Vijaya L. Simhadri

Exposing synonymous mutations

Synonymous codon changes, which do not alter protein sequence, were previously thought to have no functional consequence. Although this concept has been overturned in recent years, there is no unique mechanism by which these changes exert biological effects. A large repertoire of both experimental and bioinformatic methods has been developed to understand the effects of synonymous variants. Results from this body of work have provided global insights into how biological systems exploit the degeneracy of the genetic code to control gene expression, protein folding efficiency, and the coordinated expression of functionally related gene families. Although it is now clear that synonymous variants are important in a variety of contexts, from human disease to the safety and efficacy of therapeutic proteins, there is no clear consensus on the approaches to identify and validate these changes. Here, we review the diverse methods to understand the effects of synonymous mutations.

Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma

Synonymous mutations, which do not alter the protein sequence, have been shown to affect protein function [Sauna ZE, Kimchi-Sarfaty C (2011) Nat Rev Genet 12(10):683–691]. However, synonymous mutations are rarely investigated in the cancer genomics field. We used whole-genome and -exome sequencing to identify somatic mutations in 29 melanoma samples. Validation of one synonymous somatic mutation in BCL2L12 in 285 samples identified 12 cases that harbored the recurrent F17F mutation. This mutation led to increased BCL2L12 mRNA and protein levels because of differential targeting of WT and mutant BCL2L12 by hsa-miR-671–5p. Protein made from mutant BCL2L12 transcript bound p53, inhibited UV-induced apoptosis more efficiently than WT BCL2L12, and reduced endogenous p53 target gene transcription. This report shows selection of a recurrent somatic synonymous mutation in cancer. Our data indicate that silent alterations have a role to play in human cancer, emphasizing the importance of their investigation in future cancer genome studies.

Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A

Neutralizing antibodies (inhibitors) to replacement Factor-VIII impair the effective management of hemophilia-A1. Individuals with hemophilia-A due to major F8 gene disruptions lack antigenically cross-reactive material in their plasma (CRM-negative) and prevalence of inhibitors is >60%. Conversely, subjects with missense mutations are CRM-positive and the prevalence of inhibitors is <10%2. Individuals with the intron-22-inversion (~50% of individuals with severe hemophilia-A) should be in the former group based on the genetic defect. Although these individuals are CRM-negative, only 20% of them develop inhibitors3. Here we demonstrate the presence of comparable levels of F8 mRNA and intracellular Factor-VIII protein in B-lymphoblastoid cells and liver biopsies from healthy controls and subjects with the intron-22-inversion. These results support the hypothesis that most individuals with the intron-22-inversion are tolerized to Factor-VIII and thus do not develop inhibitors. Furthermore we developed a pharmacogenetic algorithm that permits the stratification of inhibitor risk for sub-populations by predicting immunogenicity using, as input, the number of putative T-cell epitopes in the infused FVIII and the competence of MHC-Class-II molecules to present such epitopes. The algorithm exhibited significant accuracy in predicting inhibitors in 25 unrelated individuals with the intron-22-inversion (AUC = 0.890; P = 0.001).Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma (“CRM-negative”), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ∼50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion.

Recent advances in (therapeutic protein) drug development

Therapeutic protein drugs are an important class of medicines serving patients most in need of novel therapies. Recently approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, autoimmunity/inflammation, exposure to infectious agents, and genetic disorders. The latest advances in protein-engineering technologies have allowed drug developers and manufacturers to fine-tune and exploit desirable functional characteristics of proteins of interest while maintaining (and in some cases enhancing) product safety or efficacy or both. In this review, we highlight the emerging trends and approaches in protein drug development by using examples of therapeutic proteins approved by the U.S. Food and Drug Administration over the previous five years (2011–2016, namely January 1, 2011, through August 31, 2016).

Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

Background: Immunosuppressive drug cyclosporin A (CsA) is a potent inhibitor of cyclophilin B (CypB) function. Results: CsA treatment leading to reduction in CypB levels is associated with decreased secretion of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat). Conclusion: CypB function and levels affect the secretion of ADAMTS13. Significance: A novel mechanistic explanation for CsA-induced thrombotic thrombocytopenic purpura in transplant patients is suggested. The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

Genetic determinants of immunogenicity to factor IX during the treatment of haemophilia B

Inhibitors are an impediment to the effective management of haemophilia B (HB), but there is limited understanding of the underlying genetic risk factors. In this study we aim to understand the role of F9 gene mutations on inhibitor development in patients with HB. Mutations in the F9 gene were identified and HLA typing performed for five boys with severe HB. Data from the CDC Haemophilia B Mutation Project (CHBMP) database were used to assess association between F9 gene mutation type and inhibitor development. Analysis of the CHBMP database showed that larger disruptions in the F9 gene are associated with a higher life‐time prevalence of inhibitors. We detected the following mutations in the five subjects, including four novel mutations: Nonsense in three patients (c.223 C>T; p.Arg75* in two siblings, c.553 C>T; p.Glu185*); Splice site in two patients (c.723 + 1 G>A, c.278‐27 A>G); Missense in one patient (c.580 A>G, p.Thr194Ala; c.723 G>T; p.Gln241His). Of the two siblings only one responded to immune tolerance induction (ITI). These siblings have identical F9 gene mutations but differ with respect to the HLA alleles. Interestingly, an analysis of peptide‐MHC binding affinities shows a significantly higher (one‐sided unpaired t‐test, P = 0.0018) median affinity for FIX‐derived peptides in the sibling that responded to ITI. We conclude that the nature of the F9 gene mutation may be an important risk factor for the development of inhibitors. In addition, the HLA alleles of the individual patients, in conjunction with the mutation type, could be a predictor for the development of inhibitors as well as the response to ITI.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B

Background Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%–20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. Methods We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. Results The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. Conclusions The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.

Small ncRNA Expression-Profiling of Blood from Hemophilia A Patients Identifies miR-1246 as a Potential Regulator of Factor 8 Gene

Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). Genetic mutations in the gene encoding FVIII (F8) have been extensively studied. Over a thousand different mutations have been reported in the F8 gene. These span a diverse range of mutation types, namely, missense, splice-site, deletions of single and multiple exons, inversions, etc. There is nonetheless evidence that other molecular mechanisms, in addition to mutations in the gene encoding the FVIII protein, may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-à-vis the role of nc-RNAs in the regulation of F8 expression. These hypotheses have not been exhaustively tested in this study as they require carefully curated clinical samples.

Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population

The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter. There are many avenues for how CRISPR/Cas9 technologies will be applied in vivo, including the mode of delivery. These may be expected to invoke different immunological pathways. Nonetheless, as with all protein therapeutics, it may be desirable to monitor for anti-Cas9 antibodies during clinical development. This will require the development of robust and reliable assays. Here, we describe ELISA-based assays that are capable of detecting antibodies to Cas9 from Staphylococcus aureus (SaCas9) and Streptococcs pyogenes (SpCas9) in human sera. Furthermore, using these assays to screen for pre-existing antibodies in 200 human serum samples, we found the prevalence of anti-SaCas9 and anti-SpCas9 antibodies to be 10% and 2.5%, respectively.

Single‐nucleotide variations defining previously unreported ADAMTS13 haplotypes are associated with differential expression and activity of the VWF‐cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family

Although autosomal recessive haematological disorders, such as congenital thrombotic thrombocytopenic purpura (cTTP), are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown molecular-genetic characteristics. We present an in-depth examination of ADAMTS13 haplotypes, mRNA levels and protein expression, activity, and enzyme kinetics in this case study of two Salvadoran children with cTTP – the first reported cases of this disease in individuals originally from Central America – and their parents. The propositi – a son and a daughter born to asymptomatic, non-consanguineous (but possibly distantly related) parents from the same town in El Salvador – were diagnosed with cTTP at ages 6 and 2 years, respectively. Although they developed haemolytic anaemia and thrombocytopenia 2 d (son) and 17 months (daughter) after birth, their cTTP diagnosis was not rendered until both were hospitalized simultaneously with fever, respiratory symptoms, haemolytic anaemia and thrombocytopenia. Peripheral blood smears for both demonstrated schistocytes, and their von Willebrand factor-cleaving protease (VWF-CPase) activities were <1% without ADAMTS13 IgG antibodies. Fresh frozen plasma (FFP) infusion induced rapid (within 48 h) normalization of their blood counts and resolution of the microangiopathic changes. Both children have been treated with prophylactic infusions of FFP (10 ml/kg every 2 5 weeks) without long-term neurological or renal sequelae. Following approval by institutional review boards and receipt of informed consent, blood samples were collected from the children, prior to FFP administration, and from both parents. Genomic DNA and total RNA were extracted from peripheral blood leukocytes using QIAamp DNA Blood Maxi and PAXgene Blood RNA kits, respectively (Qiagen; Germantown, MD, USA). All ADAMTS13 exons, at least 50 base pairs (bp) of each flanking intron junction, ~500 bp 5’ of the promoter, and ~200 bp of 3’-flanking genomic DNA were amplified using polymerase chain reaction (PCR), cleaned using Exo-SAP-IT (Affymetrix; Santa Clara, CA, USA), and subjected to direct bi-directional Sanger sequencing followed by capillary electrophoresis and analysis on ABI-3730 instruments. Reverse transcription was performed (Shomron et al, 2010), and plasma VWF-CPase antigen was measured using the Technozym ADAMTS13 enzyme-linked immunosorbent assay (Technoclone; Vienna, Austria) and the Victor X3 multilabel plate reader (PerkinElmer; Waltham, MA, USA). Fluorogenic FRETS-VWF73 (Peptides International; Louisville, KY, USA) was prepared and assayed (Sauna et al, 2009). The kinetic characteristics of ADAMTS13 were obtained using GraphPad Prism software. ADAMTS13 sequencing revealed both children to be homozygous and their parents to be heterozygous for the previously described, cTTP-causing, single-base-substitution mutation 20506C > T (Table IA) (Levy et al, 2001; Snider et al, 2004; Hing et al, 2013). The children’s VWF-CPase antigen and activity levels were undetectable, although steady-state levels of the ADAMTS13 mRNA were >2 5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional single-nucleotide variations (SNVs), including two previously unreported non-synonymous (ns)-SNVs: 27852C > T (c.3362C > T ⇒ 972Arg > Trp) and 33325G > A (c.3733G > A ⇒ 1096Arg > His) (Table IA). The parents’ genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA, and protein levels (Table IA). The quantitative realtime-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (P < 0 001; Table IB). Plasma VWF-CPase activity and antigen levels were ~2-fold greater in the father than in the mother (P = 0 00164 and P = 0 0633, respectively), but the specific activities of these structurally distinct VWF-CPase proteins were notably almost identical (253 5 vs. 256 2 U/lg). Initial velocity kinetic analysis using the Michelis-Menten equation demonstrated that the Vmax of the father’s VWFCPase was twice that of the mother’s (1 4 vs. 0 7; P < 0 0001) while its affinity for substrate (FRETS-VWF73) was one-third that of her VWF-CPase (Km = 0 3 vs. 0 1; P = 0 0585).