Gene Curation Example

ClinGen https://curation.clinicalgenome.org/curation-central/ee6ec3dc-0647-4038...

Close Evidence Summary

FANCF – Fanconi anemia complementation group f – Autosomal recessive inheritance

Classification owner: Hereditary Cancer Classification status: APPROVED PUBLISHED Classification Contributor(s): Illumina Date classification saved: 2021 Feb 03, 4:54 pm Date classification published: 2021 Feb 03, 4:55 pm Calculated classification: Definitive Replication Over Time: Yes Modified classification: No Modification Contradictory Evidence? Proband: No, Experimental: No Reason for modified classification: None Disease: Fanconi anemia complementation group f SOP: Gene Clinical Validity Standard Operating Procedures (SOP), Version 8

Evidence Summary

FANCF is one of the 23 FA or FA-like genes known to cause autosomal recessive Fanconi anemia (FA) characterized by bone marrow failure, developmental abnormalities, cancer predisposition, and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C. The cloning of the cDNA encoding FANCF by complementation of an FA-F cell line was first reported in 2000 (PMID:10615118). Fourteen different pathogenetic homozygous or compound heterozygous variants of FANCF account for approximately 2 to 3% of the affected individuals (PMID: 31288759). Disease-associated variants have been reported throughout the single coding exon of the FANCF gene. The most commonly seen FANCF variants are short frameshift deletions, resulting in premature termination of the protein, suggesting homozygous loss of function is the mechanism of tumorigenesis for this disorder (PMID: 10615118, 16084127, 31288759, 26033879, 27714961). This gene-disease relationship is further supported by FANCF-deficient mouse model, rescue cell culture model, protein interaction and expression studies. In summary, FANCF is definitively associated with FA complementation group F. This has been repeatedly demonstrated in both the research and clinical diagnostic settings and has been upheld over time.

Calculated Classification Matrix

Evidence Type Count Total Points Points Counted Proband with other variant type with some evidence of gene impact 0 0 0 Autosomal Dominant OR X-linked Disorder Proband with predicted or proven null variant 0 0 0 Variant is de novo 0 0 0 Variant Two variants (not predicted/proven null) with some evidence of gene impact in trans 0 0 Autosomal Recessive Disorder 12 Two variants in trans and at least one de novo or a predicted/proven null variant 6 13

Case-Level Summed LOD Family Count Candidate gene sequencing 1.45 1 Segregation 0 0 Genetic Genetic Evidence Exome/genome or all genes sequenced in linkage region 0 0 Total Summed LOD Score 1.45 Case-Control 0 0 0 Genetic Evidence Total 12 Biochemical Functions 0 0 Functional Protein Interactions 1 1.5 2 Expression 1 0.5 Patient cells 0 0 Functional Alteration 0.5 Non-patient cells 1 0.5 Non-human model organism 1 3 Models Cell culture model 0 0 Rescue in human 0 0 4

Experimental Experimental Evidence Rescue in non-human model organism 0 0 Rescue Rescue in cell culture model 0 0 Rescue in patient cells 1 1 Experimental Evidence Total 6 Total Points 18

Genetic Evidence: Case Level (variants, segregation)

Segregations

Proband Proband Proband Points Reference Proband Proband Proband # # Previous Methods of Score (default Reason for Label Variant Type Variant (PMID) Sex Age Ethnicity Proband Phenotypes Aff Unaff LOD Score Counted Sequencing Testing Detection Status points) Changed Score

Immunoprecipitation Two variants in de Winter JP, HPO term(s): and western-blot trans and at least NM_022725.4(FANCF):c.230_252del et al., 2000, Cellular Method 1: Sanger analysis showed EUFA698 one de novo or a Unknown - - - - Score 2 (2) (p.Val77GlyfsTer6) PMID: hypersensitivity to sequencing that patient cells predicted/proven 10615118 mitomycin C lacked FANCF null variant protein.

HPO term(s): This variant results Cellular in a frameshift, and hypersensitivity to is predicted to diepoxybutane cause premature Intrauterine growth termination of the Method 1: Next protein. The Two variants in retardation Nicchia E, et generation correlation between trans and at least Age of Dilation of lateral Candidate II-5 (From NM_022725.4(FANCF):c.484_485del al., 2015, Calculated: sequencing FANCF one de novo or a Female Death: 1 ventricles 3 2 Yes gene Score 2 (2) Italy) (p.Leu162fs) PMID: 1.45 panels; Method 2: c.484_485del and predicted/proven Days Aplasia/Hypoplasia sequencing 26033879 Sanger FA patients has null variant of the radius Absent thumb sequencing been reported Ventricular septal repeatedly defect (PMID:10615118, Bilateral renal PMID:16084127, hypoplasia PMID: 26033879, PMID: 27714961).

HPO term(s): Cellular hypersensitivity to diepoxybutane Duodenal stenosis Method 1: Next Two variants in Nicchia E, et Cleft palate generation trans and at least Age of II-1 (From NM_022725.4(FANCF):c.484_485del al., 2015, Growth delay Calculated: sequencing one de novo or a Female Diagnosis: Unknown 1 1 No Score 2 (2) Egypt) (p.Leu162fs) PMID: Thrombocytopenia 0.12 panels; Method 2: predicted/proven 4 Years 26033879 Cafe-au-lait spot Sanger null variant Free text: sequencing agenesis of the right kidney, aortopulmonary collateral circulation irrorating the right lung

1 of 3 2/3/21, 4:57 PM ClinGen https://curation.clinicalgenome.org/curation-central/ee6ec3dc-0647-4038...

Segregations

HPO term(s): Cellular hypersensitivity to diepoxybutane Neutropenia Bone marrow hypocellularity Method 1: Sanger Absent thumb sequencing Hypoplasia of the Description of The family history radius genotyping was significant for Microcephaly method: multiple individuals Complementation with cancer (Table Two variants in Cleft palate NM_022725.4(FANCF):c.484_485del Tryon R, et group analysis SI). This individual is trans and at least Age of Short stature (p.Leu162fs) al., 2017, demonstrated compound Patient 1 one de novo or a Female Death: 3 Unknown Cafe-au-lait spot 1 - - - Score 2 (2) NM_022725.4(FANCF):c.438_451del PMID: FA-F with heterozygous for predicted/proven Years Renal (p.Arg147Ter) 27714961 subsequent gene two variants, and null variant hypoplasia/aplasia Microtia sequencing parents were tested Microphthalmia identifying the and shown to carry Atrial septal defect c.438_451del and one of the variants Patent ductus c.484_485delCT (table 1). arteriosus mutations in the Duodenal atresia FANCF gene. Growth hormone deficiency Delayed speech and language development

HPO term(s): Cellular

hypersensitivity to Family history was diepoxybutane significant for Myelodysplasia multiple individuals Short thumb with a history of Microcephaly cancer. This Short stature individual is Sacral dimple compound Congenital giant heterozygous for Two variants in melanocytic nevus two variants, and NM_022725.4(FANCF):c.484_485del Tryon R, et trans and at least Age of Cafe-au-lait spot parents were tested (p.Leu162fs) al., 2017, Method 1: Sanger Patient 2 one de novo or a Male Diagnosis: Pelvic kidney 1 - - - Score 3 (2) and shown to carry NM_022725.4(FANCF):c.219del PMID: sequencing predicted/proven 4 Months Vesicoureteral one of the variants (p.Arg74GlyfsTer7) 27714961 reflux null variant (table 1). Another Conductive FA-F patient with hearing the same compound impairment heterozygous Microphthalmia variants Microphallus (c.484_485delCT Growth hormone and c.219delG) was deficiency reported in 2005 Hypothyroidism (PMID: 16084127). Specific learning disability

At 14 years of age, this individual showed an Method 1: Sanger abnormal absolute sequencing neutrophil count Description of and a partial 1q genotyping duplication in 25% HPO term(s): method: of metaphase cells. Two variants in Cellular Complementation NM_022725.4(FANCF):c.2T>C Tryon R, et The family history trans and at least Age of hypersensitivity to group analysis (p.Met1?) al., 2017, was significant for Patient 3 one de novo or a Male Diagnosis: Unknown diepoxybutane 1 - - - identified group F Score 2 (2) NM_022725.4(FANCF):c.698_699del PMID: multiple individuals predicted/proven 9 Years Short stature with subsequent (p.Gly233GlufsTer?) 27714961 with cancer (Table null variant Cafe-au-lait spot gene sequencing SI). This individual is Premature birth identifying the compound c.2T > C and heterozygous for c.698_699delGG two variants, and mutations in the Genetic Evidence: Case Level (family segregation information without proband data or scored proband data) parents were tested FANCF gene. and shown to carry No segregation evidence for a Family without a proband was found. one of the variants (table 1).

Total points: 13.00 Genetic Evidence: Case-Control

No scored Case-Control evidence was found.

Experimental Evidence

Score Points (default Label Experimental Category Reference Explanation Status points) Reason for Changed Score

Co-immunoprecipitation experiments showed that FANCF forms a complex with other components of the FA pathway, including FANCA, FANCC, and FANCG, in wild-type lymphoblasts. After immunoprecipitation of FANCA, FANCF was detected in wild- The protein interactions were supported by three independent Protein Interactions physical de Winter JP, et al., 2000, PMID: Co-immunoprecipitation type lymphoblasts, but not in FA-A or FA-F lymphoblasts. After Score 1.5 (0.5) experiments of co-immunoprecipitation of FANCA, FANCG and association (MI:0915) 11063725 FANCG immunoprecipitation, FANCF was detected in wild-type FANCC. and FA-D lymphoblasts. Similarly, FANCF and FANCA were co- immunoprecipitated with FANCC in wild-type and FA-D lymphoblasts.

Both the short and the long isoforms of FANCD2 were present These data show that FANCF is an essential FA core complex Functional Alteration Non-patient Bakker ST, et al., 2012, PMID: in control mouse embryonic fibroblasts (MEFs), whereas in FANCD2 ubiquitination analysis Score 0.5 (0.5) member and in its absence, the FA pathway is impaired, which cells 21915857 Fancf −/− MEFs, only the short non-monoubiquitinated isoform results in failure to maintain genome stability. (FANCD2-S) was present.

2 of 3 2/3/21, 4:57 PM ClinGen https://curation.clinicalgenome.org/curation-central/ee6ec3dc-0647-4038...

Score Points (default Label Experimental Category Reference Explanation Status points) Reason for Changed Score

Fancf-deficient mice showed an FA-like phenotype including hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC), and prone to develop tumors. Fancf-deficient primary mouse embryonic fibroblasts (MEF) cultures accumulated more This mouse model recapitalizes FA phenotype. Meanwhile, break events after MMC treatment than control MEF cultures. FANCF deficient MEF cells have impaired FANCD2 Model Systems Non-human model Bakker ST, et al., 2012, PMID: Fancf-deficient mice Moreover, Fancf-deficient animals had an increased tumor Score 3 (2) monoubiquitination, providing strong evidence for FANCF as an organism 21915857 incidence compared with control animals. Additionally, Fancf essential FA core complex member and in its absence, the FA −/− MEFs have impaired FANCD2 monoubiquitination, indicating pathway is impaired. that FANCF is an essential FA core complex member and in its absence, the FA pathway is impaired, which results in failure to maintain genome stability.

Immuno-precipitation and western- de Winter JP, et al., 2000, PMID: FANCF protein expression in the EUFA698 and EUFA121 FA-F Expression B Score 0.5 (0.5) blot analysis 10615118 patient lymphoblast cell lines was absent.

MMC hypersensitivity of FA-F cell line EUFA698 is corrected Complementation of MMC de Winter JP, et al., 2000, PMID: Rescue Patient cells after transfection of FANCF cDNA clone. FANCF protein Score 1 (1) hypersensitivity 10615118 expression in the EUFA698 FA-F lymphoblasts was lacking.

Total points: 6.50

Non-scorable Evidence

No non-scorable evidence was found.

For best printing, choose "Landscape" for layout, 50% for Scale, "Minimum" for Margins, and select "Background graphics".

Close Print PDF

3 of 3 2/3/21, 4:57 PM