Somatic frameshift mutations in exon 9 of calreticulin (CALR) gene are recognized as disease drivers in primary myelofibrosis (PMF), one of the three classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Type 1/type 1-like CALR mutations particularly confer a favorable prognostic and survival advantage in PMF patients. We report an unusual case of PMF incidentally diagnosed in a 68-year-old woman known with hepatitis C virus (HCV) cirrhosis who developed a progressive painful splenomegaly, without anomalies in blood cell counts. While harboring a type 1 CALR mutation, the patient underwent a leukemic transformation in less than 1 year from diagnosis, with a lethal outcome. Analysis of paired DNA samples from chronic and leukemic phases by a targeted next-generation sequencing (NGS) panel and single-nucleotide polymorphism (SNP) microarray revealed that the leukemic clone developed from the CALR-mutated clone through the acquisition of genetic events in the RAS signaling pathway: an increased variant allele frequency of the germline NRAS Y64D mutation present in the chronic phase (via an acquired uniparental disomy of chromosome 1) and gaining NRAS G12D in the blast phase. SNP microarray analysis showed five clinically significant copy number losses at regions 7q22.1, 8q11.1-q11.21, 10p12.1-p11.22, 11p14.1-p11.2, and Xp11.4, revealing a complex karyotype already in the chronic phase. We discuss how additional mutations, detected by NGS, as well as HCV infection and antiviral therapy, might have negatively impacted this type 1 CALR-mutated PMF. We suggest that larger studies are required to determine if more careful monitoring would be needed in MPN patients also carrying HCV and receiving anti-HCV treatment.

Inherited retinal dystrophies (IRDs) include a large chronic heterogeneity genetic disease. While many disease-causing pathogenic variants were involved in the progression of IRD, the Ceramide Kinase Like (CERKL) gene variant in Iranian patients is not well characterized. In this study, a consanguineous Iranian family with three generations was recruited whom presented with the clinical diagnosis of autosomal recessive IRD. By targeted next-generation sequencing (TGS) and Sanger sequencing, the proband was found to have a novel, pathological homozygous deletion variant c.560_568del (p.187_190del) of the CERKL gene (NM_001030311.2) that co-segregated with the disease in all affected family members. The Cerkl is highly expressed in the later four developmental retinal stages, playing a vital role in retina degeneration. Therefore, the identification of a novel, homozygous deletion CERKL variant c.560_568del (p.187_190del) in an IRD familial cohort descent provides insights into the molecular pathogenesis of IRD and facilitates genetic counseling and disease prediction.

β-Thalassemia (β-thal) is one of the most common monogenic recessive inherited diseases worldwide. The mutation spectrum of β-thal has been increasingly broadened by various genetic testing methods. The discovery and identification of novel and rare pathogenic thalassemia variants enable better disease prevention, especially in high prevalence regions. In this study, a Chinese thalassemia family with an unclear etiology was recruited to the Thalassemia Screening Program. Blood samples collected from them were primarily screened by hematology analysis and clinical routine genetic screening. Subsequently, targeted next-generation sequencing (NGS) and Sanger sequencing were performed to find and identify a novel deletion variant. The deletion, discovered by targeted NGS, was validated through real-time quantitative polymerase chain reaction (qPCR). First, a large novel β-thal deletion (3488 bp) related to a high Hb F level, NC_000011.9: g.5245533_5249020del (Chongqing deletion) (GRCh37/hg19), was found and identified in the proband and her mother. The deletion removed the entire β-globin gene and led to absent β-globin (β0). We then validated this large novel deletion in the proband and her mother by qPCR. We first discovered and identified a large novel β-thal deletion related to elevated Hb F level, it helps broaden the spectrum of pathogenic mutants that may cause β-thal intermedia (β-TI) or β-thal major (β-TM), paving the way for effective thalassemia screening. Next-generation sequencing has the potential of finding rare and novel thalassemia mutants.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophies and the most frequent cause of congenital blindness in children. To date, 25 genes have been implicated in the pathogenesis of this rare disorder. Performing an accurate molecular diagnosis is crucial as gene therapy is becoming available. This study aimed to report the molecular basis of Leber congenital amaurosis, especially novel and rare variants in 27 Polish families with a clinical diagnosis of LCA fully confirmed by molecular analyses. Whole exome sequencing or targeted next-generation sequencing (NGS) of inherited retinal dystrophies-associated (IRD) genes was applied to identify potentially pathogenic variants. Bidirectional Sanger sequencing and quantitative PCR (qPCR) were carried out for validation and segregation analysis of the variants identified within the families. We identified 28 potentially pathogenic variants, including 11 novel, in 8 LCA genes: CEP290, CRB1, GUCY2D, NMNAT1, RPGRIP1, CRX, LRAT1, and LCA5. This study expands the mutational spectrum of the LCA genes. Moreover, these results, together with the conclusions from our previous studies, allow us to point to the most frequently mutated genes and variants in the Polish cohort of LCA patients.

We aimed to reclassify a population-based cohort of 529 adult glioma patients to evaluate the prognostic impact of the 2016 World Health Organization (WHO) central nervous system tumour classification. Moreover, we evaluated the feasibility of gene panel next-generation sequencing (NGS) in daily diagnostics of 225 prospective glioma patients.
The retrospective cohort was reclassified according to WHO 2016 criteria by immunohistochemistry for IDH-R132H, fluorescence in situ hybridization for 1p/19q-codeletion and gene panel NGS. All tumours of the prospective cohort were subjected to NGS analysis up-front.
The entire population-based cohort was successfully reclassified according to WHO 2016 criteria. NGS results were obtained for 98% of the prospective patients. Survival analyses in the population-based cohort confirmed three major prognostic subgroups, that is, isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas, IDH-mutant astrocytomas and IDH-wildtype glioblastomas. The distinction between WHO grade II and III was prognostic in patients with IDH-mutant astrocytoma. The survival of patients with IDH-wildtype diffuse astrocytomas carrying TERT promoter mutation and/or EGFR amplification overlapped with the poor survival of IDH-wildtype glioblastoma patients.
Gene panel NGS proved feasible in daily diagnostics. In addition, our study confirms the prognostic role of glioma classification according to WHO 2016 in a large population-based cohort. Molecular features of glioblastoma in IDH-wildtype diffuse glioma were linked to poor survival corresponding to IDH-wildtype glioblastoma patients. The distinction between WHO grade II and III retained prognostic significance in patients with IDH-mutant diffuse astrocytic gliomas.

Previous studies showed that the fibrillin-1 gene (FBN1) is responsible for Marfan sydrome (MFS) pathogenesis. This study is conducted to screen for mutations in the FBN1 gene in Chinese families with MFS.
Eight families with MFS and related disorder were recruited in this study. All available family members underwent complete physical, ophthalmic, and cardiovascular examination. Mutation screening was performed using targeted next-generation sequencing. Candidate variants were amplified by polymerase chain reaction and verified by direct Sanger sequencing.
Four novel heterozygous mutations in FBN1, including c.2861G>T (p.R954L), c.4087G>A (p.D1363N), c.4987T>G (p.C1663G), and c.5032T>G (p.Y1678D), as well as four known mutations, c.3617G>A (p.G1206D), c.4460A>G (p.D1487G), c.4588C>T (p.R1530C), and c.718C>T (p.R240C) were identified. Affected patients from each family were found to carry one of the mutations, whereas the unaffected members and 1,086 normal controls were not. Each mutation was found to be cosegregated with MFS phenotype and related disorder in each family. Multiple sequence alignment of the human fibrillin-1 protein showed that these mutations occurred in a highly conserved region among different species.
Eight FBN1 mutations were identified in Chinese families with MFS and related disorder. These data expands FBN1 mutation spectrum and further emphasizes the role of FBN1 in the pathogenesis of MFS.

Congenital muscular dystrophies (CMD) are a group of heterogeneous disorders. Here, targeted next generation sequencing of 168 CMD-associated genes was performed on collected clinic samples to identify potential mutations. A loss-of-function mutation (c.4676-4682delGCTGCAA; p.Cys1560Thrfs*33) of the LAMA2 gene in a consanguineous family was identified and confirmed by Sanger sequencing. The second recessive mutation in SYNE1 (c.2881C>T; p.Arg961Trp) was found in the SAP motif, which was predicted to be involved in chromosomal organization. The third homozygous mutation (c.32462C>T; p.Pro10821Leu) in TTN was mapped to the third PPAK motif of the encoded protein. Muscle biopsies of the proband showed large variations in muscle fiber size, necrotic and regenerating fibers and an increase in endomysial collagen tissue. To the best of our knowledge, this is the first case with CMD and mildly enlarged heart, carrying three novel recessive mutations in LAMA2, SYNE1, and TTN.