data used in this study was retrospectively evaluated including patients who were referred to the genneka clinic ( yayasan genneka , eijkman institute for molecular biology , jakarta , indonesia ) and were diagnosed with -thal based on hematological parameters and molecular diagnosis . clinical features including age of first presentation , clinical course and physical examination were recorded . hematological analysis consisted of complete blood counts ( cbc ) , blood smear and hb analysis . the cbcs were measured using cell dyne 1700 ( abbot diagnostics , abbott park , il , usa ) , erythrocyte morphology was assesed by blood smear examination under light microscope and hb analysis was performed using high performance liquid chromatography ( hplc ) on a variant hemoglobin testing system ( -thalassemia short program ; bio - rad laboratories , hercules , ca , usa ) . genomic dna was isolated from leukocytes using modified gentra puregene blood kit ( qiagen inc . , valencia , ca , usa ) and used as a template for the polymerase chain reaction ( pcr)-based mutation detection . we performed multiplex - pcr ( m - pcr ) using primers as previously described ( 4 ) . each 25 l reaction mixture contained 5 kapa2 g gc buffer ( with mgcl2 ) , 200 m dntps ( invitrogen , life technologies corp . , carlsbad , ca , usa ) , 0.2 m of primers sa3p , xyha and xy1 , 0.1 m of primers 3r1 and 2/3p ( table 1 ) , 4.0% dmso , 0.5 units of kapa2 g robust hotstart dna polymerase ( 5 u/l ) ( kapa biosystems inc . , woburn , ma , usa ) , and 100 ng of genomic dna as template . the pcr was carried out on a veriti 96-well thermal cycler ( applied biosystems , foster city , ca , usa ) using the following conditions : initial denaturation at 95c for 3 min . ; 35 cycles at 95c for 30 seconds , 62c for 45 seconds , 72c for 2 min . ; product sizes of pcr were analyzed by electrophoresis on 1.0% agarose gel ( le agarose , roche applied science , mannheim , germany ) . the gel was photographed using molecular imager gel doc xr system ( bio - rad laboratories ) . product sizes of pcr were 1940 bp for the normal band , 2220 bp for the 3.7 kb mutant band , and 1673 bp for the 4.2 kb mutant band . for nondeletional mutation detection , we first performed dna sequencing and then developed and applied direct mutation detection [ pcr - rflp ( restriction fragment length polymorphism ) ] . list of primers used to detect single -globin gene deletions ( ivy ng , personal communication ) we carried out pcr - rflp to detect the codon 59 mutation as previously described ( 3 ) . we developed a pcr - rflp - based method to detect hb cs , codon 22 and the ivs - ii-142 mutations . for the hb cs mutation detection , 25 l reaction mixture contained 20 mm tris - hcl , 10 mm ( nh4)2so4 , 10 mm kcl , 2 mm mgso4 , 0.1% triton x-100 , ph 8.8 , 200 m dntps , 0.4 m of each primer ( table 2 ) , 0.625 units of taq dna polymerase ( new england biolabs inc , ipswich , ma , usa ) and 100 ng of genomic dna . polymerase chain reactions were carried out on a 9700 thermal cycler ( applied biosystems ) using the following conditions : initial denaturation at 95c for 5 min . ; 35 cycles at 95c for 30 seconds , 68c for 30 seconds , 72c for 1 min . ; then a final extension at 72c for 5 min . list of primers , polymerase chain reaction product sizes , restriction enzymes and restriction fragment length polymorphism product sizes to detect nondeletional -thalassemia mutations hb cs : hb constant spring . d.r . higgs ( mrc molecular haematology unit , weatherall institute of molecular medicine , john radcliffe hospital , headington , oxford , uk . ) for the detection of the codon 22 and ivs - ii-142 mutations , each 25 l reaction contained 5 phusion gc buffer containing 7.5 mm mgcl2 as 1 final concentration , 200 m dntps , 0.4 m of each primer ( table 2 ) , 6.0% dmso , 0.5 units of phusion dna polymerase ( new england biolabs inc . ) and 100 ng of genomic dna . the pcr conditions were as follows : initial denaturation at 95c for 5 min . ; 35 cycles at 95c for 1 min . , 62c for 30 seconds , 72c for 1 min . ; then a final extension at 72c for 5 min . for the detection of the rsnp 149709t < c mutation , each 25 l reaction contained 1 pcr buffer ( qiagen ) , 1 q solution ( qiagen ) , 200 m dntps , 0.2 m of each primer ( table 2 ) , 1 unit of hotstar taq polymerase ( qiagen ) and 100 ng of genomic dna . the pcr conditions were as follows : initial denaturation at 95c for 15 min . ; , 72c for 1 min . ; then a final extension at 72c for 5 min . polymerase chain reactions to detect the nondeletional mutations were carried out on a 9700 thermal cycler ( applied biosystems ) . the pcr product sizes , restriction enzymes and rflp product sizes are given in table 2 . twenty - seven cases were studied , 25 were of indonesian ethnic background and two were chinese . eighteen cases ( 67.0% ) were compound heterozygotes for hb adana and either the  ( 17 cases ) or  ( one case ) mutations ( table 3 ) , and nine cases ( 33.0% ) were compound heterozygotes for hb adana and other nondeletional mutations ( table 4 ) . hematological and clinical data of hb adana patients in combination with one gene deletion at diagnosis n.d . : not defined ; rdw : red cell distribution width ; patients 1 and 4 are transfusion - dependent ; mean and standard deviations were calculated for patients more than 1-year - old . was seen on the hplc system ( variant ; bio - rad laboratories ) ( 10 ) . 
  in transfused patients , hematology analysis was carried out after one ( or several ) blood transfusions had been received ( hb steady state ) except in patient 1 where it was carried out before he had received his first blood transfusion . the hb analyses were not done on the hplc system ( variant ; bio - rad laboratories ) . nb : all patients carry the hb adana mutation in combination with the  kb type deletion except patient 16 who has the  kb type instead . hematological , molecular and clinical data of patients carrying hb adana in combination with nondeletional -thalassemia at diagnosis rdw : red cell distribution width ; n.d . : not defined ; codon 59 : hb adana ; cs : hb constant spring . was seen on the hplc system ( variant ; bio - rad laboratories ) ( 10 ) . on hplc , hb e elutes at a similar time as hb a2 ; the parents do not carry hb e trait , maybe the blood donor carried hb e trait . although the clinical manifestations in patients with hb adana in combination with single gene deletions were mostly mild , the onset varied widely from infant to late adult ( 1 month to 49 years old ) with the steady state hb levels ranging from 6.1 to 11.9 g / dl ( 9.4  1.2 ) ( table 3 ) . only two patients required regular blood transfusions , one of whom started at 3 months of age and one at 3 years old . two patients first presented at quite a late age ( 42 and 49 years ) . several patients had a slight increase in hb f levels ( patients 3 , 4 , 6 and 7 in table 3 ) as previously described ( 1,9 ) . this phenomenon might be related with the increase of -globin gene expression , which should be further elucidated . the nondeletional mutations in trans to hb adana were hb cs ( four cases ) , codon 22 ( three cases ) , rsnp 149709t > c mutation ( one case ) and ivs - ii-142 ( one case ) ( table 4 ) . the first presentation varies from 7 days to 28 years old , with the steady state hb level ranging from 4.9 to 11.1 g / dl . most patients ( five out of nine ) required regular blood transfusions , two patients required irregular blood transfusions , and only one patient did not require any blood transfusions . one patient , a compound heterozygote for hb adana and rsnp 149709t > c required regular blood transfusions since 2 months of age , but did not require them after a splenectomy at age 9 years . two related patients ( case 6 and 7 ) with the same genotype ( hb adana and codon 22 ) showed very different clinical severity as thalassemia major and mild thalassemia intermedia this situation was also found in patients with a compound heterozygosity for hb adana and hb cs ( table 4 ) . although indonesian -thal patients who had hb adana in combination with the 3.7 kb deletion generally have mild - to - moderate anemia , a few of them could manifest as severe anemia . patients who had hb adana in combination with nondeletional mutations mostly showed more severe symptoms than those who had it with single gene deletion as we can see from their blood transfusion status , and who presented at an earlier age . the clinical severities varied widely between patients , even for those with the same genotype . the diversity of the hematology profiles and clinical manifestations might be due to genetic or non genetic factors , further research is required to explain this phenotype variation . moreover , traits of the hb adana ( 3 ) and the other nondeletional -thal mutations in this study exhibit a normal range of hb level with mild reduction of red cell indices , such as codon 22 ( mcv > 70.0 fl , mch > 25.0 pg ) , rsnp 149709t > c ( mcv > 70.0 fl , mch > 22.0 pg ) and ivs - ii-142 ( mcv > 79.0 fl , mch > 26.0 pg ) . therefore , routine dna analysis , regardless of the hematology feature , is strongly suggested for diagnosis of -thal in indonesia , especially during antenatal screening .
we describe 27 cases of mild - to - severe -thalassemia ( -thal ) syndrome caused by interaction of hb adana [ 59(e8)glyasp , ggc > gac ( 2 ) ] with deletional and nondeletional +-thal mutations in indonesian patients . 
 hematological profiles and clinical manifestations of all patients were assessed by routine procedures . 
 the genotypes were generated by a multiplex - polymerase chain reaction ( m - pcr ) , pcr - rflp ( restriction fragment length polymorphism)-based method , and dna sequencing . 
 the -thal patients who had hb adana in combination with the 3.7 kb deletion mostly have mild - to - moderate anemia . 
 in contrast , patients who were compound heterozygotes for hb adana and nondeletional mutations , generally showed a more severe anemia and it mostly presented in childhood . 
 thus , accurate diagnosis of -thal disorders is not only important for future management of these patients but also for providing proper genetic counseling to the family .