Non-coding Mutations Research Articles

Coding and noncoding somatic mutations in candidate genes in basal cell carcinoma

Basal cell carcinoma (BCC) represents the most commonly diagnosed human cancer among persons of European ancestry with etiology mainly attributed to sun-exposure. In this study we investigated mutations in coding and flanking regions of PTCH1 and TP53 and noncoding alterations in the TERT and DPH3 promoters in 191 BCC tumors. In addition, we measured CpG methylation within the TERT hypermethylated oncological region (THOR) and transcription levels of the reverse transcriptase subunit. We observed mutations in PTCH1 in 58.6% and TP53 in 31.4% of the tumors. Noncoding mutations in TERT and DPH3 promoters were detected in 59.2% and 38.2% of the tumors, respectively. We observed a statistically significant co-occurrence of mutations at the four investigated loci. While PTCH1 mutations tended to associate with decreased patient age at diagnosis; TP53 mutations were associated with light skin color and increased number of nevi; TERT and DPH3 promoter with history of cutaneous neoplasms in BCC patients. Increased reverse transcriptase subunit expression was observed in tumors with TERT promoter mutations and not with THOR methylation. Our study signifies, in addition to the protein altering mutations in the PTCH1 and TP53 genes, the importance of noncoding mutations in BCC, particularly functional alterations in the TERT promoter.

Whole-genome sequencing of 508 patients identifies key molecular features associated with poor prognosis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a poor-prognosis cancer type with limited understanding of its molecular etiology. Using 508 ESCC genomes, we identified five novel significantly mutated genes and uncovered mutational signature clusters associated with metastasis and patients’ outcomes. Several functional assays implicated that NFE2L2 may act as a tumor suppressor in ESCC and that mutations in NFE2L2 probably impaired its tumor-suppressive function, or even conferred oncogenic activities. Additionally, we found that the NFE2L2 mutations were significantly associated with worse prognosis of ESCC. We also identified potential noncoding driver mutations including hotspot mutations in the promoter region of SLC35E2 that were correlated with worse survival. Approximately 5.9% and 15.2% of patients had high tumor mutation burden or actionable mutations, respectively, and may benefit from immunotherapy or targeted therapies. We found clinically relevant coding and noncoding genomic alterations and revealed three major subtypes that robustly predicted patients’ outcomes. Collectively, we report the largest dataset of genomic profiling of ESCC useful for developing ESCC-specific biomarkers for diagnosis and treatment.

Mutations Upstream of the TBX5 and PITX1 Transcription Factor Genes Are Associated with Feathered Legs in the Domestic Chicken.

Feathered leg is a trait in domestic chickens that has undergone intense selection by fancy breeders. Previous studies have shown that two major loci controlling feathered leg are located on chromosomes 13 and 15. Here, we present genetic evidence for the identification of candidate causal mutations at these loci. This was accomplished by combining classical linkage mapping using an experimental cross segregating for feathered leg and high-resolution identical-by-descent mapping using whole-genome sequence data from 167 samples of chicken with or without feathered legs. The first predicted causal mutation is a single-base change located 25 kb upstream of the gene for the forelimb-specific transcription factor TBX5 on chromosome 15. The second is a 17.7-kb deletion located ∼200 kb upstream of the gene for the hindlimb-specific transcription factor PITX1 on chromosome 13. These mutations are predicted to activate TBX5 and repress PITX1 expression, respectively. The study reveals a remarkable convergence in the evolution of the feathered-leg phenotype in domestic chickens and domestic pigeons, as this phenotype is caused by noncoding mutations upstream of the same two genes. Furthermore, the PITX1 causal variants are large overlapping deletions, 17.7 kb in chicken and 44 kb in pigeons. The results of the present study are consistent with the previously proposed model for pigeon that feathered leg is caused by reduced PITX1 expression and ectopic expression of TBX5 in hindlimb buds resulting in a shift of limb identity from hindlimb to more forelimb-like identity.

Non-coding somatic mutations converge on the PAX8 pathway in ovarian cancer

The functional consequences of somatic non-coding mutations in ovarian cancer (OC) are unknown. To identify regulatory elements (RE) and genes perturbed by acquired non-coding variants, here we establish epigenomic and transcriptomic landscapes of primary OCs using H3K27ac ChIP-seq and RNA-seq, and then integrate these with whole genome sequencing data from 232 OCs. We identify 25 frequently mutated regulatory elements, including an enhancer at 6p22.1 which associates with differential expression of ZSCAN16 (P = 6.6 × 10-4) and ZSCAN12 (P = 0.02). CRISPR/Cas9 knockout of this enhancer induces downregulation of both genes. Globally, there is an enrichment of single nucleotide variants in active binding sites for TEAD4 (P = 6 × 10-11) and its binding partner PAX8 (P = 2×10-10), a known lineage-specific transcription factor in OC. In addition, the collection of cis REs associated with PAX8 comprise the most frequently mutated set of enhancers in OC (P = 0.003). These data indicate that non-coding somatic mutations disrupt the PAX8 transcriptional network during OC development.

The genetics of macrophage activation syndrome.

Macrophage activation syndrome (MAS), or secondary hemophagocytic lymphohistiocytosis (HLH), is a cytokine storm syndrome associated with multi-organ system dysfunction and high mortality rates. Laboratory and clinical features resemble primary HLH, which arises in infancy (1 in 50,000 live births) from homozygous mutations in various genes critical to the perforin-mediated cytolytic pathway employed by NK cells and cytotoxic CD8 T lymphocytes. MAS/secondary HLH is about ten times more common and typically presents beyond infancy extending into adulthood. The genetics of MAS are far less defined than for familial HLH. However, the distinction between familial HLH and MAS/secondary HLH is blurred by the finding of heterozygous perforin-pathway mutations in MAS patients, which may function as hypomorphic or partial dominant-negative alleles and contribute to disease pathogenesis. In addition, mutations in a variety of other pathogenic pathways have been noted in patients with MAS/secondary HLH. Many of these genetically disrupted pathways result in a similar cytokine storm syndrome, and can be broadly categorized as impaired viral control (e.g., SH2P1A), dysregulated inflammasome activity (e.g., NLRC4), other immune defects (e.g., IKBKG), and dysregulated metabolism (e.g., LIPA). Collectively these genetic lesions likely combine with states of chronic inflammation, as seen in various rheumatic diseases (e.g., still disease), with or without identified infections, to result in MAS pathology as explained by the threshold model of disease. This emerging paradigm may ultimately support genetic risk stratification for high-risk chronic and even acute inflammatory disorders. Moving forward, continued whole-exome and -genome sequencing will likely identify novel MAS gene associations, as well as noncoding mutations altering levels of gene expression.

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B.

Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.

A genomic and epigenomic atlas of prostate cancer in Asian populations.

Prostate cancer is the second most common cancer in men worldwide1. Over the past decade, large-scale integrative genomics efforts have enhanced our understanding of this disease by characterizing its genetic and epigenetic landscape in thousands of patients2,3. However, most tumours profiled in these studies were obtained from patients from Western populations. Here we produced and analysed whole-genome, whole-transcriptome and DNA methylation data for 208 pairs of tumour tissue samples and matched healthy control tissue from Chinese patients with primary prostate cancer. Systematic comparison with published data from 2,554 prostate tumours revealed that the genomic alteration signatures in Chinese patients were markedly distinct from those of Western cohorts: specifically, 41% of tumours contained mutations in FOXA1 and 18% each had deletions in ZNF292 and CHD1. Alterations of the genome and epigenome were correlated and were predictive of disease phenotype and progression. Coding and noncoding mutations, as well as epimutations, converged on pathways that are important for prostate cancer, providing insights into this devastating disease. These discoveries underscore the importance of including population context in constructing comprehensive genomic maps for disease.

Evaluation of non-coding region sequence variants and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in individuals of Sri Lankan Sinhalese ethnicity.

Mitochondrial DNA (mtDNA) mutations have been reported to be associated with various diseases, including cancer. The present study investigated the mtDNA non-coding region mutations and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in Sri Lankan Sinhalese women. Mitochondrial macro-haplogroups were determined using PCR-restriction fragment length polymorphism, whereas non-coding region sequences were determined using Sanger sequencing. The sequence of the non-coding region was also used to confirm haplogroup status. Neither the mutations in the non-coding region nor the mitochondrial haplogroups that were reported as risk factors in other populations, were determined to be potential risk factors for sporadic breast cancer in the present study. Furthermore, several novel mutations were identified in the present matched pairs case-controlled study. The M65a haplogroup with an additional mutation at position 16311 (P=0.0771) and mutations at the ori-b site (P=0.05) were considered a weak risk factor and protective factor, respectively, for sporadic breast cancer in Sinhalese women. Previous studies have indicated the use of mtDNA mutations as a biomarker; however, the present study showed that such biomarkers need to be validated for individual ethnic groups before they can be recommended for use in the prediction of disease.

Family-based whole-genome sequencing identifies compound heterozygous protein-coding and noncoding mutations in tetralogy of Fallot.

Tetralogy of Fallot (TOF) is one of most serious cyanotic congenital heart disease (CHD) and the prevalence is estimated to be 1 in 3000 live births worldwide. Though multiple studies have found genetic variants as risk factors for TOF, they could only explain a small fraction of the pathogenesis. Here, we performed whole genome sequencing (WGS) for 6 individuals derived from 2 families to evaluate pathogenic mutations located in both coding and noncoding regions. We characterized the annotated deleterious coding mutations and impaired noncoding mutations in regulatory elements by various data analysis. Additionally, functional assays were conducted to validate function regulatory elements and noncoding mutations. Interestingly, a compound heterozygous pattern with pathogenic coding and noncoding mutations was identified in probands. In proband 1, biallelic mutations (g.139409115A>T, encoding p.Asn685Ile; g.139444949C>A) in NOTCH1 exon and its regulatory element were detected. In vitro experiments revealed that the regulatory element acted as a silencer and the noncoding mutation decreased the expression of NOTCH1. In proband 2, we also found compound heterozygous mutations (g. 216235029C>T, encoding p.Val2281Met; g. 216525154A>C) which potentially regulated the function of FN1 gene. In summary, our study firstly reported an instance of newly identified noncoding mutation in regulatory element within the compound heterozygous pattern in TOF. The results provided a deeper understanding of TOF genetic architectures.

Noncoding Microdeletion in Mouse Hgf Disrupts Neural Crest Migration into the Stria Vascularis, Reduces the Endocochlear Potential, and Suggests the Neuropathology for Human Nonsyndromic Deafness DFNB39.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39 However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Hgf Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness.SIGNIFICANCE STATEMENT Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 bp deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

Novel Mutation Hotspots within Non-Coding Regulatory Regions of the Chronic Lymphocytic Leukemia Genome

Mutations in non-coding DNA regions are increasingly recognized as cancer drivers. These mutations can modify gene expression in cis or by inducing high-order chormatin structure modifications with long-range effects. Previous analysis reported the detection of recurrent and functional non-coding DNA mutations in the chronic lymphocytic leukemia (CLL) genome, such as those in the 3′ untranslated region of NOTCH1 and in the PAX5 super-enhancer. In this report, we used whole genome sequencing data produced by the International Cancer Genome Consortium in order to analyze regions with previously reported regulatory activity. This approach enabled the identification of numerous recurrently mutated regions that were frequently positioned in the proximity of genes involved in immune and oncogenic pathways. By correlating these mutations with expression of their nearest genes, we detected significant transcriptional changes in genes such as PHF2 and S1PR2. More research is needed to clarify the function of these mutations in CLL, particularly those found in intergenic regions.

Pathway and network analysis of more than 2500 whole cancer genomes

The catalog of cancer driver mutations in protein-coding genes has greatly expanded in the past decade. However, non-coding cancer driver mutations are less well-characterized and only a handful of recurrent non-coding mutations, most notably TERT promoter mutations, have been reported. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancer across 38 tumor types, we perform multi-faceted pathway and network analyses of non-coding mutations across 2583 whole cancer genomes from 27 tumor types compiled by the ICGC/TCGA PCAWG project that was motivated by the success of pathway and network analyses in prioritizing rare mutations in protein-coding genes. While few non-coding genomic elements are recurrently mutated in this cohort, we identify 93 genes harboring non-coding mutations that cluster into several modules of interacting proteins. Among these are promoter mutations associated with reduced mRNA expression in TP53, TLE4, and TCF4. We find that biological processes had variable proportions of coding and non-coding mutations, with chromatin remodeling and proliferation pathways altered primarily by coding mutations, while developmental pathways, including Wnt and Notch, altered by both coding and non-coding mutations. RNA splicing is primarily altered by non-coding mutations in this cohort, and samples containing non-coding mutations in well-known RNA splicing factors exhibit similar gene expression signatures as samples with coding mutations in these genes. These analyses contribute a new repertoire of possible cancer genes and mechanisms that are altered by non-coding mutations and offer insights into additional cancer vulnerabilities that can be investigated for potential therapeutic treatments.

Integrative pathway enrichment analysis of multivariate omics data

Multi-omics datasets represent distinct aspects of the central dogma of molecular biology. Such high-dimensional molecular profiles pose challenges to data interpretation and hypothesis generation. ActivePathways is an integrative method that discovers significantly enriched pathways across multiple datasets using statistical data fusion, rationalizes contributing evidence and highlights associated genes. As part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumor types, we integrated genes with coding and non-coding mutations and revealed frequently mutated pathways and additional cancer genes with infrequent mutations. We also analyzed prognostic molecular pathways by integrating genomic and transcriptomic features of 1780 breast cancers and highlighted associations with immune response and anti-apoptotic signaling. Integration of ChIP-seq and RNA-seq data for master regulators of the Hippo pathway across normal human tissues identified processes of tissue regeneration and stem cell regulation. ActivePathways is a versatile method that improves systems-level understanding of cellular organization in health and disease through integration of multiple molecular datasets and pathway annotations.

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1–3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10–18.

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

Identifying cancer-relevant mutations in noncoding regions is challenging due to the large numbers of such mutations, their low levels of recurrence, and difficulties in interpreting their functional impact. To uncover genes that are dysregulated due to somatic mutations in cis, we build upon the concept of differential allele-specific expression (ASE) and introduce methods to identify genes within an individual's cancer whose ASE differs from what is found in matched normal tissue. When applied to breast cancer tumor samples, our methods detect the known allele-specific effects of copy number variation and nonsense-mediated decay. Further, genes that are found to recurrently exhibit differential ASE across samples are cancer relevant. Genes with cis mutations are enriched for differential ASE, and we find 147 potentially functional noncoding mutations cis to genes that exhibit significant differential ASE. We conclude that differential ASE is a promising means for discovering gene dysregulation due to cis noncoding mutations.

Transcription Factors Involved in Tumorigenesis Are Over-Represented in Mutated Active DNA-Binding Sites in Neuroblastoma.

The contribution of coding mutations to oncogenesis has been largely clarified, whereas little is known about somatic mutations in noncoding DNA and their role in driving tumors remains controversial. Here, we used an alternative approach to interpret the functional significance of noncoding somatic mutations in promoting tumorigenesis. Noncoding somatic mutations of 151 neuroblastomas were integrated with ENCODE data to locate somatic mutations in regulatory elements specifically active in neuroblastoma cells, nonspecifically active in neuroblastoma cells, and nonactive. Within these types of elements, transcription factors (TF) were identified whose binding sites were enriched or depleted in mutations. For these TFs, a gene expression signature was built to assess their implication in neuroblastoma. DNA- and RNA-sequencing data were integrated to assess the effects of those mutations on mRNA levels. The pathogenicity of mutations was significantly higher in transcription factor binding site (TFBS) of regulatory elements specifically active in neuroblastoma cells, as compared with the others. Within these elements, there were 18 over-represented TFs involved mainly in cell-cycle phase transitions and 15 under-represented TFs primarily regulating cell differentiation. A gene expression signature based on over-represented TFs correlated with poor survival and unfavorable prognostic markers. Moreover, recurrent mutations in TFBS of over-represented TFs such as EZH2 affected MCF2L and ADP-ribosylhydrolase like 1 expression, among the others. We propose a novel approach to study the involvement of regulatory variants in neuroblastoma that could be extended to other cancers and provide further evidence that alterations of gene expression may have relevant effects in neuroblastoma development. SIGNIFICANCE: These findings propose a novel approach to study regulatory variants in neuroblastoma and suggest that noncoding somatic mutations have relevant implications in neuroblastoma development.

Enhancer Domains Predict Gene Pathogenicity and Inform Gene Discovery in Complex Disease

Non-coding transcriptional regulatory elements are critical for controlling the spatiotemporal expression of genes. Here, we demonstrate that the sizes and number of enhancers linked to a gene reflect its disease pathogenicity. Moreover, genes with redundant enhancer domains are depleted of cis-acting genetic variants that disrupt gene expression, and they are buffered against the effects of disruptive non-coding mutations. Our results demonstrate that dosage-sensitive genes have evolved a robustness to the disruptive effects of genetic variation by expanding their regulatory domains. This solves a puzzle about why genes associated with human disease are depleted of cis-eQTLs (cis-expression quantitative trait loci), suggesting that this relationship might complicate gene identification in causal genome-wide association studies (GWASs) using eQTL information, and establishes a framework for identifying non-coding regulatory variation with phenotypic consequences.

The transcription factor FLI1 promotes cancer progression by affecting cell cycle regulation.

Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects. One of them, friend leukemia integration 1 (FLI1), which belongs to the E26 transforming-specific family of transcription factors, exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumor suppressor gene's activity via opposing regulation processes. FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumors. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is downregulated in lung tumors. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner.

Beyond the coding genome: non-coding mutations and cancer.

Latest advancements in genomics involving individuals from different races and geographical locations has led to the identification of thousands of common as well as rare genetic variants and copy number variations (CNVs). These studies have surprisingly revealed that the majority of genetic variation is not present within the coding region but rather in the non-coding region of the genome, which is also termed as "Medical Genome". This short review describes how mutations/variations within; regulatory sequences, architectural proteins and transcriptional regulators give rise to the aberrant gene expression profiles that drives cellular transformations and malignancies.

Familial leukemia due to germline RUNX1 mutations: lessons learned from two decades of research and unsolved problems

The RUNX1 gene is a critical transcription factor for the generation and maintenance of hematopoietic stem cells. RUNX1 is also one of the most frequently mutated gene in sporadic leukemias. Heterozygous loss-of-function mutations of the RUNX1 gene in the germline cause a rare autosomal dominant disorder called familial platelet disorder with propensity to acute myelogenous leukemia (FPD/AML). Besides the preexisting platelet disorder in FPD/AML patients, AML also develops in 20-60% of affected individuals. Since its discovery by the Gilliland group in 1999, RUNX1 mutation in the germline has been extensively investigated in the field. The past two decades of research have taught us three important lessons: 1) patients with FPD/AML display atypical symptoms and they have a widened clinical spectrum of FPD, such as eczema and syndromic thrombocytopenia, 2) the elucidation of variant of uncertain significance (VUS) of RUNX1 have revealed their role in epigenetic functions and involvement in the Fanconi anemia DNA repair pathway, and 3) non-coding mutations of RUNX1 also causes FPD/AML. In three distinct familial cases, an enhancer for RUNX1, eR1, was either lost or disconnected from the promoter through genetic deletion or chromosomal translocation abnormalities. This experience, with congenital mutations of RUNX1, will be very useful for future research for a series of other leukemia-causing germline mutations that have been recently identified.