A Novel Likely Pathogenic Variant in the BLOC1S5 Gene Associated with Hermansky-Pudlak Syndrome Type 11 and an Overview of Human BLOC-1 Deficiencies

Hermansky-Pudlak syndrome (HPS) is a heterogeneous disorder combining oculocutaneous albinism (OCA) and a platelet function disorder of varying severity as its most prominent features. The genes associated with HPS encode for different BLOC- (biogenesis of lysosome-related organelles complex) complexes and for the AP-3 (adaptor protein-3) complex, respectively. These proteins are involved in maturation, trafficking, and the function of lysosome-related organelles (LROs) such as melanosomes and platelet δ-granules. Some patients with different types of HPS can develop additional complications and symptoms like pulmonary fibrosis, granulomatous colitis, and immunodeficiency. A new type of HPS has recently been identified associated with genetic alterations in the BLOC1S5 gene, which encodes the subunit Muted of the BLOC-1 complex. Our aim was to unravel the genetic defect in two siblings with a suspected HPS diagnosis (because of OCA and bleeding symptoms) using next generation sequencing (NGS). Platelet functional analysis revealed reduced platelet aggregation after stimulation with ADP and a severe secretion defect in platelet δ-granules. NGS identified a novel homozygous essential splice site variant in the BLOC1S5 gene present in both affected siblings who are descendants of a consanguine marriage. The patients exhibited no additional symptoms. Our study confirms that pathogenic variants of BLOC1S5 cause the recently described HPS type 11.

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Next-Generation Sequencing Improves Diagnosis, Prognosis and Clinical Management of Myeloid Neoplasms

Cancers ◽

10.3390/cancers11091364 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1364 ◽

Cited By ~ 6

Author(s):

Diego Carbonell ◽

Julia Suárez-González ◽

María Chicano ◽

Cristina Andrés-Zayas ◽

Juan Carlos Triviño ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number Variants ◽

Genetic Alterations ◽

Next Generation ◽

Pathogenic Variants ◽

Myeloid Neoplasms ◽

Diagnostic Samples ◽

Gene Panels ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Molecular diagnosis of myeloid neoplasms (MN) is based on the detection of multiple genetic alterations using various techniques. Next-generation sequencing (NGS) has been proved as a useful method for analyzing many genes simultaneously. In this context, we analyzed diagnostic samples from 121 patients affected by MN and ten relapse samples from a subset of acute myeloid leukemia patients using two enrichment-capture NGS gene panels. Pathogenicity classification of variants was enhanced by the development and application of a custom onco-hematology score. A total of 278 pathogenic variants were detected in 84% of patients. For structural alterations, 82% of those identified by cytogenetics were detected by NGS, 25 of 31 copy number variants and three out of three translocations. The detection of variants using NGS changed the diagnosis of seven patients and the prognosis of 15 patients and enabled us to identify 44 suitable candidates for clinical trials. Regarding AML, six of the ten relapsed patients lost or gained variants, comparing with diagnostic samples. In conclusion, the use of NGS panels in MN improves genetic characterization of the disease compared with conventional methods, thus demonstrating its potential clinical utility in routine clinical testing. This approach leads to better-adjusted treatments for each patient.

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One4Two®: An Integrated Molecular Approach to Optimize Infertile Couples’ Journey

Genes ◽

10.3390/genes12010060 ◽

2021 ◽

Vol 12 (1) ◽

pp. 60

Author(s):

Valeria D’Argenio ◽

Federica Cariati ◽

Rossella Tomaiuolo

Keyword(s):

Disease Genes ◽

Diagnostic Efficacy ◽

Whole Process ◽

Pathogenic Variants ◽

Limiting Step ◽

Number Of Genes ◽

Infertile Couples ◽

Next Generation Sequencing Ngs ◽

And Diffusion ◽

Generation Sequencing

The current diagnostic path of infertile couples is long lasting and often ineffective. Genetic tests, in particular, appear as a limiting step due to their jeopardized use on one side, and to the limited number of genes evaluated on the other. In this context, the development and diffusion, also in routine diagnostic settings, of next generation sequencing (NGS)-based methods for the analyses of several genes in multiple subjects at a time is improving the diagnostic sensitivity of molecular analyses. Thus, we developed One4Two®, a custom NGS panel to optimize the diagnostic journey of infertile couples. The panel validation was carried out in three steps analyzing a total of 83 subjects. Interestingly, all the previously identified variants were confirmed, assessing the analytic sensitivity of the method. Moreover, additional pathogenic variants have been identified underlying the diagnostic efficacy of the proposed method. One4Two® allows the simultaneous analysis of infertility-related genes, disease-genes of common inherited diseases, and of polymorphisms related to therapy outcome. Thus, One4Two® is able to improve the diagnostic journey of infertile couples by simplifying the whole process not only for patients, but also for laboratories and reproduction specialists moving toward an even more personalized medicine.

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A novel frequent BRCA1 recurrent variant c.5117G > A (p.Gly1206Glu) identified after 20 years of BRCA1/2 research in the Baltic region: cohort study and literature review

Hereditary Cancer in Clinical Practice ◽

10.1186/s13053-021-00168-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

P. Loza ◽

A. Irmejs ◽

Z. Daneberga ◽

E. Miklasevics ◽

E. Berga-Svitina ◽

...

Keyword(s):

Literature Review ◽

Local Spectrum ◽

Single Family ◽

Baltic Region ◽

Breast And Ovarian Cancer ◽

Pathogenic Variants ◽

Common Founder ◽

The Baltic ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background Several recent studies in the Baltic region have found extended spectrum of pathogenic variants (PV) of the BRCA1/2 genes. The aim of current study is to analyze the spectrum of the BRCA1/2 PV in population of Latvia and to compare common PV between populations of the Baltic region. Methods We present a cohort of 9543 unrelated individuals including ones with cancer and unaffected individuals from population of Latvia, who were tested for three most common BRCA1 founder PV. In second line testing, 164 founder negative high-risk individuals were tested for PV of the BRCA1/2 using next generation sequencing (NGS). Local spectrum of the BRCA1/2 PV was compared with the Baltic region by performing a literature review. Results Founder PV c.5266dupC, c.4035delA or c.181 T > G was detected in 369/9543 (3.9%) cases. Other BRCA1/2 PV were found in 44/164 (26.8%) of NGS cases. Four recurrent BRCA1 variants c.5117G > A (p.Gly1706Glu), c.4675G > A (p.Glu1559Lys), c.5503C > T (p.Arg1835*) and c.1961delA (p.Lys654fs) were detected in 18/44 (41.0%), 5/44 (11.4%), 2/44 (4.5%) and 2/44 (4.5%) cases respectively. Additionally, 11 BRCA1 PV and six BRCA2 PV were each found in single family. Conclusions By combining three studies by our group of the same cohort in Latvia, frequency of the BRCA1/2 PV for unselected breast and ovarian cancer cases is 241/5060 (4.8%) and 162/1067 (15.2%) respectively. The frequency of three “historical” founder PV is up to 87.0% (369/424). Other non-founder PV contribute to at least 13.0% (55/424) and this proportion probably will rise by increasing numbers of the BRCA1/2 sequencing. In relative numbers, c.5117G > A is currently the third most frequent PV of the BRCA1 in population of Latvia, overcoming previously known third most common founder variant c.181 T > G. In addition to three BRCA1 founder PV, a total of five recurrent BRCA1 and two recurrent BRCA2 PV have been reported in population of Latvia so far. Many of the BRCA1/2 PV reported in Latvia are shared among other populations of the Baltic region.

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Assessment of Iron Overload in a Cohort of Sri Lankan Patients with Transfusion Dependent Beta Thalassaemia and its Correlation with Pathogenic Variants in HBB, HFE, SLC40A1, and TFR2 Genes

10.21203/rs.3.rs-541471/v1 ◽

2021 ◽

Author(s):

Ruwangi Dissanayake ◽

Nayana Samarasinghe ◽

Samantha Waidyanatha ◽

Sajeewani Pathirana ◽

Vajira HW Dissanayake ◽

...

Keyword(s):

Iron Overload ◽

Serum Ferritin ◽

Sri Lankan ◽

Pathogenic Variants ◽

Increased Risk ◽

The Mean ◽

One Year ◽

Next Generation Sequencing Ngs ◽

T2 Mri ◽

Generation Sequencing

Abstract Background. Iron overload (IO) is a complication in transfusion dependent beta thalassaemmia (TDT). Pathogenic variants in genes involving iron metabolism may confer increased risk of IO. The objective of this study was to determine the magnitude of the cardiac and hepatic IO and determine whether pathogenic variants in HFE, SLC40A1 and TFR2 genes increase the risk of IO in a cohort of TDT patients in Sri Lanka.Materials and Methods. Fifty-seven (57) patients with TDT were recruited for this study. Serum ferritin was done once in 3 months for one year in all. Those who were ≥ 8 years of age underwent T2* MRI of the liver and heart. Fifty-two (52) patients underwent next generation sequencing (NGS) to identify pathogenic variants in HBB, HFE, SLC40A1 and TFR2 genes.Results. The mean age (SD) of this cohort was 9.5 (±4.6) years. It comprised of 30 (52.6%) boys and 27 (47.4%) girls. The mean serum ferritin was 3405 (±2670.5) ng/dl. Hepatic IO was seen in 38 (95%) patients and cardiac IO was seen in 17 (42.5%) patients. All patients with cardiac IO were asymptomatic and had normal echocardiogrammes. There was no statistically significant correlation between serum ferritin and hepatic or cardiac IO.32 (61.5%), 18 (34.6%), 2 (3.8%) of patients were homozygotes, compound heterozygotes and heterozygotes for pathogenic variants in the HBB gene. 9 (17.3%) and 3 (5.8%) patients were heterozygotes for pathogenic variants of HFE and SLC40A1 genes respectively. There were no pathogenic variants for the TfR2 gene. The heterozygotes of the pathogenic variants of the HFE and SLC40A1 genes were not at increased risk of IO.Conclusions. Cardiac T2* MRI helps to detect cardiac IO prior to the onset of symptoms when the echocardiogramme is normal. It is important to perform hepatic and cardiac MRI T2* to detect IO in patients with TDT.

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Genetic Variants Detected Using Cell-Free DNA from Blood and Tumor Samples in Patients with Inflammatory Breast Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21041290 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1290

Author(s):

Jennifer S. Winn ◽

Zachary Hasse ◽

Michael Slifker ◽

Jianming Pei ◽

Sebastian M. Arisi-Fernandez ◽

...

Keyword(s):

Breast Cancer ◽

Inflammatory Breast Cancer ◽

Triple Negative ◽

Genetic Profile ◽

Cell Free Dna ◽

Targeted Next Generation Sequencing ◽

Pathogenic Variants ◽

Free Dna ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%); other affected genes included PMS1, KMT2C, BRCA2, PALB2, MUTYH, MEN1, MSH2, CHEK2, NCOR1, PIK3CA, ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AFtissue ≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.

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The Application of Next-Generation Sequencing to Define Factors Related to Oral Cancer and Discover Novel Biomarkers

Life ◽

10.3390/life10100228 ◽

2020 ◽

Vol 10 (10) ◽

pp. 228

Author(s):

Soyeon Kim ◽

Joo Won Lee ◽

Young-Seok Park

Keyword(s):

Next Generation Sequencing ◽

Oral Cancer ◽

Genetic Alterations ◽

Eligibility Criterion ◽

Next Generation ◽

Novel Biomarkers ◽

Targeted Gene Therapy ◽

Next Generation Sequencing Ngs ◽

Potential Use ◽

Generation Sequencing

Despite the introduction of next-generation sequencing in the realm of DNA sequencing technology, it is not often used in the investigation of oral squamous cell carcinoma (OSCC). Oral cancer is one of the most frequently occurring malignancies in some parts of the world and has a high mortality rate. Patients with this malignancy are likely to have a poor prognosis and may suffer from severe facial deformity or mastication problems even after successful treatment. Therefore, a thorough understanding of this malignancy is essential to prevent and treat it. This review sought to highlight the contributions of next-generation sequencing (NGS) in unveiling the genetic alterations and differential expressions of miRNAs involved in OSCC progression. By applying an appropriate eligibility criterion, we selected relevant studies for review. Frequently identified mutations in genes such as TP53, NOTCH1, and PIK3CA are discussed. The findings of existing miRNAs (e.g., miR-21) as well as novel discoveries pertaining to OSCC are also covered. Lastly, we briefly mention the latest findings in targeted gene therapy and the potential use of miRNAs as biomarkers. Our goal is to encourage researchers to further adopt NGS in their studies and give an overview of the latest findings of OSCC treatment.

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Next Generation Sequencing Identifies Five Novel Mutations in Lebanese Patients with Bardet–Biedl and Usher Syndromes

Genes ◽

10.3390/genes10121047 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1047 ◽

Cited By ~ 1

Author(s):

Lama Jaffal ◽

Wissam H Joumaa ◽

Alexandre Assi ◽

Charles Helou ◽

George Cherfan ◽

...

Keyword(s):

Next Generation Sequencing ◽

Usher Syndrome ◽

Bioinformatic Analysis ◽

Next Generation ◽

Novel Mutations ◽

Pathogenic Variants ◽

Whole Exome ◽

Targeted Ngs ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Aim: To identify disease-causing mutations in four Lebanese families: three families with Bardet–Biedl and one family with Usher syndrome (BBS and USH respectively), using next generation sequencing (NGS). Methods: We applied targeted NGS in two families and whole exome sequencing (WES) in two other families. Pathogenicity of candidate mutations was evaluated according to frequency, conservation, in silico prediction tools, segregation with disease, and compatibility with inheritance pattern. The presence of pathogenic variants was confirmed via Sanger sequencing followed by segregation analysis. Results: Most likely disease-causing mutations were identified in all included patients. In BBS patients, we found (M1): c.2258A > T, p. (Glu753Val) in BBS9, (M2): c.68T > C; p. (Leu23Pro) in ARL6, (M3): c.265_266delTT; p. (Leu89Valfs*11) and (M4): c.880T > G; p. (Tyr294Asp) in BBS12. A previously known variant (M5): c.551A > G; p. (Asp184Ser) was also detected in BBS5. In the USH patient, we found (M6): c.188A > C, p. (Tyr63Ser) in CLRN1. M2, M3, M4, and M6 were novel. All of the candidate mutations were shown to be likely disease-causing through our bioinformatic analysis. They also segregated with the corresponding phenotype in available family members. Conclusion: This study expanded the mutational spectrum and showed the genetic diversity of BBS and USH. It also spotlighted the efficiency of NGS techniques in revealing mutations underlying clinically and genetically heterogeneous disorders.

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Familial and Acquired SH2B3 mutations in ALL

Blood ◽

10.1182/blood.v120.21.1326.1326 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1326-1326

Author(s):

Arianne Perez-Garcia ◽

Charles A. LeDuc ◽

Kara A. Kelly ◽

Chaim Jalas ◽

Wendy K Chung ◽

...

Keyword(s):

T Cell ◽

Cell Growth ◽

Tumor Suppressor ◽

Developmental Delay ◽

Genetic Defect ◽

Adaptor Protein ◽

Genetic Alterations ◽

Ashkenazi Jewish ◽

Wild Type ◽

Stat Signaling

Abstract Abstract 1326 Acute lymphoblastic leukemia (ALL) is a heterogeneous disease in which multiple genetic alterations contribute to the malignant transformation of lymphoid progenitor cells. Although the pathogenesis of ALL is considered to be multifactorial, few environmental factors have been conclusively associated with increased risk. In addition, although recent genome-wide association studies of childhood ALL have suggested that common variation at some genetic loci may predispose to ALL, the risk associated with these variants seems to be modest. Here we hypothesized that leukemia development in the context of rare familial inherited disorders may indicate a tumor suppressor activity for the underlying genetic defect present in these kindreds. In this context, we studied a pediatric B precursor ALL female patient of Eastern European Ashkenazi Jewish background with a history of remote consanguinity, short stature and mild developmental delay. Her family history is significant for a brother with similar growth retardation and mild developmental defects, neonatal anemia, thrombocytopenia and elevated white cell count, who was diagnosed with chronic hepatitis of probable autoimmune origin and Hashimoto's autoimmune thyroiditis. A second male sibling was healthy. We proposed that an underlying autosomal recessive genetic defect resulting in dysregulation of the development and function of the immune system could contribute to the concurrence of a lymphoid neoplasia and an autoimmune disorder in two siblings in this family. We performed homozygosity mapping and linkage analysis in the family. Whole exome sequencing of each of the two affected siblings identified a homozygous frameshift c.671insGGCCCCG p.Asp231fs mutation in the SH2B3 gene. Dideoxynucleotide sequencing of SH2B3 confirmed that both affected siblings were homozygous for this mutation, while the unaffected brother and both parents were carriers. Analysis of 2,000 normal Ashkenazi Jewish adults identified 1 SH2B3 c.671insGGCCCCG p.Asp231fs carrier and no homozygous individuals. The SH2B adaptor protein gene 3 (SH2B3) encodes an adaptor protein involved in the negative regulation of the JAK STAT signaling pathway. Western Blot analysis of immortalized lymphoblastoid cell lines from the two affected siblings and an unrelated normal control showed complete loss of SH2B3 protein expression in SH2B3 mutant cells. Consistently, analysis of JAK STAT signaling in these cells showed increased JAK2 and STAT3 phosphorylation compared with controls. Moreover, SH2B3 mutant lymphoblastoid cells showed increased proliferation and accelerated cell cycle kinetics, which was particularly pronounced in low serum conditions. Consistently, expression of wild type SH2B3 in these cells impaired cell growth and decreased JAK-STAT signaling. Analysis of a cohort of 167 additional ALLs including 119 B-precursor ALLs and 48 T-ALLs identified a homozygous c.1279insCTGTTGCCGTGTGC p.Val426fs SH2B3 mutation in a B-precursor ALL sample and a homozygous nonsense c.908C>A p.Ser303* SH2B3 mutation in a pediatric T-cell ALL. Notably, analysis of normal (remission) DNA demonstrated the somatic origin of the c.908C>A p.Ser303* SH2B3 mutation. In addition, our sequence analysis of SH2B3 in ALL identified two additional heterozygous germline single nucleotide variants resulting in amino acid substitutions (c.557G>T p.Ser186Ile and c.232G>A p.Glu78Lys). Expression of each of these variants in SH2B3 null cells resulted in partial correction of the increased cell growth phenotype and JAK-STAT signaling associated with SH2B3 loss compared with wild type controls. Notably, heterozygous somatic mutations in SH2B3 have been observed in patients with myeloproliferative neoplasms and heterozygous germline SH2B3 alleles have been linked to essential erythrocytosis, suggesting that the hypomorphic SH2B3 p.Ser186Ile and p.Glu78Lys variants found in our analysis may represent rare leukemia predisposing SH2B3 alleles. Overall these results link the loss of SH2B3 with the pathogenesis of a familial developmental delay, autoimmunity and leukemia predisposition syndrome and demonstrate a tumor suppressor role for SH2B3 in the pathogenesis of B-precursor and T-cell ALL. Disclosures: No relevant conflicts of interest to declare.

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Mutational Spectrum Analysis of Seven Genes Associated with Thyroid Dyshormonogenesis

International Journal of Endocrinology ◽

10.1155/2018/8986475 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 7

Author(s):

Xi Chen ◽

Xiaohong Kong ◽

Jie Zhu ◽

Tingting Zhang ◽

Yanwei Li ◽

...

Keyword(s):

Genetic Basis ◽

Genetic Alterations ◽

Chinese Patients ◽

Study Cohort ◽

Chinese Han ◽

Hormone Synthesis ◽

Functional Studies ◽

Pathogenic Variants ◽

Thyroid Dyshormonogenesis ◽

Generation Sequencing

Objective. Thyroid dyshormonogenesis (DH) is a genetically heterogeneous inherited disorder caused by thyroid hormone synthesis abnormalities. This study aims at comprehensively characterizing the mutation spectrum in Chinese patients with DH. Subjects and Methods. We utilized next-generation sequencing to screen for mutations in seven DH-associated genes (TPO, DUOX2, TG, DUOXA2, SLC26A4, SLC5A5, and IYD) in 21 Chinese Han patients with DH from Xinjiang Province. Results. Twenty-eight rare nonpolymorphic variants were found in 19 patients (90.5%), including 19, 5, 3, and 1 variants in DUOX2, TG, DUOXA2, and SLC26A4, respectively. Thirteen (62%) patients carried monogenic mutations, and six (28.5%) carried oligogenic mutations. Fifteen (71%) patients carried 2 or more DUOX2 (14) or DUOXA2 (1) variants. The genetic basis of DH in nine (43%) patients harboring biallelic or triallelic pathogenic variants was resolved. Seventeen patients (81%) carried DUOX2 mutations, most commonly p.R1110Q or p.K530X. No correlations were found between DUOX2 mutation types or numbers and clinical phenotypes. Conclusions. DUOX2 mutations were the most predominant genetic alterations of DH in the study cohort. Oligogenicity may explain the genetic basis of disease in many DH patients. Functional studies and further clinical studies with larger DH patient cohorts are needed to validate the roles of the mutations identified in this study.

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Using a Next Generation Sequencing Panel to Discover the Obscure Causes of Hereditary Hemolytic Anemias

Blood ◽

10.1182/blood.v128.22.2433.2433 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2433-2433 ◽

Cited By ~ 1

Author(s):

Archana M Agarwal ◽

N. Scott Reading ◽

Kimberly Frizzell ◽

Wei Shen ◽

Shelly Sorrells ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hemolytic Anemia ◽

Clinical Laboratory ◽

Single Gene ◽

Cytoskeletal Proteins ◽

Next Generation ◽

Pathogenic Variants ◽

Late Morbidity ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Hereditary hemolytic anemias are a heterogeneous group of disorders with consequences ranging from non-anemic hemolysis to severe life-threatening anemia. However, the late morbidity in patients without transfusions is often underappreciated because of erythropoietic compensatory stimulation inducing hematopoiesis by erythroferrone/hepcidin axis. Principal causes of hereditary hemolytic anemias are germline mutations of red cell cytoskeleton (e.g. hereditary spherocytosis and elliptocytosis/pyropoikilocytosis) or enzyme deficiencies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency). Routine morphological and biochemical analysis may be inconclusive and misleading particularly in transfusion-dependent infants and children. Molecular studies have not been extensively used to diagnose these disorders due to the complex genetic nature of these disorders, and multi-gene disorders. In these cases, patients may undergo multiple rounds of single gene testing, which can be very costly and time consuming. The advent of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using Sure Select kit (Agilent Technologies, Santa Clara, USA). Prior to sequencing on the Illumina Next Seq, (Illumina Inc) instrument, indexed samples are quantified using qPCR and then pooled. Samples were sequenced using 2x150 paired end sequencing. We now report the first 68 patients evaluated using our NGS panel. The age of the patients ranged from newborn to 62 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified using the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic variants in 11 patients and likely pathogenic variants in 12 others, the majority of these were novel. Many variants with unknown significance were also identified that could potentially contribute to disease. The most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits. Some complex interactions were uncovered i.e. SPTA1 mutations along with alpha LELY leading to hereditary pyropoikilocytosis; Spectrin variants along with Gilbert syndrome causing severe hyperbilirubinemia in neonates; and Spectrin variants in combination with PKLR and G6PD variants. Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes, but their clinical significance is further augmented by polymorphisms of UGT1A1 gene contributing to severe neonatal hyperbilirubinemia and its consequences. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular diagnosis, especially in instances where traditional testing failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in many cases with no prior family history. Disclosures Yaish: Octapharma: Other: Study investigator.

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