Supplementary Material for: Novel Nonsense Mutation in the <b><i>NLRP7</i></b> Gene Associated with Recurrent Hydatidiform Mole

<b><i>Aim:</i></b> This study aimed to clarify the genetic and epigenetic features of recurrent hydatidiform mole (RHM) in Japanese patients. <b><i>Methods:</i></b> Four Japanese isolated RHM cases were analyzed using whole-exome sequencing. Villi from RHMs were collected by laser microdissection for genotyping and DNA methylation assay of differentially methylated regions (DMRs). Single nucleotide polymorphisms of <i>PEG3</i> and <i>H19</i> DMRs were used to confirm the parental origin of the variants. <b><i>Results:</i></b> A novel homozygous nonsense mutation in <i>NLRP7</i> (c.584G>A; p.W195X) was identified in 1 patient. Genotyping of one of her molar tissue revealed that it was biparental but not androgenetic in origin. Despite the fact that the RHM is biparental, maternally methylated DMRs of <i>PEG3</i>, <i>SNRPN</i> and <i>PEG10</i> showed complete loss of DNA methylation. A paternally methylated DMR of <i>H19</i> retained normal methylation. <b><i>Conclusions:</i></b> This is the first Japanese case of RHM with a novel homozygous nonsense <i>NLRP7</i> mutation and a specific loss of maternal DNA methylation of DMRs. Notably, the mutation was identified in an isolated case of an ethnic background that has not previously been studied in this context. Our data underscore the involvement of <i>NLRP7</i> in RHM pathophysiology and confirm that DNA methylation of specific regions is critical

Supplementary Material for: Microhomology-Mediated Microduplication in the Y Chromosomal Azoospermia Factor a Region in a Male with Mild Asthenozoospermia

Y chromosomal azoospermia factor (AZF) regions AZFa, AZFb and AZFc represent hotspots for copy number variations (CNVs) in the human genome; yet the number of reports of AZFa-linked duplications remains limited. Nonallelic homologous recombination has been proposed as the underlying mechanism of CNVs in AZF regions. In this study, we identified a hitherto unreported microduplication in the AZFa region in a Japanese male individual. The 629,812-bp duplication contained 22 of 46 exons of <i>USP9Y</i>, encoding the putative fine tuner of spermatogenesis, together with all exons of 3 other genes/pseudogenes. The breakpoints of the duplication resided in the DNA/TcMar-Tigger repeat and nonrepeat sequences, respectively, and were associated with a 2-bp microhomology, but not with short nucleotide stretches. The breakpoint-flanking regions were not enriched with GC content, palindromes, or noncanonical DNA structures. Semen analysis of the individual revealed a normal sperm concentration and mildly reduced sperm motility. The paternal DNA sample of the individual was not available for genetic analysis. The results indicate that CNVs in AZF regions can be generated by microhomology-mediated break-induced replication in the absence of known rearrangement-inducing DNA features. AZFa-linked microduplications likely permit production of a normal amount of sperm, although the precise clinical consequences of these CNVs await further investigation