Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on short read sequencing data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n = 2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs and short read data for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions.

Transcriptome innovations in primates revealed by single-molecule long-read sequencing

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Our transcriptomes reveal thousands of novel transcripts, some of them under active translation, expanding and completing the repertoire of primate gene models. Our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n=2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions.

DDAH1 SNP rs480414 that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia results in lower nitric oxide production in neonatal cord blood-derived lymphoblastoid cell lines

BACKGROUND: Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and pulmonary hypertension (PH) is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a vasodilator and apoptotic mediator made by NO synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA), and dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Previously, in a BPD patient cohort, we identified single nucleotide polymorphism (SNP) DDAH1 rs480414 (G > A) that was protective against developing PH. This study aims to determine functional consequences of the DDAH1 SNP in lymphoblastoid cell lines (LCLs) derived from neonatal cord blood. We tested the hypothesis that DDAH1 SNP (AA) results in DDAH1 gain of function, leading to greater NO-mediated apoptosis compared to DDAH1 wild-type (GG) in LCLs. METHODS: LCLs were analyzed by Western blot (DDAH1, cleaved and total caspase-3 and -8, and β-actin), and RT-PCR (DDAH1, iNOS). Cell media assayed for nitrites with chemiluminescence NO analyzer, and conversion of ADMA to L-citrulline was measured by spectrophotometry. RESULTS: LCLs with DDAH1 SNP had similar levels of DDAH1 protein and mRNA expression, as well as DDAH activity, compared to DDAH1 WT LCLs. There were also no changes in cleaved caspase-3 and -8 protein levels. LCLs with DDAH1 SNP had similar iNOS mRNA expression. Nitrite levels in media were lower for DDAH1 SNP LCLs compared to DDAH1 WT LCLs (p < 0.05). CONCLUSION: Contrary to our hypothesis, we found that NO production was lower in DDAH1 SNP LCLs, indicative of a loss of function phenotype.

Epigenomic profiling of primate lymphoblastoid cell lines reveals the evolutionary patterns of epigenetic activities in gene regulatory architectures

Changes in the epigenetic regulation of gene expression have a central role in evolution. Here, we extensively profiled a panel of human, chimpanzee, gorilla, orangutan, and macaque lymphoblastoid cell lines (LCLs), using ChIP-seq for five histone marks, ATAC-seq and RNA-seq, further complemented with whole genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS). We annotated regulatory elements (RE) and integrated chromatin contact maps to define gene regulatory architectures, creating the largest catalog of RE in primates to date. We report that epigenetic conservation and its correlation with sequence conservation in primates depends on the activity state of the regulatory element. Our gene regulatory architectures reveal the coordination of different types of components and highlight the role of promoters and intragenic enhancers (gE) in the regulation of gene expression. We observe that most regulatory changes occur in weakly active gE. Remarkably, novel human-specific gE with weak activities are enriched in human-specific nucleotide changes. These elements appear in genes with signals of positive selection and human acceleration, tissue-specific expression, and particular functional enrichments, suggesting that the regulatory evolution of these genes may have contributed to human adaptation.

Lymphoblastoid Cell Lines as Models to Study Mitochondrial Function in Neurological Disorders

Neurological disorders, including neurodegenerative diseases, are collectively a major cause of death and disability worldwide. Whilst the underlying disease mechanisms remain elusive, altered mitochondrial function has been clearly implicated and is a key area of study in these disorders. Studying mitochondrial function in these disorders is difficult due to the inaccessibility of brain tissue, which is the key tissue affected in these diseases. To overcome this issue, numerous cell models have been used, each providing unique benefits and limitations. Here, we focussed on the use of lymphoblastoid cell lines (LCLs) to study mitochondrial function in neurological disorders. LCLs have long been used as tools for genomic analyses, but here we described their use in functional studies specifically in regard to mitochondrial function. These models have enabled characterisation of the underlying mitochondrial defect, identification of altered signalling pathways and proteins, differences in mitochondrial function between subsets of particular disorders and identification of biomarkers of the disease. The examples provided here suggest that these cells will be useful for development of diagnostic tests (which in most cases do not exist), identification of drug targets and testing of pharmacological agents, and are a worthwhile model for studying mitochondrial function in neurological disorders.

Identifying genetic modulators of statin response using subject-derived lymphoblastoid cell lines

Although statins (3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) have proven effective in reducing plasma low-density lipoprotein levels and risk of cardiovascular disease, their lipid lowering efficacy is highly variable among individuals. Furthermore, statin treatment carries a small but significant risk of adverse effects, most notably myopathy and new onset diabetes. Hence, identification of biomarkers for predicting patients who would most likely benefit from statin treatment without incurring increased risk of adverse effects can have a significant public health impact. In this review, we discuss the rationale for the use of subject-derived lymphoblastoid cell lines in studies of statin pharmacogenomics and describe a variety of approaches we have employed to identify novel genetic markers associated with interindividual variation in statin response.