Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm.

This study compares the relative effects of advancing male age on multiple genomic defects in human sperm [DNA fragmentation index (DFI), chromatin integrity, gene mutations, and numerical chromosomal abnormalities], characterizes the relationships among these defects and with semen quality, and estimates the incidence of susceptible individuals for a well characterized nonclinical nonsmoking group of 97 men (22-80 years). Adjusting for confounders, we found major associations between age and the frequencies of sperm with DFI and fibroblast growth factor receptor 3 gene (FGFR3) mutations associated with achondroplasia (P < 0.01) with no evidence for age thresholds. However, we found no associations between age and the frequencies of sperm with immature chromatin, aneuploidies/diploidies, FGFR2 mutations (Apert syndrome), or sex ratio in this cohort. There were also no consistent correlations among genomic and semen-quality endpoints, except between DFI and sperm motility (r = -0.65, P < 0.001). These findings suggest there are multiple spermatogenic targets for genomically defective sperm with substantially variable susceptibilities to age. Our findings predict that as healthy males age, they have decreased pregnancy success with trends beginning in their early reproductive years, increased risk for producing offspring with achondroplasia mutations, and risk of fathering offspring with Apert syndrome that may vary across cohorts, but with no increased risk for fathering aneuploid offspring (Down, Klinefelter, Turner, triple X, and XYY syndromes) or triploid embryos. Our findings also suggest that the burden of genomic damage in sperm cannot be inferred from semen quality, and that a small fraction of men are at increased risk for transmitting multiple genetic and chromosomal defects.

Evidence for the lack of mismatch-repair directed antirecombination during mouse meiosis.

Meiotic recombination was studied in DNA mismatch repair (MMR)-deficient mice using a strain carrying a Pms2 knockout mutation. Using single-sperm typing, recombination was analyzed over five intervals on four chromosomes in four Pms2 -/- animals. A total of 1936 meioses were studied and compared to 1848 meioses from three Pms2 +/+ controls. A smaller study was carried out on a single interval in each of two chromosomes in an MMR-deficient mouse homozygous for the Msh2 knockout mutation. A total of 792 meioses were examined in the Msh2 -/- and 880 meioses in the Msh2 +/+ animal. Recombination fractions were not significantly different in either of the MMR-deficient mouse strains when compared to MMR-proficient controls. Our results appear to conflict with mouse embryonic stem (ES) cell gene-targeting experiments where MMR plays a major role in determining the efficiency of homologous recombination between nonidentical sequences. A number of possibilities could explain the apparent lack of a significant effect on meiosis.

Unequal exchange at the Charcot-Marie-Tooth disease type 1A recombination hot-spot is not elevated above the genome average rate.

An increasing number of human diseases and syndromes are being found to result from micro-duplications or microdeletions arising from meiotic recombination between homologous repeats on the same chromosome. The first microduplication syndrome delineated, Charcot-Marie-Tooth disease type 1A (CMT1A), results from unequal crossing over between two >98% identical 24 kb repeats (CMT1A-REPs) on chromosome 17. In addition to its medical significance, the CMT1A region has features that make it a unique resource for detailed analysis of human unequal recombination. Previous studies of CMT1A patients showed that the majority of unequal crossovers occurred within a small region (<1 kb) of the REPs suggesting the presence of a recombination hot-spot. We directly measured the frequency of unequal recombination in the hot-spot region using sperm from four normal individuals. Surprisingly, unequal recombination between the REPs occurs at a rate no greater than the average rate for the male genome (approximately 1 cM/Mb) and is the same as that expected for equally aligned REPs. This conclusion extends to humans the findings in yeast that recombination between repeated sequences far apart on the same chromosome may occur at similar frequencies to allelic recombination. Finally, the CMT1A hot-spot stands in sharp contrast to the human MS32 mini-satellite-associated hot-spot that exhibits highly enhanced recombination initiation in addition to positional specificity. One possibility is that the CMT1A hot-spot may consist of a region with genome average recombination potential embedded within a recombination cold-spot.

Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export.

We present here a detailed analysis of a rat polypeptide termed Nup50 (formerly NPAP60) that was previously found to be associated with the nuclear pore complex (F. Fan et al., Genomics 40:444-453, 1997). We have found that Nup50 (and/or a related 70-kDa polypeptide) is present in numerous rat cells and tissues. By immunofluorescence microscopy, Nup50 was found to be highly concentrated at the nuclear envelope of rat liver nuclei, whereas in cultured NRK cells it also is abundant in intranuclear regions. On the basis of immunogold electron microscopy of both rat liver nuclear envelopes and NRK cells, we determined that Nup50 is specifically localized in the nucleoplasmic fibrils of the pore complex. Microinjection of anti-Nup50 antibodies into the nucleus of NRK cells resulted in strong inhibition of nuclear export of a protein containing a leucine-rich nuclear export sequence, whereas nuclear import of a protein containing a classical nuclear localization sequence was unaffected. Correspondingly, CRM1, the export receptor for leucine-rich export sequences, directly bound to a fragment of Nup50 in vitro, whereas several other import and export receptors did not significantly interact with this fragment. Taken together, our data indicate that Nup50 has a direct role in nuclear protein export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes.

Evidence for heterogeneity in recombination in the human pseudoautosomal region: high resolution analysis by sperm typing and radiation-hybrid mapping.

Accurate genetic and physical maps for the human pseudoautosomal region were constructed by use of sperm typing and high-resolution radiation-hybrid mapping. PCR analysis of 1,912 sperm was done with a manual, single-sperm isolation method. Data on four donors show highly significant linkage heterogeneity among individuals. The most significant difference was observed in a marker interval located in the middle of the Xp/Yp pseudoautosomal region, where one donor showed a particularly high recombination fraction. Longitudinal models were fitted to the data to test whether linkage heterogeneity among donors was significant for multiple intervals across the region. The results indicated that increased recombination in particular individuals and regions is compensated for by reduced recombination in neighboring intervals. To investigate correspondence between physical and genetic distances within the region, we constructed a high-resolution radiation-hybrid map containing 29 markers. The recombination fraction per unit of physical distance varies between regions ranging from 13- to 70-fold greater than the genome-average rate.

Meiotic segregation analysis of RB1 alleles in retinoblastoma pedigrees by use of single-sperm typing.

In hereditary retinoblastoma, different epidemiological studies have indicated a preferential paternal transmission of mutant retinoblastoma alleles to offspring, suggesting the occurrence of a meiotic drive. To investigate this mechanism, we analyzed sperm samples from six individuals from five unrelated families affected with hereditary retinoblastoma. Single-sperm typing techniques were performed for each sample by study of two informative short tandem repeats located either in or close to the retinoblastoma gene (RB1). The segregation probability of mutant RB1 alleles in sperm samples was assessed by use of the SPERMSEG program, which includes experimental parameters, recombination fractions between the markers, and segregation parameters. A total of 2,952 single sperm from the six donors were analyzed. We detected a significant segregation distortion in the data as a whole (P=.0099) and a significant heterogeneity in the segregation rate across donors (.0092). Further analysis shows that this result can be explained by segregation distortion in favor of the normal allele in one donor only and that it does not provide evidence of a significant segregation distortion in the other donors. The segregation distortion favoring the mutant RB1 allele does not seem to occur during spermatogenesis, and, thus, meiotic drive may result either from various mechanisms, including a fertilization advantage or a better mobility in sperm bearing a mutant RB1 gene, or from the existence of a defectively imprinted gene located on the human X chromosome.

Polymorphisms in the human DNA repair gene XPF.

DNA sequence polymorphisms were sought in the coding region and at the exon-intron boundaries of the human XPF gene, which plays a role in nucleotide excision repair. Based on a survey of 38 individuals, we found six single nucleotide polymorphisms, one in the 5' non-coding region of the XPF gene, and five in the 2751 bp coding region. At each site, the frequency of the rarer allele varies from about 0.01 to over 0.38. Except for the 5' non-coding and one coding sequence polymorphism, the rarer alleles for the remaining four polymorphisms were found only in heterozygotes. Of the five polymorphisms in the coding region, one is silent, one results in a conserved amino acid difference, and the remaining three result in non-conserved amino acid differences. Because of its biological function in nucleotide excision repair, functionally significant XPF gene polymorphisms are candidates for influencing cancer susceptibility and overall genetic stability. Nucleotide sequence diversity estimates for XPF are similar to the lipoprotein lipase and beta-globin genes.

French Machado-Joseph disease patients do not exhibit gametic segregation distortion: a sperm typing analysis.

Segregation distortion has been reported to occur in a number of the trinucleotide repeat disorders. On the basis of a sperm typing study performed in patients of Japanese descent with Machado-Joseph disease (MJD), it was reported that disease alleles are preferentially transmitted during meiosis. We performed a sperm typing study of five MJD patients of French descent and analysis of the pooled data shows a ratio of mutant to normal alleles of 379:436 (46.5:53.5%), which does not support meiotic segregation distortion. To confirm these results, sperm typing analysis was also performed using a polymorphic marker, D14S1050, closely linked to the MJD1 gene. Among 910 sperm analyzed, the allele linked to the disease chromosome was detected in 50.3% of the samples and the allele linked to the normal chromosome was found in 49.6% of the sperm. The difference in frequency of these two alleles is not significant ( P = 0.8423). Likelihood-based analysis of segregation distortion in the single sperm data using the SPERMSEG program also showed no support for segregation distortion at the gamete level in this patient population. The previous report on the Japanese patients also suggested that disease allele stability may be influenced by a trans effect of an intragenic polymorphism (987 G/C) in the wild-type allele. All of the French patients were heterozygous for this polymorphism. However, analysis of the variance in repeat number in sperm from the French MJD patients overlapped significantly with the variance in repeat number observed in the C/C homozygous Japanese patients.

Different mutator phenotypes in Mlh1- versus Pms2-deficient mice.

Deficiencies in DNA mismatch repair (MMR) result in increased mutation rates and cancer risk in both humans and mice. Mouse strains homozygous for knockouts of either the Pms2 or Mlh1 MMR gene develop cancer but exhibit very different tumor spectra; only Mlh1(-/-) animals develop intestinal tumors. We carried out a detailed study of the microsatellite mutation spectra in each knockout strain. Five mononucleotide repeat tracts at four different chromosomal locations were studied by using single-molecule PCR or an in vivo forward mutation assay. Three dinucleotide repeat loci also were examined. Surprisingly, the mononucleotide repeat mutation frequency in Mlh1(-/-) mice was 2- to 3-fold higher than in Pms2(-/-) animals. The higher mutation frequency in Mlh1(-/-) mice may be a consequence of some residual DNA repair capacity in the Pms2(-/-) animals. Relevant to this idea, we observed that Pms2(-/-) mice exhibit almost normal levels of Mlh1p, whereas Mlh1(-/-) animals lack both Mlh1p and Pms2p. Comparison between Mlh1(-/-) animals and Mlh1(-/-) and Pms2(-/-) double knockout mice revealed little difference in mutator phenotype, suggesting that Mlh1 nullizygosity is sufficient to inactivate MMR completely. The findings may provide a basis for understanding the greater predisposition to intestinal cancer of Mlh1(-/-) mice. Small differences (2- to 3-fold) in mononucleotide repeat mutation rates may have dramatic effects on tumor development, requiring multiple genetic alterations in coding regions. Alternatively, this strain difference in tumor spectra also may be related to the consequences of the absence of Pms2p compared with the absence of both Pms2p and Mlh1p on as yet little understood cellular processes.

Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing.

In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma.

High-resolution gametic map of the sheep callipyge region: linkage heterogeneity among rams detected by sperm typing.

The callipyge locus (CLPG) causing muscular hypertrophy in domestic sheep has previously been mapped to the distal part of ovine chromosome 18. In this study, an accurate multipoint linkage map consisting of six microsatellite markers in this chromosomal region was constructed based on the analysis of 1145 single sperm cells. The best supported order of markers was OARHH47-ILSTS54-MCM38-CSSM18-IDVGA30-BM S1561. The log odds against the second most likely order, which has a reversal of the closely linked markers CSSM18 and IDVGA30, was 5.026. Sperm typing can be used to examine a large number of meioses in single individuals, and therefore, was exploited to study individual variability of recombination rate in rams of different callipyge genotypes. The results revealed statistically significant linkage heterogeneity among rams (P < 0.05) for marker interval OARHH47-CSSM18, with individual recombination fractions varying from 0.209 to 0.357.

Triplet repeats form secondary structures that escape DNA repair in yeast.

Several human neurodegenerative diseases result from expansion of CTG/CAG or CGG/CCG triplet repeats. The finding that single-stranded CNG repeats form hairpin-like structures in vitro has led to the hypothesis that DNA secondary structure formation is an important component of the expansion mechanism. We show that single-stranded DNA loops containing 10 CTG/CAG or CGG/CCG repeats are inefficiently repaired during meiotic recombination in Saccharomyces cerevisiae. Comparisons of the repair of DNA loops with palindromic and nonpalindromic sequences suggest that this inefficient repair reflects the ability of these sequences to form hairpin structures in vivo.

Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic mutation mechanism.

Trinucleotide repeat disease alleles can undergo 'dynamic' mutations in which repeat number may change when a gene is transmitted from parent to offspring. By typing >3500 sperm, we determined the size distribution of Huntington's disease (HD) germline mutations produced by 26 individuals from the Venezuelan cohort with CAG/CTG repeat numbers ranging from 37 to 62. Both the mutation frequency and mean change in allele size increased with increasing somatic repeat number. The mutation frequencies averaged 82% and, for individuals with at least 50 repeats, 98%. The extraordinarily high mutation frequency levels are most consistent with a mutation process that occurs throughout germline mitotic divisions, rather than resulting from a single meiotic event. In several cases, the mean change in repeat number differed significantly among individuals with similar somatic allele sizes. This individual variation could not be attributed to age in a simple way or to ' cis ' sequences, suggesting the influence of genetic background or other factors. A familial effect is suggested in one family where both the father and son gave highly unusual spectra compared with other individuals matched for age and repeat number. A statistical model based on incomplete processing of Okazaki fragments during DNA replication was found to provide an excellent fit to the data but variation in parameter values among individuals suggests that the molecular mechanism might be more complex.

Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Disease alleles contain a trinucleotide repeat expansion of variable length, which encodes polyglutamine tracts near the amino terminus of the HD protein, huntingtin. Polyglutamine-expanded huntingtin, but not normal huntingtin, forms nuclear inclusions. We describe a Drosophila model for HD. Amino-terminal fragments of human huntingtin containing tracts of 2, 75, and 120 glutamine residues were expressed in photoreceptor neurons in the compound eye. As in human neurons, polyglutamine-expanded huntingtin induced neuronal degeneration. The age of onset and severity of neuronal degeneration correlated with repeat length, and nuclear localization of huntingtin presaged neuronal degeneration. In contrast to other cell death paradigms in Drosophila, coexpression of the viral antiapoptotic protein, P35, did not rescue the cell death phenotype induced by polyglutamine-expanded huntingtin.

Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair.

Germline mutations in the human MSH2, MLH1, PMS2 and PMS1 DNA mismatch repair (MMR) gene homologues appear to be responsible for most cases of hereditary non-polyposis colorectal cancer (HNPCC; refs 1-5). An important role for DNA replication errors in colorectal tumorigenesis has been suggested by the finding of frequent alterations in the length of specific mononucleotide tracts within genes controlling cell growth, including TGF-beta receptor type II (ref. 6), BAX (ref. 7) and APC (ref. 8). A broader role for MMR deficiency in human tumorigenesis is implicated by microsatellite instability in a fraction of sporadic tumours, including gastric, endometrial and colorectal malignancies. To better define the role of individual MMR genes in cancer susceptibility and MMR functions, we have generated mice deficient for the murine homologues of the human genes MLH1, PMS1 and PMS2. Surprisingly, we find that these mice show different tumour susceptibilities, most notably, to intestinal adenomas and adenocarcinomas, and different mutational spectra. Our results suggest that a general increase in replication errors may not be sufficient for intestinal tumour formation and that these genes share overlapping, but not identical functions.

Gene hunting without sequencing genomic clones: finding exon boundaries in cDNAs.

We propose a new experimental protocol, ExonPCR, which is able to identify exon boundaries in a cDNA even in the absence of any genomic clones. ExonPCR can bypass the isolation, characterization, and DNA sequencing of subclones of genomic DNA to determine exon boundaries: a major effort in the process of positional cloning. Given a cDNA sequence, ExonPCR uses a series of "adaptive" steps to analyze the PCR products from cDNA and genomic DNA thereby revealing the approximate positions of "hidden" exon boundaries in the cDNA. The nucleotide sequence of adjacent intronic regions is determined by ligation-mediated PCR. Primers adjacent to the "hidden" exon boundaries are used to amplify genomic DNA followed by limited DNA sequencing of the PCR product. The method was successfully tested on the 3-kb hMSH2 cDNA with 16 known exons and the 9-kb PRDII-BF1 cDNA with a previously unknown number of exons. We subsequently developed the ExonPCR algorithm and software to direct the experimental protocol using a strategy that is analogous to that used in the game "Twenty Questions." Through the use of ExonPCR, the search for disease-causing mutations can be initiated almost immediately after cDNA clones in a genetically mapped region become available. This approach would be most valuable in gene discovery strategies that focus initially on cDNA isolation.

Patchy fur, a mouse coat mutation associated with X-Y nondisjunction, maps to the pseudoautosomal boundary region.

Patchy fur is a semidominant X-linked mutation in the mouse, resulting in a sparse coat. The Paf mutation also alters the normal segregation of the X and the Y chromosomes during male meiosis by causing nondisjunction at anaphase I. Analysis of 1139 female meioses from an intersubspecific backcross using 15 PCR-based markers localizes Paf to an approximately 0.2-cM interval that includes the pseudoautosomal boundary. The meiotic nondisjunction phenotype may result from a chromosomal rearrangement that includes pseudoautosomal sequences and affects XY pairing.

The mutation properties of spinal and bulbar muscular atrophy disease alleles.

We studied the gene for the trinucleotide repeat disorder X-linked spinal and bulbar muscular atrophy (SBMA) to quantify the spectrum of mutations and gain insight into genetic anticipation. This analysis was performed using single sperm typing from an affected individual. This method allows the quantification of large numbers of meioses and therefore provides accurate information about genetic instability of the CAG repeat expansions which cause SBMA. Among 198 X chromosome-containing sperm cells, 20% had a CAG repeat number equal to the donor's somatic DNA of 49 CAG repeats, 56% were expansions, and 24% contractions. Most of the expansions (84%) and contractions (94%) were between 1 and 3 CAG repeats. These results are consistent with those obtained from one previously studied SBMA patient and reveal greater CAG repeat instability in sperm than in somatic tissue. Our results indicate that in SBMA, in contrast to sperm typing analysis of Huntington's disease, there is relative stability of the CAG repeat number during paternal transmissions and that the spectrum of mutations is narrow. These results are in agreement with the limited available clinical data and suggest that anticipation may not be a significant feature of this disease.

The effect of FMR1 CGG repeat interruptions on mutation frequency as measured by sperm typing.

Fragile X syndrome results from the unstable expansion of a CGG repeat within the FMR1 gene. Three classes of FMR1 alleles have been identified, normal alleles with 6-60 repeats, premutations with 60-200 repeats, and full mutations with > 230 repeats. Premutations are exquisitely unstable upon transmission. Normal alleles, while generally stable upon transmission, are thought to have different intrinsic mutation frequencies, such that some normal alleles may be predisposed towards expansion while others may be more resistant to such change. One variable that may account for this difference is the occurrence of one or more AGG triplets punctuating the normal CGG repeat. The AGG interruptions lead to alleles that have equivalent overall length but different lengths of perfect repeats. To test the influence of the length of perfect repeats on stability, we examined the CGG repeat of single sorted sperm from two males, each with 39 total repeats, but distinct AGG interruption patterns. Sorted sperm of each donor showed -15% variation in repeat length, consistent with previous studies of sorted sperm at other triplet repeat loci. However, when discounting the majority variation of +/-1 repeat, the male with 29 perfect repeats showed 3% expansion changes while the donor with only 19 perfect repeats had none (< 0.9%). Moreover, > 90% of all variant sperm, including all those observed with expansions, showed expansion or contraction of the 3' end of the repeat array. These data are consistent with the hypothesis that perfect repeat tracts influence the repeat stability and that changes of the FMR1 repeat exhibit polarity.