Survival Comes at a Cost: A Coevolution of Phage and Its Host Leads to Phage Resistance and Antibiotic Sensitivity of Pseudomonas aeruginosa Multidrug Resistant Strains

The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (∼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection.

MEIS2 (15q14) gene deletions in siblings with mild developmental phenotypes and bifid uvula: documentation of mosaicism in an unaffected parent

MEIS2 (Meis homeobox 2) encodes a homeobox protein in the three amino acid loop extension (TALE) family of highly conserved homeodomain-containing transcription regulators important for development. MEIS2 deletions/mutations have been associated with cleft lip/palate, dysmorphic facial features, cardiac defects, as well as intellectual disability at a variable severity. Here we report on one familial case that two affected siblings carry the same non-mosaic ~ 423 kb genomic deletion at 15q14 encompassing the entirety of CDIN1 and the last three exons (ex. 10, 11, 12) of the MEIS2 gene, while their unaffected father is mosaic for the same deletion in about 10% lymphocytes. Both siblings presented with mild developmental delay and bifid uvula, while no congenital cardiac abnormalities were identified. The elder sister also showed syncopal episodes and mild speech delay and the father had atrial septal defects. This is the first report showing multiple family members inherit a genomic deletion resulting in a MEIS2 partial truncation from a mosaic parent. Taken all together, this study has important implications for genetic counseling regarding recurrence risk and also points to the importance of offering MEIS2 gene tests covering both point mutations and microdeletions to individuals with milder bifid uvula and developmental delay.

Identification and Characterization of a Cryptic Genomic Deletion-Insertion in EYA1 Associated with Branchio-Otic Syndrome

Branchio-oto-renal spectrum disorder (BORSD) is characterized by hearing loss accompanied by ear malformations, branchial cysts, and fistulae, with (branchio-oto-renal syndrome (BORS)) or without renal abnormalities (BOS (branchio-otic syndrome)). As the most common causative gene for BORSD, dominant mutations in EYA1 are responsible for approximately 40% of the cases. In a sporadic deaf patient diagnosed as BOS, we identified an apparent heterozygous genomic deletion spanning the first four coding exons and one 5 ′ noncoding exon of EYA1 by targeted next-generation sequencing of 406 known deafness genes. Real-time PCR at multiple regions of EYA1 confirmed the existence of this genomic deletion and extended its 5 ′ boundary beyond the 5 ′ -UTR. Whole genome sequencing subsequently located the 5 ′ and 3 ′ breakpoints to 19268 bp upstream to the ATG initiation codon and 3180 bp downstream to exon 5. PCR amplification across the breakpoints in both the patient and his parents showed that the genomic alteration occurred de novo. Sanger sequencing of this PCR product revealed that it is in fact a GRCh38/hg38:chr8:g.71318554_71374171delinsTGCC genomic deletion-insertion. Our results showed that the genomic variant is responsible for the hearing loss associated with BOS and provided an example for deciphering such cryptic genomic alterations following pipelines of comprehensive exome/genome sequencing and designed verification.

A novel STS mutation and an Xp22.3 microdeletion from one Chinese family with X-linked ichthyosis

Background X-linked ichthyosis (XLI, OMIM# 308100) is a relatively common type of skin disorder, characterized by widespread, dark brown, polygonal scales and generalized dryness. Most patients (90%) are contributed to genomic deletion of the entire STS (steroid sulfatase) gene encoding steroid sulfatase, with the remaining cases being caused by point mutations or partial deletions. The patient carrying genomic deletion comprising the entire STS gene and adjacent genes often manifests neurological symptoms. Methods Herein, we reported a large four-generation Chinese family with 5 individuals with XLI with/ without mental delay. Genetic analysis was conducted by whole exome sequencing and confirmed by Sanger sequencing and quantitative Real-time PCR. Results These patients present generalized dryness and scaling of the skin with dark scales of the skin on trunk, and limbs. Two pathogenic variants, a novel STS mutation (c.1532A > G, p.E511G, NM_000351) and a ~ 2 Mb genomic microdeletion at Xp22.3 (6,451,785-8,434,424) embracing entire STS gene were identified in this family. In addition, an individual with Xp22.3 deletion has mental delay. Conclusion Our study expands the genetic profile of the STS gene in XLI patients and highlights the clinical application of exome sequencing.

Complex deletion and proximal reinsertion of a 150bp regulatory sequence in the mouse Csf1r promoter mediated by CRISPR-Cas9

This paper describes a deletion/reinsertion event encountered in a genome-editing project using CRISPR-Cas9. The objective was to delete a 150bp enhancer region in the mouse Csf1r locus using a pair of guides and a homology-dependent repair (HDR) template. The editing was successful in generating a founder pup with the anticipated precise deletion. However, the deleted fragment and a duplicated copy of part of the HDR template was reinserted around 50bp downstream. The reinsertion event was recognised because the PCR primer site used in genotyping was duplicated, so that there were three PCR products in a heterozygous animal and two in a homozygote. The event we describe is more subtle and more difficult to detect than large-scale rearrangements reported by others. We suggest that any genomic deletion mediated by CRISPR-Cas9 needs to be confirmed by assessing the copy number in the genome.

A large intragenic deletion in the CLCN1 gene causes Hereditary Myotonia in pigs

Mutations in the CLCN1 gene are the primary cause of non-dystrophic Hereditary Myotonia in several animal species. However, there are no reports of Hereditary Myotonia in pigs to date. Therefore, the objective of the present study was to characterize the clinical and molecular findings of Hereditary Myotonia in an inbred pedigree. The clinical, electromyographic, histopathological, and molecular findings were evaluated. Clinically affected pigs presented non-dystrophic recessive Hereditary Myotonia. Nucleotide sequence analysis of the entire coding region of the CLCN1 gene revealed the absence of the exons 15 and 16 in myotonic animals. Analysis of the genomic region flanking the deletion unveiled a large intragenic deletion of 4,165 nucleotides. Interestingly, non-related, non-myotonic pigs expressed transcriptional levels of an alternate transcript (i.e., X2) that was identical to the deleted X1 transcript of myotonic pigs. All myotonic pigs and their progenitors were homozygous recessive and heterozygous, respectively, for the 4,165-nucleotide deletion. This is the first study reporting Hereditary Myotonia in pigs and characterizing its clinical and molecular findings. Moreover, to the best of our knowledge, Hereditary Myotonia has never been associated with a genomic deletion in the CLCN1 gene in any other species.