Bs Haplotypes in various world populations

8/8/2019 Bs Haplotypes in various world populations

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Hum Genet (1992) 89:99-104

9 Springer-Verlag 1992

13 H a p l o t y p e s in v a r i o u s w o r l d p o p u l a t i o n sC i h a n O n e r : , A l e k s a n d a r J . D i m o v s k i 1, N a n c y F . O l i v i e ri 2 , G i n o S c h i li r o 3 , J o h n F . C o d r in g t o n : ' 4 ,S l a d d e h i n e F a t t o u m L 5 , A d e k u n l e D . A d e k i l e : ' 6 , R e y h a n O n e r 1 , G u n e s T . Y i i r e g ir 7 , (~ . A I t a y 8 , A . G u r g e y s ,R a s h i k B . G u p ta :" 9, V i n o d B . J o g e s s a r 1 ~ M i c h a e l N . K i t u n d u t' : : , D i m i t r i s L o u k o p o u l o s 12, G a b r i e l P . T a m a g n i n i 13,M . L e t ic i a S . R i b e i r o : 3, F e r d a n e K u t l a r 1 , L i - H a o G u 1 , K e n n e t h D . L a n c l o s : , a n d T i t u s H . J . H u i s m a n 1:Department of Biochemistryand Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, USA2Department of Hematology,The Hospital for Sick Children, Toronto M5G 1X8, Canada3Department of Pediatric Hematology,Universityof Catania, 1-95125 Catania, Italy4Academisch Ziekenhuis, Paramaribo, Suriname5Laboratoire de Biochimie, H6pital d'Enfants, Place Bab-Saadoun, Tunis, Tunisie6Department of Pediatrics, Obafemi Awolowo University, Ile-Ife, Nigeria7Department of Biochemistry,Universityof ffukurova, Adana, Turkey8Children's Medical Center, Hacettepe University, Ankara, Turkey9Regional Medical Center for Tribals, Jabalpur 482003, India1~ of Haematology,King Edward VIII Hospital, P.O. Congella, Durban, Republic of South Africal:Yuma Foundation Laboratories, Iramba District, Kinampanda,Tanzania:2First Department of Medicine, Universityof Athens, Laikon Hospital, GR-11527 Athens, Greece13Department of Hematology,Centro Hospitalar, P-3000 Coimbra, PortugalReceived July 15, 1991 / Revised October 29, 1991

S u m m a r y . We have determined the 13 haplotypes in 709patients with sickle cell anemia, 30 with SC disease, 91with S-[~-thalassemia, and in 322 Hb S heterozygotesfrom different countries. The methodology concernedthe detection of mutations in the promoter sequences ofthe Gy_ and Ay-globin genes through dot blot analysis ofamplified DNA with 32p-labeled probes, and an analysisof isolated HbF by reversed phase high performanceliquid chromato graphy to detect t he presence of the ATXchain [Ay75(E19) Il e~ Th r] that is characteristic for hap-lotype 17 (Cameroon). The results support previouslypublished data obtained with conventional methodologythat indicates that the 13 gene arose separately in differ-ent locations. The present methodology has the advan-tage of being relatively inexpensive and fast, allowingthe collection of a vast body of data in a short period oftime. It also offers the opportunity of identifying unusual13 haplo types that may be associated with a milder ex-pression of the disease. The numerous blood samplesobtained from many SS patients living in different coun-tries made it possible to compare their hematologicaldata. Such information is included (as average values) for395 SS patients with haplotype 19/19, for 2 with haplo-type 17/17, for 50 with haplotype 20/20, for 2 with haplo-type 3/3, and for 37 with haplotype 31/31. Some informa-tion on haplotype characteristics of normal ~A chromo-somes is also presented.

I n t r o d u c t i o nSickle cell anemia (SS) is one of the most commonheredi tary diseases. Its hematological characteristics andOffprint requests to: T. H. J. Huisman

clinical severity are variable and are influenced by anumber of factors, including the simultaneous presenceof an a-thalassemia (a-thai), variations in HbF level,and the haplotype background that is linked to the 13-globin gene (for review see Nagel and Ranney 1990).There is also convincing evidence that the ~s mutationarose in Africa and Saudi Arabia-India on at least fivedifferent types of chromosomes. These chromosomeshave distinct haplotypes that are l inked to the [3 geneand are numbered 17, 19, 20, 3, and 31, respectively,and are also known as the Cameroon, Benin, Bantu orCAR (Central African Republic), Senegal, and SaudiArabia-India types, respectively (for review see Nageland Ranney 1990; Schroeder et al. 1990).Determination of these haplotypes is based upon thepresence or absence of different restriction sites (re-viewed in Hattor i et al. 1986). We recently detected cer-tain mutations within the promoter regions of the Gy_and Ay-globin genes and in the second intervening se-quence (IVS-II) of the Ay-globin gene that are character-istic for the listed haplotypes (Lanclos et al. 1991; Di-movski et al. 1991). A [3 chromosome with haplo type 19has two specific mutations, one in each of the promotersequences, namely -3 69 (C---~G) Gy and -65 7 (G-*T)Ay, that with haplotype 20 has a double-base mutation, ahighly characteristic 6-bp dele tion in the Gy promoter,and a single base pair muta tion in the Ay promoter . A 13chromosome with haplotype 17 has none of these butcarries a T ~ C mutation at codon 75 (Ay) leading to thesynthesis of the A~T chain (75 Ile---~Thr; Ricco et al.1976). The 13s chromosome with either haplotypes 3 or31 cannot be distinguished from each o ther by this pro-cedure. Both have only the C---,T mutation at -158 inthe Gy promoter that results in a high level of Gy chains


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10 0Tab le 1 . Muta t ion s cha rac te r i s t i c for ~3 chromo som es wi th spec if i cha p l o t ype s . + , T he m u t a t i on is p r e s e n t ; - , t he m u t a t i on is a b s e n tM u t a t i o n s H a p l o t y p e s

17 19 20 3 31-1106 /05 (GC- -~TT ) (07) - - + - -6 b p d e l e t io n ( - C T T T A A ) ( 0 7) - - + - --36 9 (C---~G) (07) - + - - --15 8 (C--+T) (07) - - - + +-6 57 (G---~T) (A~/) _ q _ _ _ _-271 (C--+T) (A7) - - - - + - - - -Hb F high G7 (> 60% ) - - _ _ + +Hb F wi th ATT +2 _ _ _b _ba D ue to a T- --~C mu ta t ion a t codo n 75 of the A7 -globin geneb D i f f e r e n t i a t i on o f ha p l o t ype s 3 a nd 31 i s ba s e d on r a c i a l/ e t hn i cba c kg r ound o f t he s ub j e c t o r on t he p r e s e nc e o r a bs e nc e o f aHinclI s i te 5 ' t o t he ~ ge ne ( ha p l o t ype 31 : + : 3 : - )

( G i l m a n a n d H u i s m a n 1 98 4 ; T a b l e 1 ). D i f f e r e n t i a t i o nb e t w e e n t h e t w o i s b a s e d o n t h e e t h n i c / r a c i a l b a c k -g r o u n d o f th e i n d i v i d u a l a n d , i n c e r t a i n c a se s , o n t h ep r e s e n c e o r a b s e n c e o f t h e HinclI s i t e 5 ' t o t h e ~ g e n ea n d t h e BamHI s i te 3 ' t o t h e [ 3 - g lo b in g e n e . M o r e o v e r ,B e r g e t a l . (1 9 9 1 ) h a v e r e p o r t e d t h a t t h e + A T A / - Tc o n f i g u r a t i o n a t p o s i t i o n - 5 3 0 t o th e [ 3 -g l ob i n g e n e i sc h a r a c t e r is t i c f o r h a p l o t y p e 3 1 . T o d i s t i n g u i s h h a p l o -t y p e s 3 a n d 3 1 , a D N A s e g m e n t b e t w e e n p o s i t i o n s - 7 2 4a n d + 2 7 r e l a t i v e t o t h e C a p s i t e o f t h e [ 3 - gl o b in g e n e i sa m p l i f i e d a n d h y b r i d i z e d w i t h p r o b e s s p e c i f ic fo r t h isc h a n g e i n s e q u e n c e a n d f o r t h e n o r m a l s e q u e n c e ( f o rm e t h o d o l o g y , s ee W o n g e t a l. 1 98 9) . T h e m e t h o d o l o g yis r e l a t iv e l y f a st ; n u m e r o u s a m p l i f i e d D N A s a m p l e s a r ep l a c e d o n a n y l o n m e m b r a n e a n d a r e h y b r i d i z e d s e q u e n -t ia l ly w i th t h e p r o b e s f o r t h e f o u r m u t a t i o n s i n th e G7p r o m o t e r o r t h e t w o i n t h e a 7 p r o m o t e r . I d e n t i f i c a t io no f h a p l o t y p e 1 7 r e q u i r e s e i t h e r a n a d d i t i o n a l a m p l i f ic a -t i o n i n v o l v i n g t h e A T - g lo b i n g e n e a n d h y b r i d i z a t i o n w i t ha p r o b e s p e c if i c f o r th e T - - +C m u t a t i o n a t c o d o n 7 5 , o rt h e i s o l a t i o n o f H b F f r o m t h e r e d c e l l s a n d c h a r a c t e r i z a -

t i o n o f t h e t y p e s o f 7 ch a i n b y r e v e r s e d p h a s e h i g h p e r -f o r m a n c e l i q u id c h r o m a t o g r a p h y ( H P L C ; t h e l a t t e r a p -p r o a c h w a s u s e d i n th i s s t u d y ) . A n a d d e d b e n e f i t o f th em e t h o d o l o g y i s t h a t i t a l l o w s t h e s e l e c ti o n o f u n u s u a l [3h a p l o t y p e s t h a t d i f fe r f r o m t h e f iv e m a j o r t y p e s a n d a r eo f t e n a s s o c i a t e d w i t h a m i l d e r f o r m o f th e d i s e a se .

S i n ce t h e d e v e l o p m e n t o f t hi s m e t h o d o l o g y , w e h a v er e c o n f i r m e d s o m e o f t h e [3 h a p l o t y p e s p r e v i o u s l y d e t e r -m i n e d i n o u r l a b o r a t o r y w i t h t h e r e s tr i c ti o n e n d o n u c l e a s em e t h o d , a n d a l l c u r r e n t d a t a w e r e o b t a i n e d w i t h th e n e wa p p r o a c h . I n th e p r e s e n t c o m m u n i c a t i o n , w e d e s c r ib eo u r e x p e r i e n c e w i t h h a p l o t y p i n g a la r g e n u m b e r o f [3c h r o m o s o m e s f r o m p a t i e n t s l i v in g i n 14 d i f f e r en t c o u n -t r ie s u s i n g t h e n e w a p p r o a c h . D e t a i l s o f h e m a t o l o g y a n dc l i n i c al f e a t u r e s f o r m o s t o f t h e p a t i e n t s h a v e e i t h e r b e e np u b l i s h e d o r w il l b e p u b l i s h e d e l s e w h e r e . T h e d a t a f o rp a t i e n t s f r o m t h e U n i t e d S t a t e s ( H a t t o r i e t al . 19 8 6 ;K u t l a r e t a l. 1 9 8 5 ), K e n y a ( O j w a n g e t a l . 1 9 8 7 ) , a n dT u n i s i a ( A b b e s e t a l. 1 9 9 1 ; F a t t o u m e t al . 1 99 1 ) in t h ep r e s e n t s t u d y a r e a r e c o n f i r m a t i o n o f a l r e a d y p u b l i s h e di n f o r m a t i o n , w h i l e t h e o t h e r s r e p r e s e n t n e w a n d o n g o i n ga n a l y s e s .

M a t e r i a l s a n d m e t h o d sB l ood s a m p l e s f r om 1152 i nd i v i dua l s w e r e c o l l e c t ed i n va c u t a i ne r sw i t h E D T A a s a n t i c oa gu l a n t . T he s ub j e c t s c a m e f r om 14 d i f f e re n tcoun t r i e s and h ad SS, SC di sea se , s i ckle ce ll t r a i t (AS ) , or H b 8-]3-tha l ; t he 13-thal a l l e l e s we re n ot i dent i f i ed .T he U n i t e d S t a t e s pa t i e n t s w e r e m a i n l y A f r i c a n A m e r i c a nsf r om t he G e o r g i a a r e a o f the s ou t he a s t e r n U SA , bu t i nc l ude d oneA r a b f a m i l y . T he C a na d i a n pa t i e n t s w e r e C a r i bbe a n i m m i g r a n t s :t hos e f r om T un i s i a a nd Sy r i a w e r e A r a bs , w h i l e thos e f r om T u r ke yw e r e E t i - T u r ks f r om ~ uku r ova p r ov i nc e . T he I t a l i a n a nd G r ee i~pa t i e n t s w e r e C a uc a s i a ns f r om S i c i ly a nd A t he n s , r e s pe c t i ve l y .T he Su r i na m e s e w e r e b l a c ks w i t h va r y i ng a nc e s t r i e s , a nd t hos ef r om I nd i a be l on ge d t o t he G o nd t r i ba l g r oups f r om t he c e n t r a lpa r t o f t he c oun t r y . T he K e nya ns , T a nz a n i a ns , a nd A ngo l a ns w e r eB a n t us , w h i l e t he N i ge r i a ns w e r e r e p r e s e n t a t i ve o f va r i ous e t hn i cg r oups ( m a i n l y I bo , H a us a , a nd Y or uba ) a nd w e r e d r a w n f r ome ight d i f f e rent cente r s . A l l pa t i ent s we re a t t endin g s i ckle ce l l

T a b l e 2 . D a t a a bou t p r i m e r s a nd p r obe s .M , M u t a n t p r o b e ; N , n o r m a l p r o b e S e q u e n ce5 'Pos i t i ons Func t i on3 '

A C G T C A T A A T C T A C C A A G G T C A T GA G C T T A G G G G A T A A A C T A A T T T GG G C G T C T G G A C T A G G A G C T T A T T GT A C T T C C T [ ~ C A T G T T A A G (M )T A C T T C C T G[GCJC A T G T T A A G ( N)C A T G C T T T A A A C T A C A G G C (M )T A C A T G C T I T A A I C T T T A A I A (N )C T G G A G C T A ~ G ~ A G A C A A G A A (M )C T G G A G C T A I C IA G A C A A G A A (N )G A A A C G G T ~ T ~C C T G G C T A A A ( M)G A A A C G G T [ C J C C T G G C T A A A (N )T G A G G T A A G C A T T A G [ ~ T C T (M )T G A G G T A A G C A T T A G I G I T C T ( N)A G A G A A A A A [ ~ T G G A A T G A C (M )A G A G A A A A A C ~ J T G G A A T G A C ( N )

c7: - 1227 to - 12 04A : - - 1279 to - - 1257Ca , r +53 to +3007: -- 1114 to --10960,/ : - -407 to - -383 (M)409 t o - 391 ( N )0 7 : - 3 7 8 t o - 3 6 00 7 : - 1 4 8 t o - 1 6 6AT: - 6 7 2 t o - 6 5 4A T : - 2 8 0 t o - 2 6 2

D i r e c t p r i m e rD i r e c t p r i m e rR e ve r s e p r i m e r- 1106 /05 ( G C ~ T T )6 - bp de l e t i on( - C T T T A A )- 3 6 9 ( C - + G )- 1 5 8 ( C - - , T )

- 6 5 7 ( G -- ~ T )-271 (C---~T)


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10 1c l i n ic s i n t h e i r v a r i o u s c o u n t r i e s b u t t h e A n g o l a n s w e r e r e s i d e n t s o fP o r t u g a l a n d a t t e n d i n g t h e C e n t r a l H o s p i t a l in C o i m b r a . A l l w e r ei n s t e a d y s t a t e , i . e . , n o t i n c r i s i s o r a n y o t h e r a c u t e i l l n e s s a t t h et i m e o f b l o o d c o l l e c t i o n . L e s s t h e n 1 0 % o f t h e p a t i e n t s w e r e r e -l a t e d , b u t t h e A S i n d i v i d u a l s w e r e m o s t l y r e l a t iv e s o f t h e S Sp a t i e n t s .A l l b l o o d s a m p l e s w e r e k e p t , a s m u c h a s p o s s ib l e , a t 4 ~ f o rv a r y i n g p e r i o d s o f t im e , a n d w e r e s h i p p e d o r c a r r i e d i n w e t i c e b yf a s t a i r s e r v i c e a n d b y d i f f e r e n t a u t h o r s t o A u g u s t a , G a . M o s t s a m -p l e s r e a c h e d t h e l a b o r a t o r y m o r e t h a n 3 w e e k s a f t e r c o l l e c t i o n .H o w e v e r , c o m p l e t e b l o o d c o u n t s w e r e o b t a i n e d w i t h i n 1 d a y o fc o l l e c t io n f o r s a m p l e s fr o m t h e U n i t e d S t a t e s, C a n a d a , P o r t u g a l ,I t a ly , G r e e c e , a n d S o u t h A f r i c a . T h e s e w e r e d o n e e i t h e r l o c a l ly att h e c e n t e r s o f c o l le c t i o n o r in t h e l a b o r a t o r y i n A u g u s t a , G a .H e m a t o l o g i c a l d a t a w e r e o b t a i n e d u s i n g a n a u t o m a t e d c e l lc o u n t e r . A l l s a m p l e s w e r e a n a l y z e d b y i s o e l e c t ro f o c u s i n g ( I E F ;R i g h e t t i 1 9 86 ) a n d b y c a t i o n e x c h a n g e H P L C ( B is s 6 a n d W i e l a n d1 9 88 ; K u t l a r e t a l . 1 9 9 0 ) , fo r t h e q u a n t i t a t i o n o f H b F a n d H b A 2 .H b F w a s i s o la t e d b y D E A E - c e l l u l o s e c h r o m a t o g ra p h y ( S c h r o e d e ra n d H u i s m a n 1 98 0) a n d i t s 7 c h a in c o m p o s i t i o n w a s d e t e r m i n e d b yr e v e r s e d p h a s e H P L C ( S h e l t o n e t a l . 1 98 4 ; K u t l a r e t a l. 1 9 86 ).D N A w a s i s o l a t e d f r o m t h e b l o o d s a m p l e s w i t h t h e m e t h o d o fP o n c z e t a l . ( 19 8 2 ) . F o r t h e m a j o r i t y o f t h e s a m p l e s t h e 13 m u t a -t i o n w a s c o n f i r m e d b y d o t - b l o t a n a l y s i s , h y b r i d i z i n g a m p l i f i e dD N A w i t h a 3 2 p - la b e l e d o l i g o n u c l e o t i d e p r o b e t h a t i s s p e c i f i c f o rthe A- - - ~T mu ta t io n a t cod on 6 ( Glu- -- ~Val ) o f t he [ 3 -g lob in gene .S e g m e n t s o f a b o u t 1 3 5 0 b p o f b o t h t h e c , {_ a n d A q ,- gl ob in g e n e sw e r e a m p l i f i e d f r o m g e n o m i c D N A w i t h 5 ' d i r e c t o l i g o n u c l e o t i d ep r i m e r s s p e c i f i c f o r c ~ , a n d A7 a n d a c o m m o n 3 ' r e v e r s e p r i m e r( T a b l e 2 ). A l i q u o t s o f th e s e a m p l i f ie d D N A s a m p l e s w e r e b l o t t e do n t o n y l o n m e m b r a n e s a n d h y b r i d i z e d t o t h e s p e c i fi c 5 ' e n d - l a b e l e do l i g o n u c l e o t i d e p r o b e s l i s t e d in T a b l e 2 ; t h e l i s t i n g i n c l u d e s s e -q u e n c e s o f t h e p r o b e s , t h e i r l o c a ti o n s w i t h i n t h e s e q u e n c e s o f t h e5 ' f l a n k i n g r e g i o n s o f t h e " ~ -g lo b in g e n e s , a n d t h e m u t a t i o n s o r t h ed e l e t i o n f o r w h i c h t h e s e p r o b e s a r e s p e c i f i c . M e t h o d o l o g y h a sb e e n d e t a i l e d i n e a r l i e r s t u d ie s ( L a n c l o s e t a l. 1 9 91 ; D i m o v s k i e tal . 1991) .

ResultsSubjectsA tota l of 1861 13 chromosomes were analyzed; 509 [3chromosomes came from patients of North and South

America, 791 from Africa, 426 from Mediterraneancountries, and 135 from Indian donors. The largest groupof patients was from Nigeria, followed by the UnitedStates and Turkey. There was no significant sex differ-ence in the whole population.[ 3 H a p l o t y p e s (Table 3)Four diffe rent haplotypes were present in the North andSouth American donors; the frequency of haplotype 19was the highest in all three groups, followed by haplo-types 20, 3, and 17. The high frequency of haplotype 20among patients from Surinam was striking. The sevenchromosomes with haplotype 31 were from four mem-bers of an Arab family, described before (Kutlar et al.1985). As man y as 36 [3 chromosomes had atypical hap-lotypes; some of these are presently under study.The data from African patients fell into two groups.The Keny an, Angolan, and Tanzanian [3 chromosomeshad haplotype 20, while those of Nigerian subjects hadhaplo type 19 with 3.7% haplo type 17, 1.0% haplotype 3,and 0.8% haplo type 20. As many as 411 of the 426 [3chromosomes (96.5%) from the patients of Mediterra-nean descent had haplotype 19; 13 13 chromosomes arein need of further characterization. All but two of the135 Indian 13 chromosomes had haplotype 31; the originof two [3 chromosomes with haplotype 17 remains un-clear.f lA a n d [ 3- th a l c h r o m o s o m e sDimovski et al. (1991) reported the specific changes inthe 5' flanking regions of the G and A7 genes of 105 13Achromosomes from normal African American black in-dividuals from the southeastern United States. Theyfound that 26 chromosomes were positive for the -657G ~ T ( A ] ) mutation, while 18 of these 26 chromosomesalso had the -369 C---~G (GT) muta tion . These twochanges were specific for haplotype 19. None were posi-

Table 3 . N u m b e r o f p a t i e n t s w i t h h e m o g l o b i n o p a t h i e s p a r t i c i p a t i n g i n t h i s s t u d y a n d t h e i r 13 h a p l o t y p e sC o u n t r y S S S C A S SI3 M [ 3 H a p l o t y p e s

c h r o m o s o m e s 19 17 20 3 31 Ot her sU S A ( SE ) 147 13 41 23 371 205 13 71 56 7 19C a na da 21 11 5 3 61 30 8 7 8 0 8Sur in am 14 1 45 3 77 41 2 23 2 0 9Ke ny a 55 0 1 0 111 2 0 109 0 0 0T a nza n ia 14 0 13 0 41 0 0 41 0 0 0Nig er i a 294 2 30 3 623 576 23 5 6 0 13A ng ola a 6 0 4 0 16 2 0 14 0 0 0T un i s i a 35 2 37 6 115 109 0 0 0 0 6Sai ly ( I taly ) 18 0 19 15 70 70 0 0 0 0 0Gr ee ce 0 0 14 0 14 13 0 0 1 0 0T u r ke y 67 1 60 19 214 206 0 0 0 1 7Syr i a 2 0 5 4 13 13 0 0 0 0 0Ind ia 36 0 45 12 129 0 2 0 0 127 0Sou th Af r i ca ( I nd ian s ) 0 0 3 3 6 0 0 0 0 6 0T o ta l 709 30 322 91 1861 1267 48 270 73 141 62a F r o m s u b j e c t s li v i n g i n P o r t u g a l


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10 2Table 4 . Hem atological an d H b com posi t ion data fo r SS pat ients f rom d i f ferent countr ies wi th hom ozygosi t ies for speci fic 13 haplotyp es(average values wi th s tandard d evia t ions in paren theses )? n d, No data : Hb , hemoglob in; PCV, Packe d cel l volum e; RBC, red blood count :M C V , m ean co rpuscu l a r vo lum e ; M C H , m ean co rpuscu l a r hem og lob in ; M C H C , m ean co rpuscu l a r hem og lob in concen t ra t i onH a p lo - C o u n tr y n A g e H b P C V R B C M C V M C H M C H C A 2b F b Gyctype (years) (g/dl) Off) 10t2/l (fl) (pg) (g/dl) (% ) (% ) (% )19/19 US A 24 7.9 8.80 0.250 3.02 84.0 29.4 35.0 2.9 11.7 40.8(6.4) (1.0) (0.030) (0.57) (9.0) (4.0) (2.7) (0.5) (5.2) (4.6)

Surina m 5 10.5 8.30 0.294 2.85 102.5 28.8 28.2 3.2 9.3 42.4(4.8) (1.4) (0.048) (0.35) (8.7) (2.7) (1.8) (1.0) (4.4) (4.0)Nigeria 249 9.7 7.53 0.282 2.76 103.7 26.8 26.8 2.9 9.2 41.6(6.0) (1.4) (0.04 5) (0.65) (13.3) (2.2) (2.2) (1 .0) (5.6) (5.9tTun isia 35 14.3 8.20 0.250 2.53 98.8 32.4 32.8 2.9 7.6 36.5(6,7) (1.2) (0.041) (0.31) (8.1) (3.11 (2.6) (1.1) (2.9) (4.3)Italy 18 11.2 7.55 0.240 2.47 97.2 30.6 31.5 2.9 9.0 39.6(7.1) (1.6) (0.039) (0.37) (7.5) (3.2) (2.1) (0.7) (4.1) (3.6)Tu rke y 62 10.0 8.40 0.250 2.72 91.9 30.7 34.2 2.7 9.8 44.0(4.2) (1 .8 1 (0.060) (0.65) (7.0) (2,1) (2.0) (0.6) (4.0) (4.1)Syria 2 4 7.20 0.225 2.73 82.0 26.2 32.0 3 .7 7.8 41.120 9.20 0.255 2.61 98.0 35.2 36.1 3.1 9.2 40.0

17/17 U SA 1 12 7.90 0.225 2.89 77.7 27.3 35.1 3.2 5.4 42.7N igeria 1 . . . . . . 3.1 14.1 49.220/20 U SA 6 9 .8 8.20 0.210 2.55 84.5 31.8 37.5 2.5 12.1 41.3(4.1) (0.9) (0.020) (0.29) (7.11 (3.2) (1.4) (0 .35) (6.4) (3.8)Surin am 3 12.6 7.60 0.290 2.96 98.0 25.7 26.2 3.6 5.7 38.6(4.8) (1.5) (0.050) (0.60) (7.1) (2.11 (1.5) (0.4) (2.3) (2.9)Tan zania 12 10.7 6.42 0.247 2.27 108.8 28.8 26.0 nd 8.6 44.0(6.5) (1.3) (0.050) (0.49) (14.4) (4.9) (1.7) (3.4) (3,6)Ke nya 25 10.9 7.85 0.260 2.54 102.4 30.9 30.2 3.1 7.5 40.6(7.2) (1.3) (0.040) (0.31) (8.0) (2.8) (1.3) (0.7) (4.5) (4.9)An gol a 4 9.3 7.30 0.207 2.70 88.3 30.5 35.3 nd 2.7 nd(5.0) (0.5) ( - ) (0.23) (6.4) (2.4) (1.0) (2.3)

3/3 U SA 2 43 9.6 0.280 3.60 79.0 26.7 33.7 3.0 19.2 71.115 8.2 0.300 3.11 78.0 26.4 33.5 3.3 14.0 71.231/31 U SA 3 13.3 10.1 0.295 3.78 78.0 26.7 34.2 1.2 27.8 64.9(5.8) (1.4) (0.020) (0.12) (4.0) (2.7) (1.3) (0.3) (5.3) (1.3)

Ind ia 34 12.1 9.7 0.281 3.17 88.6 30.6 34.5 1.9 23.2 68.6(8.2) (1.8) (0.038) (0.78) (7.0) (5.2) (1.4) (0.3) (5.9) (3.7)a Th e data for the US pa t ients are f rom H at tor i e t a l . (1986) ; Kut lar e t a l . (1985)b By microc hrom atograp hy (Schle ider e t a l. 1977 ) or ca t ion exchange HP LC (Biss6 andc By reversed phase H PL C (Shel ton e t a l . 198 4; Kut lar e t a l . 1986) Wieland 1988; Kutlar et al . 1990)

t iv e f o r h a p l o t y p e 2 0 . I n t h e s a m e s t u d y i t w a s o b s e r v e dt h a t 2 3 o f 3 2 c h r o m o s o m e s w i t h a [ 3 -th al m u t a t i o n h a dh a p l o t y p e A ( w h i c h is s i m i l a r t o t h e 13 h a p l o t y p e 3 ) . F i v ec h r o m o s o m e s w i t h h a p l o t y p e B , w h i c h i s s i m i la r t o 13h a p l o t y p e 1 9 , w e r e p o s i t i v e f o r t h e - 6 5 7 G - - + T ( 1 7 ) m u -t a ti o n , b u t o n l y o n e c h r o m o s o m e c a r r ie d t h e C - - , G m u -t a t io n a t - 3 6 9 ( Gy ). O n e c h r o m o s o m e w i th a n a ty p i c alh a p l o t y p e i n th e s t u d y w a s p o s i ti v e f o r 13 h a p l o t y p e 2 0 -r e l a t e d m u t a t i o n s .

O f t h e 3 0 1 3A c h r o m o s o m e s f r o m N i g e r i a n A S i n d i-v i d u a l s in t h e p r e s e n t s t u d y , s e v e n ( 2 3 . 3 % ) w e r e p o s i t iv ef o r h a p l o t y p e 1 9 - s pe c i fi c m u t a t i o n s , w h i le o n e ( 3 . 3 % )w a s p o s i t iv e f o r h a p l o t y p e 2 0 m u t a t i o n s . T h e [ 3- th alc h r o m o s o m e , p r e s e n t i n t h r e e m e m b e r s o f o n e f a m il y ,h a d h a p l o t y p e 2 0 - s p e c i f ic m u t a t i o n s . O f t h e t h i r t e e n ~3Ac h r o m o s o m e s f r o m T a n z a n i a , f o u r h a d h a p l o t y p e 1 9 -s p e c i fi c m u t a t i o n s . I n f o r m a t i o n o n 13A c h r o m o s o m e sf r o m t h e o t h e r c o u n t r i e s is n o t a v a i l a b l e .

Hematological and hemoglobin (Hb) composition dataC o m p l e t e a n d s a t is f a c to r y d a t a c o u l d b e o b t a i n e d f o r4 8 6 S S p a t i e n t s ( T a b l e 4 ) . B e c a u s e m a n y s a m p l e s w e r ec o l l e c t e d m o r e t h a n 3 w e e k s b e f o r e a n a l y s i s (S u r i n a m ,N i g e r i a , T a n z a n i a , S y r i a , I n d i a ) , t h e P C V , M C V , a n dM C H C v a l u es s h o u l d b e v i e w e d w i th r e s e rv a t i o n . T h em o s t s e v e r e a n e m i a i s f o u n d a m o n g S S p at i en t s w i t hh a p l o t y p e 2 0 / 2 0 , f o l l o w e d b y t h e p a t ie n t s w i t h h a p l o t y p e1 9 /1 9 . T h e h e m a t o l o g i c a l d a t a f r o m 3 7 S S p a t i e n t s w i t hh a p l o t y p e 3 1 / 3 1 i n d i c a t e d m i l d t o m o d e r a t e a n e m i a w i t ha n a v e r a g e H b l e v e l o f a b o u t 1 0 .0 g /d l. H b F v a lu e s v a r -i e d c o n s i d e r a b l y b u t w e r e g e n e r a l l y b e l o w 1 0 . 0 % f o r S Sp a t i e n t s w i t h t h e 1 9 / 1 9 , 1 7 /1 7 , a n d 2 0 / 2 0 h a p l o t y p e s .H i g h e r v a l u e s w i t h h i g h ~ 7 v a l u e s w e r e o b s e r v e d i n a l lS S p a t ie n t s w i t h h a p l o t y p e 3 /3 a n d 3 1 / 3 1; d e t a i l e d i n f o r -m a t i o n a b o u t t h e I n d i a n S S p a t i e n t s w i ll b e p r e s e n t e de l s e w h e r e ( G u p t a e t a l . 1 9 9 11 .


5/6

103Di scu s s i on

T w o r e c e n t r e v i e w s ( S c h r o e d e r e t a l. 1 9 9 0; N a g e l a n dR a n n e y 1 9 90 ) h a v e s u m m a r i z e d t h e [3 h a p l o t y p e d a t aa n d h a v e e s t i m a t e d t h e a p p r o x i m a t e b o u n d a r i e s o f t h ef o u r m a j o r h a p l o t y p e s . T h e S e n e g a l t y p e 3 is p r e s e n t i nA t l a n t i c W e s t A f r i c a , t h e B e n i n t y p e ( 1 9 ) i n c e n t r a lW e s t A f r i c a , t h e B a n t u ( C A R ) t y p e ( 20 ) i n S o u t h - c e n t r a la n d E a s t e r n A f r i c a , w h i l e t h e S a u d i A r a b i a - I n d i a t y p e3 1 i s r e s t r i c t e d t o t h e I n d i a n s u b c o n t i n e n t a n d t h e e a s t -e r n s e c t i o n o f t h e A r a b i a n p e n i n s u l a . T h e f i f t h t y p e ( 1 7 )is m u c h m o r e r e s t r i c te d , b e i n g o b s e r v e d m a i n l y i n th eE t o n e t hn i c g r ou p o f C a m e r o o n ( L a p o u m e r o u l i e e t a l.1 9 8 9 ); it w a s o r i g i n a l ly o b s e r v e d a m o n g A f r i c a n A m e r i -c a n s l i v i n g i n t h e s o u t h e a s t e r n U n i t e d S t a t e s ( H a t t o r ie t a l . 1 9 8 6 ). T h e n e a r l y e x c l u s i v e o c c u r r e n c e o f h a p l o t y p e1 9 a m o n g S S p a t i e n t s in v a r io u s M e d i t e r r a n e a n c o u n t r i e ss u g g e s ts t h a t th i s [3 c h r o m o s o m e w a s i n t r o d u c e d f r o mc e n t r a l W e s t A f r i c a v i a A l g e r i a , T u n i s i a , a n d E g y p t .

T h e l a rg e b o d y o f d a t a p r e s e n t e d h e r e s u p p o r t s t h e s ec o n c l u s i o n s . T h e a l m o s t e x c l u s i v e o c c u r r e n c e o f o n e s p e -c i fi c [~s h a p l o t y p e i n p a t i e n t s f r o m d i f f e r e n t c o u n t r i e s ( 2 0i n K e n y a , A n g o l a , a n d T a n z a n i a ; 1 9 i n N i g e r ia ; 1 9 i n a l lM e d i t e r r a n e a n c o u n t r i e s ; 3 1 i n I n d i a ) i s m o s t s t r i k i n g .T h e p r e s e n c e o f h a p l o t y p e s 3 an d 1 7 a m o n g N i g e r i a n S Sp a t i e n t s i s a s e x p e c t e d ; [3 h a p l o t y p e 3 is i n t r o d u c e d f r o mt h e w e s t a n d 1 7 f r o m t h e e a s t ( C a m e r o o n ) . T h e p r e s e n c eo f h a p l o t y p e 2 0 i s al so n o t s u r p r i s i n g s i n c e it is g e n e r a l l ya g r e e d t h a t B a n t u m i g r a t i o n o r i g i n a t e d a r o u n d t h eN i g e r i a / C a m e r o o n a r e a ( N a g e l 1 9 8 4 ). L e s s e v i d e n t is t h eo r i g i n o f t h e [3 h a p l o t y p e 1 7 i n t w o I n d i a n t r i b a l p a t i e n t s .T h e v a r i a t io n i n t h e r e l a t iv e p e r c e n t a g e s o f t h e A f r i c a n~ s h a p l o t y p e s i n A m e r i c a n , C a n a d i a n ( m a i n l y i m m i -g r a n ts f r o m t h e C a r i b b e a n a r e a ) , a n d S u r i n a m p a t i e n t sl i k e l y r e f l e c t s t h e o r i g i n s o f t h e i r p r e d e c e s s o r s . A p p a r -e n t l y , m a n y s l a v e s i n S o u t h A m e r i c a w e r e B a n t u s w h oc a r r y t h e [3 c h r o m o s o m e w i t h h a p l o t y p e 2 0.

R e c e n t l y , B o u h a s s i r a e t a l . ( 1 9 8 9 ) s u g g e s t e d t h e o c -c u r r e n c e o f a g e n e c o n v e r s i o n i n t h e [3 c h r o m o s o m ew i t h h a p l o t y p e 2 0 t h a t i n vo l v e s t h e r e p l a c e m e n t o f t h eA7 s e q u e n c e b y a G7 s e q u e n c e i n th e p r o m o t e r o f t h e Av -g l o b i n g e n e . T h e 5 ' l i m i t o f t h e c o n v e r s i o n i s t h o u g h t t ob e l o c a t e d b e t w e e n p o s i t io n s - 3 0 6 a n d - 2 7 1 ( a t p o s i ti o n- 2 7 1 t h e C i s r e p l a c e d b y a T a s in G y) a n d t h e 3 ' l i m i tb e t w e e n p o s i t io n s + 2 5 ( t h e A i s r e p l a c e d b y G ) a n d+ 1 1 0 8 ( t h e T f o u n d i n A y i s r e t a i n e d ; t h e 6 y - g l o b i n g e n eh a s a n A a t th i s p o s i t i o n ) . T h e a u t h o r s o b s e r v e d t h a t t h i sc o n v e r s i o n i s h ig h l y , b u t n o t e x c l u s i v e l y , a s s o c ia t e d w i t hh a p l o t y p e 2 0 b e c a u s e s o m e n o r m a l c h r o m o s o m e s a l s oa p p e a r t o c a r r y t h is c h a n g e . M o r e o v e r , t h e f r e q u e n c y o ft h e c o n v e r s i o n , d e t e r m i n e d f o r 1 1 S S p a t i e n t s w i t h ah o m o z y g o s i t y f o r h a p l o t y p e 2 0 , w a s 0 .8 2 . T h e c h a r a c-t e r i z a t i o n o f th e 13 c h r o m o s o m e s w i th h a p l o t y p e 2 0 ,l i s te d i n T a b l e 3 , is b a s e d u p o n t h e p r e s e n c e o f c h a n g e si n t h e G y a n d a , ~ p r o m o t e r s , i n c l u d i n g t h e C -- -~ T m u t a t i o na t p o s i t i o n - 2 7 1 ( A y) ( T a b l e 2 ) . A l l 27 0 [3 c h r o m o s o m e sw i t h h a p l o t y p e 2 0 o f t h e p a t i e n t s f r o m t h e s o u t h e a s t e r nU n i t e d S t a t es , t h e C a r i b b e a n a r e a ( t h e C a n a d i a n p a -t i e n t s ) , S u r i n a m , a n d t h e A f r i c a n c o u n t r i e s c a r r i e d t h a tm u t a t i o n , w h i l e t h e o t h e r [ 3 c h r o m o s o m e s , t h e [ 3A, 13 ,a n d [ 3-th al c h r o m o s o m e s , d i d n o t .

T h e p o l y m e r a s e c h a i n r e a c ti o n ( P C R ) a m p l i f ic a t io nm e t h o d f o r m a s s s c r e e n i n g o f 13 h a p l o t y p e s h a s t h ea d v a n t a g e o v e r t h e r e s tr i c ti o n e n z y m e m e t h o d o l o g y o fb e i n g f a s t , si m p l e , a n d a c c u r a t e , a s w a s i n d i c a t e d b y t h ee x c e l le n t a g r e e m e n t b e t w e e n t h e d a t a o b t a i n e d w i t h t h et w o a p p r o a c h e s ( D i m o v s k i e t al . 1 99 1 ). A n o t h e r a d v a n -t a g e i s t h e r a p i d s e l e c t i o n o f u n u s u a l h a p l o t y p e s t h a t a r ea s s o c i a t e d w i t h a m i l d e r e x p r e s s i o n o f t h e d i s e a s e ; a ne x a m p l e i s a n e w g e n e t i c r e a r r a n g e m e n t f o r t h e h y b r i dh a p l o t y p e ( 3 / 1 9 ) f o u n d i n a T u r k i s h S S p a t i e n t w i t h m i l dd i s e a s e a n d a h i g h l e v e l o f H b F ( ( 3 n e r e t a l. 1 9 9 1 ) . O nt h e o t h e r h a n d , t h e m e t h o d f a i l e d t o d e t e c t t h e r a r e 13h a p l o t y p e o b s e r v e d a m o n g I n d i a n S S p a t i e n ts t h a t i sc l o se l y r e l a t e d t o t h e c o m m o n h a p l o t y p e 3 1 ( K u l o z i k e ta l . 1986) .Acknowledgement. This study was supported by United StatesPublic Hea lth Service research grant HLB -41544.

Re f e r en c e sAbb es S, Fattoum S, Vidaud M, Goossens M, Ro sa J (1991) Sicklecell anemia in the T unisian population: haplotyping and Hb Fexpression. Hemoglobin 15 : 1-9Berg PE, Mittleman M, Elion J , Labie D, Schechter AN (1991)Increased protein binding to a -530 mutation of the human[3-globin gene associated with decreased [3-globin synthesis.Am J Hem atol 36: 42-47Biss6 E, Wieland H (1988) High-performance liquid chromato-graphic separation of human haemoglobins: s imultaneousquantitation of foetal and glycated haemoglobins. J Chro-matogr 434 : 95-110Bouhassira EE, Lachman H, Krishnamoorthy R, Labie D, NagelRL (1989 ) A gene conversion located 5' to the Ay gene inlinkage disequilibrium with the Bantu haplotype in sickle cell

anemia. J Clin Invest 83 : 2070-2073Dimovski A J, O ner C, A garwal S, Gu Y -C, Kutlar F, LanclosKD, Huisman THJ (1991) Certain mutations observed in the5' sequences of the G?_ and AT-globingenes of ~s chromosomesare specific for chromosomes with major haplotypes. ActaHae ma tol (Basel) 85 : 79-87Fat toum S, Guem ira F, One r C, Oner R, L i H-W , Kutlar F, Huis-man TH J (1991) ~-Thalassemia, H b S-[3-thalassemia and sicklecell anemia among Tunisians. Hemoglobin 15 :11-2 1Gilman JG, Huisman T HJ (1984) Two indep enden t genetic factorsin the [3-globin gene cluster a re associated w ith high G levelsin the Hb F of SS patients . Blood 64 : 452-457Gupta RB, Tiwary RS, Pande PL, Kut lar F, Oner C, Oner R,Huisman TH J (1991) Hemog lobinopathies am ong the Gondtribal groups of Central India; interaction of u- and [3-thalas-semia w ith 13 chain v ariants. Hem oglobin 15 : 441-458Hattori Y, K ut lar F, K ut lar A, McKie VC, Huisman TH J (1986)Haplotypes of ~s chromosomes among patients w ith sickle cellanemia fro m Georgia. Hemoglobin 10 : 623-642Kulozik AE, Wainscoat JS, Serjeant GR, Kar BC, AI-Awamy B,Essan GJF, Falus i AG, Haque SK, Hila l i AM, Kate AS,Ranasinghe W AE P, W eatherall DJ (1986) Geographical sur-vey of Bs globin gene haplotypes: evidence for an inde pend entAsian origin of the sickle cell mutation. Am J Hum Genet39: 239-244Kutlar A, H attori Y, Bakioglu I, Kutlar F, Kamel K, Huisman TH J(1985) Hematological observations on Arabian SS patientswith a homozygosity or heterozygosity for a ~s chromosomewith haplotype 31. Hemoglobin 9 : 545-557Kutlar F, Kutlar A, Huisman THJ (1986) Separation of normaland abnormal hem oglobin chains by reversed phase high per-formance liquid chromatography. J Chromatogr 357:147-153


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Bs Haplotypes in various world populations