Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients [version 2; peer review: 3 approved]

Some somatic single nucleotide variants (SNVs) are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an autoantigen associated with multiple sclerosis (MS) would contain SNVs. MS patients develop antibodies to hnRNP A1 293-304 , an epitope within the M9 domain (AA 268-305 ) of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1) and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1 268-289 ) and the MS IgG epitope (hnRNP A1 293-304 ), within M9. In contrast to the nuclear localization of wild type (WT) hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, I have no further comments. The authors have addressed my points and have pointed out their view on this issue and that some of the points have to be addressed in future studies. Competing I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. an innovative set of complex studies seeking to elucidate somatic mutation variants in patients with MS; across the various clinical subtypes, and compared to normal subjects. Quite surprising this the identified a TPNO-1 binding domain change of hnRNPA1-M9; an work by Levin on the role of an immune response to hnRNP-A1 in human demyelinating disorders, including multiple sclerosis. Previously, Levin had shown that an antibody response to HTLV-I Tax cross-reacted with hnRNP-A1. Now, they go on to show that there are mutations in hnRNP-A1 that affect the localization of this protein and how it could affect neuronal survival in multiple sclerosis.In general, the experiments appear to be well performed. One might be interested to know whether testing in another cell line such as a oligodendroglial cell line would have similar effects on cell survival as in the neuroblastoma cell work discussion. This is a potentially interesting study, reporting increased presence of novel somatic single nucleotide variants (SNVs) within the RNA binding protein hnRNP A1 in peripheral blood mononuclear cells (PBMCs) in patients with multiple sclerosis (MS) in comparison to controls. The disease associated SNVs resulted in a mis-localization of hnRNP A1 into cytoplasmic stress granules and this mis-localization was associated with cellular apoptosis in cells, transfected with the respective mutants. From a technical point of view the study is well performed. The question, however, remains what the findings mean for the pathogenesis of multiple sclerosis.It is interesting to see that there is apparently a high incidence of induced SNVs in PBMCs of MS patients, which most likely not only affects the gene investigated in this study. This may be due to the fact that MS is a chronic inflammatory disease, leading to proliferation of leukocytes (in particular T-lymphocytes) in the peripheral immune system. In addition increased oxidative stress in PBMCs of MS patients has been reported before. Both conditions may lead to increased somatic mutation in these cell populations. Thus, in a study like this the inclusion of a control population with long-lasting chronic inflammation would be important in addition to the study of normal controls. According to the presented data, the particular SNVs are also likely to result in cell elimination. Whether this changes the immune response in MS patients has not been investigated in this study.Finally the authors discuss that their findings may have This study reports the discovery of somatic (acquired) mutations in the hnRNPA1 gene in multiple sclerosis patients. Unfortunately, the method for detecting somatic mutations is not valid. The authors discovered the mutations using a method that is artefact prone. They first created PCR-product of the target region, then cloned these PCR products into a plasmid vector and then sequenced bacterial colonies carrying these vectors. It is a well-known problem to all, who have done PCR-cloning that artefact mutations, created by PCR amplification, are commonly encountered. There was no validation of the mutation by another method. Therefore, there is no convincing evidence for the existence of somatic mutations in the multiple sclerosis patients in the hnRNPA1 gene. These mutations are most likely PCR-artefacts.