High Sequence Variability Among Little cherry virus Isolates Occurring in British Columbia.

The LC5 isolate of Little cherry virus (LChV-LC5) is one of at least two distinct viruses contributing to a severe disease of cherry (Little cherry disease [LChD]) in British Columbia. A near-complete nucleotide sequence of LChV-LC5 is available as well as polyclonal antibodies against LChV-LC5 coat protein produced in bacterial cells. A survey for LChV-LC5-infected trees in the Okanagan Valley and Kootenay region of British Columbia was carried out using enzyme-linked immunosorbent assays (ELISA) and LChV-LC5 antibodies. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequence analysis of four different regions of the genomes of 31 of these isolates have been conducted. A high level of sequence variability was found: nucleotide sequence divergence between LChV-LC5 and the other sequenced isolates ranged from 0 to 19.7%, and amino acid sequence divergence ranged from 0 to 9.1%. Further examination of RT-PCR and sequence data identified six discrete groups of isolates, including a group identical to LChV-LC5. The high level of divergence in LChV-LC5 isolates occurring in British Columbia suggests that caution should be used in the selection of methods used for diagnosis during surveys for this virus.

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test.

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.

The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size.

Prior studies describing the relationship between CAG size and the age at onset of Huntington disease (HD) have focused on affected persons. To further define the relationship between CAG repeat size and age at onset of HD, we now have analyzed a large cohort of affected and asymptomatic at-risk persons with CAG expansion. This cohort numbered 1,049 persons, including 321 at-risk and 728 affected individuals with a CAG size of 29-121 repeats. Kaplan-Meier analysis has provided curves for determining the likelihood of onset at a given age, for each CAG repeat length in the 39-50 range. The curves were significantly different (P < .0005), with relatively narrow 95% confidence intervals (95% CI) (+/-10%). Penetrance of the mutation for HD also was examined. Although complete penetrance of HD was observed for CAG sizes of > or = 42, only a proportion of those with a CAG repeat length of 36-41 showed signs or symptoms of HD within a normal life span. These data provide information concerning the likelihood of being affected, by a specific age, with a particular CAG size, and they may be useful in predictive-testing programs and for the design of clinical trials for persons at increased risk for HD.

Sex-dependent mechanisms for expansions and contractions of the CAG repeat on affected Huntington disease chromosomes.

A total of 254 affected parent-child pairs with Huntington disease (HD) and 440 parent-child pairs with CAG size in the normal range were assessed to determine the nature and frequency of intergenerational CAG changes in the HD gene. Intergenerational CAG changes are extremely rare (3/440 [0.68%]) on normal chromosomes. In contrast, on HD chromosomes, changes in CAG size occur in approximately 70% of meioses on HD chromosomes, with expansions accounting for 73% of these changes. These intergenerational CAG changes make a significant but minor contribution to changes in age at onset (r2 = .19). The size of the CAG repeat influenced larger intergenerational expansions (> 7 CAG repeats), but the likelihood of smaller expansions or contractions was not influenced by CAG size. Large expansions (> 7 CAG repeats) occur almost exclusively through paternal transmission (0.96%; P < 10(-7)), while offspring of affected mothers are more likely to show no change (P = .01) or contractions in CAG size (P = .002). This study demonstrates that sex of the transmitting parent is the major determinant for CAG intergenerational changes in the HD gene. Similar paternal sex effects are seen in the evolution of new mutations for HD from intermediate alleles and for large expansions on affected chromosomes. Affected mothers almost never transmit a significantly expanded CAG repeat, despite the fact that many have similar large-sized alleles, compared with affected fathers. The sex-dependent effects of major expansion and contractions of the CAG repeat in the HD gene implicate different effects of gametogenesis, in males versus females, on intergenerational CAG repeat stability.

DNA haplotype analysis of Huntington disease reveals clues to the origins and mechanisms of CAG expansion and reasons for geographic variations of prevalence.

This study of allelic association using three intra- and two extragenic markers within 150 kb of the Huntington disease (HD) mutation has provided evidence for linkage disequilibrium for four of five markers. Haplotype analysis of 67 HD families using markers in strong linkage disequilibrium with HD identified two haplotypes underlying 77.6% of HD chromosomes. Normal chromosomes with these two haplotypes had a mean number of CAG repeats significantly larger than and an altered distribution of CAG repeats compared with other normal chromosomes. Furthermore, haplotype analysis of five new mutation families reveals that HD has arisen on these same two chromosomal haplotypes. These findings suggest that HD arises more frequently on chromosomes with specific DNA haplotypes and higher CAG repeat lengths. We then studied CAG and CCG repeat lengths in the HD gene on 896 control chromosomes from different ancestries to determine whether the markedly reduced frequency of HD in Finland, Japan, China and African Blacks is associated with an altered frequency of DNA haplotypes and subsequently lower CAG lengths on control chromosomes compared to populations of Western European descent. The results show a highly significant inverse relationship between CAG and CCG repeat lengths. In populations with lowered prevalence rates of HD, CAG repeat lengths are smaller and the distribution of CCG alleles is markedly different from Western European populations. These findings suggest that, in addition to European emigration, new mutations make a contribution to geographical variation of prevalence rates and is consistent with a multistep model of HD developing from normal chromosomes with higher CAG repeat lengths.

Normal CAG repeat length in the Huntington's disease gene in senile chorea.

There is a widely held belief that most patients presenting with senile chorea have late-onset Huntington's disease (HD) with an unknown family history. We measured CAG trinucleotide repeat expansion in the HD gene in four patients with a clinical presentation of senile chorea and found that CAG repetition lengths were normal. These findings support senile chorea as being a distinct clinical entity that is nosologically separate from late-onset HD.

Proceed with care: direct predictive testing for Huntington disease.

The cloning of the Huntington disease (HD) gene allows highly accurate predictive testing using direct analysis of the CAG repeat. This new test provides results with almost complete certainty but poses unique counseling issues related to direct testing for an adult-onset disease. These include testing individuals who are at 25% risk, without the need for blood from a 50% at risk relative; the assessment of symptomatic individuals; the need for ongoing counseling despite simplification of laboratory procedures; and counseling of persons from families who represent a new mutation for HD. This paper describes protocols for direct predictive testing for adult and prenatal assessment, on the basis of the experience of the Canadian Collaborative Study on Predictive Testing (CCSPT). Over the past 8 years, we have provided > 400 results by using linked markers and, more recently, 416 results by using direct assessment of CAG expansion in the HD gene. The vast majority (86%) of requests for direct predictive testing have been from persons who have not previously received results by using linked markers. Despite the ability to now directly assess for the mutation associated with HD, we still recommend assessment of DNA from an affected relative, as this may significantly enhance the accuracy of information to be provided to the at-risk individual. Distance from a genetics center has previously limited the availability of testing, and therefore we have developed approaches to providing predictive testing in the patient's own community.

Geographical distribution of haplotypes in Swedish families with Huntington's disease.

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.

A worldwide study of the Huntington's disease mutation. The sensitivity and specificity of measuring CAG repeats.

BACKGROUND: Huntington's disease is associated with an expanded sequence of CAG repeats in a gene on chromosome 4p16.3. However, neither the sensitivity of expanded CAG repeats in affected persons of different ethnic origins nor the specificity of such repeats for Huntington's disease as compared with other neuropsychiatric disorders has been determined. METHODS: We studied 1007 patients with diagnosed Huntington's disease from 565 families and 43 national and ethnic groups. In addition, the length of the CAG repeat was determined in 113 control subjects with a family history of Alzheimer's disease (44 patients), schizophrenia (39), major depression (16), senile chorea (5), benign hereditary chorea (5), neuroacanthocytosis (2), and dentatorubropallidoluysian atrophy (2). The number of CAG repeats was also assessed in 1595 control chromosomes, with the size of adjacent polymorphic CCG trinucleotide repeats taken into account. RESULTS: Of 1007 patients with signs and symptoms compatible with a diagnosis of Huntington's disease, 995 had an expanded CAG repeat that included from 36 to 121 repeats (median, 44) (sensitivity, 98.8 percent; 95 percent confidence interval, 97.7 to 99.4 percent). There were no significant differences among national and ethnic groups in the number of repeats. No CAG expansion was found in the 110 control subjects with other neuropsychiatric disorders (specificity, 100 percent; 95 percent confidence interval, 95.2 to 100 percent). In 1581 of the 1595 control chromosomes (99.1 percent), the number of CAG repeats ranged from 10 to 29 (median, 18). In 12 control chromosomes (0.75 percent), intermediate-sized CAG sequences with 30 to 35 repeats were found, and 2 normal chromosomes unexpectedly had expanded CAG sequences, of 39 and 37 repeats. CONCLUSIONS: CAG trinucleotide expansion is the molecular basis of Huntington's disease worldwide and is a highly sensitive and specific marker for inheritance of the disease mutation.

Huntington disease without CAG expansion: phenocopies or errors in assignment?

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.

Somatic and gonadal mosaicism of the Huntington disease gene CAG repeat in brain and sperm.

Huntington disease is associated with an unstable and expanded (CAG) trinucleotide repeat. We have analysed the CAG expansion in different tissues from 12 affected individuals. All tissues examined were found to display some repeat mosaicism, with the greatest levels detected in brain and sperm. Regions within the brain showing most obvious neuropathology, such as the basal ganglia and the cerebral cortex, displayed the greatest mosaicism, whereas the cerebellar cortex, which is seldom involved, displayed the lowest degree of CAG instability. In two cases of childhood onset disease we detected differences of 8 and 13 trinucleotides between the cerebellum and other regions of the brain. Our results provide evidence for tissue specific instability of the CAG repeat, with the largest CAG repeat lengths in affected regions of the brain.

A CCG repeat polymorphism adjacent to the CAG repeat in the Huntington disease gene: implications for diagnostic accuracy and predictive testing.

The polymorphic CAG repeat that is expanded on Huntington disease (HD) chromosomes is flanked by a CCG repeat. Here we show that this CCG tract, previously assumed to be invariant at seven CCG repeats, is also polymorphic. We have identified five CCG alleles from 205 normal chromosomes, with 137 (67%) having alleles of seven repeats, five (2%) with nine repeats, 61 (30%) with 10 repeats, one (0.5%) with 11 repeats and one (0.5%) with 12 repeats. In contrast, analysis of 113 HD chromosomes revealed that the majority (105 chromosomes, 93%) contained seven CCG repeats, while the remaining eight chromosomes (7%) had allele sizes of 10 CCG repeats. Despite evidence that both CAG and CCG are polymorphic on normal chromosomes, we have found that it is only the CAG length that has a significant impact on age of onset. The discovery of larger sized CCG alleles, however, has significant implications for the assessment of CAG repeat length, particularly for persons with estimated CAG size of 36-42 repeats, since an overestimation of CAG length in this range could result in erroneous information being imparted to patients.

Precise mapping of the brain alpha 2-adrenergic receptor gene within chromosome 4p16.

The gene encoding the brain alpha 2-adrenergic receptor (ADRA2C) is located on human chromosome 4. It has been circumstantially associated with a number of human disorders, including Parkinson disease, panic disorders, and Huntington disease (HD). Using somatic cell hybrids, we localized the gene to chromosome 4p16 distal to P8 (D4S62). To investigate this locus further, we isolated several cosmid clones covering the entire gene. The gene was found to be intronless. Two (GT)n repeats in close proximity to the ADRAC2 gene were analyzed and used to define its precise location. Linkage disequilibrium studies of one microsatellite in HD families showed strong nonrandom association to the HD mutation, indicating its tight linkage to the HD gene. The investigation of families carrying recombinant chromosomes, pulsed-field analysis, and genomic walking mapped the ADRA2C gene adjacent to D4S81, 500 kb proximal to the HD gene. The newly defined microsatellites at the ADRAC2 locus, its precise localization within 4p16, and the detailed PCR conditions facilitate the identification of any defect caused by this gene.

Familial predisposition to recurrent mutations causing Huntington's disease: genetic risk to sibs of sporadic cases.

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.

Molecular analysis of late onset Huntington's disease.

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.

Attitudes toward direct predictive testing for the Huntington disease gene. Relevance for other adult-onset disorders. The Canadian Collaborative Group on Predictive Testing for Huntington Disease.

OBJECTIVE: To assess attitudes toward, and projected utilization of, direct mutation testing by individuals at risk for Huntington disease (HD). DESIGN: Prior to the cloning of the gene for HD, a questionnaire concerning the use of a definitive test was constructed and mailed to 354 participants in the Canadian Collaborative Study for HD. Completed questionnaires were received from 250 participants (response rate, 71%). Persons were asked to indicate whether they would participate in a new predictive test that was either 100% accurate (the definitive test, requiring blood only from the proband) or only 99% accurate. RESULTS: Most (72%) of the persons who had previously received a result in a predictive testing program said they would request testing in either situation. Significantly more persons would request the definitive test than the 99% accurate test (72% vs 58%; P < .02). Respondents for whom testing was uninformative in the linkage test program or who had previously received an increased-risk result were more likely to indicate they would use the test than those who received a decreased-risk result or chose not to have the original test (P = .0003). Less than half (46%) of the participants who initially chose not to have the linkage test said they would return for the new direct test. The major factor that has limited acceptance of predictive testing for this group is the concern about receiving an increased-risk result in the absence of any therapy to alter progression of the disease. CONCLUSIONS: A direct mutation test for HD will most readily be accepted by persons who wanted but could not previously receive a result in the linkage test program and those who previously received an increased-risk result. In the absence of therapy, the majority of persons who previously chose not to have predictive testing are unlikely to participate in a new test despite improved accuracy. This has implications for the expected demands for testing services for other adult-onset genetic disorders.

Molecular analysis of juvenile Huntington disease: the major influence on (CAG)n repeat length is the sex of the affected parent.

Juvenile Huntington disease (HD), characterised by onset of symptoms before the age of 20 with rigidity and intellectual decline, is associated with a predominance of affected fathers. In order to investigate the molecular basis for the observed parental effect, we have analysed the CAG trinucleotide repeat within the HD gene in 42 juvenile onset cases from 34 families. A highly significant correlation was found between the repeat length and age of onset (r = -0.86, p < 10(-7) and it was determined that the sex of transmitting parent was the major influence on CAG expansion leading to earlier onset. Neither the size of the parental upper allele, the age of parent at conception of juvenile onset child, nor the grandparental sex conferred a significant effect upon expansion. Affected sib pair analysis of CAG repeat length, however, revealed a high correlation (r = 0.91, p < 10(-7). Furthermore, analysis of nuclear and extended families showed a familial predisposition to juvenile onset disease. This study demonstrates that the sex of transmitting parent is the major influence on trinucleotide expansion and clinical features in juvenile Huntington disease.

Molecular analysis of new mutations for Huntington's disease: intermediate alleles and sex of origin effects.

Huntington's disease (HD) is associated with expansion of a CAG repeat in a novel gene. We have assessed 21 sporadic cases of HD to investigate sequential events underlying HD. We show the existence of an intermediate allele (IA) in parental alleles of 30-38 CAG repeats in the HD gene which is greater than usually seen in the general population but below the range seen in patients with HD. These IAs are meiotically unstable and in the sporadic cases, expand to the full mutation associated with the phenotype of HD. This expansion has been shown to occur only during transmission through the male germline and is associated with advanced paternal age. These findings suggest that new mutations for HD are more frequent than prior estimates and indicate a previously unrecognized risk of inheriting HD to siblings of sporadic cases of HD and their children.