Identification of a Novel Cause of RBC ... - tsh.or.th

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



HPP

HE

HS



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

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

Presenter

Presentation Notes

EVH – Extra Vascular Hemolysis Decreased free plasma haptoglobin ( Hb binding protein )

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


The typical morphology of RBCs seen in hereditary elliptocytosis are shown in this view Some of these cells exhibit a classical elongated cigar shape


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

Presenter

Presentation Notes

Analysis of erythrocyte membrane proteins. A) Coomassie blue-stained SDS-PAGE profile of erythrocyte ghosts prepared from WT (+/+) and Prkag1−/− (−/−) mice. Major membrane skeleton components α- and β-spectrin, ankyrin, band 3, proteins 4.1 and 4.2, and actin appear to be normal in −/− erythrocytes. B) Western blot of erythrocyte ghosts prepared from +/+ and −/− mice, probed with antibodies directed against phospho-adducin (Ser-724). Adjacent lane doublets separated by the line were run simultaneously on the same gel but not in neighboring lanes; they have been juxtaposed for the purposes of this figure. Data are representative of 6 mice/group.

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

Presenter

Presentation Notes

Dr. Tanphaichitr et al. reported 2 siblings with dRTA and hemolytic anemia. The patients carry a homozygous G701D missense mutation of AE1 gene causing recessively transmitted DRTA. Both patients also carry homozygous HbE/E mutation. There blood smear showed xerocytic-like dumbbell shape and cells with transverse ridge other than hypochromic RBC anisopoikilocytosis and sperocytosis in the mother who has homozygous HbE/E with heterozygousG701D mutation. [They also found that all parents and patients had normal erythroid anion transport. But when they did expression study in xenopus oocytes they found that AE1 G701D mutation results in inactive anion transport because of impaired protein trafficking which is rescued by coexpression with glycophorin A.]

Patient’s Father with SAO Morphology

Presenter

Presentation Notes


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

Presenter

Presentation Notes

Primary distal RTA is characterized by impairment of acid secretion by alpha intercalated cells in the distal nephron and collecting tubules. It can cause by abnormalities of several iron channels. The abnormality of Cl/HCO3 exchange at the basolateral membrane which is encoded by anion exchanger 1 or AE1 gene is the most common cause of dRTA in Thailand.

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

Presenter

Presentation Notes


Autosomal Recessive dRTA caused by G701D of AE1 gene

Healthy individual

Homozygous AE1 G701D mutation

Yenchitsomasus P. et al. AJKD, 2002; 40: 21-29

Presenter

Presentation Notes

A year later, Bruce et al. described 9 families of RTA patients from Malaysia and Papua new guinea. They reported 3 mutations of AE 1 causing dRTA and RBC abnormalities. Two of them are novel mutations. They also studied RBC rigidity and transport. The SAO erythrocyte is exceptionally rigid, but the abnormality does not cause hemolytic anemia. The patient who carry A858D/SAO has anemia and elliptocytes. While the pts who have A858D/V850 have anemia and microcytes, poikilocytes and elliptocytes. They also reported RTA patients with G701D/SAO who presented with anemia and splenomegaly along with classic symptoms of RTA.

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

Presenter

Presentation Notes

A year later, Bruce et al. described 9 families of RTA patients from Malaysia and Papua new guinea. They reported 3 mutations of AE 1 causing dRTA and RBC abnormalities. Two of them are novel mutations. They also studied RBC rigidity and transport. The SAO erythrocyte is exceptionally rigid, but the abnormality does not cause hemolytic anemia. The patient who carry A858D/SAO has anemia and elliptocytes. While the pts who have A858D/V850 have anemia and microcytes, poikilocytes and elliptocytes. They also reported RTA patients with G701D/SAO who presented with anemia and splenomegaly along with classic symptoms of RTA.

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

Presenter

Presentation Notes

Since both patients have hemolytic anemia and hepatosplenomegaly which was not previously reported in dRTA patients with SAO. We did Hb typing and , B globin gene analysis. We found that the first patient had 4 bp deletion at condon 41/42 compatible with heterozygous Bo-thalassemia. While the second patient had heterozygous + thalassemia dueto a single globin gene deletion. Thus we conclude that the presence of heterozygous or B thalassemia with compound heterozygous SAO/G701D mutation resulting in chronic hemolytic anemia in dRTA patients.

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

Presenter

Presentation Notes

ส่ง WES 12 cases (รวม เบญจภรณ์ หมื่นผัด no Laos/Buff และ ธีรวัฒน์ รู้หลัก) Homo Laos (HF gr.) ปฏิพล (นนทเวช) ผันผาย, ธีรวัฒน์ รู้หลัก (not confirmed DNA yet), คนไข้หมอเป้ Homo Laos (TD gr.) จิรัชญา สังฆะ (prior Dx siderobl. anemia) วาสนา สีสรรค์ (prior Dx HPP with Hx HF) วิสิทธิ์ ไสวงาม (Hx swelling placenta = HF?) Other combined SPTB mutations Laos/BKK -> HF =1 ธามไทย กันทาวงศ์; TD = 1 ดารินทร์ ภูจริงดาว Laos/Buffalo -> HF =1 ศุภาพิชญ์ ริเริ่ม; TD = 6 จิตบุณย์ สมหวัง (hetero Lely), นรากร บุญปก(หมอเป้), ธัญญาทิพย์ ปุ้งมา(หมอเจน), ภัทรนันท์ อุทุมภา, ชัยชนะ ยาจิตร, ธนิสร แก้วสีนวม

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

Presenter

Presentation Notes

A model of human PIEZO1 and associated mutations in hereditary xerocytosis. The location of PIEZO1 mutations identified in hereditary xerocytosis are shown. Reproduced with permission from Bagriantsev et al.30

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

Presenter

Presentation Notes

Peripheral blood smears in altered volume homeostasis. (Left) Hereditary xerocytosis. (Right) Hereditary hydrocytosis. (Left) A Wright-stained peripheral blood smear from a patient with hereditary xerocytosis due to a PIEZO1 mutation showing rare stomatocytes, occasional dessicytes–dense, abnormal erythrocyte forms where hemoglobin appears puddled at the periphery, and rare target cells. (Right) A Wright-stained peripheral blood smear from a patient with hereditary hydrocytosis is shown. Numerous stomatocytes, erythrocytes with a central mouth-like “stoma” are seen.

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

Identification of a Novel Cause of RBC ... - tsh.or.th

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