Longitudinal profiling identifies co-occurring BRCA1/2 reversions, TP53BP1, RIF1 and PAXIP1 mutations in PARP inhibitor-resistant advanced breast cancer.

BACKGROUND: Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. PATIENTS AND METHODS: Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. RESULTS: BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by in vitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. CONCLUSIONS: These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Structured pain management reduces patient discomfort after catheter ablation and rhythm device surgery.

BACKGROUND: The goal was to test the effectiveness of a structured pain management programme after invasive electrophysiological interventions in cardiology including ablation of atrial fibrillation (AF) or ventricular tachycardia (VT) and implantation, or explantation, of pacemakers or implantable cardioverter defibrillators. METHODS: This was a prospective study with a pre-/post-design where a post-intervention group (116 consecutive patients) was compared to a pre-intervention group (102 consecutive patients) after implementation of a structured pain-management programme using the numeric rating scale (NRS 0-10) and classified as moderate-to-severe if NRS > 3. Measurements were recorded every two hours during the first 24 h post-operatively. The location of the pain and the amount of analgesic used were also recorded. RESULTS: The proportion of patients who experienced moderate-to-severe pain after the procedure decreased after initiation of the pain-management program: 47% versus 61%; p = 0.048. This difference was driven primarily by reduced pain late (8-24 h) after the procedure; 16% versus 39%; p < 0.001. The risk to develop late (8-24 h) post-procedural pain was reduced approximately three-fold after implementation of the pain-management programme (OR = 0.32, 95% CI 0.16-0.64, p = 0.001). Multivariate analysis indicated chronic pain, early pain (0-6 h), and type of intervention were associated with late post-interventional pain. In contrast, age, diabetes mellitus, BMI, gender and procedure time were not related. CONCLUSION: The findings illustrate the potential value of a structured pain-management programme. The proportion of patients who experienced moderate-to-severe pain after these electrophysiological procedures decreased significantly. SIGNIFICANCE: This is the first exploratory study that evaluates the impact of a multidisciplinary pain-management programme after cardiac electrophysiological interventions. It demonstrates that significant quality improvement is achievable following simple rules together with patient and staff education. The programme reduces the proportion of patients with moderate-to-severe pain after electrophysiological procedures significantly.

Mutations in 3 genes (MKS3, CC2D2A and RPGRIP1L) cause COACH syndrome (Joubert syndrome with congenital hepatic fibrosis).

OBJECTIVE: To identify genetic causes of COACH syndrome BACKGROUND: COACH syndrome is a rare autosomal recessive disorder characterised by Cerebellar vermis hypoplasia, Oligophrenia (developmental delay/mental retardation), Ataxia, Coloboma, and Hepatic fibrosis. The vermis hypoplasia falls in a spectrum of mid-hindbrain malformation called the molar tooth sign (MTS), making COACH a Joubert syndrome related disorder (JSRD). METHODS: In a cohort of 251 families with JSRD, 26 subjects in 23 families met criteria for COACH syndrome, defined as JSRD plus clinically apparent liver disease. Diagnostic criteria for JSRD were clinical findings (intellectual impairment, hypotonia, ataxia) plus supportive brain imaging findings (MTS or cerebellar vermis hypoplasia). MKS3/TMEM67 was sequenced in all subjects for whom DNA was available. In COACH subjects without MKS3 mutations, CC2D2A, RPGRIP1L and CEP290 were also sequenced. RESULTS: 19/23 families (83%) with COACH syndrome carried MKS3 mutations, compared to 2/209 (1%) with JSRD but no liver disease. Two other families with COACH carried CC2D2A mutations, one family carried RPGRIP1L mutations, and one lacked mutations in MKS3, CC2D2A, RPGRIP1L and CEP290. Liver biopsies from three subjects, each with mutations in one of the three genes, revealed changes within the congenital hepatic fibrosis/ductal plate malformation spectrum. In JSRD with and without liver disease, MKS3 mutations account for 21/232 families (9%). CONCLUSIONS: Mutations in MKS3 are responsible for the majority of COACH syndrome, with minor contributions from CC2D2A and RPGRIP1L; therefore, MKS3 should be the first gene tested in patients with JSRD plus liver disease and/or coloboma, followed by CC2D2A and RPGRIP1L.

Recombination hotspot in NF1 microdeletion patients.

Neurofibromatosis type 1 (NF1) patients that are heterozygous for an NF1 microdeletion are remarkable for an early age at onset and an excessive burden of dermal neurofibromas. Microdeletions are predominantly maternal in origin and arise by unequal crossover between misaligned NF1REP paralogous sequence blocks which flank the NF1 gene. We mapped and sequenced the breakpoints in several patients and designed primers within each paralog to specifically amplify a 3.4 kb deletion junction fragment. This assay amplified a deletion junction fragment from 25 of the 54 unrelated NF1 microdeletion patients screened. Sequence analysis demonstrated that each of the 25 recombination events occurred in a discrete 2 kb recombination hotspot within each of the flanking NF1REPs. Two recombination events were accompanied by apparent gene conversion. A search for recombination-prone motifs revealed a chi-like sequence; however, it is unknown whether this element stimulates recombination to occur at the hotspot. The deletion-junction assay will facilitate the prospective identification of patients with NF1 microdeletion at this hotspot for genotype-phenotype correlation studies and diagnostic evaluation.

High levels of MHC class II allelic diversity in lake trout from Lake Superior.

Sequence variation in a 216 bp portion of the major histocompatibility complex (MHC) II B1 domain was examined in 74 individual lake trout (Salvelinus namaycush) from different locations in Lake Superior. Forty-three alleles were obtained which encoded 71-72 amino acids of the mature protein. These sequences were compared with previous data obtained from five Pacific salmon species and Atlantic salmon using the same primers. Although all of the lake trout alleles clustered together in the neighbor-joining analysis of amino acid sequences, one amino acid allelic lineage was shared with Atlantic salmon (Salmo salar), a species in another genus which probably diverged from Salvelinus more than 10-20 million years ago. As shown previously in other salmonids, the level of nonsynonymous nucleotide substitution (dN) exceeded the level of synonymous substitution (dS). The level of nucleotide diversity at the MHC class II B1 locus was considerably higher in lake trout than in the Pacific salmon (genus Oncorhynchus). These results are consistent with the hypothesis that lake trout colonized Lake Superior from more than one refuge following the Wisconsin glaciation. Recent population bottlenecks may have reduced nucleotide diversity in Pacific salmon populations.

Lack of effect of beta-naphthoflavone on induction of Nramp genes in adult rainbow trout Oncorhynchus mykiss.

Natural resistance-associated macrophage protein (Nramp) genes in rainbow trout, Oncorhynchus mykiss, were identified and characterized. The greatest mRNA level encoding these genes was in the developing ovary of rainbow trout. We evaluated the response of these genes to a certain aromatic hydrocarbon receptor (AHR) agonist. Adult rainbow trout were treated with beta-naphthoflavone (BNF) (50 and 100 mg/kg) for 48 h. Using reverse-transcriptase polymerase chain reaction with ovary and head kidney RNA and specific alpha and beta Nramp primers, a 400 bp Nramp-alpha- and a 400 bp Nramp-beta-specific cDNA were obtained. There were no changes in the alpha and beta Nramp mRNA levels in the ovary following BNF administration. CYP1A1 mRNA was increased in the ovary and kidney, suggesting the presence of AHR in rainbow trout ovary, while the AHR agonist produced no effect on Nramp mRNAs.

NF1 microdeletion breakpoints are clustered at flanking repetitive sequences.

Neurofibromatosis type 1 patients with a submicroscopic deletion spanning the NF1 tumor suppressor gene are remarkable for an early age at onset of cutaneous neurofibromas, suggesting the deletion of an additional locus that potentiates neurofibromagenesis. Construction of a 3.5 Mb BAC/PAC/YAC contig at chromosome 17q11.2 and analysis of somatic cell hybrids from microdeletion patients showed that 14 of 17 cases had deletions of 1.5 Mb in length. The deletions encompassed the entire 350 kb NF1 gene, three additional genes, one pseudogene and 16 expressed sequence tags (ESTs). In these cases, both proximal and distal breakpoints mapped at chromosomal regions of high identity, termed NF1REPs. These REPs, or clusters of paralogous loci, are 15-100 kb and harbor at least four ESTs and an expressed SH3GL pseudogene. The remaining three patients had at least one breakpoint outside an NF1REP element; one had a smaller deletion thereby narrowing the critical region harboring the putative locus that exacerbates neurofibroma development to 1 Mb. These data show that the likely mechanism of NF1 microdeletion is homologous recombination between NF1REPs on sister chromatids. NF1 microdeletion is the first REP-mediated rearrangement identified that results in loss of a tumor suppressor gene. Therefore, in addition to the germline rearrangements reported here, NF1REP-mediated somatic recombination could be an important mechanism for the loss of heterozygosity at NF1 in tumors of NF1 patients.

Comparative analysis of two Nramp loci from rainbow trout.

Innate resistance to intracellular parasites is controlled in part by Nramp1 (Natural resistance-associated macrophage protein 1) in mammals and birds. To isolate Nramp homologs from rainbow trout, a combination of library screening and rapid amplification of cDNA ends was performed. Two closely related Nramp loci, designated OmNramp alpha and OmNramp beta, were cloned and characterized. OmNramp alpha and OmNramp beta encode two highly conserved proteins of 585 and 558 amino acids, respectively. Deduced amino acid seqences showed that the OmNramp alpha and OmNramp beta proteins share 90% of their residues and contain all of the signature features of the Nramp family of proteins: 12 transmembrane domains, two N-linked glycosylation sites, and a conserved transport motif. Phylogenetic analysis supported a close relation to Nramp2 proteins, a related member of the Nramp family. Despite this relation, juvenile trout expressed OmNramp alpha in a manner consistent with an Nramp1 homolog and OmNramp beta similar to an Nramp2 locus. Both trout loci were expressed at relatively high amounts in the ovaries of juveniles, a finding not reported in the investigations of previously characterized mammalian and avian homologs. These results suggest a role for Nramp loci in the follicular development of teleost fishes, as well as in mammals. Because salmonid fishes are ancestral tetraploids, fragments of OmNramp alpha and OmNramp beta were isolated from smelt, a diploid relative, to determine whether the trout loci represent duplicates of a single gene. Homologous sequences for both loci were found in smelt, supporting the hypothesis that OmNramp alpha and OmNramp beta are indeed independent loci that were present before the chromosomal duplication of salmonids. The isolation of Nramp loci from rainbow trout may eventually produce a genetic tool for the control of disease in aquaculture operations. Determining the involvement of trout homologs in innate immunity may also provide insight regarding the evolution of host resistance to pathogens.

[Histological studies of tympanic membrane rim defects in chronic otitis media mesotympanalis]. / Histologische Untersuchungen des Trommelfelldefektrandes bei der chronischen Otitis media mesotympanalis.

In the present study we investigated resected tympanic membranes taken during tympanoplasty. Tissue from 111 patients with chronic otitis media was analyzed after being embedded in paraffin and stained with hematoxylin and eosin. In 67 patients (60%) the tympanic membrane epidermis did not extend beyond the margin of the perforation rim, so that no epithelial migration was observed on the inner side of the tympanic membrane. In 27 specimens (24%) we found an epithelial migration on the inner side of the tympanic membrane, but this did not extend to the margins of the excised tissue. In 17 tissue specimens (16%) epithelial migration extended to the margins of the resected tissue. Clinically, these patients were found to have non-functioning Eustachian tubes. The size of the tympanic membrane perforation was not found to impact on epithelial migration. However, there was a correlation between the extent of the epithelial migration seen in the specimens and the occurrence of a permanent tympanic membrane perforation after tympanoplasty. Of 17 patients with these findings, 4 (23%) had consistent reperforations. The tympanic membrane rims of these patients were completely covered with squamous epithelium. Patients with no or only little epithelial migration to the inner side of the tympanic membrane were found to have a significantly lower postoperative rate of recurrent infection and drum reperforation.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): taxonomic implications for the Great Lakes species flock.

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Characteristics of two salmonid repetitive DNA families in rainbow trout, Oncorhynchus mykiss.

DNA sequence and genomic location of two repetitive DNA families in rainbow trout (Oncorhynchus mykiss) were investigated to develop molecular markers for chromosome identification. DNA fragments with sequences similar to the tandem and interspersed elements described in other salmonids were isolated. One clone showed differential hybridization to 12 pairs of chromosomes and should be a useful marker for physical mapping.