SPRED1 Human

What is the structural composition of SPRED1 protein?

SPRED1 is a 50-kDa protein comprising an N-terminal Ena/VASP Homology 1 (EVH1) domain and a C-terminal Sprouty-related domain (SPR) separated by a central c-Kit binding domain. The EVH1 domain functions as a PH-like protein-protein interaction module that binds to proline-rich sequence stretches of the type FPPPP. Most interaction partners of SPRED1 involve binding to the C-terminal SPR domain, though the precise function of these interactions remains under investigation .

What is the primary function of SPRED1 in cellular signaling?

SPRED1 specifically inhibits the Ras/MAPK pathway in response to growth factor-, cytokine-, and chemokine-induced ERK activation. It acts as a neurofibromin recruitment factor, binding to the GAP-related domain. This inhibitory role in the Ras/MAPK pathway explains its tumor suppressor function and its involvement in developmental disorders when mutated .

What conditions are associated with SPRED1 mutations?

The primary condition associated with germline SPRED1 mutations is Legius Syndrome, a rare developmental disorder sharing clinical features with Neurofibromatosis-1 (NF1). Most Legius Syndrome-associated mutations result in premature stop codons with truncated translation products. The majority of identified nontruncating missense mutations are located within the EVH1 domain . Additionally, SPRED1 acts as a tumor suppressor in pediatric acute myeloblastic leukemia, and its dysregulation has been implicated in other cancer types .

What are the current approaches for SPRED1 genetic testing?

Multiple approaches exist for SPRED1 genetic testing, depending on the research or clinical question:

• RNA-based testing: Particularly useful for identifying deep intronic splice variants through their observed effect on splicing, which might be missed in exon-by-exon DNA sequencing. This approach has identified over 65 different locations harboring deep intronic splice variants, accounting for 2.5% of all pathogenic variants identified in the NF1 UAB cohort .

• DNA-based Next-Generation Sequencing (NGS): Involves sequencing and deletion/duplication analysis of the entire coding SPRED1 region. This method uses customized and optimized Agilent HaloPlex capture probes, followed by sequencing of overlapping amplicons using 300bp paired-end Illumina sequencing chemistry .

• Combined NF1/SPRED1 testing: For patients with pigmentary features but no neurofibromas and no NF1 variants, SPRED1 gene analysis is conducted as a reflex test, including sequencing and deletion/duplication analysis .

How can SPRED1 expression be quantified in research settings?

Several methods are employed to quantify SPRED1 expression:

• ELISA: SPRED1 protein concentrations can be determined using enzyme-linked immunosorbent assay kits, with optical density read at 450 nm using a microtiter plate reader .

• Quantitative PCR: The relative expression level of SPRED1 mRNA can be calculated using the 2^-ΔΔCt method, comparing expression between different groups .

• FISH analysis: Fluorescence in situ hybridization using SPRED1-specific probes allows for visualization and analysis of SPRED1 gene copy number alterations .

What experimental approaches can be used to model SPRED1 deficiency?

Researchers have employed several strategies to model SPRED1 deficiency:

• shRNA knockdown: Using pLKO shRNA lentiviral vectors with specific hairpin sequences targeting SPRED1 .

• CRISPR-based inactivation: LentiCRISPR lentiviral vectors with specific gRNAs can be used to inactivate SPRED1 in human cell lines. For example:

  • SPRED1#1: 5′-GCGGTGAGGGAAAGATGAGCG-3′

  • SPRED1#2: 5′-GGTGGATGGTTACCACTTGG-3′

  • SPRED1#3: 5′-GTGGTTACCACTTGGAGGGAG-3′

• Animal models: CRISPR MiniCoopR vectors have been used to inactivate spred1 in zebrafish melanomas, with the gRNA: 5′-GGCGTCCGCCGGGCTCTGGA-3′ .

How does SPRED1 loss affect MAPK inhibition resistance in melanoma?

Research has demonstrated that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma confers resistance to BRAF V600E inhibition. When BRAF-driven A375 cells were suboptimally transfected with CRISPR vectors targeting SPRED1 and subjected to long-term BRAF inhibition, there was rapid emergence of clones resistant to dabrafenib treatment specifically in cultures transfected with SPRED1 gRNAs. The proportion of SPRED1 mutant alleles sharply increased in dabrafenib-treated cultures while remaining constant in vehicle-treated cultures. Deep sequencing revealed that the vast majority of these CRISPR variants exhibited frameshift mutations. SPRED1 protein levels remained constant over five passages in the absence of drugs but dropped within three passages of dabrafenib treatment, indicating rapid selection for SPRED1-deficient cells .

The SPRED1 knockout cells displayed residual MAPK signaling under dabrafenib treatment compared with control cells, suggesting that SPRED1 loss confers a selective advantage under continuous pharmacologic BRAF inhibition by increasing cell proliferation and/or survival in vitro .

What is the significance of SPRED1 expression in acute myeloid leukemia?

Studies have found significantly decreased SPRED1 mRNA expression in AML patients compared to ALL patients and healthy controls. This suggests potential utility as a prognostic biomarker. The measurement of SPRED1 expression has been conducted using various methods:

The reduced expression of SPRED1 in AML appears to correlate with its function as a tumor suppressor, supporting its role in disease progression and potential as a therapeutic target.

How are novel SPRED1 mutations identified and characterized?

Novel SPRED1 mutations are identified and characterized through comprehensive genetic analysis approaches:

• PCR amplification and sequencing: The exons and flanking areas of the SPRED1 gene are amplified by PCR using specific primers. Sequencing is then carried out with ddNTP terminator reaction and analyzed on genetic analyzers. Mutations are identified via comparison between sample sequences and reference sequences using specialized software like Mutation Surveyor .

• Functional classification: Mutations are classified based on their effect on protein function, with categories including:

  • '+' indicating the variant affects function

  • '+?' probably affects function

  • '-' does not affect function

  • '-?' probably does not affect function

  • '?' effect unknown

  • '.' effect was not classified

• Variant databases: Resources like the Global Variome shared LOVD (Leiden Open Variation Database) catalog variants in SPRED1, providing information about their clinical significance and frequency .

What types of mutations are most commonly observed in SPRED1?

The most common pathogenic mutations in SPRED1 include:

• Premature stop codons: The majority of Legius Syndrome-associated mutations result in premature stop codons, presumably associated with truncated translation products .

• Missense mutations: Most of the identified nontruncating missense mutations are located within the EVH1 domain .

• Deep intronic splice variants: These affect splicing and have been identified in over 65 different locations in comprehensive RNA-based testing. Together they account for 2.5% of all pathogenic variants identified in the NF1 UAB cohort, and would not be detected if a "simple" exon-by-exon DNA-based sequencing approach were used .

Two recently identified novel mutations include:

• A frameshift mutation causing a stop codon (p.Ile60Tyrfs*18) identified in an Italian family

• A missense variation identified in one sporadic Italian case

How can inconsistencies between SPRED1 genomic analysis and protein expression be resolved?

Resolving inconsistencies between genomic analysis and protein expression requires a multi-faceted approach:

• Integrated analysis: Combine RNA-based testing with protein expression analysis to identify variants affecting splicing or protein stability that may not be apparent from genomic sequencing alone.

• Tissue-specific analysis: Since SPRED1 may show tissue-specific expression patterns, analyze both affected tissues (e.g., café-au-lait macules, neurofibromas) and blood samples.

• Mosaic detection: For cases with suspected mosaicism, analyze multiple biopsies. If no variants are identified despite full analysis on 2 biopsies with successful cultures, (mosaic) NF1/Legius syndrome is very unlikely (<0.2%) .

• Epigenetic regulation: Consider epigenetic modifications that might affect SPRED1 expression without altering the genomic sequence.

What mechanisms underlie SPRED1-mediated resistance to MAPK pathway inhibitors?

Several mechanisms have been proposed for SPRED1-mediated resistance to MAPK pathway inhibitors:

• Restoration of MAPK signaling: SPRED1 knockout cells displayed residual MAPK signaling under BRAF inhibitor treatment compared with control cells, indicating that SPRED1 loss allows bypass of pharmacological inhibition .

• Selective pressure: Under continuous BRAF inhibition, SPRED1-deficient cells are rapidly selected for, suggesting that SPRED1 loss confers a survival advantage under drug pressure .

• Genomic evolution: In transfected cultures under drug pressure, the genotype evolves from mostly wild type to almost completely knockout for SPRED1 within five passages, demonstrating the strong selective advantage of SPRED1 loss .

• Alternative pathway activation: Loss of SPRED1 may allow activation of alternative signaling pathways that compensate for MAPK inhibition.

How might differential diagnosis between NF1 and Legius Syndrome be improved?

Improving differential diagnosis between NF1 and Legius Syndrome requires sophisticated approaches:

• Comprehensive genetic testing: A combined approach using both DNA-based and RNA-based testing for NF1 and SPRED1 provides the most accurate diagnosis. This should include:

  • Next-Gen Sequencing and Deletion/Duplication analysis of NF1 and SPRED1

  • RNA-based testing to identify deep intronic variants

• Tissue-specific analysis: For patients with only pigmentary features (CALMs with/without skinfold freckling but no neurofibromas), and no NF1 variants found in melanocytes, SPRED1 gene analysis should be performed as a reflex test .

• Biomarker development: Research into specific biomarkers that differ between the two conditions could enhance diagnostic accuracy.

• Imaging correlations: Correlation of genetic findings with advanced imaging techniques might reveal subtle phenotypic differences not apparent through clinical examination alone.

What therapeutic strategies targeting SPRED1 might be developed?

Potential therapeutic strategies targeting SPRED1 include:

• Restoration of SPRED1 function: For cancers where SPRED1 is downregulated or lost, gene therapy approaches to restore SPRED1 expression might suppress tumor growth.

• Combination therapies: For MAPK inhibitor-resistant melanomas with SPRED1 loss, combination therapies targeting alternative pathways might overcome resistance.

• Synthetic lethality: Identifying genes that, when inhibited, cause selective death in SPRED1-deficient cells could provide novel therapeutic targets.

• Small molecule modulators: Development of small molecules that mimic SPRED1 function or enhance residual SPRED1 activity in partially functional mutants.

How might single-cell technologies advance our understanding of SPRED1 function?

Single-cell technologies offer several advantages for SPRED1 research:

• Heterogeneity analysis: Single-cell RNA sequencing could reveal cell-specific effects of SPRED1 mutations or expression changes, particularly in heterogeneous tissues like tumors.

• Spatial transcriptomics: This could map SPRED1 expression patterns within tissues, providing insights into its context-dependent functions.

• Clonal evolution tracking: In drug resistance studies, single-cell approaches could track the evolution of SPRED1-deficient clones under treatment pressure.

• Protein-protein interaction networks: Single-cell proteomics might uncover cell type-specific interaction partners of SPRED1, leading to a more nuanced understanding of its function.