The Flow Cytometric Osmotic Fragility Test Is an Effective Screening Method for Dehydrated Hereditary Stomatocytosis

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 929-929
Author(s):  
Taiju Utsugisawa ◽  
Takuya Iwasaki ◽  
Takako Aoki ◽  
Yoshio Okamoto ◽  
Takahiro Kawakami ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Screening Method ◽  
Osmotic Fragility ◽  
Target Cells ◽  
Next Generation ◽  
Causative Gene ◽  
Rbc Membrane ◽  
Flow Cytometric ◽  
Congenital Hemolytic Anemia

Abstract Introduction: Dehydrated hereditary stomatocytosis (DHSt) or hereditary xerocytosis (HX) is a form of congenital hemolytic anemia characterized by red blood cell (RBC) dehydration. Heterozygous mutations in PIEZO1, a mechanically-activated ion channel, cause DHSt. Recently, KCNN4, which encodes the Gardos channel, has been found to be the second pathogenic gene for DHSt. DHSt is characterized by an alteration in the RBC morphology in target cells, stomatocytes, and/or echinocytes, and RBC deformability assessments by ektacytometry as well as RBC ion flux measurements are currently the standard laboratory tests for DHSt, but their use in laboratories is limited. The flow cytometric osmotic fragility (FCM-OF) test is a useful diagnostic test for hereditary spherocytosis (HS) and also for hereditary elliptocytosis (HE). In this study, we showed that the FCM-OF test could also successfully diagnose DHSt. Subjects: A total of 46 cases of RBC membrane disorders were examined, and tentative diagnoses were made based on the RBC morphology, acid glycerol lysis time, and eosin 5'-maleimide binding tests, resulting in HS (n=31), HE (n=6), and DHSt (n=9). Methods: The number of RBCs in isotonic and hypotonic buffers were measured by flow cytometry. The degree of osmotic fragility was expressed as the "percentage residual RBCs (%RRC)". We confirmed the DHSt diagnosis by the massively paralleled sequencing using our custom panels targeting 68 hemolytic anemia-related genes with the next-generation sequencer. Results: Both HS and HE patients showed a decrease in %RRC; HS (18.0±8.9%, p<0.001) and HE (41.8±15.7%, p<0.001) compared to normal control (66.7±1.5%). DHSt patients showed a significant increase (112.6±34.5%, p<0.001) in FCM-OF. Additionally, next-generation sequencing revealed consistent causative gene mutations for DHSt; PIEZO1 (p.R2488Q and p.E2496ELE) or KCNN4 (p.P204R, p.A279T and p.R352H). Discussion: We examined 77 patients with congenital hemolytic anemia recently, and 59 cases were confirmed by diagnostic tests (76.6%). The results were as follows: 48 cases of RBC membrane abnormality (62.3%), 6 cases of RBC enzymopathy (7.8%), and 5 cases of hemoglobinopathy (6.5%). Of the cases of RBC membrane disorders, 31 cases of HS, 9 cases of DHSt, and 8 cases of HE were identified. These observations suggest that DHSt is the second-most common RBC membranopathy in Japan, and that the FCM-OF test and targeted sequencing efficiently discriminate DHSt from other RBC membrane disorders. Disclosures No relevant conflicts of interest to declare.

COL4A1 is a Novel Causative Gene Responsible for Congenital Hemolytic Anemia, Representing Characteristic Clinical Course in Infants

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 934-934
Author(s):  
Hiromi Ogura ◽  
Shouichi Ohga ◽  
Takako Aoki ◽  
Taiju Utsugisawa ◽  
Hidehiro Takahashi ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Small Vessel Disease ◽  
Gene Mutations ◽  
Basement Membranes ◽  
Red Cell ◽  
Missense Mutations ◽  
Causative Gene ◽  
Congenital Hemolytic Anemia

Abstract We have been working on the differential diagnosis of congenital hemolytic anemia, but even with extensive analysis of hemoglobin, red cell membrane and enzymes, approximately 40% of patients remained to be diagnosed. In this study, we analyzed 17 undiagnosed hemolytic anemia subjects under the age of 1 by whole-exome sequencing, and identified COL4A1 gene mutations in 5 cases (29.4%). All patients were de novo cases without family histories and exhibited moderate to severe neonatal hemolytic anemia: Hgb, 5.2-9.3 g/dl; MCV, 90.0-126.9; MCHC, 29.9-32.7; and reticulocyte count, 9.2-33.0%. Either schizocytes or poikilocytes were observed in peripheral blood smears of 3 cases, suggesting that the microangiopathy was attributable to hemolysis. Previous reports showed that mutation of COL4A1 accounts for brain small-vessel disease characterized by stroke and eye abnormalities and the most characteristic complications of the present cases were congenital anomaly in the central nervous system, such as porencephaly, schizencephaly, congenital hydrocephalus, cataracts or paraventricular calcification, as reported previously. Hemolytic anemia became less severe within 2 months after birth, and all cases no longer required red cell transfusion after Day 50. COL4A1 encodes subtype 1 of type IV collagen, which is most abundantly expressed in basement membranes, including the vasculature. The COL4A1 gene mutations identified in the cases were all novel missense mutations except one, located in exons 26, 27, 37, 38 and 51. Although the pathophysiological significance of the mutations remains unclear, COL4A1 is the first identified causative gene responsible for congenital hemolytic anemia without intrinsic defects of red blood cells, and mutation of COL4A1 is the most prevalent cause of neonatal hemolytic anemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Novel Missense Mutation of GATA1 Caused Red Cell Adenosine Deaminase Overproduction Associated with Congenital Hemolytic Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 400-400
Author(s):  
Hiromi Ogura ◽  
Toshiyuki Yamamoto ◽  
Taiju Utsugisawa ◽  
Takako Aoki ◽  
Takuya Iwasaki ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Missense Mutation ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Mean Value ◽  
Blood Type ◽  
Target Cells ◽  
Red Cell ◽  
Aberrant Expression ◽  
Congenital Hemolytic Anemia

Abstract Red cell adenosine deaminase (ADA) overproduction (OMIM 102730) is a rare form of congenital hemolytic anemia. To date, only four independent families have been reported. Recently, we examined the red cell enzyme activities of an 18-year-old male Japanese patient with congenital hemolytic anemia, and diagnosed a new case of ADA overproduction. His ADA activity was measured as 39.7 IU/gHb, representing an over 30-fold elevation of the normal mean value. The patient's mother also showed a high red cell ADA, 7.40 IU/gHb, and the father had normal ADA activity. To elucidate the molecular basis of the elevated ADA activity, we performed a target-captured sequencing focusing on the 67 congenital-anemia related genes. The results showed that there was no structural mutation of ADA gene, and that the proband had a novel missense mutation of GATA1, c.920G>A, p.R307H. The proband was hemizygous, and the mother was heterozygous for the mutation. Subsequently, we examined a previously reported case of ADA overproduction, and identified the identical missense mutation of GATA1. These two cases had clinical similarities, such as low birth weight with hypospadias, splenomegaly, and slightly decreased platelet counts, suggesting that these cases could be categorized as X-linked anemia with or without neutropenia and/or platelet abnormality (XLANP, OMIM#300835). The previous case showed a rare blood type, Lu(a-b-) and a low beta/alpha globin synthetic ratio, whereas the present case depicted abnormal red cell morphology, such as stomatocytosis and target cells. Taken together, the missense mutation of GATA1 might cause the aberrant expression of erythroid-genes, inducing a short life-span of red cells. Disclosures No relevant conflicts of interest to declare.

Flow Cytometric Osmotic Fragility Test; A Sensitive and Quantitative Diagnostic Approach for Hereditary Spherocytosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5151-5151
Author(s):  
Ye Jee Shim ◽  
Dong Il Won ◽  
Joon Ho Moon ◽  
Sang Kyun Sohn ◽  
Eun Sil Park ◽  
...  
Keyword(s):  
Disease Severity ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Osmotic Fragility ◽  
Diagnostic Methods ◽  
Diagnostic Approach ◽  
P Value ◽  
Red Cell ◽  
Healthy Patient ◽  
Flow Cytometric

Abstract Abstract 5151 Background & Aim: Hereditary spherocytosis (HS) is the most common cause of congenital inherited hemolytic anemia. Traditional osmotic fragility test (OF) using a series of hypotonic solutions of NaCl, the most widely used diagnostic approach, has relatively low sensitivity. Further the ‘positive’ result cannot quantify the disease severity. We have performed OF using flow cytometric method (FCM OF) to make a diagnosis of HS in Kyungpook National University Hospital (Daegu, South Korea) from September 2008 until now. In this new test, deionized water (a hemolysis-inducing agent) is spiked to a red cell suspension during acquisition and the count of red cell is measured sequentially in real-time FCM. The healthy/patient ratio of %residual red cell over 3. 0 is considered ‘positive’. The aim of this study is to investigate the usefulness of FCM OF (from September 2008 to July 2012) in comparison with the traditional one (January 2002 to August 2008). Methods: The HS patients' laboratory results were divided into two groups based on the diagnostic methods (FCM OF vs. traditional one). The values were described as ‘mean ± standard deviation’. Statistical analyses were conducted using SPSS ver. 19. 0 (Chicago, IL, USA). The independent T-test was performed to compare inter-group differences for the disease severity variables at the time of test - hemoglobin, hematocrit, reticulocyte, corrected reticulocyte (c-reticulocyte), spherocyte percentage in peripheral blood (PB), and total bilirubin. To determine the factors which influence the healthy/patient ratio of %residual red cell, Pearson or Spearman correlation were performed according to the aforementioned severity variables in the subjects who underwent FCM OF. Absolute values of rho > 0. 3 and p < 0. 05 were considered statistically significant. Results: Nineteen HS patients (male: female = 8: 11) underwent a total of 23 times of OF (FCM OF: traditional one = 11: 12). Their mean age at the time of diagnosis was 8. 8 years (range, 0–72). The hemoglobin and hematocrit were higher in FCM OF group than in traditional one (both, p = 0. 038). The mean value of severity variables and respective p-value are summarized in table 1. And sixteen subjects (male: female = 8: 8) underwent a total of 19 times of FCM OF (positive: negative = 11: 8). A negative correlation was observed between the healthy/patient ratio of %residual red cell and hemoglobin (p = 0. 039). We also observed a positive correlation between The healthy/patient ratio of %residual red cell and the reticulocyte/c-reticulocyte/spherocyte percentage in PB (respectively, p = 0. 040, 0. 014, and 0. 018). The rho and p-value are described in table 2. Conclusions: Considering the higher level of hemoglobin and hematocrit at the time of diagnosis in FCM OF group than those in the case of traditional one, we supposed that less severe cases could be diagnosed as HS by using this new test. Furthermore, the value of healthy/patient ratio of %residual red cell correlated with the severity of the disease. Thus FCM OF could be an useful first line screening test for HS due to its sensitivity and quantitative advantage. Disclosures: No relevant conflicts of interest to declare.

scholarly journals COMPARISON BETWEEN FLOW CYTOMETERIC METHOD AND OTHER METHODS FORRETICULOCYTE COUNT IN DIAGNOSIS OF PHENYLHYDRAZINE-INDUCED HEMOLYTIC ANEMIA

2021 ◽  
Vol 9 (02) ◽  
pp. 938-952
Author(s):  
Rania Mohammed Baker ◽  
◽  
Fatma Abdel-Monem Gad ◽  
Khalid Mostafa Fararh ◽  
◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Blood Cells ◽  
Reticulocyte Count ◽  
Osmotic Fragility ◽  
Flow Cytometer ◽  
Control Group ◽  
Albino Rats ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
Manual Methods

Reticulocyte count is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, the reticulocyte counting is a challenge for clinical laboratoriesmainly for the ordinary ones, which still use the manual method.This study was designed to evaluate the performance of flow cytometer for reticulocytes counting comparing to traditional and optimized manual methods which helpful in diagnosis of phenylhydazine-induced anemia.For that 45 male white Albino rats were divided into 5 groups, control group,phenylhydrazine group (PHZ) which injected by phz(20 mg/kg b.w, I/P),quercetin+phz group (quercetin, 50 mg/kgb.w per os), silymarin+phz group (silymarin, 100 mg/kgb.w per os) and quercetin group. Whole blood samples of these groups were collected at day 3, 5 and 10 after 1st injection of phz which used for reticulocyte counts by flow cytometeric method and other manual methods in addition to measurement ofCBC and osmotic fragility. Analysis of the results showed that phenylhydrazine injection induced hemolytic anemia with significant reticulocytosis and using of flow cytometer in reticulocyte count more precise, easy and fast than traditional and optimized manual methods. Furthermore, degree of hemolysis was significantly increases in phz group comparing to other groups. Therefore, we concluded that flow cytometric method for reticulocyte counts was simple, fast and highly reliable comparable to traditional and optimized manual methods. Also optimized manual showed that more perfect than traditional manual method and nearly to accuracy of flow cytometeric method.

HbS-Oman Heterozygote: A New Dominant Sickle Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4375-4382 ◽  
Author(s):  
Ronald L. Nagel ◽  
Shahina Daar ◽  
Jose R. Romero ◽  
Sandra M. Suzuka ◽  
David Gravell ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Clinical Picture ◽  
Clinical Syndrome ◽  
Moderate Intensity ◽  
Target Cells ◽  
Rbc Membrane ◽  
Gardos Channel ◽  
Abnormal Hemoglobin ◽  
Hb S ◽  
Clear Increase

Abstract Hemoglobin (Hb) S-Oman has two mutations in the β-chains. In addition to the classic βS mutation (β6 Glu → Val), it contains a second mutation in the same chain (β121 Glu → Lys) identical to that of HbOARAB. We have studied a pedigree of heterozygous carriers of HbS-Oman that segregates into two types of patients: those expressing about 20% HbS-Oman and concomitant −/ thalassemia and those with about 14% of HbS-Oman and concomitant −/− thalassemia. The higher expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in Oman. In addition, the higher expressors exhibit a unique form of irreversibly sickled cell reminiscent of a “yarn and knitting needle” shape, in addition to folded and target cells. The CSAT of S-Oman is identical to that of S-Antilles, another supersickling hemoglobin, whose carriers express the abnormal hemoglobin at 40% to 50%, with a very similar clinical picture to HbS-Oman. Because the level of expression is so different and the clinical picture so similar, and based on the hemolysates CSAT’s, we conclude that HbS-Oman produces pathology beyond its sickling tendencies. A clue for this additional pathogenesis is found in the fact that homozygous HbOARAB, which has the same second substitution as S-Oman, has a moderately severe hemolytic anemia; when HbOARAB is combined with HbS, it makes the phenotype of this double heterozygote as severe as SS. Properties of HbS-Oman red blood cells (RBCs) include reticulocytes that are much denser than normal (similar to those of SC and CC disease), a decrease in the Km for Ca2+ needed to activate the Gardos’ channel (making this transporter more sensitive to Ca2+), increased association of HbS-Oman with the RBC membrane, the presence of dense cells by isopycnic gradient, the presence of folded cells, and abundant nidus of polymerization under the membrane. Other properties include a clear increase in volume and N-ethylmaleimide–stimulated K:Cl cotransport in RBCs expressing more than 20% HbS-Oman. We conclude that the pathology of heterozygous S-Oman is the product of the sickling properties of the β6 Val mutation which are enhanced by the second mutation at β121. In addition, the syndrome is further enhanced by a hemolytic anemia induced by the mutation at β121. We speculate that this pathology results from the abnormal association of the highly positively charged HbS-Oman (3 charges different from normal hemoglobin) with the RBC membrane.

Phenotypic Evaluation of a Family Cohort with Hemolytic Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3214-3214
Author(s):  
Brett L. Houston ◽  
Donald S. Houston ◽  
Sara J. Israels ◽  
Gail Coghlan ◽  
Bernie N. Chodirker ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Serum Ferritin ◽  
Autosomal Dominant ◽  
Family Study ◽  
Reticulocyte Count ◽  
Family Members ◽  
Osmotic Fragility ◽  
Glycolytic Enzymes ◽  
Target Cells ◽  
The Mean

Abstract Abstract 3214 Background: We have identified a kindred in Manitoba and Saskatchewan, Canada, affected by non-immune hemolytic anemia. Red cell morphology, an elevated MCHC and decreased osmotic fragility are consistent with hereditary xerocytosis, a rare hemolytic disorder, for which the causative genetic mutation is unknown. Objectives: To describe the clinical phenotype and inheritance of an uncharacterized chronic hemolytic disorder in a large kindred. Methods: With assistance from each consenting family member, a pedigree was constructed. A focused history was taken and the presence of splenomegaly was assessed by physical examination. Laboratory analysis included a CBC, reticulocyte count, osmotic fragility and peripheral blood film. Biochemical measurements of LDH, ALT, bilirubin, ferritin, haptoglobin, plasma hemoglobin and methemoglobin were performed. Glycolytic enzymes were evaluated in a subset of patients to rule out other rare causes of hemolysis. Results: The family pedigree captured the genetic relations of 342 individuals spanning 5 generations. Consent to participate in the detailed family study was obtained from 137 family members. The average age of the study population was 29 years (range 8 months to 76 years). Laboratory specimens were collected from 26 unrelated spouses and 111 related individuals. Males represented 48% of the studied population. The distribution of reticulocyte counts was distinctly bimodal with no overlap between the two populations, allowing classification of individuals as phenotypically affected or non-affected. The mean percent reticulocyte count of non-affected subjects (related family members and unrelated spouses) was 1.1% (± 0.4, range 0.5–2.3%). Affected subjects had a mean percent reticulocyte count of 9.7% (± 2.6, range 5.3–14.6%). Using this classification, the hemolytic process segregated in an autosomal dominant fashion with complete penetrance. A history of anemia (46 vs. 8%), jaundice (45 vs. 4%), red or brown urine (45 vs. 1%), and either gallstones or cholecystectomy (41 vs. 4%) was more prevalent in affected than unaffected individuals. Episodes of anemia tended to be associated with illness or stress. There was no association between the hemolytic phenotype and neuromuscular, cardiovascular, pulmonary, renal, hepatic, or endocrine disorders. Despite a mean percent reticulocyte count of 9.7% in affected individuals, the mean hemoglobin concentration was not statistically different between affected and unaffected individuals (13.5 ± 1.2 g/dL vs. 13.8 ± 1.4 g/dL, p=0.26). The MCV (96.7 ± 5.5 fL vs. 87.3 ± 5.2 fL, p<0.01) and MCHC (36.6 ± 0.6 g/dL vs. 33.8 ± 0.9 g/dL, p<0.01) were significantly elevated among affected individuals. Morphologically, target cells and stomatocytes were increased among affected individuals. Affected individuals had significantly elevated indirect bilirubin and decreased haptoglobin compared to unaffected or unrelated individuals. Serum ferritin was elevated in all age tertiles in affected individuals compared to non-affected or unrelated individuals, and 7/29 affected individuals had a serum ferritin >900 μg/L. Osmotic fragility performed on 4 affected individuals was decreased. Glycolytic enzymes, screens for unstable hemoglobins and hemoglobinopathies were normal in those tested. Conclusions: In this family study, elevated percent reticulocyte counts were used to characterize the presence of a well compensated, autosomal dominant hemolytic process associated with an elevated MCHC and decreased osmotic fragility. Clinically this condition is associated with gallstones and progressive iron loading. Features are consistent with hereditary xerocytosis. Molecular analysis is currently underway to locate the causative gene and identify the underlying mutation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Red cell calcium leak in congenital hemolytic anemia with extreme microcytosis

Blood ◽  
1976 ◽  
Vol 47 (2) ◽  
pp. 197-210 ◽  
Author(s):  
JS Wiley ◽  
FM Gill
Keyword(s):  
Hemolytic Anemia ◽  
Cell Morphology ◽  
Osmotic Fragility ◽  
Red Cells ◽  
Red Cell ◽  
Fresh Blood ◽  
Ouabain Binding ◽  
Red Cell Membrane ◽  
Membrane Area ◽  
Congenital Hemolytic Anemia

Abstract A child with congenital hemolytic anemia, extreme microcytosis and bizarre red cell morphology has been studied. Splenectomy at the age of 21 mo greatly improved the hemolytic anemia, although red cell morphology was unchanged. Aniso- and poikilocytosis were marked on a stained smear, and there were many small hyperchromatic cells of irregular shape. The MCV of 25 cu mu was very low and the MCHC was normal. Osmotic fragility of fresh blood was increased, and postsplenectomy blood showed a fraction of extremely fragile cells. Concentration and fluxes of Na+ and K+ were normal, except K+ efflux, which was stimulated by external Ca2+. Inward Ca2+ movement into the patient's red cells was elevated three- to fourfold above red cells of the same mean age. Red cell Ca2+ concentration was raised 2.5 times normal and most of the Ca2+ was localized in the stroma. Red cell lipid, sialic acid, and ouabain-binding sites, all per milliliter of cells, were increased by 16%-23%, and, since these substances estimate the amount of membrane, it was likely that Ca2+ content per unit of membrane area was at least twice normal. Deformability of the cells, as judged by their filterability was markedly impaired. It was concluded that the red cell membrane was defective, and an increased membrane Ca2+ content was associated with reduced deformability, hemolysis, and distorted red cell morphology in this syndrome.

Dehydration response of sickle cells to sickling-induced Ca++ permeabilization

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2578-2585 ◽  
Author(s):  
Virgilio L. Lew ◽  
Zipora Etzion ◽  
Robert M. Bookchin
Keyword(s):  
Sickle Cell ◽  
Fragility Curves ◽  
Initial Density ◽  
Osmotic Fragility ◽  
Sickle Cells ◽  
Rbc Membrane ◽  
Flow Cytometric ◽  
Gardos Channel ◽  
Cell Dehydration

Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability (“Psickle”) to (at least) Na+, K+, Ca2+, and Mg2+. Resulting changes in [Ca2+] and [H+] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca2+-sensitive K+ (“Gardos”) channel (KCa) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of Psickle expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca2+ pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca2+)–induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their Psickle values. Psickle-mediated dehydration was inhibited by clotrimazole, verifying the role of KCa, and also by elevated [Ca2+]o, above 2 mM. Very high Psickle values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of Psickle documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in Psickle-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.

scholarly journals Use of Next Generation Sequencing Panel for Routine Diagnosis of Hereditary Hemolytic Anemias

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2325-2325 ◽  
Author(s):  
Archana M Agarwal ◽  
Jay L Patel ◽  
Adam Clayton ◽  
Noel Scott Reading
Keyword(s):  
Genetic Counseling ◽  
Next Generation Sequencing ◽  
Hemolytic Anemia ◽  
Erythroid Cell ◽  
Red Cell ◽  
Next Generation ◽  
Novel Mutations ◽  
Ugt1a1 Gene ◽  
Rbc Membrane ◽  
Generation Sequencing

Abstract Hereditary hemolytic anemia (HHA) are a heterogeneous group of disorders due to germline mutations of the red cell cytoskeleton (e.g. hereditary spherocytosis (HS) and hereditary elliptocytosis/pyropoikilocytosis (HE/HPP)) or enzyme deficiencies (e.g. glucose 6 phosphate dehydrogenase deficiency (G6PD) and pyruvate kinase deficiency (PKD). Routine morphological and biochemical analysis may be inconclusive in neonates due to the physiological nature of erythroid cell maturation and can also be misleading in transfusion-dependent patients. Additionally, there has been increasing awareness of inherited red cell membrane disorders that are not easily identified by routine laboratory approaches. For example, clinically insignificant defects of RBC membrane genes (e.g. alpha LELY and alpha LEPRA in SPTA1), which can be present in the parents without significant hemolysis, may result in compound heterozygosity in the offspring, causing severe morbidity or even mortality due to significant hemolysis. Awareness of these low expression alleles is important for genetic counseling purposes. Molecular studies, although becoming more mainstream, have not been used extensively to diagnose these disorders. This is most likely due to the complex genetic nature of these disorders (e.g. large genes with multiple exons involved, and multi-gene disorders (i.e. hyperbilirubinemia due to HS as well as involvement of genes involved in bilirubin metabolism). The accessibility of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our current 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 NGS are performed using a Sure Select kit (Agilent). Indexed samples are quantified using qPCR and then pooled prior to sequencing on the Illumina NextSeq or HiSeq instruments. Samples are sequenced using 150 bp paired-end sequencing. This panel includes genes responsible for RBC membrane defects, enzyme deficiencies, as well as bilirubin uridine diphosphate glucuronosyltransferase (UGT1A) genes that have a distinct role in hyperbilirubinemia. We now report the first 268 patients evaluated using our NGS panel between 2015-2018. These patients were evaluated using an Institutional Review Board Protocol (IRB - 00077285). The age of the patients ranged from newborn to 68 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified according to the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic and likely pathogenic variants in 64/268 (24%) patients that were clearly responsible for the disease phenotype (e.g. moderate to severe hemolytic anemia). Approximately half of them were novel mutations. Moreover, 29/268 (11%) of patients were homozygous for a promoter polymorphism in the UGT1A1 gene A(TA)7TAA (UGT1A1*28), which may lead to reduced expression of the UGT1A1 gene and Gilbert's syndrome. Furthermore, 4/29 UGT1A1 polymorphism cases were associated with pathogenic spectrin mutations, likely increasing the severity of the clinical phenotype in these patients. Overall, the most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits, followed by PKLR and ANK1 (Table 1). Complex interactions between variants in the SPTA1 gene and the common alpha-LELY and alpha-LEPRA alleles were predicted to be associated with HPP and autosomal recessive HS in 12/64 patients. Furthermore, 23/268 (9%) patients had mutations that were predicted to cause moderate to severe anemia if inherited with another mutation, making them important for genetic counseling purposes (data not shown). Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes. Many variants of unknown significance were also identified that could potentially contribute to disease. To conclude, the use of NGS provides a cost-effective and comprehensive method to assist in the diagnosis of hemolytic anemias, especially in instances where complex gene-gene interactions are suspected. Disclosures No relevant conflicts of interest to declare.

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