Recombinant Human Uncharacterized protein C10orf71 (C10orf71), partial

What is C10orf71 and what is its significance in cardiac research?

C10orf71 is a recently characterized gene located on chromosome 10q11.23 that encodes an intrinsically disordered protein specifically expressed in cardiomyocytes. Its significance in cardiac research emerged when frameshift variants in this gene were identified as causal factors for dilated cardiomyopathy (DCM) . Previously considered an uncharacterized protein, C10orf71 has now been established as a critical contributor to cardiac function through comprehensive genetic and functional studies. The gene exhibits no linkage disequilibrium with surrounding regions, and the closest gene, DRGX (approximately 36.6 Kbp away), has no reported link to DCM .

The protein encoded by C10orf71 demonstrates several characteristic features of intrinsically disordered proteins: it has almost no regular secondary structure, remains in a disordered state throughout its entire length, exhibits high hydrophilicity, and is classified as unstable with an instability index of 65.67 according to the ProtParam database . This discovery represents a significant advancement in our understanding of the genetic landscape of DCM, as it adds to the repertoire of known DCM-associated genes and provides new insights into disease mechanisms.

What is the expression pattern of C10orf71 in normal tissues?

C10orf71 demonstrates a highly specific expression pattern that is predominantly restricted to cardiac and skeletal muscle in humans, as evidenced by GTEx (V8) data . In mice, the homologous gene (3425401B19Rik, referred to as mC10orf71) exhibits a similar tissue-specific expression profile, further confirming the conserved nature of this expression pattern across species .

The temporal expression of mC10orf71 in murine cardiac development follows a progressive increase during embryonic stages. Analysis of serial embryonic cardiac structures at four in utero stages (E10.5, E13.5, E16.5, and E19.5) and cardiac tissue at 3 days after birth revealed that mC10orf71 expression increases significantly during heart development, mirroring the expression pattern of the established cardiac marker Tnnt2 . This expression pattern is also observed during myogenic differentiation, suggesting that C10orf71 plays a role in both the development and maturation of muscle tissue .

At the cellular level, single-nucleus RNA sequencing data of human hearts demonstrates that C10orf71 is specifically expressed in cardiomyocytes, with even higher specificity than well-established cardiomyocyte markers such as TNNT2 and MYH7 . Further single-cell sequencing analysis has revealed that C10orf71 is predominantly expressed in ventricular cardiomyocytes . Immunofluorescence studies in mouse cardiomyocytes have shown that mC10orf71 colocalizes with markers of the Z-disc (Actn2) and myofibers (Actc1), a finding that was further validated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) .

How were the frameshift variants in C10orf71 initially discovered?

The discovery of frameshift variants in C10orf71 followed a methodical genetic investigation initially centered on a large family with eight patients affected by dilated cardiomyopathy (DCM). Using whole-exome sequencing (WES), researchers examined the proband (diagnosed at age 12 with moderately diminished left ventricular systolic function), four affected family members (father, two paternal uncles, and a paternal aunt), and two unaffected members (mother and a paternal aunt) . The initial variant segregation analysis, performed in 2018 by GeneDx, did not identify pathogenic variants in known DCM causal or candidate genes, prompting further investigation .

The breakthrough came with the identification of a C10orf71 frameshift variant (NM_001135196.1: c.1057_1072del; p.(D353YfsTer41)) present in all affected family members but absent in unaffected members. The genotypes were confirmed with Sanger sequencing on separate DNA samples from additional family members, resulting in a log odds (LOD) score of 3.0 for the association between genotype and phenotype, with no other functional variant cosegregating with DCM in this family .

Following this discovery, researchers screened for C10orf71 loss-of-function (LOF) variants in two independent sporadic DCM cohorts of Chinese ancestry: the Beijing Anzhen DCM cohort (220 patients) and the Wuhan Tongji DCM cohort (363 patients, with 91 excluded due to deleterious variants in known DCM genes) . This screening identified three additional frameshift variants (c.1095delT, c.1913delA, and c.348dupC) in the Beijing cohort and one heterogeneous stop gain variant (c.G3053A) in the Wuhan cohort. The three frameshift variants in the Beijing cohort were confirmed to be de novo through parental genotyping . All five identified predicted LOF variants were absent in gnomAD and in over 1,000 in-house control individuals of Chinese descent without evidence of cardiomyopathy .

What molecular mechanisms explain how C10orf71 deficiency leads to dilated cardiomyopathy?

At the functional level, C10orf71-null cardiomyocytes exhibit impaired contractile function while maintaining normal sarcomere structure. This suggests that C10orf71 contributes directly to the contractile apparatus of cardiomyocytes without affecting the structural organization of sarcomeres . The specific mechanisms may involve altered expression and splicing of contractile cardiac genes, as observed in C10orf71-KO mice .

Notably, the contractile defects in C10orf71-KO mice could be rescued with omecamtiv mecarbil, a cardiac myosin activator, further supporting the hypothesis that C10orf71 plays a crucial role in cardiac contractility . This evidence collectively suggests that C10orf71 deficiency leads to DCM primarily through impaired cardiac contractile function, potentially offering therapeutic targets for addressing this specific pathophysiological mechanism.

What are the genotype-phenotype correlations observed in patients with C10orf71 variants?

The genotype-phenotype correlations in patients with C10orf71 variants reveal consistent patterns of cardiac pathology despite some variations in clinical presentation. The following table summarizes the clinical information of the five probands identified with C10orf71 variants across the multigenerational family and two sporadic DCM cohorts:

The clinical data reveals several noteworthy patterns. First, the age of diagnosis varies considerably, from pediatric cases (7-12 years) to adult-onset disease (31-38 years) . This age variation suggests possible influences of modifier genes or environmental factors on disease penetrance and expressivity. Second, all patients demonstrated the hallmark features of DCM: ventricular dilation and reduced ejection fraction, though the severity varied across cases .

A significant finding is the presence of left ventricular noncompaction (LVNC) in the two 7-year-old probands from the Beijing cohort, suggesting a potential developmental component to the disease when C10orf71 variants are present early in life . This correlates with the embryonic cardiac abnormalities observed in C10orf71-KO mice, particularly the ventricular noncompaction seen in approximately half of the KO embryos .

The correlation between genotype and phenotype is further supported by the family study, where the LOD score of 3.0 for the association between the C10orf71 variant and DCM phenotype provides strong evidence for causality . Additionally, all five identified LOF variants were absent in control populations, including gnomAD and over 1,000 in-house controls of Chinese descent without cardiomyopathy, reinforcing the pathogenic nature of these variants .

How do C10orf71 frameshift variants affect protein expression and function?

C10orf71 frameshift variants significantly impact protein expression and function through multiple mechanisms. The direct effects of these variants were confirmed through site-directed mutagenesis experiments in human C10orf71 cDNA subcloned in the expression vector pCMV3 . Compared to wild-type plasmid, the mutant plasmids produced truncated protein products, demonstrating the immediate impact of frameshift mutations on protein structure .

A critical question in understanding the functional consequences of frameshift variants is whether the mutant mRNA undergoes nonsense-mediated decay (NMD), a cellular quality control mechanism that degrades mRNAs containing premature termination codons. Since patient-derived cardiomyocytes were unavailable for direct testing, researchers generated C10orf71-mutant human induced pluripotent stem cells (hiPSCs) to obtain cardiomyocytes with endogenously expressed mutant C10orf71 .

Using two hiPSC lines from healthy individuals (WT1 and WT2), researchers created two C10orf71-mutant hiPSC lines (Mut1: c.173dupA and Mut2: c.172_182del) . Although these introduced mutations differed from those found in patients, they were also frameshift mutations located in the front of the only exon coding for C10orf71 protein, similar to the mutations carried by patients. Quantitative PCR and transcriptome sequencing demonstrated that the level of frameshift variant mRNA was significantly decreased compared to the corresponding wild-type control, suggesting that the frameshift variants acted as functional nulls through NMD-mediated degradation .

Functionally, cardiomyocytes and heart organoids derived from hiPSCs with C10orf71 frameshift variants exhibited contractile defects despite normal electrophysiological activity . This indicates that while the electrical conduction system remains intact, the mechanical function of the heart is compromised by C10orf71 deficiency, consistent with the protein's colocalization with Z-disc (Actn2) and myofiber (Actc1) markers observed in immunofluorescence studies .

These findings collectively demonstrate that C10orf71 frameshift variants result in functional protein nulls through truncation and mRNA degradation, leading to specific contractile defects in cardiomyocytes that ultimately manifest as dilated cardiomyopathy.

What are the optimal methods for generating and validating C10orf71 knockout models?

The optimal methods for generating and validating C10orf71 knockout models involve a comprehensive approach spanning genomic editing, molecular verification, and functional assessment. Based on the research methodologies described in the search results, the following protocol has proven effective:

For generating C10orf71 knockout models, CRISPR/Cas9 technology has been successfully employed to create a mC10orf71-KO mouse model that simulates the functional deletion of the C10orf71 gene caused by loss-of-function variants . This approach involves designing guide RNAs targeting specific regions of the C10orf71 gene, followed by Cas9-mediated genomic editing to create frameshift mutations or large deletions that render the gene non-functional.

Validation of successful knockout requires multi-level confirmation:

• Genomic verification: PCR-based genotyping to confirm the presence of the intended genetic modification, followed by Sanger sequencing to verify the exact nature of the mutation .

• Transcriptional validation: Quantitative PCR (qPCR) to confirm the absence or significant reduction of C10orf71 mRNA expression in the knockout model compared to wild-type controls .

• Protein validation: Western blotting using specific antibodies against C10orf71 to confirm the absence of protein expression in knockout tissues . The research demonstrated this approach using heart tissue from mC10orf71-/- mice and wild-type littermate controls to confirm knockout efficiency at both mRNA and protein levels .

For human cell models, generating C10orf71-mutant human induced pluripotent stem cells (hiPSCs) provides a valuable complementary approach. This can be achieved by introducing frameshift mutations in the C10orf71 gene using CRISPR/Cas9 in established hiPSC lines from healthy individuals . The researchers created two C10orf71-mutant hiPSC lines (Mut1: c.173dupA and Mut2: c.172_182del) from two wild-type hiPSC lines (WT1 and WT2) . These hiPSC models can then be differentiated into cardiomyocytes to study the effects of C10orf71 deficiency in a human cellular context.

Functional validation should assess the phenotypic consequences of C10orf71 knockout, including:

What techniques are most effective for studying C10orf71's role in cardiac contractility?

Studying C10orf71's role in cardiac contractility requires a multi-faceted approach combining molecular, cellular, and physiological techniques. Based on the methodologies described in the search results, the following techniques have proven most effective:

• Cardiac-specific gene expression analysis: Quantitative PCR (qPCR) and transcriptome sequencing provide insights into the expression patterns of C10orf71 during cardiac development and in adult cardiomyocytes. The research demonstrated that C10orf71 expression increases significantly during heart development, similar to established cardiac markers like Tnnt2 , suggesting its importance in cardiac maturation and function.

• Single-cell and single-nucleus RNA sequencing: These techniques have revealed the cell-type specificity of C10orf71 expression, demonstrating its predominant expression in cardiomyocytes, particularly ventricular cardiomyocytes . This high-resolution expression data helps establish the context in which C10orf71 functions within the heart.

• Protein localization studies: Immunofluorescence analysis using specific antibodies against C10orf71 and established markers of cardiac structures (e.g., Z-disc marker Actn2 and myofiber marker Actc1) can reveal the subcellular localization of C10orf71 within cardiomyocytes . The colocalization of mC10orf71 with these structural markers in mouse cardiomyocytes and hiPSC-derived cardiomyocytes provides insights into its potential functional roles .

• Functional assays in knockout models: Assessing cardiac function in C10orf71-knockout mice using echocardiography allows for the evaluation of various parameters including left ventricular internal diameter at end-systole (LVID s) and end-diastole (LVID d), as well as left ventricular ejection fraction (LVEF) . These measurements directly quantify the impact of C10orf71 deficiency on cardiac contractile function.

• Isolated cardiomyocyte contractility measurements: Direct assessment of contractile properties in cardiomyocytes isolated from C10orf71-knockout mice or derived from C10orf71-mutant hiPSCs provides cellular-level insights into contractile defects . The research demonstrated that C10orf71-null cardiomyocytes exhibited impaired contractile function despite having normal sarcomere structure .

• Heart organoid models: Generating heart organoids from hiPSCs with C10orf71 frameshift variants offers a three-dimensional tissue-level system to study contractile function . These organoids can be assessed for contractile activity using video microscopy and quantitative analysis of beating patterns.

• Pharmacological rescue experiments: Testing the effects of cardiac contractility modulators, such as the cardiac myosin activator omecamtiv mecarbil, on C10orf71-deficient models provides insights into the specific contractile pathways affected . The successful rescue of contractile function in C10orf71-KO mice with omecamtiv mecarbil suggests that C10orf71 deficiency primarily affects cardiac myosin function .

How can researchers effectively characterize intrinsically disordered proteins like C10orf71?

Characterizing intrinsically disordered proteins (IDPs) like C10orf71 presents unique challenges due to their lack of stable three-dimensional structure and dynamic conformational properties. Based on the methodologies employed in the C10orf71 research, the following approaches are effective for IDP characterization:

What are the potential therapeutic implications of C10orf71 research for dilated cardiomyopathy patients?

The discovery of C10orf71 as a causal gene for dilated cardiomyopathy (DCM) opens several promising therapeutic avenues for patients with C10orf71 mutations. These therapeutic implications span from immediate clinical applications to long-term research directions:

• Targeted pharmacological interventions: The research demonstrated that omecamtiv mecarbil, a cardiac myosin activator, successfully restored contractile function in C10orf71-KO mice . This finding suggests that patients with C10orf71 mutations might specifically benefit from myosin-targeting therapies. Omecamtiv mecarbil has undergone clinical trials for heart failure, making it a potentially available treatment option for DCM patients with C10orf71 mutations . This represents a significant advancement toward mutation-specific, precision medicine approaches in cardiomyopathy treatment.

• Genetic diagnosis and risk stratification: The identification of C10orf71 as a DCM-causing gene expands the panel of genes that should be screened in genetic testing for cardiomyopathy patients . Including C10orf71 in genetic screening panels would improve diagnostic yield and provide more accurate genetic diagnoses for patients with DCM. Early identification of individuals with C10orf71 mutations, particularly within affected families, could enable proactive monitoring and intervention before the development of severe cardiac dysfunction.

• Gene therapy approaches: The specific expression of C10orf71 in cardiomyocytes and its relatively simple genetic structure (containing only one exon) make it a potentially suitable candidate for gene replacement therapies . Adeno-associated virus (AAV)-mediated delivery of functional C10orf71 to cardiomyocytes could theoretically restore protein expression and function in patients with loss-of-function mutations.

• mRNA stabilization therapies: For patients with frameshift mutations that lead to nonsense-mediated mRNA decay, therapies aimed at stabilizing mutant mRNA or promoting read-through of premature termination codons could be explored . Compounds that suppress nonsense-mediated decay or promote translational read-through of premature stop codons have shown promise in other genetic diseases and could potentially be applied to C10orf71-related DCM.

• Contractility-enhancing therapies: Given that C10orf71 deficiency primarily affects cardiac contractile function rather than sarcomere structure, various contractility-enhancing agents beyond omecamtiv mecarbil could be investigated as potential therapies . These might include other myosin activators, calcium sensitizers, or compounds that modulate the regulatory proteins involved in cardiomyocyte contraction.

• Regenerative approaches: The findings regarding C10orf71's role in cardiomyocyte proliferation during development suggest potential applications in cardiac regeneration strategies . Modulating C10orf71 expression or function might enhance cardiomyocyte proliferation in adult hearts following injury, potentially promoting cardiac repair in DCM patients.

The successful rescue of cardiac function in C10orf71-KO mice with omecamtiv mecarbil represents a proof-of-concept for mutation-specific therapy in C10orf71-related DCM . This targeted approach aligns with the emerging paradigm of precision medicine in cardiology, where treatment strategies are tailored to the specific genetic and molecular causes of disease rather than solely addressing symptoms or general pathophysiological processes.

How might understanding C10orf71 function advance personalized medicine approaches for cardiomyopathies?

Understanding C10orf71 function significantly advances personalized medicine approaches for cardiomyopathies through several interconnected mechanisms that bridge basic science discoveries with clinical applications:

• Genotype-directed therapeutic selection: The identification of C10orf71 as a dilated cardiomyopathy (DCM) causal gene with a specific pathophysiological mechanism—impaired contractile function without affecting sarcomere structure—allows for more precise therapeutic targeting . Patients with C10orf71 mutations could specifically benefit from contractility-enhancing agents, as demonstrated by the successful rescue of contractile function in C10orf71-KO mice with omecamtiv mecarbil . This represents a shift from the current one-size-fits-all approach to heart failure treatment toward genotype-specific interventions.

• Predictive phenotyping: The research has begun to establish genotype-phenotype correlations for C10orf71 variants, including variations in age of onset (from childhood to adulthood) and the presence of specific cardiac features such as left ventricular noncompaction in some patients . As more patients with C10orf71 variants are identified and characterized, clinicians will be able to predict disease course, severity, and specific complications more accurately, enabling personalized monitoring and intervention strategies.

• Risk stratification and preventive interventions: The identification of C10orf71 as a DCM causal gene enables cascade genetic testing in families with identified mutations . Relatives found to carry pathogenic C10orf71 variants can be enrolled in preventive monitoring programs before clinical manifestations appear. The varying age of onset observed in C10orf71-related DCM (7 to 38 years in the reported cases) underscores the importance of long-term monitoring for mutation carriers .

• Biomarker development: The characterization of C10orf71's expression, localization, and function in cardiomyocytes opens possibilities for developing specific biomarkers that reflect C10orf71 activity or its downstream effects . Such biomarkers could be used to monitor disease progression, predict outcomes, and assess treatment responses in patients with C10orf71 mutations.

• Drug response prediction: The specific molecular mechanism of C10orf71-related DCM—impaired contractile function—provides a rational basis for predicting which pharmacological agents might be most effective in these patients . Beyond omecamtiv mecarbil, other drugs that enhance cardiac contractility through various mechanisms could be systematically evaluated for their efficacy in C10orf71-deficient models, leading to personalized pharmacotherapy recommendations.

• Gene therapy customization: The identification of specific frameshift variants in C10orf71 enables the design of personalized gene therapy approaches targeting these specific mutations . Whether through gene replacement, mRNA stabilization, or gene editing strategies, therapeutic interventions can be tailored to the specific genetic defect in each patient.

• iPSC-based personalized medicine: The successful generation of C10orf71-mutant human induced pluripotent stem cells (hiPSCs) and their differentiation into cardiomyocytes demonstrates the feasibility of creating patient-specific cellular models to test drug responses in vitro . This "clinical trial in a dish" approach could eventually be applied to individual patients with C10orf71 mutations to determine optimal therapeutic strategies before administering treatments.

The evidence that a cardiac myosin activator can rescue the phenotype in C10orf71-deficient models exemplifies the potential of personalized medicine in DCM . It suggests that understanding the specific pathophysiological mechanism associated with each genetic cause of cardiomyopathy can lead to targeted, mechanism-based treatments rather than generic symptomatic management.