HIST1H1E heterozygous protein-truncating variants cause a recognizable syndrome with intellectual disability and distinctive facial gestalt: A study to clarify the HIST1H1E syndrome phenotype in 30 individuals.

Histone Gene Cluster 1 Member E, HIST1H1E, encodes Histone H1.4, is one of a family of epigenetic regulator genes, acts as a linker histone protein, and is responsible for higher order chromatin structure. HIST1H1E syndrome (also known as Rahman syndrome, OMIM #617537) is a recently described intellectual disability (ID) syndrome. Since the initial description of five unrelated individuals with three different heterozygous protein-truncating variants (PTVs) in the HIST1H1E gene in 2017, we have recruited 30 patients, all with HIST1H1E PTVs that result in the same shift in frame and that cluster to a 94-base pair region in the HIST1H1E carboxy terminal domain. The identification of 30 patients with HIST1H1E variants has allowed the clarification of the HIST1H1E syndrome phenotype. Major findings include an ID and a recognizable facial appearance. ID was reported in all patients and is most frequently of moderate severity. The facial gestalt consists of a high frontal hairline and full lower cheeks in early childhood and, in later childhood and adulthood, affected individuals have a strikingly high frontal hairline, frontal bossing, and deep-set eyes. Other associated clinical features include hypothyroidism, abnormal dentition, behavioral issues, cryptorchidism, skeletal anomalies, and cardiac anomalies. Brain magnetic resonance imaging (MRI) is frequently abnormal with a slender corpus callosum a frequent finding.

Dileucine signal-dependent and AP-1-independent targeting of a lysosomal glycoprotein in Trypanosoma brucei.

Sorting of trans-membrane proteins destined for the lysosome is achieved by selective inclusion into post-Golgi transport vesicles. In higher eukaryotes sorting may be mediated by a peptidic motif, principally acidic clusters and tyrosine- or dileucine-based cytoplasmic signals or by inclusion of mannose-6-phosphate (M6P) into the N-glycans of lysosomal proteins. In African trypanosomes a major lysosomal trans-membrane protein is CB-1/p67. The cytoplasmic domain of p67 lacks tyrosine and lysine, but does contain a canonical dileucine sequence embedded within an acidic region. AP-1, -3 and -4 adaptin complexes, which recognise tyrosine- and dileucine-sorting signals, are encoded by the trypanosome genome, but the genes for M6P-receptors or activities required to produce M6P are absent, suggesting that lysosomal delivery of p67 is most likely adaptin-mediated. By construction of p67 reporter constructs we show that the dileucine signal is necessary and sufficient for efficient lysosomal delivery of a trans-membrane protein in bloodstream stage trypanosomes. However, this targeting does not require AP-1, as knockdown of the trypanosome gamma-adaptin subunit by RNAi has no detectable effect on the location or maturation of p67. These data suggest that p67 is targeted to the lysosome by dileucine-dependent but AP-1-independent mechanisms.

Both of the Rab5 subfamily small GTPases of Trypanosoma brucei are essential and required for endocytosis.

Endocytosis is an essential process in Trypanosoma brucei and all evidence suggests it is exclusively clathrin-mediated. The trypanosome genome encodes two Rab5 proteins, small GTPases that play a role in very early stages of endocytosis. In the mammalian bloodstream stage TbRAB5A localises to compartments containing internalised antibody, variant surface glycoprotein (VSG) and transferrin, whilst TbRAB5B localises to compartments containing the transmembrane protein ISG(100). Dominant-active forms of TbRAB5A stimulate endocytosis in procyclic forms and alter the kinetics of anti-VSG antibody and transferrin turnover in bloodstream stages. Similar mutants of TbRAB5B increase fluid phase uptake in procyclic cells but do not significantly affect endocytosis in bloodstream forms. Here, we use RNA interference to evaluate the relative importance of TbRAB5A and TbRAB5B and show that both GTPases are essential in the bloodstream form. Depletion of either TbRAB5A or TbRAB5B results in morphological abnormalities, including enlargement of the flagellar pocket, consistent with a potent block to endocytosis. Also, RNAi compromises transferrin accumulation in both cases but induces distinct patterns of mislocalisation of endosomal markers. Finally, RNAi of either TbRAB5A or TbRAB5B results in a decrease in levels of clathrin. Taken together, these data indicate that both TbRAB5A and TbRAB5B are required for endocytosis in trypanosomes and demonstrate that there are multiple essential endocytic routes in this organism.

Perturbation of local endogenous expression by insertion of Pol I expression constructs into the genome of Trypanosoma brucei.

Manipulation of gene expression is a common tool for the elucidation of biological function. Here we investigated the effects of over-expression in trypanosomes of a small GTPase, TbRABX1, using 2D gel electrophoresis and mass-spectrometry. An over-expression construct was targeting to the tubulin locus of chromosome I for stable integration and expression. Unexpectedly we observed alterations to the expression of gene products, i.e., tubulin, from surrounding regions of the genome; this effect was shown to be general and not dependent on the identity of the ectopic gene being expressed. These data suggest that local perturbation of the genome by insertion of DNA constructs can have wider impacts on gene expression, which need to be monitored.

Clathrin-mediated endocytosis is essential in Trypanosoma brucei.

In Trypanosoma brucei, the plasma membrane is dominated by glycosylphosphatidylinositol (GPI)-anchored proteins. Endocytic activity correlates with expression levels of the clathrin heavy chain TbCLH, and additional evidence suggests that rapid endocytosis may play a role in evasion of the immune response. TbCLH is present on both endocytic vesicles and post-Golgi elements, suggesting a similar range of functions in trypanosomes to higher eukaryotes. We have assessed the role of TbCLH using RNA interference (RNAi). Suppression of TbCLH expression results in rapid lethality in the bloodstream stage, the form most active for endocytosis. The flagellar pocket, the site of both endocytosis and exocytosis, becomes massively enlarged, suggesting that membrane delivery is unaffected but removal is blocked. Endocytosis in TbCLHRNAi cells is essentially undetectable, suggesting that clathrin-mediated mechanisms are the major route for endocytosis in T.brucei and hence that GPI-anchored proteins are endocytosed by clathrin-dependent pathways in trypanosomes. In contrast, a massive internal accumulation of vesicles and significant alterations to trafficking of a lysosomal protein were observed in the procyclic stage, indicating developmental variation in clathrin function in trypanosomes.

New approaches to the microscopic imaging of Trypanosoma brucei.

Protozoan parasites are fearsome pathogens responsible for a substantial proportion of human mortality, morbidity, and economic hardship. The principal disease agents are members of the orders Apicomplexa (Plasmodium, Toxoplasma, Eimeria) and Kinetoplastida (Trypanosomes, Leishmania). The majority of humans are at risk from infection from one or more of these organisms, with profound effects on the economy, social structure and quality of life in endemic areas; Plasmodium itself accounts for over one million deaths per annum, and an estimated 4 x 10(7) disability-adjusted life years (DALYs), whereas the Kinetoplastida are responsible for over 100,000 deaths per annum and 4 x 10(6) DALYs. Current control strategies are failing due to drug resistance and inadequate implementation of existing public health strategies. Trypanosoma brucei, the African Trypanosome, has emerged as a favored model system for the study of basic cell biology in Kinetoplastida, because of several recent technical advances (transfection, inducible expression systems, and RNA interference), and these advantages, together with genome sequencing efforts are widely anticipated to provide new strategies of therapeutic intervention. Here we describe a suite of methods that have been developed for the microscopic analysis of T. brucei at the light and ultrastructural levels, an essential component of analysis of gene function and hence identification of therapeutic targets.