Daily Archives: December 6, 2020

Abstract

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

The study of histones and their role in epigenetics is a rapidly expanding field. Histones are nuclear proteins that associate with DNA to facilitate packaging into condensed chromatin. Histones are dynamically decorated with posttranslational modifications (PTMs), which regulate these processes as DNA repair, gene expression, mitosis, and meiosis. Dysregulation of PTMs leads to cancer, neurodevelopmental syndromes, psychiatric disorders, and even cardiovascular disease (14). The vast array of diseases that stem from histone dysregulation makes understanding histone biology vital to understanding the pathophysiology of many diseases and developing treatments. Histones are highly evolutionarily conserved, and only a few germline disease-causing variants in the histones themselves have been found (5). Rather, most disease-causing variants affecting histone regulation are found in histone-modifying proteins or histone chaperones. Somatic mutations in histone H3.3 (H3.3), encoded by both H3F3A and H3F3B, have recently been identified in pediatric glia and other tumors. This discovery revolutionized the field of epigenetic changes in tumors and how they lead to cancer progression (6). There has been one case report suggesting a relationship between a neurodevelopmental syndrome and H3F3A, but no additional patients or functional data have been published previously (7). Studying how histone mutations cause disease can provide the key to understanding how mutations in the histone network lead to disease.

Histone 3 family 3 (H3F3) histones (H3.3) mark active genes, maintain epigenetic memory, and maintain heterochromatin and telomeric integrity. Every nucleosome contains a version of H3. H3.1 and H3.2 are canonical histones that are replication dependent and, therefore, added to chromatin only during DNA replication. H3.3, however, is a replication-independent variant, which differs from the canonical H3.1 and H3.2 by only five and four amino acids, respectively (8). H3F3A and H3F3B code for an identical protein product, H3.3, a 135amino acid protein (after cleavage of the first methionine) with a histone tail, four helices, and two loop domains. These genes, however, contain different regulatory and coding sequences, which lead to different expression patterns and levels for H3F3A and H3F3B. Both H3F3B and H3F3A are expressed ubiquitously, during development and throughout life, with relatively high levels of expression in the brain, testes, ovaries, and uterus (9).

Because of the biologic importance of H3F3A and H3F3B, their orthologs have been studied extensively in multiple model organisms. When both H3f3a and H3f3b are knocked out in mice, it causes embryonic lethality at embryonic day 6.5, and reduced expression leads to sterility, growth retardation, and increased neonatal lethality (10). Disrupting either His3.3A or His3.3B in Drosophila is tolerated; however, disrupting both leads to sterility and increased lethality (11). While knockout models have been studied in mice and Drosophila, germline missense mutations have not. There have been many studies in yeast that show the lethality of various missense mutations, but it is difficult to infer whether that means missense mutations in humans would also have a profound effect (12).

In this work, we have identified a cohort of 46 unrelated patients bearing de novo heterozygous germline missense mutations in H3F3A or H3F3B with a core phenotype of progressive neurologic dysfunction and congenital anomalies. Notably, although all known H3.3 mutations in humans cause cancer, none of the patients in our cohort have cancer. These mutations are distinct and appear to be acting, although it is a completely different pathogenic mechanism than the cancer-causing mutations. The mutations are spread throughout the coding sequence, and neither the location of the mutation nor whether the mutation is in H3F3A or H3F3B appears to affect the phenotype. The breadth of these previously unidentified disease-causing mutations in H3F3A and H3F3B provides evidence for how sensitive the neural-related functions of H3.3 are to small variation.

Our patient cohort consists of 46 unrelated patients bearing de novo germline missense mutations in H3F3A or H3F3B (Fig. 1). Both H3F3A and H3F3B code for H3.3. Therefore, a heterozygous mutation in either gene would affect only about 25% of the total H3.3 in the cell, depending on different expression levels from the two genes. None of the patients have malignancies, but they share a phenotype of developmental delay, usually severe and often progressive, with mostly minor congenital anomalies (Table 1). These patients were identified through exome or genome sequencing performed for the indication of neurodevelopmental delays and/or congenital anomalies (Table 1, fig. S1, and table S1). There is substantial phenotypic heterogeneity in the patients, and future individuals are likely to be identified using unbiased molecular testing. Notably, 9 of the 46 patients (21%) have demonstrated clinical neurologic degeneration, which suggests that this may be a progressive disorder. Multiple patients (26% of the cohort) have cortical atrophy on brain magnetic resonance imaging (MRI), even without intractable epilepsy. For example, the oldest patient in this cohort (32 years) developed seizures at the age of 14 years and a progressive spastic paraparesis starting at the age of 29 years.

N, 1, 2, and 3 refer to the N-Helix, Helix-1, Helix-2, and Helix-3 of H3.3, respectively. Upper mutations are encoded by H3F3B, and lower mutations are encoded by H3F3A. Red arrows indicate mutations found in two or more unrelated patients. A few variants, p.N108S, p.P121R, and p.Q125R, were found in the same position in both H3F3A and H3F3B. *p.S146X is only present in an H3F3B alternate transcript not shown here.

Developmental delay was seen in varying degrees in the majority of patients; hypotonia, poor growth, oculomotor abnormalities, seizures, abnormal skull shape, and microcephaly were also commonly seen in the patients.

The severity of the phenotype does not correlate with the location of the mutation or whether the mutation is in H3F3A or H3F3B. Four unrelated patients have the p.T45I mutation and exhibit the full range of the phenotype severity. The age at sitting for patients with the p.T45I mutation ranges from 8 months to not achieved within 4.5 years. The age at walking was 21 months, 2 years, and 3 years and not achieved at 4.5 years. One exhibited developmental regression, while the other three did not, and only one has seizures. Even the tone abnormalities are inconsistent with three exhibiting hypotonia, while one has hypertonia. This strongly suggests that the variation in phenotype is not due to the location of the mutation but rather due to either modifying alleles or environmental factors.

Five mutations [H3F3A (NM_002107.4) p.R17G, p.T45I, p.A114G, p.Q125R, and H3F3B (NM_005324.4) p.P121R] were detected in two or more unrelated patients. p.Q125R was found in H3F3A in three individuals and in a different individual in H3F3B, while p.P121R was found in H3F3A in one individual and in H3F3B in two other individuals. Notably, in two patients, the mutations (H3F3A p.Q125R and p.V117L) were identified only by trio genome sequencing after negative exome sequencing, as the last exon of H3F3A is currently not covered by some exome capture kits. We speculate that similar mutations in H3F3A may be underdiagnosed in current exome studies. It should be noted for nomenclature consistency purposes that, historically, many publications on H3.3 exclude the initiator methionine in the residue count. In this publication, we will use the traditional histone nomenclature and exclude the first methionine. However, we will include traditional human genetics nomenclature in the clinical table of mutations and include this first residue in the mutation notation.

One mutation in H3F3B (p.S146X) is only present in the alternate transcript. It is p.V117V in the canonical transcript. Although the mutation is only in the alternate transcript, the patient phenotype is consistent with the rest of the cohort, and the patient cells have the same phenotype as the other patient cells analyzed. The role of the alternate transcript is currently unknown and requires additional study.

These 37 unique missense mutations in 46 patients are all de novo and, with one exception (H3F3A c.362 T > A; p.M120K, 1 incidence), are not found in a large database of 138,632 controls, the Genome Aggregation Database (gnomAD) (13). Upon closer view of the raw data for this p.M120K variant, it may be a technical mapping error, as it is only present on one strand and did not meet the previous Exome Aggregation Consortium (ExAC) quality control criteria for reporting. In the general population, both genes have a very low rate of missense variants; the gnomAD missense Z score is 3.21 for H3F3A and 2.95 for H3F3B (>2 is significant) (13).

It is likely that these variants are pathogenic through various different mechanisms, as they are found throughout the entire coding sequence in different domains. To explore these mechanisms further, we used molecular modeling of all 37 of the variants reported here. These 37 variants were mapped to a total of 25 loci distributed over several experimental structures. Eleven locations were in the structure of H3.3 in the nucleosome; at the sequence level, they spread from central positions to near the C-terminal end of H3.3. The remaining 14 locations (as well as H39, which was also present in the nucleosome) were mapped to different complexes between H3.3 and epigenetic regulators. At the sequence level, they concentrated in the N-terminal tail of H3.3.

In Fig. 2 (top), we show the location of the variants in the H3.3 structure (for clarity, they are shown only in one H3.3 monomer) and (Fig. 2, bottom) a summary of the amount and type of interatomic contacts at each mutation locus. Together, these data suggest three broad scenarios for the variants impact. In the first one, variants are likely to disrupt the H3.3-DNA interaction because the native residue is involved in a large number of contacts with the DNA. For instance, this would be the case of p.R83C because the arginine residue penetrates the DNA minor groove. In the second scenario, variants are more likely to disrupt the histone octamer, either because they affect the intramonomer contacts of H3.3 (e.g., p.Q125R) or because they may alter the interaction of H3.3 with other nucleosomal histones (e.g., p.L48R). In the third scenario, the variants disrupt histone-protein binding such as the p.G90R mutation disrupting chaperone binding. In summary, through different mechanisms, the variants in this section are likely to affect the formation, the deposition, or the stability of the nucleosomes containing H3.3 or of the nucleosome pairing. This may result in a generalized loosening of chromatin structure in those processes requiring the incorporation of H3.3 to the nucleosome.

At the top of the figure, we show the structure of the nucleosome with the H3.3 variants identified with spheres; the H3.3 monomer carrying them is colored in dark emerald green. The coloring of the variants reflects the predominating interactions at each location: DNA binding (magenta), intramonomer contacts (light orange), and contacts with other histones (dark blue). The same color code is used in the histogram below the structure, where we show the amount of the three interaction types at each location. Note that we use different y axis for these interactions: The y axis to the left corresponds to the H3.3-DNA binding contacts (magenta bars), and the y axis to the right corresponds to the intramonomer (light orange bars) and intermonomer contacts (dark blue bars).

The available complexes between H3.3 and epigenetic regulators (Fig. 3A) involve short stretches of the H3.3 tail bound to sites of different shapes (Fig. 3B). The number of interprotein contacts at the variant locus may vary substantially. For example, p.H39 has 33 and 4 contacts with SETD2 (SET Domain Containing 2, Histone Lysine Methyltransferase) and ZMYND11 (Zinc Finger MYND-Type Containing 11) domains, respectively. We find that residues p.R8, p.S31, p.K36, and p.H39 are involved in more than 10 contacts across epigenetic regulators, suggesting that their mutation may disrupt one or more biologically relevant interactions. For the remaining residues, the number of interprotein contacts decreases rapidly, limiting our ability to interpret mutation impact. For example, visual analysis of the H3.3-BRD4 (Bromodomain-containing protein 4) complex shows that p.A15 barely participates in the complex between both proteins; contact analysis shows that p.A15 has only one interatomic contact with Brd4. Consequently, we conclude that destabilization of the H3.3-Brd4 complex is an unlikely explanation for pathogenic variants. A general mechanism that may also be valid for variants in the histone tail is that they may disrupt the internucleosome packing, an important interaction in which H3 tails are involved (14). In summary, most of the variants in this section are likely to affect the interaction between H3.3 and epigenetic regulators with consequences that will depend on the biological role of each complex, or they may loosen chromatin structure by disrupting internucleosome packing.

Variant locations could be mapped to the experimental structure of different complexes involving H3.3. In (A), we show the variants mapped and the gene names of the H3.3 partners in the corresponding complex. The same color is used for the lines originating in the same variant These complexes include CARM1 (Coactivator Associated Arginine Methyltransferase 1), ZMYND11, SETD2, NSD3 (Histone-lysine N-methyltransferase NSD3), MORC3 (MORC Family CW-Type Zinc Finger 3), MLLT3 (MLLT3 Super Elongation Complex Subunit), KDM1B (Lysine Demethylase 1B), and BRD4. In (B), we show the total amount of interatomic interactions at each location, for each H3.3-epigenetic regulator complex. To help interpretation, we give three examples where we can see the histone tail (blue spheres) interacting with its partner (continuous surface in light orange); the histone residue at the variant location is shown in magenta.

To quantify this histone PTM dysregulation, patients Epstein-Barr virustransformed lymphoblasts (H3F3A p.A15G, H3F3A p.R17G, H3F3A p.T45I, H3F3B p.A29P, and H3F3B p.P121R) and primary fibroblasts (H3F3A p.R17G, H3F3A p.G90R, and H3F3A p.T45I) were obtained from patients and age- and ethnicity-matched controls. Histones were extracted and analyzed by nanoliquid chromatographytandem mass spectrometry (LC-MS/MS) as previously described (15). This allowed for a rigorous and robust quantification of histone PTM levels, providing insight into the global epigenetic state in each cell type. For reference, the average of the control lymphoblasts is depicted in Fig. 4A. Notably, analysis of coefficients of variation both within and across patients and controls revealed that the distribution of histone PTM abundances was very similar (Fig. 4B). Although these data show modest changes compared to cells that express p.K27M- or p.K36M-mutant H3.3, which display a near complete loss of di- and trimethylation at K27 or K36, a dominant negative effect cannot be ruled out (16).

(A) Average profile of PTMs on canonical histones H3 and H4 across control lymphoblasts. Error bars represent SD (n = 9 donors; 3 biological replicates each). (B) Tukey boxplot depicting the coefficients of variation of 73 modified histone H3 and H4 peptides detected by nanoLC-MS/MS (biological variance: across all 14 donors; patient variance: across five patients; control variance: across nine controls). (C) Average histone H3.3 protein abundance (relative to total histone H3) in patient and control lymphoblasts. Error bars represent SD. (D) Volcano plot demonstrating significantly altered histone PTMs in patients versus controls. Dotted line represents P < 0.05 significance threshold. (E) K9 and K14 PTM abundances were compared between (i) protein transcribed from the mutant p.A15G allele from patient cells, (ii) protein transcript from the wild-type (WT) allele from the same patient cells, and (iii) protein transcribed from the WT alleles from a control. Note that the peptide from amino acids 9 to 17 is indistinguishable between canonical H3 and H3.3, so the WT peptide encompasses both. (F) A29P is the only mutation occurring on the same peptide that distinguishes H3.3 from H3. PTMs that fall on this peptide are compared across the mutant peptide, the WT peptide from the mutant sample, and the average profile of the peptide from control samples. *P < 0.05, **P < 0.01, and ***P < 0.001. This shows notable local deregulation of PTMs on the mutant peptide. qMS, quantitative mass spectrometry.

It was also observed that the overall histone PTM variation was slightly greater in healthy donors than in patients. Nonetheless, some histone PTMs were reproducibly altered when comparing patients to controls (Fig. 4D). Although all patients share a common phenotype of developmental delay, only some of them developed major congenital malformations (i.e., cardiac and cranial anomalies). Further study of the specific local dysregulation during development may lead to insights into the transcriptional control in these processes (i.e., cardiac and cranial development). In general, the magnitude of the changes was modest and may reflect the cell types studied and developmental timing.

The tails of H3.3 and the other H3 histones (1 to 89 amino acids) are identical except for residue 31, where they contain alanine and serine, respectively. Because peptides are generated by cleavage at arginine residues, it is technically impossible to assign PTMs to a specific H3 histone, except in the case of p.A29P. For this case, two PTM abundances (K27 and K36) were compared between (i) protein transcribed from the mutant p.A29P allele from patient cells, (ii) protein transcript from the wild-type (WT) allele from the same patient cells, and (iii) protein transcribed from the two WT alleles from a control (Fig. 4E). These data show notable local deregulation of PTMs on the mutant protein, while the WT protein from the affected patient shows fewer differences from the control. This is a more minor effect than that of published somatic oncogenic mutations; however, the difference neither confirms nor refutes a dominant negative effect (17).

Together, these data suggest that the mutant histones can be incorporated into the nucleosome, cause marked local deregulation of chromatin state, and modestly alter the global control of histone modifications. Of greatest interest are the local chromatin changes induced by mutant histone deposition: H3.3 is known to have roles in diverse functions, including gene expression and repression, chromatin stability, DNA damage repair, and differentiation. These mutant proteins and their aberrant PTM states could disrupt any of these processes to lead to the observed phenotype.

To evaluate which biological pathways were differentially perturbed in the patients, we performed RNA sequencing (RNA-seq) on fibroblast cells derived from patients and age- and ethnic-matched controls. All reads were aligned with the STAR (Spliced Transcript Alignment to a Reference) aligner, and Cufflinks was used for performing differential expression analysis. In fibroblast cells (three mutations listed above versus three controls), the H3.3 transcripts (H3F3A and H3F3B) contribution to total histone H3 expression ranged from 45.1 to 72.6% in cases and 64.1 to 81.7% in controls (table S2). We found 323 genes to be differentially expressed with at least two fold changes in fibroblast cells (P < 0.05; table S3). Of these 323 genes, 166 were up-regulated, and 157 were down-regulated in cases. Differentially expressed genes were analyzed through David Functional Annotation Resource, and we found no significant biological signal in the genes with lower expression in cases but showed significant enrichment for up-regulated genes important in mitotic cell cycle process [Benjamini-Hochbergcorrected P (PBH-corrected) = 7.8 1014], mitotic nuclear division (PBH-corrected = 5.8 1010), cell division (PBH-corrected = 7.5 1010), and many other mitosis-related processes (table S4).

To assess whether up-regulation of mitosis-related genes alters cells proliferation, we quantified the cellular proliferation capacity of five patient fibroblast lines compared to six age- and sex-matched control fibroblast lines. Patient lines had increased cell proliferation, notably at 72 and 96 hours (Fig. 5A). Furthermore, all five patient lines shared similar viability to the six control lines (Fig. 5B). Cell cycle analyses showed that H3F3A p.G90R and H3F3A p.T45I had a similar cell cycle profile to the control lines, while H3F3A p.R17G showed a decrease in cells in G1 phase and an increase in cells in S phase compared to all three control lines (Fig. 5C).

(A) Five H3F3A/B patient fibroblast lines (H3F3B: p.G34V; H3F3A: p.R17G; H3F3A: p.G90R; H3F3A: p.T45I; and H3F3B: p.V117V or p.S146X in alternate transcript) demonstrated increased proliferation over six matched controls. **P < 0.005 and ***P < 0.0005. Data represent means SEM of three biological replicates using three technical replicates each. (B) The same five H3F3A/B patient fibroblasts and six controls show no major differences in cell viability. The data represent the means SEM of four biological replicates using two technical replicates each. (C) Cell cycle analysis showed differences in the S (P = 0.0127) and G2 (P = 0.0338) phase in the same five patient cell line compared to the six control fibroblast lines. Data represent the means SEM of four biological replicates using two technical replicates each.

A dominant zebrafish model (p.D123N) derived from a forward genetic screen has been previously reported with craniofacial abnormalities, which were replicated in our patient cohort (Fig. 6A) (18). This heterozygous missense variant replicates the dominant inheritance observed in humans and is only two amino acids away from a mutation identified in an affected patient (p.Q125R). Further investigation of this model also reveals a defect in foxd3-positive neural crestderived glia, as well as melanocytes and xanthophores (Fig. 6, B to D.) The loss of glial cells may relate to the hypomyelination phenotype that is noted on the brain MRIs of over one-third of the H3.3 cohort as glial cells are the cell type responsible for myelination.

(A) Ventral whole-mount views of larval zebrafish heads at 5 dpf stained with Alcian Blue. Homozygous h3f3adb1092 mutants display complete loss of neural crestderived jaw cartilages (n = 10/10). (B to D) In situ hybridization of zebrafish embryos for markers of glia (foxd3; 24 hpf), melanocytes (dct; 27 hpf), and xanthophores (xdh; 27 hpf). Homozygous h3f3adb1092 mutants injected at the one-cell stage with a control mCherry RNA show partial reductions in cranial glia (n = 5), melanocytes (n = 4), and xanthophores (n = 3), while those injected with dominant-negative H3f3a RNA to further reduced H3.3A function show complete loss of melanocytes (n = 5) and severe reductions of glia (n = 6) and xanthophores (n = 4) throughout cranial and trunk regions.

The discovery of missense mutations in H3.3 that cause a neurodevelopmental disorder, but not cancer, has profound implications for future research in histone biology. Until now, H3.3 mutations have only been directly linked to cancer (6). These specific mutations that cause a neurodevelopmental phenotype are distinct from the ones that cause cancer. The cancer mutations are mostly in lysines in the histone tail, while the only lysine mutation in our cohort is p.K36E. When this lysine-36 is mutated in cancer, it is mutated to methionine and not glutamate (19). The patient with the p.K36E mutation was 32 years old when he was last evaluated and still had no signs of cancer, suggesting that the p.K36E mutation does not lead to a strongly increased risk of malignancy. The only other variant in our cohort that is similar to a cancer-linked variant is the p.G34V mutation in H3F3B. This p.G34V variant in H3F3A has been shown to cause cancer only when combined with mutations in ATRX/DAXX (ATRX Chromatin Remodeler/Death Domain Associated Protein) and TP53 (TAR-DNA binding protein-43) (19). Understanding why the somatic p.G34V variant causes cancer, but germline variants cause a neurodevelopmental phenotype, is currently unknown and requires further study.

Notably, although the mutations are spread throughout the coding sequence, they all converge on a similar phenotype. Each mutation affects different specific interactions but converge on the same phenotype. This suggests that the phenotype is due to dysregulation of H3.3 in general and not limited to a single mechanism. Molecular modeling revealed that mutations in the histone core likely affect nucleosome stability, while the mutations in the tail affect various protein-protein interactions. In addition to molecular modeling data, previous work in other species such as yeast can provide insights into the pathogenic mechanism of these variants. Of particular interest, two of the five amino acids that differentiate H3.3 from canonical H3 (p.S31 and p.G90) are mutated in our patient cohort. Both p.S31 and p.G90 are essential for proper recognition of H3.3 by other proteins. Mutagenic analysis in yeast shows that mutations at p.G90 prevent H3.3-specific chaperones DAXX and UBN1 (Ubinuclein 1) from binding (20). The serine at position 31 is required for recognition of H3.3 by ZMYND11 (21). Mutations in ZMYND11 cause an autosomal dominant neurodevelopmental phenotype similar to that seen in our patient cohort, including hypotonia, seizures, dysmorphic facial features, and developmental delay (22). A second mutation in our patient cohort, p.G34V, has also been shown to disrupt ZMYND11 binding to H3.3 (21). Other variants may disrupt histone octamer formation, nucleosome sliding, and chaperone binding based on mutagenic analysis of both H3 and H3.3 in model organisms (2325). We hypothesized that additional missense mutations in our cohort induce epigenetic dysregulation of histone PTMs. These histones PTMs within the nucleosome affect chromatin state, mitotic initiation, protein-chromatin interactions, and gene expression (2630). Specific PTMs of unincorporated histones also mediate chaperone recognition before incorporation into the nucleosome (31).

The fact that all of these mutations are heterozygous is particularly noteworthy. Since there are two genes that produce H3.3, a heterozygous mutation in one of them means that 75% of the alleles are WT. A missense mutation in only 25% of the alleles is sufficient to cause global developmental delay, hypomyelination, cortical atrophy, and craniofacial anomalies. H3F3A and H3F3B are not expressed at the same levels so the amount of mutant protein could be more or less than 25% in any individual patient or tissue at any particular time; however, we observe the same phenotype regardless of whether the mutation is in H3F3A or H3F3B so a higher expression level from one of the genes cannot explain the severity of the phenotype. The fact that a single missense mutation in one allele causes such a severe phenotype strongly supports the importance of tight regulation of histones and histone PTMs. Furthermore, since our studies showed that the changes in the PTMs are in cis on the mutant H3.3 and not in trans on the WT H3.3 in the same nucleosome as is observed in the cancer causing mutations, the severity of the phenotype cannot be explained by a global dysregulation of PTMs.

Although the exact mechanism of the cellular pathology in these patients is unclear, H3.3 is vital for normal neurologic functioning. A recent study showed that H3.3 begins to replace H3.1 and H3.2 in postnatal mouse and human brains in a time-dependent manner and displaces these canonical H3 variants almost completely in adulthood. The important role of H3.3 over time may explain the unique neurodegenerative phenotype, as mice with decreased H3.3 expression in the hippocampus have impaired long-term memory (32). Humans with major depressive disorder have increased percentages of H3.3 in the nucleus accumbens, which is modulated by antidepressant therapy (33). This suggests that H3.3 modulators may represent potential targeted therapies for H3.3-related disorders and perhaps associated neuropsychiatric conditions in the general population. Future studies in patient-derived induced pluripotent stem cell neural cell types may further elucidate the underlying pathology.

Although these are the first germline variants associated with histone H3, germline variants in histones H1 and H4 with similar features have been reported. The overgrowth and neurodevelopmental delay associated with Rahman syndrome (MIM 617537) are caused by truncating variants in H1 Histone Family, Member E (HIST1H1E). (34) Recently, two specific germline variants in histone H4, which caused delayed growth and neurodevelopment, have been described in two families (35). In addition, there are many neurodevelopmental disorders associated with the histone lysine methylases and demethylases. These histone-related disorders provide a unique window into the role of histones in the control of development and growth.

In conclusion, we show here that heterozygous de novo missense variants in H3F3A and H3F3B, coding for H3.3, are associated with a previously undescribed phenotype of developmental delay, neurodegeneration, epilepsy, facial dysmorphism, and congenital anomalies. The functional effects of these mutations appear to be different from those that are well-studied in cancer and may offer a target for therapy for these and other patients.

The Institutional Review Boards of Columbia University, the University of Michigan, Childrens Hospital of Eastern Ontario, University Medical Center Hamburg-Eppendorf, and the Childrens Hospital of Philadelphia approved this study. Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article.

Genomic DNA was extracted from whole blood from the affected children and their parents. Exome or genome sequencing was performed with a variety of standard capture kits and the general assertion criteria for variant classification following ACMGG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) guidelines (36). There were no other variants in these patients that survived filtration and analysis using either dominant or recessive models and could explain the phenotypes.

Six healthy control cells lines that were matched to available patient cells for passage number, age, and sex were obtained from the Coriell Institute for Medical Research tissue bank. All these fibroblast samples were derived from skin biopsies performed either on the arm or on the leg of the patient. Only a portion of the patients who participated in our study agreed to donate cellular material, and the patient mutations that were analyzed reflect the sum of the viable donations.

Histones were purified in acid as previously described by Karch et al. (37). Acid-extracted histones (15 to 25 g) were resuspended in 100 mM ammonium bicarbonate (pH 8), derivatized using propionic anhydride, and digested with trypsin as previously described (37). The resulting histone peptides were desalted using the C18 Stage Tips, dried using a centrifugal evaporator, and reconstituted using 0.1% formic acid in preparation for nanoLC-MS analysis.

Nano-LC was performed using the Thermo Fisher Scientific Easy nLC 1000 System equipped with a 75 m by 20 cm in-house packed column using Reprosil-Pur C18-AQ (3 m; Maisch GmbH, Germany). Buffer A was 0.1% formic acid, and buffer B was 0.1% formic acid in 100% acetonitrile. Peptides were resolved using a two-step gradient from 0 to 26% B over 45 min and then from 26 to 98% B over 10 min at a flow rate of 300 nl/min. The high-performance LC was coupled online to an Orbitrap Elite mass spectrometer operating in the positive mode using a Nanospray Flex ion source (Thermo Fisher Scientific) at 2.40 kV. MS was performed using data-independent acquisition (DIA) as previously described with slight modifications (37). Briefly, two full MS scans [mass/charge ratio (m/z), 300 to 100) were acquired in the Orbitrap with a resolution of 120,000 (at 200 m/z) every eight DIA MS/MS events using isolation windows of 50 m/z each (e.g., 300 to 350, 350 to 400 650 to 700). The full MS scan is performed twice within the same duty cycle to allow for a more resolved definition of the precursor peak profile. MS/MS events were acquired in the ion trap operating in normal mode. Fragmentation was performed using collision-induced dissociation in the ion trap mass analyzer with a normalized collision energy of 35. Automatic gain control (AGC) target and maximum injection time were 10 106 and 50 ms for the full MS scan and 10 104 and 150 ms for the MS/MS scan, respectively.

Data were searched using EpiProfile. The peptide relative ratio was calculated using the total area under the extracted ion chromatograms of all peptides with the same amino acid sequence (including all of its modified forms) as 100%. For isobaric peptides, the relative ratio of two isobaric forms was estimated by averaging the ratio for each fragment ion with different mass between the two species. Two to three biological replicates were analyzed per condition, and the relative abundance of each peptide modification was averaged across the runs.

Isolated histones were separated on a 4 to 12% NuPAGE bis-tris gel and transferred to a 0.2-m nitrocellulose blotting membrane. Membranes were blocked in TBS-T [50 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] containing 5% milk powder and probed for H3.3 (Thermo Fisher Scientific, MA5-24667; 1:5000), H3 (Abcam, ab1791; 1:5000), and H4 (Abcam, ab7311; 1:5000) primary antibodies diluted in TBS-T containing 5% milk powder. This was followed by anti-rabbit horseradish peroxidase (Abcam, ab99697; 1:10,000) and visualized using enhanced chemiluminesence prime Western blotting detection chemiluminescent reagent.

RNA was extracted using the RNeasy Mini Kit (QIAGEN) on the six samples at the same time. Extracted RNA samples underwent quality control assessment using Bioanalyzer (Agilent, Santa Clara, CA, USA) and were quantified using NanoDrop from NanoDrop Technologies (Wilmington, DE, USA). RNA libraries were prepared using the Illumina TruSeq RNA sample prep V2 with ribosomal RNA depletion and were sequenced using HiSeq 2500 sequencer (Illumina Inc., San Diego, CA, USA) at the Center for Applied Genomics at the Childrens Hospital of Philadelphia per standard protocols (paired-end 100 base pairs). The RNA-seq data were aligned on the hg19 reference genome using STAR (www.encodeproject.org/software/star/) and processed using Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/). For each gene, we compared the expression levels between cases and controls. A gene was considered differentially expressed if the P value is less than 0.05 and fragments per kilobase of transcript per million mapped reads has at least twofold change. To identify overrepresented functional categories among genes that are differentially expressed, we performed annotation analysis using the David Functional Annotation Resource (https://david.ncifcrf.gov/). Multiple testing was adjusted using the Benjamini-Hochberg approach, and enrichment was declared if the adjusted P value is less than 0.05.

Cellular proliferation was assayed with five patients and six control fibroblast cell lines; they were plated at 3 104 cells per well and then were manually counted at baseline, 24, 48, 72, and 96 hours. Then, the means SEM of three biological replicates using three technical replicates each were analyzed. Cell viability was analyzed with three patients and three control fibroblast cell lines; the cells were plated and grown until ~80% confluent. Then, they were stained for annexin V/propidium iodide (PI), and fluorescence-activated cell sorting (FACS) was performed on Accuri C6 (BD Biosciences). Then, the means SEM of four biological replicates using two technical replicates each were analyzed. Cell cycle analysis was performed with three patients and three control fibroblast cell lines; the cells were plated and grown until ~80% confluent. They were then stained for PI, and FACS was performed on Calibur and analyzed by FlowJo. Then, the means SEM of four biological replicates using two technical replicates each were analyzed.

All zebrafish experiments were approved by the University of Southern California Institutional Animal Care and Use Committee (protocol #20258). h3f3adb1092 mutants were genotyped, and control mCherry or dominant-negative D123N H3f3a mRNAs were prepared and injected at 900 ng/l into one-cell stage embryos as described (18). Acid-free cartilage staining with Alcian Blue was performed as described (38). In situ hybridization was performed with the probes and procedures described in (18). Images were captured on a Leica DM2500 compound microscope.

For each H3.3 variant location, we analyzed its pattern of interatomic contacts (see below) using experimental structural information. The list of known structures was retrieved from the UniProt record of H3.3 (P84243). In all cases, the structure of H3.3 was incomplete, that is, only a fragment in complex with other proteins is described. For each variant, we identified the Protein Data Bank (PDB) entries that provided information about its location. We provide a table of the PDB files used in table S5.

For each variant, we computed the network of interatomic interactions of the native residue. This network was obtained following a simple protocol: (i) retrieve the corresponding PDB, (ii) compute the network of interactions for all the protein residues using Ring, (iii) extract the interaction data corresponding to the native residue, and (iv) organize these interaction data into three groups [intramonomer (within the same H3.3 monomer), intermonomer (between H3.3 monomer and other proteins), and H3.3-DNA] (39). Intramonomer interactions were only kept when the atoms involved came from residue pairs (i,j) separated by more than two residues in sequence, i.e., |i j| > 2. Ring computations were executed with default parameters, except for interaction type, which was set to all. H3.3-DNA contacts were only included if their distance was lower than 5.5 .

Acknowledgments: We thank the families for contribution. We also thank J. F. Deleuze, A. Boland, and V. Meyer from the CNRGH for genome sequencing and data primary analysis. Funding: Funding was provided by the French Foundation for Rare Diseases (Fondation maladies rares). This work was also supported in part by a grant from the from SFARI (to W.K.C.), JPB Foundation (to W.K.C.), and the Morton S. and Henrietta K. Sellner Professorship in Human Genetics (to J.W.I.), as well as an NIDDK T35 training grant (T35DK093430) (to E.F.J.). E.J.B. was supported by a K12 training grant (K12HD043245-14) and the Roberts Collaborative. D.M. was supported by a T32 training grant (T32GM008275). Funding from NIH grants GM110174 and CA196539 to B.G. is acknowledged. This research was funded in part by the Estonian Science Foundation grant PUT0355 and PRG471. Analysis for one patient was provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and was funded by the National Human Genome Research Institute, the National Eye Institute, and the National Heart, Lung, and Blood Institute grant UM1 HG008900 to D. MacArthur and H. Rehm. Support also provided by NIH T32 HD07466 (to M.H.W.), Alabama Genomic Health Initiative F170303004 through University of Alabama at Birmingham IRB (to A.C.H., J.D., and M.T.) The Toronto-based authors (to G.C., M.S.M., and D.C.) acknowledge support from the Centre for Genetic Medicine, The Centre for Applied Genomics, and the Norman Saunders Complex Care Initiative. The Care4Rare Canada Consortium is funded by Genome Canada, the Canadian Institutes of Health Research, the Ontario Genomics Institute, Ontario Research Fund, Genome Quebec, and Childrens Hospital of Eastern Ontario Foundation. This work was also supported by Dipartimenti di Eccellenza 20182022 Project code D15D18000410001 to A.B. and Fondazione Bambino Ges (Vite Coraggiose) and Italian Ministry of Health (CCR-2017-23669081) to M.T. Author contributions: A.B., A.C.H., A.J.Y., A.L., A.P.A.S., A.Z., B.F., C.F., C.G.S.P., C.T.R.M.S., D.H., D.N., E.Bri., E.Bro., E.V., F.C.R., H.K., G.B.F., G.G., J.A.M.-A., J.C.P., J.M.T., J.Ne., J.No., J.S.S., K.E.S., K.M., K.T., K.Wie., K.Wil., L.B., L.U., L.W.L., M.B., M.G.M., M.I.V.A., M.S., M.Ta., M.Th., N.H.P., N.L., R.J.L., R.R.L., S.A.S.V., S.F.N., S.J.C.S., S.M.-A., S.S.C., T.H., T.P., W.K.C., E.J.B., M.J.Lyo., M.J.Lar., W.C., E.She., E.Sel., K.J.W., A.W., G.M.S.M., N.P.-H., J.v.d.K., T.G., John Dean, J.Den., Joy Dean, A.R., H.P.C., M.C.W., L.M., C.N., A.A., D.Les., T.K., J.Win., J.Wan., C.B.C., A.Vol., A.Van., E.F.J., J.L.S., J.W.I., A.P., S.L., H.D., J.H., H.R., B.K., O.S., S.O., S.M., K.Ret., K.Rad., K.., P.I., A.M.I., G.C., M.S.M., D.Chi., D.Car., M.M.W., and E.Z. contributed to the clinical evaluation of the affected individuals. R.B., J.A.L., B.R., Z.P., M.C., R.S., D.Les., M.W., K.Ret., K.Rad., T.M.S., K.G.M., R.G., K.R., C.M., K.L.H., B.C., S.Bez., S.Bar., T.B., S.K., R.R., M.H.W., K.D.K., Care4Rare Canada Consortium, CAUSES Study, DDD Study, and Undiagnosed Disease Program contributed to the molecular evaluation of the affected individuals. E.J.B., D.Les., H.H.H., M.E.M., B.G., D.M., K.J., Z.-F.Y., P.L., C.S., J.G.C., X.d.l.C., N.P., S.G.C., K.Wil., and D.Li contributed the functional evaluation of the variants found in the affected individuals. Competing interests: K.R. and K.M. are employees of GeneDx. W.K.C. and M.C. were employees of GeneDx. The authors declare that they have no other competing interests. Data and materials availability: Patient variants will be available in ClinVar. All patient materials may be obtained through a material transfer agreement. All other data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

Go here to read the rest:
Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients...

When applying genetics to the study of COVID-19, scientists are learning a lot. Our DNA codes for proteins, some of which are required for SARS-CoV-2 to interact with and infect a host cell, others that are implicated in the downstream effects of viral infection, such as inflammatory responses. But how do scientists choose which genes to study?A new study by Dr Thomas Stoeger, a postdoc at North Western University, suggests there is a historical bias involved; scientists are studying human genes that have already been heavily investigated, independent of COVID-19.

In this installment of Teach Me in 10, Stoeger expands on the key points of this study and the implications of bias in scientific research.

Full research publication: Meta-Research: COVID-19 research risks ignoring important host genes due to pre-established research patterns.

For more Teach Me in 10 videos, check out our hub page.

Are you a Facebook user? Like the Teach Me in 10 Facebook page to engage and network with the video audience.

See original here:
Teach Me in 10 Why COVID-19 Genetics Research May Be Biased With Dr Thomas Stoeger - Technology Networks

Bathinda: Central University of Punjab, Bathinda (CUPB) under the patronage of Vice Chancellor Prof. Raghavendra P. Tiwari organised a Webinar on Population Genomics and Public Health. On this occasion, Dr. Kumarswamy Thangraj, Director, Centre for Fingerprinting and Diagnostics (CDFD), Hyderabad was the keynote speaker.

The programme commenced with the Welcome Address by Prof. Anjana Munshi, Dean Research, where she put a light on the topic of the webinar. While introducing the keynote speaker, she mentioned that Dr. Kumarswamy Thangraj is an acclaimed senior geneticist of India who is known for his contributions in the field of population and medical genetics.

In his keynote address, Dr. Kumarswamy Thangraj talked about how population genomics, the study of evolution of humans, and the genetics of modern human helps in understanding the human health. While putting a light on the evolution of human beings, he mentioned that earlier human species diverged from their homogenates like Chimpanzee about 7.5 Million Years Ago.(MYA). He also discussed different stages of evolution of human beings including Hominidae, Ardipithecus Ramidus, Australopithecus Afarensis, Homo Hebilis, Homo Erectus, Homo Heidelbergensis, Homo Sapiens and pointed out that during these stages the human brain has developed over a period of time and its size has increased as compared to the predecessors. He stated that Neanderthals were the early archaic human with larger brain size who emerged around 200 thousand years ago but they were replaced by early modern humans.

Dr. Kumarswamy Thangraj informed that several geological tools including the draught of Malwai Lake have indicated that modern humans originated in Africa and migrated to different parts of the world. He also shared that the recent studies have shown that enigmatic tribal populations of Andaman and Nicobar Islands are the first modern humans who migrated out of Africa through the Southern Coastal Route about 65,000 years ago.

He stated that India is a genetic hotspot and there are more than 4500 anthropologically well-defined population groups and genetic identity of different ethnic groups helps in identifying the health status of that population. He added that due to the practice of endogamy, the disease pattern is restricted to that particular population. Dr. Kumarswamy Thangraj asserted that IBD Scores (Identity by dissent scores) helps the researchers to find the level of identical DNA and know about their ancestors of several decades back. Further, the level of similarity of gene mutation pattern helps in identifying the health risk level of that ethnic group. During this webinar, various genetic diseases including mitochondrial diseases were discussed and it was suggested that a community genetic testing programme helps in identifying the health risk factor of a population.

Prof. R.K. Wusirika, Dean Incharge Academics, appreciated the keynote speaker for scholastic discourse. Towards the end, Dr. Sandeep Singh delivered the formal vote of thanks. This Webinar was attended by faculty and students of the university.

Continued here:
Central University of Punjab organized Webinar on Population Genomics and Public Health' - India Education Diary

Gitanjali Rao occupied the top spot on the trends list on Friday after TIME Magazine named her as its first-ever Kid of the Year 2020. The 15-year-old Indian-American is an aspiring scientist and inventor, and she was among 5,000 nominees, pitted against each other for the distinction.

We will tell you more about Gitanjali. But, before that, do you know Gitanjali appeared as a guest speaker on the second season of TED Talks India: Nayi Baat with Shah Rukh Khan in 2019? On the show, Gitanjali discussed her passion for science and also demonstrated experiments in front of Shah Rukh Khan.

The superstar was absolutely impressed with the "simply incredible" Gitanjali and applauded her after she said that she intends to make the world a better place.

Here are 10 things that Gitanjali Rao told Shah Rukh Khan on TED Talks India: Nayi Baat in November last year:

HOW ARE SCIENTISTS DIFFERENT FROM SUPERHEROES?

"In our minds, superheroes can jump tall buildings, have technology gadgets. Have superpowers such as flying and robotic hands. But what do they all have in common? The ability to save lives and the magical thing is that they show up exactly at the right time to save lives. If superheroes were real, we would give them all of the support in the world. Wouldn't we? Well, how are real, breathing, living scientists different from the superheroes in movies and comics? No matter where they are, scientists come up with solutions to help people."

I WANT TO BE A SCIENTIST SUPERHERO

"I love science and I want to be a scientist superhero, solving real world problems and saving lives."

IT'S NEVER TOO EARLY TO START MAKING A DIFFERENCE

"I like to think of solutions every time I see a problem in the society. It's never too early to start making a difference and I can prove of that by sharing some of my journey towards innovation."

WHAT IS TETHYS?

"The health effects of lead in water ranges from anywhere. From just headaches and nausea to possible seizures and even death. I invented my device Tethys to detect lead levels in water. By dipping it into the water sample, it can speedily detect the lead content, how people take preventive measures such as switching to a filtered-water source, and maybe even save lives."

PROTOTYPE BASED ON HUMAN GENETICS

"I am currently working on another prototype that is based on human genetics and can detect the growing problem of prescription drug addiction. I have chosen to develop an easy to use portable and efficient device that physicians can use to tell if their patients are at the onset of addiction."

FORTUNATE TO HAVE INCREDIBLE MENTORS

"I am fortunate to have some incredible mentors who believe in me, inspire me and mentors whom I have learn from, and will continue to learn from."

I AM LEARNING EVERY SINGLE DAY

"I am learning every single day. Learning about the problems around us. Learning about new technology options. Learning about ways to solve them, and how me, and other young innovators like me, can come up with solutions."

I AM JUST 13 BUT THAT'S GOOD NEWS

"I may only be 13, but that's good news because hopefully, I have many years ahead of me to be a scientist, to solve problems, to save lives, and to make the world a better place for me, and for children who haven't been born yet."

WHAT IS THE PURPOSE OF SCIENCE?

"I believe that the purpose of science is to make a difference. We can all do our part in society by mentoring, volunteering and raising funds."

WE CAN ALL BE SUPERHEROES

"I don't think there should be an age barrier to solving problems. I hope that with the right focus and approach to continuous learning, we can all be superheroes, saving lives."

WHO IS GITANJALI RAO?

Based in Colorado, US, Gitanjali Rao is a scientist, who aspires to study genetics and epidemiology at Massachusetts Institute of Technology. She won the Discovery Education 3M Young Scientist Challenge in 2017 and was also recognised as Forbes 30 U 30 for her innovations. In September 2018, Gitanjali Rao was awarded the United States Environmental Protection Agency President's Environmental Youth Award.

ALSO READ: Indian American Gitanjali Rao, 15, named first ever TIME Kid of the Year 2020

ALSO READ: Myanmar monk creates refuge for snakes at monastery. Viral pics

Go here to see the original:
10 things Kid Of The Year Gitanjali Rao told Shah Rukh Khan on TED Talks in 2019 - India Today

Researchers from Germanys Max Planck Institute of Molecular Cell Biology and Genetics in Germany and Japans Central Institute for Experimental Animals introduced a specifically human gene,ARHGAP11B, into the fetus of a common marmoset monkey, causing the enlargement of its brains neocortex. The scientistsreported their findings in Science.

The neocortex is the newest part of the brain to evolve. Its in the nameneo meaning new, and cortex meaning, well, the bark of a tree. This outer shell makes up more than 75 percent of the human brain and is responsible for many of the perks and quirks that make us uniquely human, including reasoning and complex language.

The scientists call these human-monkey hybrids transgenic non-human primates, which may be enough to ring the alarm of any doomsdayer. It certainly raises a lot of ethical questions when doing experiments on primates, let alone when introducing human genes into other animals.

For this reason, the researchers limited their study to monkey fetuses, which were taken out by C-section after growing for 100 days. Allowing the experiment to go past the fetal phase and let the human gene-carrying monkeys to be born would beirresponsible and unethical, study coauthor Wieland Huttner said in the press release.

Read the original post

Here is the original post:
Planet of the Apes redux? Human brain gene inserted into monkey fetuses enlarged their brains, raising ethical concerns - Genetic Literacy Project

Results presented at Foundation for Angelman Syndrome Therapeutics (FAST) Global Summit

Additional data, including EEG findings, support prior initial indications of activity and there were no new adverse events

SARASOTA, Fla. and NOVATO, Calif., Dec. 05, 2020 (GLOBE NEWSWIRE) -- GeneTx Biotherapeutics LLC and Ultragenyx Pharmaceutical Inc. (NASDAQ: RARE), companies partnered in the development of intrathecally administered GTX-102, an investigational treatment for Angelman syndrome, today announced the presentation of data from the Phase 1/2 study of GTX-102 at the Foundation for Angelman Syndrome Therapeutics (FAST) Global Summit. Details regarding the scientific basis for GTX-102 targeting in Angelman syndrome were presented along with additional supportive clinical data on EEG and other endpoints, along with further description of the safety events. Additional nonclinical study data were included showing substantial silencing activity at low repeat doses along with chronic nonclinical safety data at higher doses compared to dosing in the human study. Presentations were made by Scott Stromatt, M.D., Chief Medical Officer of GeneTx and Elizabeth M. Berry-Kravis, M.D., Ph.D. Professor of Pediatrics, Neurological Sciences and Biochemistry at Rush University on Friday December 4th, and by Emil D. Kakkis, M.D., Ph.D., Chief Executive Officer and President of Ultragenyx, on Saturday, December 5th.

I am excited by the preliminary findings presented at the FAST scientific symposium. A tremendous amount of work was put into understanding the UBE3A-AS transcript and developing GTX-102, so it is great to see how those efforts have translated into initial indications of effect in the clinical study in patients with Angelman syndrome, said Scott V. Dindot, Ph.D., Associate Professor, Texas A&M University, and Executive Director, Molecular Genetics at Ultragenyx. I am grateful to be a part of this endeavor, and I look forward to seeing what the future holds for the Angelman syndrome community.

GTX-102 demonstrates a paternal UBE3A gene targeting strategy can result in substantial clinical activity and in a more rapid time frame than we expected, stated Dr. Scott Stromatt. We better understand the serious adverse events reported with GTX-102 at higher doses and we see a way forward to redose patients and to enroll new patients into the clinical trial. We are working with FDA to reach agreement on a modified trial design.

Study Design and Dosing

Five patients in three dose cohorts were enrolled who all had deletions in the UBE3A locus as the cause of Angelman syndrome and were treated with a monthly intrathecal dose of GTX-102 that increased for each of the first four doses provided to each patient. Two patients in cohort 1 received a monthly ascending dose sequence of 3.3 mg, 10 mg, 20 mg, and 36 mg, with the first patient receiving one additional fifth dose in an extension amendment at the 36 mg level. Two patients in cohort 2 received three sequential monthly doses of 10 mg, 20 mg, and 36 mg. One patient in cohort 3 received a single dose of 20 mg. Further dosing was stopped once the first serious adverse event occurred, as previously described.

Pharmacokinetic results indicate that plasma levels of GTX-102 were dose proportional. GTX-102 was not detectable in the blood or cerebrospinal fluid (CSF) in samples taken one month after the last dose and prior to subsequent monthly doses, indicating that the drug did not accumulate in the blood or CSF.

Interim Efficacy Results

Previously disclosed improvements in the Clinical Global Impression of Improvement Scale for Angelman Syndrome (CGI-I-AS) were presented along with detailed individual results for both global scores and individual domains. The mean change was +2.4 in the CGI-I-AS global score and all patients had at least 3 domains of improvement and 2 domains of much improved or very much improved at this interim assessment.

CommunicationCommunication was one of the most impaired functions in these five patients based on baseline scores and is the most important disease domain for families according to a recently published disease concept model1. Detailed scores from the communication domain of the CGI-I-AS showed much improved or very much improved scores in four of five subjects along with supportive detailed data from other scores. In the Bayley Scales of Infant and Toddler Development (Bayley-4), multiple patients improved on receptive or expressive communication sub-scales. In the Observed Reported Communication Ability (ORCA) measure of expressive, receptive, and pragmatic communication, three patients, ages 5, 10, and 15, demonstrated clinically relevant increases at day 128 and two patients did not have notable changes.

EEG and SeizuresAt baseline, all patients had stable seizure control per protocol requirements and did not have reports of seizures as adverse events during the study. Blinded independent central electroencephalogram (EEG) readings were conducted at baseline and day 128 (day 86 for patient 5) for four of five treated patients to assess delta waves and epileptiform discharges among other findings common in Angelman syndrome. Qualitative readings of the EEGs indicate decreases in the prevalence of notched delta waves in three of the four evaluated patients with patient 1 showing minimal change or a slight increase. Decreases in the prevalence of epileptiform discharges were also observed in three of the four evaluated patients with patient 5 showing minimal change or a slight increase. Quantitative analysis of the EEGs completed to date in the first two patients showed decreases in relative delta power (2-4 Hz) in both evaluated patients after beginning GTX-102. These are preliminary findings and, due to normal variability in EEG tracings, the assessments will be repeated after longer-term treatment with GTX-102.

Gross Motor and Fine MotorPreliminary readings from the ActiMyo device that measures hourly distance walked, stride length, and stride speed, support the utility of this functional measure. One patient, who initially had a decrease in distance walked due to the lower extremity weakness SAE, later was able to exhibit a meaningful increase from baseline as the SAE resolved. Other improvements in fine motor function previously disclosed were presented.

Length of EffectThe clinical changes observed appear to last at least 3 to 5 months from the last dose. To date, most of the subjects have retained many caregiver-reported clinical changes observed but some patients are observed to be experiencing some loss of effect. The long period of observed clinical response post-dose would support use of a maintenance dosing regimen of every 3 months, if an appropriate and safe dosing regimen is identified.

Additional Interim Safety Results

As previously reported, all patients had a grade 1 or 2 serious adverse event (SAE) of lower extremity weakness associated with local inflammation in the region of intrathecal administration in the lower back at the higher doses of GTX-102. The SAE has fully resolved in all five patients.

The SAE occurred between 6 and 30 days after the last infusion of 36 mg in four patients and 20 mg in one patient. In patient 1, the SAE was not observed until after the second dose at the 36 mg level. Clinical improvements observed in the study have been sustained beyond resolution of the SAE and the negative impact of the SAE on gross motor function in certain patients has recovered with resolution of the SAE.

No new adverse events have been reported since the last update. No patients have withdrawn from the study.

Additional Nonclinical Data

Results from additional non-human primate (NHP) studies were also reported including both single dose and repeat dose studies conducted for as long as six months. Toxicology assessments indicated acute clinical observations including sporadic transient lower limb weakness generally resolving by 24 hours after dosing. There was no observation throughout these studies of delayed-onset weakness similar to the human study SAE, which included single doses as high as 10 mg (equivalent to a dose in humans of approximately 110 mg) or at repeat monthly doses as high as 5 mg (human equivalent of approximately 56 mg per dose). No kidney or platelet toxicities were observed in the NHP studies.

The NHP studies also assessed knockdown of the UBE3A-antisense (UBE3A-AS) transcript, the RNA that inhibits expression of the paternal UBE3A allele in Angelman syndrome. Monthly dosing of GTX-102 showed substantial reduction of the UBE3A-AS transcript at monthly doses of 1, 2, and 3 mg. UBE3A-AS reduction occurred in multiple brain regions relevant to Angelman syndrome.

Scientific Detail on GTX-102 Targeting

Detail was presented on the GTX-102 target region in the UBE3A-AS transcript. Dr. Dindots work on understanding the molecular genetics of the antisense transcripts allowed the discovery of a more potent place to target an antisense oligonucleotide (ASO) for the knockdown of the repressive antisense RNA transcripts to induce more UBE3A expression. The manuscript describing the work performed by Dr. Dindots laboratory is currently under review.

Update on Clinical Study Next Steps

The companies will propose a plan to the FDA to resume enrollment and dosing in the study which is currently on hold. The proposed plan is to amend the dosing and titration regimen to start at a low dose and titrate individually, based on patient age and response to GTX-102. The maximum dose will be below 20 mg, which is the lowest dose at which the lower extremity weakness SAE was observed. Also, a new administration procedure will be used to minimize duration of exposure at the injection site. The companies aim to resume enrollment as soon as possible following receipt of guidance and approval from the FDA.

1: Willgoss, T.et al.Measuring What Matters to Individuals with Angelman Syndrome and Their Families: Development of a Patient-Centered Disease Concept Model.Child Psychiatry Hum Dev(2020). https://doi.org/10.1007/s10578-020-01051-z

About Angelman Syndrome

Angelman syndrome is a rare, neurogenetic disorder caused by loss-of-function of the maternally inherited allele of the UBE3A gene. The maternal-specific inheritance pattern of Angelman syndrome is due to genomic imprinting of UBE3A in neurons of the central nervous system, a naturally occurring phenomenon in which the maternal UBE3A allele is expressed and the paternal UBE3A is not. Silencing of the paternal UBE3A allele is regulated by the UBE3A antisense transcript (UBE3A-AS), the intended target of GTX-102. In almost all cases of Angelman syndrome, the maternal UBE3A allele is either missing or mutated, resulting in limited to no protein expression. This condition is typically not inherited but instead occurs spontaneously. It is estimated to affect 1 in 12,000 to 1 in 20,000 people globally.

Individuals with Angelman syndrome have developmental delay, balance issues, motor impairment, and debilitating seizures. Some individuals with Angelman syndrome are unable to walk and most do not speak. Anxiety and disturbed sleep can be serious challenges in individuals with Angelman syndrome. While individuals with Angelman syndrome have a normal lifespan, they require continuous care and are unable to live independently. Angelman syndrome is not a degenerative disorder, but the loss of the UBE3A protein expression in neurons results in abnormal communications between neurons. Angelman syndrome is often misdiagnosed as autism or cerebral palsy. There are no currently approved therapies for Angelman syndrome; however, several symptoms of this disorder can be reversed in adult animal models of Angelman syndrome suggesting that improvement of symptoms can potentially be achieved at any age.

About GTX-102

GTX-102 is an investigational antisense oligonucleotide designed to target and inhibit expression of UBE3A-AS. Nonclinical studies show that GTX-102 reduces the levels of UBE3A-AS and reactivates expression of the paternal UBE3A allele in neurons of the CNS. Reactivation of paternal UBE3A expression in animal models of Angelman syndrome has been associated with improvements in some of the neurological symptoms associated with the condition. GTX-102 has been granted Orphan Drug Designation, Rare Pediatric Disease Designation, and Fast Track Designation from the U.S. Food and Drug Administration (FDA). In August 2019, GeneTx and Ultragenyx announced a partnership to develop GTX-102, with Ultragenyx receiving an exclusive option to acquire GeneTx.

About GeneTx Biotherapeutics

GeneTx Biotherapeutics LLC is a startup biotechnology company singularly focused on developing and commercializing a safe and effective antisense therapeutic for the treatment of Angelman syndrome. GeneTx was launched by FAST, a patient advocacy organization and the largest non-governmental funder of Angelman syndrome research. GeneTx licensed the rights to antisense technology intellectual property from the Texas A&M University System in December 2017.

About Ultragenyx

Ultragenyx is a biopharmaceutical company committed to bringing novel products to patients for the treatment of serious rare and ultra-rare genetic diseases. The company has built a diverse portfolio of approved therapies and product candidates aimed at addressing diseases with high unmet medical need and clear biology for treatment, for which there are typically no approved therapies treating the underlying disease.

The company is led by a management team experienced in the development and commercialization of rare disease therapeutics. Ultragenyxs strategy is predicated upon time- and cost-efficient drug development, with the goal of delivering safe and effective therapies to patients with the utmost urgency.

For more information on Ultragenyx, please visit the companys website at http://www.ultragenyx.com.

Forward-Looking Statements

Except for the historical information contained herein, the matters set forth in this press release, including statements related to Ultragenyx's expectations and projections regarding its business plans and objectives for GTX-102, the therapeutic potential and clinical benefits of GTX-102, expectations regarding the safety and tolerability of GTX-102, and future clinical developments for GTX-102 are forward-looking statements within the meaning of the "safe harbor" provisions of the Private Securities Litigation Reform Act of 1995. Such forward-looking statements involve substantial risks and uncertainties that could cause our clinical development programs, collaboration with third parties, future results, performance or achievements to differ significantly from those expressed or implied by the forward-looking statements. Such risks and uncertainties include, among others, the Companys ability to successfully develop GTX-102 at lower doses, including the resolution of adverse events that were seen at higher doses, whether lower doses of GTX-102 are sufficiently effective to support the continued development of the program, the effects from the COVID-19 pandemic on the companys commercialization activities, business and operating results, smaller than anticipated market opportunities for the companys products and product candidates, manufacturing risks, competition from other therapies or products, uncertainties related to insurance coverage and reimbursement status of the companys newly approved products, the companys evolving integrated commercial organization, and other matters that could affect sufficiency of existing cash, cash equivalents and short-term investments to fund operations, the companys future operating results and financial performance, the timing of clinical trial activities and reporting results from same, and the availability or commercial potential of Ultragenyxs products and drug candidates. Ultragenyx undertakes no obligation to update or revise any forward-looking statements. For a further description of the risks and uncertainties that could cause actual results to differ from those expressed in these forward-looking statements, as well as risks relating to the business of Ultragenyx in general, see Ultragenyx's Quarterly Report on Form 10-Q filed with theSecurities and Exchange CommissiononOctober 27, 2020, and its subsequent periodic reports filed with theSecurities and Exchange Commission.

ContactsUltragenyx Pharmaceutical Inc.Investors & MediaJoshua Higa415-660-0951

GeneTxPaula Evans630-639-7271Paula.Evans@GeneTxBio.com

Read the original:
GeneTx and Ultragenyx Announce Presentation of Phase 1/2 Data on Investigational GTX-102 in Patients with Angelman Syndrome - GlobeNewswire