Discrimination of Deletion and Duplication Subtypes of the Deleted in Azoospermia Gene Family in the Context of Frequent Interloci Gene Conversion

<div><p>Due to its palindromic setup, AZFc (Azoospermia Factor c) region of chromosome Y is one of the most unstable regions of the human genome. It contains eight gene families expressed mainly in the testes. Several types of rearrangement resulting in changes in the cumulative copy number of the gene families were reported to be associated with diseases such as male infertility and testicular germ cell tumors. The best studied AZFc rearrangement is gr/gr deletion. Its carriers show widespread phenotypic variation from azoospermia to normospermia. This phenomenon was initially attributed to different gr/gr subtypes that would eliminate distinct members of the affected gene families. However, studies conducted to confirm this hypothesis have brought controversial results, perhaps, in part, due to the shortcomings of the utilized subtyping methodology. This proof-of-concept paper is meant to introduce here a novel method aimed at subtyping AZFc rearrangements. It is able to differentiate the partial deletion and partial duplication subtypes of the Deleted in Azoospermia (DAZ) gene family. The keystone of the method is the determination of the copy number of the gene family member-specific variant(s) in a series of sequence family variant (SFV) positions. Most importantly, we present a novel approach for the correct interpretation of the variant copy number data to determine the copy number of the individual DAZ family members in the context of frequent interloci gene conversion.Besides DAZ1/DAZ2 and DAZ3/DAZ4 deletions, not yet described rearrangements such as DAZ2/DAZ4 deletion and three duplication subtypes were also found by the utilization of the novel approach. A striking feature is the extremely high concordance among the individual data pointing to a certain type of rearrangement. In addition to being able to identify DAZ deletion subtypes more reliably than the methods used previously, this approach is the first that can discriminate DAZ duplication subtypes as well.</p></div

Flowchart showing the steps involved in the elaboration and application of variant ratio analysis.

<p>The numbered arrows stand for the processes applied, the rectangles symbolize the result of the respective process. The green rectangle shows the desired end-result of the analysis. Brown rectangles symbolize calibration data used to help derive variant ratios from AUC ratios. Red rectangles stand for input data required for stage 2 or, if there are marker associations, stage 3 analysis. Samples with different partial rearrangement types are indicated by three different shades of gray. The dashed arrow means that only deletion and duplication samples undergo stage 2 or stage 3 analysis. The large rectangle with transparent grey background contains the steps that are required for a complete analysis. The steps outside the large rectangle were used for the elaboration of the method. All processes are listed below. 1/ Aligning the sequences of the four DAZ genes extracted from the human reference genome in order to select amplifiable fragments which i) consist of four amplicons belonging to the four DAZ genes, respectively, and ii) contain as many SFV positions as possible (Fragments I and II) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.g002" target="_blank">Fig 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s001" target="_blank">S1 Fig</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s007" target="_blank">S5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s009" target="_blank">S7</a> Files, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s011" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s012" target="_blank">S2</a> Tables). 2/ Amplification of selected regions in all individual samples to get Fragments I and II. 3/ Sequencing Fragments I and II in all individual samples. 4/ Preparing control plasmid mixtures by cloning Fragment I amplified from samples selected to contain every DAZ1, DAZ2, DAZ3 and DAZ4-specific variant in order to mimic wild-type, AZFc partial deletion and AZFc partial duplication samples having <i>known</i> variant ratios at each SFV position (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s014" target="_blank">S4 Table</a>). 5/ Amplification of selected regions to get Fragment I in control mixtures. 6/ Sequencing Fragment I in control mixtures. 7/ Measuring AUCs by ImageJ software and calculating the AUC ratio at each SFV position in every control mixture. 8/ Correlating the AUC ratios measured in control mixtures with known variant ratios (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s015" target="_blank">S5 Table</a>). 9/ Measuring AUCs and calculating the AUC ratio at each SFV position in all individual samples. 10/ Plotting AUC ratios throughout all samples for each studied position to visualize AUC ratio clustering (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.g003" target="_blank">Fig 3</a>). 11/ Assigning a variant ratio to each SFV position in all individual samples. 12/ Determining the horizontal variant ratio distribution in all individual samples. 13/ Grouping samples according to AZFc partial deletion/duplication status based on their horizontal variant ratio distribution (Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t004" target="_blank">4</a>). The results of this step were validated by a generally accepted DAZ dosage test (not shown) and two multiplex PCRs amplifying six sY markers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s002" target="_blank">S2 Fig</a>). 14/ Specifying the variant ratios that remained ambiguous on the basis of the AUC ratio (electropherogram picture) in step 11 (0:2x, x:x and 2x:0 positions). 15/ Deducing the copy number of the specific variant at each SFV position in all individual samples from the relevant variant ratio. 16/ Cloning Fragments I and II in four selected control samples to separate the four amplicons derived from the four DAZ family members, respectively. 17/ Sequencing an appropriate number of colonies in order to study the co-segregation of DAZ family member-specific variants, i.e. the fulfillment of requirement (c) imposed on an ideal marker (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s016" target="_blank">S6 Table</a>).18/ Determining the vertical variant ratio distribution throughout all control samples at each studied SFV position. 19/ Determining p1 and p2 values on the basis of the vertical variant ratio distribution at each SFV position within the control panel in order to study the fulfillment of requirements (a) and (b) also imposed on an ideal marker (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t004" target="_blank">Table 4</a>, the equations are seen in the text). 20/ Classifying family member-specific variants on the basis of p1 and p2, which results in the distinction of class I, class II/a, class II/b and class III markers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t005" target="_blank">Table 5</a>). 21/ Determining the relationship between the copy number of a gene family member-specific variant and the copy number of the corresponding gene family member for class II/a and class II/b markers. It results in the “restricted” applicability of the markers that will be utilized at stage 2 and stage 3 analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s003" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s004" target="_blank">S2</a> Files, Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t007" target="_blank">7</a>). 22/ Searching for perfect associations between variants specific to different DAZ family members in the control panel in order to re-evaluate (extend) the applicability of some markers if possible (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t004" target="_blank">Table 4</a>). 23/ Determining the relationship between the copy number of the members of the associated marker pairs and the copy number of the corresponding gene family members for the relevant class II/a and class II/b markers, which results in their “extended” applicability that will be utilized at stage 3 analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s005" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s006" target="_blank">S4</a> Files, Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t008" target="_blank">8</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t009" target="_blank">9</a>). 24/ Evaluating partial deletion and partial duplication samples identified in step 13 using the classified markers with restricted applicability (stage 2 and 3) or, if exists, extended applicability (stage 3) to determine the samples’ deletion and duplication subtype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s018" target="_blank">S8</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s019" target="_blank">S9</a> Tables, Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.t003" target="_blank">3</a>). 25/ Comparing the series of the variant ratios (variant ratio haplotype, VRH) of the deletion and duplication samples with the VRHs observed in the control panel in order to support the existence of the assigned rearrangement subtypes in the studied population (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s017" target="_blank">S7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s021" target="_blank">S11</a> Tables). 26/ Checking if the assigned rearrangement subtypes are concordant with or might even be indicated by the variant ratios determined at positions also examined but not utilized for stage 2 and stage 3 analyses for some reason (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s020" target="_blank">S10 Table</a>).</p

Clustering of AUC ratios at two SFV positions in fifty-two samples.

<p>The fifty-two samples sequenced form five distinct clusters according to the AUC ratio measured at SVF positions 1926 (<b>A</b>) and 1702 (<b>C</b>). Samples 1–5 are the duplication samples (referred to as Ydup_01–05 in the text), whereas samples 6–13 are the deletion samples (referred to as Ydel_06–13). Samples 14–52, each having one copy of all four DAZ family members, constitute the control panel. Representative electropherogram pictures of SVF positions 1926 (<b>B</b>) and 1702 (<b>D</b>) obtained in samples belonging to the distinct clusters are shown. The average (AUC ratio_av) and standard deviation (StDev) of the AUC ratio values were calculated from all samples belonging to a cluster. Comparing with the AUC ratio–variant ratio relationship determined in control mixes, a variant ratio was assigned to each cluster at both positions. The AUC ratio (presented here as percentage) calculation is described in the Methods section.</p

Structure of the AZFc region with the eight gene families.

<p>The colored arrows show the direct and inverted repeats of AZFc. The colored arrowheads indicate the members of the eight gene families located in the region. The two rectangles enclose the gene family members eliminated by gr/gr deletion with two different breakpoints (g1/g2 versus r2/r4 deletion), respectively. The STS markers that have usually been analyzed in search for deletions are also shown.</p

Schematic representation of the four DAZ family members along with the studied markers.

<p>Panel A: DAZ1 and DAZ2. Panel B: DAZ3 and DAZ4. The figure was captured from the UCSC Genome Browser after uploading an appropriate BED file (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163936#pone.0163936.s009" target="_blank">S7 File</a>). Below the scale, chromosome Y coordinates according to the human genome build NCBI36/hg18 are shown. Below the RefSeq Genes label, the structure of the DAZ family members are illustrated with the exons indicated by perpendicular lines. The aligned arrowheads in the thick green line show the direction of the coding sequence. Variants presented in red and blue colors are specific and non-specific for the corresponding DAZ family members, respectively. For non-specific variants, a digit in square brackets indicates the DAZ family member for which the relevant SFV position contains a specific variant. The numbers in the variants' name indicate the variants' location in Fragments I or II.</p

The human reference genome-based characteristics of the studied SFV positions and their variant ratios found in eight partial deletion samples.

<p>The human reference genome-based characteristics of the studied SFV positions and their variant ratios found in eight partial deletion samples.</p

Classification of DAZ family member-specific markers.

<p>Classification of DAZ family member-specific markers.</p

Restricted applicability of class II/a markers.

<p>Restricted applicability of class II/a markers.</p