Mammalian artificial chromosome pilot production facility: large-scale isolation of functional satellite DNA-based artificial chromosomes.

BACKGROUND: A pilot production facility has been established to isolate mammillian artificial chromosomes at high purity by using flow cytometric techniques. Dicentric chromosomes have been generated by the targeted amplification of pericentric heterochromatic and centromeric DNA by activating the "megareplicator." Breakage of these dicentric chromosomes generates satellite DNA-based artificial chromosomes (SATAC) from 60 to 400 megabases. METHODS: For large-scale production, we have developed cell lines capable of carrying one or two SATACs. A SATAC, because of a high adenine-thymine (AT) composition, is easily identified and sorted by using chromomycin A3 and Hoechst 33258 stains and a dual laser high-speed flow cytometer. A prototype SATAC (60 megabases) has been characterized. The prototype SATAC has been isolated from an original rodent/human hybrid cell line and transferred by using modified microcell fusion into a CHO production cell line. RESULTS: Metaphase chromosomes from this production cell line were isolated in a modified polyamine buffer, stained, and sorted by using a modified sheath buffer that maintains condensed chromosomes. SATACs are routinely sorted at rates greater than 1 million per hour. Sorted SATACs have been transferred to a variety of cells by using microcell fusion technology and were found to be functional. CONCLUSIONS: By developing new SATAC containing cell lines with fewer numbers of chromosomes in conjunction with operating a high speed flow sorter we have effectively generated an efficient production facility geared purely for the isolation of SATACs.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

Contribution of DNA sequence and CAG size to mutation frequencies of intermediate alleles for Huntington disease: evidence from single sperm analyses.

New mutations for Huntington disease (HD) arise from intermediate alleles (IAs) with between 29 and 35 CAG repeats that expand on transmission through the paternal germline to 36 CAGs or greater. Using single sperm analysis, we have assessed CAG mutation frequencies for four IAs in families with sporadic HD (IANM) and IAs ascertained from the general population (IAGP) by analyzing 1161 single sperm from three persons. We show that IANM are more unstable than IAGP with identical size and sequence. Furthermore, comparison of different sized IAs and IAs with different sequences between the CAG and the adjacent CCG tracts indicates that DNA sequence is a major influence on CAG stability. These studies provide estimates of the likelihood of expansion of IANM and IAGP to > or = 36 CAG repeats for these individuals. For an IA with a CAG of 35 in this family with sporadic HD, the likelihood for siblings to inherit a recurrent mutation > or = 36 CAG is approximately 10%. For IAGP of a similar size, the risk of inheriting an expanded allele of > or = 36 CAG through the paternal germline is approximately 6%. These risk estimates are higher than previously reported and provide additional information for counselling in these families. Further studies on persons with IAs will be needed to determine whether these results can be generalized to other families.

Increased instability of intermediate alleles in families with sporadic Huntington disease compared to similar sized intermediate alleles in the general population.

We have directly compared intergenerational stability of intermediate alleles (IAs) derived from new mutation families (IANM) for Huntington disease (HD) with IAs in the general population (IAGP) which occur in approximately 1 in 50 persons. Analysis of meiotic events in blood and sperm reveals that IANM are significantly more unstable than IAGP despite similar size. However, for both IANM and IAGP CAG changes were small and risks for inheriting an expansion into the HD affected range were low. Sequence analysis reveals that the CAG tract is generally interrupted by a penultimate CAA in IAGP, IANM and alleles in the affected range. In one new mutation family, however, two A-->G mutations result in a pure CAG tract which is associated with very marked instability. These mutations alter the predicted DNA hairpin structure with a predicted increase in the likelihood of large expansion, supporting the model that hairpin loop formation plays an important role in trinucleotide instability.

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.

Somatic mosaicism in sperm is associated with intergenerational (CAG)n changes in Huntington disease.

We have analysed the CAG repeat in the Huntington disease (HD) gene in sperm and blood from 20 unrelated HD patients. Although the CAG repeat displayed significant mosaicism in sperm from all individuals, there were marked differences in the degree of repeat instability. Individuals who had either inherited or transmitted an expanded CAG repeat displayed the highest levels of repeat mosaicism, whereas individuals who had inherited or transmitted a contracted repeat had very limited CAG mosaicism in sperm. A strong association between intergenerational change in CAG allele size and the level of sperm repeat mosaicism was determined (P = 0.019). In contrast, neither blood CAG size nor repeat mosaicism in blood, were significantly associated with intergenerational CAG changes. These data suggest the presence of a cis-acting factor, separate from CAG size, that strongly influences the intergenerational behaviour of the CAG repeat. Additional studies are needed to determine whether analysis of CAG mosaicism in sperm is useful for assessing an individual's risk for transmitting large expansions or contractions to his offspring.

Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization.

Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 [inv dup(15)], including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality.

The molecular genetics of Huntington's disease.

The past year has witnessed outstanding developments in research on Huntington's disease (HD). A gene was identified that contains an expanded CAG trinucleotide repeat on HD chromosomes. Patterns of expression of this gene and the nature of two transcripts were identified. CAG repeat size ranges between 36 and 121 in affected persons, and it is highly sensitive and specific marker for HD. A correlation between CAG repeat size and the age of onset of HD was demonstrated. Identification of this mutation has facilitated direct approaches to predictive testing for HD. The new mutation rate, previously deemed to be exceedingly rare, is now shown to be responsible for up to 3% of affected persons. Although the mechanism by which CAG repeat length induces neuronal death is not known, there is evidence that the pathogenesis involves a gain of function in the HD gene.

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.

Tetrasomy 15q: two marker chromosomes with no detectable alpha-satellite DNA.

Two patients with specific and similar phenotypes were both found to have an unusual marker chromosome present in 70%-80% of their lymphocytes at routine cytogenetic examination. The marker chromosomes were isolated by flow sorting and were amplified by degenerate oligonucleotide-primed PCR. These libraries and a cosmid probe located at 15q26 were used to characterize the marker chromosomes by FISH. Both marker chromosomes were found to consist of duplicated chromosome material from the distal part of chromosome 15q and were identified as inv dup(15) (qter-->q23::q23-->qter) and inv dup(15) (qter-->q24::q24-->qter), respectively. Hence, the markers did not include any known centromere region, and no alpha-satellite DNA could be detected at the site of the primary constriction. Tetrasomy 15q may be a new syndrome, associated with a specific type of marker chromosome. In addition, further analyses of this type of marker chromosome might give new insight into the structure and function of the mammalian centromere.

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.

Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato.

We obtained DNA clones specific to tomato chromosome 2 from a small number of chromosomes collected by flow sorting. Suspensions of metaphase chromosomes were prepared from 3-month-old tomato cell cultures of Lycopersicon pennellii. Isolated chromosomes stained with chromomycin A3 and Hoechst 33258 were analyzed on an EPICS 753 flow cytometer using a UV laser to excite Hoechst fluorescence and a 458 nm laser to excite chromomycin A3 fluorescence. Chromosomes from well-resolved peaks on a bivariate flow karyotype were sorted directly onto membrane filters for spot-blot analysis. The filters were processed and hybridized with chromosome-specific repetitive DNA probes. In this way tomato chromosome 1 and chromosome 2 were assigned to peaks in the bivariate flow karyotypes. One thousand copies of the putative chromosome 2 were flow-sorted directly into microfuge tubes. DNA specific to chromosome 2 was amplified by a polymerase chain reaction (PCR) technique using universal 22mer degenerate oligonucleotide primers (DOP) sequences. DOP-PCR yields a smear of fragments of various sizes from 250 to 1600 bp. Amplified products were cloned into the Bluescript plasmid vector. Approximately 11% of the clones contained sequences with highly repetitive elements, and 85% contained only low-copy-number sequences. Eleven clones containing low-copy-number sequences that detect restriction fragment length polymorphisms were placed on the molecular linkage map of tomato. All showed linkage to chromosome 2.

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.

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.

Preparation of chromosome-specific paints and complete assignment of chromosomes in the pig flow karyotype.

Sorted chromosomes from each of the 20 clusters of the male porcine bivariate flow karyotype were amplified and biotinylated using DOP-PCR. The chromosomes comprising each cluster were identified by fluorescence in situ hybridization (FISH) of the 20 probes to R-banded male pig metaphase spreads. A standard flow karyotype for the pig is presented.

The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease.

Huntington's disease (HD) is associated with the expansion of a CAG trinucleotide repeat in a novel gene. We have assessed 360 HD individuals from 259 unrelated families and found a highly significant correlation (r = 0.70, p = 10(-7)) between the age of onset and the repeat length, which accounts for approximately 50% of the variation in the age of onset. Significant associations were also found between repeat length and age of death and onset of other clinical features. Sib pair and parent-child analysis revealed that the CAG repeat demonstrates only mild instability. Affected HD siblings had significant correlations for trinucleotide expansion (r = 0.66, p < 0.001) which was not apparent for affected parent-child pairs.