Study design and population
We conducted a cross-sectional study between December 2016 and March 2019 recruiting patients previously diagnosed with SCD from seven thalassaemia centres in Sri Lanka. The thalassaemia centres were located in the districts of Mahara, Kurunegala, Anuradhapura, Hambantota, Monaragala, Ampara and Batticaloa, All SCD patients were eligible for the study and there were no exclusion criteria. All patients were examined by the study physician and clinical details were obtained using a pre-tested interviewer-administered questionnaire.
Haematological and haemoglobin analyses
A Five ml venous blood sample was collected into EDTA (Ethylenediaminetetraacetic acid) from each participant. Routine haematological measurements were conducted using a Coulter counter Ac•T 5diff OV (Beckman Coulter, Inc., Brea, California, United States). Haemoglobin phenotype was determined by capillary electrophoresis (CE) using Capillarys 2 flex piercing analyzer (Sebia, France). DNA for genetic analyses was extracted by QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and stored at -20 0C until further use.
Basic Genetic analyses
Classical β- globin haplotyping was performed. Six regions around and within the β globin gene cluster were amplified by the polymerase chain reaction (PCR), using primers from Integrated DNA Technologies, Inc., Iowa, United States. Primer sequences were those referenced by [13]. PCR products of each patient were treated with appropriate restriction enzymes (from Thermofisher scientific) according to manufactures instructions and the resulting fragments were separated on 2% agarose gel. Bands were visualized and photographed by UVP BioDoc-It® Imaging System. Six polymorphic restriction sites were studied; 5’ to ε gene by Hind II, 5, to Gγ gene by Xmn I, within IVS 2 of the Gγ and Aγ genes by Hind III, 3’ to ψβ by Hind II, and IVS 2 of the β gene by Ava II. When the Restriction Fragment Length Polymorphism (RFLP) pattern was heterozygous, the sickle haplotype was determined based on the assumption that common sickle haplotypes were present [14]. Common α + globin gene deletions (3.7 and 4.2 kb) were studied by multiplex GAP polymerase chain reaction [15]. Beta- thalassaemia mutations of the SBT patients were determined by Amplification Refractory Mutation System (ARMS) [16].
Sequencing analyses of Hb SS patients
New generation sequencing (NGS) was done using a customized panel which sequenced 5 regions of the genome of all the Hb SS patients reported in study including; Chromosome 2 (hg 19 Grch build 37) - chr2:60,575,685 − 60,753,050, Chromosome 6 (hg 19 Grch build 37) - chr6:135,281,347 − 135,540,835, Chromosome 11 (hg 19 Grch build 37) - chr11:3,779,641–7,224,114, Chromosome 16 (hg 19 Grch build 37) - chr16: 575,307- 2,619,179 and Chromosome X (hg 19 GrCh build 37) - chrX:11,253,922 − 11,377,717 using Illumina platform (Illumina Miseq). Variations found were annotated with Integrative Genomic Viewer version 2.6 (Broad Institute) using GRCh37 - hg19 - Genome – Assembly by NCBI (National Centre for bio-informatics) as the reference sequence.
Genotyping of Foetal Haemoglobin (Hb F) modifiers among SCD patients
Four known Hb F modifiers (rs1427407 and rs6545816 in BCL11A, rs66650371 in HMIP-2A and rs9402686 in HMIP-2B) were genotyped by Taqman assay real time PCR using Viia 7 Applied Biosystems. One Hb F modifier (rs7482144 in Xmn1-HBG2) was genotyped by RFLP.