ORIGINAL ARTICLE

Hb G-SAN JOSE VARIANT LEVELS CORRELATEWITH a-THALASSEMIA GENOTYPES

Giuseppina Lacerra,1 Mirella Fiorito,1 Leonilde Pagano,2

Rosario Testa,3 Giovanni Li Volti,3 Carmelo Magnano,4

Emilia Medulla,4 and Clementina Carestial,*

1Istituto di Genetica e Biofisica ‘‘Adriano Buzzati Traverso,’’

Consiglio Nazionale delle Ricerche, via Guglielmo Marconi 12,

I-80125 Napoli, Italia2Ospedale ‘‘Antonio Cardarelli,’’ Sezione di Microcitemia

‘‘A. Mastrobuoni,’’ I-80131 Napoli, Italia3Dipartimento di Pediatria, Universita degli Studi,

I-95123 Catania, Italia4Servizio di Thalassemia, Presidio Ospedaliero Garibaldi,

I-95124 Catania, Italia

ABSTRACT

Hb G-San Jose or b7(A4)Glu!Gly has been reported in Southern Italian or

Mexican families. We have studied four families from Sicily and Campania,

Southern Italy. In six carriers, the hemoglobin variant level ranged from 32 to

38%. In four double heterozygotes for Hb G-San Jose and a-thalassemia the

variant level showed a strong correlation with the a-thalassemia genotype. In

fact, the variant level was 15% when interacting with the � (a)20.5=aa, 19.6%

with the aa=aPoly Aa, and 24.8% with aa=a�5 nta genotypes. In two double

heterozygotes for Hb G-San Jose and bþ -IVS-I-6 (T!C) the hemoglobin

variant level was 67%. These data show that the reduced synthesis of a chains

causes drastic reduction of probability to form Hb G-San Jose in favor of the

formation of Hb A. Moreover, this reduction, (i) correlates with the type of

a-thalassemia genotype and with the degree of the a chain deficiency, and

(ii) is, most probably, more marked than the degree of a chain reduction. The

minor affinity of the b chain variant for the a chains associated with the

HEMOGLOBIN, 26(1), 59–66 (2002)

59

Copyright # 2002 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author. Fax: þ39-81-7257243; E-mail: carestia@iigb.na.cnr.it

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reduced synthesis of the a chains is probably the principal cause of the variant

hemoglobin reduction. Moreover, the rapid removal of the abnormal chains by

proteolytic enzymes must have an essential role in order to reduce the chain

variant pool. These conclusions are in agreement with the results obtained in

reticulocyte and in vitro recombination experiments.

INTRODUCTION

Hb G-San Jose [b7(A4)Glu!Gly] was first characterized in Californian

family of Italian origin in 1957 (1,2). The variant is rare and shows an exclusive

epidemiological distribution; in fact, it has only been reported in a few families of

Southern Italian (3–7) or of Mexican heritage (8,9). Carriers have no hematolo-

gical disorders (3,4,6,7). Studies of functional properties confirmed that the Bohr

effect and the oxygen affinity of the hemoglobin (Hb) variant are identical to

normal Hb A (10). The variant is slightly less stable than Hb A in isopropanol (4,5)

and heat stability (5,10) tests.

The percentage of Hb variant ranged from 32 to 37% in the carriers (3,4,6,7).

Interaction with Hb Fukuoka [b2(NA2)His!Tyr] did not change the Hb G-San

Jose concentration; in fact, the level of the two variants were 58 and 33%,

respectively (9). By contrast, interaction with b- or a-thalassemia (thal), reported in

a Sicilian family (5), determined pronounced variations. In a healthy com-

pound heterozygote with b0-thal, the variant level was 91% and the Hb level

was 13.0 g=dL; in two double heterozygotes with a-thal the variant range was

13–14% (5).

This article provides new data giving more insights into the role of a-thal

mutants in determining the level of Hb G-San Jose in peripheral blood.

MATERIALS AND METHODS

Hematological parameters were obtained by standard methods. Qualitative

and quantitative analysis of Hb was performed by cation exchange high perfor-

mance liquid chromatography (HPLC) (DIAMATTM or VARIANTTM Systems,

Bio-Rad Laboratories, Hercules, CA, USA).

DNA from white blood cells was purified with a standard method. Point

mutations of the b- and a-globin genes were detected by DNA polymerase chain

reaction (PCR)-direct sequencing performed as previously reported (11). a-Thal

deletions were tested with gap-PCR (12). Point mutations of the a-globin genes

were identified by means of denaturing gradient gel electrophoresis (DGGE) (13)

and confirmed with DNA sequencing [AATAAA!AATAAG of the a2 gene

(polyadenylation signal or poly A)] or HphI digestion [5 nucleotide (5 nt) deletion

at the IVS-I donor site (GAGGTGAGG!GAGG- - - - -) in the a2 gene]. b-Thal

mutations were identified by PCR-amplification refractory mutation system

(ARMS) (14). Oligonucleotides used as primers are reported in Table 1.

60 LACERRA ET AL.

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RESULTS

We studied four unrelated families (Table 2) originating from Campania

(Families 1 and 4) and from East Sicily (Families 2 and 3), two regions of Southern

Italy. The four probands and some members of their families were carriers of a Hb

variant with the chromatographic features of Hb G-San Jose, values ranging from

15 to 67% of the total Hb (Table 2). Moreover, they showed microcytosis and

hypochromia associated with increased Hb A2 levels ranging from 3.5 to 5.0%

(Table 2).

DNA sequencing of the first exon of the b-globin gene of all probands led to

the detection of the GAG!GGG change in codon 7 (Fig. 1), corresponding to the

amino acid substitution Glu!Gly present in the Hb G-San Jose mutation (15).

The gap-PCR analysis of the a-globin genes revealed that the proband

of Family 1 and one of the two sisters were carriers of the a0-thal deletion

�(a)20.5 (16).

DGGE indicated that the probands of Families 2 and 3 were carriers of the

poly A (AATAAA!AATAAG) (17,18) and the �5 nt (19,20) mutations,

respectively. The first mutation was confirmed by DNA sequencing; the second

by HphI digestion of the relative amplified PCR fragment.

Molecular screening forb-thal mutations revealed that the proband of Family 4,

the mother and two sisters, were carriers of the bþ-IVS-I-6 (T!C) mutation.

DISCUSSION

We studied four unrelated families and encountered six carriers of Hb G-San

Jose, four double heterozygotes for the Hb variant and a-thal, two compound

heterozygotes for the variant and bþ-thal. In the six carriers the Hb variant level

ranged from 32 to 38% (average 35%); these values are in agreement with those

Table 1. Oligonucleotide-Primers Used for Sequencing (1,2), for gap-PCR (3), and for PCR

Amplification and HphI Restriction Digestion (4)

Mutations Primer Sequences (50 ! 30) References

Hb G-San Jose Forward: CCAATCTACTCCCAGGAGCA This study

Reverse: CACTCAGTGTGGCAAAGGTG

aPoly Aa Forward: TGACCCTCTTCTCTGCACAGCTC This study

Reverse: GTCTGAGACAGGTAAACACCTCCAT

� (a)20.5 Forward: GGCAAGCTGGTGGTGTTACACA 12

Reverse: GAGGCCCAAGGGGCAAGAAGCAT

a�5 nta Forward: GCGGCCCGGCACTCTTCTG This study

Reverse A: GTCTGAGACAGGTAAACACCTCCAT

Reverse B: AAGCAGAGTGAGGGGTGGGG

Hb G-SAN JOSE AND a-THALASSEMIA 61

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62 LACERRA ET AL.

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reported in the literature (3,4,6,7). In the four double heterozygotes for a-thal and

Hb G-San Jose the variant level ranged from 15 to 24.8%.

In Family 1, the proband and her son showed 15% of the Hb variant, that is

about 45% of the average value found in the carriers. Both patients were double

heterozygotes for the Hb variant and for the �(a)20.5 deletion that causes a defect

similar to a null mutation of a single locus gene. It produces an a=b chain

biosynthetic ratio of about 0.5 (21), that means about a 50% deficit of the a chain

synthesis.

In Family 2, the proband had 19.6% of the Hb variant, that is slightly higher

than 50% of the average value in the carriers. He was a double heterozygote for the

Hb variant and for the poly A (a2) (AATAAA!AATAAG) mutation that causes

synthesis of extended transcripts with a new signal 30 to the normal poly A site,

and production of nonfunctional, unstable mRNA (17,18). Even if this mutation

alters only one of the two a-globin genes of a single cluster, it produces a defect of

intermediate severity between aþ- and a0-thal, leading to mild phenotype altera-

tions in carriers, and to an anemic syndrome with hematological and clinical

features similar to Hb H (b4) disease in the homozygote (18,21).

In Family 3, the proband had 24.8% of the Hb variant, that is about 70% of

the average value in the carriers. He was a double heterozygote for the 5 nt deletion

at the IVS-I donor site of the a2 gene that causes abnormal RNA splicing, and

likely produces a most mild defect of synthesis of a chains (21), certainly more

minor than that due to the poly A (a2) (AATAAA!AATAAG) mutation found in

Family 2. This was confirmed by the fact that this mutation can cause Hb H disease

only in trans to an a0-thal mutation.

Figure 1. Sequence (sense) of exon 1 of the b-globin gene. The mutation at codon 7

(GAG!GGG) is indicated with the corresponding amino acid substitution (Glu!Gly). The

asterisk indicates the sequence of the codon 2 polymorphism.

Hb G-SAN JOSE AND a-THALASSEMIA 63

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In Family 4, there were two patients who were double heterozygotes for the

bþ-IVS-I-6 (T!C) and Hb G-San Jose mutations. In these cases the Hb variant

increased to 67%. This is due to the decreased synthesis of the b chains that leads

to a relative excess of the a chains.

In the Hb G-San Jose carriers the average value of the Hb variant is 35%. The

minor affinity of the b chain variant for the a chains most likely plays an important

role in controlling the Hb variant level. The slight instability (4,5) may have only a

marginal role, considering that a homozygous patient did not show hemolytic

anemia syndrome and had 14.5 g=dL of total Hb (4). The results reported here

clearly indicate that, in the double heterozygote patients of Families 1, 2, and 3,

the reduced synthesis of a chains causes drastic reduction of probability to form

Hb G-San Jose. Moreover, these data document that the degree of reduction of

probability, (i) correlates with the degree of the a chains deficiency, and (ii) is,

most probably, more marked than the degree of the a chain reduction. The

reduction of the Hb variant is firstly due to the minor affinity of the b chain

variant for the a chains. Moreover, the rapid removal of the abnormal chains by

proteolytic enzymes must have an essential role in order to reduce the chain variant

pool. These conclusions confirm in vivo the results obtained in reticulocytes and in

vitro recombination experiments that indicated that the Hb G-San Jose chains

exhibit a minor affinity (relative to bA chains) for complementary a chains, and

that preferential removal of abnormal chains might play an important role (22,23).

ACKNOWLEDGMENTS

We acknowledge Mr. Romeo Prezioso, from Istituto di Genetica e Biofisica

‘‘Adriano Buzzati Traverso,’’ CNR, Napoli, Italia, for his skillful aid in collecting

and transferring experimental results and family pedigree data in an electronic

database, and in the elaboration of a specific software. This study was supported

by funds given by TELETHON Project E.00l, Roma, Italia.

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Hb G-SAN JOSE AND a-THALASSEMIA 65

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Received September 9, 2001

Accepted September 26, 2001

66 LACERRA ET AL.

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