Nondisjunction of chromosome 21 is the leading cause of Down syndrome. Two risk factors for maternal nondisjunction of chromosome 21 are increased maternal age and altered recombination. In order to provide further insight on mechanisms underlying nondisjunction, we examined the association between these two well established risk factors for chromosome 21 nondisjunction. In our approach, short tandem repeat markers along chromosome 21 were genotyped in DNA collected from individuals with free trisomy 21 and their parents. This information was used to determine the origin of the nondisjunction error and the maternal recombination profile. We analyzed 615 maternal meiosis I and 253 maternal meiosis II cases stratified by maternal age. The examination of meiosis II errors, the first of its type, suggests that the presence of a single exchange within the pericentromeric region of 21q interacts with maternal age-related risk factors. This observation could be explained in two general ways: 1) a pericentromeric exchange initiates or exacerbates the susceptibility to maternal age risk factors or 2) a pericentromeric exchange protects the bivalent against age-related risk factors allowing proper segregation of homologues at meiosis I, but not segregation of sisters at meiosis II. In contrast, analysis of maternal meiosis I errors indicates that a single telomeric exchange imposes the same risk for nondisjunction, irrespective of the age of the oocyte. Our results emphasize the fact that human nondisjunction is a multifactorial trait that must be dissected into its component parts to identify specific associated risk factors.

We have previously examined characteristics of maternal chromosomes 21 that exhibited a single recombination on 21q and proposed that certain recombination configurations are risk factors for either meiosis I (MI) or meiosis II (MII) nondisjunction. The primary goal of this analysis was to examine characteristics of maternal chromosomes 21 that exhibited multiple recombinant events on 21q to determine whether additional risk factors or mechanisms are suggested. In order to identify the origin (maternal or paternal) and stage (MI or MII) of the meiotic errors, as well as placement of recombination, we genotyped over 1,500 SNPs on 21q. Our analyses included 785 maternal MI errors, 87 of which exhibited two recombinations on 21q, and 283 maternal MII errors, 81 of which exhibited two recombinations on 21q. Among MI cases, the average location of the distal recombination was proximal to that of normally segregating chromosomes 21 (35.28 vs. 38.86 Mb), a different pattern than that seen for single events and one that suggests an association with genomic features. For MII errors, the most proximal recombination was closer to the centromere than that on normally segregating chromosomes 21 and this proximity was associated with increasing maternal age. This pattern is same as that seen among MII errors that exhibit only one recombination. These findings are important as they help us better understand mechanisms that may underlie both age-related and nonage-related meiotic chromosome mal-segregation.

Fragile X-associated disorders are caused by a CGG trinucleotide repeat expansion in the 5′-untranslated region of the FMR1 gene. Expansion of the CGG trinucleotide repeats to >200 copies (that is, a full mutation) induces methylation of the FMR1 gene, with transcriptional silencing being the eventual outcome. Previous data have shown that FMR1 premutation carriers (individuals with 55–199 repeats) have increased FMR1 mRNA levels with decreased protein (fragile X mental retardation protein (FMRP)) levels. However, the point at which this translational inefficiency occurs, given the increased transcription mechanism, has not yet been explored and remains to be elucidated. We examined the repeat length group, FMR1 transcript and FMRP levels in 74 males with a wide range of repeat lengths using analysis of covariance to better characterize this association. Results showed that the mean FMRP level among carriers with 80–89 repeats was significantly higher than the mean levels among lower (54–79) and higher (90–120) premutation carriers, in spite of the increasing transcript level with repeat length. Taken together, these results suggest that the 80–89-repeat group may lead to different properties that increase the efficiency of translation compared with other premutation repeat size groups.

Trisomy 21, resulting in Down Syndrome (DS), is the most common autosomal trisomy among live-born infants and is caused mainly by nondisjunction of chromosome 21 within oocytes. Risk factors for nondisjunction depend on the parental origin and type of meiotic error. For errors in the oocyte, increased maternal age and altered patterns of recombination are highly associated with nondisjunction. Studies of normal meiotic events in humans have shown that recombination clusters in regions referred to as hotspots. In addition, GC content, CpG fraction, Poly(A)/Poly(T) fraction and gene density have been found to be significant predictors of the placement of sex-averaged recombination in the human genome. These observations led us to ask whether the altered patterns of recombination associated with maternal nondisjunction of chromosome 21 could be explained by differences in the relationship between recombination placement and recombination-related genomic features (i.e., GC content, CpG fraction, Poly(A)/Poly(T) fraction or gene density) on 21q or differential hot-spot usage along the nondisjoined chromosome 21. We found several significant associations between our genomic features of interest and recombination, interestingly, these results were not consistent among recombination types (single and double proximal or distal events). We also found statistically significant relationships between the frequency of hotspots and the distribution of recombination along nondisjoined chromosomes. Collectively, these findings suggest that factors that affect the accessibility of a specific chromosome region to recombination may be altered in at least a proportion of oocytes with MI and MII errors.

Previous studies on relatively small samples of individuals with trisomy 21 caused by paternally derived errors have shown that: (1) advanced paternal age is not a risk factor for chromosome 21 nondisjunction (NDJ), (2) absence of recombination, but not the location of recombination is associated with paternal NDJ and (3) there is an excess of males among live-births with paternally derived trisomy 21. An excess of males is also observed among all individuals with trisomy 21. Using 128 families that had a child with trisomy 21 due to a paternally derived error, we examined: paternal age, recombination and the male/female sex ratio. We genotyped STRs along 21q to identify the origin of the error and the location of recombination on the paternal chromosome. Results showed that 32% of paternal meiotic errors occurred in meiosis I (MI) and 68% in meiosis II (MII). We confirmed the lack of a paternal age association with either type of error (mean paternal age for controls, MI, and MII errors: 31.3 ± 6.6, 32.2 ± 6.3, 30.6 ± 6.5, respectively). However, contrary to previous findings, we did not find altered patterns of recombination among paternal MI or MII errors. We found an increased male/female sex ratio among paternal (1.28, 95% CI: 0.68–1.91) and maternal (1.16, 95% CI: 1.02–1.33) meiotic errors. While the sex ratio among individuals with paternal errors was not statistically significant, these findings suggest that selection against female fetuses with trisomy 21 may contribute to the excess of males observed among all individuals with trisomy 21.

Both a lack of maternal folic acid supplementation and the presence of genetic variants that reduce enzyme activity in folate pathway genes have been linked to meiotic nondisjunction of chromosome 21; however, the findings in this area of research have been inconsistent. To better understand these inconsistencies, we asked whether maternal use of a folic acid-containing supplement before conception reduces risk for chromosome 21 nondisjunction. Using questionnaire data from the National Down Syndrome Project, a population-based case-control study, we compared the use of folic acid-containing supplements among mothers of infants with full trisomy 21 due to maternal nondisjunction (n=702) and mothers of infants born with no major birth defects (n=983). Using logistic regression, adjusting for maternal age, race/ethnicity, and infant age at maternal interview, we found no evidence of an association between lack of folic acid supplementation and maternal nondisjunction among all case mothers (OR=1.16; 95% CI: 0.90–1.48). In analyses stratified by meiotic stage and maternal age (<35 years or ≥ 35 years), we found an association among older mothers experiencing meiosis II nondisjunction errors (OR=2.00; 95% CI: 1.08–3.71). These data suggest that lack of folic acid supplementation may be associated specifically with MII errors in the aging oocyte. If confirmed, these results could account for inconsistencies among previous studies, as each study sample may vary by maternal age structure and proportion of meiotic errors.

Variation in the zinc finger-binding domain (ZFBD) of the protein PR Domain-Containing Protein 9 (PRDM9) is associated with altered placement of recombination in the human genome. As both the absence and altered placement of recombination are observed among chromosomes 21 that nondisjoin, we genotyped the PRDM9 ZFBD among mothers of children with Trisomy 21 in efforts to determine if variation within this region is associated with the recombination-related risk for chromosome 21 nondisjunction (NDJ). In our approach, PCR was used to amplify the ZFBD of PRDM9 and products were then subjected to bi-directional Sanger sequencing. DNA sequencing reads were aligned and compared to the sequence of the PRDM9 alleles previously identified. Chi-Square analysis was used to compare allele frequencies between cases (N=235, mothers of children with maternally-derived Trisomy 21) and controls (N=48, fathers of children with maternally-derived Trisomy 21). Results of our analysis showed that the frequency of PRDM9 ZF minor alleles is significantly increased among women displaying NDJ of chromosome 21 and no recombination on 21q (p=0.02). Even more, when compared to those for the PRDM9 major A-allele, these minor alleles displayed fewer predicted binding sites on 21q. These findings suggest that allelic variation in the ZF of PRDM9 may play a role in the risk for chromosome 21 NDJ by leading to reduced recombination on 21q.

An R package implementing TroX is available for download at http://przeworski.uchicago.edu/.