Hotspot Variants Research Articles

Detection of Hybrid Fusion Transcripts, Aberrant Transcript Expression, and Specific Single Nucleotide Variants in Acute Leukemia and Myeloid Disorders with Recurrent Gene Rearrangements

Introduction: A variety of gene rearrangements and molecular alterations are key drivers in the pathobiology of acute leukemia and myeloid disorders; current classification systems increasingly incorporate these findings in diagnostic algorithms. Therefore, clinical laboratories require versatile tools, which can detect an increasing number and variety of molecular and cytogenetic alterations of clinical significance. Methods: We validated an RNA-based next-generation sequencing (NGS) assay that enables the detection of: (i) numerous hybrid fusion transcripts (including rare/novel gene partners), (ii) aberrantly expressed EVI1 (MECOM) and IKZF1 (Del exons 4–7) transcripts, and (iii) hotspot variants in KIT, ABL1, NPM1 (relevant in the context of gene rearrangement status). Results: For hybrid fusion transcripts, the assay showed 98–100% concordance for known positive and negative samples, with an analytical sensitivity (i.e., limit of detection) of approximately 0.8% cells. Samples with underlying EVI1 (MECOM) translocations demonstrated increased EVI1 (MECOM) expression. Aberrant IKZF1 (Del exons 4–7) transcripts detectable with the assay were also present on orthogonal reverse transcription PCR. Specific hotspot mutations in KIT, ABL1, and NPM1 detected with the assay showed 100% concordance with orthogonal testing. Lastly, several illustrative samples are included to highlight the assay’s clinically relevant contributions to patient workup. Conclusion: Through its ability to simultaneously detect various gene rearrangements, aberrantly expressed transcripts, and hotspot mutations, this RNA-based NGS assay is a valuable tool for clinical laboratories to supplement other molecular and cytogenetic methods used in the diagnostic workup and in clinical research for patients with acute leukemia and myeloid disorders.

Results of combined newborn hearing and deafness gene screening in Yuncheng area of Shanxi Province

To analyze the clinical significance of combined newborn hearing and deafness gene screening in Yuncheng area of Shanxi Province. Results of audiological examinations, including transient evoked otoacoustic emission and automatic discriminative auditory brainstem evoked potentials, for 6 723 newborns born in Yuncheng area from January 1, 2021 to December 31, 2021, were retrospectively analyzed. Those who failed one of the tests were considered to have failed the examination. A deafness-related gene testing kit was used to detect 15 hot spot variants of common deafness-associated genes in China including GJB2, SLC26A4, GJB3, and mtDNA12S rRNA. Neonates who had passed the audiological examinations and those who had not were compared using a chi-square test. Among the 6 723 neonates, 363 (5.40%) were found to carry variants. These have included 166 cases (2.47%) with GJB2 gene variants, 136 cases (2.03%) with SLC26A4 gene variants, 26 cases (0.39%) with mitochondrial 12S rRNA gene variants, and 33 cases (0.49%) with GJB3 gene variants. Among the 6 723 neonates, 267 had failed initial hearing screening, among which 244 had accepted a re-examination, for which 14 cases (5.73%) had failed again. This has yielded an approximate prevalence of hearing disorder of 0.21% (14/6 723). Among 230 newborns who had passed the re-examination, 10 (4.34%) were found to have carried a variant. By contrast, 4 out of the 14 neonates (28.57%) who had failed the re-examination had carried a variant, and there was a significant difference between the two groups (P < 0.05). Genetic screening can provide an effective supplement to newborn hearing screening, and the combined screening can provide a best model for the prevention of hearing loss, which can enable early detection of deafness risks, targeted prevention measures, and genetic counseling to provide accurate prognosis for the newborns.

The genetic and clinical spectrum in a cohort of 39 families with complex inherited peripheral neuropathies.

With complicated conditions and a large number of potentially causative genes, the diagnosis of a patient with complex inherited peripheral neuropathies (IPNs) is challenging. To provide an overview of the genetic and clinical features of 39 families with complex IPNs from central south China and to optimize the molecular diagnosis approach to this group of heterogeneous diseases, a total of 39 index patients from unrelated families were enrolled, and detailed clinical data were collected. TTR Sanger sequencing, hereditary spastic paraplegia (HSP) gene panel, and dynamic mutation detection in spinocerebellar ataxia (SCAs) were performed according to the respective additional clinical features. Whole-exome sequencing (WES) was used in patients with negative or unclear results. Dynamic mutation detection in NOTCH2NLC and RCF1 was applied as a supplement to WES. As a result, an overall molecular diagnosis rate of 89.7% was achieved. All 21 patients with predominant autonomic dysfunction and multiple organ system involvement carried pathogenic variants in TTR, among which nine had c.349G > T (p.A97S) hotspot variants. Five out of 7 patients (71.4%) with muscle involvement harbored biallelic pathogenic variants in GNE. Five out of 6 patients (83.3%) with spasticity reached definite genetic causes in SACS, KIF5A, BSCL2, and KIAA0196, respectively. NOTCH2NLC GGC repeat expansions were identified in all three cases accompanied by chronic coughing and in one patient accompanied by cognitive impairment. The pathogenic variants, p.F284S and p.G111R in GNE, and p.K4326E in SACS, were first reported. In conclusion, transthyretin amyloidosis with polyneuropathy (ATTR-PN), GNE myopathy, and neuronal intranuclear inclusion disease (NIID) were the most common genotypes in this cohort of complex IPNs. NOTCH2NLC dynamic mutation testing should be added to the molecular diagnostic workflow. We expanded the genetic and related clinical spectrum of GNE myopathy and ARSACS by reporting novel variants.

Tumour purity as an underlying key factor in tumour mutation detection in colorectal cancer

Tumour purity as an underlying key factor in tumour mutation detection in colorectal cancer

The spectrum of phenylalanine hydroxylase variants and genotype–phenotype correlation in phenylketonuria patients in Gansu, China

BackgroundPhenylketonuria (PKU) is a common, congenital, autosomal recessive, metabolic disorder caused by Phenylalanine hydroxylase (PAH) variants.Methods967 PKU patients from Gansu, China were genotyped by Sanger sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing. We analyzed the variants of PAH exons, their flanking sequences, and introns.ResultsThe detection of deep intronic variants in PAH gene can significantly improve the genetic diagnostic rate of PKU. The distribution of PAH variants among PKU subtypes may be related to the unique genetic background in Gansu, China.ConclusionThe identification of PAH hotspot variants will aid the development of large-scale neonatal genetic screening for PKU. The five new PAH variants found in this study further expand the spectrum of PAH variants. Genotype–phenotype correlation analysis may help predict the prognosis of PKU patients and enable precise treatment regimens to be developed.

WFS1 autosomal dominant variants linked with hearing loss: update on structural analysis and cochlear implant outcome

BackgroundWolfram syndrome type 1 gene (WFS1), which encodes a transmembrane structural protein (wolframin), is essential for several biological processes, including proper inner ear function. Unlike the recessively inherited Wolfram syndrome, WFS1 heterozygous variants cause DFNA6/14/38 and wolfram-like syndrome, characterized by autosomal dominant nonsyndromic hearing loss, optic atrophy, and diabetes mellitus. Here, we identified two WFS1 heterozygous variants in three DFNA6/14/38 families using exome sequencing. We reveal the pathogenicity of the WFS1 variants based on three-dimensional (3D) modeling and structural analysis. Furthermore, we present cochlear implantation (CI) outcomes in WFS1-associated DFNA6/14/38 and suggest a genotype-phenotype correlation based on our results and a systematic review.MethodsWe performed molecular genetic test and evaluated clinical phenotypes of three WFS1-associated DFNA6/14/38 families. A putative WFS1–NCS1 interaction model was generated, and the impacts of WFS1 variants on stability were predicted by comparing intramolecular interactions. A total of 62 WFS1 variants associated with DFNA6/14/38 were included in a systematic review.ResultsOne variant is a known mutational hotspot variant in the endoplasmic reticulum (ER)-luminal domain WFS1(NM_006005.3) (c.2051 C > T:p.Ala684Val), and the other is a novel frameshift variant in transmembrane domain 6 (c.1544_1545insA:p.Phe515LeufsTer28). The two variants were pathogenic, based on the ACMG/AMP guidelines. Three-dimensional modeling and structural analysis show that non-polar, hydrophobic substitution of Ala684 (p.Ala684Val) destabilizes the alpha helix and contributes to the loss of WFS1-NCS1 interaction. Also, the p.Phe515LeufsTer28 variant truncates transmembrane domain 7–9 and the ER-luminal domain, possibly impairing membrane localization and C-terminal signal transduction. The systematic review demonstrates favorable outcomes of CI. Remarkably, p.Ala684Val in WFS1 is associated with early-onset severe-to-profound deafness, revealing a strong candidate variant for CI.ConclusionsWe expanded the genotypic spectrum of WFS1 heterozygous variants underlying DFNA6/14/38 and revealed the pathogenicity of mutant WFS1, providing a theoretical basis for WFS1-NCS1 interactions. We presented a range of phenotypic traits for WFS1 heterozygous variants and demonstrated favorable functional CI outcomes, proposing p.Ala684Val a strong potential marker for CI candidates.

PD46-03 UTILITY OF URINARY TUMOR DNA DETECTION IN TREATMENT-NA&amp;IUML;VE UTUC

You have accessJournal of UrologyCME1 Apr 2023PD46-03 UTILITY OF URINARY TUMOR DNA DETECTION IN TREATMENT-NA&IUML;VE UTUC Heather Huelster, Facundo Davaro, Kyle Rose, Billie Gould, Tiantian Zheng, Prithvi Murthy, Shreyas Naidu, Lucia Camperlengo, Pan Du, Shidong Jia, Aram Vosoughi, and Roger Li Heather HuelsterHeather Huelster More articles by this author , Facundo DavaroFacundo Davaro More articles by this author , Kyle RoseKyle Rose More articles by this author , Billie GouldBillie Gould More articles by this author , Tiantian ZhengTiantian Zheng More articles by this author , Prithvi MurthyPrithvi Murthy More articles by this author , Shreyas NaiduShreyas Naidu More articles by this author , Lucia CamperlengoLucia Camperlengo More articles by this author , Pan DuPan Du More articles by this author , Shidong JiaShidong Jia More articles by this author , Aram VosoughiAram Vosoughi More articles by this author , and Roger LiRoger Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urine-based molecular biomarkers correlate with tumor variants in urothelial carcinoma of the bladder, showing promise for noninvasive detection of newly diagnosed tumors. However, the utility of next-generation sequencing (NGS) to detect urinary tumor DNA (utDNA) in upper tract urothelial carcinoma (UTUC) remains to be explored. Herein, we prospectively sequenced tumors and utDNA at the time of and serially after radical nephroureterectomy (RNU) to examine the feasibility of using utDNA in detection and surveillance of UTUC. METHODS: Fourteen systemic therapy naïve patients with high-grade, clinically-localized UTUC undergoing RNU were prospectively accrued. Urine was collected preoperatively on the day of surgery and urinary supernatant and cell pellets (UCP) processed for extraction of utDNA. NGS used shallow whole-genome sequencing (WGS) to detect genome-wide copy number changes and a 152-pancancer panel (PredicineCARE) to profile single nucleotide variants, small insertions/deletions, and gene-level copy number changes. Variants with mutation allele frequency ≥0.25% in supernatant, ≥0.3% in UCP, and cancer hotspot variants >0.1% were reported. RESULTS: Prior to RNU, utDNA variants were detected in 11/11 (100%) of urinary supernatants and 9/14 (64%) of UCPs using NGS. The most common alterations detected in the urine were in TP53 (81%), TERT promoter (55%), FGFR3 (55%), and ERBB2 (45%). More tumor variants were detected in urine supernatant than UCP (22.9 vs 2.4, p=0.004). Use of a bespoke tumor-informed approach to adjust MAF thresholds for calling variants increased utDNA detection by a mean 0.5 (range 0-3) variants in urinary supernatant and mean 2.3 (range 0-7) variants in UCP. There was no significant difference in the number of variants detected in urine of patients with UTUC staged as <pT2 compared to ≥pT2 or pN+. CONCLUSIONS: UTUC tumor variants are detectable in the urine of treatment-naïve patients undergoing RNU for UTUC using both a panel-based approach and WGS. utDNA detection and dynamics may present an opportunity for personalized monitoring of UTUC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1170 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Heather Huelster More articles by this author Facundo Davaro More articles by this author Kyle Rose More articles by this author Billie Gould More articles by this author Tiantian Zheng More articles by this author Prithvi Murthy More articles by this author Shreyas Naidu More articles by this author Lucia Camperlengo More articles by this author Pan Du More articles by this author Shidong Jia More articles by this author Aram Vosoughi More articles by this author Roger Li More articles by this author Expand All Advertisement PDF downloadLoading ...

Novel variants and phenotypic heterogeneity in a cohort of 11 Chinese children with Wiedemann-Steiner syndrome.

Objective: Wiedemann-Steiner syndrome (WSS) is a rare autosomal dominant disorder caused by deleterious heterozygous variants of the KMT2A gene. This study aims to describe the phenotypic and genotypic features of Chinese WSS patients, and assess therapeutic effects of recombinant human growth hormone (rhGH). Methods: Eleven Chinese children with WSS were enrolled in our cohort. Their clinical, imaging, biochemical and molecular findings were analyzed retrospectively. Moreover, the phenotypic features of 41 previously reported Chinese WSS patients were reviewed and included in our analysis. Results: In our cohort, the 11 WSS patients presented with classic clinical manifestations, but with different frequencies. The most common clinical features were short stature (90.9%) and developmental delay (90.9%), followed by intellectual disability (72.7%). The most frequent imaging features were patent ductus arteriosus (57.1%) and patent foramen ovale (42.9%) in cardiovascular system, and abnormal corpus callosum (50.0%) in the brain. In the set comprising 52 Chinese WSS patients, the most common clinical and imaging manifestations were developmental delay (84.6%), intellectual disability (84.6%), short stature (80.8%) and delayed bone age (68.0%), respectively. Eleven different variants, including three known and eight novel variants, of the KMT2A gene were identified in our 11 WSS patients without a hotspot variant. Two patients were treated with rhGH and yielded satisfactory height gains, but one developed acceleration of bone age. Conclusion: Our study adds 11 new patients with WSS, reveals different clinical characteristics in Chinese WSS patients, and extends the mutational spectrum of the KMT2A gene. Our study also shares the therapeutic effects of rhGH in two WSS patients without GH deficiency.

KRAS, NRAS, BRAF, HER2 and MSI Status in a Large Consecutive Series of Colorectal Carcinomas

This study aimed to analyze clinical and regional factors influencing the distribution of actionable genetic alterations in a large consecutive series of colorectal carcinomas (CRCs). KRAS, NRAS and BRAF mutations, HER2 amplification and overexpression, and microsatellite instability (MSI) were tested in 8355 CRC samples. KRAS mutations were detected in 4137/8355 (49.5%) CRCs, with 3913 belonging to 10 common substitutions affecting codons 12/13/61/146, 174 being represented by 21 rare hot-spot variants, and 35 located outside the "hot" codons. KRAS Q61K substitution, which leads to the aberrant splicing of the gene, was accompanied by the second function-rescuing mutation in all 19 tumors analyzed. NRAS mutations were detected in 389/8355 (4.7%) CRCs (379 hot-spot and 10 non-hot-spot substitutions). BRAF mutations were identified in 556/8355 (6.7%) CRCs (codon 600: 510; codons 594-596: 38; codons 597-602: 8). The frequency of HER2 activation and MSI was 99/8008 (1.2%) and 432/8355 (5.2%), respectively. Some of the above events demonstrated differences in distribution according to patients' age and gender. In contrast to other genetic alterations, BRAF mutation frequencies were subject to geographic variation, with a relatively low incidence in areas with an apparently warmer climate (83/1726 (4.8%) in Southern Russia and North Caucasus vs. 473/6629 (7.1%) in other regions of Russia, p = 0.0007). The simultaneous presence of two drug targets, BRAF mutation and MSI, was observed in 117/8355 cases (1.4%). Combined alterations of two driver genes were detected in 28/8355 (0.3%) tumors (KRAS/NRAS: 8; KRAS/BRAF: 4; KRAS/HER2: 12; NRAS/HER2: 4). This study demonstrates that a substantial portion of RAS alterations is represented by atypical mutations, KRAS Q61K substitution is always accompanied by the second gene-rescuing mutation, BRAF mutation frequency is a subject to geographical variations, and a small fraction of CRCs has simultaneous alterations in more than one driver gene.

Incidence and genetic variants of inborn errors of metabolism identified through newborn screening: A 7-year study in eastern coastal areas of China.

The incidence of inborn errors of metabolism (IEM) varies across countries and areas. Currently, there are no studies on IEM using newborn screening (NBS) in eastern coastal areas of China. We aimed to estimate the incidence and genetic variants of IEM and understand the spectrum of diseases caused by IEM and variants among them in this area. The NBS performed by tandem mass spectrometry (MS/MS) from 2016 to 2021 was retrospectively reviewed. Heel blood was collected from all newborns 72 h after birth. Targeted massively parallel sequencing was performed for genetic analysis. Among 245,194 newborns, 95 were diagnosed with IEM, the overall incidence observed was-IEM: 1/2581; amino acid metabolism disorder: 1/4715; organic acid metabolism disorder: 1/11676; and fatty acid metabolism disorder: 1/11145. The incidence of different IEM was in the range of 1/245194 to 1/6452. Phenylketonuria (PKU, 1/7211) was the most common IEM, followed by methylmalonic acidemia (MMA, 1/27244), short-chain acyl-CoA dehydrogenase deficiency (SCADD, 1/30649), and citrin deficiency (CD, 1/35028). For genetic variants, the common hotspot variants found were-PAH gene for PKU: c.728G > A, c.442-1G > A, c.611A > G, c.721C > T; PTS gene for non-classical PKU: c.259C > T; MMACHC gene for MMA: c.658_660delAAG, c.609G > A; MMUT gene for MMA: c.1663G > A; ACADS gene for SCADD: c.1031A > G and c.1130C > T; and SLC25A13 gene for CD: c.1638_1660dup, c.852_855del. This study displayed the diseases and varied spectrum of IEM in eastern coastal areas of China. Implementing NBS for IEM by MS/MS combined with massively parallel sequencing can offer an improved plan for NBS to detect IEM.

Cross-Ancestry Genome-Wide Association Study Defines the Extended CYP2D6 Locus as the Principal Genetic Determinant of Endoxifen Plasma Concentrations.

The therapeutic efficacy of tamoxifen is predominantly mediated by its active metabolites 4-hydroxy-tamoxifen and endoxifen, whose formation is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6). Yet, known CYP2D6 polymorphisms only partially determine metabolite concentrations in vivo. We performed the first cross-ancestry genome-wide association study with well-characterized patients of European, Middle-Eastern, and Asian descent (n=497) to identify genetic factors impacting active and parent metabolite formation. Genome-wide significant variants were functionally evaluated in an independent liver cohort (n=149) and in silico. Metabolite prediction models were validated in two independent European breast cancer cohorts (n=287, n=189). Within a single 1-megabase (Mb) region of chromosome 22q13 encompassing the CYP2D6 gene, 589 variants were significantly associated with tamoxifen metabolite concentrations, particularly endoxifen and metabolic ratio (MR) endoxifen/N-desmethyltamoxifen (minimal P=5.4E-35 and 2.5E-65, respectively). Previously suggested other loci were not confirmed. Functional analyses revealed 66% of associated, mostly intergenic variants to be significantly correlated with hepatic CYP2D6 activity or expression (ρ=0.35 to -0.52), and six hotspot regions in the extended 22q13 locus impacting gene regulatory function. Machine learning models based on hotspot variants (n=12) plus CYP2D6 activity score (AS) increased the explained variability (~ 9%) compared with AS alone, explaining up to 49% (median R2 ) and 72% of the variability in endoxifen and MR endoxifen/N-desmethyltamoxifen, respectively. Our findings suggest that the extended CYP2D6 locus at 22q13 is the principal genetic determinant of endoxifen plasma concentration. Long-distance haplotypes connecting CYP2D6 with adjacent regulatory sites and nongenetic factors may account for the unexplained portion of variability.

Unique mutation spectrum of progressive pseudorheumatoid dysplasia in the Chinese population: a retrospective genotype–phenotype analysis of 105 patients

BackgroundProgressive pseudorheumatoid dysplasia (PPRD) is a rare genetic disease with autosomal recessive inheritance. There was a lack of genotype–phenotype correlation data from the Chinese population. This study aimed to identify the genotype and phenotype characteristics of Chinese PPRD patients and to conduct a genotype–phenotype analysis of Chinese PPRD patients.MethodsGenetic analysis was performed for suspected PPRD patients from Peking Union Medical College Hospital. Medical records were collected from the electronic medical record system and patient-held portable health records. Published Chinese PPRD cases were gathered from both international and Chinese local databases. We collected demographic information, genetic variants, clinical manifestations, and imaging characteristics for further analysis.ResultsWe included 105 Chinese PPRD patients in the current study. Thirty-three variants, including nine novels and five hotspot variants, were identified, with 26/33 (79%) variants exclusively seen in the Chinese population. Chinese PPRD patients share a phenotype similar to that in international reports. Joint involvement may progress with age (R2 = 0.2541). Long bone shortening and severe deformities occur in three patients with biallelic null variants, of which at least one variant is located in exon 2. Among hotspot variants, c.624dupA (p.C209Mfs*21) were associated with later onset and more involved joints. Elbow joints were more likely to be affected in patients carrying c.624dupA (p.C209Mfs*21) and c.866dupA (p.S209Efs*13). Shoulder joints are more likely to be involved in patients with biallelic null variants (P = 0.027).ConclusionsChinese PPRD patients share a unique mutation spectrum. Among the five hotspot variants, c.624dupA is associated with later onset of disease, more extensive joint involvement, and a tendency to affect elbow joints. Biallelic null variants with at least one variant in exon 2 could be a likely cause of long bone shortening and severe deformities.

Genetic abnormalities assist in pathological diagnosis and EBV-positive cell density impact survival in Chinese angioimmunoblastic T-cell lymphoma patients.

To explore the application of genetic abnormalities in the diagnosis of angioimmunoblastic T-cell lymphoma (AITL) and the reliable pathological prognostic factors. This study included 53 AITL cases, which were reviewed for morphological patterns, immunophenotypes, presence of Hodgkin and Reed-Sternberg (HRS)-like cells, and co-occurrence of B cell proliferation. The Epstein-Barr virus (EBV)-positive cells in tissues were counted, and cases were classified into "EBV encoded RNA (EBER) high-density" group if >50/HPF. Targeted exome sequencing was performed. Mutation data can assist AITL diagnosis: 1) with considerable HRS-like cells (20 cases): RHOA mutated in 14 cases (IDH2 co-mutated in 3 cases, 4 cases with rare RHOA mutation), TET2 was mutated in 5 cases (1 case co-mutated with DNMT3A), and DNMT3A mutated in 1 case; 2) accompanied with B cell lymphoma (7 cases): RHOA mutated in 4 cases (1 case had IDH2 mutation), TET2 mutated in 2 cases and DNMT3A mutated in 1 case; 3) mimic peripheral T cell lymphoma, not otherwise specified (5 cases): RHOA mutated in 2 cases (IDH2 co-mutated in 1 case), TET2 mutated in 3 cases, and DNMT3A mutated in 1 case; 4) pattern 1 (1 case), RHOA and TET2 co-mutated. Besides RHOAG17V (30/35), rare variant included RHOAK18N, RHOAR68H, RHOAC83Y, RHOAD120G and RHOAG17del, IDH2R172 co-mutated with IDH2M397V in one case. There were recurrent mutations of FAT3, PCLO and PIEZO1 and genes of epigenetic remodeling, T-cell activation, APC and PI3K/AKT pathway. EBER high-density independently indicated adverse overall survival and progression-free survival (P=0.046 and P=0.008, Kaplan-Meier/log-rank). Over half AITL cases might be confused in diagnosis for certain conditions without mutation data. Targeted exome sequencing with a comprehensive panel is crucial to detect both hot-spot and rare mutation variants for RHOA and IDH2 and other recurrent mutated genes in addition to TET2 and DNMT3A. EBER high-density independently indicated adverse survival.

Clinical and Molecular Features of a Chinese Cohort With Syndromic and Nonsyndromic Retinal Dystrophies Related to the CEP290 Gene

To reveal the clinical and genetic features of 54 Chinese pedigrees with syndromic or nonsyndromic retinal dystrophies related to CEP290 and to explore the genotype-phenotype correlation. Retrospective cohort study. Patients diagnosed with nonsyndromic inherited retinal dystrophy (IRD) or syndromic ciliopathy (SCP) were enrolled. We identified 61 patients from 54 families carrying biallelic pathogenic CEP290 variants using next-generation sequencing, Sanger sequencing, and co-segregation validation. Genotype-phenotype correlation was evaluated. This study included 37 IRD patients from 32 families and 24 patients with SCP from 22 pedigrees. Four retinal dystrophy phenotypes were confirmed: Leber congenital amaurosis (LCA, 46/61), early-onset severe retinal dystrophy (EOSRD, 4/61), retinitis pigmentosa (RP, 10/61), and cone-rod dystrophy (CORD, 1/61). The SCP phenotypes included Joubert syndrome (JS) (23/24) and Bardet-Biedl syndrome (BBS) (1/24). We detected 73 different CEP290 variants, of which 33 (45.2%) were not previously reported. Two novel copy number variations (CNVs) and 1 novel pathogenic synonymous change were identified. The most recurrent alterations in the IRD and SCP were p.Q123* (6/64, 9.4%) and p.I556Ffs*17 (10/44, 22.7%), respectively. IRD patients carried more stop-gain alleles (25/64, 39.1%), whereas SCP patients carried more frameshift alleles (23/44, 52.3%). LCA was the most common retinal dystrophy phenotype, and JS was the most prevalent syndrome in CEP290 patients; RP/CORD and BBS may be present in early adulthood. The hot spot variants and distribution of genotypes were distinct between IRD and SCP. Our study expands the CEP290 variant spectrum and enhances the current knowledge of CEP290 heterogeneity.

Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants.

Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls' isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.

High Deliverability of a Midostaurin Triplet Regimen Incorporating Venetoclax and Low Dose Cytarabine in Non-Adverse Cytogenetic Risk Acute Myeloid Leukaemia: A Sub-Analysis of the Australasian Leukaemia Lymphoma Group (ALLG) Intervene Study

Background Adaptive resistance mechanisms leading to treatment failure in older patients (pts) receiving frontline venetoclax (VEN) combination therapies include emergence/expansion of kinase activating mutations (principally, FLT3-ITD) in pts with non-adverse (NON-ADV) karyotype AML. In both the RATIFY and QuANTUM-First trials, the FLT3 inhibitor was scheduled sequentially after chemotherapy, with no evidence that this approach augmented marrow toxicity. We conducted the Phase 1b/2 study, INTERVENE, to explore the deliverability and preliminary efficacy of a novel triplet regimen incorporating the FLT3/multi-kinase inhibitor midostaurin (MIDO) delivered sequentially after low dose cytarabine (LDC), in tandem combination with VEN. We hypothesized that this triplet schedule would suppress emergence of kinase-activating mutations on VEN-based therapy. MethodsEligibility: Pts with treatment naïve AML (excluding APL), aged ≥60 years and unfit for intensive chemotherapy. Prior hypomethylating agents for antecedent myeloid neoplasms were permitted. Pts with NON-ADV karyotype were enrolled to receive VEN-LDC-MIDO. Treatment (safety run-in): LDC 20 mg/m2 SC D1-10, MIDO 50 mg BD commenced on D11 and continued to D28. After a dose ramp-up in cycle 1 (C1), VEN D1-28 was administered at two dose levels (1= 400 mg, 2= 600 mg). Azole antifungals were prohibited during C1 but allowed from C2 with VEN dose reduced to 100 mg. Molecular studies: NGS using a custom 48-gene Roche KAPA HyperCap panel (sensitivity 1%) and FLT3-ITD PCR-NGS (sensitivity 10-4-10-5) were performed on serial bone marrow samples. RT-qPCR was used to detect NPM1 measurable residual disease (MRD) (sensitivity 10-6). Results First pt enrolled: 7SEP2020. Data cut-off: 31MAY2022. Eighteen pts were enrolled to receive VEN-LDC-MIDO in the run-in phase. Median age was 77 years (range 73-87, 84% age≥75). 44% had secondary AML, including 11% t-AML. 2 pts withdrew consent within the first 7 days due to non-therapy related reasons and were not response evaluable. 30-day mortality was 0%. There were no dose limiting toxicities observed. With a median follow up of 18 months (m), median overall survival (OS) was not reached (95% CI 8.02-NR). Overall response rate (CR+CRi+CRh) by intention-to-treat was 77.8%, with 44.4% CR. Median relapse free survival was 11.7m. To assess treatment deliverability, we evaluated the inter-cycle time for the first 12 cycles among pts achieving CR/CRi/CRh (Fig 1A). The median inter-cycle times ranged from 28-35 days, with majority commencing the next cycle in <42 days. There was no evidence of cumulative lengthening of inter-cycle time with successive treatment cycles, suggesting that MIDO did not promote marrow toxicity when combined with VEN-LDC. Pts received a median number of 9 cycles (range 1-16), with 7 completing ≥12 cycles (Fig 1B). Of these, 3 electively ceased therapy (at C12, C14, C16), with a treatment-free remission (TFR) lasting 3.3-5.7m, from which one relapsed. For pts opting for therapy continuation (TC) >12m (4/7), one has relapsed. Of the remaining 5 (2 TFR, 3 TC) pts with durable remissions, 4 had either NPM1 or IDH2 mutations at enrolment and all achieved undetectable MRD in CR. One additional patient with NPM1 mutationat diagnosis relapsed after C5 with NPM1 negative AML. Molecular data and treatment outcomes are shown in Fig 1B. Two of 16 response-evaluable pts had FLT3 mutations at baseline (1=FLT3-ITD, 1=TKD). Using PCR-NGS to enhance detection of FLT3-ITD microclones, an additional 3 FLT3-ITDs were identified in 2 pts. During VEN-LDC-MIDO, all 4 FLT3-ITDs were suppressed to undetectable levels by PCR-NGS. No pts relapsed with FLT3 variants at the time of progression. Other kinase variants extinguished by VEN-LDC-MIDO include NRAS G12D and KRAS G12D. In contrast, 4 pathways of adaptive resistance were observed at time of treatment failure: emergence of 1) TP53 hotspot variants (N=2), 2) PPM1D frameshift variants (N=2), 3) BAX inactivating variant (N=1), and 4) evolution of KRAS Q61 mutations (N=2), Conclusion After a median follow up of 18m, we report that VEN-LDC-MIDO, with MIDO delivered sequentially after LDC, is tolerable with excellent treatment deliverability. Preliminary efficacy of this triplet is promising, with molecular evidence of FLT3-ITD suppression in all cases so far. A randomized phase 2 comparison of VEN-LDC-MIDO versus VEN-LDC is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Somatic IDH1 Hotspot Variants in Chinese Patients With Pheochromocytomas and Paragangliomas.

IDH1 is a pheochromocytoma/paraganglioma (PPGL) susceptibility gene; however, its role, especially in the Chinese population, has not been characterized. To determine the prevalence of somatic IDH1 hotspot variants in a large cohort of Chinese patients with PPGLs and to summarize associated phenotypes. This retrospective cross-sectional study was based on a main cohort of 1141 patients with PPGLs from 2 tertiary-care centers in China. We included 50 cases with urinary bladder paragangliomas (UBPGLs), of whom 29 were part of the main cohort and 21 were from other centers. Two additional cases with IDH1 hotspot variants not part of the main cohort were also included for summarizing IDH1-associated phenotypes. Next-generation sequencing of tumor DNA was used to analyze a customized panel of genes. The overall prevalence of IDH1 hotspot variants in the main cohort was 0.5% (6/1141). Among those PPGLs without mutations in 15 common driver genes, the prevalence of IDH1 variants was 0.9% (4/455). When restricted to paraganglioma (PGL) without mutations, the prevalence reached 4.7% (4/86). Among UBPGLs, IDH1 hotspot variants accounted for 8% (4/50). Together, all 10 patients (9 PGLs and 1 pheochromocytoma) with IDH1 hotspot variants, including 3 females with concurrent EPAS1 hotspot variants, had apparently sporadic tumors, without metastasis or recurrence. There were 3 patients with biochemical data, all showing a non-adrenergic phenotype. The somatic IDH1 hotspot variants cause PPGL development in some Chinese patients, especially among those apparently sporadic PGLs with a non-adrenergic phenotype and without mutations in major PPGL driver genes.

Clinical characteristics and long-term outcomes of 12 children with vitamin D-dependent rickets type 1A: A retrospective study.

Vitamin D-dependent rickets type 1A (VDDR1A) is a rare autosomal recessive disorder caused by deficiency of the CYP27B1 gene. This study aims to investigate the phenotypic and genotypic features of VDDR1A children in southern China and evaluate the long-term therapeutic effects. Twelve children from southern China with VDDR1A were enrolled in this study. Their clinical, radiological, biochemical, and molecular findings were analyzed retrospectively. The rickets severity score (RSS), biochemical parameters, and height standard deviation score (HtSDS) were used to evaluate clinical outcomes. Six males and six females were included in this VDDR1A cohort. The age of onset was from 6 months to 1.8 years, and the age at diagnosis was 2.1 ± 0.8 years. The most common clinical symptoms at diagnosis were delayed walking (10/12) and severe growth retardation (9/12). HtSDS at diagnosis was negatively associated with age (p < 0.05). All patients presented with hypocalcemia, hypophosphatemia, increased serum alkaline phosphatase and parathyroid hormone, and high RSS at diagnosis. Two allelic variants of the CYP27B1 gene were identified in all patients, including nine different variants, four known and five novel, with c.1319_1325dupCCCACCC(p.Phe443Profs*24) being the most frequent. All patients were treated with calcitriol and calcium after diagnosis, and all patients but one were followed-up from 6 months to 15.6 years. HtSDS, RSS, and biochemical parameters were found to be improved during the first few years of the treatment. However, only five patients had good compliance. Although RSS and biochemical parameters were significantly improved, the HtSDS change was not significant from the time of diagnosis to the last visit, and seven patients remained of a short stature (HtSDS < -2). Our study extends the mutational spectrum of VDDR1A and finds a hotspot variant of the CYP27B1 gene in southern China. The results reconfirm the importance of early diagnosis and treatment compliance and reveal the challenge of height improvement in VDDR1A patients.

Deep Molecular and In Silico Protein Analysis of p53 Alteration in Myelodysplastic Neoplasia and Acute Myeloid Leukemia.

Mutation of the TP53 gene is one of the major drivers of myelodysplastic neoplasias (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MR). TP53 mutations present in these hematopoietic malignancies form a distinct molecular genetic cluster with a worse prognosis than without the alteration. However, besides well-characterized hot-spot variants, a significant proportion of TP53 alterations are of uncertain clinical significance. To enlighten so far unknown aspects, bone-marrow samples from altogether 77 patients are analyzed retrospectively with the diagnosis of AML-MR (26 cases), MDS-IB (12 cases), and MDS-LB (39 cases) according to WHO 2022 guidelines. Next-generation sequencing results are correlated with histological, cytogenetic, and survival data. Twenty out of the 30 TP53 mutation types detected by NGS are not categorized in current public databases; thus, their clinical significance remained mysterious. Because of the interpretation difficulties and the absence of clinical correlations, pathogenicity is established based on in silico approaches. The 12 pathogenicity classification systems, as well as protein stability, protein-DNA, protein-protein interaction, and post-translational modification analyses are applied. We found statistically significant differences between AML/MDS groups considering p53 pathogenicity, protein structural changes, and overall survival. The largest number of abnormalities with the most severe consequences are found in AML-MR cases. These molecular and in silico protein data further support that MDS with increased-blast (MDS-IB) is an intermediate group between AML-MR and MDS with low-blast (MDS-LB) patients, which frequently progresses to AML and is therefore considered a pre-leukemic condition.

Clinical and genetic characteristics of amyotrophic lateral sclerosis patients with ANXA11 variants.

Increasing genetic evidence supports the hypothesis that variants in the annexin A11 gene (ANXA11) contribute to amyotrophic lateral sclerosis pathogenesis. Therefore, we studied the clinical aspects of sporadic amyotrophic lateral sclerosis patients carrying ANXA11 variants. We also implemented functional experiments to verify the pathogenicity of the hotspot variants associated with amyotrophic lateral sclerosis-frontotemporal dementia. Korean patients diagnosed with amyotrophic lateral sclerosis (n = 882) underwent genetic evaluations through next-generation sequencing, which identified 16 ANXA11 variants in 26 patients. We analysed their clinical features, such as the age of onset, progression rate, initial symptoms and cognitive status. To evaluate the functional significance of the ANXA11 variants in amyotrophic lateral sclerosis-frontotemporal dementia pathology, we additionally utilized patient fibroblasts carrying frontotemporal dementia-linked ANXA11 variants (p.P36R and p.D40G) to perform a series of in vitro studies, including calcium imaging, stress granule dynamics and protein translation. The frequency of the pathogenic or likely pathogenic variants of ANXA11 was 0.3% and the frequency of variants classified as variants of unknown significance was 2.6%. The patients with variants in the low-complexity domain presented unique clinical features, including late-onset, a high prevalence of amyotrophic lateral sclerosis-frontotemporal dementia, a fast initial progression rate and a high tendency for bulbar-onset compared with patients carrying variants in the C-terminal repeated annexin homology domains. In addition, functional studies using amyotrophic lateral sclerosis-frontotemporal dementia patient fibroblasts revealed that the ANXA11 variants p.P36R and p.D40G impaired intracellular calcium homeostasis, stress granule disassembly and protein translation. This study suggests that the clinical manifestations of amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia spectrum patients with ANXA11 variants could be distinctively characterized depending upon the location of the variant.