KMT2D Variants Research Articles

Investigation of genetic and phenotypic heterogeneity in 37 Turkish patients with Kabuki and Kabuki-like phenotype.

Kabuki syndrome (KS) is a rare disorder characterized by distinct face, persistent fingertip pads, and intellectual disability (ID) caused by mutation in KMT2D (56%-76%) or KDM6A (5%-8%). Thirty-seven children aged 1-16 years who followed for median of 6.8 years were included in this study, which aimed to investigate the genetic and clinical characteristics of KS patients. KMT2D and KDM6A were evaluated by sequencing and multiplex-ligation-dependent probe amplification in 32 patients. Twenty-one pathogenic variants in KMT2D, of which 17 were truncated and nine were novel, one frame-shift novel variant in KDM6A were identified. The molecular diagnosis rate was 68.7% (22/32). In the whole-exome sequencing analysis performed in the remaining patients, no pathogenic variant that could cause any disease was detected. All patients had ID; 43.2% were severe and moderate. We observed that facial features that became more prominent with age were enough for a possible diagnosis of KS in infancy. The frequencies of facial features, cardiac and renal anomalies, short stature, microcephaly, and epilepsy did not differ depending on whether they had truncating or nontruncating variants or were in variant-negative KS-like group. This study has expanded clinical features of the disease, as well as identified new variants in genes causing KS.

Sleep disturbance is a common feature of Kabuki syndrome.

Kabuki syndrome (KS) is a rare epigenetic disorder caused by heterozygous loss of function variants in either KMT2D (90%) or KDM6A (10%), both involved in regulation of histone methylation. While sleep disturbance in other Mendelian disorders of the epigenetic machinery has been reported, no study has been conducted on sleep in KS. This study assessed sleep in 59 participants with KS using a validated sleep questionnaire. Participants ranged in age from 4 to 43 years old with 86% of participants having a pathogenic variant in KMT2D. In addition, data on adaptive function, behavior, anxiety, and quality of life were collected using their respective questionnaires. Some form of sleep issue was present in 71% of participants, with night-waking, daytime sleepiness, and sleep onset delay being the most prevalent. Sleep dysfunction was positively correlated with maladaptive behaviors, anxiety levels, and decreasing quality of life. Sleep issues were not correlated with adaptive function. This study establishes sleep disturbance as a common feature of KS. Quantitative sleep measures may be a useful outcome measure for clinical trials in KS. Further, clinicians caring for those with KS should consider sleep dysfunction as an important feature that impacts overall health and well being in these patients.

Molecular characterization of an embryonal rhabdomyosarcoma occurring in a patient with Kabuki syndrome: report and literature review in the light of tumor predisposition syndromes

Kabuki syndrome is a well-recognized syndrome characterized by facial dysmorphism and developmental delay/intellectual disability and in the majority of patients a germline variant in KMT2D is found. As somatic KMT2D variants can be found in 5–10% of tumors a tumor predisposition in Kabuki syndrome is discussed. So far less than 20 patients with Kabuki syndrome and a concomitant malignancy have been published. Here we report on a female patient with Kabuki syndrome and a c.2558_2559delCT germline variant in KMT2D who developed an embryonal rhabdomyosarcoma (ERMS) at 10 years. On tumor tissue we performed DNA-methylation profiling and exome sequencing (ES). Copy number analyses revealed aneuploidies typical for ERMS including (partial) gains of chromosomes 2, 3, 7, 8, 12, 15, and 20 and 3 focal deletions of chromosome 11p. DNA methylation profiling mapped the case to ERMS by a DNA methylation-based sarcoma classifier. Sequencing suggested gain of the wild-type KMT2D allele in the trisomy 12. Including our patient literature review identified 18 patients with Kabuki syndrome and a malignancy. Overall, the landscape of malignancies in patients with Kabuki syndrome was reminiscent of that of the pediatric population in general. Histopathological and molecular data were only infrequently reported and no report included next generation sequencing and/or DNA-methylation profiling. Although we found no strong arguments pointing towards KS as a tumor predisposition syndrome, based on the small numbers any relation cannot be fully excluded. Further planned studies including profiling of additional tumors and long term follow-up of KS-patients into adulthood could provide further insights.

Whole-genome sequencing reveals de-novo mutations associated with nonsyndromic cleft lip/palate

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact protein-altering DNMs that contribute to the risk of nsCL/P, we conducted whole-genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

Abstract 82: Molecular characterization of gallbladder cancer

Abstract Introduction: Gallbladder carcinomas (GBC) are rare and aggressive neoplasms. Previous studies in small GBC cohorts have suggested potential molecular alterations associated with poor prognosis, however, a comprehensive understanding of the recurrent genetic events in this cancer type is very limited. In this study, we aim to characterize in detail the recurrent molecular alterations in GBC and their association with pathologic and clinical characteristics. Material and Methods: We studied the prevalence of somatic mutations and copy number alterations (CNA) in n=244 GBC samples (54% primary, 56% metastatic), collected from 2014 to 2021, and sequenced with the targeted NGS panel MSK-IMPACT. We assessed the correlation of the recurrent genomic variants with several pathologic (e.g., T stage, N stage, grade, histologic subtypes) and clinical characteristics (e.g., clinical stage) using Fisher's exact test. We also used Cox Proportional-Hazards modeling to assess the correlation between genetic variants and patient overall survival (OS). Results: The most common histologic subtypes in this GCB cohort included adenocarcinomas NOS (83%), carcinomas with squamous differentiation (9%), high-grade carcinomas (4%), and carcinomas with neuroendocrine differentiation (3%). Most patients were diagnosed with stage IV (65%) and stage III (11%) disease at the time of biopsy/resection, and the mean OS survival was 29.4 months. The most commonly mutated genes were TP53 (59%), SMAD4 (21%), ARID1A (19%), PIK3CA (10%), KRAS (7%), and ERBB2 (7%). Potentially recurrent oncogenic CNAs included deep deletions in CDKN2A (14%) and CDKN2B (14%), and amplifications in MDM2 (12%), ERBB2 (10%), CCNE1 (9%), MYC (7%), and KRAS (7%). RB1, PBRM1, and CTNNB1 variants were more common in cases with neuroendocrine differentiation, whereas alterations in IKZF1 and AGO2 were enriched in cases with squamous differentiation. The most significant event associated with shorted OS was chromosome 12q13-15 amplification i.e., CDK4 p=0.03 HR=2 [95% CI 1-3.6] and MDM2 p=0.05 HR=1.6 [1-2.5], which associated with a median OS of 20 months (vs. 34 months in the wild-type). Genomic variants associated with longer OS included ERBB2 (p=0.006 HR=0.2 [0.06-0.6]), KMT2C (p=0.03 HR=0.2 [0.1-0.9]) and KMT2D (p=0.01 HR=0.2 [0.04-0.7]) mutations, and CDK12 amplification (p=0.04 HR=0.4 [0.2-0.96]). Although ERBB2 amplification was not associated with prognosis, co-amplification of CDK12 and ERBB2 (chromosome 17q12) was frequently observed (Pearson correlation r=0.8). Conclusions: This large-scale genomic analysis reveals recurrent genomic events potentially associated with prognosis in GBC, including single nucleotide variants in ERBB2, KMT2C, and KMT2D, in addition to CNAs in chromosome 12q13-15 and 17q12 regions. This effort will continue to include a detailed analysis of recurrent structural variants, loss of heterozygosity loci, and analysis of the microbiota in this GBC cohort. Citation Format: Nicolas A. Giraldo, Abhinita Mohanty, Chad Vanderbilt, Rose Brannon, Ryma Benayed, Efsevia Vakiani, Ghassan Abou-Alfa, James Harding, Imane El Dika, Nikolaus Schultz, Bob Li, Michael F. Berger, Marc Ladanyi, Eileen O'Reilly, William Jarnagin, Olca Basturk, Maria Arcila. Molecular characterization of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 82.

Abstract 1254: Actionable fusions detected by RNA-seq co-occur with PD-L1 expression and driver mutations in solid tumors patients

Abstract Background: Incorporating gene fusions into a comprehensive profile is critical not only because very effective therapies targeting oncogenic fusion proteins exist but they may also negate the response to therapy of an actionable SNV and InDel mutations. We examined the co-occurrence of actionable gene fusions detected by RNA-seq with actionable SNVs/InDels and with the immunotherapy response biomarker, PD-L1. Methods: In 2021, 5341 FFPE samples were analyzed by our clinical laboratory using a novel hybridization-based RNA sequencing assay. DNA (SNV/Indels) mutations were detected with a clinical grade NGS assay and PD-L1 protein expression was determined by IHC using an appropriate LDT or FDA approved assay. De-identified data were analyzed following an approved IRB protocol. Results: Of the 5341 patients tested for gene fusions only 0.7% were profiled with a comprehensive fusion detection panel for 250 clinically relevant fusion genes. Conversely, 67% of all patients were tested only for NTRK gene fusions. The prevalence of the following most relevant fusions was: ALK=3.16% (in particular EML4-ALK), NTRKs=0.85%, FGFR2=5.60%, RET=1.34% and ROS1=1.05%. Among NTRK fusions, NTRK3 was detected in 52.9% of the positive cases; in particular, dominant fusions are ETV6-NTRK3 (26%) and EML4-NTRK3 (20%). In the 163 fusion positive cases, 37 cases had available DNA mutation testing and PD-L1 expression testing. These patients presented 24 different actionable fusions, including ALK (2), FGFR1/2 (5), NTRK 1/2/3 (10), NRG1 (3), RET (3), and ROS1 (3) fusions. Interestingly, while 73% (27/37) of those tumors were PD-L1 positive, similar to the 75% found on the fusion negative samples, PD-L1 was positive in 88% (15/17) of the lung samples with pathogenic mutations from this subset. This was strikingly higher than the 45% (192/424) found in the fusion negative cohort. Finally, excluding TP53 mutations, NTRK fusions frequently co-occurred with RNF43, FBXW7, TERT promoter, and ARID1A pathogenic mutations. FGFR fusions co-occurred with BAP1, PBRM1, or KRAS mutations, which correlates with the fact that both FGFR2 fusions and swi/snf alterations are enriched in intrahepatic cholangiocarcinoma, where the FGFR fusions were detected. RET fusions co-occurred with ARID1A and TERT. ROS1 fusions co-occurred with SMARCA4 and KRAS pathogenic mutations simultaneously. NRG1 fusions co-occurred with ARID1A and FBXW7 mutations. EML4-ALK fusions co-occurred with FGFR2 and KMT2D variants of unknown significance. Conclusion: Actionable cancer-driving gene fusions were detected by RNA-sequencing and co-occurred with other biomarkers that can guide the selection of therapies, such as immune checkpoint inhibitors (ICI) and olaparib. The data suggest that gene fusion testing is an important addition to the genomic profiling for therapy selection in solid tumors. Citation Format: Miyoung Shin, Paris Petersen, Steven Lau-Rivera, Segun Jung, Sally Agersborg, Jacyln Hechtman, Fernando Lopez-Diaz, Vincent Funari. Actionable fusions detected by RNA-seq co-occur with PD-L1 expression and driver mutations in solid tumors patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1254.

Comparison of methylation episignatures in KMT2B- and KMT2D-related human disorders.

Aim & methods: To investigate peripheral blood methylation episignatures in KMT2B-related dystonia (DYT-KMT2B), the authors undertook genome-wide methylation profiling of ∼2M CpGs using a next-generation sequencing-based assay and compared the findings with those in controls and patients with KMT2D-relatedKabuki syndrome type 1 (KS1). Results: A total of 1812 significantly differentially methylated CpG positions (false discovery rate <0.05) were detected in DYT-KMT2B samples compared with controls. Multi-dimensional scaling analysis showed that the 10 DYT-KMT2B samples clustered together and separately from 29 controls and 10 with pathogenic variants in KMT2D. The authors found that most differentially methylated CpG positions were specific to one disorder and that all (DYT-KMT2B) and most (Kabuki syndrome type 1) methylation alterations in CpG islands were gain of methylation events. Conclusion: Using sensitive methylation profiling methodology, the authors replicated recent reports of a methylation episignature for DYT-KMT2B. These findings will facilitate the development of episignature-based assays to improve diagnostic accuracy.

MP40-16 EXPRESSION ANALYSIS AND MUTATIONAL STATUS OF HISTONE METHYLTRANSFERASE KMT2D IN UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 May 2022MP40-16 EXPRESSION ANALYSIS AND MUTATIONAL STATUS OF HISTONE METHYLTRANSFERASE KMT2D IN UPPER TRACT UROTHELIAL CARCINOMA Ekaterina Laukhtina, Ursula Lemberger, Andreas Bruchbacher, Dafina Ilijazi, David D’Andrea, Martin Susani, Dmitry Enikeev, Eva Compérat, Shahrokh F. Shariat, and Melanie R. Hassler Ekaterina LaukhtinaEkaterina Laukhtina More articles by this author , Ursula LembergerUrsula Lemberger More articles by this author , Andreas BruchbacherAndreas Bruchbacher More articles by this author , Dafina IlijaziDafina Ilijazi More articles by this author , David D’AndreaDavid D’Andrea More articles by this author , Martin SusaniMartin Susani More articles by this author , Dmitry EnikeevDmitry Enikeev More articles by this author , Eva CompératEva Compérat More articles by this author , Shahrokh F. ShariatShahrokh F. Shariat More articles by this author , and Melanie R. HasslerMelanie R. Hassler More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002600.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The gene coding for histone methyltransferase KMT2D is found among the top mutated genes in upper tract urothelial carcinoma (UTUC); however, there is a lack of data regarding its association with clinicopathologic features as well as survival outcomes. Therefore, we aimed to investigate KMT2D expression and mutation patterns and their utility as prognostic biomarkers in patients with UTUC. METHODS: A retrospective single-center study was conducted on tumor specimens from 51 patients treated with radical nephroureterectomy (RNU). Analysis of KMT2D expression on the protein level was performed by immunohistochemistry (IHC). Customized Next Generation Sequencing (NGS) was used to assess alterations in KMT2D exons. Cox-regression analyses were used to correlate KMT2D protein expression and mutational status with survival outcomes such as disease-free and overall survival. RESULTS: KMT2D expression was increased in patients with a previous history of bladder cancer (25% vs. 0%, p=0.02). NGS analysis of KMT2D exons in 27 UTUC tumors revealed a significant association between pathogenic KMT2D variants and tumor location (p=0.02). Pathogenic KMT2D variants were predominantly found in patients with non-pelvic or multifocal tumors (60% vs. 14%), while the majority of patients with pelvic tumor location (81% vs. 20%) did not harbor pathogenic KMT2D alterations. Both IHC and NGS analyses of KMT2D failed to detect a statistically significant association between KMT2D protein or KMT2D gene alteration status and clinical variables such as stage/grade of the disease as well as survival outcomes (all p >0.05). CONCLUSIONS: In this study, KMT2D protein expression and KMT2D mutational status did not emerge as prognostic markers for UTUC. However, KMT2D alterations and protein expression were associated with features of biologically aggressive UTUC such as multifocality, ureteral location, and previous bladder cancer. Therefore, KMT2D seems to hold the potential of improving our clinical decision in UTUC regarding perioperative systemic therapy and/or additional intravesical therapy. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e681 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ekaterina Laukhtina More articles by this author Ursula Lemberger More articles by this author Andreas Bruchbacher More articles by this author Dafina Ilijazi More articles by this author David D’Andrea More articles by this author Martin Susani More articles by this author Dmitry Enikeev More articles by this author Eva Compérat More articles by this author Shahrokh F. Shariat More articles by this author Melanie R. Hassler More articles by this author Expand All Advertisement PDF DownloadLoading ...

Expression Analysis and Mutational Status of Histone Methyltransferase KMT2D at Different Upper Tract Urothelial Carcinoma Locations.

The gene coding for histone methyltransferase KMT2D is found among the top mutated genes in upper tract urothelial carcinoma (UTUC); however, there is a lack of data regarding its association with clinicopathologic features as well as survival outcomes. Therefore, we aimed to investigate KMT2D expression, mutation patterns, and their utility as prognostic biomarkers in patients with UTUC. A single-center study was conducted on tumor specimens from 51 patients treated with radical nephroureterectomy (RNU). Analysis of KMT2D protein expression was performed using immunohistochemistry (IHC). Customized next-generation sequencing (NGS) was used to assess alterations in KMT2D exons. Cox regression was used to assess the relationship of KMT2D protein expression and mutational status with survival outcomes. KMT2D expression was increased in patients with a previous history of bladder cancer (25% vs. 0%, p = 0.02). The NGS analysis of KMT2D exons in 27 UTUC tumors revealed a significant association between pathogenic KMT2D variants and tumor location (p = 0.02). Pathogenic KMT2D variants were predominantly found in patients with non-pelvic or multifocal tumors (60% vs. 14%), while the majority of patients with a pelvic tumor location (81% vs. 20%) did not harbor pathogenic KMT2D alterations. Both IHC and NGS analyses of KMT2D failed to detect a statistically significant association between KMT2D protein or KMT2D gene alteration status and clinical variables such as stage/grade of the disease or survival outcomes (all p > 0.05). KMT2D alterations and protein expression were associated with UTUC features such as multifocality, ureteral location, and previous bladder cancer. While KMT2D protein expression and KMT2D mutational status do not seem to have prognostic value in UTUC, they appear to add information to improve clinical decision-making regarding the type of therapy.

Kidney and urinary tract findings among patients with Kabuki (make-up) syndrome.

Kabuki syndrome (KS) is a genetic disorder caused mainly by de novo pathogenic variants in KMT2D or KDM6A, characterized by recognizable facial features, intellectual disability, and multi-systemic involvement, including short stature, microcephaly, hearing loss, cardiac defects, and additional congenital anomalies. While congenital anomalies of the kidneys and urinary tract (CAKUT) are known manifestations of this disorder, studies focused solely on kidney involvement are scarce, and its prevalence is most likely underestimated. This study aimed to describe the prevalence and nature of CAKUT and other renal manifestations, in a cohort of KS patients followed at a single tertiary center. All patients who were evaluated at the Sheba Medical Center and received a clinical and/or molecular diagnosis of KS, over a 16-year period (2004-2020), were included. Digital medical records, including ultrasound studies, were reviewed by a team of pediatric nephrologists. Thirteen patients were included in the study, at ages ranging from the neonatal period to 20years. In eight patients, a pathogenic variant in KMT2D was established. CAKUT were detected in 8/13 (61.5%) of patients and varied from hypospadias, hydronephrosis, or double collecting systems to pelvic kidney, kidney asymmetry, horseshoe kidney, or kidney agenesis. One patient experienced kidney failure necessitating transplantation at 20years of age. Our findings underscore the high prevalence of CAKUT and genitourinary involvement in patients with KS and suggest that assessment by pediatric nephrology specialists is warranted as part of the routine multidisciplinary evaluation of newly diagnosed patients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Genetic features of B-cell lymphoblastic lymphoma with TCF3-PBX1.

BackgroundLymphoblastic lymphoma (LBL) and acute lymphoblastic leukemia (ALL) are categorized as the same entity under precursor lymphoid neoplasms in the World Health Organization classification. However, compared to B‐cell ALL, the molecular genetic makeup of B‐cell LBL remains to be understood, mainly due to its rarity. We performed whole exome sequencing (WES) on seven patients with TCF3‐PBX1‐positive B‐cell LBL.MethodsWES was performed using DNA extracted from tumor specimens and paired blood samples at remission for six patients, and tumor‐only analysis was performed for one patient whose remission sample was not available. For one patient, a relapsed sample was also analyzed.Results KMT2D variants and 6q LOH were found as recurrent alterations. Somatic variants of KMT2D were identified in three of the seven patients. Of note, the two patients with heterozygous nonsense variant of KMT2D were at stage III, without bone marrow infiltration. 6q LOH was also identified in two others, out of the seven patients. The common 6q deleted region of the two patients ranged from 6q12 to 6q16.3. Both patients had bone marrow infiltration. Analysis of recurrent case also revealed that the relapsed clone might be derived from a minor clone of the bone marrow at diagnosis.ConclusionIn this study, through WES for seven patients with TCF3‐PBX1‐positive B‐LBL, we identified KMT2D mutations and 6q LOH as recurrent alterations. In order to elucidate the relationship between these recurrent alterations and disease specificity or outcomes, further studies comparing with TCF3‐PBX1‐positive B‐ALL are required.

Genomic Profiling Reveals Spatial Intra-Tumor Heterogeneity in Follicular Lymphoma

Background and Aims: Follicular lymphoma (FL) is an incurable B cell malignancy characterized by advanced stage disease and a heterogeneous clinical course. Despite recent genomic studies focused on profiling single FL biopsies over several time-points, little is known about the spatial heterogeneity as multi-site sampling is rarely conducted in lymphomas. However, in solid cancers, this approach has demonstrated profound spatial intra-tumor heterogeneity (ITH), impacting mechanisms of drug resistance and compromising precision-based strategies. As the majority of FL patients manifest disseminated tumor involvement, we sought to characterize the extent and clinical importance of spatial genetic heterogeneity in FL.Methods: Our study cohort comprised 9 FL patients (SP1-9) from which 22 synchronously removed spatially-separated samples from several tissue sources (lymph node (LN), bone marrow (BM), skin (SK), pleural effusion (PE), ascites (AS), spleen (SP)) were collected. For patients SP3 and SP4, both spatial and longitudinal samples at 2 time-points; at FL and histological transformation, were included. FL B-cells and normal T-cells were enriched by fluorescence activated cell sorting (FACS) in most cases. Each sample was subjected to whole exome sequencing and we defined somatic variants, copy number alterations (CNAs) and mutation signatures using established bioinformatic tools. We employed a statistical clustering model (mclust) for clonal density estimation and together with ultra-deep targeted sequencing, reconstructed each tumor's subclonal architecture.Results: Whilst spatially-separated FL tumors shared identical BCL2 - IGH breakpoints, we observed variable degrees of ITH. On average, 82% (range 50%-99%) of variants were shared between sites with two patterns of spatial ITH observed: high (SP1, SP8, SP9) and low (SP5, SP6, SP7). Copy number analyses showed a high level of concordance in most cases, except for SP4, SP7 and SP8. The overall distribution of known mutational signatures between spatially shared and discordant variants was different. Shared variants were predominantly enriched for mutational signature 1, representing age-related changes. In contrast signatures 6 and 24, both suggestive of defective DNA mismatch repair, were more pronounced in the spatially-discordant variants (χ2 test, p <0.0001), indicating that distinct mutagenic processes operate at different times during a tumor's evolution. Clonal architecture analyses revealed that some tumors comprised multiple competing subclones, with distinct clusters of mutations demonstrating differential expansions within spatially-separated sites, exemplified in SP2 (Figure 1). Importantly, for cases where spatial tumors were examined at two time-points, the degree of heterogeneity markedly increased with transformation. Detailed mutation analysis showed that apart from CREBBP mutations, that were clonally maintained throughout spatially separated biopsies, there were several instances of site-specific alterations in genes encoding proteins known to activate signaling pathways amenable to targeted therapies such as, TNFAIP3 (SP3 and SP9), TNFRSF14 (SP1), PIK3CD (SP4) and copy number loss of PTEN (SP8). In depth characterization of these variants using deep-sequencing techniques confirmed their discordant nature, suggesting ongoing genetic diversification driving evolution after widespread tumor dissemination.Conclusion: Collectively, our results demonstrate that spatial ITH is prevalent in FL. The existence of site-specific aberrations suggests a single biopsy insufficiently captures the full genomic complexity present and these spatial variations need to be considered in biomarker-led clinical studies.Figure 1: Dot plot demonstrating variants allele frequencies (VAFs) of all non-synonymous variants in SP2. Three distinct subclones circled in purple, orange and green can be inferred. The purple cluster includes ATP 6V1B2 and KMT2D variants classified as clonal in the BM and subclonal in the LN. The orange cluster includes different variants in KMT2D classified as subclonal in the BM and clonal in the LN. The green cluster includes variants in EZH2 and BCL2 that have a higher VAF in the LN compared to the BM. VAFs for selected variants in each of these putative subclones are shown in the horizontal bar graphs. [Display omitted] DisclosuresAuer:Janssen: Consultancy. Gribben:Kite: Honoraria; Karyopharm: Honoraria; Pharmacyclics: Honoraria; TG Therapeutics: Honoraria; Celgene: Honoraria; Acerta: Honoraria; Janssen: Honoraria; Genentech/Roche: Honoraria; Abbvie: Honoraria.

Ophthalmic manifestations in Kabuki (make-up) syndrome: A single-center pediatric cohort and systematic review of the literature

Kabuki syndrome (KS) is a genetic disorder caused by pathogenic variants in KMT2D or KDM6A, and manifesting with multi-systemic involvement, including recognizable facial features, developmental delay and multiple congenital anomalies. Ophthalmological involvement has been described in varying rates in several studies.We aimed to evaluate the prevalence and nature of ophthalmological findings in a cohort of KS patients in Israel.Medical records of all patients diagnosed with KS in our tertiary center between 2004 and 2020 were retrospectively reviewed. Data collected included physical examination findings, molecular analysis as well as comprehensive ophthalmic characteristics including visual acuity, ocular alignment and motility, ocular adnexa, anterior segments and dilated fundus exams. Finally, an updated systematic review of the literature was performed.Thirteen unrelated patients were included in the study, diagnosed at an age raging from the first months of life to 20 years. Of these, three (23%) showed significant ophthalmological abnormalities, beyond the characteristic structural findings of long palpebral fissures and lower eyelid eversion. These included bilateral posterior colobomata in the first patient; bilateral ptosis, hypermetropia, esotropia, blue sclera and anisocoria in the second; and bilateral congenital cataracts in the third.To conclude, our findings underscore the importance of a comprehensive ophthalmological evaluation as part of the routine multidisciplinary assessment of children suspected/diagnosed with KS.

Clinical exome sequencing data reveal high diagnostic yields for congenital diaphragmatic hernia plus (CDH+) and new phenotypic expansions involving CDH

BackgroundCongenital diaphragmatic hernia (CDH) is a life-threatening birth defect that often co-occurs with non-hernia-related anomalies (CDH+). While copy number variant (CNV) analysis is often employed as a diagnostic test for...

Clinical and molecular characterization study of Chinese Kabuki syndrome in Hong Kong.

Kabuki syndrome (OMIM #147920 and 300867) is a rare genetic disorder characterized by a distinctive facial gestalt, intellectual disability and multiple congenital anomalies. We summarized the clinical features and molecular findings of the Kabuki syndrome (KS) patients diagnosed in Hong Kong between January 1991 and December 2019. There were 21 molecularly confirmed KS. Twenty of them were due to pathogenic KMT2D variants and one patient had KDM6A deletion. Nine KMT2D variants were novel. The clinical phenotype of our Chinese KS patients was largely comparable with that reported in patients of other ethnicities. This study expands the mutation spectrum but also provide important natural history information of Chinese KS in literature.

Update of the genotype and phenotype of KMT2D and KDM6A by genetic screening of 100 patients with clinically suspected Kabuki syndrome.

Kabuki syndrome is characterized by a variable degree of intellectual disability, characteristic facial features, and complications in various organs. Many variants have been identified in two causative genes, that is, lysine methyltransferase 2D (KMT2D) and lysine demethylase 6A (KDM6A). In this study, we present the results of genetic screening of 100 patients with a suspected diagnosis of Kabuki syndrome in our center from July 2010 to June 2018. We identified 76 variants (43 novel) in KMT2D and 4 variants (3 novel) in KDM6A as pathogenic or likely pathogenic. Rare variants included a deep splicing variant (c.14000-8C>G) confirmed by RNA sequencing and an 18% mosaicism level for a KMT2D mutation. We also characterized a case with a blended phenotype consisting of Kabuki syndrome, osteogenesis imperfecta, and 16p13.11 microdeletion. We summarized the clinical phenotypes of 44 patients including a patient who developed cervical cancer of unknown origin at 16 years of age. This study presents important details of patients with Kabuki syndrome including rare clinical cases and expands our genetic understanding of this syndrome, which will help clinicians and researchers better manage and understand patients with Kabuki syndrome they may encounter.

Comparative meta-analysis of Kabuki syndrome with and without hyperinsulinaemic hypoglycaemia.

Kabuki syndrome (KS), caused by pathogenic variants in KMT2D or KDM6A, is associated with hyperinsulinaemic hypoglycaemia (HH) in 0.3%-4% of patients. We characterized the clinical, biochemical and molecular data of children with KS and HH compared to children with KS without HH in a multicentre meta-analysis. Data of seven new and 17 already published children with KS and HH were compared to 373 recently published KS patients without HH regarding molecular and clinical characteristics. Seven new patients were identified with seven different pathogenic variants in KDM6A (n=4) or KMT2D (n=3). All presented with HH on the first day of life and were responsive to diazoxide. KS was diagnosed between 9months and 14years of age. In the meta-analysis, 24 KS patients with HH had a significantly higher frequency of variants in KDM6A compared to 373 KS patients without HH (50% vs 11.5%, P<.001), and KDM6A-KS was more likely to be associated with HH than KMT2D-KS (21.8% vs. 3.5%, P<.001). Sex distribution and other phenotypic features did not differ between KS with and without HH. The higher incidence of HH in KDM6A-KS compared to KMT2D-KS indicates that KDM6A loss of function variants predispose more specifically to beta cell dysfunction compared to KMT2D variants. As difficulties to assign syndromic characteristics to KS in early infancy often lead to delayed diagnosis, genetic testing for KS should be considered in children with HH, especially in the presence of other extrapancreatic/syndromic features.

A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct fromKabuki syndrome

PurposeTo investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). MethodsMultiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. ResultsThe consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. ConclusionKMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

Phenotypic expansion of KMT2D-related disorder: Beyond Kabuki syndrome.

Pathogenic variants in KMT2D, which encodes lysine specific methyltransferase 2D, cause autosomal dominant Kabuki syndrome, associated with distinctive dysmorphic features including arched eyebrows, long palpebral fissures with eversion of the lower lid, large protuberant ears, and fetal finger pads. Most disease-causing variants identified to date are putative loss-of-function alleles, although 15-20% of cases are attributed to missense variants. We describe here four patients (including one previously published patient) with de novo KMT2D missense variants and with shared but unusual clinical findings not typically seen in Kabuki syndrome, including athelia (absent nipples), choanal atresia, hypoparathyroidism, delayed or absent pubertal development, and extreme short stature. These individuals also lack the typical dysmorphic facial features found in Kabuki syndrome. Two of the four patients had severe interstitial lung disease. All of these variants cluster within a 40-amino-acid region of the protein that is located just N-terminal of an annotated coiled coil domain. These findings significantly expand the phenotypic spectrum of features associated with variants in KMT2D beyond those seen in Kabuki syndrome and suggest a possible new underlying disease mechanism for these patients.

Identification of KMT2D and KDM6A variants by targeted sequencing from patients with Kabuki syndrome and other congenital disorders

Kabuki syndrome (KS) is a rare congenital disorder characterized by distinctive facies, postnatal growth deficiency, cardiac defects and skeletal anomalies. Studies have determined that pathogenic variants of the lysine-specific methyltransferase 2D (KMT2D) and lysine-specific demethylase 6A (KDM6A) genes are the major causes of KS. The two genes encode different histone-modifying enzymes that are found in the same protein complex that is critical for cell differentiation during development. Here we report the results from next-generation sequencing of genomic DNA from 13 patients who had a clinical diagnosis of KS based on facial dysmorphism and other KS-specific cardinal phenotypes. Nine of the 13 patients were confirmed to be carrying heterozygous pathogenic KMT2D variants, seven of which were truncating and two were missense substitutions. Overall, we uncovered 11 novel variants – nine in KMT2D and two in KDM6A. Seven of the novel variants (all KMT2D) were likely causative of the KS phenotype. Our study expands the number of naturally occurring KMT2D and KDM6A variants. The discovery of novel pathogenic variants will add to the knowledge on disease-causing variants and the relevance of missense variants in KS.