Targeted Next Generation Sequencing of the Five Hemochromatosis Genes in Italian Patients with Iron Overload and Non-Diagnostic First Level Genetic Test: A Pilot Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4030-4030
Author(s):  
Sadaf Badar ◽  
Fabiana Busti ◽  
Giampiero Zamperin ◽  
Alberto Ferrarini ◽  
Paolo Bozzini ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pilot Study ◽  
Iron Overload ◽  
Genetic Test ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Autosomal Recessive Disorder ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Generation Sequencing

Abstract Background and Aim: Molecular diagnosis of HFE-related hereditary hemochromatosis (HH) is typically made by searching for the C282Y and H63D mutations (first level genetic test). However, in the Mediterranean area up to one third of patients with HH phenotype do not have the “diagnostic” genotypes (C282Y homozygosity, or C282Y/H63D compound heterozygosity). This pilot study was designed to develop a “second level” next generation sequencing (NGS)-based test for rapid and simultaneous analysis of the five HH genes (HFE, HFE2, HAMP, TFR2 and SLC40A1). Methodology: we studied 61 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HH. The five HH genes were captured by Halo-Plex™ technology, and then sequenced using aNGS platform (Illumina HiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease. Results: In IO patients a large number of new non-synonymous variants (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Many of them were relatively frequent and detected also in controls, thus being considered likely “non-pathogenic”, unless clearly enriched in patients. On the other hand, some rare variants (i.e. limited to a single or very few individuals), particularly in SCL40A1, TFR2, and HFE, were found exclusively in patients, and could be considered “potentially pathogenic”. Conclusions: The combination of the Halo-Plex™ approach with NGS platform and GoldenHelix™ algorithm appears a suitable approach for a better molecular characterization of patients with unexplained HH phenotype, and could represent a good option for second level genetic testing in referral centers. However, establishing the clinical relevance of NGS-detected “novel” genetic variants in a prevalently autosomal recessive disorder like HH remains a difficult task, requiring further functional studies and national/international collaborative efforts. Disclosures No relevant conflicts of interest to declare.

scholarly journals Next-generation sequencing of adrenocortical carcinoma reveals new routes to targeted therapies

2014 ◽  
Vol 67 (11) ◽  
pp. 968-973 ◽  
Author(s):  
J S Ross ◽  
K Wang ◽  
J V Rand ◽  
L Gay ◽  
M J Presta ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Adrenocortical Carcinoma ◽  
Single Case ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Modest Improvement ◽  
Aggressive Form ◽  
Comprehensive Genomic Profiling ◽  
Cytotoxic Therapies ◽  
Generation Sequencing

AimsAdrenocortical carcinoma (ACC) carries a poor prognosis and current systemic cytotoxic therapies result in only modest improvement in overall survival. In this retrospective study, we performed a comprehensive genomic profiling of 29 consecutive ACC samples to identify potential targets of therapy not currently searched for in routine clinical practice.MethodsDNA from 29 ACC was sequenced to high, uniform coverage (Illumina HiSeq) and analysed for genomic alterations (GAs).ResultsAt least one GA was found in 22 (76%) ACC (mean 2.6 alterations per ACC). The most frequent GAs were in TP53 (34%), NF1 (14%), CDKN2A (14%), MEN1 (14%), CTNNB1 (10%) and ATM (10%). APC, CCND2, CDK4, DAXX, DNMT3A, KDM5C, LRP1B, MSH2 and RB1 were each altered in two cases (7%) and EGFR, ERBB4, KRAS, MDM2, NRAS, PDGFRB, PIK3CA, PTEN and PTCH1 were each altered in a single case (3%). In 17 (59%) of ACC, at least one GA was associated with an available therapeutic or a mechanism-based clinical trial.ConclusionsNext-generation sequencing can discover targets of therapy for relapsed and metastatic ACC and shows promise to improve outcomes for this aggressive form of cancer.

Next‐generation sequencing assay in salivary gland cytology: A pilot study

2020 ◽  
Vol 49 (10) ◽  
pp. 1037-1043
Author(s):  
Shuhei Yamamoto ◽  
Yuma Sakamoto ◽  
Satsuki Nakano ◽  
Keiichiro Fujii ◽  
Kaori Ueda ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Next Generation Sequencing ◽  
Pilot Study ◽  
Next Generation ◽  
Generation Sequencing

Targeted Next-Generation Sequencing in Men with Metastatic Prostate Cancer: a Pilot Study

2018 ◽  
Vol 13 (4) ◽  
pp. 495-500 ◽  
Author(s):  
Pedro C. Barata ◽  
Prateek Mendiratta ◽  
Brandie Heald ◽  
Stefan Klek ◽  
Petros Grivas ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Next Generation Sequencing ◽  
Pilot Study ◽  
Metastatic Prostate Cancer ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

scholarly journals British kindred with dominant FMF associated with high incidence of AA amyloidosis caused by novel MEFV variant, and a review of the literature

Rheumatology ◽  
2019 ◽  
Author(s):  
Dorota M Rowczenio ◽  
Taryn Youngstein ◽  
Hadija Trojer ◽  
Ebun Omoyinmi ◽  
Anna Baginska ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Index Case ◽  
Autosomal Recessive Disorder ◽  
Index Patient ◽  
Sequencing Analysis ◽  
Inherited Disease ◽  
Next Generation ◽  
Aa Amyloidosis ◽  
Laboratory Assessment ◽  
Generation Sequencing

Abstract Objectives Hereditary systemic autoinflammatory diseases are rare genetic disorders, which if untreated, can be complicated by AA amyloidosis leading to renal failure and premature death. Our objective was to find a genetic cause in a British family with a dominantly inherited autoinflammatory disease complicated by AA amyloidosis. Methods The index patient and his sister underwent comprehensive clinical and laboratory assessment including the next-generation sequencing panel targeting autoinflammatory genes. Subsequently, other relatives underwent clinical evaluation and genetic testing. Screening of the SAA1 gene was performed in all symptomatic cases. Results The index case and his sister presented with proteinuria due to AA amyloidosis. They have been suffering from episodes of fever accompanied by severe abdominal and chest pain, arthritis and erythema since childhood. Their father died aged 52 years from complications following a cadaveric renal transplantation. The post-mortem examination demonstrated AA amyloidosis. The index case’s grandmother, two paternal cousins and two of their children described similar symptoms. All symptomatic individuals had excellent responses to colchicine. Next-generation sequencing analysis identified a single MEFV p.P373L variant in the index case, his sister and subsequently, in symptomatic family members. Sequencing of the SAA1 gene revealed all cases were heterozygous for the SAA1.1 allele. Conclusion Typically FMF is an autosomal recessive disorder; nonetheless rare cases of dominantly inherited disease have previously been described. Here we report a novel MEFV variant p.P373L, causing dominant FMF complicated by AA amyloidosis in four generations of a British family.

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.

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.

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