Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

Development of mAb-based polyglutamine-dependent and polyglutamine length-independent huntingtin quantification assays with cross-site validation.

Huntington's disease (HD) is caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin gene that results in expression of a mutant huntingtin protein (mHTT) containing an expanded polyglutamine tract in the amino terminus. A number of therapeutic approaches that aim to reduce mHTT expression either locally in the CNS or systemically are in clinical development. We have previously described sensitive and selective assays that measure human HTT proteins either in a polyglutamine-independent (detecting both mutant expanded and non-expanded proteins) or in a polyglutamine length-dependent manner (detecting the disease-causing polyglutamine repeats) on the electrochemiluminescence Meso Scale Discovery detection platform. These original assays relied upon polyclonal antibodies. To ensure an accessible and sustainable resource for the HD field, we developed similar assays employing monoclonal antibodies. We demonstrate that these assays have equivalent sensitivity compared to our previous assays through the evaluation of cellular and animal model systems, as well as HD patient biosamples. We also demonstrate cross-site validation of these assays, allowing direct comparison of studies performed in geographically distinct laboratories.

CRISPRing future medicines.

Introduction: The ability to engineer mammalian genomes in a quick and cost-effective way has led to rapid adaptation of CRISPR technology in biomedical research. CRISPR-based engineering has the potential to accelerate drug discovery, to support the reduction of high attrition rate in drug development and to enhance development of cell and gene-based therapies.Areas covered: How CRISPR technology is transforming drug discovery is discussed in this review. From target identification to target validation in both in vitro and in vivo models, CRISPR technology is positively impacting the early stages of drug development by providing a straightforward way to genome engineering. This property also attracted attention for CRISPR application in the cell and gene therapy area.Expert opinion: CRISPR technology is rapidly becoming the preferred tool for genome engineering and nowadays it is hard to imagine the drug discovery pipeline without this technology. With the years to come, CRISPR technology will undoubtedly be further refined and will flourish into a mature technology that will play a key role in supporting genome engineering requirements in the drug discovery pipeline as well as in cell and gene therapy development.

NEK11 as a candidate high-penetrance melanoma susceptibility gene.

BACKGROUND: A proportion of patients diagnosed with cutaneous melanoma reports a positive family history. Inherited variants in CDKN2A and several other genes have been shown to predispose to melanoma; however, the genetic basis of familial melanoma remains unknown in most cases. The objective of this study was to provide insight into the genetic basis of familial melanoma. METHODS: In order to identify novel melanoma susceptibility genes, whole exome sequencing (WES) analysis was applied in a Dutch family with melanoma. The causality of a candidate variant was characterised by performing cosegregation analysis in five affected family members using patient-derived tissues and digital droplet PCR analysis to accurately quantify mutant allele frequency. Functional in-vitro studies were performed to assess the pathogenicity of the candidate variant. RESULTS: Application of WES identified a rare, nonsense variant in the NEK11 gene (c.1120C>T, p.Arg374Ter), cosegregating in all five affected members of a Dutch family. NEK11 (NIMA-related Kinase 11) is involved in the DNA damage response, enforcing the G2/M cell cycle checkpoint. In a melanoma from a variant carrier, somatic loss of the wildtype allele of this putative tumour suppressor gene was demonstrated. Functional analyses showed that the NEK11 p.Arg374Ter mutation results in strongly reduced expression of the truncated protein caused by proteasomal degradation. CONCLUSION: The NEK11 p.Arg374Ter variant identified in this family leads to loss-of-function through protein instability. Collectively, these findings support NEK11 as a melanoma susceptibility gene.

A novel germline variant in the DOT1L gene co-segregating in a Dutch family with a history of melanoma.

A proportion of patients diagnosed with melanoma has a positive family history. Despite increasing knowledge on the genes responsible for familial clustering, the genetic basis in the majority of the families with an inherited predisposition to melanoma remains to be clarified. To identify novel melanoma-susceptibility genes, we applied whole-exome sequencing on DNA from two members of a family with four melanoma cases, not explained by established high penetrance melanoma-susceptibility genes. Whole-exome sequencing identified 10 rare, co-segregating, predicted deleterious missense gene variants. Subsequent co-segregation analysis revealed that only variants in the DOT1L (R409H) and the SLCO4C1 (P597A) genes were present in the other two affected members of this family. DOT1L is a methyltransferase that methylates histone H3 lysine 79 (H3K79). It is involved in maintenance of genomic stability, since mutations in the DOT1L gene have been previously reported to compromise the removal of ultraviolet photoproducts in ultraviolet-irradiated melanocytes, thereby enhancing malignant transformation. We hypothesized that the presence of DOT1L R409H variant might be associated with an increased risk of melanoma, since we found co-segregation of the DOT1L mutation in all four melanoma-affected family members. However, this missense variant did neither lead to detectable loss-of-heterozygosity nor reduction of histone methyltransferase activity in melanoma samples from mutation carriers nor altered ultraviolet-survival of mouse embryonic stem cells containing an engineered homozygous DOT1L R409H mutation. Although functional analysis of this rare co-segregating variant did not reveal compromised histone methyltransferase activity and ultraviolet exposure sensitivity, the role of DOT1L as melanoma susceptibility gene deserves further study.

Genetics of skin color variation in Europeans: genome-wide association studies with functional follow-up.

In the International Visible Trait Genetics (VisiGen) Consortium, we investigated the genetics of human skin color by combining a series of genome-wide association studies (GWAS) in a total of 17,262 Europeans with functional follow-up of discovered loci. Our GWAS provide the first genome-wide significant evidence for chromosome 20q11.22 harboring the ASIP gene being explicitly associated with skin color in Europeans. In addition, genomic loci at 5p13.2 (SLC45A2), 6p25.3 (IRF4), 15q13.1 (HERC2/OCA2), and 16q24.3 (MC1R) were confirmed to be involved in skin coloration in Europeans. In follow-up gene expression and regulation studies of 22 genes in 20q11.22, we highlighted two novel genes EIF2S2 and GSS, serving as competing functional candidates in this region and providing future research lines. A genetically inferred skin color score obtained from the 9 top-associated SNPs from 9 genes in 940 worldwide samples (HGDP-CEPH) showed a clear gradual pattern in Western Eurasians similar to the distribution of physical skin color, suggesting the used 9 SNPs as suitable markers for DNA prediction of skin color in Europeans and neighboring populations, relevant in future forensic and anthropological investigations.

Allele-specific transcriptional regulation of IRF4 in melanocytes is mediated by chromatin looping of the intronic rs12203592 enhancer to the IRF4 promoter.

The majority of significant single-nucleotide polymorphisms (SNPs) identified with genome-wide association studies are located in non-coding regions of the genome; it is therefore possible that they are involved in transcriptional regulation of a nearby gene rather than affecting an encoded protein's function. Previously, it was demonstrated that the SNP rs12203592, located in intron 4 of the IRF4 gene, is strongly associated with human skin pigmentation and modulates an enhancer element that controls expression of IRF4. In our study, we investigated the allele-specific effect of rs12203592 on IRF4 expression in epidermal skin samples and in melanocytic cells from donors of different skin color. We focused on the characteristics and activity of the enhancer, and on long-range chromatin interactions in melanocytic cells homozygous and heterozygous for rs12203592. We found that, irrespective of the trans-activating environment, IRF4 transcription is strongly correlated with the allelic status of rs12203592, the activity of the rs12203592 enhancer and that the chromatin features depend on the rs12203592 genotype. Furthermore, we demonstrate that the rs12203592 enhancer physically interacts with the IRF4 promoter through an allele-dependent chromatin loop, and suggest that subsequent allele-specific activation of IRF4 transcription is stabilized by another allele-specific loop from the rs12203592 enhancer to an additional regulatory element in IRF4. We conclude that the non-coding SNP rs12203592 is located in a regulatory region and affects a wide range of enhancer characteristics, resulting into modulation of the enhancer's activity, its interaction with the IRF4 promoter and subsequent allele-specific transcription of IRF4. Our findings provide another example of a non-coding SNP affecting skin color by modulating enhancer-mediated transcriptional regulation.

Human skin color is influenced by an intergenic DNA polymorphism regulating transcription of the nearby BNC2 pigmentation gene.

Single nucleotide polymorphisms (SNPs) found to be statistically significant when associated with human diseases, and other phenotypes are most often located in non-coding regions of the genome. One example is rs10765819 located in the first intron of the BNC2 gene previously associated with (saturation of) human skin color. Here, we demonstrate that a nearby intergenic SNP (rs12350739) in high linkage disequilibrium with rs10756819 is likely the causal DNA variant for the observed BNC2 skin color association. The highly conserved region surrounding rs12350739 functions as an enhancer element regulating BNC2 transcription in human melanocytes, while the activity of this enhancer element depends on the allelic status of rs12350739. When the rs12350739-AA allele is present, the chromatin at the region surrounding rs12350739 is inaccessible and the enhancer element is only slightly active, resulting in low expression of BNC2, corresponding with light skin pigmentation. When the rs12350739-GG allele is present however, the chromatin at the region surrounding rs12350739 is more accessible and the enhancer is active, resulting in a higher expression of BNC2, corresponding with dark skin pigmentation. Overall, we demonstrate the identification of the functional DNA variant that explains the BNC2 skin color association signal, providing another important step towards further understanding human pigmentation genetics beyond statistical association. We thus deliver a clear example of how an intergenic non-coding DNA variant modulates the regulatory potential of the enhancer element it is located within, which in turn results in allele-dependent differential gene expression affecting variation in common human traits.

Genetic variation in regulatory DNA elements: the case of OCA2 transcriptional regulation.

Mutations within the OCA2 gene or the complete absence of the OCA2 protein leads to oculocutaneous albinism type 2. The OCA2 protein plays a central role in melanosome biogenesis, and it is a strong determinant of the eumelanin content in melanocytes. Transcript levels of the OCA2 gene are strongly correlated with pigmentation intensities. Recent studies demonstrated that the transcriptional level of OCA2 is to a large extent determined by the noncoding SNP rs12913832 located 21.5 kb upstream of the OCA2 gene promoter. In this review, we discuss current hypotheses and the available data on the mechanism of OCA2 transcriptional regulation and how this is influenced by genetic variation. Finally, we will explore how future epigenetic studies can be used to advance our insight into the functional biology that connects genetic variation to human pigmentation.

A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.

In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 µL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 µL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present.

HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter.

Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases.

mRNA-based skin identification for forensic applications.

Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous studies, in skin samples and in other forensically relevant tissues. We identified mRNA transcripts from three genes CDSN, LOR and KRT9, showing strong over-expression in skin samples relative to samples from forensic body fluids, making them suitable markers for skin identification. Out of the candidate reference genes tested, only ACTB showed similarly high expression in skin and body-fluid samples, providing a suitable reference marker for quantitative real-time PCR (qPCR) analysis of skin. Analyses of palmar and thumbprint skin samples indicate that our qPCR approach for the three skin-targeted mRNA markers, as well as the reference mRNA marker ACTB, is highly sensitive, allowing successful detection of minute amounts of skin material including full, half and quarter thumbprints, albeit with decreased success in decreasing print material. Furthermore, thumbprints stored for 6.5 months provided similar results relative to freshly analysed samples implying reasonable time-wise stability of the three skin-targeted mRNAs as well as the ACTB reference mRNA. Our study represents the first attempt towards reliable mRNA-based skin identification in forensic applications with particular relevance for future trace/touched object analyses in forensic case work. Although the approach for skin identification introduced here can be informative when applied on its own, we recommend for increased reliability the integration of (one or more of) the skin-targeted mRNA markers presented here into multiplex assays additionally including mRNA markers targeting alternative cell types expected in forensic samples.

Apoptin: therapeutic potential of an early sensor of carcinogenic transformation.

The avian virus-derived protein apoptin induces p53-independent apoptosis in a tumor-specific way. Apoptin acts as a multimeric complex and forms superstructures upon binding to DNA. In tumor cells, apoptin is phosphorylated and mainly nuclear, whereas in normal cells it is unphosphorylated, cytoplasmic, and becomes readily neutralized. Interestingly, apoptin phosphorylation, nuclear translocation, and apoptosis can transiently be induced in normal cells by cotransfecting SV40 large T oncogene, indicating that apoptin recognizes early stages of oncogenic transformation. In cancer cells, apoptin appears to recognize survival signals, which it is able to redirect into cell death impulses. Apoptin targets include DEDAF, Nur77, Nmi, Hippi, and the potential drug target APC1. Apoptin-transgenic mice and animal tumor models have revealed apoptin as a safe and efficient antitumor agent, resulting in significant tumor regression. Future antitumor therapies could use apoptin either as a therapeutic bullet or as an early sensor of druggable tumor-specific processes.