Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability.

The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.

Late-Onset Autosomal Dominant Macular Degeneration Caused by Deletion of the CRX Gene.

PURPOSE: To characterize the phenotype observed in a case series with macular disease and determine the cause. DESIGN: Multicenter case series. PARTICIPANTS: Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. METHODS: Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. MAIN OUTCOME MEASURES: Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. RESULTS: All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. CONCLUSIONS: Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.

Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients.

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.

Comparison of 2SC, AKR1B10, and FH Antibodies as Potential Biomarkers for FH-deficient Uterine Leiomyomas.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome caused by germline fumarate hydratase (FH) mutations and characterized by uterine and cutaneous leiomyomas and renal cell cancer. Currently, there is no generally approved method to differentiate FH-deficient uterine leiomyomas from other leiomyomas. Here, we analyzed 3 antibodies (S-(2-succino)-cysteine [2SC], aldo-keto reductase family 1, member B10 [AKR1B10], and FH) as potential biomarkers. The study consisted of 2 sample series. The first series included 155 formalin-fixed paraffin-embedded uterine leiomyomas, of which 90 were from HLRCC patients and 65 were sporadic. The second series included 1590 unselected fresh frozen leiomyomas. Twenty-seven tumors were from known HLRCC patients, while the FH status for the remaining 1563 tumors has been determined by copy number analysis and Sanger sequencing revealing 45 tumors with monoallelic (n=33) or biallelic (n=12) FH loss. Altogether 197 samples were included in immunohistochemical analyses: all 155 samples from series 1 and 42 available corresponding formalin-fixed paraffin-embedded samples from series 2 (15 tumors with monoallelic and 7 with biallelic FH loss, 20 with no FH deletion). Results show that 2SC performed best with 100% sensitivity and specificity. Scoring was straightforward with unambiguously positive or negative results. AKR1B10 identified most tumors accurately with 100% sensitivity and 99% specificity. FH was 100% specific but showed slightly reduced 91% sensitivity. Both FH and AKR1B10 displayed also intermediate staining intensities. We suggest that when patient's medical history and/or histopathologic tumor characteristics indicate potential FH-deficiency, the tumor's FH status is determined by 2SC staining. When aberrant staining is observed, the patient can be directed to genetic counseling and mutation screening.

Uterine leiomyomas in hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome can be identified through distinct clinical characteristics and typical morphology.

INTRODUCTION: Hereditary leiomyomatosis and renal cell cancer (HLRCC) constitute a tumor susceptibility syndrome caused by germline mutations in the fumarate hydratase (FH) gene. The most common features are leiomyomas of the uterus and the skin. The syndrome includes a predisposition to early-onset, aggressive renal cell cancer. It is important to identify women with HLRCC among other uterine leiomyoma patients in order to direct them to genetic counseling and to identify other affected family members. MATERIAL AND METHODS: We conducted a nationwide historical study to identify typical clinical characteristics, uterine leiomyoma morphology, and immunohistochemistry for diagnosing HLRCC. The study included 20 women with a known FH germline mutation and 77 women with sporadic uterine leiomyomas. The patient records of all women were reviewed to obtain clinical details regarding their leiomyomas. Uterine leiomyoma tissue specimens from 43 HLRCC-related leiomyomas and 42 sporadic leiomyomas were collected and prepared for histology analysis. A morphologic description was performed on hematoxylin & eosin-stained tissue slides, and immunohistochemical analysis was carried out for CD34, Bcl-2, and p53 stainings. RESULTS: The women with HLRCC were diagnosed with uterine leiomyomas at a young age compared with the sporadic leiomyoma group (mean 33.8 years vs. 45.4 years, P < 0.0001), and their leiomyomas occurred as multiples compared with the sporadic leiomyoma group (more than four tumors 88.9% vs. 30.8%, P < 0.0001). Congruently, these women underwent surgical treatment at younger age compared with the sporadic leiomyoma group (mean 37.3 years vs. 48.3 years, P < 0.0001). HLRCC leiomyomas had denser microvasculature highlighted by CD34 immunostaining when compared with the sporadic leiomyoma group (112.6 mean count/high-power field, SD 20.8 vs. 37.4 mean count/high-power field, SD 21.0 P < 0.0001) and stronger anti-apoptotic protein Bcl-2 immunostaining when compared with the sporadic leiomyoma group (weak 4.7%, moderate 44.2%, strong 51.2% vs. 26.2%, 52.4%, 21.4%, respectively, P = 0.003). No differences were observed in p53 staining. CONCLUSIONS: Women with HLRCC may be identified through the distinct clinical characteristics: symptomatic and numerous leioymyomas at young age, and morphologic features of FH-mutant leiomyomas, aided by Bcl-2 and CD34 immunohistochemistry. Further, distinguishing individuals with a germline FH mutation enables proper genetic counseling and regular renal monitoring.

Characterization of MED12, HMGA2, and FH alterations reveals molecular variability in uterine smooth muscle tumors.

Uterine smooth muscle tumors range from benign leiomyomas to malignant leiomyosarcomas. Based on numerous molecular studies, leiomyomas and leiomyosarcomas mostly lack shared mutations and the majority of tumors are believed to develop through distinct mechanisms. To further characterize the molecular variability among uterine smooth muscle tumors, and simultaneously insinuate their potential malignant progression, we examined the frequency of known genetic leiomyoma driver alterations (MED12 mutations, HMGA2 overexpression, biallelic FH inactivation) in 65 conventional leiomyomas, 94 histopathological leiomyoma variants (18 leiomyomas with bizarre nuclei, 22 cellular, 29 highly cellular, and 25 mitotically active leiomyomas), and 51 leiomyosarcomas. Of the 210 tumors analyzed, 107 had mutations in one of the three driver genes. No tumor had more than one mutation confirming that all alterations are mutually exclusive. MED12 mutations were the most common alterations in conventional and mitotically active leiomyomas and leiomyosarcomas, while leiomyomas with bizarre nuclei were most often FH deficient and cellular tumors showed frequent HMGA2 overexpression. Highly cellular leiomyomas displayed the least amount of alterations leaving the majority of tumors with no known driver aberration. Our results indicate that based on the molecular background, histopathological leiomyoma subtypes do not only differ from conventional leiomyomas, but also from each other. The presence of leiomyoma driver alterations in nearly one third of leiomyosarcomas suggests that some tumors arise through leiomyoma precursor lesion or that these mutations provide growth advantage also to highly aggressive cancers. It is clinically relevant to understand the molecular background of various smooth muscle tumor subtypes, as it may lead to improved diagnosis and personalized treatments in the future.

Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry.

MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5' end nonsense mutation (c.97G>T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin-α and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis. METHODS: MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA). RESULTS: Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours. CONCLUSIONS: Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers.

Uterine leiomyomas are common benign smooth muscle tumors that impose a major burden on women's health. Recent sequencing studies have revealed recurrent and mutually exclusive mutations in leiomyomas, suggesting the involvement of molecularly distinct pathways. In this study, we explored transcriptional differences among leiomyomas harboring different genetic drivers, including high mobility group AT-hook 2 (HMGA2) rearrangements, mediator complex subunit 12 (MED12) mutations, biallelic inactivation of fumarate hydratase (FH), and collagen, type IV, alpha 5 and collagen, type IV, alpha 6 (COL4A5-COL4A6) deletions. We also explored the transcriptional consequences of 7q22, 22q, and 1p deletions, aiming to identify possible target genes. We investigated 94 leiomyomas and 60 corresponding myometrial tissues using exon arrays, whole genome sequencing, and SNP arrays. This integrative approach revealed subtype-specific expression changes in key driver pathways, including Wnt/ß-catenin, Prolactin, and insulin-like growth factor (IGF)1 signaling. Leiomyomas with HMGA2 aberrations displayed highly significant up-regulation of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1), suggesting that HMGA2 promotes tumorigenesis through PLAG1 activation. This was supported by the identification of genetic PLAG1 alterations resulting in expression signatures as seen in leiomyomas with HMGA2 aberrations. RAD51 paralog B (RAD51B), the preferential translocation partner of HMGA2, was up-regulated in MED12 mutant lesions, suggesting a role for this gene in the genesis of leiomyomas. FH-deficient leiomyomas were uniquely characterized by activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes, supporting the hypothesis that accumulation of fumarate leads to activation of the oncogenic transcription factor NRF2. This study emphasizes the need for molecular stratification in leiomyoma research and possibly in clinical practice as well. Further research is needed to determine whether the candidate biomarkers presented herein can provide guidance for managing the millions of patients affected by these lesions.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms.

BACKGROUND: Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. METHODS: To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. RESULTS: In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. CONCLUSIONS: Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Somatic MED12 mutations are associated with poor prognosis markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. We performed systematic database search and identified highly specific MED12 mutations in CLL patients. To study this further, we collected three independent sample series comprising over 700 CLL samples and screened MED12 exons 1 and 2 by direct sequencing. Mutations were identified at significant frequency in all three series with a combined mutation frequency of 5.2% (37/709). Positive mutation status was found to be associated with unmutated IGHV and ZAP70 expression, which are well-known poor prognosis markers in CLL. Our results recognize CLL as the first extrauterine cancer type where 5'terminus of MED12 is mutated at significant frequency. Functional analyses have shown that these mutations lead to dissociation of Cyclin C-CDK8/19 from the core Mediator and to the loss of Mediator-associated CDK kinase activity. Additional studies on the role of MED12 mutation status as a putative prognostic factor as well as mutations' exact tumorigenic mechanism in CLL are warranted.

MED12 mutation frequency in unselected sporadic uterine leiomyomas.

OBJECTIVE: To determine the frequency of mediator complex subunit 12 (MED12) mutations in well-documented, prospectively collected, unselected series of sporadic uterine leiomyomas to better understand the contribution of MED12 mutations in leiomyoma genesis. DESIGN: Mutation analysis of two prospectively collected sample series. SETTING: Department of gynecology in university hospital and medical genetics research laboratory. PATIENT(S): 164 uterine leiomyomas from 28 patients (13 consecutive and 15 unselected patients) undergoing hysterectomy. INTERVENTION(S): MED12 mutation screening by direct sequencing, and clinical data collection. MAIN OUTCOME MEASURE(S): MED12 mutation status and various clinical variables. RESULT(S): MED12 mutations were found in 73 (83.0%) of 88 and 65 (85.5%) of 76 of uterine leiomyomas from the consecutive and unselected patient series, respectively. Smaller tumor size and a larger number of tumors correlated with positive MED12 mutation status. CONCLUSION(S): The frequency of MED12 mutations in our prospectively collected uterine leiomyoma sets was higher than in previous works. This is in keeping with the concept that MED12 mutation-positive tumors tend to be smaller in size than MED12 mutation-negative tumors. The results highlight the central role of MED12 mutations in uterine leiomyoma genesis.

Mutations in Exon 1 highlight the role of MED12 in uterine leiomyomas.

Mediator regulates transcription by connecting gene-specific transcription factors to the RNA polymerase II initiation complex. We recently discovered by exome sequencing that specific exon 2 mutations in mediator complex subunit 12 (MED12) are extremely common in uterine leiomyomas. Subsequent screening studies have focused on this mutational hot spot, and mutations have been detected in uterine leiomyosarcomas, extrauterine leiomyomas and leiomyosarcomas, endometrial polyps, and colorectal cancers. All mutations have been missense changes or in-frame insertions/deletions. Here, we have analyzed 611 samples representing all above-mentioned tumor types for possible exon 1 mutations. Five mutations were observed, all of which were in-frame insertion/deletions in uterine leiomyomas. Transcriptome-wide expression data revealed that MED12 exon 1 and exon 2 mutations lead to the same unique global gene expression pattern with RAD51B being the most upregulated gene. Immunoprecipitation and kinase activity assays showed that both exon 1 and exon 2 mutations disrupt the interaction between MED12 and Cyclin C and CDK8/19 and abolish the mediator-associated CDK kinase activity. These results further emphasize the role of MED12 in uterine leiomyomas, show that exon 1 and exon 2 exert their tumorigenic effect in similar manner, and stress that exon 1 should be included in subsequent MED12 screenings.

Uterine leiomyoma-linked MED12 mutations disrupt mediator-associated CDK activity.

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.

Characterization of uterine leiomyomas by whole-genome sequencing.

BACKGROUND: Uterine leiomyomas are benign but affect the health of millions of women. A better understanding of the molecular mechanisms involved may provide clues to the prevention and treatment of these lesions. METHODS: We performed whole-genome sequencing and gene-expression profiling of 38 uterine leiomyomas and the corresponding myometrium from 30 women. RESULTS: Identical variants observed in some separate tumor nodules suggested that these nodules have a common origin. Complex chromosomal rearrangements resembling chromothripsis were a common feature of leiomyomas. These rearrangements are best explained by a single event of multiple chromosomal breaks and random reassembly. The rearrangements created tissue-specific changes consistent with a role in the initiation of leiomyoma, such as translocations of the HMGA2 and RAD51B loci and aberrations at the COL4A5-COL4A6 locus, and occurred in the presence of normal TP53 alleles. In some cases, separate events had occurred more than once in single tumor-cell lineages. CONCLUSIONS: Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).

MED12 exon 2 mutations in histopathological uterine leiomyoma variants.

Uterine leiomyomas, or fibroids, are the most common human tumors. Based on histopathology, they can be divided into common leiomyomas and various relatively rare subtypes that mimic malignancy in one or more aspects. Recently, we showed that exon 2 of mediator complex subunit 12 (MED12) is mutated in up to 70% of common fibroids. To investigate the frequency of MED12 exon 2 mutations in histopathological uterine leiomyoma variants, we screened altogether 206 lesions, including 69 histopathologically common leiomyomas, 59 cellular (23 cellular and 36 highly cellular), 18 atypical and 26 mitotically active leiomyomas, as well as 34 uterine fibroid samples from 14 hereditary leiomyomatosis and renal cell cancer patients with a heterozygous germ line mutation in fumarate hydratase (FH). The uterine leiomyoma variants harbored MED12 exon 2 mutations significantly less frequently than common leiomyomas (P=2.93 × 10(-8)). In all, 6 mutations were detected among cellular fibroids (6/67; 8.96%), 3 among atypical fibroids (3/18; 16.67%) and 10 among mitotically active fibroids (10/26; 38.46%). Only mitotically active fibroids displayed a mutation frequency that was not statistically different from common leiomyomas (P=0.11). Three MED12 exon 2 mutations were detected among 34 tumors with a heterozygous germ line FH mutation (P=5.28 × 10(-7)). None of these tumors displayed biallelic inactivation of FH. Our results suggest that MED12 mutation positivity is a key characteristic of common leiomyomas. Cellular and atypical fibroids, in particular, may arise through different molecular mechanisms. The results also propose that MED12 and biallelic FH mutations may be mutually exclusive.

Breast tumors from CHEK2 1100delC-mutation carriers: genomic landscape and clinical implications.

INTRODUCTION: Checkpoint kinase 2 (CHEK2) is a moderate penetrance breast cancer risk gene, whose truncating mutation 1100delC increases the risk about twofold. We investigated gene copy-number aberrations and gene-expression profiles that are typical for breast tumors of CHEK2 1100delC-mutation carriers. METHODS: In total, 126 breast tumor tissue specimens including 32 samples from patients carrying CHEK2 1100delC were studied in array-comparative genomic hybridization (aCGH) and gene-expression (GEX) experiments. After dimensionality reduction with CGHregions R package, CHEK2 1100delC-associated regions in the aCGH data were detected by the Wilcoxon rank-sum test. The linear model was fitted to GEX data with R package limma. Genes whose expression levels were associated with CHEK2 1100delC mutation were detected by the bayesian method. RESULTS: We discovered four lost and three gained CHEK2 1100delC-related loci. These include losses of 1p13.3-31.3, 8p21.1-2, 8p23.1-2, and 17p12-13.1 as well as gains of 12q13.11-3, 16p13.3, and 19p13.3. Twenty-eight genes located on these regions showed differential expression between CHEK2 1100delC and other tumors, nominating them as candidates for CHEK2 1100delC-associated tumor-progression drivers. These included CLCA1 on 1p22 as well as CALCOCO1, SBEM, and LRP1 on 12q13. Altogether, 188 genes were differentially expressed between CHEK2 1100delC and other tumors. Of these, 144 had elevated and 44, reduced expression levels.Our results suggest the WNT pathway as a driver of tumorigenesis in breast tumors of CHEK2 1100delC-mutation carriers and a role for the olfactory receptor protein family in cancer progression. Differences in the expression of the 188 CHEK2 1100delC-associated genes divided breast tumor samples from three independent datasets into two groups that differed in their relapse-free survival time. CONCLUSIONS: We have shown that copy-number aberrations of certain genomic regions are associated with CHEK2 mutation 1100delC. On these regions, we identified potential drivers of CHEK2 1100delC-associated tumorigenesis, whose role in cancer progression is worth investigating. Furthermore, poorer survival related to the CHEK2 1100delC gene-expression signature highlights pathways that are likely to have a role in the development of metastatic disease in carriers of the CHEK2 1100delC mutation.

Five polymorphisms and breast cancer risk: results from the Breast Cancer Association Consortium.

Previous studies have suggested that minor alleles for ERCC4 rs744154, TNF rs361525, CASP10 rs13010627, PGR rs1042838, and BID rs8190315 may influence breast cancer risk, but the evidence is inconclusive due to their small sample size. These polymorphisms were genotyped in more than 30,000 breast cancer cases and 30,000 controls, primarily of European descent, from 30 studies in the Breast Cancer Association Consortium. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) as a measure of association. We found that the minor alleles for these polymorphisms were not related to invasive breast cancer risk overall in women of European descent: ECCR4 per-allele OR (95% CI) = 0.99 (0.97-1.02), minor allele frequency = 27.5%; TNF 1.00 (0.95-1.06), 5.0%; CASP10 1.02 (0.98-1.07), 6.5%; PGR 1.02 (0.99-1.06), 15.3%; and BID 0.98 (0.86-1.12), 1.7%. However, we observed significant between-study heterogeneity for associations with risk for single-nucleotide polymorphisms (SNP) in CASP10, PGR, and BID. Estimates were imprecise for women of Asian and African descent due to small numbers and lower minor allele frequencies (with the exception of BID SNP). The ORs for each copy of the minor allele were not significantly different by estrogen or progesterone receptor status, nor were any significant interactions found between the polymorphisms and age or family history of breast cancer. In conclusion, our data provide persuasive evidence against an overall association between invasive breast cancer risk and ERCC4 rs744154, TNF rs361525, CASP10 rs13010627, PGR rs1042838, and BID rs8190315 genotypes among women of European descent.

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.