Selection of human chromosome 21-specific DNA probes for genetic analysis in Alzheimer's dementia and Down syndrome

Summary We used a mouse-human somatic cell hybrid to construct a chromosome 21-enriched library in phage vector EMBL4. In all, 35 phage clones containing human inserts were identified by differential screening with total human and mouse DNA. Whole recombinant phages were regionally mapped on chromosome 21 by Southern blot analysis using competitive hybridisation conditions to block repetitive sequences. Ten phage clones mapped proximal to a translocation breakpoint in band 21q21.2, while 25 mapped distal to this point. Three of the phage clones identify restriction fragment length polymorphisms. Polymorphic chromosome 21 markers may be useful in the genetic analysis of Alzheimer's dementia and Down syndrome.     

Cloned DNA probes regionally mapped to human chromosome 21 and their use in determining the origin of nondisjunction.

A number of unique sequence recombinant DNA clones were isolated from a recombinant DNA library constructed from DNA enriched for chromosome 21 by flow sorting. Of these, five were mapped to chromosome 21 using a somatic cell hybrid. Regional mapping of these probes and of a probe previously assigned to chromosome 21, was carried out with the aid of chromosome 21 rearrangements using both chromosome sorting and a somatic cell hybrid. Three probes were shown to be located on either side of the breakpoint 21q21.2. Two of the probes were shown to identify restriction fragment length polymorphisms (RFLPs) with high rare-allele frequencies (0.46 and 0.43). A Bgl II RFLP revealed the parental origin of non-disjunction in three of ten families with Down's syndrome.     

Centromeric genotyping and direct analysis of nondisjunction in humans: Down syndrome

In species with chiasmate meioses, alterations in genetic recombination are an important correlate of nondisjunction. In general, these alterations fall into one of two categories: either homologous chromosomes fail to pair and/or recombine at meiosis I, or they are united by chiasmata that are suboptimally positioned. Recent studies of human nondisjunction suggest that these relationships apply to our species as well. However, methodological limitations in human genetic mapping have made it difficult to determine whether the important determinant(s) in human nondisjunction is absent recombination, altered recombination, or both. In the present report, we describe somatic cell hybrid studies of chromosome 21 nondisjunction aimed at overcoming this limitation. By using hybrids to “capture” individual chromosomes 21 of the proband and parent of origin of trisomy, it is possible to identify complementary recombinant meiotic products, and thereby to uncover crossovers that cannot be detected by conventional mapping methods. In the present report, we summarize studies of 23 cases. Our results indicate that recombination in proximal 21q is infrequent in trisomy-generating meioses and that, in a proportion of the meioses, recombination does not occur anywhere on 21q. Thus, our observations indicate that failure to recombine is responsible for a proportion of trisomy 21 cases. Received: 12 January 1997; in revised form: 16 February 1998 / Accepted: 19 February 1998     

Distribution of meiotic recombination along nondisjunction chromosomes 21 in Down syndrome determined using cytogenetics and RFLP haplotyping

Summary Ten families (Down syndrome children and their parents) showing evidence of meiotic recombination between intraparental chromosomes transmitted after nondisjunction were studied. Cytogenetic polymorphisms and a cassette of RFLP markers distributed along chromosome 21 were used to analyze these families to localize the regions of meiotic recombination. Results indicated that only one crossover occurred per meiotic division and that nine of ten nondisjunctions appeared to be of maternal origin. In one family the crossover had taken place in the pericentromeric region, proximal to marker D21S13, which is quite exceptional. A chance of meiotic recombination within region 21q21, flanked by marker D21S72 and the amyloid gene, could be demonstrated in seven of the ten families. Most strikingly, this chance significantly decreased distal to q21, with frequencies of 0.3 and 0.1 in regions q22.2 and q22.3-qter, respectively. It is hypothesized that decreased chiasmata formation in the most distal part of chromosome 21q might promote nondisjunction. Furthermore, data from the ten crossovers made it possible to map provisionally two previously undefined markers, D21S24 and D21S82, to regions q21-qter and q22.1-qter, respectively.     

Trisomy 21 (Down syndrome): studying nondisjunction and meiotic recombination by using cytogenetic and molecular polymorphisms that span chromosome 21.

By combining molecular and cytogenetic techniques, we demonstrated the feasibility and desirability of a comprehensive approach to analysis of nondisjunction for chromosome 21. We analyzed the parental origin and stage of meiotic errors resulting in trisomy 21 in each of five families by successfully using cytogenetic heteromorphisms and DNA polymorphisms. The 16 DNA fragments used to detect polymorphisms spanned the length of the long arm and detected recombinational events on nondisjoined chromosomes in both maternal meiosis I and maternal meiosis II errors. The meiotic stage at which errors occurred was determined by sandwiching the centromere between cytogenetic heteromorphisms on 21p and an informative haplotype constructed using two polymorphic DNA probes that map to 21q just below the centromere. This study illustrates the necessity of combining cytogenetic polymorphisms on 21p with DNA polymorphisms spanning 21q to determine (1) the source and stage of meiotic errors that lead to trisomy 21 and (2) whether an association exists between nondisjunction and meiotic recombination.     

Generation of a chromosome-22-specific c-DNA library as confirmed by FISH analysis

We have recently developed a strategy for the rapid enrichment of c-DNA fragments from selected human chromosomes. Heteronuclear RNA (hn-RNA) is isolated from a somatic cell hybrid that retains a single human chromosome in a rodent background. Following c-DNA synthesis, human sequences are selectively amplified by the Alu polymerase chain reaction (Alu-PCR). Here we have applied this protocol for the selective isolation of novel c-DNAs encoded by chromosome 22. Fluorescence in situ hybridization has been used to confirm the chromosome-22-specific origin of the c-DNA fragments. Controls show DNAse-free RNase-treated hn-RNA results in no c-DNAs or Alu-PCR products. As demonstrated by competitive in situ suppression hybridization (CISS), the majority of the Alu-PCR products from hybrid GM 10027 are located on chromosome 22. Without competition, hybridization signals have also been identified on other human chromosomes. These unspecific hybridization signals result from Alu sequences and can successfully be reduced by competition with cot 1 DNA. This is the first report of the use of CISS for the localization of chromosome-specific c-DNAs.     

Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-l cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with beta-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognized by the antibodies. At least two previously unrecognized epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-l) cells cause expression of human p53 recognized by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.     

Identification of chromosome 21 DNA polymorphisms for genetic studies in Alzheimer's disease and Down syndrome

Summary Linkage studies in families with presenile onset of Alzheimer's disease (AD) indicated the presence of a predisposing gene on the proximal long arm of chromosome 21. We mapped four new loci in the candidate AD region using somatic cell hybrids. For three of the four loci, several restriction fragment length polymorphisms were found; for one locus, a multiallelic (CA)_n dinucleotide polymorphism was detected. Preliminary genetic mapping of the new polymorphic loci relative to the AD-linked loci was obtained in a reference pedigree. In addition, we used the (CA)_n dinucleotide polymorphism to reconstruct the non-disjunction event in a Down syndrome (DS) patient whose mother died of familial AD.     

Telomere length is associated with types of chromosome 21 nondisjunction: a new insight into the maternal age effect on Down syndrome birth

Advanced maternal age is a well-documented risk factor of chromosome 21 nondisjunction in humans, but understanding of this association at the genetic level is still limited. In particular, the state of maternal genetic age is unclear. In the present study, we estimated maternal genetic age by measuring telomere length of peripheral blood lymphocytes among age-matched mothers of children with Down syndrome (cases: N = 75) and mothers of euploid children (controls: N = 75) in an age range of 18–42 years. All blood samples were taken within 1 week of the birth of the child in both cases and controls. The telomere length estimation was performed by restriction digestion—Southern blot hybridization method. We stratified the cases on the basis of centromeric STR genotyping into maternal meiosis I (N = 48) and maternal meiosis II (N = 27) nondisjunction groups and used linear regression to compare telomere length as a function of age in the euploid, meiosis I and meiosis II groups. Our results show that all three groups have similar telomere length on average for younger mothers. As age increases, all groups show telomere loss, but that loss is largest in the meiosis II mother group and smallest in the euploid mother group with the meiosis I mother group in the middle. The regression lines for all three were statistically significantly different from each other (p < 0.001). Our results do not support the theory that younger women who have babies with Down syndrome do so because are ‘genetically older’ than their chronological age, but we provide the first evidence that older mothers who have babies with Down syndrome are “genetically older” than controls, who have euploid babies at the same age. We also show for the first time that telomere length attrition may be associated in some way with meiosis I and meiosis II nondisjunction of chromosome 21 and subsequent Down syndrome births at advanced maternal age.     

Multiple recurrence of trisomy 21 Down syndrome

Summary A young family with four chromosomally documented cases of trisomy 21, at least two, further cases of Down syndrome diagnosed only clinically, and no healthy children among their offspring is reported. No sign of mosaicism was found in studies of lymphocytes and skin fibroblasts from the parents. In a biopsy from the mother's ovary a trisomic cell line was found, thus giving some, but not a complete explanation for this extraordinary family. Additional explanations are suggested.     

Non-disjunction of chromosome 21, alphoid DNA variation, and sociogenetic features of Down syndrome

The analysis of non-disjunction of chromosome 21 and alphoid DNA variation by using cytogenetic and molecular cytogenetic techniques (quantitative fluorescence in situ hybridization) in 74 nuclear families was performed. The establishment of possible correlation between alphoid DNA variation, parental age, environmental effects, and non-disjunction of chromosome 21 was made. The efficiency of techniques applied was found to be 92% (68 from 74 cases). Maternal non-disjunction wasfound in 58 cases (86%) and paternal non-disjunction - in 7 cases (10%). Post-zygotic mitotic non-disjunction was determined in 2 cases (3%) and one case was associated with Robertsonian translocation 46,XX,der(21;21)(q10;q10), +21. Maternal meiosis I errors were found in 43 cases (64%) and maternal meiosis II errors--in 15 cases (22%). Paternal meiosis I errors occurred in 2 cases (3%) and paternal meiosis I errors--in 5 cases (7%). The lack of the correlation between alphoid DNA variation and non-disjunction of chromosome 21 was established. Sociogenetic analysis revealed the association of intensive drug therapy of infectious diseases during the periconceptual period and maternal meiotic non-disjunction of chromosome 21. The correlation between non-disjunction of chromosome 21 and increased parental age as well as exposure to irradiation, alcohol, tobacco, mutagenic substances was not found. The possible relevance of data obtained to the subsequent studies of chromosome 21 non-disjunction is discussed.     

The meiotic stage of nondisjunction in trisomy 21: Determination by using DNA polymorphisms

We have studied DNA polymorphisms at loci in the pericentromeric region on the long arm of chromosome 21 in 200 families with trisomy 21, in order to determine the meiotic origin of nondisjunction. Maintenance of heterozygosity for parental markers in the individual with trisomy 21 was interpreted as resulting from a meiosis I error, while reduction to homozygosity was attributed to a meiosis II error. Nondisjunction was paternal in 9 cases and was maternal in 188 cases, as reported earlier. Among the 188 maternal cases, nondisjunction occurred in meiosis I in 128 cases and in meiosis II in 38 cases; in 22 cases the DNA markers used were uninformative. Therefore meiosis I was responsible for 77.1% and meiosis II for 22.9% of maternal nondisjunction. Among the 9 paternal nondisjunction cases the error occurred in meiosis I in 2 cases (22.2%) and in meiosis II in 7 (77.8%) cases. Since there was no significant difference in the distribution of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular of maternal ages between maternal I error versus maternal II error, it is unlikely that an error at a particular meiotic stage contributes significantly to the increasing incidence of Down syndrome with advancing maternal age. Although the DNA polymorphisms used were at loci which map close to the centromere, it is likely that rare errors in meiotic-origin assignments may have occurred because of a small number of crossovers between the markers and the centromere.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down syndrome: increased frequency of maternal meiosis I nondisjunction during the transitional stages of the ovulatory seasons

Summary In a month-of-birth study of Down syndrome (DS) individuals, we found—in agreement with a previous collaborative European study (Jongbloet et al. 1982)—a distribution of maternal meiosis I (Mat MI)-DS births, both graphically and statistically differing from that of both total births (P<0.05) and from that of the three other (Mat MII+Pat MI+Pat MII)-DS subcategories (P<0.001). In four subgroups, namely the DS individuals born in the years 1960–1979 and 1980–1983 and those from mothers ≤30 years, and ≥31 years, analogous results were found. In addition, the Mat MI-DS individuals were shown to have been conceived more frequently during the “strong” change rate periods. i.e. the transitional stages of ovulatory breakthrough and breakdown, and less during the “weak” change rate periods, i.e. the periods of a rather stable ovulatory rate (P<0.05). The same results were found in two subgroups of individuals born 1960–1970 (P<0.055) and from mothers≥31 years (P<0.02). This interdependence estimated by linear regression in the total group (r _s =0.503; P<0.055) and in the subgroup ≥31 years (r _s =0.556; P<0.05), supports the hypothesis that Mat MI nondisjunctions occur more frequently during the transitional stages of the ovulatory seasons, i.e. they are caused by seasonal pre-ovulatory overripeness ovopathy (SPOO).     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA

A specific and efficient method is presented for the conversion of 2′-deoxyuridine to thymidine via formation and reduction of the intermediate 5-hydroxymethyl derivative. The method has been used to generate both thymidine and 5-methyl-2′-deoxycytidine containing the stable isotopes ²H, ¹³C and ¹⁵N. Oligodeoxyribonucleotides have been constructed with these mass-tagged bases to investigate sequence-selectivity in hydroxyl radical reactions of pyrimidine methyl groups monitored by mass spectrometry. Studying the reactivity of 5-methylcytosine (5mC) is difficult as the reaction products can deaminate to the corresponding thymine derivatives, making the origin of the reaction products ambiguous. The method reported here can distinguish products derived from 5mC and thymine as well as investigate differences in reactivity for either base in different sequence contexts. The efficiency of formation of 5-hydroxymethyluracil from thymine is observed to be similar in magnitude in two different sequence contexts and when present in a mispair with guanine. The oxidation of 5mC proceeds slightly more efficiently than that of thymine and generates both 5-hydroxymethylcytosine and 5-formylcytosine but not the deaminated products. Thymine glycol is generated by both thymine and 5mC, although with reduced efficiency for 5mC. The method presented here should be widely applicable, enabling the examination of the reactivity of selected bases in DNA.