Mutation Screening Research Articles

P20.03.A PREDICTING THE CLINICAL PHENOTYPE INNF2 RELATED SCHWANNOMATOSIS PATIENTS USING NEW GENETIC SCORES

Abstract BACKGROUND NF2 related Schwannomatosis (NF2 -SCHW MIM # 101000) is a rare autosomal dominant familial cancer syndromes caused by mutations in the NF2 gene, mainly due to NF2 point mutations or intragenic deletions. NF2 patients. In 95% of patients, bilateral vestibular schwannomas are present, but NF2 -SCHW patients can develop other tumors and show ophthalmic and dermatological signs. Several studies established a genotype-phenotype correlations; the UK NF2 Reference Group proposed a Genetic Severity Score (GSS) based on the type and location of NF2 germline variant observed. Catasus et al. after validation of the score in a Spanish cohort suggested a new score based also on a functional assay of Merlin and its downstream pathways (FGSS). Recently Teranish et al. used targeted deep sequencing to predict functional prognosis in those patients. MATERIAL AND methods 70 patients (median age 40) were identified by scanning the electronic NF2-SCHW patient database at Fondazione IRCCS C. Besta, where the patients had undergone full neurological, ophthalmic, and audiological assessment. The median follow-up was 14 yrs. Clinical diagnosis was established following the Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis”. All patients underwent NF2, SMARCB1 and LZTR1 mutational screening by NGS and MPLA analysis using blood DNA or the tissue of two different tumors when available.NF2 gene germline pathogenic were found in 45 subjects (64%), in most cases NF2 point mutations, but in 4 cases and whole gene deletions was observed and in 2 a ring NF2 chromosome was identified, in four cases a mosaic NF2-SCH was identified and in 21 (30%) patients no pathogenic variant was found. We assessed NF2-SCHW clinical phenotype in relation to the UK GSS and FGSS to validate their use as clinical and research tools, despite the significant phenotypic variability observed. RESULTS The disease outcome differed significantly depending both on clinicaland genetic factors. Among these factors,” Age of symptom onset” “Truncating”, “Mosaic”, and “whole gene deletion” had the most significant effects on functional disability. Conclusion GSS and FGSS showed significant correlation with several measures, allowing stratification of patients with severe and mild disease but not with moderate phenotype. Furthermore, we provided evidence on correlation between whole NF2 deletion extent and phenotype severity. Large cohorts of NF2-SCHW patients are needed to identify more accurate scores, becoming useful tools for patient management.

Frequency and prognostic influence of ASXL1 mutations and its potential association with BCR-ABL1 transcript type and smoke in chronic myeloid leukemia patients

BackgroundHeterogeneous response to tyrosine kinase inhibitors (TKIs) and progress to advance phases, still is a significant clinical problem. These are attributed to additional mutations in mutated non-ABL1 genes. we aimed to determine prognostic effects of ASXL1 mutations as a biomarker for diverse treatment response and disease progression to aid clinical management. MethodsWe performed ASXL1 gene mutational screening in 80 Ph+CML patients at different phases and 10 healthy control by direct sequencing method. Multiplex and qRT-PCR, standard chromosome banding analysis were used to determine BCR-ABL1 transcript type, molecular and cytogenetic responses respectively. Resultsoverall, four type mutations were identified in 11.25% of the patients. There was significant difference regarding mutation frequency between chronic and advance phases (P = 0.0002), sokal risk score (P = 0.0001), smoking (P = 0.02) and mean of during time of imatinib treatment (P = 0.009) between patients with and without ASXL1 mutations. ASXL1 mutations frequency had a bias toward younger than older and women than men, but no significant (P > 0.05). ASXL1 mutations were found more recurrently in patients carrying ABL1 KD mutations (P = 0.003). The risk of increasing resistance and disease progression in patients with ASXL1 mutations was 32 and 63 fold higher than those without mutations respectively (P = 0.01; P = 0.0002). The risk of ASXL1 mutations presence in patients with b2a2 transcript type was much higher than b3a2 type (P = 0.02, OR = 10). ConclusionOur findings suggest that ASXL1 mutations may be favorable predictive biomarkers to determine the best TKI for each patient, and to prevent CML progression.

Akut Miyeloid Lösemide FLT3 Mutasyonlarının Araştırılması: Hasta Sonuçları ve Tedavi Stratejileri Üzerine Tek Merkezli Gerçek Yaşam Verisi

Acute Myeloid Leukemia (AML) is a complex hematological malignancy with considerable genetic heterogeneity. Fms-like tyrosine kinase 3 (FLT3) mutations are associated with poor prognosis and occur in nearly 30% of AML cases. This study delves into the prevalence of FLT3 mutations, their impact on clinical outcomes, and the efficacy of various treatment approaches in a cohort of AML patients. We examined 157 de novo non-acute promyelocytic leukemia AML patients aged 20-95 years, screening for FLT3-ITD and FLT3-TKD mutations. We tailored chemotherapy based on age, ECOG performance status, and FLT3 mutation presence. Our research revealed that 27.3% of patients harbored FLT3 mutations, with 65% FLT3-ITD and 35% FLT3-TKD mutations. Those with FLT3 mutations exhibited higher mortality rates compared to patients without mutations. Age, FLT3 mutation status, and relapsed/refractory disease emerged as independent risk factors for mortality. Patients treated with midostaurin faced a lower mortality risk than those administered sorafenib. This study underscores the significance of FLT3 mutations in AML, their influence on clinical outcomes, and the advantages of targeted therapies. Our findings stress the urgency for further investigation aimed at enhancing the prognosis for AML patients with FLT3 mutations.

Concomitant Coronary Artery Disease in Identical Twins: Case Report and Systematic Literature Review.

Coronary artery disease (CAD) is multifactorial and strongly affected by genetic, epigenetic and environmental factors. Several studies have reported development of concomitant CAD in identical twins. We report a case in which a pair of Caucasian male monozygotic twins presented almost concomitantly with acute coronary syndrome (ACS) and had concordant coronary anatomy and identical site of occlusion. We performed a systematic literature review of PubMed, Web Of Science and Scopus databases from inception until 28 February 2023 of case reports/case series reporting the concomitant development of CAD in monozygotic twins. We found 25 eligible case reports with a total of 31 monozygotic twin pairs (including the case from our center) suffering from CAD and presenting (most of them simultaneously) with ACS (mean age of presentation: 45 ± 12 years, males: 81%). Coronary angiograms demonstrated lesion and anatomy concordance in 77% and 79% of the twin pairs, respectively. Screening for disease-related genetic mutations was performed in six twin pairs leading to the identification of five CAD-related genetic polymorphisms. This is the first systematic literature review of studies reporting identical twin pairs suffering from CAD. In summary, there is high concordance of coronary anatomy and clinical presentation between monozygotic twins. Future monozygotic twin studies-unbiased by age effects-can provide insights into CAD heritability being able to disentangle the traditional dyad of genetic and environmental factors and investigate the within-pair epigenetic drift.

Schwannomatosis: a Realm Reborn: year one.

In 2022, an international consensus recommendation revised the nomenclature for neurofibromatosis type 2 (NF2) and Schwannomatosis (SWN), now grouped under the umbrella term Schwannomatosis, and defined new diagnostic criteria. This review describes the molecular criteria for diagnosis of schwannomatosis and the subsequent diagnosis strategy, while setting out the most recent advances in our understanding of the natural history, pathology, molecular biology and treatment of schwannomatosis-associated tumors, including schwannomas, meningiomas and ependymomas. Somatic mutation screening should become a new standard for the diagnosis of NF2-, LTZTR1-, SMARCB1- and 22q-schwannomatosis to discriminate those conditions. Constitutional events in NF2-Schwannomatosis have a major influence on disease severity and justifiably motivate ongoing efforts on gene replacement therapy research. On the other hand, underlying mechanisms of disease severity and associated pain remain largely unknown in non-NF2-SWN and independent of germline mutation. Research efforts therefore focus on pain relief in ongoing trials and the discovery of new molecular mechanisms underlying schwannoma tumorigenesis/pain/neuropathies.

A multi-tier computational screening framework to effectively search the mutational space of SARS-CoV-2 receptor binding motif to identify mutants with enhanced ACE2 binding abilities.

SARS-CoV-2 gained crucial mutations at the receptor binding domain (RBD) that often changed the course of the pandemic leading to new waves with increased case fatality. Variants are observed with enhanced transmission and immune invasion abilities. Thus, predicting future variants with enhanced transmission ability is a problem of utmost research interest. Here, we have developed a multi-tier exhaustive SARS-CoV-2 mutation screening platform combining MM/GBSA, extensive molecular dynamics simulations, and steered molecular dynamics to identify RBD mutants with enhanced ACE2 binding capability. We have identified four RBM mutations (F490K, S494K, G504F, and the P499L) with significantly higher ACE2 binding abilities than wild-type RBD. Compared to wild-type RBD, they all form stable complexes with more hydrogen bonds and salt-bridge interactions with ACE2. Our simulation data suggest that these mutations allosterically alter the packing of the RBM interface of the RBD-ACE2 complex. As a result, the rupture force required to break the RBD-ACE2 contacts is significantly higher for these mutants.

Characterization of Mutation of BRCA1 Gene at Chromosome 17q21 in Bangladeshi Women With Familial Breast Cancer

Breast cancer is the most common cancer among women in Bangladesh. About 69% cancer death of women is due to breast cancer. In Bangladesh the incidence rate of breast cancer is about 22.5 per 100000 female. Female who carries mutation in the BRCA1 gene have an 80% chance of developing breast cancer during their lifetime. Due to its high prevalence, genetic testing for BRCA1 mutation has become an integral part of clinical practice. This study was aimed to characterize the mutation at chromosome 17q21 by targeting BRCA1 gene among Bangladeshi women with familial breast cancer. This descriptive case series study was conducted in the Department of Biochemistry, Dhaka Medical College, Dhaka, from July 2017 to June 2018. In this study, thirty diagnosed breast cancer patients with family history breast cancer were selected according to the selection criteria from the Department of Surgery and Department of Radiotherapy, Dhaka Medical College, Dhaka. Mutations were detected by Next Generation Sequencing method. Characterization and clinical significance were verified from NCBI genome database. The study parameters were presence of mutation, types of mutation, clinical significance, frequency (%) of mutation. In this study, sixteen distinct mutations were found. Three pathogenic mutations (10%) were found among the patients which were nonsense mutations. Pathogenic mutations were c.5251C>T, c.2158G>T and c.1059G>A. Three mutations of uncertain significance with missense variant, five benign mutations of missense variant and five benign mutations of silent variant were also found among thirty patients. In this study we found that, Bangladeshi women with familial breast cancer had 10% pathogenic mutations. Early screening for BRCA1 gene mutations might provide better information for health to those who have family history of breast cancer.
 Bangladesh J Med Biochem 2018; 11(2): 17-23

CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication.

The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.

Factor V Leiden (FVL), Prothrombin G20210A and MTHFR C677T Mutations among Patients of Budd-Chiari Syndrome

Abstract Background Many studies ask whether screening Factor V Leiden (FVL), Prothrombin G20210A and Methyl Tetra Hydro Folate Reductase (MTHFR) C677T Mutations should be routinely performed in patients with Budd–Chiari syndrome (BCS). However, the prevalence of these mutations in such patients is substantially variable. This is the first study in clinical pathology department in Ain Shams University to evaluate the importance of this mutational screening in patients with Budd-Chiari syndrome to detect occult genetic susceptibility to thrombophilia. Aim of the Work This study aims to detect Factor V Leiden (FVL), Prothrombin G20210A and MTHFR C677T Mutations among Patients with Budd-Chiari Syndrome. Patients and Methods This study was a cross sectional study conducted on 35 newly of Budd Chiari syndrome patients who were attending tropical department at Ain Shams University Hospitals during the period from April 2020 to January 2021. Informed consent was obtained from all patients in the study. Results results of our study revealed that JAK2 mutation it was detected in 17.1% of patients (6 patients), While PGM was detected in 2.9% of our patients. Furthermore Factor V Leiden Mutation was detected in 65.7%of patients and MTHFR mutation was detected in 48.6% of patients. The present study found no significant statistical difference between patients with Factor V Leiden Mutation & patients with normal Factor V Leiden regarding CBC parameters, levels of ANA, ACL IgM, ACL IgG, PC Deficiency, PS Deficiency and AT III Deficiency. In addition, our study revealed that there was no effect of MTHFR Mutation & JAK2 Mutation on levels of ANA, ACL IgM, ACL IgG, PC Deficiency, PS Deficiency and AT III Deficiency. Also there was no effect for MTHFR Mutation & JAK2 Mutation on outcome of patients, survival and response to treatment. Conclusion BCS is associated usually with genetic thrombophilia. Most commonly Factor V Leiden Mutation in 65.7%, followed by MTHFR mutation in 48.6% of our patients. Although, only Factor V Leiden Mutation has an effect on patients Outcome, Survival and Response to treatment through its effect on CBC parameters, levels of ANA, ACL IgM, ACL IgG, PC Deficiency, PS Deficiency and AT III Deficiency. Otherwise, JAK2 Mutation have significant effect on hemopoiesis.

EnzyHTP Computational Directed Evolution with Adaptive Resource Allocation.

Directed evolution facilitates enzyme engineering via iterative rounds of mutagenesis. Despite the wide applications of high-throughput screening, building "smart libraries" to effectively identify beneficial variants remains a major challenge in the community. Here, we developed a new computational directed evolution protocol based on EnzyHTP, a software that we have previously reported to automate enzyme modeling. To enhance the throughput efficiency, we implemented an adaptive resource allocation strategy that dynamically allocates different types of computing resources (e.g., GPU/CPU) based on the specific need of an enzyme modeling subtask in the workflow. We implemented the strategy as a Python library and tested the library using fluoroacetate dehalogenase as a model enzyme. The results show that compared to fixed resource allocation where both CPU and GPU are on-call for use during the entire workflow, applying adaptive resource allocation can save 87% CPU hours and 14% GPU hours. Furthermore, we constructed a computational directed evolution protocol under the framework of adaptive resource allocation. The workflow was tested against two rounds of mutational screening in the directed evolution experiments of Kemp eliminase (KE07) with a total of 184 mutants. Using folding stability and electrostatic stabilization energy as computational readout, we identified all four experimentally observed target variants. Enabled by the workflow, the entire computation task (i.e., 18.4 μs MD and 18,400 QM single-point calculations) completes in 3 days of wall-clock time using ∼30 GPUs and ∼1000 CPUs.

Genetic screening of the LRRK2 G2019S mutation in the population of the South-West of Morocco

Genetic screening of the LRRK2 G2019S mutation in the population of the South-West of Morocco

Retracted: Mutational Screening of Skeletal Genes in 14 Chinese Children with Osteogenesis Imperfecta Using Targeted Sequencing.

[This retracts the article DOI: 10.1155/2022/5068523.].

A novel strategy for screening mutations in the voltage-gated sodium channel gene of Aedes albopictus based on multiplex PCR-mass spectrometry minisequencing technology

BackgroundThe current prevention and control strategy for Aedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene in Ae. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology.MethodsWe established a new strategy for detecting mutations in the VGSC gene in Ae. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples of Ae. albopictus. A total of 70 wild-collected Ae. albopictus were used to evaluate the performance of the method by comparing it with other methods.ResultsThree target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization–time-of-flight mass spectrometry, achieving a detection limit of 20 fg/μl. We applied this method to 70 wild-collected Ae. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method.ConclusionsMPCR-MS minisequencing technology provides a sensitive and high-throughput approach to Ae. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.Graphical

The clinical relevance of broad mutational screening of myeloproliferative neoplasms at diagnosis.

Myeloproliferative neoplasm (MPN) is a heterogenous group of hematological malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F is the most frequent driver mutation in all three entities, but in PMF and ET mutations in CALR and MPL are also frequent. Mutations seen in additional genes are also often the same regardless of subtype of MPN. The aim of this study was to analyze a population based MPN cohort for genetic variants with prognostic value that can guide clinical decisions. MPN patients from Western Sweden diagnosed between 2008-2013 (n=248) were screened for mutations in 54 genes associated with myeloid malignancy. Mutations in the genes SRSF2 and U2AF1 correlated significantly with impaired overall survival but did not correlate to increased risk for vascular events, neither before nor after diagnosis. Rather, mutations in these genes showed an association with disease transformation. Several recurrent gene variants with allele frequency close to 50% were confirmed to be germline. However, none of these variants was found to have an earlier onset of MPN. In conclusion, we identified gene mutations to be independent markers of impaired survival in MPN. This indicates the need for more individualized assessment and treatment of MPN patients and a wider gene mutation screening already at diagnosis. This could ensure the identification of patients with high-risk mutations early on. In addition, several genetic variants were also identified as germline in this study but gave no obvious clinical relevance. To avoid conclusions from non-informative genetic variants, a simultaneous analysis of normal cell DNA from patients at diagnosis should be considered.

Molecular Studies for the Early Detection of Philadelphia-Negative Myeloproliferative Neoplasms.

JAK2 V617F is the predominant driver mutation in patients with Philadelphia-negative myeloproliferative neoplasms (MPN). JAK2 mutations are also frequent in clonal hematopoiesis of indeterminate potential (CHIP) in otherwise "healthy" individuals. However, the period between mutation acquisition and MPN diagnosis (known as latency) varies widely between individuals, with JAK2 mutations detectable several decades before diagnosis and even from birth in some individuals. Here, we will review the current evidence on the biological factors, such as additional mutations and chronic inflammation, which influence clonal expansion and may determine why some JAK2-mutated individuals will progress to an overt neoplasm during their lifetime while others will not. We will also introduce several germline variants that predispose individuals to CHIP (as well as MPN) identified from genome-wide association studies. Finally, we will explore possible mutation screening or interventions that could help to minimize MPN-associated cardiovascular complications or even delay malignant progression.

Genetic screening of an endemic mutation in the DYSF gene in an isolated, mountainous population in the Republic of Dagestan.

Dysferlinopathy has a high prevalence in relatively isolated ethnic groups where consanguineous marriages are characteristic and/or the founder effect exists. However, the frequency of endemic mutations in most isolates has not been investigated. The prevalence of the pathological DYSF gene variant (NM_003494.4); c.200_201delinsAT, p. Val67Asp (rs121908957) was investigated in an isolated Avar population in the Republic of Dagestan. Genetic screenings were conducted in a remote mountainous region characterized by a high level of consanguinity among its inhabitants. In total, 746 individuals were included in the screenings. This pathological DYSF gene variant causes two primary phenotypes of dysferlinopathy: limb-girdle muscular dystrophy (LGMD) type R2 and Miyoshi muscular dystrophy type 1. Results indicated a high prevalence of the allele at 14% (95% confidence interval [CI]: 12-17; 138 out of 1518 alleles), while the allele in the homozygous state was detected in 29 cases-3.8% (CI: 2.6-5.4). The population load for dysferlinopathy was 832.3 ± 153.9 per 100,000 with an average prevalence of limb-girdle muscular dystrophies ranging from 0.38 ± 0.38 to 5.93 ± 1.44 per 100,000. A significant burden of the allele was due to inbreeding, as evidenced by a deficiency of heterozygotes and the Wright fixation index equal to 0.14 (CI 0.06-0.23).

Identification of tumor mutations in plasma based on mutation variant frequency change (MVFC).

To overcome the dependency of strategies utilizing cell-free DNA (cfDNA) on tissue sampling, the emergence of sequencing panels for non-invasive mutation screening was promoted. However, cfDNA sequencing with panels still suffers from either inaccuracy or omission, and novel approaches for accurately screening tumor mutations solely based on plasma without gene panel restriction are urgently needed. We performed unique molecular identifier (UMI) target sequencing on plasma samples and peripheral blood mononuclear cells (PBMCs) from 85 hepatocellular carcinoma (HCC) patients receiving surgical resection, which were divided into an exploration dataset (20 patients) or an evaluation dataset (65 patients). Plasma mutations were identified in pre-operative plasma, and the mutation variant frequency change (MVFC) between post- and pre-operative plasma was then calculated. In the exploration dataset, we observed that plasma mutations with MVFC < 0.2 were enriched for tumor mutations identified in tumor tissues and had frequency changes that correlated with tumor burden; these plasma mutations were therefore defined as MVFC-identified tumor mutations. The presence of MVFC-identified tumor mutations after surgery was related to shorter relapse-free survival (RFS) in both datasets and thus indicated minimum residual disease (MRD). The combination of MVFC-identified tumor mutations and Alpha Fetoprotein (AFP) could further improve MRD detection (P < 0.0001). Identification of tumor mutations based on MVFC was also confirmed to be applicable with a different gene panel. Overall, we proposed a novel strategy for non-invasive tumor mutation screening using solely plasma that could be utilized in HCC tumor-burden monitoring and MRD detection.

Mutation spectrum of retinoblastoma patients in Vietnam.

Retinoblastoma (RB), an intraocular malignancy commonly diagnosed in children, is mostly caused by inactivating mutations of both alleles of the RB1 gene. Early genetic screening for RB1 gene mutations would greatly improve treatment outcomes and patient management. In this study, both somatic and germline mutations were detected in blood and tumour samples of 42 RB patients using direct sequencing and multiplex ligation-dependent probe amplification. In total, 34 different mutations were found in 36 patients, including 1 SNP, 4 large deletions, 5 splicing sites, 1 missense, 7 frameshifts and 17 nonsense mutations. There were five novel mutations and one unreported which have not been found in large databases such as Leiden Open Variation Database (LOVD) and ClinVar. A higher rate of RB patients carrying heterozygous germline mutation and highly prevalent with pathogenic truncated mutation, hence, early detection of RB is essential for vision salvation and genetic counselling.

Uncovering a new SASH1 mutation associated with dyschromatosis universalis hereditaria using whole-exome-sequencing: A case report.

Dyschromatosis universalis hereditaria (DUH) is an uncommon form of pigmented genodermatosis that is typically inherited autosomally and dominantly. In the previous study, the pathogenic genes of DUH have been identified in ATP-binding cassette subfamily B, member 6 and SASH1. However, the mutational screening of the causative gene remains incomplete and still lacks sufficient proof in the etiology. A 2-generation Chinese family clinically diagnosed with DUH were enrolled. They showed pigmented spots from their childhood and came to the hospital for medical advice and genetic analysis. We found a novel mutation c.1757T > C (p.I586T) of SASH1 in 3 affected family members by whole-exome sequencing. Genetic outcomes and clinical examinations confirmed the diagnosis of DUH in 3 family members with lentiginous syndrome. Using whole-exome sequencing and sanger sequencing technologies, we identified a novel mutation c.1757T > C (p.I586T) of SASH1 that co-segregated in 3 afflicted family members but not in the normal individuals. Significantly, c.1757T > C (p.I586T) is a novel mutation which had not been previously reported. The same codon position in SASH1 (c.1758C > G, p.I586M) has been reported in a Japanese man, and he showed identical phenotype compared to our study participants. Our study broadens the spectrum of DUH mutations and provides more genetic characteristics of DUH in understanding its etiology. Furthermore, we demonstrated the diagnostic accuracy of whole-exome sequencing for inherited skin diseases and provided new information for etiological study.

Reliability of flow-cytometry in diagnosis and prognostic stratification of myelodysplastic syndromes: correlations with morphology and mutational profile

Diagnosis and prognostic stratification of myelodysplastic syndromes (MDS) have been complemented by new techniques, including flow cytometry and NGS. To analyze the relationship between molecular and cytofluorimetric data, we enrolled in this retrospective study, 145 patients, including 106 diagnosed with MDS and 39 controls. At disease onset, immunophenotypic (IF), cytogenetic tests, and cytomorphological (CM) examination on bone marrow were carried out in all patients, while NGS was performed in 58 cases. Ogata score presented a specificity of 100% and a sensitivity of 59%. The detection of at least two phenotypic aberrancies in Ogata negative patients increased the sensitivity to 83% and specificity to 87%. Correlations were identified between IF aberrancies and mutations, including positive Ogata>2 and mutations in SRSF2 (p=0.035), CD15 and U2AF1 (0.032), CD56 and DNMT3A (p=0.042), and CD38 and TP53 (p=0.026). In multivariate analysis, U2AF1 mutations, associated with del(20q) and/or abnormalities of chromosome 7 (group 4 as defined by the EuroMDS score), significantly correlated with an inferior overall survival (p=0.019). These parameters and Ogata score>2 also showed a significant correlation with inferior event-free survival (p=0.023 and p=0.041, respectively). Both CM and FC features correlated with prognosis and mutational patterns. In an integrated MDS work-up, these tools may guide indications for mutational screening for optimal risk stratification.