Molecular Genetics Reports Research Articles

Tumor-Associated Retinal Pigmentation in Choroidal Melanoma

To report a previously unrecognized choroidal melanoma clinical feature termedtumor-associated retinal pigmentation(TARP) and determine any correlation with tumor biology. Imaging and histologic analysis of a retrospective cohort of patients. Patients with choroidal melanoma identified as having TARP on funduscopy at the Liverpool Ocular Oncology Centre (LOOC), United Kingdom, from January 2020 through January2023. Clinical and imaging characteristics of patients diagnosed with choroidal melanoma and exhibiting TARP on fundoscopy were documented. Details of these choroidal melanomas were collated and correlated with histopathology and molecular genetic reports. The chromosome 3 status of each tumor was assessed. In enucleated samples, immunostaining was undertaken to determine the nature of the TARP using specific markers (CD68 and MelanA). Features of TARP on widefieldfundus color imaging, fundus autofluorescence (FAF), and OCT were described. Tumor chromosome 3 status and the immunoprofile of the TARP also were collated. Tumor-associated retinal pigmentationhad a prevalence rate of 7.47 per 100 cases of choroidal melanoma at the LOOC. Twenty-three eyes with TARP were analyzed, with a mean age of 71.4 years (range, 51-88 years). The median largest basal diameter was 16.10 mm (range, 9.17-21.32 mm), and the mean tumor thickness was 8.04 mm (range, 1.40-13.80 mm).Tumor-associated retinal pigmentationwas observed on widefield color fundus imaging, with hypofluorescence on FAF images and represented hyperreflective foci located in intraretinal and subretinal spaces on OCT scans. Seventeen patients (73.9%) underwent enucleation, and 6 patients (26.1%) underwent globe-sparing treatment. Molecular genetic analysis of 20 choroidal melanomas (after enucleation or radiotherapy biopsy) revealed monosomy 3 in 18 tumors (90%). Immunostaining of the TARP in enucleated eyes showed CD68+ melanophages in all 17 patients appearing as scattered cells and aggregates; MelanA findings were negative. Tumor-associated retinal pigmentationrepresents tumor-associated macrophages, not melanocytes, within intraretinal and subretinal spaces of larger choroidal melanomas. Radiation treatments need not involve this area in the treatment plan, minimizing radiation-related complications. This novel clinical sign seems to be linked to tumors of high metastatic-risk clinical and genetic characteristics, with a preponderance having monosomy 3 anomalies. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Interpretation and actionability of genetic variants in cardiomyopathies: a position statement from the European Society of Cardiology Council on cardiovascular genomics.

This document describes the contribution of clinical criteria to the interpretation of genetic variants using heritable Mendelian cardiomyopathies as an example. The aim is to assist cardiologists in defining the clinical contribution to a genetic diagnosis and the interpretation of molecular genetic reports. The identification of a genetic variant of unknown or uncertain significance is a limitation of genetic testing, but current guidelines for the interpretation of genetic variants include essential contributions from clinical family screening that can establish a de novo assignment of the variant or its segregation with the phenotype in the family. A partnership between clinicians and patients helps to solve major uncertainties and provides reliable and clinically actionable information.

S2635 Abnormal Liver Enzymes Attributed to HNF-1b Associated Disease

Introduction: Hepatocyte nuclear factor 1 beta (HNF-1b) associated diseases are genetic conditions caused by an error in the HNF1b gene. This gene regulates early development of organs such as the kidney, genital tract, and pancreas. Common signs/symptoms include kidney findings like cysts, mature onset of diabetes at a young age (MODY), genital abnormalities, as well as abnormal pancreas/liver function. We present a case of abnormal liver enzymes secondary to HNF-1b associated disease. Case Description/Methods: A 22-year-old man was referred for abnormal liver enzymes and suspected NAFLD. Past medical history included surgery for an undescended testicle at age 6 and new onset DM with relative weight loss. Due to DM, weight loss, and a family history of pancreatic cancer he was evaluated by GI and had labs in 1/2020 showing ALK PHOS (254 U/L), AST (98 U/L), ALT (251 U/L), and total bilirubin (1.4 mg/L). MRI/MRCP in 3/2020 was overall unremarkable and upper EUS 6/2020 showed echogenic liver and increased fullness of the pancreas with non-hyperechoic stranding throughout. Detailed serologic evaluation was without specific abnormalities other than ongoing elevated liver enzymes in a mixed pattern. He had a liver biopsy in 1/2021, in which native bile ducts were absent in < 50% of portal tracts, while other portal tracts contained small ducts relative to size of the tract. Portal vein abnormalities were noted including atrophy and fragmentation. No features of NAFLD or chronic liver diseases. He underwent a genetic cholestasis panel showing a heterozygous ∼1.51 Mb deletion that was pathogenic for HNF-1b associated disease. He has been referred to a genetic counselor and maintains follow up with his specialty teams. Discussion: The HNF1B gene is associated with functional and transcriptional regulation in hepatobiliary, pancreatic B-cells, and urogenital tract tissues. Pathogenic deficiencies are related to point mutations or deletions. The combination of MODY 5 diabetes plus renal cysts or urogenital abnormality has led this finding to be called RCAD (renal cysts and diabetes). There have been case reports with classic syndrome plus varying hepatobiliary findings. To our knowledge, this is the first case of HNF-1B associated disease without the classic finding of renal pathology. It is suggested that for young patients with early onset of diabetes with abnormal hepatobiliary and/or pancreatic testing, HNF-1B disease be considered in the differential diagnosis.Figure 1.: Prevention Genetics - Cholestasis Panel Molecular Genetic Report.

Risk of cardiac manifestations in adult mitochondrial disease caused by nuclear genetic defects

ObjectiveRegular cardiac surveillance is advocated for patients with primary mitochondrial DNA disease. However, there is limited information to guide clinical practice in mitochondrial conditions caused by nuclear DNA defects. We...

Accurate Prediction of Gene Mutations with Flow Cytometry Immune-Phenotyping By Machine Learning Algorithm

Introduction Identification of gene mutation status prior treatment has improved our capability in risk stratifying acute myeloid leukemia (AML) patients greatly, but it is really a labor-exhausting work to identify these mutations. In this this study, we hypothesize immunophenotyping could predict gene mutation and we aim to develop machine learning algorithms that could predict the AML gene mutation status with the immunophenotype by clinical flow cytometry. Method Retrospective clinical data of patients with AML, including demographic (age &amp; gender), molecular genetics, cytogenetics as well as flow cytometry (FC) data at National Taiwan University Hospital were collected. A total of 529 newly diagnosed de novo AML from 2009 to 2019 enrolled this study. The median age at diagnosis was 58 years (Table 1). In total, 428 NPM1, 415 FLT3, 331 CEBPA and 338 RUNX1 gene testing results and a total of 529 initial diagnostic FC data from these patients were used in developing the gene mutation prediction models. Each FC data sample contained 100,000 cells acquired on FACSCantoII machine 6 fluorescent channels with multiple fluorescent markers. The markers measured are listed in Table 2. There were 19 combinations of markers and fluorescent channels which were served as feature inputs of the machine learning framework. Our proposed machine learning framework can be divided as a phenotype representation learning paradigm and a classification model. To derive the phenotype representation, we trained a multivariate Gaussian Mixture Model (GMM) on the 19-dimension FC data to capture the training data distribution and characteristics in a probabilistic unsupervised manner. Then, a Fisher-scoring method was used to vectorize each sample as a high dimensional representation via differential computation in terms of the learned GMM parameters. This Fisher vectorization method transformed samples to a high dimensional feature space as phenotype vectors. We performed analysis of variance (ANOVA)-based feature selection on these representations which were finally fed into the support vector machine (SVM) classifier. To alleviate the negative effects of imbalance classes in gene mutation identification tasks, we applied synthetic minority oversampling technique (SMOTE) algorithm which augmented the minority class by interpolating samples near support vectors. We train independent SVM models to detect the occurrences of the four gene mutation. The algorithm is evaluated by randomly divided 5-fold cross validation which separates 80% data for training and 20% for testing. Results This gene mutation rate of this cohort for NPM1, FLT3, CEBPA and RUNX1 were 22.2% (95/428), 25.1% (104/415), 20.2% (67/331), and 13.6% (46/338), respectively. The average accuracies (ACC) of the prediction model performance for NPM1, FLT3, CEBPA and RUNX1 were 82.6%, 76.3%, 84.2% and 84.1%, respectively, whereas the area under the ROC curve (AUC) were 77.9%, 63.4%, 80.7% and 67.7%, respectively (Table. 3). Conclusions We demonstrated the potential of the correlation of recurrent AML gene mutation status with immunophenotype of AML through our preliminary gene mutation prediction model. Further study with larger cohorts followed by external validation are needed to further evaluate the feasibility of using machine learning based algorithm as one of triage tools to support physicians in aggressive AML clinical decision before receiving molecular genetic reports. Disclosures Ko: Roche: Honoraria.

NEW GENES AND DISEASES / NGS & RELATED TECHNIQUES: P.328 Neuromuscular disease variant of unknown significance (VUS) resolution using muscle biopsy evaluation: The Iowa experience

Whole exome sequencing and large next generation sequencing panels are being performed frequently in muscular dystrophy (MD) and other neuromuscular disease patients. These tests often reveal numerous variants of unknown significance (VUS) that result in diagnostic uncertainty. A potential means to resolve the pathogenicity of VUS is to evaluate muscle biopsy pathology. The purpose of this study was to investigate the efficacy of our extended muscle biopsy workup in interpreting VUS. Muscle biopsies accessioned at the University of Iowa between November 2015 and December 2019 for the purpose of evaluating VUS were reviewed. The 144 cases included in the study came from 31 states or provinces of the USA and Canada. A total of 91 different genes had a VUS. Individual patients had 1 to 8 VUS, with an average of 1.8 VUS/patient. Thirty-three MD genes had a VUS, 9 of which were dystroglycanopathy-related. Genes causing non-dystrophic myopathies (17) and mitochondrial diseases (9) were also commonly represented. The most frequent VUS involved RYR1 (18) and CAPN3 (15) with COL6A3, DYSF, and TTN having 14 each. Variants in the three genes encoding components of collagen VI made up the largest single disease group with 33 cases. The workup to evaluate VUS was tailored to the individual patient history, muscle biopsy histopathology, and genetic testing data, and included histochemistry, immunostaining, electron microscopy, and/or western blotting. Using these techniques, 33% of cases showed evidence that one of the VUS was likely to be disease-causing. Overall, VUS in CAPN3, DMD, and RYR1 were the most frequently solved as pathogenic (9, 7, and 4 cases, respectively). In 35% of cases, the workup suggested that the VUS were not likely to be pathogenic. Interestingly, in 12% of cases, a genetic MD or other myopathy was suggested that was completely unrelated to the VUS provided. For example, dystrophinopathy was diagnosed in 4 cases, despite molecular genetics reports of VUS in other unrelated genes. In the remaining 20% of cases, the workup was unable to provide guidance about the VUS. Our data suggest that muscle biopsy testing can provide information critical to determining pathogenicity in 80% of neuromuscular disease cases with a VUS.

A computer-assisted method for pathogenicity assessment and genetic reporting of variants stored in the Australian Inherited Retinal Disease Register.

The assignment of pathogenicity to variants suspected of causing an inherited retinal disease and the subsequent creation of molecular genetic reports sent to clinical geneticists and ophthalmologists has traditionally been time-consuming and subject to error and ambiguity. The purpose of this paper is to describe a computer-assisted method we have developed for (1) assessment of the predicted pathogenicity of genetic variants identified in patients diagnosed with an inherited retinal disease and (2) the incorporation of these results into the Australian Inherited Retinal Disease Register and DNA Bank's databases, for the production of molecular genetics reports. This method has significantly accelerated the assessment of variant pathogenicity prediction and subsequent patient report generation for the Australian Inherited Retinal Disease Register and DNA Bank, and has reduced the potential for human error. The principles described in this paper may be applied in any situation where genetic variants and patient information are stored in a well-organised database.

Why genes extending lifespan in model organisms have not been consistently associated with human longevity and what it means to translation research

A recent paper by Deelen et al. (2014) in Human Molecular Genetics reports the largest genome-wide association study of human longevity to date. While impressive, there is a remarkable lack of association of genes known to considerably extend lifespan in rodents with human longevity, both in this latest study and in genetic association studies in general. Here, I discuss several possible explanations, such as intrinsic limitations in longevity association studies and the complex genetic architecture of longevity. Yet one hypothesis is that the lack of correlation between longevity-associated genes in model organisms and genes associated with human longevity is, at least partly, due to intrinsic limitations and biases in animal studies. In particular, most studies in model organisms are conducted in strains of limited genetic diversity which are then not applicable to human populations. This has important implications and, together with other recent results demonstrating strain-specific longevity effects in rodents due to caloric restriction, it questions our capacity to translate the exciting findings from the genetics of aging to human therapies.

Clinicogenetic Study of Turkish Patients With Syndromic Craniosynostosis and Literature Review

BackgroundFibroblast growth factor receptor 2 mutations have been associated with the craniosynostotic conditions of Apert, Crouzon, Pfeiffer, Saethre-Chotzen, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes in various ethnic groups. MethodsThirty-three unrelated Turkish patients (12 with Apert syndrome, 14 with Crouzon syndrome, six with Pfeiffer syndrome, and one with Saethre-Chotzen syndrome) and 67 nonsyndromic craniosynostosis patients were screened for mutations in exons IIIa and IIIc of the FGFR2 gene by denaturing high-performance liquid chromatography and confirmed by direct sequencing. ResultsWe detected several pathogenic mutations in 11/33 (33%) patients with Apert syndrome (four with p.Pro253Arg; seven with p.Ser252Trp) and 8/33 (24%) patients with Crouzon syndrome (three with p.Trp290Arg, one with p.Cys342Tyr, p.Cys278Phe, p.Gln289Pro, and a novel p.Tyr340Asn mutation) and five (15%) with Pfeiffer syndrome (p.Cys342Arg, p.Pro253Arg, p.Trp290Arg, and p.Ser351Cys). No FGFR2 gene mutation was detected in any of the patients with Saethre-Chotzen syndrome and nonsyndromic craniosynostosis. ConclusionsOur results indicate that the majority of Turkish patients with syndromic craniosynostosis have detectable genetic changes with an overall frequency of 72.7%. Because this is the first molecular genetic report from a Turkish cohort, the identified spectrum profile of FGFR2 mutations of the syndromic craniosynostotic patients would be very helpful for understanding the genotype–phenotype relationship and has a great value for diagnosis, prognosis, and genetic counseling.

Is Prevalence of Retinopathy Related to the Age of Onset of Diabetes? Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular Genetic Report No. 5

Aim: To compare the prevalence of diabetic retinopathy (DR) and risk factors in patients with a known onset of diabetes before 40 years and after 40 years of age. Methods: This is a population-based study for which 1,414 diabetics were recruited. The fundi were photographed using 45-degree 4-field stereoscopic digital photography. The diagnosis of DR was based on Klein’s classification of the Early Treatment Diabetic Retinopathy Study scales. Results: The prevalence of DR was 33.3% (95% confidence interval, CI: 26.6–39.9) in known onset of diabetes (≤40 years) compared to 15.6% (95% CI: 13.6–17.6) in those with late onset (>40 years; p < 0.0001). In the group with age of known onset of diabetes ≤40 years, the risk factors, associated with any DR, were poor glycemic control (odds ratio, OR: 1.36 for every g% increase in glycosylated hemoglobin), insulin use (OR: 4.21), increasing known duration of diabetes (OR: 1.10 for increase of every year in known duration of diabetes) and presence of macroalbuminuria (OR: 13.39). In the late onset of diabetes group, besides the above-mentioned risk factors, the presence of microalbuminuria (OR: 2.08), male gender (OR: 1.67), presence of anemia (OR: 1.89) and increased systolic blood pressure (OR: 1.01) were the risk factors for DR. Conclusion: The prevalence of DR was almost twice more in those subjects who developed diabetes before the age of 40 years than those who developed it later.

Chromophobe renal cell carcinoma with microcystic and adenomatous arrangement and pigmentation—a diagnostic pitfall. Morphological, immunohistochemical, ultrastructural and molecular genetic report of 20 cases

We present clinical, morphological, immunohistochemical, ultrastructural and molecular genetic features of 20 cases of a peculiar form of chromophobe renal cell carcinoma (CRCC) with morphology differing from that of conventional CRCC. Microscopically, the typical features of the tumors were microcystic arrangement and formation of adenomatous structures. Microcystic areas were composed of smaller eosinophilic and bigger pale cells having cytological appearance typical of conventional CRCC. Cytological features of the adenomatous structures were mostly different from those of conventional CRCC. They had a typical columnar arrangement with nuclei positioned at the base of the glandular structures and a small amount of a deeply eosinophilic cytoplasm often endowed with brush border facing the lumen of the glands. In addition, all the tumors showed a brown pigmentation. The pigmentation was located mostly extracellularly, where it formed pools of heavy deposits. Microscopic calcifications present in all cases formed psammoma bodies or else the calcifications were more extensive and amorphous in shape. Ultrastructurally, the cells showed features characteristic of CRCC: typical cytoplasmic vesicles were 100-700 nm in size and mitochondria had tubulovesicular, lamellar or circular cristae. Some tumor cells contained dark, variously sized electron-dense pigment granules. Neither melanosomes nor membrane-bound neurosecretory granules were seen. Using fluorescence in-situ hybridization probes for chromosomes 1, 2, 6, 10, 13, 17 and 21, the tumors revealed massive loss of tested chromosomes typical for conventional CRCC. Monosomy of chromosomes 1, 2, 6, 10, 13 and 21 was found in 100, 36, 91, 82, 82, 82 and 64% of cases, respectively. None of the cases showed mutation of exons 9, 11, 13 and 17 of the c-kit gene. The important feature of pigmented microcystic chromophobe renal cell carcinoma is a relatively benign biological behavior and the absence of distant metastases and sarcomatoid transformation.

The early treatment of craniosynostosis

Fibroblast growth factor receptor 2 mutations have been associated with the craniosynostotic conditions of Apert, Crouzon, Pfeiffer, Saethre-Chotzen, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes in various ethnic groups.Thirty-three unrelated Turkish patients (12 with Apert syndrome, 14 with Crouzon syndrome, six with Pfeiffer syndrome, and one with Saethre-Chotzen syndrome) and 67 nonsyndromic craniosynostosis patients were screened for mutations in exons IIIa and IIIc of the FGFR2 gene by denaturing high-performance liquid chromatography and confirmed by direct sequencing.We detected several pathogenic mutations in 11/33 (33%) patients with Apert syndrome (four with p.Pro253Arg; seven with p.Ser252Trp) and 8/33 (24%) patients with Crouzon syndrome (three with p.Trp290Arg, one with p.Cys342Tyr, p.Cys278Phe, p.Gln289Pro, and a novel p.Tyr340Asn mutation) and five (15%) with Pfeiffer syndrome (p.Cys342Arg, p.Pro253Arg, p.Trp290Arg, and p.Ser351Cys). No FGFR2 gene mutation was detected in any of the patients with Saethre-Chotzen syndrome and nonsyndromic craniosynostosis.Our results indicate that the majority of Turkish patients with syndromic craniosynostosis have detectable genetic changes with an overall frequency of 72.7%. Because this is the first molecular genetic report from a Turkish cohort, the identified spectrum profile of FGFR2 mutations of the syndromic craniosynostotic patients would be very helpful for understanding the genotype–phenotype relationship and has a great value for diagnosis, prognosis, and genetic counseling.