A stepwise strategy for rapid and cost-effective RB1 screening in Indian retinoblastoma patients.

India has the highest number of retinoblastoma (RB) patients among the developing countries owing to its increasing population. Of the patients with RB, about 40% have the heritable form of the disease, making genetic analysis of the RB1 gene an integral part of disease management. However, given the large size of the RB1 gene with its widely dispersed exons and no reported hotspots, genetic testing can be cumbersome. To overcome this problem, we have developed a rapid screening strategy by prioritizing the order of exons to be analyzed, based on the frequency of nonsense mutations, deletions and duplications reported in the RB1-Leiden Open Variation Database and published literature on Indian patients. Using this strategy for genetic analysis, mutations were identified in 76% of patients in half the actual time and one third of the cost. This reduction in time and cost will allow for better risk prediction for siblings and offspring, thereby facilitating genetic counseling for families, especially in developing countries.

Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma.

BACKGROUND: The spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB. METHODS: Thirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families. RESULTS: Using our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study. CONCLUSIONS: We demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.

Genomic differences and species delimitation: a case for two species in the zoonotic cestode Dipylidium caninum.

Dipylidium caninum (Linnaeus, 1758) is a common zoonotic cestode of dogs and cats worldwide. Previous studies have demonstrated the existence of largely host associated canine and feline genotypes based on infection studies, genetic differences at the nuclear 28S rDNA gene and complete mitochondrial genomes. There have been no comparative studies at a genome-wide scale. Here, we sequenced the genomes of a dog and cat isolate of Dipylidium caninum from the United States using the Illumina platform and conducted comparative analyses with the reference draft genome. Complete mitochondrial genomes were used to confirm the genotypes of the isolates. D. caninum canine and feline genomes generated in this study had mean coverage depths of 45x and 26x and an average identity of 98% and 89% respectively when compared to the reference genome. SNPs were 20 times higher in the feline isolate. Comparison and species delimitation using universally conserved orthologs and protein coding mitochondrial genes revealed that the canine and feline isolates are different species. Data from this study builds a base for future integrative taxonomy. Further genomic studies from geographically diverse populations are necessary to understand implications for taxonomy, epidemiology, veterinary clinical medicine, and anthelmintic resistance.

Comparative Genomic Analysis and Species Delimitation: A Case for Two Species in the Zoonotic Cestode Dipylidium caninum.

Dipylidium caninum (Linnaeus, 1758) is a common zoonotic cestode of dogs and cats worldwide. Previous studies have demonstrated the existence of largely host-associated canine and feline genotypes based on infection studies, differences at the 28S rDNA gene, and complete mitochondrial genomes. There have been no comparative genome-wide studies. Here, we sequenced the genomes of a dog and cat isolate of Dipylidium caninum from the United States using the Illumina platform at mean coverage depths of 45× and 26× and conducted comparative analyses with the reference draft genome. Complete mitochondrial genomes were used to confirm the genotypes of the isolates. Genomes of D. caninum canine and feline genotypes generated in this study, had an average identity of 98% and 89%, respectively, when compared to the reference genome. SNPs were 20 times higher in the feline isolate. Comparison and species delimitation using universally conserved orthologs and protein-coding mitochondrial genes revealed that the canine and feline isolates are different species. Data from this study build a base for future integrative taxonomy. Further genomic studies from geographically diverse populations are necessary to understand implications for taxonomy, epidemiology, veterinary clinical medicine, and anthelmintic resistance.

Retinoblastoma discordance in families with twins.

Retinoblastoma has an increased inheritance risk of germline RB1 mutations in offspring and siblings, especially twins. Three families, each having one retinoblastoma-affected twin, were selected for genetic analysis and DNA profiling. Germline RB1 mutations were found in all probands. DNA profiling carried on similar-looking twins of families I and II, proved them to be fraternal. This study demonstrates the importance of genetic analysis of RB1 gene for risk prediction in retinoblastoma families. It also emphasizes that DNA profiling is a mandate for genetic screening of families with twins, thus adding a new dimension in counseling of retinoblastoma.