When do you have to perform the molecular biology in the hemoglobinopathies diagnosis

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1 When do you have to perform the molecular biology in the hemoglobinopathies diagnosis Maria Domenica Cappellini MD, FRCP;FACP Fondazione Ca Granda Policlinico IRCCS University of Milan

2 Disclosure Member of Advisory Board: - Novartis - Genzyme/Sanofi - Celgene - Shire - Merganser Biotech - Isis

3 Agenda Definition and patophysiology of Thalassemias Classification Hematological Diagnosis Molecular diagnosis

4 Classification of the thalassaemias Group of inherited haemoglobin disorders Absence or reduced synthesis of α chains of Hb α-thalassaemias Absence or reduced synthesis of β chains of Hb β-thalassaemias Muncie HL, Campbell JS. Am Fam Physician. 2009;80:

5 Globin genes cluster Chromosome 16 ζ α 2 α 1 Chromosome 11 ε Gγ Aγ δ β Embryonics: ζ 2 ε 2 Gower 1 ζ 2 γ 2 Portland α 2 ε 2 Gower 2 Fetals: α G o A 2 γ 2 HbF Adults α 2 β 2 HbA α 2 δ 2 HbA2

6 Globin synthesis at various stages of embryonic and fetal development Megaloblast Macrocyte Normocyte S ite of cell erythro-type poies is Liver Bone Marrow 50 Yolk sac α Spleen α 40 γ β ζ ε β δ γ B irth Post-conceptual age (weeks) Postnatal age (weeks)

7 Thalassemias are a group of inherited hemoglobinopathies Absence or reduced synthesis of α chains of Hb Absence or reduced synthesis of β chains of Hb α thalassemias 1 α thalassemia silent carrier (single α gene deletion) α thalassemia trait minor (double α gene deletion) Hb constant spring (reduced output of α globin) Hb H disease (triple α gene deletion) Hb Barts Hydrops (absence of α genes) 1. Muncie HL and Campbell JS. Am Fam Physician 2009;80: ; 2. Galanello R and Origa R. Orphanet J Rare Dis 2010;5:11. β thalassemias 2 β thalassemia minor (silent or carrier ) β thalassemia intermedia β thalassemia minor β thalassemia with Hb anomalies Hb C/β thalassemia Hb E/β thalassemia Hb S/β thalassemia Hereditary Hb F and β thalassemia β-thalassemia associated with Trichothiodystrophy X-linked thrombocytopenia

8 Thalassemia has a broad clinical spectrum, complicating diagnosis and management NTDT patients do not require regular red cell transfusions but may require occasional transfusions for growth failure, pregnancy, infections and other specific situations 1 4 NTDT β thalassemia intermedia Mild/moderate Hb E/β thalassemia Hb H disease (α thalassemia) Hb S β thalassemia Hb C thalassemia Transfusions seldom required Occasional transfusions required (eg surgery, pregnancy, infection) Intermittent transfusions required (eg poor growth and development, specific morbidities) Regular, lifelong transfusions required for survival Transfusions not required α thalassemia trait β thalassemia minor 1.Taher AT et al. Br J Haematol 2011;152: ; 2. Galanello R and Origa R. Orphanet Journal of Rare Diseases 2010;5:11; 3. Vichinsky E. Hematology Am Soc Hematol Educ Program 2007;79 83; 4. Muncie HL and Campbell JS. Am Fam Physician 2009;80: ; 5. Figure adapted from Musallam KM et al. Haematologica 2013;98: Transfusion-dependent thalassemia (TDT) β thalassemia major Severe Hb E/β thalassemia Hb Barts hydrops (α thalassemia major).

9 Agenda Definition and patophysiology of Thalassemias Classification Hematological Diagnosis Molecular analysis

10 Several assessments can assist diagnosis Family history Laboratory exams Diagnosis should incorporate as much information as possible, utilizing family history, and laboratory examinations 1. Taher A et al. Guidelines for the management of NTDT. 2013;TIF Publication No. 19; 2. Weatherall DJ. Blood Rev 2012;26S:S3 S6.

11 Diagnostic work-up of Thalassemias Primary hematology tests Full medical history Family history (screen families in high risk areas) Complete blood cell count with erythrocyte indices (use an automated blood cell counter) Blood smear/bcb staining + Low MCV (<80 fl) ± Low MCH (<27 pg) Microcytosis Hypochromia Target cells ± Inclusion bodies (Hb H) Serum ferritin >12 ng/ml _ Consider other causes of anemia Serum ferritin 12 ng/ml Consider iron deficiency anemia Adequate iron supplement for 3 months Screen for type of thalassemia Hb A 2 4% Hb F 0.1 5% Hb electrophoresis and HPLC Hb A 2 <4% Hb F <1% Hb A 2 4% Hb F >5 50% Hb H <5 25% Hb A 2 <4% ±Hb CS/PS No improvement + Other normal Hb variant Genetic testing β thalassemia trait α thalassemia traits and related disorders β thalassemia intermedia Hb H disease Hb E disorders Hb S disorders Hb C disorders Others DNA analysis for α and β globin mutations BCB, brilliant cresyl blue; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin Viprakasit V et al. Personal communication.

12 Formulas based on parameters from the CBC The most commonly used formulas are the following: Mentzer index (MI) = MCV/RBC Discriminant factor (DF) = MCV x (RDW/Hb 9 x100) Shine and Lal Index (S&L) = MCV x (MCH/100) Srivastava Index (SI) = MCH/RBC; RDW index (RDWI) = (MCV 9 RDW)/RBC Chatterjee T, Chakravarty A, Chakravarty S. Hemoglobin 2015;39:384 8.

13 HPLC and electrophoresis examples Hb electrophoresis of Hb H disease Vip Viprakasit. EHA Educational Book 2013.

14 Agenda Definition and patophysiology of Thalassemias Classification Hematological Diagnosis Molecular diagnosis

15 Diagnosis of NTDT is challenging due to the diversity of genotype interactions and limited understanding of the disease Weaker understanding of milder forms Diagnosis can be more difficult NTDT Excellent understanding of severe forms such as β thalassemia major Diagnosis is clear TDT Weaker Moderate Strong Early identification is key to ensure the most appropriate monitoring and treatment 1. Musallam KM. Haematologica 2013;98:

16 Several laboratory tests are available which together contribute to an NTDT diagnosis Hb levels Blood smear Other RBC findings HPLC electrophoresis DNA analysis (not always available) Diagnosis should incorporate as much information as possible, utilizing patient history, physical and laboratory examinations HPLC, high-performance liquid chromatography; RBC, red blood cells 1. Taher AT et al. Blood Cells Mol Dis 2006;37:12 20; 2. Galanello R and Origa R. Orphanet J Rare Dis 2010;5:11; 3. Harteveld C and Higgs D. Orphanet J Rare Dis 2010;5:13.

17 Laboratory examination: parameters that can help distinguish and diagnose different forms of NTDT β TI Hb E/β thalassemia Hb H Hb levels ~7 10 g/dl 1 Blood smear HPLC electrophoresis DNA analysis Basophilic stippling 6 Nucleated RBC 6 Hb F 10 50% (up to 100%) 5 Hb A 2 >4% 5 Genetic analysis should be performed in event of abnormal hematology findings 2 Mild 2 Moderately Severe 2 Severe g/dl 6 7 g/dl 4 5 g/dl Target cells 7 Red cell hypochromia 7 Microcytes 7 Nucleated RBC 7 hemolysis 4 Hb E and F 6 Hb A 2 9 To distinguish between different Hb E disorders g/dl 3 Microcytosis 8 Hypochromia 8 Target cells 8 Inclusion bodies 8 Irregularly crenated RBC 8 Increased reticulocytes (5 10%) 8 α/β-globin chain synthesis ratio measurement 3 RBC indices 3 Hb A 2 3 Variable Hb H (0.8 40%) and occasional Hb Barts hydrops 3 Gap-PCR developed for seven common α thalassemia 3 For unknown rearrangements, Southern Blotting or MLPA analysis required 3 MLPA, multiplex ligation-dependent probe amplification PCR, polymerase chain reaction 1. Taher AT et al. Blood Cells Mol Dis 2006;37:12 20; 2. Galanello R et al. Orphanet J Rare Dis 2010;5:11; 3. Harteveld C et al. Orphanet J Rare Dis 2010;5:13; 4.Vichinsky E. Hematology Am Soc Hematol Educ Program 2007;79 83; 5. Thalassaemia International Federation. Guidelines for the Clinical Management of thalassaemia, 2nd edition revised 2008; 6. Yaish HM et al Fucharoen S et al. Clin Chem 1998;44:

18 Globin Chain Synthesis Before the DNA era, the globin chain synthesis analysis,introduced more than 30 years ago, was utilized to identify the severity of globin chain Imbalance and consequently to predict the clinical severity. This method has been used in the late 1970s for prenatal diagnosis At present, it remains a sensitive diagnostic tool limited to define some complex or atypical forms of thalassemia.

19 Molecular Analysis The molecular diagnosis was applied in hemoglobinopathies and thalassemias an early stage of DNA era These diseases have been used as a prototype for the development of new techniques for molecular abnormalities. At present, there are many different PCR-based techniques used to diagnose the known globin gene mutations as well as other methods to identify the unknown mutations

20 Molecular Analysis There are more than 300 known b-thalassemia mutations, the majority being single nucleotide substitutions,insertions, or short deletions Fortunately, a limited number of point mutations are prevalent in different ethnic group; therefore, for any given ethnic region, a PCR method designed to detect the common-specific mutation simultaneously is initially used With this approach, more than 80% of cases for most ethnic groups could be identify.

21 Other Molecular Techniques Reverse-dot-blotting technique, in which amplified DNA is hybridized to a panel of mutation-specific probes fixed to a nylon strip The amplification refractory mutation system: It is rapid, cost-effective, and convenient to test multiple mutations simultaneously Denaturing gradient gel electrophoresis is an alternative approach to the above-mentioned methods, and it is convenient in countries where a very large spectrum of b-mutations occur

22 DNA sequencing The improvement and cost reduction of DNA sequencing rendered the direct sequence of b-gene the most widely use in many laboratories allowing the analysis of the exact nucleotide sequence of the gene or the gene area under evaluation The original method described by Sanger et al. in 1977 has been modified and today several type of automatic sequencer is available Direct sequencing allows also the identification of unknown mutation

23 Alpha Genes The amplification of sequences in the a -globin gene cluster is more difficult than that of the b-globin gene cluster due to a considerable sequence homology within the a-globin gene cluster. For the unknown a-deletion, the Southern blotting technique is still used in some laboratories More recently, the multiplex ligation dependent probe amplification have been introduced: sensitive, reproducible, no radioactivity need

24 Next-Generation Sequencing Next-generation sequencing (NGS) represents a new principle of sequencing technology; it increases the sequencing capacity from a few hundred base pairs to several thousands in a single analysis NGS is based on massively parallel sequencing of clonally amplified DNA molecules, coupled with sufficient computational power and appropriate software for efficient data analysis It has brought genetic diagnostics into clinical practice at an affordable price

25 Conclusions DNA analysis for globin gene mutations is not required to diagnose most of the carrier status It is advisable to confirm the diagnosis of TDT, knowing the parents mutations It is advisable in NTDT cases were the coinheritance of different globin gene defects may beresponsible for different phenotypes Some cases are hematologically and clinically difficult to be diagnosed even with the family hematological evaluations

26 J. (10 yrs) and E. (6 yrs) J. Transfusion dependent from the age of 6 yrs E. Occasional transfusions Hb: 8,6 g/dl J. E. Hb: 8.8 g/dl Father Hb: 12,8 g/dl Hb: 10,3 g/dl Mother MCV: 62,6 fl MCH: 20.3 pg MCV: 59,2 fl MCH: 19.3 pg HbA 2 : 4,8% HbA 2 : 5,10% HbF: 14,60% HbF: 13,10 % Anisocitosis ipochromia Anisocitosis anisochromia MCV: 63 fl MCH: 19,9 pg MCV: 72,8 fl MCH: 23,3 pg HbA 2 : n.d. HbA 2 : 3.1% HbF: n.d. HbF: 0,9 % Anisocitosi,s ipochromia Microcitosis and mild ipochromia

27 β Splicing mutation causing a premature stop codon Triplication ααα No functional protein IVSI-I heterozigosity HBB: c.[92+1g>a];[=] Triplication 3.7 ααα anti3.7 /αα IVSI-i heterozigosity HBB: c.[92+1g>a];[=] Triplication 3.7 ααα anti3.7 /αα Trans Allele of α3.7 deletion

28 Hb: 8,6 g/dl J. E. Hb: 8.8 g/dl Madre Hb: 10,3 g/dl Hb: 13,9 g/dl ααα/αα MCV: 62,6 fl MCH: 20.3 pg MCV: 59,2 fl MCH: 19.3 pg HbA 2 : 4,8% HbA 2 : 5,10% HbF: 14,60% HbF: 13,10 % Anisocitosis ipocrhomia Anisocitosis anisochromia MCV: 72,8 fl MCV: 80 MCH: 23,3 pg MCH: 28 HbA 2 : 3.1% HbA 2 : 2,4% HbF: 0,9 % HbF: 0,8% Microcitosis and emild ipochromia n.a. Something not clear with the mum

29 MLPA Multiplex-Ligation Probe-dependent Amplification Identification of chnges in copy number deletion ) (duplication and

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32 MLPA CLUSTER ALFA GLOBINICO Quadruplication of alpha genes αααα/αα α 2 α 2 α 2 α 1 /α 2 α 1 α 2 α 1 2 α 1 2 α 1 /α 2 α 1

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34 Quadruplication alpha αααα/αα αα/αα quadruplication αααα/αα IVSI-I in eterozigosi HBB: c.[92+1g>a];[=] Triplication+ Quadruplication αααα/ααα IVSI-I in eterozigosi HBB: c.[92+1g>a];[=] Triplication+Quadruplication αααα/ααα

35 Principles of prenatal diagnosis: molecular approach Mutated alleles in both parents must be identified before prenatal testing can be performed Prenatal diagnosis for pregnancies at increased risk could be done through: chorionic villi sampling at around 11 weeks' gestation analysis of DNA extracted from fetal cells obtained by amniocentesis, usually performed at approximately weeks' gestation 1. McPhee J. Science Creative Quarterly. 2011;6. Available from: Accessed March

36 Centro Anemie Congenite e Medicina Interna 1A Policlinico di Milano Maria.cappellini@unimi.it