Analysis of DAZ gene expression in a partial AZFc deletion of the human Y chromosome Byunghyuk Kim A , Wonkyung Lee A , Kunsoo Rhee A,C , Soo Woong Kim B and Jae-Seung Paick B,C A Department of Biological Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-747, Korea. B Department of Urology, College of Medicine, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul 110-799, Korea. C Corresponding authors. Emails: rheek@snu.ac.kr; jspaick@snu.ac.kr Abstract. The azoospermia factor c (AZFc) region of the Y chromosome consists of repetitive amplicons and is therefore highly susceptible to structural rearrangements, such as deletions and duplications. The b2/b3 deletion is a partial AZFc deletion that is conventionally determined by the selective absence of sY1191 in sequence-tagged site polymerase chain reaction (PCR) and is generally believed to retain two of the four deleted in azoospermia (DAZ) genes on the Y chromosome. In the present study we determined the copy number and expression of DAZ genes in sY1191-negative individuals. Using a DAZ dosage PCR assay and Southern blot analysis we evaluated the expression of four DAZ genes in five of six sY1191-negative individuals. Furthermore, cloning and immunoblot analyses revealed that three or more DAZ genes are expressed in sY1191-negative testes with germ cells. The results indicate that the selective absence of sY1191 not only means b2/b3 deletion with two DAZ genes, but also includes another AZFc configuration with four DAZ genes. These results exemplify the prevalence of variations in the AZFc region of the human Y chromosome. Additional keywords: male infertility, microdeletion, testis. Received 6 September 2012, accepted 10 January 2013, published online 20 February 2013 Introduction The azoospermia factor c (AZFc) locus of the human Y chromosome contains massive sequence repeats called ampli- cons (Kuroda-Kawaguchi et al. 2001). The amplicons are organised in palindromic domains that harbour a cohort of genes for spermatogenesis (Kuroda-Kawaguchi et al. 2001). Because the ampliconic domains are highly susceptible to intra- and inter-chromosomal recombinations, microdeletions frequently occur in the AZFc region, often leading to spermatogenic failure in men (Kuroda-Kawaguchi et al. 2001; Skaletsky et al. 2003). For example, total deletion of AZFc results from recombination of the b2 and b4 domains, and spermatogenic failure is fre- quently observed in individuals with b2/b4 deletions (Reijo et al. 1995; Kuroda-Kawaguchi et al. 2001). The gr/gr deletion eliminates 1.6 Mb of sequence in the AZFc region and is con- sidered to be a significant risk factor for impaired spermato- genesis (Repping et al. 2003). The b2/b3 deletion is another frequent deletion pattern in which an inversion and homologous recombination may occur sequentially within the AZFc locus (Fernandes et al. 2004; Repping et al. 2004). However, the effects of partial deletions on male fertility remain contentious. For example, some researchers have found an association between the b2/b3 deletion and infertility (Wu et al. 2007; Lu et al. 2009; Eloualid et al. 2012), whereas others have not (Repping et al. 2004; Hucklenbroich et al. 2005). Sequence redundancy in AZFc makes it difficult to analyse the deletions with direct sequencing. Instead, unique sequence- tagged sites (STSs) at the AZFc region have been widely used to detect partial deletions within the region. For example, the gr/gr and b2/b3 deletions are determined in individuals with a selec- tive absence of STS markers sY1291 and sY1191, respectively (Repping et al. 2003, 2004). The AZFc configuration resulting from these deletions has been further supported by the results of interphase fluorescent in situ hybridisation (FISH), Southern blot analyses and sequence family variant (SFV)-based assays (Fernandes et al. 2002, 2004; Repping et al. 2003, 2004; Lin et al. 2005, 2006). In this context, several association studies for the phenotypic consequences of partial AZFc deletions have relied largely on STS-PCR assays to detect such deletions (Hucklenbroich et al. 2005; Lynch et al. 2005; Ravel et al. 2006; Eloualid et al. 2012). However, this simple method may not provide information as to exactly which genes or gene copies are deleted in the chromosomes. Deleted in azoospermia (DAZ) is a well known male infertil- ity gene in the AZFc region of the Y chromosome (Saxena et al. 1996). DAZ and two autosomal homologues, namely deleted in CSIRO PUBLISHING Reproduction, Fertility and Development, 2014, 26, 307–315 http://dx.doi.org/10.1071/RD12290 Journal compilation Ó CSIRO 2014 www.publish.csiro.au/journals/rfd