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Prof. Vip Viprakasit, M.D., D.Phil. (Oxon) Department of Pediatrics and Thalassemia Center Faculty of Medicine, Siriraj Hospital Mahidol University, THAILAND
Identification of a Novel Cause of RBC Membranopathy in Thailand Through NGS
The 52th Thailand Society of Hematology Annual Meeting “Hematology 2018: The Nuts and Bolts”
The Centara World, Bangkok, Thailand; 6 March 2018
“Can we find a new disease condition in Thailand or in Thai patients?”
Reticulocyte count*
Decrease
Nutritional deficiency Iron deficiency
Bone marrow diseases Chronic renal failure
Increase
Hemolysis: Acute: AIHA Chronic: Thalassemia RBC membrane defects Enzymopathies Blood loss
Anemic patients
*Normal Reticulocyte count = 2%
Simple Approach to Anemia
Intracorpuscular cause - Hemoglobin disorders - Rbc enzymopathies - RBC membrane disorder
β major
β thal/Hb E Hb H
Approach to Hemolytic anemia:
At birth
3 mths
Approach to Hemolytic anemia:
Intracorpuscular cause - Hemoglobin disorders - Rbc enzymopathies - RBC membrane disorder
HPP
HE
HS
Approach to Hemolytic anemia:
Intracorpuscular cause - Hemoglobin disorders - Rbc enzymopathies - RBC membrane disorder
Before Genomic Era Conventional molecular medicine
Genomic Era Precision medicine
Approach to Hereditary Hemolytic Anemia (HHA)
How RBCs pass through narrow slits of the microcirculation
Structure of RBC membrane: Deformability secret
HE/HO/HPP
HS
Pathophysiology of Hereditary Spherocytosis
Gallagher, P. G. Hematology 2005;2005:13-18
An 8 year old male who was being evaluated for an upper respiratory infection was noted to be mildly anemic on his CBC
An 8 year old male who was being evaluated for an upper respiratory infection was noted to be mildly anemic on his CBC
micro-spherocytes without central pallor
RBC Morphology in Hereditary Sperocytosis
Causes of Spherocytosis
• Hereditary Spherocytosis • Immunohemolytic Anemia
– Isoimmune: ABO incompatibility – Autoimmune warm antibody
» Idiopathic » Secondary to collagen vascular disease, viral illness,
drugs, lymphoma, hypogammaglobulinemia – Autoimmune cold antibody
» Idiopathic » Secondary to lymphoma, Mycoplasma infection
• Membrane Damage » Clostridial toxin » Severe burns » Wilson disease (Cu++)
Pathophysiology in Hereditary Spherocytosis
HS patients
Chronic hemolysis
Anemia Hyperbilirubinemia
↑Intestinal absorption
Blood transfusion
Growth retardation
Iron overload
Jaundice Hepatic Overload
Gall stone
Diagnosis parameters for HS
Bolton-Maggs PH et al Blood 2004
Parameter Feature
Clinical features splenomegaly almost always Laboratory red cell indices
↓Hb, MCV (N – ↓Inc), ↑MCHC, ↑ % hyperdense cells, ↑ retic.
Blood film spherocytes
Direct antiglobulin test negative
Evidence of hemolysis Raised bilirubin; reticulocytosis reduced haptoglobin
Hematological profile of HS Hb Dec (Reflects EVH)
Red Cell Count Dec (Reflects EVH) Hct Dec (Reflects EVH)
WBC N - Inc Plt N - Inc
MCV N - Inc Microspherocytes have normal vol.
MCH N - Inc MCHC Increase
Haemoglobinisation Normochromic Anisocytosis ++ (Polychromatic Macrocytes) Poikilocytosis ++ - +++ (Microspherocytes)
Immature Forms Polychromasia +→+++ (Nucleated RBCs if severe)
EVH = extra vascular hemolysis
Please do not forget that 40% of Thais are thalassemia carriers
Osmotic fragility of red blood cells in HS
(Segel GB, Hirsh MG, & Feig SA. Pediatr Rev, 2002)
http://erasmeinfo.ulb.ac.be
Normal
Increased
Classical Feature of Hereditary Sperocytosis (HS)
EMA-binding technique
– The maleimide moiety binds covalently with Lys-430 in the first extracellular loop of band 3 protein, and eosin lies in a pocket deep within the transmembrane core of band 3
– detect HS patients with a
deficiency of band 3, spectrin and protein 4.2, but not ankyrin
– Can use for HS screening test
Zhang, D. et al. Blood 2000;96:2925-2933
Southeast Asian J Trop Med & Pub Health 2009
EMA-binding technique for HS & SAO
This method’s advantages: 1. Highly specific for membrane abnormalities 2. Results produced quickly (within 2h of receiving samples) 3. Only 5 ml of packed red cells required (suitable for neonatal and pediatric cases) 4. Availability of a flow cytometer in most haematology laboratories
EMA-binding technique for HS & SAO
Cutoff at 91.5 by ROC analysis with AUC =0.99
HS SAO
Southeast Asian J Trop Med & Pub Health 2009
EMA binding assay in Thai patients with different hemolytic anemia
Problems: several cases HS had normal EMA other forms of RBC membranopathy had variable results
Classification of spherocytosis and indications for splenectomy HS trait Mild Moderate Moderately Severe **
Hb (g/dl) Normal 11 - 15 8 - 12 6 - 8 < 6
Retic. (%) < 3 3 - 8 > 8 > 10 > 10
Bilirubin (mg/dl) 0 - 1 1 - 2 > 2 2 - 3 > 3
Blood smear Normal Mild spherocytosis spherocytosis Poikilocytosis
OF
Normal N or ↑
↑ ↑
↑ ↑
↑ ↑ Fresh
Incubated Slightly ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ Splenec. none unusual During school age
before puberty
Necessary-delay till 6 yrs of age
Necessary-delay till 6 yrs of age
** By definitions, patients with severe disease have life-threatening anemia and are transfusion dependent.
Modified from Gallagher PG & Forget BG. Williams Hematology 6th edition, 2001
Management of Hereditary Spherocytosis (HS)
Prapa Aroonyadet, Kalaya Tachavanich, Voravarn S. Tanphaichitr, Vip Viprakrasit**
Division of Hematology & Oncology, Department of Pediatrics, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok,
Thailand 1070.
Clinical manifestation and natural history of red cell membrane disorders in Thai pediatric patients
ISH 2008
Background
• RBC membrane disorders includes hereditary spherocytosis (HS), elliptocytosis/ovalocytosis (HE/HO), stomatocytosis (HSt), pyropoikilocytosis (HPp) causing one of the most common inherited chronic hemolytic anemia in Caucasians.
• Their clinical manifestations are heterogeneous, ranging from severe transfusion-dependent anemia to well-compensated chronic hemolysis.
• However, the clinical data and disease progression in non-Caucasian is less well-documented.
• In Thailand, There is no comprehensive study for such disease before.
Objective
• To evaluate clinical manifestation and natural history of red cell membrane disorders in Thai pediatric patients who have been followed up at Siriraj hospital, Mahidol University, Bangkok, Thailand.
Materials & Methods
• A retrospective study was conducted in 67 patients who have been diagnosed with red cell membrane disorders from 1974 to 2007. (now = 115 cases in 2018)
• Previously, definitive diagnosis was made by simple blood smear and/or osmotic fragility.
• Since 2004 we performed eosine-maliamide binding assay (EMA) using flow cytometry to document RBC defects.
• Clinical & demographic data including their hematology and growth development were collected and analyzed.
Result-1 • There were 55 HS (82.1%, M:F 24:31), 2 HO (3%, M:F
1:1), 4 SAO (6%, M:F 2:2), 1 HPp (1.5%, M:F 1:0), 1 dehydrated xerocytosis and 4 unspecified phenotype.
• Mean age onset of anemia was 1.6 yrs (0-13 yrs) with • Mean age at diagnosis was 3.9 yrs (0-14.9 yrs). • Hemoglobin (Hb) and hematocrit (Hct) at diagnosis was 8.9 g/dl (3-13) and 26.2% (10.1-39.2). • 51/67 patients (76.1%) had neonatal jaundice in whom 36
(55.4%) and 17 (25.8%) required phototherapy and exchange transfusion respectively.
• Using family study in 97 parents, 61.2% of patients had a dominant mode of inheritance.
• In HS patients, they had 14 hemolytic crisis events per total 942 follow up years (0.03/yr).
• 27/67 patients (40.3%) underwent splenectomy (26 of 27 were HS) with average age of splenectomy was 6.9 yrs.(2.5-14.2).
• In splenectomized cases, all improves after the operation; their hematocrit levels increased significantly and all became independent from blood transfusion with normal growth afterward.
• None had overwhelming infection, thrombosis complication, nor pulmonary hypertension during our follow-up period.
Result-2
A longitudinal weight Z-score after splenectomy in Thai patients with RBC membranopathy (n=27, mainly HS)
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Years after splenectomy (1974-2007)
Sustainable response to splenectomy in Thai patients with RBC membranopathy (n=27, mainly HS)
Mean packed cell volume (PCV or Hct; %)
Summary of RBC membranopathy study in Thailand
1. Hereditary Spherocytosis (HS) is the most common and clinically significant red cell membrane disorders in Thailand.
2. As several affected individuals have mild to moderate hemolysis with history of neonatal jaundice, it is expected that many more cases might have been under-diagnosed due to non-availability of laboratory tests.
3. The presence of spherocytic cell is non-specific and this could be found in other causes of hemolytic anemia.
4. Autosomal dominant is the major mode of inheritance in RBC membranopathy in Thailand
How about other forms of RBC membranopathy beside HS ?
At a higher magnification, the predominance of the elliptical shaped RBCs is even more apparent
An 8 year old male who was being evaluated for an upper respiratory infection was noted to be mildly anemic on his CBC
The typical morphology of RBCs seen in hereditary elliptocytosis are shown in this view Some of these cells exhibit a classical elongated cigar shape
An 8 year old male who was being evaluated for an upper respiratory infection was noted to be mildly anemic on his CBC
Hereditary pyropoikilocytosis is usually double heterozygosity of genes for hereditary elliptocytosis. RBCs are tiny fragments with low MCV (50 fL) and severe hemolytic anemia.
Siblings: Hereditary Pyropoikilocytosis (HPP)
What is the molecular basis of RBC membranopathy in Thailand ?
Analysis of erythrocyte membrane proteins using Coomassie blue-stained SDS-PAGE profile of erythrocyte ghosts
Marc Foretz et al. FASEB J 2011;25:337-347 Copyright Federation of American Societies for Experimental Biology (FASEB). Please see the journal's copyright policies at www.fasebj.org to obtain permission to use this image.
Abnormal protein profiles: - decreased intensity - aberrant banding
Further protein evaluation - Mass spect & aa seq. - Western Blot - Immuno assay/Flow
Normal ratio (membrane study)
(Reinhart WH et al 1994)
Subjects Ratio
Controls (n=8)
Alpha spectrin/band 3 0.61 ± 0.10
Beta spectrin/band 3
0.80 ± 0.11
Band 3/band 3 1
Band 4.1/band 3 0.19 ± 0.03
Band 4.2/band 3
0.38 ± 0.24
Table : Membrane skeleton proteins normalized for the transmembranous membrane protein band 3
Figure : Schematic representation of band 3, ankyrin, β-spectrin, and α-spectrin peptides with positions of known mutations causing red blood cell membrane disorders indicated beneath each protein (From Tse WT & Lux SE. Br J Haematol, 1999)
Molecular study of HS
Hereditary Stomatocytosis Syndromes
Stomatocytes
Antenatal care was uneventful Negative for all TORCH infection Screening for thalassaemia was negative The fetus had a sign of severe anemia, tachypnea CBC: Hb 9.5 g/dL, Hct 30%, MCV 92 fL, MCH 28 pg, MCHC 30 pg/dL
A 38 week GA newborn baby presented with anemia at birth
Index Patient with SAO Morphology
Patient’s Father with SAO Morphology
EMA Binding Assay in Patients with SAO
Patient
Father
Mother
β Thal/Hb E
Vip Viprakasit, unpublished data
MFI 84.38 %
MFI 66.64 %
MFI 100.39%
MFI 98.55 %
Laosombat V et al, Int J Hematol 2005
Neonatal anemia associated with Southeast Asian Ovalocytosis
SLC4A1 or AE1 gene
Molecular basis of AE-1 or Band 3 mutation causing SAO
Acid Secretion by Alpha-intercalated Cells at Distal Tubules of Nephron
Novel AE1 mutations in recessive distal renal tubular acidosis: Loss-of-function is rescued by glycophorin A
Tanphaichitr et al. J Clin Invest 1998; 102: 2173-9.
Homozygous G701D (Band 3 Bangkok I) mutation with HbE/E mutation
Mother: HbE/E Heterozygous G701D
Patients: HbE/E and G701D/G701D
Xerocytic-like dumbbell and cells with transverse ridges
Autosomal Recessive dRTA caused by G701D of AE1 gene
Healthy individual
Homozygous AE1 G701D mutation
Yenchitsomasus P. et al. AJKD, 2002; 40: 21-29
Band 3 mutations; RTA and SAO in Malaysia and Papua New Guinea
SAO/N A858D/SAO A858D/∆V850 Bruce JL. et al. Biochem J (2000) 350, 41-51
Case 1: 3 yrs old boy
Chronic hemolytic anemia due to AE-1 mutations (AE-1 disease: SAO/G701D) +thalassemia
• Hb 8-9.3 g/dL Hct 27-28.6% • MCV 72.2 fL • RDW 20.6% SF 98 ng/mL • Hb typing: AA2 (A2=4%) HbF 0.3% • DNA = β41/42/β + αα/αα
Case 2: 9 yrs old boy
• Hb 9.0 g/dL Hct 29-30 % • MCV 85-89 fL • RDW 17% SF 107 ng/mL • Hb typing: AA2 (A2=3%) • DNA = β/β + αα/-α
The majority of molecular basis underlying RBC membranopathy remains unknown ?
Before Genomic Era Conventional molecular medicine
Genomic Era Precision medicine
Approach to Hereditary Hemolytic Anemia (HHA)
13 yrs old girl from Rangung, Myanmar
G1P1A0 GA 34 weeks: hydropic changes in utero
Case study-6
Before… After…
107,946 births at Siriraj hospital in 12 years (2005-2016)
83 Hydrops fetalis (HF)
82 Non-immune HF
1 Immune HF
41 Unresolved HF
19 Available DNA for study
1500 in pediatric hematology transfusion clinic
300 Known cause
350 Hemolytic anemia
Unknown cause 50 Transfusion dependent
Homozygous SPTB Laos N = 6
107,864 Non-hydrops fetalis
2 CDA type1 2 Fetal anomaly 7 Others
20 Severe thalassemia 4 Twin pregnancy 4 Infection 2 Genetic (DS)
10 pending WES result
N=3
10 Available DNA for study
N=3
Other compound heterozygotes with SPTB Laos, N = 9
N=2
N=7
To be continue… • Spectrin with HE/HO/HPP is the most clinical important cause of
HHA in Thailand • Spectrin α causes mainly mild to moderate anemia and respond well
to splenectomy Common mutations; spectin α-Lely, Leon, Yanggon • Spectrin β causes more severe phenotype; TD to hydrops fetalis Common mutations; spectin β-Laos, Buffalo, Bangkok • A combination of spectrin α and β have been identified and this
genetic interaction is mimic that of globin mutations • Carrier frequency of spectrin mutation range: 0.1-0.5 in Thai
population (n= 4000 in Bangkok and Sriracha)
Summery • Hereditary hemolytic anemia (HHA) can result in severe
intra uterine anemia and fetal hydrops • Several causes of HHA identified in Thailand are due to
genetic mutation of globin, KLF-1, membranopathy and enzymopathy
• Diagnosis of HHA is now based on genome analysis • Some genetic mutations such as alpha thalassemia, KLF1
and Spectrin are common in Thailand • A comprehensive carrier screening can provide a better
prevention of such cases
13 yrs old girl from Rangung, Myanmar She has been diagnosed with β thal intermedia since 3 yrs of age She received infrequent blood transfusion during the first 9 yrs In the last two years she became more anemic and required frequent blood transfusions She has developed all- and autoantibody and the doctors in Myanmar could not find any blood Tx for her She arrived at SiPH with Hb of 4.5 g/dL and dependent on oxygen therapy
Case study-5
13 yrs old girl from Rangung, Myanmar Blood smear revealed mix blood picture Hemoglobin typing: not possible DAT + IAT : Positive 4+ Serology: Negative All laboratory evaluation for SLE, systemic vasculitis: Negative What would you do in this case??
DNA analysis for α and β globin genes; Negative
Family study in both parents: Normal including EMA binding assay and 7 RBC enzyme analyses
c.5572C>G (p.Leu1858Val): SPTA1 rs3737515, SIFT score=0.005,0.009
Dehydrated xerocytosis +/- pseudohyperkalemia/perinatal edema
WES revealed a compound heterozygotes of PIEZ
Hereditary xerocytosis
• The hereditary xerocytosis (HX) syndromes are the most commonly encountered disorders of erythrocyte hydration.
• The primary defect in HX erythrocytes is the passive loss of intracellular K in excess of accumulation of intracellular Na, leading to a gradual decrease in total intracellular cations accompanied by obligate water loss to maintain osmotic balance.
Father Mother
A model of human PIEZO1 and associated mutations in hereditary xerocytosis.
Patrick G. Gallagher Hematology 2015;2015:392-399
©2015 by American Society of Hematology
Peripheral blood smears in altered volume homeostasis
Patrick G. Gallagher Hematology 2015;2015:392-399
©2015 by American Society of Hematology
Hereditary Xerocytosis Hereditary Hydrocytosis
Summary of RBC membranopathy study in Thailand
1. Hereditary Spherocytosis (HS) is the most common and clinically significant red cell membrane disorders in Thailand.
2. As several affected individuals have mild to moderate hemolysis with history of neonatal jaundice, it is expected that many more cases might have been under-diagnosed due to non-availability of laboratory tests.
3. The presence of spherocytic cell is non-specific and this could be found in other causes of hemolytic anemia.
1. Hereditary ovalo-elliptocytosis (HS) is the most common and clinically significant red cell membrane disorders in Thailand.
2. As several affected individuals have more severe hemolysis with history of neonatal hydrops, it is expected that many more cases might have been under-diagnosed due to non-availability of laboratory tests.
3. Diagnosis is difficult to make since most severe patients are transfusion dependent
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