LETTERS TO THE EDITOR Y-chromosomemicrodeletions are not associated with SHOX haploinsufficiency

Dear Sir, Dr Chianese et al. (2013) expressed concerns about their results reported in ‘Y-chromosome microdeletions are not associated with SHOX haploinsufficiency’ and our earlier publication ‘Aberrations in pseudoautosomal regions (PARs) found in infertile men with Y-chromosome microdeletions’ (Jorgez et al., 2011). We wish to respond to several of their statements regarding our work in hopes of clarifying our findings. Deletions of Yq and isodicentric Y-chromosomes are two relatively common structural abnormalities affecting male fertility. Deletions of distal Yq frequently include pseudoautosomal region 2 (PAR2) while the isodicentric-Y is characterized by duplication of PAR1 and sex determining region of the Y-chromosome (SRY) and deletion of PAR2 (Lange et al., 2009).We evaluated 87menwith Y-chromosomemicrodeletions by array comparative genomic hybridization (aCGH) and/or qPCR for PARs abnormalities. Thirteenmen (15%) had an abnormal karyotype involving the Y-chromosome; they had PAR abnormalities. In addition, seven men with a normal karyotype also displayed PAR abnormalities. The Chianese et al.’s study included 11 patients (6%) with abnormal karyotypes, but PAR-copy number variations (CNVs) were reported in five patients. If the karyotype of the remaining six involved Y-chromosome aberrations, then PAR-CNVs should be evident. Also, if an isodicentric Y-chromosome is present, then CNVs should be present in both PARs. Since PAR-CNVs were not reported, the sensitivity of the methods employed is a concern and may explain the difference in the results since the majority of their patients were analyzed by qPCR only. Chianese et al. stated that their discrepancy with our data may be due to methodological issues although no specific problems were mentioned. Our study included a complete analysis of high density Xand Y-chromosomeaCGHperformed in 25menwith non-obstructive azoospermia (NOA).We confirmed the CNVs detected by aCGH and identified CNVs using qPCR for at least three genes in each PAR and the SRY gene in the additional patients. Chianese et al. analyzed only SHOX gene CNVs in the majority of samples studied. Therefore, it is possible that PAR microdeletions or microduplications not encompassing SHOX were missed. Notably, one of our patients had a PAR1 duplication that did not encompass SHOX and this was confirmed by both aCGH and SHOX qPCR-analysis. A sub-set of NOA-men in our study with Y-microdeletion andnormal karyotype (5.4%,4/74)hadPAR1deletions that included SHOX (height not available). Chianese et al. identified men with a normal karyotype (2/177), normal height and SHOXCNVs (duplications) but at a lower frequency (1.1%). The normal height of these two individuals is not surprising as individuals with SHOX duplications have variable height. Chianese et al.mentioned that our patients’ phenotype was not fully described. Our paper states that patients were evaluated according to the American Urological Association Practice Guidelines (Gangel, 2002).Diagnosiswas idiopathicNOAprior toY-chromosomemicrodeletion testing. Our paper focused on SHOX aberrations as a co-existing genomic syndromebecause height is an easilymeasured physical characteristic. Dosage changes of other PAR genes are associated with clear phenotypic anomalies, such as intellectual disability, but these characteristics may be difficult to ascertain. In our study, height was noted in the charts for only seven patients, with three men ,10th percentile and one male .95th percentile (outside defined ranges of normalcy). One Y-isodicentric man displayed the typical body habitus and arm deformities characteristic of SHOX syndrome. Over 200 individualswhowere not infertile (C.J. Jorgez andD.J. Lamb, unpublished) and 112 normospermic, but apparently infertile, controls by Chianese et al. did not exhibit CNVs in SHOX. SHOX-CNVs were present only in Y-chromosome microdeleted patients. The Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER database) (https://decipher.sanger.ac.uk/) indicates that 35 of 29 626 subjects (0.11%) have SHOX CNVs. Of those patients with a phenotype described, 89% have intellectual disability.OneDECIPERmalewith a PAR1-gain andYq-deletion had azoospermia, as well as glaucoma and abnormalities of pyramidal motor function. Thus, SHOXCNVs rarely occur in the general population, which further supports the significance of identifying PAR-CNVs in Y-chromosome microdeleted patients. In conclusion,wearepleased that the findingsofChianese et al., essentially confirm our earlier publication. Molecular studies using aCGH and CNV-qPCR of patients with Y-chromosome microdeletions allow the identification of additional, previously unrecognized Y structural variations in NOA men. Additional Y-chromosome microdeletion men should be tested for PAR CNVs to define the incidence with certainty. Although the function of many PAR genes is under investigation, there may be consequences for male offspring of NOA men with PAR rearrangements who conceive using assisted reproductive technologies. Accordingly, further studies are needed to better understand the risks that may be present and this is an area where Chianese et al. and our laboratory certainly agree.