thus, the absence of trinucleotide repeat amplification does not necessarily rule out the diagnosis. This implies that for presymptomatic testing, it is important to first document the presence of a repeat amplification in an affected family member, which then verifies the underlying mechanism of disease.

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