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In Apis mellifera , csd is the primary gene involved in sex determination: haploid hemizygous eggs develop as drones, while females develop from eggs heterozygous for the csd gene. If diploid eggs are homozygous for the csd gene, diploid drones will develop, but will be eaten by worker bees before they are born. Therefore, high csd allelic diversity is a priority for colony survival and breeding. This study aims to investigate the variability of the hypervariable region (HVR) of the csd gene in bees sampled in an apiary under a selection scheme. To this end, an existing dataset of 100 whole-genome sequences was analyzed with a validated pipeline based on de novo assembly of sequences within the HVR region. In total, 102 allelic sequences were reconstructed and translated into amino acid sequences. Among these, 47 different alleles were identified, 44 of which had previously been observed, while 3 are novel alleles. The results show a high variability in the csd region in this breeding population of honeybees.

Sexual regulation in Apis mellifera is controlled by the complementary sex-determiner (csd) gene: females (queens and workers) are heterozygous at this locus and males (drones) are hemizygous. When homozygous diploid drones develop, they are eaten by worker bees. High csd allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate csd variability in the genomic sequence of the hypervariable region (HVR) of the csd gene in honeybee subspecies sampled in Italy. During the summer of 2017 and 2018, worker bees belonging to 125 colonies were sampled. The honeybees belonged to seven different A. mellifera subspecies: A. m. ligustica , A. m. sicula , A. m cecropia , A. m. carnica , A. m. mellifera , Buckfast and hybrid Carnica. Illumina genomic resequencing of all samples was performed and used for the characterization of global variability among colonies. In this work, a pipeline using existing resequencing data to explore the csd gene allelic variants present in the subspecies collection, based on de novo assembly of sequences falling within the HVR region, is described. On the whole, 138 allelic sequences were successfully reconstructed. Among these, 88 different alleles were identified, 68 of which match with csd alleles present in the NCBI GenBank database.

This work describes the application of NGS for molecular diagnosis of RP in a family with a history of severe hypovision. In particular, the proband received a clinical diagnosis of RP on the basis of medical, instrumental examinations and his family history. The proband was subjected to NGS, utilizing a customized panel including 24 genes associated with RP and other retinal dystrophies. The NGS analysis revealed a novel missense variant (c.668T > A, I223N) in PRPH2 gene, which was investigated by segregation and bioinformatic analysis. The variant is located in the D2 loop domain of PRPH2, which is critical for protein activity. Bioinformatic analysis described the c.668T > A as a likely pathogenic variant. Moreover, a 3D model prediction was performed to better characterize the impact of the variant on the protein, reporting a disruption of the α-helical structures. As a result, the variant protein showed a substantially different conformation with respect to the wild-type PRPH2. The identified variant may therefore affect the oligomerization ability of the D2 loop and, ultimately, hamper PRPH2 proper functioning and localization. In conclusion, PRPH2 _c.668T > A provided a molecular explanation of RP symptomatology, highlighting the clinical utility of NGS panels to facilitate genotype-phenotype correlations.