Clinical Spectrum and Molecular Pathophysiology of Shwachman-Diamond Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-34-SCI-34
Author(s):  
Akiko Shimamura
Keyword(s):  
Next Generation Sequencing ◽  
Autosomal Recessive ◽  
Conflicts Of Interest ◽  
Diagnostic Algorithm ◽  
Next Generation ◽  
Recessive Mutations ◽  
Pancreatic Dysfunction ◽  
Shwachman Diamond Syndrome ◽  
Molecular Pathophysiology ◽  
Generation Sequencing

Abstract Shwachman-Diamond Syndrome (SDS) is an inherited marrow failure syndrome associated with exocrine pancreatic dysfunction and leukemia predisposition. SDS patients may also manifest additional non-hematologic abnormalities. Autosomal recessive mutations in the SBDS gene are found in over 90 percent of patients fitting the classical clinical phenotype of SDS. The advent of genetic testing has revealed an unexpectedly broad range of SDS phenotypes. Through the Shwachman-Diamond Syndrome Registry, we found that diagnosis may be obscured by cryptic or non-classical presentations of SDS. The timely diagnosis of SDS carries profound ramifications for medical management and treatment. We are developing assays utilizing massively parallel next generation sequencing to address this challenging diagnostic problem. Clinical applications of next generation sequencing to the diagnostic algorithm for marrow failure or myelodysplastic syndrome and implications for medical treatment will be explored. Disclosures: No relevant conflicts of interest to declare.

Diagnostic value of a combination of next-generation sequencing, chorioretinal imaging and metabolic analysis: lessons from a consanguineous Chinese family with gyrate atrophy of the choroid and retina stemming from a novel OAT variant

2018 ◽  
Vol 103 (3) ◽  
pp. 428-435 ◽  
Author(s):  
Junting Huang ◽  
Jiewen Fu ◽  
Shangyi Fu ◽  
Lisha Yang ◽  
Kailai Nie ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Low Income ◽  
Autosomal Recessive ◽  
Diagnostic Value ◽  
Consanguineous Marriage ◽  
Chinese Family ◽  
Next Generation ◽  
Gyrate Atrophy ◽  
Metabolic Analysis ◽  
Generation Sequencing

Background/AimGyrate atrophy of the choroid and retina (GACR) is an extremely rare autosomal recessive inherited disorder characterised by progressive vision loss. To identify the disease-causing gene in a consanguineous Chinese pedigree with GACR, we aimed to accurately diagnose patients with GACR through a combination of next-generation sequencing (NGS) genetic diagnosis, clinical imaging and amino acid metabolic analysis.MethodsA consanguineous Chinese pedigree with GACR, including two patients, was recruited and a comprehensive ophthalmological evaluation was performed. DNA was extracted from a proband and her family members, and the sample from the proband was analysed using targeted NGS. Variants ‎detected by NGS were confirmed by Sanger sequencing and subjected to segregation analysis. Tandem mass spectrometry (MS/MS) was subsequently performed for metabolic assessment.ResultsWe identified a ‎novel, deleterious, homologous ornithine aminotransferase (OAT) variant, c.G248A: p.S83N, which contributes to ‎the progression of GACR in patients. Our results showed that the p.S83N autosomal recessive ‎variant of OAT is most likely ‎pathogenic, with changes in protein stability drastically decreasing functionality. MS/MS verified that ornithine levels in patients were significantly elevated.ConclusionsRecruitment of a third-degree first cousin consanguineous marriage family with GACR allowed us to identify a novel pathogenicOATvariant in the Chinese population, broadening the mutation spectrum. Our findings reported the diagnostic value of a combination of NGS, retinal imaging and metabolic analysis of consanguineous marriage pedigrees in low-income/middle-income and low-incidence countries, including China, and may help to guide accurate diagnosis and ‎treatment of this disease.

Identification and Characterization of a Novel Parvovirus-Like Virus in Seronegative Hepatitis Patients by Next Generation Sequencing

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 273-273
Author(s):  
Baoyan Xu ◽  
Ning Zhi ◽  
Gangqing Hu ◽  
Zhihong Wan ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Capsid Protein ◽  
Conflicts Of Interest ◽  
Virus Titer ◽  
The United States ◽  
Western China ◽  
Healthy Controls ◽  
Next Generation ◽  
Specific Antibodies ◽  
Generation Sequencing

Abstract Abstract 273 Seronegative hepatitis—non-hepatitis A, non-B, non-C, non-E—is poorly characterized but strongly associated with serious complications, especially aplastic anemia and fulminant hepatitis of childhood. Seronegative hepatitis is rare in the United States but more prevalent in Asia, constituting about 10–20% of acute cases. We applied next-generation sequencing to blood samples of patients from western China with seronegative hepatitis for virus discovery. A total of 92 plasma specimens were collected at Chongqing, China, between 1999 and 2007. Twenty-seven patients were diagnosed as having acute hepatitis by clinical and laboratory characteristics. Sixty-five patients had biopsy-proven chronic aggressive hepatitis, ten of which had cirrhosis. Serologic assays for hepatitis viruses A, B, C, E, HIV, Epstein-Barr virus and cytomegalovirus were all negative. Additional tests for antinuclear antibody, rheumatoid factor, anti-mitochondrial antibody also were normal. Ten plasma pools derived from 93 specimens of the patients were screened by Solexa deep sequencing. We discovered a 3780-bp contig present in all ten-pools that yielded tBLASTx E scores of 0.003 to 1.5 against parvoviruses. The sequence of the in silico assembled 3780-bp contig was confirmed by overlapping PCRs, indicating the contig that contained the nearly complete new virus genome indeed existed in the patient samples rather than being artificially generated by misassembly. The new virus is provisionally designated NIH-CQV. Further analysis revealed that the contig was composed of two major open reading frames (ORF). Protein Blast showed that ORF1 encoded a protein that contained a conserved P-loop NTPase domain, homologous to the replication-associated protein of bat circovirus (E score=4e-04). ORF2 was homologous to capsid protein of porcine parvovirus (E scores=7e-06). Phylogenetic analysis indicated that the NIH-CQV represents a new subfamily of parvovirus, located at the interface of Parvoviridae and Circoviridae (Figure 1). Prevalence of the NIH-CQV in hepatitis patients was investigated by qPCR. Sixty three out of 92 (69%) patient samples were positive, while all 45 healthy controls were negative. The average virus titer in the patients was 1.28 E4 copies/ul, and the highest one was 3.2 E4 copies/ul. Specific antibodies against NIH-CQV were sought by immunoblot using a recombinant capsid protein. No cross reactivity was detected between the capsid protein of NIH-CQV and other major human parvoviruses. Eighty five percent (78/92) of patients were positive for IgG, and 32% (29/92) of them were positive for IgM. In contrast, 78% (35/45) of healthy controls were positive for IgG and 16% (7/45) were positive for IgM. Viral particles were purified from IgM-positive patient plasma by ultracentrifugation through a 40% sucrose cushion and examined by electron microscopy: spherical, naked, parvovirus-like particles approximately 26–29 nm in diameter were visualized. There was no correlation between clinical diagnosis and the presence or absence of the viral DNA or specific antibodies. Although more work is needed to determine the etiologic role of NIH-CQV in human disease, our data indicate that a novel parvovirus-like virus is highly prevalent in a cohort of patients with seronegative hepatitis. Figure 1, whole-proteome tree of the new parvovirus and members of the families Parvoviridae and Circoviridae. Figure 1,. whole-proteome tree of the new parvovirus and members of the families Parvoviridae and Circoviridae. Disclosures: No relevant conflicts of interest to declare.

Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1703-1703
Author(s):  
Kankana Ghosh ◽  
Parsa Hodjat ◽  
Priyanka Priyanka ◽  
Beenu Thakral ◽  
Keyur P. Patel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Myelodysplastic Syndrome ◽  
Mutation Analysis ◽  
Conflicts Of Interest ◽  
International Prognostic Scoring System ◽  
P Value ◽  
Newly Diagnosed ◽  
Next Generation ◽  
Generation Sequencing

Abstract INTRODUCTION Myelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R). MATERIALS AND METHODS We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment naïve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients. RESULTS Median age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μ L (5-652), WBC: 2.8 K/μ L (0.4-20.8), ANC 1.3 K/μ L (0.0 -12.0), AMC 0.2 K/μ L (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score. CONCLUSIONS: MDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R. Table 1. Number of cases % cases TP53 46 23 TET2 33 16.5 RUNX1 27 13.5 ASXL1 25 12.5 DNMT3A 17 8.5 EZH2 12 6 IDH2 8 4 IDH1 7 3.5 NRAS 7 3.5 JAK2 5 2.5 FLT3 4 2 PTPN11 3 1.5 EGFR 2 1 MPL 2 1 WT1 2 1 GATA2 1 0.5 KIT 1 0.5 KRAS 1 0.5 MYD88 1 0.5 NPM1 1 0.5 BRAF 1 0.5 Table 2. Mutated genes p value WBC ASXL1 <0.042 AEC TET2 <0.016 BM blast % RUNX1, CEBPA <0.008, p<0.02 BM myelocyte % TP53, TET2, RUNX1, DNMT3A <0.014, <0.014, <0.015, <0.038 AEC: absolute eosinophil count, BM: bone marrow Disclosures No relevant conflicts of interest to declare.

scholarly journals Assessment of the Application and Clinical Value of Next-Generation Sequencing for the Diagnosis of Inherited Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4993-4993
Author(s):  
Miao Jiang ◽  
Qi Wang ◽  
Yiming Zhao ◽  
Ziqiang Yu ◽  
Suning Chen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Conflicts Of Interest ◽  
Gene Sequencing ◽  
Hereditary Diseases ◽  
Next Generation ◽  
Clinical Value ◽  
Sequencing Platform ◽  
Pathogenic Variants ◽  
Inherited Thrombocytopenia ◽  
Generation Sequencing

Abstract Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 182 patients with thrombocytopenia, we screened 78 patients with hereditary features. For the blood samples of these 78 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 23 of 78 patients (29.5%) were diagnosed with inherited thrombocytopenia. In addition, 29 pathogenic variants, including 11 previously unreported variants, were identified in these patients. In summary, NGS could play more important role in the molecular pathology diagnosis of inherited thrombocytopenia. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Compound Heterozygous Mutations in TMC1 and MYO15A Are Associated with Autosomal Recessive Nonsyndromic Hearing Loss in Two Chinese Han Families

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Pengcheng Xu ◽  
Jun Xu ◽  
Hu Peng ◽  
Tao Yang
Keyword(s):  
Hearing Loss ◽  
Next Generation Sequencing ◽  
Genetic Diagnosis ◽  
Compound Heterozygous ◽  
Next Generation ◽  
Chinese Han ◽  
Targeted Next Generation Sequencing ◽  
Recessive Mutations ◽  
Compound Heterozygous Mutations ◽  
Generation Sequencing

Genetic hearing loss is a common sensory disorder, and its cause is highly heterogeneous. In this study, by targeted next-generation sequencing of 414 known deafness genes, we identified compound heterozygous mutations p.R34X/p.M413T in TMC1 and p.S3417del/p.R1407T in MYO15A in two recessive Chinese Han deaf families. Intrafamilial cosegregation of the mutations with the hearing phenotype was confirmed in both families by the Sanger sequencing. Auditory features of the affected individuals are consistent with that previously reported for recessive mutations in TMC1 and MYO15A. The two novel mutations identified in this study, p.M413T in TMC1 and p.R1407T in MYO15A, are classified as likely pathogenic according to the guidelines of ACMG. Our study expanded the mutation spectrums of TMC1 and MYO15A and illustrated that genotype-phenotype correlation in combination with next-generation sequencing may improve the accuracy for genetic diagnosis of deafness.

Characterization of Chromosome 20q Deletions In Myeloproliferative Neoplasms Using Microarray Karyotyping and Next-Generation Sequencing

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4099-4099
Author(s):  
Roland Jäger ◽  
Tiina Berg ◽  
Ashot Harutyunyan ◽  
Thorsten Klampfl ◽  
Francesco Passamonti ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Single Gene ◽  
Next Generation ◽  
Large Deletions ◽  
Cytogenetic Aberrations ◽  
A Genome ◽  
Generation Sequencing

Abstract Abstract 4099 Deletions on chromosome 20q (del20q) are among the most frequent cytogenetic aberrations in myeloid disorders. Mapping of common deleted regions (CDRs) has the potential to link often large deletions to single gene defects. In the past years 20q CDRs from several patient cohorts have been reported, most of them combining patients with different myeloid malignancies, such as myeloproliferative neoplasms (MPN), chronic myeloid leukemia and myelodysplastic syndrome. However, it is not clear whether the same genes within del20q are relevant for pathogenesis in the different myeloid diseases. We aimed to delineate a common deleted region exclusively based on a substantial number of MPN patients. Using a copy number assay, we screened for del20q in a total of 822 MPN patients combined from three independent MPN patient cohorts. Del20q was present in granulocyte DNA from 11 patients (1.1%). We consequently mapped the 11 deletions using Affymetrix 6.0 microarray karyotyping, resulting in a common deleted region of 6.4 Mb size, comprising 82 transcribed genes. In a next step, we aimed to investigate the remaining undeleted allele for the presence of somatic mutations. We applied a next generation sequencing approach and sequenced 774 coding exons within our del20q CDR in 11 patients with del20q. We prepared a DNA pool containing equal amounts of granulocyte DNA from each patient and amplified 774 exons in separate PCR reactions. The PCR reactions were pooled, concatemerized by ligation, refragmented, and prepared for a single-end 36bp sequencing read on a Genome Analyzer IIx (Illumina). The single-end read delivered a coverage between 1000- and 5000-fold per exonic base. We identified five putative variants in the genes SGK2, SEMG1, SEMG2, WFDC9 and SLC13A3 that were non-synonymous and were not annotated in any of the public SNP databases. Further validation in the single patients using classical capillary sequencing identified heterozygous calls in SGK2, SEMG1 and SEMG2 as false positives. The putative variants in WFDC9 (position chr20:43670771; G/A; NCBI36/hg18) and SLC13A3 (position chr20:44654462; C/G; NCBI36/hg18) could be confirmed in granulocyte DNA from a subset of the del20q patients. However, the variants could also be detected in DNA from a control non-myeloid tissue indicating their germ line origin. The variants were present in healthy individuals at variable frequencies, identifying them as novel SNPs presumably not involved in MPN pathogenesis. From the absence of mutations on the undeleted allele in patients with del20q we can conclude that haploinsufficiency is the most likely functional consequence of tumor suppressor inactivation within the del20q CDR in myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

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