Transformation of t(14;18)-negative follicular lymphoma to plasmablastic lymphoma: a case report with analysis of genetic evolution.

BACKGROUND: Follicular lymphoma (FL) is characterized by t(14;18)(q32;q21) involving the IGH and BCL2 genes. However, 10-15% of FLs lack the BCL2 rearrangement. These BCL2-rearrangement-negative FLs are clinically, pathologically, and genetically heterogeneous. The biological behavior and histological transformation of such FLs are not adequately characterized. Here, we report the first case of t(14;18)-negative FL that rapidly progressed to plasmablastic lymphoma (PBL). CASE PRESENTATION: A previously healthy 51-year-old man presented with leg swelling. Computed tomography (CT) showed enlarged lymph nodes (LNs) throughout the body, including both inguinal areas. Needle biopsy of an inguinal LN suggested low-grade B-cell non-Hodgkin lymphoma. Excisional biopsy of a neck LN showed proliferation of centrocytic and centroblastic cells with follicular and diffuse growth patterns. Immunohistochemical analysis showed that the cells were positive for CD20, BCL6, CD10, and CD23. BCL2 staining was negative in the follicles and weak to moderately positive in the interfollicular areas. BCL2 fluorescence in situ hybridization result was negative. Targeted next-generation sequencing (NGS) revealed mutations in the TNFRSF14, CREBBP, STAT6, BCL6, CD79B, CD79A, and KLHL6 genes, without evidence of BCL2 or BCL6 rearrangement. The pathologic and genetic features were consistent with t(14;18)-negative FL. Two months after one cycle of bendamustine and rituximab chemotherapy, the patient developed left flank pain. Positron emission tomography/CT showed new development of a large hypermetabolic mass in the retroperitoneum. Needle biopsy of the retroperitoneal mass demonstrated diffuse proliferation of large plasmablastic cells, which were negative for the B-cell markers, BCL2, BCL6, and CD10; they were positive for MUM-1, CD138, CD38, and C-MYC. The pathologic findings were consistent with PBL. The clonal relationship between the initial FL and subsequent PBL was analyzed via targeted NGS. The tumors shared the same CREBBP, STAT6, BCL6, and CD79B mutations, strongly suggesting that the PBL had transformed from a FL clone. The PBL also harbored BRAF V600E mutation and IGH::MYC fusion in addition to IGH::IRF4 fusion. CONCLUSIONS: We propose that transformation or divergent clonal evolution of FL into PBL can occur when relevant genetic mutations are present. This study broadens the spectrum of histological transformation of t(14;18)-negative FL and emphasizes its biological and clinical heterogeneity.

Identification of a Novel Indel Variant in the DARS2 Gene in Russian Patients with Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is an inherited disease caused by pathogenic biallelic variants in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. This disease is characterized by slowly progressive spastic gait, cerebellar symptoms, and leukoencephalopathy with brainstem and spinal cord involvement. CASE PRESENTATION: Peripheral blood samples were collected from four patients from four unrelated families to extract genomic DNA. All patients underwent partial exon analysis of the DARS2 gene using Sanger sequencing, which detected the c.228-21_228-20delinsC variant in a heterozygous state. Further DNA from three patients was analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies, and one of the patients underwent whole genome sequencing. We identified a novel pathogenic variant c.1675-1256_*115delinsGCAACATTTCGGCAACATTCCAACC in the DARS2 gene. Three patients (patients 1, 2, and 4) had slowly progressive cerebellar ataxia, and two patients (patients 1 and 2) had spasticity. In addition, two patients (patients 2 and 4) showed signs of axonal neuropathy, such as decreased tendon reflexes and loss of distal sensitivity. Three patients (patients 1, 2, and 3) also had learning difficulties. It should be noted the persistent presence of characteristic changes in brain MRI in all patients, which emphasizes its importance as the main diagnostic tool for suspicion and subsequent confirmation of LBSL. Conclusions: We found a novel indel variant in the DARS2 gene in four patients with LBSL and described their clinical and genetic characteristics. These results expand the mutational spectrum of LBSL and aim to improve the laboratory diagnosis of this form of leukodystrophy.

A chorea-acanthocytosis patient with novel mutations in the VPS13A gene without acanthocyte.

Chorea-acanthocytosis (ChAc) is a rare clinical genetic disorder of the nervous system, which is characterized by choreiform movement disorder, cognitive decline, and psychiatric disorders. ChAc is mostly diagnosed based on its typical clinical manifestations and the increased number of acanthocytes in peripheral blood smears. Here, we report a patient, who has the characteristic clinical manifestations of ChAc with limb choreiform movements, involuntary lip and tongue bites, seizures, and emotional instability. However, her blood smear was negative for acanthocytes with scanning electron microscopy. We later identified two novel pathogenic mutations in the patient's vacuolar protein sorting homolog 13 A (VPS13A) on chromosome 9q21 by targeted gene sequencing, and she was definitively diagnosed with "ChAc." After treatment with carbamazepine, haloperidol, the patient's symptoms gradually improved. We consider that an acanthocyte negative blood smear cannot rule out ChAC diagnosis, and genetic testing is the "gold standard" for the diagnosis. Through a review of previous research, it is rare for a patient to have a clear diagnosis of ChAc by genetic testing, but whose blood smear is negative for acanthocytes with electron microscopy. In addition, in this report, we discovered two novel pathogenic mutations, which have not been reported previously, and extended the genetic characteristics of ChAc.

Four Novel Disease-Causing Variants in the NOTCH3 Gene in Russian Patients with CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited disease with unknown mechanisms and a broad phenotypic spectrum. It is caused by pathogenic variants in the NOTCH3 gene. The symptoms of the disease mainly include recurrent strokes with vascular risk factors, migraine with aura, dementia, and mood disturbances. CASE PRESENTATION: Peripheral blood samples were collected from five patients from four unrelated families to extract genomic DNA. In four patients, analysis of exons 2, 3, 4, 5, 6 and adjacent intronic regions of the NOTCH3 gene was made via Sanger sequencing. Two previously undescribed nucleotide variants were identified in two patients: missense variant c.208G>T, (p.Gly70Cys) in exon 1 and splice-site variant c.341-1G>C in intron 3. Further DNA of two other patients were analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies. Two novel missense variants in the NOTCH3 gene were identified, c.1136G>A, (p.Cys379Tyr) in exon 7 and c.1547G>A, (p.Cys516Tyr) in exon 10. The pathogenic variant c.1547G>A, (p.Cys516Tyr) was confirmed in the fifth patient (family case) by Sanger sequencing. All patients had a history of headaches, transient ischemic attacks, memory impairment, and characteristics of MRI results. Three patients had strokes and two patients had psychiatric symptoms. CONCLUSION: We found four previously undescribed pathogenic variants in the NOTCH3 gene in five patients with CADASIL and described their clinical and genetic characteristics. These results expand the mutational spectrum of CADASIL.

Combined Single Gene Testing and Genome Sequencing as an Effective Diagnostic Approach for Anophthalmia and Microphthalmia Patients.

Anophthalmia and microphthalmia (A/M) are among the most severe congenital developmental eye disorders. Despite the advancements in genome screening technologies, more than half of A/M patients do not receive a molecular diagnosis. We included seven consanguineous families affected with A/M from Pakistani cohort and an unknown molecular basis. Single gene testing of FOXE3 was performed, followed by genome sequencing for unsolved probands in order to establish a genetic diagnosis for these families. All seven families were provided with a genetic diagnosis. The identified variants were all homozygous, classified as (likely) pathogenic and present in an A/M-associated gene. Targeted FOXE3 sequencing revealed two previously reported pathogenic FOXE3 variants in four families. In the remaining families, genome sequencing revealed a known pathogenic PXDN variant, a novel 13bp deletion in VSX2, and one novel deep intronic splice variant in PXDN. An in vitro splice assay was performed for the PXDN splice variant which revealed a severe splicing defect. Our study confirmed the utility of genome sequencing as a diagnostic tool for A/M-affected individuals. Furthermore, the identification of a novel deep intronic pathogenic variant in PXDN highlights the role of non-coding variants in A/M-disorders and the value of genome sequencing for the identification of this type of variants.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis.

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Identification of a novel mutation in the ALDOB gene in hereditary fructose intolerance.

OBJECTIVES: Hereditary fructose intolerance (HFI) is caused by aldolase B enzyme deficiency. There has been no report about HFI from Iran and the type of mutations has not been reported in the Iranian population so far. CASE PRESENTATION: Herein we report a 2 year old girl presented with failure to thrive, hepatomegaly, and liver dysfunction. The primary impression has been hepatic glycogen storage disease type 1 or 6. This diagnosis was not confirmed by laboratory data and liver biopsy. Therefore, targeted-gene sequencing (TGS) covering 450 genes involved in inborn errors in metabolic diseases was performed. The results of TGS showed a rare novel homozygous pathogenic variant c.944del (p.Gly315ValfsTer15) in the ALDOB gene. CONCLUSIONS: This report introduces a novel variant that expands the mutational spectrum of the ALDOB gene in patients with HFI.

A Case Report of Burkholderia mallei Infection Leading to Pneumonia.

BACKGROUND: Glanders is a rare zoonotic disease caused by Burkholderia mallei. Humans can be infected by B. mallei, which causes cutaneous lymphadenitis and pneumonia, leading to sepsis and death in severe cases. CASE PRESENTATION: We report a case of a 60-year-old male who was diagnosed with glanders. The patient who had a history of diabetes presented with cough, expectoration, and fever. Computed tomography (CT) imaging showed B. mallei infection in the right upper lobe of the lung with mediastinal lymph node involvement and the lingual segment of the left lung. Moreover, the posterior basal segment of the lower lobe of both lungs had inflammation. Subsequently, B. mallei infection was confirmed by lymph node biopsy and bronchoalveolar lavage multiplex PCR-based targeted gene sequencing. After meropenem treatment, the patient was discharged, and CT imaging showed reduced absorption of pulmonary inflammatory lesions. CONCLUSIONS: Glanders is a rare disease that can cause skin infection, lymphadenitis, and pneumonia, and in severe cases, it can be life-threatening. The diagnosis of this disease mainly relies on microbiological culture and pathological biopsy. Diagnosis is also facilitated by multiplex PCRbased targeted gene sequencing. Glanders is treated with cephalosporins, carbapenems, and other sensitive antibiotics.

Genetic Causes, Clinical Features, and Survival of Underlying Inborn Errors of Immunity in Omani Patients: a Single-Center Study.

PURPOSE: Early identification of inborn errors of immunity (IEIs) is crucial due to the significant risk of morbidity and mortality. This study aimed to describe the genetic causes, clinical features, and survival rate of IEIs in Omani patients. METHODS: A prospective study of all Omani patients evaluated for immunodeficiency was conducted over a 17-year period. Clinical features and diagnostic immunological findings were recorded. Targeted gene testing was performed in cases of obvious immunodeficiency. For cases with less conclusive phenotypes, a gene panel was performed, followed by whole-exome sequencing if necessary. RESULTS: A total of 185 patients were diagnosed with IEIs during the study period; of these, 60.5% were male. Mean ages at symptom onset and diagnosis were 30.0 and 50.5 months, respectively. Consanguinity and a family history of IEIs were present in 86.9% and 50.8%, respectively. Most patients presented with lower respiratory infections (65.9%), followed by growth and development manifestations (43.2%). Phagocytic defects were the most common cause of IEIs (31.9%), followed by combined immunodeficiency (21.1%). Overall, 109 of 132 patients (82.6%) who underwent genetic testing received a genetic diagnosis, while testing was inconclusive for the remaining 23 patients (17.4%). Among patients with established diagnoses, 37 genes and 44 variants were identified. Autosomal recessive inheritance was present in 81.7% of patients with gene defects. Several variants were novel. Intravenous immunoglobulin therapy was administered to 39.4% of patients and 21.6% received hematopoietic stem cell transplantation. The overall survival rate was 75.1%. CONCLUSION: This study highlights the genetic causes of IEIs in Omani patients. This information may help in the early identification and management of the disease, thereby improving survival and quality of life.

A retrospective single-centered, comprehensive targeted genetic sequencing analysis of prognostic survival using tissues from Korean patients with metastatic renal cell carcinoma after targeted therapy.

PURPOSE: To identify candidate gene mutations to significantly predict the risk of survival prognosis after treatment with systemic first-line targeted therapy (TT) in metastatic renal cell carcinoma (mRCC) patients. MATERIALS AND METHODS: Between 2005 and 2017, 168 triplet-tissue block samples from 56 mRCC patients were selected for targeted gene sequencing (TGS). Fifty-six patients' medical records including overall survival (OS) and progression-free survival (PFS) at the time of mRCC diagnosis were evaluated. The patients were grouped into favorable (>12 months/>3 years), intermediate (3-12/12-36 months), and poor groups according to their PFS/OS (<3 months/<12 months). We identified any significant therapeutic targeted genes relating to the survival with a significance at p<0.050. RESULTS: The first line therapeutic response showed 1.8% complete remission, 14.2% partial response, 42.9% stable disease, and 41.1% progressive disease. Among the overall TGS results, the cumulative effect of CDH1, and/or PTK2 genes significantly reflected the therapeutic responses in terms of PFS/OS; CDH1 and PTK2 mutations were associated with poor prognostic outcomes (p<0.050). Among only triplet-quality check passed tissues, the SGO2, BRAF, URB1, and NEDD1 mutated genes significantly correlated with OS. Regarding metastasis, patients with liver metastasis had the worst OS (p=0.050). The combinational mutation number from these two candidate genes in the liver metastatic samples with mutated EGFR2 and FABP7 also showed a significantly worse OS than those with other metastatic lesions (p<0.050). CONCLUSIONS: This study reports several significant mutated genes related to the survival prognosis in mRCC patients treated with first-line TT.

Using single molecule Molecular Inversion Probes as a cost-effective, high-throughput sequencing approach to target all genes and loci associated with macular diseases.

Macular degenerations (MDs) are a subgroup of retinal disorders characterized by central vision loss. Knowledge is still lacking on the extent of genetic and nongenetic factors influencing inherited MD (iMD) and age-related MD (AMD) expression. Single molecule Molecular Inversion Probes (smMIPs) have proven effective in sequencing the ABCA4 gene in patients with Stargardt disease to identify associated coding and noncoding variation, however many MD patients still remain genetically unexplained. We hypothesized that the missing heritability of MDs may be revealed by smMIPs-based sequencing of all MD-associated genes and risk factors. Using 17,394 smMIPs, we sequenced the coding regions of 105 iMD and AMD-associated genes and noncoding or regulatory loci, known pseudo-exons, and the mitochondrial genome in two test cohorts that were previously screened for variants in ABCA4. Following detailed sequencing analysis of 110 probands, a diagnostic yield of 38% was observed. This established an ''MD-smMIPs panel," enabling a genotype-first approach in a high-throughput and cost-effective manner, whilst achieving uniform and high coverage across targets. Further analysis will identify known and novel variants in MD-associated genes to offer an accurate clinical diagnosis to patients. Furthermore, this will reveal new genetic associations for MD and potential genetic overlaps between iMD and AMD.

[Application of modern approaches in the screening and early diagnosis programs for the orphan diseases]. / Primenenie sovremennykh tekhnologii v programmakh rannei diagnostiki orfannykh boleznei v Rossii i za rubezhom.

Orphan diseases have a prevalence ranging one patient per 10.000 population in the Russian Federation to one per 1500-2000 individuals in Australia and the USA. Many orphan diseases lead to a severe decrease in quality of life and high mortality. In this article, we discuss the problem of early diagnosis in orphan diseases in the Russian Federation, which has lagged behind global trends towards improved recognition and treatment of orphan diseases. We identify the need for improved focus at the level of national healthcare, while discussing relevant issues arising from the international experience. We review national and regional health programs and healthcare practices of Australia, Germany, Denmark, China, Norway, Slovenia, UK, and the United States, with a focus on screening and diagnosis of orphan disease. We also present a review on the state of affairs in the Russian Federation. Orphan diseases are amenable to current molecular-genetic and other diagnostic technologies, including targeted, whole exome and whole genome sequencing (targeted NGS, WES, WGS) using next generation sequencing technologies (next generation sequencing, NGS) and tandem mass spectrometry (TMS, MS/MS). We conclude with a call for major measures aimed at improving the diagnosis of orphan diseases, in particular through the expansion of the neonatal screening program, the creation of a network of orphan disease referral centers, and centralized management of patients registers.

Identification of a De Novo Heterozygous Missense ACTB Variant in Baraitser-Winter Cerebrofrontofacial Syndrome.

Baraitser-Winter cerebrofrontofacial syndrome (BWCFF, OMIM: 243310) is a rare autosomal-dominant developmental disorder associated with variants in the genes ACTB or ACTG1. It is characterized by brain malformations, a distinctive facial appearance, ocular coloboma, and intellectual disability. However, the phenotypes of BWCFF are heterogenous, and its molecular pathogenesis has not been fully elucidated. In the present study, we conducted detailed clinical examinations on a Chinese patient with BWCFF and found novel ocular manifestations including pseudoduplication of the optic disc and nystagmus. Targeted gene panel sequencing and Sanger sequencing identified a de novo heterozygous missense c.478A > G (p.Thr160Ala) variant in ACTB. The mRNA and protein expression of ACTB was assessed by quantitative reverse transcription PCR and Western blots. Furthermore, the functional effects of the pathogenic variant were analyzed by protein structure analysis, which indicated that the variant may affect the active site for ATP hydrolysis by the actin ATPase, resulting in abnormal filamentous actin organization in peripheral blood mononuclear cells. This discovery extends the ACTB variant spectrum, which will improve genetic counseling and diagnosis, and may contribute to understanding the pathogenic mechanisms of actin-related diseases.

Heterogeneous clinical features in Cockayne syndrome patients and siblings carrying the same CSA mutations.

BACKGROUND: Cockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC6/CSB or ERCC8/CSA that participate in the transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage. CS patients display a large heterogeneity of clinical symptoms and severities, the reason of which is not fully understood, and that cannot be anticipated in the diagnostic phase. In addition, little data is available for affected siblings, and this disease is largely undiagnosed in North Africa. METHODS: We report here the clinical description as well as genetic and functional characterization of eight Tunisian CS patients, including siblings. These patients, who belonged to six unrelated families, underwent complete clinical examination and biochemical analyses. Sanger sequencing was performed for the recurrent mutation in five families, and targeted gene sequencing was done for one patient of the sixth family. We also performed Recovery RNA Synthesis (RRS) to confirm the functional impairment of DNA repair in patient-derived fibroblasts. RESULTS: Six out of eight patients carried a homozygous indel mutation (c.598_600delinsAA) in exon 7 of ERCC8, and displayed a variable clinical spectrum including between siblings sharing the same mutation. The other two patients were siblings who carried a homozygous splice-site variant in ERCC8 (c.843+1G>C). This last pair presented more severe clinical manifestations, which are rarely associated with CSA mutations, leading to gastrostomy and hepatic damage. Impaired TC-NER was confirmed by RRS in six tested patients. CONCLUSIONS: This study provides the first deep characterization of case series of CS patients carrying CSA mutations in North Africa. These mutations have been described only in this region and in the Middle-East. We also provide the largest characterization of multiple unrelated patients, as well as siblings, carrying the same mutation, providing a framework for dissecting elusive genotype-phenotype correlations in CS.

Identification of a novel de novo variant in OTX2 in a patient with congenital microphthalmia using targeted next-generation sequencing followed by prenatal diagnosis.

BACKGROUND: Next-generation sequencing has been proven to be a reliable method for the detection of genetic causes in heterogeneous ocular disorders. In this report an NGS-based diagnostic approach was taken to uncover the genetic etiology in a patient with coloboma and microphthalmia, a highly heterogeneous disease with intrafamilial phenotypic variability. MATERIALS AND METHODS: Next generation sequencing using a targeted panel of 316 genes, was carried out in the proband. Prioritized variants were then identified and confirmed using Sanger sequencing. Prenatal diagnosis of the detected variant was then performed in the family. RESULTS: A novel de novo frameshift variant c.157_164delTTCACTCG (p.Phe53fs) in OTX2, leading to a truncated protein, was identified. Prenatal diagnosis identified the same variant in the fetus. CONCLUSIONS: This report demonstrates the importance of genetic counseling and underscores the efficiency and effectiveness of targeted NGS as a means of detecting variants in inherited eye disorders.

An unusual case of microsatellite instability-high/deficient mismatch repair (MSI-H/dMMR) diffuse large B-cell lymphoma revealed by targeted gene sequencing.

Microsatellite instability-high/deficient mismatch repair (MSI-H/dMMR) status has been approved as a tissue-agnostic biomarker for immune checkpoint inhibitor therapy in patients with solid tumors. We report the case of an MSI-H/dMMR diffuse large B-cell lymphoma (DLBCL) identified by targeted gene sequencing (TGS). A 90-year-old female who presented with vaginal bleeding and a large mass in the upper vagina was diagnosed with germinal center-B-cell-like DLBCL, which recurred at the uterine cervix at 9 months after chemotherapy. Based on TGS of 121 lymphoma-related genes and the LymphGen algorithm, the tumor was classified genetically as DLBCL of EZB subtype. Mutations in multiple genes, including frequent frameshift mutations, were detected by TGS and further suggested MSI. The MSI-H/dMMR and loss of MLH1 and PMS2 expression were determined in MSI-fragment analysis, MSI real-time polymerase chain reaction, and immunohistochemical tests. This case demonstrates the potential diagnostic and therapeutic utility of lymphoma panel sequencing for DLBCL with MSI-H/dMMR.

Identification of a novel mutation in the PHKA2 gene in a child with liver cirrhosis.

OBJECTIVES: Glycogen storage diseases (GSDs) are heterogeneous disorders caused by various enzyme deficiencies. GSD type IX α2, the most common subtype of GSD IX, is due to a deficiency of hepatic phosphorylase kinase. Herein we will report a novel mutation in this disease with an unusual presentation. CASE PRESENTATION: we describe a 3-year-old boy who suffered from hepatomegaly, fatty liver disease, and liver cirrhosis. The cause of cirrhosis at a young age was unknown based on the laboratory data and liver biopsy, so we performed a targeted-gene sequencing (TGS) covering 450 genes involved in inborn metabolic diseases consisting of glycogen storage disorders genes with hepatic involvement. He was found out to have a rare novel pathogenic variant in the PHKA2 gene. CONCLUSIONS: This novel variant c.2226+2T > C expands the mutational spectrum of the PHKA2 gene. Also, severe liver damage (cirrhosis) in patients with GSD- IX α2 has rarely been reported, which needs further discussion. We hypothesize that unidentified PHKA2 variants may be a rare cause of childhood liver cirrhosis.

The landscape of PD-L1 expression and somatic mutations in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver, and becoming the third-leading cause of cancer-related mortality worldwide. Despite the immune checkpoint inhibitors and molecular targeted therapies have shown preferable efficacy in HCC, large number of HCC patients do not respond effectively to anti-PD-1 reagents. Besides, the accumulation of genetic mutations in cancer cells may lead to the therapy resistant. Hence, there are clinical gaps between genetic and transcriptomic biomarkers for the HCC treatment. METHODS: To investigate the genetic mapping of liver cancer, targeted deep sequencing (TDS) and bioinformatics analysis were performed on hepatocellular carcinoma (HCC) tumor tissues and matched blood samples. Furthermore, copy number variants (CNVs) and Tumor mutation burden (TMB) were calculated. Immunohistochemistry was applied to determine the PD-L1 expression in HCC tumor tissues. Clinical characteristic, PD-L1 expression, and the TMB were analyzed in 32 HCC patients. RESULTS: This study indicated that the PD-L1 positive patients exhibited a lower TMB compared to the PD-L1 negative group, and PD-L1 positive patients were more likely to suffer from aggressive clinicopathologic features than PD-L1 negative patients. We also verified the top 30 mutated genes, including TP53, CTNNB1, KMT2D, AXIN1, ALK, and NOTCH1, in our dataset. Our results indicated that PD-L1 positive patients possessed more tumors with vascular invasion and advanced CCLC stage. Moreover, PD-L1 positive patients exhibited a lower TMB compared to the PD-L1 negative group. CONCLUSIONS: These findings could improve our understanding of the effects of immune checkpoint therapies on prognosis, and could facilitate the monitoring of somatic mutations in HCC.

Targeted-Gene Sequencing and Bioinformatics Analysis of Patients with Pancreatic Mucoepidermoid Carcinoma: A Case Report and Literature Review.

BACKGROUND: Primary pancreatic mucoepidermoid carcinoma (MEC) is an extremely rare malignant tumor with unclear etiology and pathogenesis. There are only eleven reported cases in English papers published from 1959 to 2020. MEC generally occurs in the salivary gland, but cases in the pancreas have also been reported. Although being considered as a low-grade indolent carcinoma, pancreatic MEC always invades the surrounding lymph node and metastasizes. The prognosis of pancreatic MEC is unsatisfactory. To date, the genetic alterations, mechanistic relationships among mutated genes and signaling pathways of pancreatic MEC are unclear. PATIENT AND METHODS: This paper presents a case of rare primary pancreatic MEC in a 56-year-old male patient with liver metastasis. Radical surgery of distal pancreatectomy and radiofrequency ablation (RFA) of two liver metastatic lesions is conducted. Targeted-gene sequencing and bioinformatics analysis tools, including STRING, DAVID, cBioPortal, DGidb and Human Protein Atlas database (HPA), are used to clarify the biological functions and features of mutated genes in pancreatic MEC. RESULTS: Eight gene mutations (TP53, KRAS, ATR, FLI1, FLT4, MAGI2, RBM10, and TNFAIP3) were observed, and a tumor mutation burden (TMB) of 5.6 muts/Mb was calculated in the pancreatic MEC using targeted-gene sequencing. The patient subsequently underwent adjuvant chemotherapy and died three months after surgery. Gene-gene interaction network was constructed, which showed the significant interactions among eight mutated genes. In terms of the functions and pathways of eight gene mutations based on GO and KEGG, 20 tumor-related results are presented in this paper, Furthermore, the biological functions and features of pancreatic MEC are further compared with pancreatic ductal adenocarcinoma. CONCLUSION: Pancreatic MEC requires early and effective treatment, and lymph node metastases and multiple organ metastases were unfavorable prognostic factors. Pancreatic MEC can be compared with other pancreatic cancers that have characteristic clinical phenotype, molecular alterations, functional information and enrichment pathway.