Linkage disequilibrium between rare mutations

The statistical associations between mutations, collectively known as linkage disequilibrium (LD), encode important information about the evolutionary forces acting within a population. Yet in contrast to single-site analogues like the site frequency spectrum, our theoretical understanding of linkage disequilibrium remains limited. In particular, little is currently known about how mutations with different ages and fitness costs contribute to expected patterns of LD, even in simple settings where recombination and genetic drift are the major evolutionary forces. Here, I introduce a forward-time framework for predicting linkage disequilibrium between pairs of neutral and deleterious mutations as a function of their present-day frequencies. I show that the dynamics of linkage disequilibrium become much simpler in the limit that mutations are rare, where they admit a simple heuristic picture based on the trajectories of the underlying lineages. I use this approach to derive analytical expressions for a family of frequency-weighted LD statistics as a function of the recombination rate, the frequency scale, and the additive and epistatic fitness costs of the mutations. I find that the frequency scale can have a dramatic impact on the shapes of the resulting LD curves, reflecting the broad range of time scales over which these correlations arise. I also show that the differences between neutral and deleterious LD are not purely driven by differences in their mutation frequencies, and can instead display qualitative features that are reminiscent of epistasis. I conclude by discussing the implications of these results for recent LD measurements in bacteria. This forward-time approach may provide a useful framework for predicting linkage disequilibrium across a range of evolutionary scenarios.

HDL Is Not Dead Yet

High-density lipoprotein cholesterol (HDL-C) levels are inversely correlated with coronary heart disease (CHD) in multiple epidemiological studies, but whether HDL is causal or merely associated with CHD is unclear. Recent trials for HDL-raising drugs were either not effective in reducing CHD events or, if beneficial in reducing CHD events, were not conclusive as the findings could be attributed to the drugs’ LDL-reducing activity. Furthermore, the first large Mendelian randomization study did not causally relate HDL-C levels to decreased CHD. Thus, the hypothesis that HDL is protective against CHD has been rightfully challenged. However, subsequent Mendelian randomization studies found HDL characteristics that are causally related to decreased CHD. Many aspects of HDL structure and function, especially in reverse cholesterol transport, may be better indicators of HDL’s protective activity than simply measuring HDL-C. Cholesterol efflux capacity is associated with lower levels of prevalent and incident CHD, even after adjustment for HDL-C and apolipoprotein A-1 levels. Also, subjects with very high levels of HDL-C, including those with rare mutations that disrupt hepatic HDL uptake and reverse cholesterol transport, may be at higher risk for CHD than those with moderate levels. We describe here several cell-based and cell-free in vitro assays of HDL structure and function that may be used in clinical studies to determine which of HDL’s functions are best associated with protection against CHD. We conclude that the HDL hypothesis may need revision based on studies of HDL structure and function, but that the HDL hypothesis is not dead yet.

Analysis of coding variants in the human FTO gene from the gnomAD database

Single nucleotide polymorphisms (SNPs) in the first intron of the FTO gene reported in 2007 continue to be the known variants with the greatest effect on adiposity in different human populations. Coding variants in the FTO gene, on the other hand, have been little explored, although data from complete sequencing of the exomes of various populations are available in public databases and provide an excellent opportunity to investigate potential functional variants in FTO. In this context, this study aimed to track nonsynonymous variants in the exons of the FTO gene in different population groups employing the gnomAD database and analyze the potential functional impact of these variants on the FTO protein using five publicly available pathogenicity prediction programs. The findings revealed 345 rare mutations, of which 321 are missense (93%), 19 are stop gained (5.6%) and five mutations are located in the splice region (1.4%). Of these, 134 (38.8%) were classified as pathogenic, 144 (41.7%) as benign and 67 (19.5%) as unknown. The available data, however, suggest that these variants are probably not associated with BMI and obesity, but instead, with other diseases. Functional studies are, therefore, required to identify the role of these variants in disease genesis.

Local occurrence and fast spread of B.1.1.7 lineage: A glimpse into Friuli Venezia Giulia

In-depth study of the entire SARS-CoV-2 genome has uncovered many mutations, which have replaced the lineage that characterized the first wave of infections all around the world. In December 2020, the outbreak of variant of concern (VOC) 202012/01 (lineage B.1.1.7) in the United Kingdom defined a turning point during the pandemic, immediately posing a worldwide threat on the Covid-19 vaccination campaign. Here, we reported the evolution of B.1.1.7 lineage-related infections, analyzing samples collected from January 1st 2021, until April 15th 2021, in Friuli Venezia Giulia, a northeastern region of Italy. A cohort of 1508 nasopharyngeal swabs was analyzed by High Resolution Melting (HRM) and 479 randomly selected samples underwent Next Generation Sequencing analysis (NGS), uncovering a steady and continuous accumulation of B.1.1.7 lineage-related specimens, joined by sporadic cases of other known lineages (i.e. harboring the Spike glycoprotein p.E484K mutation). All the SARS-CoV-2 genome has been analyzed in order to highlight all the rare mutations that may eventually result in a new variant of interest. This work suggests that a thorough monitoring of the SARS-CoV-2 genome by NGS is essential to contain any new variant that could jeopardize all the efforts that have been made so far to resolve the emergence of the pandemic.

Involvement of Rare Mutations of SCN9A, DPP4, ABCA13, and SYT14 in Schizophrenia and Bipolar Disorder

Rare mutations associated with schizophrenia (SZ) and bipolar disorder (BD) usually have high clinical penetrance; however, they are highly heterogeneous and personalized. Identifying rare mutations is instrumental in making the molecular diagnosis, understanding the pathogenesis, and providing genetic counseling for the affected individuals and families. We conducted whole-genome sequencing analysis in two multiplex families with the dominant inheritance of SZ and BD. We detected a G327E mutation of SCN9A and an A654V mutation of DPP4 cosegregating with SZ and BD in one three-generation multiplex family. We also identified three mutations cosegregating with SZ and BD in another two-generation multiplex family, including L711S of SCN9A, M4554I of ABCA13, and P159L of SYT14. These five missense mutations were rare and deleterious. Mutations of SCN9A have initially been reported to cause congenital insensitivity to pain and neuropathic pain syndromes. Further studies showed that rare mutations of SCN9A were associated with seizure and autism spectrum disorders. Our findings suggest that SZ and BD might also be part of the clinical phenotype spectra of SCN9A mutations. Our study also indicates the oligogenic involvement in SZ and BD and supports the multiple-hit model of SZ and BD.

Identification of a Rare Novel KMT2C Mutation That Presents with Schizophrenia in a Multiplex Family

Schizophrenia is a complex genetic disorder involving many common variants with modest effects and rare mutations with high penetrance. Rare mutations associated with schizophrenia are highly heterogeneous and private for affected individuals and families. Identifying such mutations can help establish the molecular diagnosis, elucidate the pathogenesis, and provide helpful genetic counseling for affected patients and families. We performed a whole-exome sequencing analysis to search for rare pathogenic mutations co-segregating with schizophrenia transmitted in a dominant inheritance in a two-generation multiplex family. We identified a rare missense mutation H1574R (Histidine1574Arginine, rs199796552) of KMT2C (lysine methyltransferase 2C) co-segregating with affected members in this family. The mutation is a novel deleterious mutation of KMT2C, not reported before in the literature. The KMT2C encodes a histone 3 lysine 4 (H3K4)-specific methyltransferase and involves epigenetic regulation of brain gene expression. Mutations of KMT2C have been found in neurodevelopmental disorders, such as Kleefstra syndrome, intellectual disability, and autism spectrum disorders. Our finding suggests that schizophrenia might be one of the clinical phenotype spectra of KMT2C mutations, and KMT2C might be a novel risk gene for schizophrenia. Nevertheless, the co-segregation of this mutation with schizophrenia in this family might also be due to chance; functional assays of this mutation are needed to address this issue.

Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity

Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10−7 bp) and G:C > C:G (2 × 10−7 bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10−7 bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10−7 bp and 0.12 × 10−7 bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10−7 bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens.

DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes.

RNA sensing via LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency

RNA editing by the enzyme Adenosine Deaminase Acting on RNA 1 (ADAR1) is an important mechanism by which cells avoid innate immune responses to some endogenous RNAs. In ADAR1-deficient cells, unedited self RNAs can form base-paired structures that resemble viral RNAs and inadvertently activate antiviral innate immune pathways that lead to the induction of type I interferon (IFN). Rare mutations in ADAR1 cause Aicardi-Goutieres Syndrome (AGS), a severe childhood autoinflammatory syndrome that is characterized by chronic and excessive type I IFN production and developmental delay. Conversely, ADAR1 dysfunction and consequent type I IFN production helps restrict tumor growth and potentiates the activity of some chemotherapy drugs. Induction of type I IFN in ADAR1-deficient cells is thought to be due to triggering of the cytosolic RIG-I-like receptor (RLR), MDA5, by unedited self RNAs. Here, we show that another RLR, LGP2, also has an essential role. We demonstrate that ADAR1-deficient human cells fail to mount a type I IFN response in the absence of LGP2 and this involves the canonical function of LGP2 as an RNA sensor and facilitator of MDA5-dependent signaling. Further, we show that the sensitivity of tumor cells to ADAR1 loss requires the presence of LGP2. Finally, we find that type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics is fully dependent on the expression of LGP2. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications for the treatment of AGS as well as for the potential application of ADAR1-directed anti-tumor therapy.