Human FCHO1 deficiency reveals role for clathrin-mediated endocytosis in development and function of T cells

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans.


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Policy information about availability of computer code Data collection For data collection following software was used: whole-exome sequencing (WES) -BWA software, version 0.7.15; flow cytometry data -FACS diva software (BD); confocal microscopy -Zen Blue software, version 2.0x (Zeiss); Western Blot collection -Image Lab software (Bio-Rad laboratories).

Data analysis
For data analysis following software was used: whole-exome sequencing (WES) -BWA software (version 0.7.15) for short sequence alignment, GATK pipeline (version 3.6) to call the variants and recalibrate, VEP release 85 to annotate the final variants, effects of filtered variants on protein were predicted with SIFT and PolyPhen2; crystal structure of FCHO1 domains were modeled using PyMol software with mutalyzer wizard; Western Blot pictures -Quantity One or Image Lab software (bith Bio-rad laboratories); confocal microscopy pictures and movies -Zen Blue software, version 2.0x (Zeiss); flow cytometry data -FlowJo Software v.8 or 9 (TreeStar); statistical analysis -GraphPadPrism software v.6; to assess Pearson correlaion and colocalization coefficient on selected cell fragments Zen Blue software, version 2.0x For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The identified FCHO1 mutations have been submitted to the ClinVar database with accession numbers XXX. According to current regulatory frameworks, exome sequencing data cannot be made publicly available.

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Sample size
Sample size was determined by number of individuals carrying the rare FCHO1 variants. All individuals who were identified to carry rare homozygous FCHO1 variants are included in manuscript. Quantification of Pearson correlation or colocalization coefficient of FCHO1 wild-type and all tested mutants with EPS15, adaptin and clathrin was preformed on pooled data of two to three independent experiments. Each symbol represents one region of 25μm2. Up to three regions per cells were quantified, minimum 50 data points were collected for each FCHO1 variant. For statistical analysis of the confocal microscopy pictures minimum 50 measures per each FCHO1 variant were collected. Data are pooled of two to three independent experiments. Pearson correlation of FCHO1 and clathrin from individual movies are pooled data from three independent experiments. Each symbol represents one square region of 25μm2. Up to three regions per cells were quantified, minimum 50 regions were assessed.
Data exclusions For confocal microscopy analysis only cell with low to moderate expression of FCHO1 were used, as high expression of FCHO1 may results in dominant-negative effects on CCP dynamic. For analysis of fixed samples, cells which partially detached from cover slip or appeared in anyway damaged were excluded from analysis. Those criteria were set before the experiment started. Otherwise there was not data exclusion.

Replication
In order to assure reproducibility, following measures were taken. 1/ To minimize the risk of accidental mutagenesis introduced during CRISPR/Cas9 modification and subsequent (sub)clonal selection, minimum five independent (sub)clones of cells sufficient or deficient for FCHO1 were taken for analysis. Whenever possible (i.e. Ca2+ release or TCR internalization assay performed on Jurkat cells) multiple (from three to five) clones of both wt and ko genotype were analyzed in one experiment. For confocal microscopy assessments different clones of SK-MEL cells deficient for FCHO1 were used interchangeably.

Blinding
For assessment of confocal microscopy images blinding was not possible, as the effects of tested FCHO1 variants were obvious even for researchers not involved in the studies.

Validation
Specificity of polyclonal Ab targeted against human FCHO1 eptiopes were tested in Western blot using both positive and negative controls. Cells transfected with WT FCHO1 construct served as positive control whereas cells with genetically disrupted FCHO1 locus (CRISPR/Cas/9 mediated modification were resulting in frame-shitf mutations in both chromosomes) were used as negative control. Specificity of other antibodies used in WB were controlled using protein size "ladder" and by comparison of band pattern with those provided by manufacturer and other publications. For confocal microscopy experiments only the Ab which give minimal unspecific background in WB analysis were used. All antibodies used for flow cytometric analysis were monoclonal, provided by well-established manufacturers and staining pattern obtained from healthy volunteers was as expected.

Mycoplasma contamination
All cell lines were routinely tested for mycoplasma and were mycoplasma negative throughout the study.

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All relevant information is provided in Table 1 and Sup Table 1 Recruitment Patients were referred to the clinical and scientific team of Professor Christoph Klein for further investigations.

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Informed consent/assent was obtained from all legal representatives and patients. Genetic and functional studies on biosamples from patients and their relatives were performed under the framework of a scientific project entitled "Genetic characterization of congenital bone marrow failure and immunodeficiency syndromes". This study has been approved in 2011 by the ethics committee at LMU (438-11).
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