Abstract Background and aims Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are genetically heterogeneous kidney diseases that manifest with kidney involvement, AS also with ...hearing and visual impairment. Electron microscopic examination shows characteristic ultrastructural changes in the glomerular basement membrane (GBM) in AS and TBMN, but cannot reliably distinguish between the two entities. Therefore, molecular genetic studies and the identification of individual pathogenic variants in the target genes are crucial to confirm the diagnosis. The aim of our study is to determine the significance of the genetic variants associated with the ultrastructural changes of GBM found in the renal biopsy. Method We retrospectively analyzed records of patients from the University Medical Centre Ljubljana in which ultrastructural GBM changes characteristic for AS and TBMN were found in the renal biopsy, including their clinical, laboratory and histological features. Molecular genetic investigations were made using the new generation sequencing method (NGS) to determine germline pathogenic variants in genes directly related to AS and TBMN (COL4A3, COL4A4 and COL4A5) and in genes that are important in syndromes with a related clinical picture (COL4A6, CD151, LMX1B, MYH9, FN1 and MYO1E). The pathogenicity of variants was determined according to the Clinical Molecular Genetic Society best practice guidelines. Results The study included 73 patients with ultrastructurally confirmed thin GBM. Indications for kidney biopsy were chronic nephritis syndrome in 33 patients (45%), acute worsening of renal function in 16 (22%), nephrotic syndrome in 9 (12%), nephrotic proteinuria and chronic kidney disease in 6 (8%), isolated proteinuria in 5 (7%) and suspected vasculitis in 4 patients (6%). At the time of kidney biopsy average age was 50 ± 16 years, average creatinine 112 ± 70 µmol/L and average estimated glomerular filtration rate (eGFR) 65 ± 24 ml/min/1.73 m2, 7 patients had eGFR below 30 ml/min/1.73 m2. Average daily proteinuria was 2.4 ± 3.3 gr and 88% of patients had hematuria. 38% of them have hearing impairment and 43% had positive familiar history of kidney disease. We recorded 33 patients (45%) with genetic variants associated with AS or TBMN: 22 patients (67%) with pathogenic variants, 2 patients (6%) with likely pathogenic and 9 patients (27%) with variants of uncertain significance. The presence of a positive genetic analysis was associated with a higher level of hematuria (p = 0.003), a lower level of proteinuria (p = 0.004) and a positive family history of kidney disease (p < 0.001). It was also associated with hearing loss, but the association was not statistically significant (p = 0.099). Patients with genetic variants had similar eGFR to those with negative genetics (63.9 ml/min/1.73 m2 (58.3-69.4) vs 66.2 ml/min/1.73 m2 (60.0-72.3)), p = 0.698. We also found no differences in GBM thickness in patients without compared to those with identified gene variants (213 nm (160-315) vs. 209 nm (140-410)), p = 0.761. 32 patients (44%) had only focal chronic changes and ultrastructural changes of thin GBM present on kidney biopsy and 41 patients (56%) had also been diagnosed with other renal pathology. When comparing these two groups, patients without associated pathology had lower daily proteinuria (p = 0.048), higher level of hematuria (p = 0.021), frequent hearing impairment (p = 0.008) and also positive genetics analysis (p = 0.016). Conclusion We have shown that in patients with proven ultrastructural alterations in GBM, the presence of so far recognized genetic variants associated with AS and TBMN is 45%, and corresponds to the frequency of other phenotypic characteristics of AS, including hearing impairment and positive family history. In the future, additional clinical tools will be required to accurately determine the role of coexistent thin GBM on the prognosis of patients with other relevant kidney pathology and other genetic tools beyond NGS should be employed to explore other pathologic genetic variants associated with the AS spectrum.
Chondromyxoid fibroma (CMF) is a rare benign bone tumour. While CMF located entirely on the surface of a bone (i.e. juxtacortical CMF) has been well characterised, CMF has not so far been ...convincingly documented to arise in soft tissues without connection to an underlying bone.
We report a subcutaneous CMF in a 34-year-old male, located on the distal medial aspect of the right thigh without any connection with the femur. The tumour measured 15 mm, it was well-circumscribed and displayed typical morphological features of a CMF. At the periphery, there was a small area of metaplastic bone. Immunohistochemically, the tumour cells were diffusely positive for smooth muscle actin and GRM1, and negative for S100 protein, desmin and cytokeratin AE1AE3. Whole transcriptome sequencing revealed a novel
PNISR
::
GRM1
gene fusion.
Our case indicates that CMF should be included in the differential diagnosis of soft tissue (including subcutaneous) tumours composed of spindle/ovoid cells, with a lobular architecture and chondromyxoid matrix. The diagnosis of CMF arising in soft tissues can be confirmed by identifying a
GRM1
gene fusion or GRM1 expression by immunohistochemistry.
ATP2A2 encodes the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA2) and has been identified as a defective gene in Darier disease (DD). It is an autosomal dominant genodermatosis, which is ...characterized by loss of adhesion between suprabasal epidermal keratinocytes (acantholysis) and abnormal keratinization (dyskeratosis). We examined 28 Slovenian patients with DD (the cohort of patients represents over 50% of all DD patients in Slovenia) and screened genomic DNA for ATP2A2 mutations and RNA for splice site mutations. We identified 7 different ATP2A2 mutations, 4 of which are novel: A516P, R559G, 544+1del6, and 1762-6del18. We also found two previously described polymorphisms 2741+54 G>A in intron XVIII and 2172 G>A (A724A) in exon 15, with allele frequencies of 64.2% and 11.3%, respectively. The mutations are scattered throughout the gene and affect the actuator, phosphorylation, stalk and transmembrane domains of SERCA2. A P160L mutation in a Slovene patient with severe DD and a history of deafness is another consistent genotype-phenotype correlation. It seems that mutations of the ATP2A2 gene may also play a role in the pathogenesis of deafness, which seems to be a new phenotypic characteristic of DD patients.
A novel PTPRZ1‐ETV1 fusion in gliomas Matjašič, Alenka; Zupan, Andrej; Boštjančič, Emanuela ...
Brain pathology,
March 2020, Letnik:
30, Številka:
2
Journal Article
Recenzirano
Odprti dostop
The aggressive nature of malignant gliomas and their genetic and clinical heterogeneity present a major challenge in their diagnosis and treatment. Development of targeted therapy brought attention ...on detecting novel gene fusions, since they represent promising therapeutic targets (eg, TRK inhibitors in NTRK fusion‐positive tumors). Using targeted next‐generation sequencing, we prospectively analyzed 205 primary brain tumors and detected a novel PTPRZ1‐ETV1 fusion transcript in 11 of 191 (5.8%) gliomas, including nine glioblastomas, one anaplastic oligodendroglioma and one pilocytic astrocytoma. PTPRZ1‐ETV1 fusion was confirmed by RT‐PCR followed by Sanger sequencing, and in‐silico analysis predicted a potential driver role. The newly detected fusion consists of the PTPRZ1 promoter in frame with the highly conserved DNA‐binding domain of ETV1 transcription factor. The ETV1 and PTPRZ1 genes are known oncogenes, involved in processes of tumor development. ETV1 is a member of the ETS family of transcription factors, already known oncogenic drivers in Ewing sarcoma, prostate cancer and gastrointestinal stromal tumors, but not in gliomas. Its overexpression contributes to tumor growth and more aggressive tumor behavior. PTPRZ1 is already considered to be a tumor growth promoting oncogene in gliomas. In 8%–16% of gliomas, PTPRZ1 is fused to the MET oncogene, resulting in a PTPRZ1‐MET fusion, which is associated with poorer prognosis but is also a positive predictive biomarker for treatment with kinase inhibitors. In view of the oncogenic role that the two fusion partners, PTPRZ1 and ETV1, exhibit in other malignancies, PTPRZ1‐ETV1 fusion might present a novel potential therapeutic target in gliomas. Although histopathological examination of PTPRZ1‐ETV1 fusion‐positive gliomas did not reveal any specific or unique pathological features, and the follow‐up period was too short to assess prognostic value of the fusion, careful monitoring of patients and their response to therapy might provide additional insights into the prognostic and predictive value of this novel fusion.
Aneurysmal bone cyst (ABC) is a benign but locally aggressive neoplasm, with a tendency for local recurrence. In contrast to other bone tumors with secondary cystic change, ABC is characterized by ...USP6 gene rearrangement. There is a growing list of known USP6 fusion partners, characterization of which has been enabled with the advent of next‐generation sequencing (NGS). The list of known fusion partners includes CDH11, CNBP, COL1A1, CTNNB1, EIF1, FOSL2, OMD, PAFAH1B1, RUNX2, SEC31A, SPARC, STAT3, THRAP3, and USP9X. Using NGS, we analyzed a series of 11 consecutive ABCs and identified USP6 fusions in all cases, providing further evidence that USP6 fusions are universally present in primary ABCs. We identified four novel fusion partners in five ABCs and confirmed them by RT‐PCR and Sanger sequencing, ASAP1, FAT1, SAR1A, and TNC (in two cases). Because of high sensitivity and specificity, detection of a USP6 fusion by NGS may assist in differentiating between ABC and its mimics, especially in small biopsy samples when a definite diagnosis cannot be achieved on morphological grounds alone. Further studies with a large number of cases and follow‐up are needed to determine whether different fusion partners are associated with specific clinical and pathologic features of ABCs.
Medullary thyroid carcinoma (MTC) is a rare endocrine malignancy with distinctive features separating it from other thyroid cancers. Cancer may be sporadic or occur as a consequence of the hereditary ...syndrome called multiple endocrine neoplasia type 2 (MEN2) with three distinct phenotypes in MEN2A, MEN2B and FMTC. Each variant of MEN2 results from different RET gene mutations, with a good genotype–phenotype correlation. The goal of the study was to develop a fast and accurate screening method for a reliable detection of hot-spot RET germline and sporadic tumor mutations. From a cohort of 191 patients with MTC and their relatives, 38 tested positive and 31 tested negative for a germline or somatic tumor RET mutation were selected. A positive HRM mutation pattern was detected in all mutation-positive patients and altogether the method was able to clearly differentiate between twenty different genotypes. A novel germline variant p.Ala639Thr was detected in MTC patient, which was determined to be likely benign. Analytical specificity was determined to be 98.6% and a sensitivity threshold was determined to be 30%. The fast and accurate HRM method reduces the turnaround time providing fast and important information, especially when targeted anti-tyrosine kinase therapy on tumor samples is considered. Overall, we developed a high-throughput, accurate and cost-effective approach for the detection of RET germline and sporadic tumor mutations.
•The development of high resolution melt analysis method for RET mutation detection•Novel germline RET proto-oncogene variant c.1915G>A (p.Ala639Thr)•A novel, high-throughput, accurate and cost-effective approach with faster turnaround time
To investigate differences in genotype distributions of single nucleotide polymorphisms within genes, encoding inflammatory mediators, among patients with rhegmatogenous retinal detachment (RRD) and ...patients with proliferative vitreoretinopathy (PVR).
A genetic association study was performed on 191 Slovenian patients, divided into 2 groups: 113 RRD patients with PVR and 78 RRD patients without PVR. Genotype distributions were investigated within the following 13 single nucleotide polymorphisms: rs3760396 (CCL2), rs9990554 (FGF2), rs17561 (IL1A), rs2069763 (IL2), rs1800795 (IL6), rs1800871 (IL10), rs3008 (JAK3), rs2229094 (LTA), rs1042522 (TP53), rs7656613 (PDGFRA), rs7226855 (SMAD7), rs1800471 (TGFB1), and rs1800629 (TNF).
Differences in genotype distributions between patients with RRD with or without PVR were detected in rs1800795 (IL6) (P = 0.04), rs1800871 (in the vicinity of the IL10) (P = 0.034), and rs1800471 (TGFB1) (P = 0.032). After adjustment none of the 13 analyzed single nucleotide polymorphisms showed statistically significant associations in single nucleotide polymorphism genotype distributions between patients with RRD with and without PVR.
Further research is needed, particularly expanded multicentric population-based studies, to clarify the issue of genetic contribution to PVR from different genetic, clinical, and population-based aspects.
Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable ...based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.
Glioblastoma (GB) is the most common primary malignant brain tumor, characterized by resistance to therapy. Despite aggressive treatment options, GB remains an incurable disease. Invasiveness and ...heterogeneity are key GB features that cannot be studied in preclinical in vitro models. In this study, we investigated the effects of standard therapy using patient-derived GB organoids (GBOs). GBOs reflect the complexity and heterogeneity of the original tumor tissue. No significant effect on GBO viability or invasion was observed after irradiation and temozolomide treatment. E3 ubiquitin-protein ligase (MDM2), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the serine/threonine kinases ATM and ATR were upregulated at the gene and protein levels after treatment. Our results show that the p53 pathway and DNA-damage response mechanisms were triggered, suggesting that GBOs recapitulate GB therapy resistance. GBOs thus provide a highly efficient platform to assess the specific responses of GB patients to therapy and to further explore therapy resistance.
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•GBOs retain cellular diversity, heterogeneity, and transcriptional profile of GBs•GBOs recapitulate GB therapy resistance•Expression of ATM, ATR, MDM2, and CDKN1A is increased in GBOs
Cellular physiology; Cellular toxicology; In vitro toxicology including D culture; Technical aspects of cell biology; Cancer
Abstract
Background: The Slovenian territory is geographically positioned between the Alps, the Adriatic Sea, the Pannonian basin and the Dinaric Mountains and, as such, has served as a passageway ...for different populations over different periods of time. Turbulent historic events and the diverse geography of the region have produced a diverse contemporary population whose genetic analysis could provide insight into past demographic events.
Aim: The aim of this study was to analyse Y-chromosome biallelic and STR markers in a Slovenian population from five different regions.
Subjects and methods: A total of 42 Y-chromosomal biallelic markers and 17 Y-STRs were genotyped in 399 individuals from five different Slovenian regions.
Results: The analysis of Y-chromosome markers revealed 29 different haplogroups in the Slovenian population, with the most common being R1a1a, R1b, I2a1 and I1. Analysis of the genetic affiliations between different populations revealed strong affiliations of the Slovenian gene pool with West Slavic populations.
Conclusion: Analysis of Y-chromosomal markers in five Slovenian regions revealed a diverse genetic landscape. Slovenian population display close genetic affiliations with West Slavic populations. The homogenous genetic strata of the West Slavic populations and the Slovenian population suggest the existence of a common ancestral Slavic population in central European region.
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