A Diversity Outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors

: Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding 8 inbred founder strains, and CC mice are recombinant inbred mice generated from the same 8 founders. We generated 207 DOB6F1 mice representing 48 DO Dams and demonstrated that these mice reliably accept the C57BL/6 syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.

Characterization of Synthetic Wheat Line Largo for Resistance to Stem Rust

Resistance breeding is an effective approach against wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The synthetic hexaploid wheat line Largo (pedigree: durum wheat ‘Langdon’ × Aegilops tauschii PI 268210) was found to have resistance to a broad spectrum of Pgt races including the Ug99 race group. To identify the stem rust resistance (Sr) genes, we genotyped a population of 188 recombinant inbred lines developed from a cross between the susceptible wheat line ND495 and Largo using the wheat Infinium 90 K SNP iSelect array and evaluated the population for seedling resistance to the Pgt races TTKSK, TRTTF, and TTTTF in the greenhouse conditions. Based on genetic linkage analysis using the marker and rust data, we identified six quantitative trait loci (QTL) with effectiveness against different races. Three QTL on chromosome arms 6AL, 2BL, and 2BS corresponded to Sr genes Sr13c, Sr9e, and a likely new gene from Langdon, respectively. Two other QTL from PI 268210 on 2DS and 1DS were associated with a potentially new allele of Sr46 and a likely new Sr gene, respectively. Additionally, Sr7a was identified as the underlying gene for the 4AL QTL from ND495. Knowledge of the Sr genes in Largo will help to design breeding experiments aimed to develop new stem rust-resistant wheat varieties. Largo and its derived lines are particularly useful for introducing two Ug99-effective genes Sr13c and Sr46 into modern bread wheat varieties. The 90 K SNP-based high-density map will be useful for identifying the other important genes in Largo.

Mutational Analysis of a Familial Adenomatous Polyposis Pedigree with Bile Duct Polyp Phenotype

A large number of colorectal cancers have a genetic background in China. However, due to insufficient awareness, the diagnostic rate remains low and merely 5-6% of colorectal cancer patients are diagnosed with hereditary colorectal cancer. Familial adenomatous polyposis (FAP) is an autosomal dominant genetic disease caused by mutations in the adenomatous polyposis coli (APC) gene. Different mutation sites in APC are associated with the severity of FAP, risks of carcinogenesis, and extraintestinal manifestations. We used next-generation sequencing (NGS) and capture techniques to screen suspected mutation points in the proband in this pedigree. Using modified Sanger sequencing, we identified members of the family who were carriers of this variant and whether this segregated well with disease occurrence. FAP family members had multiple adenomatous polyps in their gastrointestinal tracts, some of which developed into cancer with age. Two subjects presented a rare common bile duct polyp phenotype. No extraintestinal manifestations were observed. A heterozygous frameshift mutation in APC exon 16 (NM_000038.6) was observed in the proband and in other patients: c.3260_3261del (p.Leu1087GlnQfs ∗ 31) (rs587782305); the variant call format was CCT/C. Due to the deletion of two bases, a stop codon appeared after 31 amino acids, and the protein was truncated prematurely, which affected the conformation of the protein. Pedigree genetic linkage analysis showed that the clinical phenotype cosegregated with the APC mutation p.L1087fs. This mutation may be the pathogenic in this FAP family and responsible for this rare common bile duct polyp.

Human Genetic Research in Pakistan: Challenges and Way forward

The 90s ushered in the era of molecular genetics in Pakistan. Our centuries’ old tradition of consanguineous marriages, resulting in the availability of large, inbred families with inherited disorders, proved to be a goldmine for geneticists, working to discover new genes and their functions. A multitude of novel genes with previously unknown functions were discovered through genetic linkage analysis, a technique that compares the segregations of DNA markers in normal and affected family members to pinpoint the region that contains the suspect gene. The first few landmark publications on linkage analysis in genetic diseases, from Pakistan, identified only the regions harboring the suspected disease genes.1,2 As the field advanced, disease causing mutations segregating in affected family members were identified through Sanger DNA sequencing of candidate genes in the linked region. In the last decade, the advent of high throughput techniques such as whole exome analysis and animal disease modeling, functional genomics studies became an integral of part of such genetic studies. In addition, bioinformatics tools were developed for predictive modeling of the effect of mutations on protein structure. The analysis of a single large multigenerational family with a genetic disorder could provide the same information that would be obtained from many small nuclear families, as usually found in developed countries. This allowed Pakistani researchers to attract collaborators from Universities around the world. DNA samples of numerous Pakistani families were sent to labs across the world for analysis, many times accompanied by a PhD student who would typically spend around six months working on those families. Universities developed efficient pipelines, whereby students would find families with genetic diseases, extract DNA and carry out linkage analysis and, in some cases, identify the gene mutations using Sanger sequencing. This led to a significant increase in the number of publications on genetics, from Pakistan. However, despite all the good work carried in the country, no credible effort had been made to build national capacity to carry the work beyond initial mutation screening. We lack the ability to conduct good quality high through put –omics analysis and animal model studies within the country. There are several reasons for this. Firstly, the cost of such research is prohibitive. While some institutes have invested in next generation sequencing platforms, these machines are largely underutilized due to high cost of consumables. Lack of adequate funding for reagents, required for genomic, transcriptomic and proteomic studies, is a major roadblock. Secondly, the ease in attracting foreign collaborators to carry out the functional studies in their labs, has made many Pakistani researchers complacent, often, they are happy to be co-authors in research papers without having to go through the pains to set up the required laboratory facilities. Lastly, the bureaucratic processes and red tape, especially in public sector Universities, make procurement of chemicals and consumables, tedious and time consuming, resulting in demotivation of researchers and faculty. There is a need to address these issues, if we wish to move towards self-sufficiency in conducting high level functional genomics and stem the flow of biological samples from Pakistan. While genetic studies in Pakistan have resulted in the discovery of many novel genes, the benefit of these studies is yet to trickle down to the patients. The willingness of the affected families to participate in these studies is crucial. Many affected families belong to far flung rural areas and have no understanding of their disease, how it is inherited or the implications of participating in such research. It is unlikely that families participating in these studies have any immediate benefits and they should be informed at the time of recruitment using common language which they are able to understand. The role of Institutional Ethics and Review Boards, for the protection of study participants and their rights, needs to be strengthened. It is important not to abandon these families after sample collection but to relay the results of the study and counsel them regarding their future options. Despite the rapid advances in genetic medical knowledge, our population has yet to reap its benefits. A small step in right direction is the Compulsory Blood Test of the Relatives of Thalassemia Patient Bill-2017, which makes it compulsory for couples to get tested before marrying. However, the scarcity of gene testing facilities and healthcare professionals trained in clinical genetics is an impediment to the implementation of this bill in the true spirit. It is becoming imperative to educate our healthcare professionals regarding the application of genetics to medical practice. Clinical genetics and its related competencies need to be recognized as medical specialties in the country, before they can be introduced into mainstream clinical practice to improve health outcomes of our affected families.

Huntington’s Disease Pathogenesis: Two Sequential Components

Historically, Huntington’s disease (HD; OMIM #143100) has played an important role in the enormous advances in human genetics seen over the past four decades. This familial neurodegenerative disorder involves variable onset followed by consistent worsening of characteristic abnormal movements along with cognitive decline and psychiatric disturbances. HD was the first autosomal disease for which the genetic defect was assigned to a position on the human chromosomes using only genetic linkage analysis with common DNA polymorphisms. This discovery set off a multitude of similar studies in other diseases, while the HD gene, later renamed HTT, and its vicinity in chromosome 4p16.3 then acted as a proving ground for development of technologies to clone and sequence genes based upon their genomic location, with the growing momentum of such advances fueling the Human Genome Project. The identification of the HD gene has not yet led to an effective treatment, but continued human genetic analysis of genotype-phenotype relationships in large HD subject populations, first at the HTT locus and subsequently genome-wide, has provided insights into pathogenesis that divide the course of the disease into two sequential, mechanistically distinct components.

Obelisc: an identical-by-descent mapping tool based on SNP streak

Motivation Genetic linkage analysis has made a huge contribution to the genetic mapping of Mendelian diseases. However, most previously available linkage analysis methods have limited applicability. Since parametric linkage analysis requires predefined model of inheritance with a fixed set of parameters, it is inapplicable without fully structured pedigree information. Furthermore, the analytical results are dependent on the specification of model parameters. While non-parametric linkage analysis can avoid these problems, the runs of homozygosity (ROH) mapping, a widely used non-parametric linkage analysis method, can only deal with recessive inheritance. The implementation of non-parametric linkage analyses capable of dealing with both dominant and recessive inheritance has been required. Results We have developed the Obelisc (Observational linkage scan), a flexibly applicable user-friendly non-parametric linkage analysis tool, which also provides an intuitive visualization of the analytical results. Obelisc is based on the SNP streak approach, which does not require any predefined inheritance model with parameters. In contrast to the ROH mapping, the SNP streak approach is applicable to both dominant and recessive traits. To illustrate the performance of Obelisc, we generated a pseudo-pedigree from the publicly available BioBank Japan Project genome-wide genotype dataset (n > 180 000). By applying Obelisc to this pseudo-pedigree, we successfully identified the regions with inherited identical-by-descent haplotypes shared among the members of the pseudo-pedigree, which was validated by the population-based haplotype phasing approach. Availability and implementation Obelisc is feely available at https://github.com/qsonehara/Obelisc as a python package with example datasets. Supplementary information Supplementary data are available at Bioinformatics online.

Identification of key modules and hub genes regulating drought stress response in rice drought sensitive line PY6 by weighted gene co-expression network analysis

Background: Drought stress is an adverse factor with deleterious effects on several facets of rice growth. However, the mechanism underlying drought resistance in rice remains unclear. In order to genetically understand the potential molecular mechanism for drought response in rice, a drought sensitive Chromosome Segment Substitution Line (CSSL) PY6, which was constructed by the introgression of genomic segments of drought sensitive variety LAMBAYEQUE1 into drought-resistance variety PR403 via backcrossing, was used to map the QTL locus dss-1 for its sensitive phenotype, and to reveal the impact of dss-1 on the transcriptional profiling of PY6 via RNA-seq and WGCNA (weighted gene co-expression network analysis) analysis. Results: The genetic linkage analysis showed that dss-1 was located on the short arm of chromosome 1 of rice. In contrast to PR403, the over-accumulation of H 2 O 2 and MDA that might result in drought sensitive phenotype was observed in PY6 under drought stress. In the analysis of RNA-seq data, the identified differentially expressed genes (DEGs) mainly enriched in photosynthesis-related GO terms and exhibited a down-regulation pattern of their expressions in both PY6 and PR403 in response to drought stress, indicating that the photosynthesis was greatly inhibited in rice. Further WGCNA analysis constructed a co-expression network with 26 gene modules in which 4 and 3 modules that were highly correlated with H 2 O 2 and MDA, respectively. Likewise, the GO analysis of the differentially expressed hub genes (DEHGs) enriched in H 2 O 2 -correlated modules showed that the photosynthesis related GO terms were consistently over-represented. Furthermore, functional annotation of DEHGs in H 2 O 2 and MDA correlated modules revealed a cross talk between abiotic and biotic stresses. This was reflected by the differential expression alterations of hub genes which were annotated as encoding MYBs, laccases, WRKYs, and PRs family proteins, and ZFP36 were notably observed between PY6 and PR403 in response drought stress. Conclusions: Collectively, we speculated that drought-induced the inhibition of photosynthesis lead to the accumulation of H 2 O 2 and MDA that can trigger the reprogramming the profiling of transcriptome in rice. This included the differential regulation of hub genes that involve in ROS eliminated pathways to prevent the damage of rice plants from oxidative stress.

Genome Sequence Analysis of Auricularia heimuer Combined with Genetic Linkage Map

Auricularia heimuer is one of the most popular edible fungi in China. In this study, the whole genome of A. heimuer was sequenced on the Illumina HiSeq X system and compared with other mushrooms genomes. As a wood-rotting fungus, a total of 509 carbohydrate-active enzymes (CAZymes) were annotated in order to explore its potential capabilities on wood degradation. The glycoside hydrolases (GH) family genes in the A. heimuer genome were more abundant than the genes in the other 11 mushrooms genomes. The A. heimuer genome contained 102 genes encoding class III, IV, and V ethanol dehydrogenases. Evolutionary analysis based on 562 orthologous single-copy genes from 15 mushrooms showed that Auricularia formed an early independent branch of Agaricomycetes. The mating-type locus of A. heimuer was located on linkage group 8 by genetic linkage analysis. By combining the genome sequence analysis with the genetic linkage map, the mating-type locus of A. heimuer was located on scaffold45 and consisted of two subunits, α and β. Each subunit consisted of a pair of homeodomain mating-type protein genes HD1 and HD2. The mapping revealed conserved synteny at the whole mating-type loci and mirror symmetry relations near the β subunit between A. heimuer and Exidia glandulosa. This study proposed the potential for the bioethanol production by consolidated bioprocessing of A. heimuer. It will promote understanding of the lignocellulose degradation system and facilitate more efficient conversion of the agricultural wastes used for mushroom cultivation. It also will advance the research on the fruiting body development and evolution of A. heimuer.

Clinical and genetic analysis of two wolfram syndrome families with high occurrence of wolfram syndrome and diabetes type II: a case report

Background Mutations of the WFS1 gene are responsible for most cases of Wolfram syndrome (WS), a rare, recessively inherited neurodegenerative disorder characterized by juvenile-onset non-autoimmune diabetes mellitus and optic atrophy. Variants of WFS1 are also associated with non-syndromic hearing loss and type-2 diabetes mellitus (T2DM). Our study adds to literature significant associations between WS and T2DM. Case presentation In this study, we analyzed the clinical and genetic data of two families with high prevalence of WS and T2DM. Genetic linkage analysis and DNA sequencing were exploited to identify pathogenic variants. One novel pathogenic variant (c.2243-2244insC) and one known pathogenic (c.1232_1233delCT) (frameshift) variant were identified in exon eight of WFS1 gene. Conclusions The mutational and phenotypic spectrum of WS is broadened by our report of novel WFS1 mutation. Our results reveal the value of molecular analysis of WFS1 in the improvement of clinical diagnostics for WS. This study also confirms the role of WFS1 in T2DM.