Neuregulin 1 Gene (NRG1). A Potentially New Targetable Alteration for the Treatment of Lung Cancer

Oncogenic gene fusions are hybrid genes that result from structural DNA rearrangements, leading to unregulated cell proliferation by different mechanisms in a wide variety of cancer. This has led to the development of directed therapies to antagonize a variety of mechanisms that lead to cell growth or proliferation. Multiple oncogene fusions are currently targeted in lung cancer treatment, such as those involving ALK, RET, NTRK and ROS1 among many others. Neuregulin (NRG) gene fusion has been described in the development of normal tissue as well as in a variety of diseases, such as schizophrenia, Hirschsprung’s disease, atrial fibrillation and, most recently, the development of various types of solid tumors, such as renal, gastric, pancreatic, breast, colorectal and, more recently, lung cancer. The mechanism for this is that the NRG1 chimeric ligand leads to aberrant activation of ERBB2 signaling via PI3K-AKT and MAPK cellular cascades, leading to cell division and proliferation. Details regarding the incidence of these gene rearrangements are lacking. Limited case reports and case series have evaluated their clinicopathologic features and prognostic significance in the lung cancer population. Taking this into account, NRG1 could become a targetable alteration in selected patients. This review highlights how the knowledge of new molecular mechanisms of NRG1 fusion may help in gaining new insights into the molecular status of lung cancer patients and unveil a novel targetable molecular marker.

Validation of methodology for efficient genotyping of CYP2D6 and CYP2C19

There are some data associating variants in the CYP2D6 and/or CYP2C19 genes with concentration-to-dose ratios, efficacy, and retention in treatments. However, much of the above arises from relatively small studies or large datasets with limited genotyping methodologies. Our aim was to develop and validate comprehensive and accurate genotyping methodology for these two genes to facilitate regenotyping in large datasets and hence the generation of more accurate clinical associations. TaqMan copy number variant (CNV) assays for CYP2D6 were used to identify samples from a relevant large dataset (GENDEP study, N = 853) with particularly challenging genotypes to call. These and those representing as broad a range of CYP2D6 and CYP2C19 genotypes as possible by prior available data (AmpliChip CYP450 and TaqMan CYP2C19*17) were chosen for further analysis (N = 96). Genotyping techniques employed were: Luminex CYP2D6 xTAGv3 and Luminex CYP2C19 xTAGv3, PharmacoScan, the Ion S5 AmpliSeq Pharmacogenomics Panel, TaqMan single nucleotide variant (SNV) assays, and, for the CYP2D6 hybrids, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing. Agena was also used for CYP2C19. The TaqMan SNV assays were able to assist with identifying which gene was duplicated or in tandem for multiple copy variants. A multiplex assay was adaptable for analysis of CYP2D6 hybrid genes, with Sanger sequencing data being consistent with the data arising; we provide these data for efficient genotyping of such CYP2D6 hybrid genes with adaptable multiplex methods. Consensus genotypes generated to date resulted in revision of assigned enzyme activity score for 28/96(29%) and 2/93 samples (2.2%) for CYP2D6 and CYP2C19, respectively.

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst

Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.

GRAde: a long-read sequencing approach to efficiently identifying the CYP11B1/CYP11B2 chimeric form in patients with glucocorticoid-remediable aldosteronism

Background Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11β‐hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA. Results We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis. Conclusions PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde.

Transcriptional network of the industrial hybrid Saccharomyces pastorianus reveals temperature-dependent allele expression bias and preferential orthologous protein assemblies

Saccharomyces pastorianus is an industrial natural yeast evolved from different hybridisation events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic or antagonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% have their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes express preferentially one parental allele over the other, and that S. eubayanus-like alleles are significantly over-represented among the genes involved in cold acclimatisation. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes established in the hybrid are likely to be either exclusively chimeric or uni-specific, and that the redundancy is discouraged, a scenario which fits well with the stoichiometric balance-hypothesis. This study offers a first overview of the transcriptional pattern of S. pastorianus and provide a rationalisation for its unique industrial traits at expression level.

Mutation accumulation in cancer genes relates to nonoptimal outcome in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm accounting for ∼15% of all leukemia. Progress of the disease from an indolent chronic phase to the more aggressive accelerated phase or blast phase (BP) occurs in a minority of cases and is associated with an accumulation of somatic mutations. We performed genetic profiling of 85 samples and transcriptome profiling of 12 samples from 59 CML patients. We identified recurrent somatic mutations in ABL1 (37%), ASXL1 (26%), RUNX1 (16%), and BCOR (16%) in the BP and observed that mutation signatures in the BP resembled those of acute myeloid leukemia (AML). We found that mutation load differed between the indolent and aggressive phases and that nonoptimal responders had more nonsilent mutations than did optimal responders at the time of diagnosis, as well as in follow-up. Using RNA sequencing, we identified other than BCR-ABL1 cancer-associated hybrid genes in 6 of the 7 BP samples. Uncovered expression alterations were in turn associated with mechanisms and pathways that could be targeted in CML management and by which somatic alterations may emerge in CML. Last, we showed the value of genetic data in CML management in a personalized medicine setting.

Intragenic and structural variation in the SMN locus and clinical variability in spinal muscular atrophy

Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1–4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1–SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.

Frequency of Mia (MNS7) and Classification of Mia-Positive Hybrid Glycophorins in an Australian Blood Donor Population

Background: MNS blood group system genes GYPA and GYPB share a high degree of sequence homology and gene structure. Homologous exchanges between GYPA and GYPB form hybrid genes encoding hybrid glycophorins GP(A-B-A) and GP(B-A-B). Over 20 hybrid glycophorins have been characterised. Each has a distinct phenotype defined by the profile of antigens expressed including Mia. Seven hybrid glycophorins carry Mia and have been reported in Caucasian and Asian population groups. In Australia, the population is diverse; however, the prevalence of hybrid glycophorins in the population has never been determined. The aims of this study were to determine the frequency of Mia and to classify Mia-positive hybrid glycophorins in an Australian blood donor population. Method: Blood samples from 5,098 Australian blood donors were randomly selected and screened for Mia using anti-Mia monoclonal antibody (CBC-172) by standard haemagglutination technique. Mia-positive red blood cells (RBCs) were further characterised using a panel of phenotyping reagents. Genotyping by high-resolution melting analysis and DNA sequencing were used to confirm serology. Result: RBCs from 11/5,098 samples were Mia-positive, representing a frequency of 0.22%. Serological and molecular typing identified four types of Mia-positive hybrid glycophorins: GP.Hut (n = 2), GP.Vw (n = 3), GP.Mur (n = 5), and 1 GP.Bun (n = 1). GP.Mur was the most common. Conclusion: This is the first comprehensive study on the frequency of Mia and types of hybrid glycophorins present in an Australian blood donor population. The demographics of Australia are diverse and ever-changing. Knowing the blood group profile in a population is essential to manage transfusion needs.

Genus level analysis of PKS-NRPS and NRPS-PKS hybrids reveals their origin in Aspergilli

Background Filamentous fungi produce a vast amount of bioactive secondary metabolites (SMs) synthesized by e.g. hybrid polyketide synthase-nonribosomal peptide synthetase enzymes (PKS-NRPS; NRPS-PKS). While their domain structure suggests a common ancestor with other SM proteins, their evolutionary origin and dynamics in fungi are still unclear. Recent rational engineering approaches highlighted the possibility to reassemble hybrids into chimeras — suggesting molecular recombination as diversifying mechanism. Results Phylogenetic analysis of hybrids in 37 species – spanning 9 sections of Aspergillus and Penicillium chrysogenum – let us describe their dynamics throughout the genus Aspergillus. The tree topology indicates that three groups of PKS-NRPS as well as one group of NRPS-PKS hybrids developed independently from each other. Comparison to other SM genes lead to the conclusion that hybrids in Aspergilli have several PKS ancestors; in contrast, hybrids are monophyletic when compared to available NRPS genes — with the exception of a small group of NRPSs. Our analysis also revealed that certain NRPS-likes are derived from NRPSs, suggesting that the NRPS/NRPS-like relationship is dynamic and proteins can diverge from one function to another. An extended phylogenetic analysis including bacterial and fungal taxa revealed multiple ancestors of hybrids. Homologous hybrids are present in all sections which suggests frequent horizontal gene transfer between genera and a finite number of hybrids in fungi. Conclusion Phylogenetic distances between hybrids provide us with evidence for their evolution: Large inter-group distances indicate multiple independent events leading to the generation of hybrids, while short intra-group distances of hybrids from different taxonomic sections indicate frequent horizontal gene transfer. Our results are further supported by adding bacterial and fungal genera. Presence of related hybrid genes in all Ascomycetes suggests a frequent horizontal gene transfer between genera and a finite diversity of hybrids — also explaining their scarcity. The provided insights into relations of hybrids and other SM genes will serve in rational design of new hybrid enzymes.