Ulutas Med J The Ulutas Medical Journal Ulutas Med J 2149-0430 2149-388X Hayriye Zehra Ulutaş 10.5455/umj.20170608074047 268871 Review Article Addiction Medicine Screening for the Known Αlpha Thalassemia Deletions by Polymerase Chain Reaction https://orcid.org/0000-0002-6802-2666 Bukhari Kiran Tauseef Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 1 https://orcid.org/0000-0002-8818-4208 Noor Nouman Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 2 https://orcid.org/0009-0004-9974-6220 Humayoun Sadaf Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 3 https://orcid.org/0000-0003-2104-3862 Zafar Humaira Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 4 Lakhnana Noor Khan Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 5 https://ror.org/04663jx74 Pathology Consultant Haematologist, Al Nafees Med College, Isra University Islamabad Campus, Pakistan Operative Dentistry, Rawal Dental Collage, Rawal Institue of Health Sciences, Islamabad, Pakistan Senior Registrar, Rawal Dental Collage, Rawal Institute of Health Sciences, Islamabad, Pakistan https://ror.org/04d4bt482 Pathology Consultant Microbiologist, Al Nafees Med College, Isra University, Islamabad Campus, Pakistan https://ror.org/04d4bt482 Pathology Consultant Histopathologist, Al Nafees Med College, Isra University, Islamabad Campus, Pakistan 27 12 2017 2017 3 4 59 64 08 06 2017 02 10 2017 12 11 2017 © 2017 The Author(s) 2017 https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article distributed under the CC BY-NC 4.0 licence.

Thalassemia is the quantitatively inherited disorder of haemoglobin polypeptide chain synthesis. Awareness about the exact frequency of disease in our country, Pakistan, along with the appropriate knowledge of diagnostic and prevention approach will be a step forward to combat thalassemia in Pakistan. The proposed review article will provide updated information regarding the exact frequency and distribution of thalassemia in neonates in Pakistan and Internationally. All this ultimately provide a great help in establishing the policies for thalassemia prevention and control programs in Pakistan. Thalassemia is the quantitatively inherited disorder of hemoglobin polypeptide chain synthesis. Awareness about the exact frequency of disease in our country, Pakistan, along with the appropriate knowledge of diagnostic and prevention approach will be a step forward to combat thalassemia in Pakistan. The proposed review article will provide updated information regarding the exact frequency and distribution of thalassemia in neonates in Pakistan and Internationally. All this ultimately provide a great help in establishing the policies for thalassemia prevention and control programs in Pakistan.

 Alpha Thalassemia Deletions Polymerase chain reaction Diagnosis Prevention No financial support was used for the study. Introduction

The inherited Mendellian blood disorder ‘Thalassemia’ is an autosomal recessive disorder characterized by the abnormalities in alpha (α) or beta (β) globin or the polypeptide chains of Haemoglobin molecule (Hb)( 1 , 2 ).Based upon the underlying pathology and the clinical mani- festations, these can well be classified as alpha or beta thalassemias( 3 ).More than 95% of heterozygous, alpha thalassemia can be manifested as deletion in one or both the alpha globin genes from chromosome 16p13.3( 1 , 4 , 5 ). Thalassemia unlike other qualitative haemoglobino- pathies are considered to be the quantitative polypeptide globin chain abnormalities of hae- moglobin. However, the alpha thalassemias are manifested due to deletion of one or more alpha chain genes, usually located on short arm of chromosome 16. While, the severity of disease is considered to be directly related to the number of genes deleted. The classification of alpha thalassemias into four categories is mentioned below ( 6 ):

i. Hb bar's or the Hydrops fetalis (homozygous state): It is due to the deletion in all four alpha genes. Hence, due to total suppression of all four alpha globin chain synthesis, it is consi- dered as the most severe form ( 3 , 6 ).

ii. HbH disease: It results from the deletion in three alpha chain and thus produces a beta globin chain tetramer (β 4 ) i.e HbH. This form is usually is precipitated inside the sffected red cells as Heinz bodies ( 3 , 6 ).

iii. α-thalassemia trait: This form is the result of two alpha gene deletion.

iv. α-thalassemia trait (carrier): In this type only one alpha gene deletion is seen ( 3 ).

The heterozygous state of alpha-thalassemia-2 is usually not associated with any of the hematological or clinical abnormality. However, in some heterozygous alpha-thalassemia-1 or homozygous alpha-thalassemia-2, the patients might present with the mild form of microcytic and hypochromic RBC morphology. Such pa- tients usually remain clinically silent throughout their life time. The crisis begins only when the interaction with other thalassemia genes or diseases begins, which ultimately alters the disease phenotype and hence become clinically important. Haemoglobin H(HbH) disease is one of the important examples of severe α tha- lassemia. This is usually an outcome of interaction between alpha-thalassemia-1 and alpha-thalassemia-2 ( 7 , 8 ).

Therefore, this disease can well be cured by correctly introducing the defective gene into the haemopoietic compartments or by single stem cell transplants. A study report by Raja etal; in 2012, had shown the unsuccessful results of gene transfer because of limitations in the available gene transfer vectors ( 4 ).

The decreased Hb production will ultimately result in characteristic signs and symptoms of disease. Most commonly and highlighted amongst those all includes, erythroid hyper- plasia, severe form of tissue hypoxia, hemolytic anemia, iron overload, hepatomegaly, etc ( 6 ). The diagnosis of individuals who are at high risk of having off springs with severe forms of thalassemia always remained a key weapon to reduce the miseries of thalassemia patients ( 9 ). Zorai et al; in 2002, concluded from his study that simple blood complete picture along with Hb electrophoresis study provides a valuable information regarding diagnosis of thalassemia ( 10 ). Pornprasert S etal; in 2011, enlightened the necessity of screening all the high risk carrier thalassemic couples ( 11 ). Munkongdee T etal; 2010, Chamras U etal; in 2009, and Prathomtanapong P etal, in 2009, carried out molecular diagnostic studies, to assess the genetic muta- tions in thalassemia, amongst different South Asian regions of the world ( 12 – 14 ). They concluded that, for the said purpose Polymerization chain reaction (PCR) studies will be of great help ( 11 , 15 ).

The prenatal diagnosis, transfusion therapy and allogenic bone marrow transplant (BMT) based upon human leucocyte antigen (HLA) typing, ( 16 ) are considered to be the commonest management options. However, various studies had also highlighted that the success rates for thalassemia free survival can only be improve by establishing the standard protocols for specific localities based upon WHO guide-lines ( 17 – 20 ).

Thalassemia like many regions of the world is also considered to be amongst the serious health issues in Pakistan as weel. A study report by Khateeb B et al; in 2000, concluded that the policies regarding diagnosis of carriers, prenatal diagnosis and genetic counseling are the dire need for thalassemia control and prevention in Pakistan ( 21 ).

 Literature Review

A high prevalence of alpha thalassemia was observed in Sub Saharan regions of Africa, Middle East, Mediterranean Basin, Pakistan, South East Asia and United States (US)( 22 – 24 ). The estimated prevalence of alpha thalassemia in US was 30% ( 22 ). A mild form of microcytic hypochromic anemia is a usually seen as a clinical manifestation in homozygous indivi- duals. While serious conditions i.e HbH and Hb Bart hydrops fetalis syndrome are usually present in Western US ( 25 ). In Hb Bart’s hydrops fetalis syndrome, the individuals have very little or no alpha globin chains. This usually results in severe form of anemia which will ultimately be responsible for high neonatal mortality rates. Gapor SA etal; 2009, carried out a prevalence study in states of Perak and Kelantan, Northeast of Peninsular Malaysia. He found out that Malay population is having a high prevalence (9.25%) of α -thalassemia-2 with 3.7 deletion gene dele- tions, when compared with other ethnic groups. Out of which n=34 (8%) were heterozygous for α- 3.7 deletion, n=1 (0.25%) was heterozygous for α 4.2 deletion, and n=2 (0.5%) were hetero- zygous for SEA type deletion. He also described that α -thalassemia with 3.7 deletions is the commonest type of mutation on chromosome 16, seen around the globe ( 26 , 27 ). Mouélé R etal; in 2000, carried out a study in Brazil and narrated the similar finding of 3.7 deletion, a commonest one in alpha thalassemia ( 28 ).

In two more prevalence studies by Sriroong- rueng W etal; 1997 and Zygulska M etal; 1996, had shown that α thalassemia with 3.7gene deletions were noticed in 16% heterozygous and 1.2% homozygous carriers. The incidence of alpha-thal 2 and alpha-thal 1 traits however were observed to be 12.0% and 4.3%, in the Southern Thailand ( 29 , 30 ).

Khan SA et al; in 2003, carried out a study in Pakistan and described the findings that alpha (4.2) allele was detected in Sindhis. However, alphaalphaalpha (anti3.7) allele were detected in Sindhis Balouchis, and Punjabis ( 31 ). Similar findings were observed by Akbari MT etal; in 2012. He also described that –α3.7, -α4.2, - α20.5 and -MED are the responsible genes ( 32 ). Verma IC et al; in 2007, studied 325 patients of thalassemia intermedia from Pakistan, Iran and India and concluded that the most common mutations are the ‘alpha-thal’ ( 33 ).

Finlayson J et al; in 2012, carried out a study in Australlia and concluded that two frameshift mutations i.e HBA2:c, located on codon 43 and second HBA2:c located on codon 47 are responsible for alpha thalassemia trait ( 34 ). The incidence of thalassemia is more in the developing countries. However, the literature review was found to be very deficient on exact frequency and exact mutation of thalassemia in various regions of the world ( 5 ).

 Conclusion

More than 95% of heterozygous, alpha tha- lassemia can be manifested as deletion in one or both the alpha globin genes from chromo- some 16p13.3.

PCR is considered as a reliable test for accurate detection of thalassemia mutations and hence diagnosis.

Recommendations ; • Premarital and prenatal screening protocols by PCR (polymerization chain reactions) will be helpful for reducing the miseries of thalassemia sufferers.

• Genetic counselling for disease prevention is necessary premaritally

Conventional PCR Procedure

DNA Extraction: µltra Gene TM (Anagen Techno- logies,Inc.,U.S.A) or any other good quality kit. From each patient 2.5 ml of venous blood is drawn in EDTA tube and used for DNA extraction as per protocol in the kit for DNA purification from whole blood. DNA is stored at –70°c till further use.

 PCR

The PCR is run in thermocycler as per HLA programme for 2 hours or so which consisted of following basic steps. Time of different steps may vary.

a. Initial denaturation at 94 0 C for 5 min b. Denaturation at 94 0 C for 30 sec c. Annealing at 62 0 C for 1 min d. Extension at 72 0 C for 1 min e. Repeated step b-d 30 times f. Final extension at 72 0 C for 10 min

 Agarose Gel Preparation

2% Agarose gel is prepared by mixing 2 grams agarose powder Scharlau AG 0032 Agarose High EEO electrophoresis grade (Scharlau Chemie S.A, Barcelona, Spain) with to 10 ml of 10X TBE buffer in 100ml of distilled water. Aga- rose gel is prepared by heating in microwave until it was transparent. This solution was poured on agarose gel casting tray with combs already placed. Combs are removed after 20 minutes when agarose gel had solidified. 1 µl of amplified DNA product is loaded into the agarose gel wells.

 Agarose Gel Electrophoresis

The electrophoresis tank is filled with 10 times diluted 10X TBE buffer connected with power supply and loaded agarose gel is run at 150 V for around 25 minutes.

Staining is done with 0.01% ethidium bromide solution for 15 minutes to visualize absence or presence of amplified PCR products by agarose gel electrophoresis.

 Visualization of Amplified PCR Products on Agarose Gel Electropho. by UV Illuminator

After staining with 0.01% ethidium bromide, gel is visualized for bands on UV Transilluminator and alleles assigned according to the presence of bands of amplified PCR products .

 Preparation of Reagents for Research

• Primer Designing Primer Designing is done by the help of WEBSITE NCVI, TOOL USED is db SNP. After designing primers of your required gene, order is placed to Fermantas company to manufacture and deliver the primers to you.

 Primer Reconstitution

Primer reconstitution is adjusted to 20 pmol, Formula used: 4/OD x 300=X (µl) Where X (µl) was volume of primer to be used for making up to 300µl primer with distil water and OD is the absorbance of primer reconstituted.OD is written on the primer vial.

dNTP Preparation (lypholized form) o 10 µl of each of four dNTPs (Adenine, Guanine, Thymine, Cytosine) o 460 µl distilled water o Mix the two

 Control primers

In each PCR a control primer pair is included that amplifies a known gene not of ur interest or not under study.. These primers amplify known not required allelic sequences and thus functioned as internal positive amplification control.

 TBE Buffer

o Tris 108 g o Boric acid 55 g o Na2EDTA 9.3 g o Distilled water 1 litre TBE Buffer for electropheresis tank (10 times diluted of 10X TBE buffer) o 10X TBE buffer 150 ml o Distilled water 1350 ml Ethidium bromide solution used as stain o Ethidium bromide powder 20 µl o Distil water 200ml Preparation of PCR Mix for Research Purpose To prepare PCR mix (1 test) following reagents is used. Some variations are possible.

a. 10X Taq Buffer 30µl b. MgCl 2 24µl c. 2mMdNTPs 30µl d. Distilled water 200µl e. control primer C 3 35µl f. control primer C 4 35µl g. bromophenol blue dye 2µl To the PCR mix following were added; a. DNA 50µl b. Taq 6µl

PCR Master Mix (Commercially available) PCR Master Mix are also available Commer- cially. Commonly used Master mix is of Solis bioDyne. To it we add Dna of patient previously isolated and Primers of gene want to amplify. Pcr program is run on the Thermal cycler. And gel visualized for gene bands on uv illuminator.

 References Stephen Raja JV, Rachchh MA, Gokani RH. Recent advances in gene therapy for thalassemia. J Pharm Bioallied Sci. 2012; 4(3):194-201. Panyasai S, Sringam P, Fucharoen G, Sanchaisuriya K, Fucharoen S. A simplified screening for α -thalassemia 1 (SEA type) using a combination of a modified osmotic fragility test and a direct PCR on whole blood cell lysates. 2002; 108(2): 74-8. Neishabury M, Poorfathollah AA, Kahrizi l. Alpha thalassemia: deletion analysis in Iran.Archives Iran Med.2001; 4(4):160-4. Fucharoen S, Winichagoon P, Thonglairoam V, Siriboon W, Siritanaratkul N, Kanokpongsakdi S, et al. Prenatal diagnosis of thalassemia and hemoglobinopathies in Thailand: experience from 100 pregnancies. Southeast Asian J Trop Med Public Health. 1991;22:16-29. Pornprasert S, Wiengkum T, Srithep S, Chainoi I, Singboottra P, Wongwiwatthananukit S.Detection of α-thalassemia- 1 Southeast Asian and Thai type deletions and β-thalassemia 3.5-kb deletion by single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting analysis. Korean J Lab Med. 2011 Jul;31(3):138-42. Munkongdee T, Vattanaviboon P, Thummarati P, Sewamart P, Winichagoon P, Fucharoen S, et al. Rapid diagnosis of alpha-thalassemia by melting curve analysis. J Mol Diagn. 2010;12:354-358. Chamras U, Sukunthamala K, Pornprasert S. SYTO9 and SYBR GREEN1 with high resolution melting analysis for molecular confirmatory testing of the common Southeast Asian β -thalassemia mutations.Hemoglobin.2009;33:53-45. Prathomtanapong P, Pornprasert S, Phusua A, Suanta S, Saetung R, Sanguansermsri T. Detection and identification of β-thalassemia 3.5 kb deletion by SYBR Green1 and high resolution melting analysis. Eur J Haematol. 2009;82:159–160. Pornprasert S, Phusua A, Suanta S, Saetung R, Sanguansermsri T. Detection of alpha-thalassemia- 1 Southeast Asian type using real-time gap-PCR with SYBR Green1 and high resolution melting analysis. Eur J Haematol. 2008;80:510-14. Gapor SA, Bakar AMZ, Farouk AFA. Explaining ethnic relations in Malaysia through the “concentric circle model”: case studies of the states of Perak and Kelantan, Malaysia. European Journal of Social Sciences. 2009;12(2):252-258. Sriroongrueng W, Pornpatkul M, Panich V, Fucharoen S. α-thalassemia incidence in southern Thailand by restriction endonuclease analysis of globin DNA from placental blood at Songklanagarind hospital. Southeast Asian Journal of Tropical Medicine and Public Health. 1997;28(supplement 3):93–96 Zygulska LM, Eigel A, Helbig B, Sanguansermsri T, Horst J, Flatz G. Prevalence of α-thalassemias in northern Thailand. Human Genetics. 1996;98(3):345–347. Rosnah B, Rosline H, Ain FA. Detection of Common Deletional Alpha-Thalassemia Spectrum by Molecular Technique in Kelantan, Northeastern Malaysia. ISRN Haematol. 2012;46(2):989. Mohan H. The haematopoietic system. In: Mohan H, Mohan P, Mohan T, editors. The textbook of pathology. 5th ed. New Αlpha Thalassemia Deletions by PCR Bukhari, et al. Delhi: Jaypee Brothers Medical Publishers; 2008. pp. 397 – 402. Mouélé R, Pambou O, Feingold J, Galactéros F. α-thalassemia in Bantu population from Congo-Brazzaville: its interaction with sickle cell anemia. Human Heredity. 2000;50(2):118– 125. Burgess-Beusse B, Farrell C, Gaszner M, Litt M, Mutskov V, Recillas-Targa F, et al. The insulation of genes from external enhancers and silencing chromatin. Proc Natl Acad Sci U S A. 2002;99(Suppl 4):16433–7. Calmels B, Ferguson C, Laukkanen MO, Adler R, Faulhaber M, Kim HJ, et al. Recurrent retroviral vector integration at the Mds1/Evi1 locus in nonhuman primate hematopoietic cells. Blood. 2005;106:2530– 3. Carey BW, Markoulaki S, Hanna J, Saha K, Gao Q, Mitalipova M, et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A. 2009;106:157–62. Chin JY, Kuan JY, Lonkar PS, Krause DS, Seidman MM, Peterson KR, et al. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids. Proc Natl Acad Sci U S A. 2008;105:13514–9. Dave UP, Akagi K, Tripathi R, Cleveland SM, Thompson MA, Yi M, et al. Murine leukemias with retroviral insertions at Lmo2 are predictive of the leukemias induced in SCID-X1 patients following retroviral gene therapy. PLoS Genet. 2009;5:e1000491. Bernini LF. Geographic distribution of alpha thalassemia. Cambridge Univ Press. 2001;878:94. Beutler E, West C. Haemoglobin differences between African-Americans and whites; the role of iron deficiency and alpha thalassemia on haemoglobin levels and mean corpuscular volume. Blood. 2005;106:740-5. Vichinsky EP, Macklin EA, Waye JS. Changes in epidemiology of thalassemia in North America: a new minority disease. Pediatrics. 2005;116:818-25. Steensma DP, gibbons RJ, Higgs DR. Acquired alpha thalassemia in association with myelodysplastic syndrome and other haematological malignancies. Blood. 2005;105(2): 443-52. Harteveld CL, Higgs DR. Alpha thalassemia. Orphanet J Rare Dis. 2010;5:13. Chi DH,Waye JS. Hydrops fetalis caused by alpha thalassemia, an emerging health proble. Blood. 1998; 91: 2213-22. Gibbons RJ, Pellagatti A, Garrick D, Wood WG, Malik N, Ayyub H et al. Identification of acquired somatic mutations in the gene encoding chromatin remodeling factor ATXR in the alpha thalassemia myelodysplasia syndrome (ATMDS). Nat Genet. 2003; 334: 446-9. Khan SN, Hasan F, Sollaino C, Perseu L, Riazuddin S. Molecular characterization of alpha-thalassemia in Pakistan. Hemoglobin. 2003;27(3):161-6. Usman M, Moinuddin M, Ghani R, UsmanS. Screening of Five Common Beta Thalassemia Mutations in the Pakistani Population A basis for prenatal diagnosis. Sultan Qaboos Univ Med J. 2009; 9(3): 305-10. Khateeb B, Moatter T, Shaghil AM, Haroon S, Kakepoto GN.Genetic diversity of beta-thalassemia mutations in Pakistani population. J Pak Med Assoc. 2000;50(9):293-6. Zorai A, Harteveld CL, Bakir A, Van DP, Falfoul A, Delalgi K, et al. Molecular spectrum of alpha thalassemia in Tunisia: epidemiology and detection at birth. Hemoglobin. 2002; 26: 353-62. Verma IC, Kleanthous M, Saxena R, Fucharoen S, Winichagoon P, Raizuddin S et al. Multicenter study of the molecular basis of thalassemia intermedia in different ethnic populations. Hemoglobin. 2007;31(4):439-52. Finalyson J, Ghassemifar R, Holmes P, Grey D, Newbound C, Pell N et al. ???????????? thalassemia trait caused by frameshift mutations in exon 2 of the alpha-2 globin gene: HBA2:c.131 deIT and HBA2:c.143delA. Haemoglobin. 2012;36(5):511-5. Akbari MT, Hamid M. Identification of α globin gene variants: a report from Iran.Arach Iran Med. 2012;15(9):564-7.