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Several molecular mechanisms of thalidomide embryopathy (TE) have been investigated, from anti-angiogenesis to oxidative stress to cereblon binding. Recently, it was discovered that thalidomide and its analogs, named immunomodulatory drugs (IMiDs), induced the degradation of C2H2 transcription factors (TFs). This mechanism might impact the strict transcriptional regulation of the developing embryo. Hence, this study aims to evaluate the TFs altered by IMiDs, prioritizing the ones associated with embryogenesis through transcriptome and systems biology-allied analyses. This study comprises only the experimental data accessed through bioinformatics databases. First, proteins and genes reported in the literature as altered/affected by the IMiDs were annotated. A protein systems biology network was evaluated. TFs beta-catenin (CTNNB1) and SP1 play more central roles: beta-catenin is an essential protein in the network, while SP1 is a putative C2H2 candidate for IMiD-induced degradation. Separately, the differential expressions of the annotated genes were analyzed through 23 publicly available transcriptomes, presenting 8624 differentially expressed genes (2947 in two or more datasets). Seventeen C2H2 TFs were identified as related to embryonic development but not studied for IMiD exposure; these TFs are potential IMiDs degradation neosubstrates. This is the first study to suggest an integration of IMiD molecular mechanisms through C2H2 TF degradation.

Background: HAND2 is a transcription factor important for embryonic development, required for limbs and cardiovascular development. Thalidomide is a drug responsible to a spectrum of congenital anomalies known as Thalidomide Embryopathy (TE), which includes mainly limb and heart defects. It is known that HAND2 interaction with TBX5, an important protein for limbs and heart development, is inhibited by Thalidomide. The aim of this study was to evaluate and characterize HAND2 in the context of TE, and to evaluate its variability in TE individuals., Methods: DNA from 35 TE subjects was extracted from saliva samples and PCR was performed for amplification and Sanger sequencing of HAND2 coding sequence., Results: The analysis showed only one variant; a synonymous variant p.P51 (rs59621536) in exon 1 found in three individuals. Further in silico evaluation confirmed highly HAND2 conservation, being the 3'UTR the most polymorphic region of the gene. Additional computational analyses classified the variant as neutral, without alteration in splicing and miRNA sites. In silico predictions pointed to alteration of two CpG islands adjacent to the variant; however, we did not observe any alterations on the methylation pattern of HAND2 gene in our sample. Moreover, alteration of the binding site of MeCP2, a nuclear protein involved in DNA methylation, was predicted along with alteration in HAND2 mRNA structure., Conclusions: Considering HAND2 being a well conserved gene, further studies with a larger sample should be performed to evaluate the role this gene on genetic susceptibility to TE., (© 2022 Wiley Periodicals LLC.)

The Cereblon-CRL4 complex has been studied predominantly with regards to thalidomide treatment of multiple myeloma. Nevertheless, the role of Cereblon-CRL4 in Thalidomide Embryopathy (TE) is still not understood. Not all embryos exposed to thalidomide develop TE, hence here we evaluate the role of the CRL4-Cereblon complex in TE variability and susceptibility. We sequenced CRBN, DDB1, CUL4A, IKZF1, and IKZF3 in individuals with TE. To better interpret the variants, we suggested a score and a heatmap comprising their regulatory effect. Differential gene expression after thalidomide exposure and conservation of the CRL4-Cereblon protein complex were accessed from public repositories. Results suggest a summation effect of Cereblon variants on pre-axial longitudinal limb anomalies, and heatmap scores identify the CUL4A variant rs138961957 as potentially having an effect on TE susceptibility. CRL4-Cereblon gene expression after thalidomide exposure and CLR4-Cereblon protein conservation does not explain the difference in Thalidomide sensitivity between species. In conclusion, we suggest that CRL4-Cereblon variants act through several regulatory mechanisms, which may influence CRL4-Cereblon complex assembly and its ability to bind thalidomide. Human genetic variability must be addressed not only to further understand the susceptibility to TE, but as a crucial element in therapeutics, including in the development of pharmacogenomics strategies.

Thalidomide is widely used for several diseases; however, it causes malformations in embryos exposed during pregnancy. The complete understanding of the mechanisms by which thalidomide affects the embryo development has not yet been obtained. The phenotypic similarity makes TE a phenocopy of syndromes caused by mutations in ESCO2, SALL4 and TBX5 genes. Recently, SALL4 and TBX5 were demonstrated to be thalidomide targets. To understand if these genes act in the TE development, we sequenced them in 27 individuals with TE; we verified how thalidomide affect them in human pluripotent stem cells (hPSCs) through a differential gene expression (DGE) analysis from GSE63935; and we evaluated how these genes are functionally related through an interaction network analysis. We identified 8 variants in ESCO2, 15 in SALL4 and 15 in TBX5. We compared allelic frequencies with data from ExAC, 1000 Genomes and ABraOM databases; eight variants were significantly different (p < 0.05). Eleven variants in SALL4 and TBX5 were previously associated with cardiac diseases or malformations; however, in TE sample there was no association. Variant effect prediction tools showed 97% of the variants with potential to influence in these genes regulation. DGE analysis showed a significant reduction of ESCO2 in hPSCs after thalidomide exposure.

Erythema Nodosum Leprosum (ENL) occurs due to the immunological complication of multibacillary leprosy and is characterized by painful nodules and systemic compromising. It is usually recurrent and/or chronic and has both physical and economic impact on the patient, being a very important cause of disability. In addition, ENL is a major health problem in countries where leprosy is endemic. Therefore, adequate control of this condition is important. The management of ENL aims to control acute inflammation and neuritis and prevent the onset of new episodes. However, all currently available treatment modalities have one or two drawbacks and are not effective for all patients. Corticosteroid is the anti-inflammatory of choice in ENL but may cause dependence, especially for chronic patients. Thalidomide has a rapid action but its use is limited due the teratogenicity and neurotoxicity. Clofazimine and pentoxifylline have slow action and have important adverse effects. Finally, there is no pattern or guidelines for treating these patients, becoming more difficult to evaluate and to control this condition. This review aims to show the main drugs used in the treatment of ENL and the challenges in the management of the reaction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using β-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide., (© 2017 Anatomical Society.)

Thalidomide is a drug used worldwide for several indications, but the molecular mechanisms of its teratogenic property are not fully understood. Studies in animal models suggest the oxidative stress, the inhibition of angiogenesis, and the binding to E3-ubiquitin ligase complex as mechanisms by which thalidomide can change the expression of genes important to embryonic development. In this study, seven polymorphisms in genes related to development (FGF8, FGF10, BMP4, SHH, TP53, TP63, and TP73) were analyzed in people with thalidomide embryopathy (TE) and compared to people without malformations. The sample consisted of 36 people with TE and 135 unrelated and nonsyndromic people who had their DNA genotyped by PCR real-time. Although no allelic or genotypic differences were observed between the groups, we hypothesized that other regions in these genes and related genes may play an important role in thalidomide teratogenesis, which is known to have a genetic contribution. Identifying such molecular mechanisms is essential for the development of a molecule that will be analogue to thalidomide but safe enough to avoid the emergence of new cases of TE., (© 2017 Wiley Periodicals, Inc.)

Thalidomide embryopathy (TE) has affected more than 10,000 babies worldwide. The hypothesis of antiangiogenesis as the teratogenic mechanism of thalidomide has been investigated in several experimental models; but, in humans, it has only been accessed by in vitro studies. Here, we hypothesized the effect of thalidomide upon angiogenesis-related molecules or proteins, previously identified in human embryonic cells, through the in silico STRING-tool. We also investigated ten polymorphisms in angiogenesis-related genes in 38 Brazilian TE individuals and 136 non-affected Brazilians. NOS2, PTGS2, and VEGFA polymorphisms were chosen for genotyping. The STRING-tool suggested nitric oxide and β-catenin as the central angiogenesis-related molecules affected by thalidomide's antiangiogenic property. We did not identify a significant difference of allelic, genotypic or haplotypic frequencies between the groups. We could not predict a risk allele or a protective one for TE in NOS2, PTGS2, or VEGFA, although other genes should be analyzed in larger samples. The role of nitric oxide and β-catenin must be further evaluated, regarding thalidomide teratogenesis complex etiology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although the thalidomide tragedy occurred more than 50 years ago, the medication is still being used worldwide for different reasons, and several aspects regarding its teratogenicity remain unsolved. Despite the strict regulation implemented, new cases of thalidomide embryopathy (TE) are still being registered in Brazil. Furthermore, the molecular processes that lead to malformations when the embryo is exposed to thalidomide have not yet been fully identified. In this article, we perform a critical analysis of thalidomide's history in Brazil, highlighting aspects of the occurrence of TE over the decades. Finally, we present the main perspectives and challenges for ongoing surveillance and prevention of TE in Brazil. The effective control of dispensing thalidomide, especially in areas where leprosy is endemic, is one of the most important and challenging points. Furthermore, the emergence of thalidomide analogues is fast approaching, and their availability would pose additional concerns. The understanding of the molecular mechanisms and targets of thalidomide in both experimental and human models is essential for generating new insights into teratogenic mechanisms, so that safer thalidomide analogues can be developed., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Thalidomide causes Thalidomide Embryopathy (TE), but is largely used to treat several conditions. Investigations with Cereblon, a thalidomide target protein encoded by CRBN gene, have helped to understand thalidomide therapeutic and teratogenic properties. We sequenced CRBN-thalidomide binding region in 38 TE individuals and 136 Brazilians without congenital anomalies, and performed in silico analyses. Eight variants were identified, seven intronic and one in 3'UTR. TE individuals had rare variants in higher frequency than the non-affected group (p=0.04). The genotype rs1620675 CC was related to neurological anomalies in TE individuals (p=0.004). Bioinformatics analysis suggested this genotype leads to potential alterations in splicing sites and binding to transcription factors. Comparison of the Cereblon-thalidomide binding domains in mammals demonstrated that CRBN is highly conserved across species. All the variants require evaluation in functional assays in order to understand their role in Cereblon-thalidomide binding and complex interactions that lead to TE., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Antiangiogenic properties of thalidomide have created an interest in the use of the drug in treatment of cancer. However, thalidomide is responsible for thalidomide embryopathy (TE). A lack of knowledge regarding the mechanisms of thalidomide teratogenesis acts as a barrier in the aim to synthesize a safer analogue of thalidomide. Recently, our group detected a higher frequency of alleles that impair the pro-angiogenic mechanisms of endothelial nitric oxide synthase (eNOS), coded by the NOS3 gene. In this study we evaluated variable number tandem repeats (VNTR) functional polymorphism in intron 4 of NOS3 in individuals with TE (38) and Brazilians without congenital anomalies (136). Haplotypes were estimated for this VNTR with previously analyzed polymorphisms, rs2070744 (-786C > T) and rs1799983 (894T > G), in promoter region and exon 7, respectively. Haplotypic distribution was different between the groups (p = 0.007). Alleles -786C (rs2070744) and 4b (VNTR), associated with decreased NOS3 expression, presented in higher frequency in TE individuals (p = 0.018; OR = 2.57; IC = 1.2-5.8). This association was not identified with polymorphism 894T > G (p = 0.079), which influences eNOS enzymatic activity. These results suggest variants in NOS3, with pre-transcriptional effects as susceptibility factors, influencing the risk TE development. This finding generates insight for a new approach to research that pursues a safer analogue.

Thalidomide is one of the most potent teratogens known to humans. It is currently used for many clinical situations such as treatment of leprosy reactions and multiple myeloma. However, the teratogenic mechanisms by which it produces morphological defects still remain unclear. One of the hypotheses is the blockage of angiogenesis by reduction of nitric oxide (NO). In this study, we evaluated two functional polymorphisms of the endothelial nitric oxide synthase (eNOS) gene which is a constitutively expressed enzyme responsible for production of NO. The promoter -786T>C exon 7 (896G>T) polymorphisms were genotyped using real-time PCR for 28 individuals with thalidomide embryopathy (TE), 27 first-degree relatives of these individuals, and 68 individuals from the general population. Their allele, genotypic, and haplotypic frequencies were compared. A significant difference was observed in the -786T>C polymorphism genotypes (p=0.03) between the groups affected by TE and those unaffected (non-relatives). The TT genotype of the 896G>T polymorphism was observed in 10.7% of those affected and 2.9% of those unaffected, but the difference was not statistically significant (p=0.09). The haplotypic analysis indicated that the wild haplotype -786T/896G was distributed differently in the affected and unaffected groups (p=0.004). These results indicate that the individuals with TE have a higher frequency of alleles associated with lower expression of eNOS, indicating that this may be a genotype susceptible to TE., (Copyright © 2013 Elsevier Inc. All rights reserved.)