High rates of de novo 15q11q13 inversions in human spermatozoa

Clone selection and positioning

Three BAC clones were selected using the resources of the Genome Browser database (UCSC Assembly; February 2009) [19] for triple-color FISH experiments on decondensed sperm-nuclei: one proximal BAC clone (RP11-1122J3) mapping between the LCR15-1 and LCR15-2, and two distal clones (RP11-322N14 and RP11- 230M20) on either side of the LCR15-3 (Figure 2a).

The positions of the selected probes were verified on metaphase spreads. Every single probe showed specific signals on the pericentromeric area of chromosome 15 (identified by DAPI banding), and no cross-hybridization was observed.

Experimental design

Our design implies that two probes would potentially change their positions (RP11-1122J3 and RP11-322N14) while the third probe remains in place (RP11-230M20). This allowed us to unequivocally discriminate between normal and inverted haplotypes. According to the distribution of signals in the informative sperm nuclei, the following genotypes were assigned:

The presence of normal and inverted haplotypes was confirmed by confocal laser- scanning microscopy (Figure 2).

Sperm-FISH results

The hybridization efficiency of every single probe in sperm nuclei was higher than 98%.

A total of 10,292 informative sperm nuclei from the control population were analyzed (Table 2). The mean frequency of Type-1 inversions was 3.59%, ranging from 1.29% to 5.32%, with a standard error of the mean (S.E.M) of 0.46%. The mean frequency of Type-2 inversions was 9.51% (± 0.87% SEM), ranging from 6.47% to 14.06%.

In fathers of PWS individuals, a total of 16,545 informative sperm nuclei were analyzed (Table 3). The mean frequency of Type-1 inversions was 3.06% (± 0.33% SEM), ranging from 1.37% to 5.73%. The mean frequency of Type-2 inversions was 10.07% (± 0.73% SEM), ranging from 5.39% to 15.02%.

Frequencies of Type-2 inversions were significantly higher than the frequencies of Type-1 inversions in both the control population (P = 0.005) and in the PWS fathers (P = 0.0001). Moreover, a significant correlation was observed between the frequencies of Type-1 and Type-2 inversions (r = 0.55; P = 0.004) (Figure 3a).

No significant differences were observed for either the frequencies of Type-1 or Type-2 inversions between the PWS fathers and the controls (P > 0.05) (Figure 3b). Nevertheless, individual comparisons showed higher incidences of inversions in four PWS fathers due to increases of Type-2 inversions: PW-5, PW-10 and PW-13 (P < 0.05), or increases in both types of inversions: PW-1 (P < 0.05) (Table 3) (Figure 3c). It deserves to be mentioned that significant increases of 15q11q13 deletions were previously reported in all these four individuals [18]. No significant correlation was observed between the frequencies of 15q11q13 inversions and age (P > 0.05).

When considering the frequency of the different stable products resulting from intrachromatid NAHR events, that is, inversions and deletions (data previously reported [18]), no correlation was observed in either the control population (P = 0.121) or in the PWS fathers. However, a tendency to correlate (r = 0.69; P = 0.062) was observed in the PWS fathers.

Experimental design

Sperm-FISH analyses have been widely used for cytogenetic studies in spermatozoa [20] and have been mainly focused on evaluating the genetic reproductive risk of carriers of abnormal karyotypes [21] and infertile patients [22]. Furthermore, and more recently, we have demonstrated the reliability of this methodology to assess the rates of deletions and duplications in spermatozoa [18]. In all of these approaches, the chromosomal constitution of the sperm nuclei is inferred by the evaluation of the presence or absence of a given FISH signal.

In the present work, the potential of sperm-FISH to detect the inversions has been evaluated. Our results confirm that this approach is feasible and realistic if an accurate experimental design is used, taking into consideration the relation between the genetic distances and the physical distances of FISH signals in the interphase nucleus.

Our experimental design has led to the observation of two types of signal distributions: two plus one (2+1) and associations of three. As the images of the three signals in association could be the consequence of chromatin loops resulting from high-order packaging of interphase DNA, all of the nuclei showing this distribution were classified as uninformative and were discarded for haplotype evaluation. The 2+1 distributions were assigned to three different haplotypes: normal, Type-1 and Type-2 inversions. In any case, the two associated probes were always located closer than 1 Mb and the probe apart was separated by a distance longer than 5 Mb. It is well-known that the separation of interphase FISH signals increases as the genomic distance between the probes becomes larger, and this correlation is consistent for genomic separations up to 2 Mb [23]. As a consequence, the three signals in our experimental design will appear as specific distributions of 2 associated dots plus 1 separated dot.

Types of 15q11q13 inversions

The results obtained point to the existence of two types of inversions that involve the segments corresponding to the reported Class-I and Class-II 15q11q13 deletions. Our data is in agreement with the existence of the 15q11q13 inversions between the LCR15-2 and 3 (Type-2) previously described by other authors [12]. Furthermore, our experimental approach allowed us to describe a new inversion involving the segment between LCR15-1 and 3 (Type-1 inversion). As in the case of deletions, the most frequent inversion was the Type-2 inversion. Altogether, these data suggest that the LCR15-2 could harbor longer stretches of homology than LCR15-1 with the LCR15-3, making the first more susceptible to NAHR events as was previously inferred by Makoff and Flomen [9]

Susceptibility to generate 15q11q13 inversions

Unexpectedly high rates of 15q11q13 inversions were found in spermatozoa from the two populations studied. Two pieces of evidence indicate that these inversions are generated de novo: 1) Since the frequency of inversions was not close to 50% in any case, the possibility of having any constitutional heterozygote inversion carrier can be ruled out. 2) Another possibility is the presence of a mosaicism for the inversion restricted to the germ line. Two pieces of data make such a possibility unlikely. First, inversions are considered to be a predisposing haplotype that increases the risk of secondary rearrangements such as deletions and duplications. While the incidence of inversion was consistently high in all control and PWS fathers, most of them did not show increases of deletions. Second, if mosaicism is present in testicular tissue, we would expect that the degree would be different among individuals generating different percentages of sperm inversions. Instead, as mentioned above, the percentage of sperm inversions was consistent (Tables 2 and 3).

The high rate of 15q11q13 inversions in spermatozoa suggests that a certain percentage of heterozygous carriers are present among the general population. Taking into account the frequency of sperm inversions observed in the present study (Type-1 and Type-2 inversions: 13.1%) and assuming that the frequency of these inversions would be the same in oocytes, we can infer that the frequency of heterozygous carriers in the general population would be 22.6% (2 × 13/100 × 87/100). These results are in agreement with the 9% of inv(15)(q11q13) heterozygous carriers (4 out of 44 individuals) found in the control population assessed by Gimelli et al.(2003) [12]. This situation has already been described in population studies analyzing other regions with similar features such as 7q11.23 and 8p23.1, where inversions were observed in 5.8% and 79%, respectively [24, 25]. In this sense, it seems likely that the frequency of inversions in regions with a genomic architecture characterized as being flanked by LCR could be as high as the one described in the 15q11-q13 region, and this would strengthen the hypothesis that the frequencies of inversions are underestimated within the great deal of structural variants of the human genome [6].

This situation might have important implications at a population level. As was previously suggested, the presence of an inverted chromosome in heterozygosity originates an unpaired region at pachytene making the region prone to misalignment and NAHR. Thus, the risk for a secondary rearrangement affecting the offspring could be increased [6, 12, 13]. Consequently, heterozygote carriers of any of these types of inversions could have an increased risk of transmission of 15q11q13 deletions in descendants.

Intrachromatid NAHR is the major NAHR mechanism

Results obtained in the present work showed higher frequencies of 15q11q13 inversions than the frequencies of deletions and duplications previously reported in controls and PWS fathers [18], thus indicating that intrachromatid NAHR is the most frequent mechanism. Some PWS fathers showed significantly increased rates of inversions, as compared with the control population (Table 3). Strikingly, all of them were previously reported to have significant increases of 15q11q13 deletions [18]. These data point out the important participation of intrachromatid NAHR in the generation of 15q11-q13 anomalies. As a whole, our results suggest that individuals at risk (those showing significant increases of 15q11q13 anomalies in spermatozoa) have a disrupted intrachromatid NAHR mechanism, as only the products derived from this mechanism are significantly increased.

Since no constitutional heterozygous carriers of inversions were detected in any of the subjects analyzed, and different outcomes were found to be increased in spermatozoa of some PWS fathers (15q11q13 deletions or both 15q11q13 deletions and inversions), differences in the genomic architecture of the LCRs flanking this region might exist. Some authors have reported structural variation in the LCRs flanking some regions involved in genomic disorders [26, 27]. They suggested that some specific haplotypes within these LCRs could predispose those regions to misalignment between paralogous copies and thus predispose the region to NAHR, increasing the risk of transmission of genomic disorders. Our results suggest that, in fact, differences in the genomic architecture of the LCR15s predispose these individuals to intrachromatid NAHR, thus producing different rearrangements. Some individuals would show haplotypes with longer stretches of direct homology between paralogous LCRs, thus increasing the rates of deletions in spermatozoa, whereas other individuals might have haplotypes with higher degrees of direct and inverted homologies with their own paralogous copies, thus increasing both the rates of de novo deletions and inversions in spermatozoa. In this sense, the availability of methodologies that enable studying the genomic architecture of specific LCRs would allow for identifying possible predisposing haplotypes that would explain these differences and that would potentially increase the risk of transmission of 15q11q13 anomalies.

Biological samples

Semen samples from 10 control donors aged between 24 and 50, and 16 PWS fathers from 32 to 60 years of age were obtained. Donors were volunteers recruited from the general population. All subjects had normal karyotypes and were normozoospermic. To our knowledge, none of them had been exposed to genotoxic agents, and no history of chemotherapy, radiotherapy or chronic illness was recorded.

All subjects gave their informed consent in writing to participate in the study and the protocol was approved by the Institutional Ethics Committee of the Universitat Autònoma de Barcelona.

Slide preparation

Samples were processed as described previously by our group [28]. Briefly, the sperm fraction was resuspended in hypotonic solution (0.075 M KCl) for 30 minutes at 37°C and fixed in methanol:acetic acid (3:1). Spermatozoa were spread on a slide and kept at -20°C until processed.

Probes

Three BAC clones were selected using the resources of the Genome Browser database (UCSC Assembly; February 2009). All clones were obtained from the Children's Hospital Oakland Research Institute, BACPAC resources (Oakland, CA 94609 USA). BAC DNA extraction was performed using the QIAprep Miniprep kit (Qiagen GmbH; Hilden, Germany) following manufacturer's instructions.

BAC clones RP11-1122J3, RP11-322N14 and RP11-230M20 were fluorescently labeled with Spectrum Green-dUTPs (Abbott Molecular; Abbott Park, IL, USA), Spectrum Orange-dUTPs (Abbott Molecular) and DEAC-dUTPs (Perkin Elmer Inc; Boston, USA), respectively, by Nick Translation (Roche; Mannheim, Germany). Probes were mixed with 10 μg of Cot 1 DNA (Invitrogen; Carlsbad, USA), ethanol precipitated and resuspended in hybridization buffer (50% Formamide, 1xSSC and 10% dextran sulphate) (Abbott Molecular).

Probe positions were verified on lymphocyte metaphase chromosomes. Hybridization in spermatozoa was determined by the evaluation of 1,000 sperm nuclei per probe in three different FISH experiments.

Fluorescence in situ Hybridization on sperm (sperm-FISH)

Prior to hybridization, sperm nuclei were decondensed by slide incubation at 37°C in Tris buffer containing 25 mmol/ml dithiothreitol and 1% Triton X-100 for 45 minutes.

A triple-color FISH using the three BAC clones differentially labeled was performed following standard procedures. Briefly, probes were denatured at 80°C for 8 minutes and pre-annealed at 37°C for 15 minutes. Sperm nuclei were denatured at 73°C in 70% formamide in 2xSSC for 5 minutes. Hybridization was carried out by adding 5 μl of the corresponding probe mixture (200 ng of each probe) to the sperm preparation and incubating the slides in a moist chamber at 37°C for 48 hours. Post-hybridization washes were performed in 1xSSC with 0.3% NP-40 at 73°C followed by 2xSSC with 0.1% NP-40 at room temperature, for one minute in each solution. Slides were mounted with antifade solution (Abbott Molecular).

Analyses were performed using an Olympus BX-60 epifluorescence microscope equipped with a triple-band pass filter, and specific filters for Aqua, FITC and Cy3.

A minimum of 1,000 informative sperm nuclei were analyzed per sample.

Confocal Laser-Scanning Microscopy

To confirm the existence of normal and inverted haplotypes, 80 sperm-nuclei were captured and analyzed using a confocal-laser scanning TCS-SP5 AOBS microscope (Leica Microsystems, Heidelberg Gmbh; Mannheim, Germany).

Spectrum Orange fluorochromes were excited with the 561-nm line of a DPSS laser and observed in the red channel at an emission range of 569-nm to 671-nm. Spectrum Green fluorochromes were excited with the 405-nm line of a diode laser and viewed in the green channel at 502-nm to 551-nm. Finally, DEAC fluorochromes were excited with the 488-nm line of an Argon laser and observed in the blue channel at an emission range of 444-nm to 500-nm. Stacks of 16 to 20 sections every 0.3 μm were acquired.

Image combining and processing were performed with the IMARIS software package version 2.7 (Bitplane AG; Zürich, Switzerland).

Data Analyses

Data obtained were statistically analyzed using SPSS version 15.0 (SPSS Inc; Chicago, IL, USA) under the advice of the statistical service of the Universitat Autònoma de Barcelona.

To assess the relationship between the two inverted haplotypes, the following analyses were performed:

To assess the susceptibility of the 15q11-q13 region to generate inversions, the following statistical tests were performed:

To assess a possible relationship between the frequency of inversions and the frequency of deletions previously found by our group [18]:

Differences and correlations were considered statistically significant when P < 0.05.