Rearrangements Related to Classical Deletions

AZFa Duplication and HERV Homogenization

If NAHR is the causal mechanism of a recurrent deletion, it is commonly presumed that the reciprocal event, a duplication, will also be generated (21). The finding of this duplication is often regarded as proof that NAHR is indeed the mutational mechanism underlying both rearrangements. Duplications are often harder to detect than their reciprocal deletions, as they are more rarely pathogenic and require a quantitative rather than a qualitative assay. Duplications of the AZFa interval that appear to be compatible with fertility have been identified during a population survey of simple tandem repeats (STRs) diversity on the Y chromosome (22). In two unrelated individuals, STRs within the AZFa interval exhibited unusual electrophero-grams suggestive of their being duplicated relative to other YSTRs outside the AZFa interval. Junction sequences that could only derive from a hybrid HERV at the duplication breakpoint were also identified in the same individuals. As these duplications were identified in a survey of population diversity, there was no phenotypic information available. However, the duplicated STR haplotypes within the two AZFa intervals on the same chromosome were not identical, which suggests that sufficient time must have elapsed for these haplotypes to diverge, implying that, unlike their reciprocal deletions, these duplications are compatible with fertility. It is worth noting that on a constitutively haploid chromosome such as the Y, duplications must be caused by NAHR between sister chromatids.

In addition to the deletion and duplication events within the AZFa interval, NAHR has been involved in the homogenization of the HERV sequences that promote the rearrangements (14,23). In two independent lineages of the Y chromosome phylogeny, the maximal length of absolute identity that is shared between the HERVs has been expanded fivefold as a result of homogenization of the approx 4.5 kb of sequence between the two NAHR hotspots (14). This homogenization process most likely occurred because of an AZFa deletion on a chromosome that had previously undergone an AZFa duplication. This dramatic polymorphism for the absolute length of sequence identity between the HERVs, and the apparent relationship between the length of sequence identity and hotspots of NAHR activity, together suggest that some males may have higher rates of AZFa deletion/duplication than others.

Partial AZFc Deletions Predicted From the Reference Sequence: b1/b3, gr/gr, and Their Phenotypic Consequences

Although early studies (11) and several subsequent analyses had provided evidence for partial deletions and duplications within the AZFc region, their structures and phenotypic consequences were unclear. The availability of the sequence and an understanding of the mechanism of the AZFc (b2/b4) deletion led to the prediction that two partial deletions might arise by homologous recombination between other direct repeats: b1/b3, or the various green and red repeats, now collectively abbreviated as gr/gr (24) (Fig. 3). A search strategy based on the presence or absence of STSs spanning amplicon boundaries and thus providing some specificity in this highly repeated region was used to characterize 689 men (25). STS patterns indicative of both of the predicted deletions were found: one b1/b3 deletion and 22 gr/gr deletions, and all those that could be tested (1/1 and 20/22, respectively) were confirmed to be deletions by fluorescent in situ hybridization. In addition, two of the gr/gr deletions showed duplications interpreted as b2/b4 events (Fig. 3). The spermatogenic phenotype of the man with the b1/b3 deletion was not reported, but gr/gr deletions were found in 9/246 men with total sperm counts less than 2 x 107 (or <107/mL) compared with 0/148 with total sperm counts more than 4 x 107, a difference that achieves statistical significance (p < 0.014, Fisher exact test). This finding led the authors to suggest that the gr/gr deletion, which removes some but not all members of three protein-coding and five non-coding gene families, exists within the population at a lower than expected frequency as a result of mutation/ selection balance. However, statistical significance would be lost if the 149th man with normal spermatogenesis were to carry the deletion (9/246 vs 1/149,p > 0.05), a conservative consideration often applied in the statistical interpretation of forensic evidence. This factor and the prevalence of the deletion in the general population mean that the association of the gr/gr deletion with spermatogenic failure must be considered uncertain until more data are available, particularly in the light of results from other deletions discussed next. Irrespective of the phenotype associated with the gr/gr deletions, their genomic properties are of considerable interest. The Y chromosome outside the pseudoautosomal regions evolves along stable lineages that are not disrupted by recombination and can be identified and organized into a phylogenetic tree using neutral polymorphisms. gr/gr deletions were found in 14 branches of a tree that resolved 43 lineages and had apparently arisen independently in each of these branches, although the locations of the recombination events within the gr repeats are unknown and could differ between lineages: for example, some could remove DAZ1/DAZ2, wereas others could remove DAZ3/DAZ4. The deleted chromosomes made up a small proportion of most lineages, but in one, D2b, all 12 chromosomes examined carried the deletion, and this lineage is known to be present in approx 30% of men in Japan (26). Such a frequency can be achieved by a neutral marker by genetic drift, but is unlikely to be attained by a lineage that is selected against even to the extent of 1% (27), the selective disadvantage estimated by the authors. Possible explanations are that: (1) some or all gr/gr deletions have no significant effect on spermatogenesis; (2) they reduce sperm numbers but have no significant effect on fertility; or (3) a compensatory mutation has arisen. Such a mutation would be most effective if it was located on the Y chromosome and was, thus, permanently linked to the deletion, and the b2/b4

duplication reported in 2/30 gr/gr deletions (see Fig. 3) provides a candidate for such a mutation since it restores gene copy numbers to their original levels or higher (25). Unfortunately, it is not yet known whether or not D2b gr/gr deletions carry a b2/b4 duplication.

Ironically, although the gr/gr deletion may not be in mutation/selection balance, the AZFc deletion characterized by the same authors provides a clear example of such a balance. The incomplete penetrance of the AZFc deletion leads to its infrequent but recurrent transmission from father to son (see The AZFa Deletion) and results in the AZFc deletion reaching a population frequency higher than its de novo mutation rate, but being kept in check by the deletion's substantial fitness costs.

Partial AZFc Deletions Not Predicted From the Reference Sequence: g1/g3 (=b2/b3)

The detection in the population of deletions predicted from the sequence provides a powerful illustration of the usefulness of sequence data. Further surveys, however, have discovered additional partial AZFc deletions that were not predicted. A major class lacks two apparently discontinuous regions, one including u3 (and thus 50f2/C) and the other the DAZ3 and DAZ4 genes, which lie in the distal DAZ cluster (20,28). Neither deletion could arise by homologous recombination from the reference sequence, but a combination of two events, an inversion followed by a deletion, involving both the b2/b3 and g1/g3 repeats, could remove all the missing sequences by either of two pathways (see Fig. 3). This deletion product has been identified in four lineages, but most deleted chromosomes belong to one of them, N, where it appears to be fixed. The N lineage is even more widespread than D2b, predominating in northern Asia and making up 12% of Y chromosomes in one worldwide survey (29). The proposed mechanism of deletion gains credence from the detection of both of the possible intermediate inverted structures in nondeleted chromosomes, with b2/b3 inversions showing at least two independent origins in a 44-branch tree and gr/gr inversions 5; additional duplications were found in 3 of the 14 deleted chromosomes (20). Genes removed by the deletion (known either as g1/g3 or b2/b3) include some but not all members of three protein-coding and five non-coding gene families, but with the loss of more family members than the gr/gr deletion. The success of the N lineage is easily understood under the hypothesis that such partial deletions are neutral events—it is entirely owing to chance genetic drift—but requires more convoluted explanations involving compensating mutations other than the known duplications under the hypothesis that such partial deletions are associated with spermatogenic failure.

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