This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Yong-hui Jiang
Department of Genetics
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6188-8806
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, brittle bones, and dysmorphic facies. De novo heterozygous mutations in H1-4 (HIST1H1E) encoding the linker histone H1.4 are found in patients with RMNS; however, the underlying mechanisms causing the pronounced neurological manifestations are not understood. The majority of reported mutations in H1-4 are small insertions or deletions that create a shared frameshift, resulting in an H1.4 protein that is both truncated and possessing an abnormal C-terminal tail.
Methods: Seven Rahman syndrome subjects with C-terminal frameshift mutations as well as three patients with heterozygous null mutations in H1-4 were described in detail. Lymphoblastoid cells from these patients were used to identify transcriptional abnormalities in RMNS. Wildtype or mutant frameshifted human H1.4 protein was exogenously expressed in primary rat hippocampal neurons, and neuronal structure and function were assessed using immunohistochemistry and multi-electrode array recordings.
Results: Individuals with heterozygous null variants in the H1-4 gene lack several key RMNS phenotypes, supporting the hypothesis that RMNS is due to a gain-of-function of the frameshift mutant H1.4 protein. In cultured rat hippocampal neurons, H1.4 was localized to the nucleus, though the frameshift mutant H1.4 had a distinct subnuclear distribution and enlarged nuclei. Overexpression of frameshift mutant H1.4 had minimal effects on dendritic morphology; however, it significantly reduced neuronal firing rate relative to neurons overexpressing wildtype human H1.4.
Limitations: Given the small number of RMNS cases worldwide, the true breadth of phenotypes remains unknown. Though our data do not show robust differential expression of any genes, larger cohorts for clinical and molecular studies will be needed to gain reliable data.
Conclusions: These data are the first to characterize the consequence of frameshift mutant H1.4 in neurons. These data provide new insights into the breadth of phenotypes and causes of neurological dysfunction in RMNS and highlight the need for future studies on the function of histone H1.4 in neurons.
Keywords
Rahman syndrome (RMNS), HIST1H1E, H1-4, H1.4, linker histone, intellectual disability (ID)
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalFigure1Tremblay.pdf
Supplemental Figure 1 (PDF): Predicted peptide length for indels in H1-1 through -5 We utilized R to systematically create one base pair (bp) duplications (left) or 1bp deletions (right) throughout the length of the H1-1, H1-5, H1-2, H1-3, and H1-5 genes and translated the sequences until a stop codon was reached. Black bars represent nucleotide positions for which conserved, continuous peptides can be generated.
- SupplementalFigure2Tremblay.pdf
Supplemental Figure 2 (PDF): Validation of H1 qRT-PCR primers siRNA was used to knock down individual H1s in HEK293T cells and performed qPCR to validate knockdown and primer specificity. H1-2 p=0.105, H1-3 p=0.028, H1-4 p=0.0023. Unpaired T-test, *=<0.05, **=<0.005
- SupplementalFigure3Tremblay.pdf
Supplemental Figure 3 (PDF): Lentiviral validation in human control LCLs We utilized lentiviral spinocculation to exogenously express FLAG-myc-WT H1.4, frameshift mutant FLAG-myc-c.430dupG H1.4, or GFP in control LCLs obtained from Coriell Cell Repositories. Whole cell lysates run on 12% polyacrylamide gel and probed with anti-FLAG antibody. Actin used as loading control, gel cropped to show only actin bands, as it was re-probed from the same blot.
- SupplementalFile1Tremblay.xlsx
Supplemental File 1 (.xls): Detailed phenotypes of subjects in this study For subjects 1-9 in this study, health history and self-reporting questionnaires were obtained. The results are summarized in this table.
- SupplementalFile2Tremblay.xlsx
Supplemental File 2 (.xls): Summary information for indels, CNV, and frameshift mutations in H1 family members The Baylor Clinical ES database was queried for mutations in H1-1, H1-5, H1-2, H1-3, and H1-5 that were comorbid with a diagnosis of ASD or ID. This file contains separate tabs outlining (1) All frameshift mutants in H1 genes, (2) a summary of all large CNVs which contained the H1-4 gene (HIST1H1E), (3) mutations in H1-1, (4) mutations in H1-5, (5) mutations in H1-2, (6) mutations in H1-3, and (7) mutations in H1-4
- SupplementalTable1Tremblay.xlsx
Supplemental Table 1 (.xls): Primers used in this study Nucleotide sequences of primers used in this study, as well as the application each primer pair was used for.
- SupplementalTable2Tremblay.pdf
Supplemental Table 2 (PDF): Nomenclature of select H1 family members Nomenclature data obtained from NCBI (https://www.ncbi.nlm.nih.gov/gene). For each gene, human and mouse gene and protein names are listed.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Yong-hui Jiang
Department of Genetics
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6188-8806
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Dec, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Neurobiology of Disease
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, brittle bones, and dysmorphic facies. De novo heterozygous mutations in H1-4 (HIST1H1E) encoding the linker histone H1.4 are found in patients with RMNS; however, the underlying mechanisms causing the pronounced neurological manifestations are not understood. The majority of reported mutations in H1-4 are small insertions or deletions that create a shared frameshift, resulting in an H1.4 protein that is both truncated and possessing an abnormal C-terminal tail.
Methods: Seven Rahman syndrome subjects with C-terminal frameshift mutations as well as three patients with heterozygous null mutations in H1-4 were described in detail. Lymphoblastoid cells from these patients were used to identify transcriptional abnormalities in RMNS. Wildtype or mutant frameshifted human H1.4 protein was exogenously expressed in primary rat hippocampal neurons, and neuronal structure and function were assessed using immunohistochemistry and multi-electrode array recordings.
Results: Individuals with heterozygous null variants in the H1-4 gene lack several key RMNS phenotypes, supporting the hypothesis that RMNS is due to a gain-of-function of the frameshift mutant H1.4 protein. In cultured rat hippocampal neurons, H1.4 was localized to the nucleus, though the frameshift mutant H1.4 had a distinct subnuclear distribution and enlarged nuclei. Overexpression of frameshift mutant H1.4 had minimal effects on dendritic morphology; however, it significantly reduced neuronal firing rate relative to neurons overexpressing wildtype human H1.4.
Limitations: Given the small number of RMNS cases worldwide, the true breadth of phenotypes remains unknown. Though our data do not show robust differential expression of any genes, larger cohorts for clinical and molecular studies will be needed to gain reliable data.
Conclusions: These data are the first to characterize the consequence of frameshift mutant H1.4 in neurons. These data provide new insights into the breadth of phenotypes and causes of neurological dysfunction in RMNS and highlight the need for future studies on the function of histone H1.4 in neurons.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalFigure1Tremblay.pdf
Supplemental Figure 1 (PDF): Predicted peptide length for indels in H1-1 through -5 We utilized R to systematically create one base pair (bp) duplications (left) or 1bp deletions (right) throughout the length of the H1-1, H1-5, H1-2, H1-3, and H1-5 genes and translated the sequences until a stop codon was reached. Black bars represent nucleotide positions for which conserved, continuous peptides can be generated.
- SupplementalFigure2Tremblay.pdf
Supplemental Figure 2 (PDF): Validation of H1 qRT-PCR primers siRNA was used to knock down individual H1s in HEK293T cells and performed qPCR to validate knockdown and primer specificity. H1-2 p=0.105, H1-3 p=0.028, H1-4 p=0.0023. Unpaired T-test, *=<0.05, **=<0.005
- SupplementalFigure3Tremblay.pdf
Supplemental Figure 3 (PDF): Lentiviral validation in human control LCLs We utilized lentiviral spinocculation to exogenously express FLAG-myc-WT H1.4, frameshift mutant FLAG-myc-c.430dupG H1.4, or GFP in control LCLs obtained from Coriell Cell Repositories. Whole cell lysates run on 12% polyacrylamide gel and probed with anti-FLAG antibody. Actin used as loading control, gel cropped to show only actin bands, as it was re-probed from the same blot.
- SupplementalFile1Tremblay.xlsx
Supplemental File 1 (.xls): Detailed phenotypes of subjects in this study For subjects 1-9 in this study, health history and self-reporting questionnaires were obtained. The results are summarized in this table.
- SupplementalFile2Tremblay.xlsx
Supplemental File 2 (.xls): Summary information for indels, CNV, and frameshift mutations in H1 family members The Baylor Clinical ES database was queried for mutations in H1-1, H1-5, H1-2, H1-3, and H1-5 that were comorbid with a diagnosis of ASD or ID. This file contains separate tabs outlining (1) All frameshift mutants in H1 genes, (2) a summary of all large CNVs which contained the H1-4 gene (HIST1H1E), (3) mutations in H1-1, (4) mutations in H1-5, (5) mutations in H1-2, (6) mutations in H1-3, and (7) mutations in H1-4
- SupplementalTable1Tremblay.xlsx
Supplemental Table 1 (.xls): Primers used in this study Nucleotide sequences of primers used in this study, as well as the application each primer pair was used for.
- SupplementalTable2Tremblay.pdf
Supplemental Table 2 (PDF): Nomenclature of select H1 family members Nomenclature data obtained from NCBI (https://www.ncbi.nlm.nih.gov/gene). For each gene, human and mouse gene and protein names are listed.
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