Genetic and clinical profiles of subjects with H1-4 mutations
Seven individuals with RMNS C-terminal frameshift mutations and three individuals with loss-of-function of wild type (WT) H1-4 were recruited for this study (Figure 1) (see Supplemental Table 1 for histone H1 family gene nomenclature). Similar to other reports, most variants in the H1-4 gene were clustered in a 100bp region between c.360 and c.450 in the C-terminal domain of the transcript and result in long frameshift mutations predicted to have a strongly negative net charge compared to WT H1.4 protein. Six of these mutations are recurrent and have been reported by other groups, but four mutations (c.392dupC in subject #4, c.1A>G in subject #8, c.100_101insT #9, c.265delA #10) are newly reported here. The duplication variant in subject #4 is consistent with a RMNS associated frameshift mutation. By contrast, the c.1A>G variant is novel and predicted to disrupt the ATG start codon of H1-4 and result in loss-of-function, as the alternative protein product from an alternative ORF is a small 17 amino acid peptide. Similarly, c.100_101insT results in a peptide of 34 WT amino acids followed by a frameshift of 13 amino acids (p.K34Ifs*13), and c.265delA results in p.S89Afs*140, both mutations result in a loss-of-function of the WT C-terminal tail.
The clinical features we collected from a review of medical records and parent-completed questionnaires are summarized in Figure 2A and Supplemental File 1. RMNS subjects with H1-4 mutations were originally characterized as having a syndrome of “Intellectual disability and overgrowth”(1). However, except for two subjects with macrocephaly in our cohort, we did not find a significant deviation above normal growth parameters for newborns nor in current measures of head circumference and height, per Centers of Disease Control and Prevention (CDC) clinical growth charts (26) (Figure 2B). The major features of RMNS subjects carrying C-terminal frameshift mutations included dysmorphic facies (7/7, 100%), ID (7/7, 100%); ASD (3/4, 75%); motor delay (7/7, 100%); vision issue (6/6, 100%). Overall, the features of these subjects in our cohort are consistent with previous reports with large RMNS cohorts (2, 5).
Summary of variants in H1-4
To better understand the array of H1-4 mutations associated with an ASD or ID phenotype, we conducted a systemic review of the Baylor Genetics database of ~17,000 individuals undergoing clinical exome sequencing (ES). This revealed 15 variants in the UTR, 11 in frame amino acid deletions, 403 missense mutations, and 94 silent mutations in addition to the identified H1-4 frameshift indels described in Figure 1. Among individuals undergoing clinical chromosomal microarray analysis, we also identified three individuals with phenotypes of developmental delay or autism who had large CNV gains of 565-602kb in regions including the H1-4 gene.
The contribution of haploinsufficiency resulting from heterozygous H1-4 mutations has not previously been described. The lack of H1-4 CNV losses in this clinical database is consistent with the dominant negative/gain-of-function hypothesis for the frameshift mutant H1.4 protein in RMNS, rather than a loss-of-function.
Identification of novel loss-of-function H1-4 alleles
The de novo c.1A>T variant in subject #8 from our cohort is predicted to disrupt H1.4 protein expression as it occurs in the start codon and there is no alternative ATG downstream within the same reading frame. In the unlikely event that an alternative reading frame is utilized, the only alternative methionine results in a small peptide of 17 amino acids (MTWRRTTAASSWVSRAW in the +1 codon reading frame shift). The clinical features of the subject with the c.1A>T variant are summarized in Figure 2A (and Supplemental File 1). This individual has dysmorphic features including broad nasal tip, thick alae nasi, accentuated Cupid's bow, large and fleshly earlobes bilaterally, and small mouth leading to mild dental crowding. Together, this set of dysmorphic features is distinct from those in individuals carrying C-terminal frameshift mutations in H1-4 and diagnosed with RMNS. Other minor anomalies include bilateral fifth finger clinodactyly and fetal finger pads. Her language development was delayed as she spoke her first word at 18 months, two words together at 3 years, and short sentences at 4 years. She had a clinical diagnosis of ASD based on DSM-5 criteria due to her deficits in social communication and interaction, as well as repetitive behaviors. She has family history of ASD, with a sister who also has ASD and does not carry a H1-4 variant. In addition, she has flat affect, impairments in social-emotional reciprocity and nonverbal communication, and hyperreactivity to sensory input. In contrast, she did not have history of hypotonia or motor delay, as she walked at 13 months. She also did not have vision problems, bone concerns, ADHD, or anxiety. While this individual is a single case, her features are overlapping but clearly distinct clinically from the subjects with frameshift mutations in the C-terminus of H1-4.
The clinical presentation of subject #9 with a maternally inherited c.100_101insT mutation in H1-4 is also distinct from RMNS phenotypes. In the event that this transcript eludes nonsense mediated decay, the protein product is a peptide of 47 amino acids (p.K34Ifs*13), likely resulting in a loss-of-function of WT H1.4 C-terminus. This individual presents with chronic kidney disease, of which the cause is homozygous NPHP1 deletion [OMIM: 607100] identified by CMA comprehensive. However, this individual also presents with a history of delayed speech and language development (he did not speak until he was 4 years old) for which he received speech therapy. He did not have any other delays in childhood and was formally screened for developmental delay, of which he did not meet diagnostic criteria for any other delays or disorders. This subject does not have ID and has reached academic milestones as expected with typical development. As this variant is maternally inherited, we briefly investigated the phenotype of the mother. The mother of subject #9 did not have any delays during her development and of note, was enrolled in gifted programs. Neither subject #9 nor his mother have dysmorphic facial features.
We also identified an individual with a C-terminal tail distinct from the tail found in RMNS subjects. Subject #10 carries a de novo c.265delA which results in p.S89Afs140* if the transcript escapes nonsense mediated decay. While the predicted protein product contains a long tail, this frameshift tail is in the alternative reading frame from that of RMNS subjects. This individual presents in the clinic with short stature, ankylosis of both jaw joints (resolved by surgical release), retromicrognathia, and bilateral conductive hearing loss. With regards to neurological development, he had a speech delay early in life. However, he currently speaks in broken sentences, and has a below average IQ test score or 86. However, he has not met diagnostic criteria for ID or ASD. He did not have infantile hypotonia and was able to walk without support at 12 months of age. In addition, his facies are distinct from RMNS individuals in that the sole dysmorphic feature is the locked jaw. He has a family history of global developmental delay, speech delay, motor delay, and depression presenting in two male siblings who we previously reported to have a different disease caused by homozygosity for a THUMPD1 variant (27), which was not identified in the patient presented here.
The phenotype of these individuals further supports the hypothesis that the frameshift mutations clustered in the C-terminus of H1-4 operate via a gain-of-function mechanism, rather than through H1-4 haploinsufficiency or loss-of-function of the H1.4 C-terminal tail.
Conserved frameshifts and net charge changes in H1 family
The cluster of RMNS associated C-terminal frameshift mutations in H1-4 raises the question of why similar disease-associated mutations are not found in other Histone H1 family members. To investigate the consequence of indels in H1-1 through -5, we computationally introduced 1 base pair indels throughout the length of the H1-1, H1-2, H1-3, H1-4, and H1-5 gene sequences and determined the consequence of these frameshift mutations on the both the length of the protein translated and the change in charge of the resulting peptide compared with the WT protein (Figure 3). We found that all histone H1 family members could produce out-of-frame proteins of various sizes if indels were introduced at any one of a number of positions (Supplemental Figure 3). However, the frameshift mutations in the H1-4 RMNS hotspot were unique for their ability to result in peptides with a large negative change in the charge of the C-terminal tail (Figure 3). We also observed that insertions in a similar region of H1-5 could have a smaller but potentially significant effect on protein charge. However, when we queried the clinical ES database at Baylor College of Medicine (one of the largest and non-public clinical ES databases) for frameshift mutations in H1-5 which were associated with an ID phenotype, we found none. There were 9 individuals with frameshifts in H1-1 (3 of these mutations were inherited, 5 were not tested for origin), and two individuals with frameshift mutations in H1-2 (SupplementalFile 2), however these mutations had minimal effects on net protein charge. Taken together these data are consistent with the hypothesis first proposed by Tatton-Brown et al. (2017) that pathogenic mutations in H1-4 are those that disrupt the positively charged C-terminal tail, impairing the ability of this H1 histone to interact with negatively charged DNA (11, 13).
Transcriptional characterization of RMNS patient-derived cells
Although H1.4 is a histone linker protein, its exact transcriptional functions remain poorly understood. To determine whether there were gene expression changes in our H1-4 frameshift mutant cohort, we generated immortalized LCLs from three subjects and compared RNA expression in these cells to LCLs from age, sex, and ethnically matched controls (Figure 4A, B). Prior studies from knockout mice have shown that loss of a single histone H1 gene can be associated with compensatory upregulation of other histone H1 family members (12), thus we first assessed relative expression of H1 family genes between the LCL lines. We validated primers to detect H1-2, H1-3, and H1-4 in human cells (Supplemental Figure 2) and used these to measure H1 family gene expression. We did not find any significant differences in H1-2, H1-3 or H1-4 expression in H1-4 mutant LCLs compared with control (Figure 4C). These data show that the RMNS-associated H1-4 frameshift mutations in our cohort do not induce compensation via transcriptional induction of other H1 family members.
We then took an approach to examine the expression of candidate genes suggested from previous studies to be associated with H1.4 function. These candidate genes fall into four classes. First, we assessed mRNA encoding proteins that have been shown to physically interact with H1.4 in mouse cells (18-20, 28-30) (Figure 5A). Second, we examined the expression of epigenetic readers, writers, or erasers of DNA methylation, which is a process that has been reported to be regulated by H1 and aberrant in H1-4 frameshift mutant fibroblasts obtained from RMNS individuals (14, 31) (Figure 5B). Third, we looked at the expression of components of the complement system, because one complement factor, C3, was previously identified as being differentially methylated in H1-4 mutant skin fibroblasts (14) (Figure 5C). Alterations in the complement system would be of particular interest in ID because this pathway has been implicated in synaptic pruning in neurons (32) and linked to neurodevelopmental disorders such as ASD (33-35), thus finally we also examined the expression of genes that link complement cascades to synapses (Figure 5D).
The only genes we observed to be significantly different between patient and control LCLs were the complement components C3, CR1, and CR2 (p=0.0439, 0.0021, 0.0130 respectively) (Figure 5C). However, because LCLs are oligo-clonal cell lines derived from cells of the immune system, it is possible that differences in complement component expression between these lines could reflect clonal responses to serum factors in the media rather than a specific effect of the frameshift mutant H1.4 expression. We reasoned that if complement C3 was dysregulated in the patient LCLs due to the expression of mutant H1.4, then if we recreated the same H1.4 expression pattern in control cells by expressing either WT or frameshift mutant H1.4, we should generate the same difference in complement gene regulation. Similar to constructs used in prior studies(18-20) we generated N-terminal dual FLAG-myc-tagged lentiviral constructs to exogenously express either WT or frameshift mutant (c.430dupG) H1.4 in the WT control LCL cells. Western blotting against the FLAG tag in LCL lysates verified exogenous expression of the WT and mutant FLAG-myc-tagged H1.4 protein in the control lines (Supplemental Figure 3). Nonetheless, we found no difference in transcript abundance of C3, CR1, or CR2 between any of the conditions in the three control lines (p=0.449, 0.265, 0.286 respectively) (Figure 5E). These data suggest that exogenous expression of frameshift mutant H1.4 alone is not sufficient to drive upregulation of complement expression. Future studies will be required to determine whether other genetic factors interact with mutant H1.4 to alter complement component expression in LCLs from RMNS patients.
Altered distribution of H1.4 mutant protein in nuclei of rat hippocampal neurons
Frameshift mutations in H1-4 are strongly associated with neurodevelopmental impairments in humans. However, the consequences of frameshifted mutant H1.4 expression on the structure or function of neurons is unknown. Importantly, there is limited evidence of consistently prevalent and conserved gross structural malformations of brains of these individuals (Supplemental File 1 and (2, 5)), which raises the possibility that expression of frameshift mutant H1.4 may disrupt functional aspects of neuronal physiology. To elucidate the possible impact of frameshift mutant H1.4 protein on neurons, we compared the consequences of overexpressing either WT or mutant H1.4 protein in cultured primary embryonic rat neurons (Figure 6A). Immunostaining of the Myc-tag of the dual FLAG-myc-H1.4 constructs showed that most cells expressed the exogenous human proteins (Figure 6B). Thus, our model mimics the germline heterozygous expression of H1-4 frameshift mutant protein in human RMNS. Consistent with previous experiments in other cell types (5), we found that both WT H1.4 and frameshift mutant H1.4 protein were detected exclusively in the nucleus. (Figure 6B). However, the percent overlap between H1.4 and Hoescht was lower in neurons expressing the frameshift mutant H1.4 (Figure 6C) suggesting there was a difference either in nuclear morphology or in the distribution of mutant histone H1.4 compared with WT within the nucleus. Indeed, the nuclear size of neurons expressing H1.4 mutant protein is significantly larger than that of WT (p=0.0015) (Figure 6D). Yet even controlling for size, we found that the H1.4 frameshift mutant protein has a greater localization towards the periphery of the nucleus compared with the distribution of the WT protein (Figure 6E). These results indicate a potential large-scale change in chromatin or nuclear organization associated with the expression of mutant H1.4 protein.
Frameshift mutant H1.4 disturbs actional potential frequency and synchrony in hippocampal neurons
To determine if the expression of H1.4 frameshift mutant protein results in functional changes in neuronal physiology, we first assessed dendritic morphology, which determines key aspects of synapse formation between neurons. We did not find any difference in length and number of primary or secondary neurites at DIV7 between neurons expressing either exogenous WT or frameshift mutant H1.4 protein (Figure 6F). In addition, Sholl analysis performed on this data did not note a difference in number of crossings of cell neurites for the neurons expressing H1.4 mutant protein compared with neurons expressing WT protein (p=0.2344) (Figure 6G).
To determine if there is a functional consequence of H1.4 frameshift mutant protein for neural network activity, we measured action potential spike frequency and synchrony in cultured hippocampal neurons plated on 16 electrode multielectrode arrays (Figure 7A). We detected spiking with similar waveforms regardless of whether neurons were infected with the WT H1.4, frameshift mutant H1.4, or GFP (Figure 7B, C). For neurons expressing either WT H1.4 or GFP as control, both firing rate detected at a single electrode and the synchrony of firing between electrodes increased from DIV 8-13, which are consistent with increased connectivity and synaptic maturation over this period (Figure 7D). However, neurons expressing H1.4 frameshift mutant protein showed lower levels of firing (mixed effects, p=0.0006) with a delayed rise to later days in culture and significantly reduced synchrony (mixed effects, p=0.0017) when compared to WT H1.4 and GFP groups (Figure 7D). Thus, these data provide the first evidence that frameshift mutant H1.4 protein disrupts neuronal activity