Quote from Johannes on February 10, 2022, 6:53 am

I’ve previously posted about vaccines and Vitamin A and discovered data anomalies that I thought were evidence of data manipulation. I’ve been in contact with different scientists about these anomalies, and no one I’ve spoken to was really able to explain them. I uncovered some more details, so in this post I’ll try to start from the beginning and explain everything in detail. The key point is that, even though data was manipulated, I think the anomalies could be real and a sign of infertility. Vitamin A could play a role in the pathogenesis of vaccine-induced infertility.

Background

Sex chromosomes differ between mammalian females and males. Males have one X and one Y chromosome whereas females have two X chromosomes. During the development of female embryos, about 80% of the genes on one X chromosome are inactivated (X-chromosome inactivation, XCI) to prevent excess transcription of these genes (Fang, Disteche et al. 2019). The long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) plays a role in X-inactivation and is only found on the inactivated X chromosome.

Consequently, biological males express genes located on the Y-chromosome (Y-linked genes) but not XIST, whereas biological females express XIST but not Y-linked genes.

The study

32 volunteers were recruited for the study and and treated with the COVID-19 vaccine BNT162b2 (Arunachalam, Scott et al. 2021). I attached a table with full participant and anomaly details to this post. Whole blood RNA-Sequencing (RNA-Seq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) of peripheral blood mononuclear cells (PBMCs; these are usually white blood cells) was performed on different days. The first vaccine dose was given before sampling on Day 1 and the second dose was given before sampling on Day 22.

In the RNA-Seq data, the reported sex of 12 participants (37.5%) differs from the sex that is apparent from the data. Since the apparent and reported sexes are in agreement for the CITE-Seq data, but not for the RNA-Seq data, it must be assumed that a single participant ID identifies distinct people in both datasets. Additionally, very little data is available for Day 1. Notwithstanding these problems with the study, the phenomenon appears to be real and not an artifact of data manipulation.

The phenomenon

The biological sex of some participants appears to briefly switch to the opposite sex. In PBMCs, only on Day 1 (i.e. after the first vaccine dose), 1 of 1 female participant (100%) expressed the Y-linked genes RPS4Y1, DDX3Y, EIF1AY, UTY, PRKY, USP9Y, KDM5D, ZFY, TTTY14, TMSB4Y and TTTY10 for the first time, but did not stop expressing XIST. Also on Day 1, 1 of 2 male participants (50%) expressed XIST and its antisense transcript TSIX for the first time, but did not stop expressing Y-linked genes. Some, but not all sexually dimorphic genes on autosomes were also affected, for example DDIT4 and CTSW but not H2AC17. Sexually dimorphic mitochondrial genes such as MT-ATP8 were not affected.

In whole blood, 2 men and 2 women (12.5%) were affected by a similar phenomenon. In contrast to PBMCs, affected female participants not only began expressing Y-linked genes, they also stopped expressing XIST. Similarly, affected male participants expressed XIST and no longer expressed Y-linked genes. In 3 of 4 affected participants the phenomenon is only evident on one day, however in the remaining male participant the phenomenon first appeared on Day 22/23 (after second vaccine dose) and was still present on Day 28, the last sample day of the study.

Thus, the total number of affected, unique participants is 6 of 32 (19%), and the phenomenon may have been permanent in 1 of 32 (3%) participants. To the best of my knowledge, such a phenomenon has not previously been described in literature. Especially puzzling is the question of how a biological female could begin transcribing Y-linked genes, since Y chromosomes are absent in females.

Adverse events linked to the phenomenon

It’s a bit difficult to link this phenomenon to known adverse events, since something like this hasn’t been described in literature and no one really knows how such gene expression would present in terms of clinical symptoms. The least controversial association would probably be with menstrual cycle changes, since it isn’t hard to see how a woman expressing men’s genes might become affected by menstrual cycle disturbances. Since 19% of women were affected by the phenomenon in the vaccine study, and about 10–15% of women self-reported menstrual cycle changes following vaccination (Trogstad 2022), it isn’t very hard to see a potential connection.

I would also imagine that a woman expressing men’s genes might have a hard time conceiving children. The youngest affected female participant was reported as 24 years old. We have some more evidence in that respect from male infertility studies. The Wikipedia page for azoospermia factor (AZF) notes, without providing a reference, that “deletions in the USP9Y gene, which is located within AZF1, are usually associated with inability to form sperm.” Probable ubiquitin carboxyl-terminal hydrolase FAF-Y (USP9Y) is one of the affected Y-linked genes, for example participant 2027 (biological male) completely stopped expressing USP9Y in whole blood after the second vaccine dose, and mRNA of USP9Y was still not quantifiable five days later, suggesting that the vaccine may have caused permanent infertility in this participant. If anyone wants to go deeper down the rabbit hole, this post might be a good place to start.

Hypothetical mechanism to explain the phenomenon

There is mounting evidence to suggest that the lipophilic Vitamin A (retinol) plays a role in the pathogenesis of COVID-19 (reviewed by Mawson, Croft et al. 2021). In plasma, retinol is transported in complex with retinol-binding protein 4 (RBP4) and transthyretin (TTR), and delivered to cells expressing the transmembrane receptor for retinol uptake STRA6 (Berry, O'Byrne et al. 2012). The interaction between retinol-RBP4-TTR and STRA6 leads to activation of tyrosine-protein kinase JAK2 and further downstream effects. Additionally, retinol is delivered from the extracellular RBP4-TTR complex to cytosolic retinol-binding protein 1 (RBP1).

SARS-CoV-2 spike glycoprotein (S) contains a fatty acid-binding pocket which was predicted to accept retinol as a ligand (Shoemark, Colenso et al. 2021). Furthermore S is predicted to interact with STRA6 (Mahmoud, Tamer et al. 2021). STRA6 is differentially expressed in human PBMC subsets (Barrett 2019). It is unclear whether this interaction results in dissociation of retinol from the S fatty acid-binding pocket, and if it does occur, whether retinol is delivered to RBP1 (which transports it to storage), a different transport protein or possibly even to cytosolic S. S has been reported to localize to nucleus (Jiang and Mei 2021). It is possible that retinol could be transferred from extracellular S to cytosolic S, which then translocates to nucleus, transporting retinol into the cell’s nucleus.

The modified S in COVID-19 vaccines is cleaved into S₁ and S₂ fragments, and only S₁ enters circulation. S₁ is quantifiable in plasma for about 7 days following vaccination (Ogata, Cheng et al. 2021). Since the fatty acid-binding pocket is located in S₁, this mechanism would require an interaction between retinol-S₁ in systemic circulation and cytosolic full length apo-S that was very recently synthesized (recent enough to not have gotten cleaved yet). The interaction would be mediated by STRA6. Ligand binding of linoleic acid was reported to stabilize S (Toelzer, Gupta et al. 2020), so the delivery of retinol to full-length S may stabilize it, protect it from intracellular cleavage or even induce nuclear translocation. Furthermore there are two specific serine sites in S that, if phosphorylated, protect S from furin cleavage (Örd, Faustova et al. 2020). Since STRA6 is found in complex with JAK2, it shouldn’t be ruled out that JAK2 could interact with S. JAK2 phosphorylates different substrates including STRA6 and  STRA6-bound signal transducer and activator of transcription 5A (STAT5A). Even though JAK inhibitors have been proposed as a treatment strategy for COVID-19 (Seif, Aazami et al. 2020), a possible direct interaction between Janus kinases and S has not been investigated to date. It is also unclear whether the interaction between S and STRA6 leads to phosphorylation of JAK2. Activation of JAK2 is required for phosphorylation of STRA6/STAT5A but not for transfer of retinol to RBP1.

The oxidative retinol metabolite retinoic acid (RA) was shown to be required for initiation of the mitosis-to-meiosis transition in mice (Koubova, Menke et al. 2006). Furthermore, RA has a variety of specific and nonspecific genomic effects. As a transcription factor ligand, it induces dimerization of retinoic acid receptors (RAR) and retinoic acid receptors X (RXR) and transcription of genes from the RA response element (RARE). Nonspecifically, RA was shown to cause DNA damage including DNA fragmentation and micronucleation (Alakhras, Stephanou et al. 2014), (Murata and Kawanishi 2000). RA is also capable of altering chromatin structure (reviewed by Gudas 2013).

RA was reported to reverse methylations catalyzed by N-lysine methyltransferase SETD6 (Binda, Sevilla et al. 2013), which acts on Histone H2A.Z (H2AZ). H2AZ localized near transcription start sites of active genes is found acetylated (acH2A.Z) whereas H2AZ localized near inactive genes is not acetylated (Valdés-Mora, Song et al. 2012). Methylation of H2AZ at lysine 7 (H2AZK7me1) is required for the maintenance of embryonic stem cell self-renewal (Binda, Sevilla et al. 2013). H2AZ is ubiquitinated at lysines 120 and 121 on the inactive X chromosomes of female cells (Sarcinella, Zuzarte et al. 2007). Ubiquitination is mediated by putative E3 ubiquitin-protein ligase RING1b, and de-ubiquitination is mediated by ubiquitin carboxyl-terminal hydrolase 10 (USP10) (Draker, Sarcinella et al. 2011).

Since RA is capable of affecting H2AZ methylation status, it should be explored whether RA is capable of affecting H2AZ ubiquitination status. If RA were to reverse K120ub1 and K121ub1, or modulate expression of RING1B/USP10, it could explain transient expression of previously inactive X-linked genes.

Therefore, if vaccination, by introduction of S, causes transport of retinol or RA into cellular nuclei via S, the unusual gene expression seen in the data would not be entirely impossible. Uncontrolled delivery of retinol into nuclei would likely lead to undefined behavior, and may explain the broad range of adverse events reported after vaccination. Furthermore plasma retinol concentrations and hepatic ester deposits are highly variable (a function of lifetime Vitamin A consumption) and this would explain why only some vaccine recipients are affected.

Vitamin A-repressor BHLHE40

I also discovered that class E basic helix-loop-helix protein 40 (BHLHE40), formerly known as stimulated by retinoic acid gene 13 (STRA13) is differentially regulated between vaccine doses in PBMCs, and downregulation of BHLHE40 coincided with data anomalies in both PBMCs and whole blood. In PBMCs, BHLHE40 is down significantly (p < 0.05) on Day 1, highly variable on Day 22 and up significantly (p < 0.01) on Day 28. Both anomalies occurred on Day 1. In whole blood, BHLHE40 consistently decreases, reaching its lowest point on Day 21 (p < 0.01) before recovering. The first anomaly occurred on Day 21, another on Day 22/23 and two more on Day 28. Individually, BHLHE40 was down versus Day 0 in 5 of 6 participants affected by anomalies across both datasets. In the remaining male participant, BHLHE40 was up 20-fold versus Day 0 and 6-fold versus Day 21 on the day of the anomaly (Day 28).

BHLHE40 is part of the circadian clock pathway where it acts as a transcriptional repressor (UniProt). It participates in chromatin remodeling by regulating chromatin binding of transcriptional repressor CTCF (Hu, Dong et al. 2020). In activated B cells and T follicular cells, BHLHE40 restrains the germinal center reaction and prevents lymphomagenesis (Rauschmeier, Reinhardt et al. 2021). It is also highly expressed in the hippocampus, where loss of BHLHE40 was associated with decreased synaptic plasticity (Hamilton, Wang et al. 2018). If I’m understanding the data from (Schwartz, Farley et al. 2019) correctly, it would seem that exposure to RA causes downregulation of BHLHE40 and Fos-related antigen 2 (FOSL2) in induced regulatory T cells (iTreg). This is a remarkable coincidence, since I discovered that the top three genes most correlated with BHLHE40 were FOSL2, DNA damage-inducible transcript 3 protein (DDIT3) and γ-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1). This correlation was consistent in whole blood as well. I will see whether I can show that vaccination activates the same genetic program that is also activated by RA.

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