Population-based genetic screening

Population-based Antonio

Cao, Maria

genetic

Cristina

Universita

Rosatelli

s;tudi Cagliari,

screening and Renzo

Cagliari,

Galanello

Italy

A preventive genetic programme aimed to control j3-thalassemia in the Sardinian population is based on a combination of increased awareness of the population, carrier screening genetic counselling and prenatal diagnosis. As a result, the registry of thalassemia major demonstrated a profound decline in the incidence of this disease from 1 per 250 to 1 per 1200 live births, with 90% of cases effectively prevented. Current

Opinion

in Genetics

Preventive genetic programmes,basedon carrier screening in combination with genetic counsellingand prenatal diagnosis,have been proposed as a method to control an autosomal recessivediseaseat the popu!ation level [ 1I. Critical prerequisitesfor the application of this approach are as follows: the diseaseshould be common, very severe and potentially fatal; simple methods for carrier identification should be available; and fetal diagnosisshould be possible. A programme based on carrier screening, genetic counsellingand prenatal diagnosismet a high degree of successin the prevention of Tay-Sachs diseasein the American Jewish population [1] In this review, we will describe the design, features and effectivenessof a preventive genetic programme, consisting of increasingthe awarenessand level of education of the population as well as carrying out carrier screening and prenatal diagnosis.The aim of the programme,which was initiated in 1975,has been to prevent thalassemiamajor in the Sardinian population [2,3].

and types of thaiassemias in

j3-thalassemia is a very common genetic disorder in the Sardinian population. One person in eight is a carrier, and in one out of every 60 couples, both parents are carriers. One newborn in every 250 live births is affected by homozygous j3-thalassemia (Table 1) [3]. In this population, a-thakrssemia(deletion of a single a-globin gene) is also rather common (35.9%), and b-thalassemiais not exceptional [4]. Double heterozygosity of different thalassemiagenes is, therefore, not uncommon and, as de-

C&cystic

1991, 1:48-53

scribed later on, may confuse the diagnosisof the pthalassemiacarrier state (Table 2) 131.

Introduction

Incidence Sardinia

and Development

CFTR-cystic fibrosis Hb-haemoglobin; M-intervening, fibrosis;

transmembrane sequence; @

Current

Table

Population

(x

Incidence

of P-thalassemia

in the

1.7

106)

of child-bearing

Heterozygote Couples

characteristics

population.

Couples

age

at risk expected of homozygous

1.6 X 105-1.8

X lo5

0.13

frequency

2500-2700 state

I in 250

Increased awareness of the population increased awareness(sensitization) of the population was achieved mostly via the mass media: namely, local newspapers, radio, TV and magazines.A more personal approach, consisting of lectures given to the general public in factories and large stores or shops, was also frequently used. Family doctors, obstetricians, pediatricians, midwives and nurseswere all trained in this new field of preventive genetics. Postersand informative booklets were available at critical areassuch as places of marriage registry, general practitioners surgeries, obstetricians’ and pediatricians’ offices, as well as family-planning clinics. Critical evaluation of our sensitization programme showed that the most widely used channels of information were the massmedia (informing 44% of the population), followed by general practitioners (31%), obstetricians (23%) and midwives (2%). At the beginning of our programme in the mid-seventies,the largest class of people requesting screening were pregnant women,

Abbreviations conductance MCH-mean Biology

1. Demographic

Sardinian

regulator; DCCE-direct gradient cellular haemoglobin; MCV-mean

Ltd ISSN

0959-437X

gel electrophoresis; cell volume.

Population-based

whereas in the past few years, the number of couples without a pregnancy in progress presenting at our genetic service has been increasing continuously. This indicates a marked improvement in information on thalassemia and its prevention at the population level. 2. Carrier

frequency

of thalassemias

in the Sardinian

population.

3. Overall

35.9

Subjects

g-Thalassemia

13.0

Percentage

&Thalassemia

0.7

Carriers

GP-Thalassemia

0.2

Prospective

6-+

0.2

‘Single

a-globin

gene

deletion,

tdouble

heterozygote

Cao, Rosatelli, Calanello

results

of P-thalassemia

carrier

screening

in

Sardinia.

a-Thalassemia’

P-Thalassemiat

screening

most part we have used extended family testing (inductive screening) whenever a P-thalassemia carrier has been identied. Carrier screening is carried out in eight genetic units located in different areas of the island. - Table

Table

genetic

171779

tested of population

10

tested 31455

detected at -risk

couples

detected

1912

for g- and

j3-thalassemia.

Technical Genetic counselling

Genetic counselling was carried out in a non-directive manner according to well established principles: namely, accurate diagnosis, truth and conIidentialiry. It was directed to both members of the couple at risk. In the counselling sessions, the reproductive choices were discussed in detail, i.e. birth control, adoption, artificial insemination, mate selection and fetal testing. Full information was given regarding the risk of fetal mortality, the possible failure to obtain sufficient fetal material for diagnosis, the risk of misdiagnosis, and the mortality or morbidity rate for any anticipated therapeutic abortion. In the late seventies and early eighties, fetal diagnosis was based on the analysis of fetal blood obtained by placental aspiration or fetoscopy. This procedure may be carried out only during advanced gestation (18 weeks >, and is associated with a high risk of fetal mortality (6%). Mostly because of fear of complications, a consistent proportion of the couples counselled (7.4%) decided against prenatal diagnosis, and instead continued the pregnancy. In the late eighties, when first trimester prenatal diagnosis by chorionic vilk sampling was introduced, almost ail couples counselled were in favour of prenatal testing.

Carrier

screening

In order to reduce the cost of screening, one member of each couple only is tested initially, the other being examined if their spouse is found to be a carrier. To date, we have screened only 10% of the Sardinian population of child-bearing age. Nevertheless, we have been able to detect 1912 of the 2500-2700 couples at risk expected on the basis of the carrier rate. By adding the number of at-risk couples already known to this figure (i.e. with affected children), 90% of the couples at risk have been identified so far (Table 3). The high efficiency of the screening programme is related to the fact that for the

aspects of screening

Because of the high frequency of a-thakissemia, 35.9% of the @halassemia heterozygotes in the Sardinian population are also heterozygous for a-thalassemia.A proportion of these double heterozygotes may have red blood cells with normal mean cell volume (MCV) and mean hemoglobin concentration per cell, denoted as mean cellular haemoglobin (MCH), and may thus be missed in carrier screening based on MCV-MCH determination [ 51. In order not to miss these double heterozygotes, we included haemoglobin (Hb)A2 quantitation as an initial screening measure, in addition to MCV-MI-K evaluation. The second technical problem we encountered in j3-thalassemia carrier screening was the identification of heterozygous j3-thalassemia in individuals doubly heterozygous for 6- and j3-thalassemia, who may have normal HbAZ levels and hence be confused with a-thalassemia carriers [6,7]. The presence of such double heterozygosity may be suspected on the basis of family studies, which may demonstrate segregation of j3- and b-thalassemia, the latter being characterized by very low HbAz levels. Globin chain synthesis analysis may be used to identify the presence of g-thalassemia definitively. In order to improve the ability to define &thalassemia, we have recently cloned the &th+ssemia gene from a number of S- and p-thalassemia double heterozygotes. It was found that in the great majority of cases, a single nucleotide substitution (G to T) in codon 27 of the 8-globin gene had occurred. More rarely, a 7.6 kb deletion of the \Ir@globin region was detected (Fig. 1) [8,9**]. Following these studies, we now define the presence of 6-thalassemia by dot-blot analysis, using okgonucleotides complementary to these two &thalassemIa mutations to probe an amplified 6globin gene region. Other mutations that are associated with reduced MCH-MCV values but normal HbA2 levels, and thus should be differentiated from a-thalassemia, are r&3thahssemias, deletion Gj3-thalassemia and ‘sardInian’ Spthalassemia. The rare y6j3-thalassemia, which is commonly caused by a large deletion of the j3-globin gene

49

50

Genetics of disease

1 7.2 kb deletion (2%) 1 Fig. 1. &Thalassemia Sardinian population.

cluster, and deletion G~thalassemia, may be defined by globin chain and Southern blot analyses. Sardinian Spthalassemia is a’relatively common complex thalassemia determinant in the Sardinian population, characterized phenotypically by microcytosis, normal to low HbA2 levels and increased HbF. This determinant results from the presence of two mutations in the same chromosome, one of which is a P-globin cdon 39 nonsense mutation (see below), and the other a C to T substitution at position - 196 relative to the cap site of the A7 gene. The continuous production of y-chains in adult life from the mutated A gene [lO,ll] partially compensates the absence of P-chain production, thus explaining the mild clinical picture associated with Sardinian G@halassemia. The 6globin gene in the Sardinian GP-thalassemia determinant has a normal sequence and is probably downregulated because of the high expression of the cis Ar gene. Sardinian G@halassemia may be identiEed by carrying out quantitation of HbF, which consititutes around 13% of the total Hb, and globin chain synthesis analysis [12].

Molecular characterization the Sardinian population

of P-thalassemia in

Molecular characterization of /3-thalassemia in the Sardinian population began in the late seventies, when Kan and his co-workers [13] cloned and sequenced the /3globin gene from a patientaffected by thalassemia major. A C+T transversion was identified at codon 39, which substitutes the glutamate codon (CAG) for an ambertermination codon (TAG), known as the codon 39 nonsense mutation. More recently, dot-blot analysis of amplified DNA using oligonucleotides complementary to a set of the most frequent J3thalassemia mutations in the Mediterranean populations has shown that the codon 39 nonsense mutation is the most frequent b-thalassemia mutation in the Sardinian population, accounting for 95% of cases 1141 (Table 4). The second most common de feet is a frameshift at codon 6, which removes 1 bp and has been detected in 2% of P-thalassemia genes.

Table

4. o-Thalassemia

mutations

Type of mutations Codon

39 (C-T)

mutations in the kens are shaded.

in Sardinians.

Number

Percentage

2072

95.7

Frameshift

6 (- 1 bp)

66

2.2

Frameshift

76 (- 1 bp)

20

0.7

IVSI ntll0

(C&A)

14

0.5

(C-G

12

0.4

7

0.2

IVSI nt.6 Tr+C)

3

0.1

Frameshift

3

0.1

IVSII nt745 - 87 K-G)

IVY

1 (- 1 bp)

ntl

(C-+A)

1

0.03

IVSII ntl

(C-A)

1

0.03

Unknown A total

of 3000

chromosomes

were

investigated.

IVS, intervening

sequence.

Prenatal diagnosis

The procedure used for prenatal diagnosis has evolved over time. In the late seventies and early eighties, prenatal testing was carried out by globin chain synthesis analysis, using fetal blood obtained by placental aspiration, fetoscopy or cordocentesis. As soon as the molecular bases of P-thalassemia in the Sardinian population had been defined [ 141, the P-thalassemia mutations were directly detected by hybridization of oligonucleotides to electrophoretically separated DNA fragments. [ 151. In the past few years, we have used dot-blot analysis to probe amplified DNA with 32P. or horseradish peroxidaseIabelled allele-specific oligonucleotides. Globin chain synthesis analysis has been shown to be a very reliable procedure, with only two r&diagnoses (0.2%) out of 1131 cases monitored (Table 5). The procedure is associated with a very high risk of fetal mortality, however (6%). As previously mentioned, it was not

Population-based

genetic

screening

Cao, Rosatelli, Galanello

easily accepted by the prospective parents, either because of the risk or the late stage of gestation at which it has to be performed. Table 5. Overall results of prenatal Sardinia (3051 cases).

:b) Pregnancies monitored Homozygous fetuses Failures Misdiagnosis Fetal losses

k)

Normal

,:’

3 z

4

C C A C T C codon cI G T C C c G T A c T 5'

Mutation

a T C C A C 76 T [ c c T C C G G T A G T 5'

=Lm

Fig. 2. Characterization of a P-thalassemia mutation (frameshift at codon 76, p76-1 bp) by denaturing radient gel electrophoresis (DGGE) and direct sequencing of the t globin gene. (a) Schematic diagram of the P-globin gene, denoting the localization of the p” 76 f-1 bp) mutation. The space bar indicates the region amplified using primers. (b) DGGE: lane 1, normal DNA showing a single band; lanes 2 and 3, heterozygotes for the p” 76 f-1 bp) mutation showing the two homoduplexes and two heteroduplexes. (cl Direct sequencing of amplified DNA showing the loss of a nucleotide K), resulting in a frameshift mutation.

diagnosis

of g-thalassemia

in

Chronic villi samples

AFC

Fetal blood

1717

203

1131

447

56

206

1 (0.06%)

6 (3%)

10 (0.9%)

-

2 (0.2%)

33 (1.9%)

4 (2%)

60 (6%)

Oligonucleotide hybridization on electrophoreticallyseparated DNA fragments has been widely used and has also proved to be a reliable procedure; no rnisdiagnoses have yet occurred. The main disadvantages are the long period required to obtain the results (approximately 2 weeks), and the need to use radioactive material. These drawbacks have been overcome by the use of enzymatically amplified DNA, which enable results to be obtamed within two days even though using non-radioactive probes [16,17-l. At present, the mutation present for each couple at risk is detined by dot-blot analysis, using three oligonucleotide probes complementary to the most common g-thalassemia mutations in Sardinians, i.e., the codon 39 nonsense mutation, the frameshift at codon 6 and a frameshift at codon 76 [18*=]. Unknown mutations are defined by denaturing gradient gel electrophoresis (DGGE) [ W.1 and direct sequencing of amplified DNA [20], as shown in Fig. 2 for a g-thalassemia mutation very rare in Mediterranean populations, the frameshift at codon 76. This mutation has, however, been detected very recently by our group in chromosomes from 20 Sardinian g-thalassemia heterozygotes. The chorionic villi samples required were obtained by the transabdominal route. In our hands, this procedure has a very high success rate (close to lOO%), and is associated with a very low risk of fetal mortality (2%) [21=]. Prenatal diagnosis by- analysis of amplified DNA has turned out to be a very reliable procedure. Co-amplilication of maternal sequences, which may cause confusion, has been detected by us in only four cases. This potential pitfall may be avoided by the careful dissection of maternal decidua from the fetal trophoblast, the use of not less than 3 ug of trophoblast DNA, and the reduction of the number of amplification cycles to 20. III order to monitor for the presence of maternal DNA it may be useful to co-amplify a polymorphic DNA’sequence [22**]. overall, in the 3051 prenatal tests we have carried out so far, 789 fetuses homozygous for g-thalassemia have been detected, which would have come to term in the absence of prevention. Following prenatal diagnosis of an

51

52

Genetics

of disease

affected fetus, the large majority (99.9%) of the couples decided to interrupt the pregnancy. Those few (0.1%) that opted for continuation of the pregnancy took this decision for ethical reasons, because of strict adherence to the Catholic religion.

Effectiveness

of the programme

In order to monitor the efficacy of our programme, we have organized a registry of new cases of thalassemia major in the Saidinian population. As shown in Fig. 3, before prevention, one newborn in every 250 ~LZ a&-c& by thalassemia major. In absolute terms, it meant that in our population, 100 new cases of homozygous P-thalassemia arose each year. Nowadays, only ten cases arise per year (1 per 1200 live births), with 90% of cases effectively prevented (Fig. 3).

Occurence of homozygotes oer 1000 births

Years 3. Fall thalassemia

in the birth in Sardinia.

rate

of

babies

with

homozygous

The control of P-thalassemia by carrier screening and prenatal diagnosis may represent a model for the organization and delivery of preventive programs for other common autosomal recessive disorders, primarily cystic fibrosis. Following the cloning and sequencing of the cystic fibrosis transmembrane conductance regulator (CFTR), it has been shown that 50-80% of the cases of cystic fibrosis (CF), depending on the population examined, results from a single mutation, i.e. a 3 bp deletion designed APhe508, which removes Phe508 from the CFTR molecule [24]. The remaining cases have turned out to be produced by a large number of very rare mutations, however. Because of this marked heterogeneity, it would be difficult to be able to screen the population at large for CF using a simple procedure. Carrier screening for the common CF mutation may be able to identify only a proportion of the couples at risk and, in our opinion, may create mistrust of the preventive program. We believe, therefore, that CF carrier screening should be initiated only when approximately 90% of the couples at risk can be identified by a simple procedure. In the meantime, pilot programmes designed to identify the best approach could be organized. On the basis of our experience, we strongly believe that such a preventive program should be preceded by an extensive educational targeted at the population at large, in order to inform them of the state of the art in this field of preventive genetics. A fmal critical prerequisite for the success of such a genetic preventive program is the education of doctors, especially obstetricians, about the technical, psychological and social aspects of this kind of preventive medicine.

00 1975 76 77 78 79 80 81 82 83 84 85 86 87 88 89 Fig.

Turkish Cypriots, Greeks and continental Italians [23]. The major challenge in the future will be the organization of such programmes in the Middle East, Indian subcontinent and Far East, where the resources, level of education and state of development seem to preclude any effort in this direction at preserit.

p-

Acknowledgements The main reason for residual cases of thalassemia major in the Sardinian population is a complete lack of parental knowledge about thalassemia and the procedure for its prevention, followed by the refusal of prenatal diagnosis, the refusal of abortion when an tiected fetus is identified, and false paternity.

We thank Rita Loi for editorial by a grant from the Assessorato Legge regionale n.11 30 Aprile

References Conclusions

and implications

The results described herein indicate that programmes based on carrier screening, genetic counselling and prenatal diagnosis are very effective for preventing an autosomal recessive disease, such as P-thalassemia, al the population level. Indeed, very similar results have been obtained in other Catholic as well as orthodox Christian and Muslim populations, composed of Greek Cypriots,

assistance. lgiene 1990.

and recommended

Papers of special interest, published have been highlighted as: . of interest .. of outstanding interest

within

This work was e Sanit2 Regione

supported Sardegna

reading the annual

period

of review,

1.

KALMK MM, GEIGER R5. REYNOUX LW: Approaches to the Control and Prevention of Tay-Sachs Disease. In Progress in Medica/ Genetics edited by Steinhberg AG, Beam AG [book]. New York: Grune and Stratton, 1974. pp 103134.

2.

CA0 4 PNIW L, LECCA U, OLLA G, Cossu P, ROSATEU C, GALANEUO R: Control of Homozygous P-thalassemia by Car-

Population-based rier and 26:12-22.

Antenatal

Diagnosis

in Sardinians.

Clin

Gensl

16.

Cno A. ROSATEUJ C, GAIANEW P, RKIXLDI M: The Prevention Clin Gene! 1989, 36~277-285.

4.

PIIWXU M, Ltx KY, DOZY AM, KAN YW, S‘IXMA~OYANNOPOULOS G, HADJIMINAS MG, ZACHARLADE~ Z, ANGIUS A, FUHBEITA M, ROSATW C, CAO A: Alpha-Thalassemia in Two Mediterranean Populations. Blood 1982, 60:509-512.

R, MONNI G, OLLA G, COW of Thalassemia in Sardinia.

5.

MEUS MA, PIRWX M, GAIANEUD R, FURREITA M, TWERI T, CAO A: Phenotypic Effect of Heterozygous a- and P-Thalassemia Interaction. Blood 1983, 62~226-229.

6.

GALWELLO FURBEITA Thalassemia: Mutations.

R, PAGIKITI E, ADDIS M, MEI~S MA, TWERI M, CAO A: Pitfalls in Genetic Counseiling for An Individual with 4 Daerent Thalassemia Clin Gene! 1988, 33:15l-155.

PIRASTU M. GAIANELLO CAO A, KAN YW: 6+ 62:341-345.

R, MEUS MA, Thalasscmia

B~ANCATI in Sardinia

MOI P, PAGUETII E, SANNA 4 BaAxxn GAIANEIJD R. CAO A, PWTU M: Delineation Basis of 6 and Normal Hb A2P-Thalassemia. 72:530-533.

T, p-

PIRASTU M, KAN VW, GAIANEIKJ tions Produce &%Thaktssemia 223:929-930.

C, TAGARWI A, Blood 1983,

C, TAGAREW A, of the Molecule Blood 1988,

11.

OITOI.IING~II C, SERRA 4 lassemia, a Position Bkizi 1987,

12.

CAO A, MEUS MA, GALANELLO GIORDANO P, BERNINI LF: 6 beta / Med Gener 1981, 19:184-192.

13.

TIwXnN FURBETA is Caused 68:1012-1017.

R, CAO A: Multiple in Sardinia. Science

Muta1984,

S, GIGUONI B, PLIWZINI A, CO~II P, C~IASCHEILI GUERROSIO A, SAGUO G: Sardinian GPO-ThaFurther Example of a C to T Substitution at 196 of the A Gamma Globin Gene Promoter. 69:1058-1061. R, ANCIUS A, (F)-Thalassemia

FU~ETTA M, in Sardinia.

RF, LIEFIHABER SA, CHANG JC, LEE KY, Kw YW, M, ANGIUS 4 CAO A: PO Thaktssemia in Sardinia by a Nonsense Mutation. / C/in Intesf 1981,

14.

ROSATEUJ C, LEONOI CAO A: P-Thalassemia for Prenatal Diagnosis.

15.

ROSATEUJ TLICCI A,

GB, TWERI T, SCAL.IS MT, DI TUCCI Mutations in Sardinians: implication / Med Gene! 1987, 24:97-100.

C, FALCHI AM, Tmwu MONNI G, CAO AZ Prenatal

T.

SCA~AS Diagnosis

SAIKI KR, BUGAWAN lysis of Enzymatically with Allele-Specitic 324:163166.

screening the

53

Cao, Rosatelli, Calanello

Synthetic-Otigomer

Technique.

Lance1

TL, HORN GT, MULLIS KB, EIUICH HA: AnaAmpIitied P-globin and HLA-DQa DNA Oligonucleotide Probes. Nafure 1986,

KAXWN HH: The Thalassemia sis and Prenatal Diagnosis in 27:209-228. Review of the spectrum of P-thalassemia tions and methods for their detection. 17. ..

Syndromes: 1990. Semin mutations

Molecular Hem&of

in different

Ba1990, popula-

18. ..

CAO A, ROSATELL~ C, LEONI GB, TUVERI T, SCALQ MT, MONNI OLIA G, G-u.0 R: Antenatal Diagnosis of /%Thakxssemia in Sardinia. Ann NYAcucl Sci 1990, 612:215-225. Review of the methodologies of prenatal diagnosis by DNA analysis the Sardinian population.

G,

in

CAI SP, KAN YW: Identification of the Multiple P-Thalassemia Mutations by Denaturing Gradient Gel Electrophoresis. / Clin huesi 1990, 85:55&553. Study describing the application of DGGE to detect the P-thalassemia mutations in the Chinese population. By this method, which has the distinct advantage of not using molecular probes, all the 12 known pthalassemia mutations in the Chinese population are distinguished on the basis of homoduplex and heteroduplex mobility. 19. ..

GALQIELLO R, MEUS MA, PODDA A, MONNE M, PERSEU l, t.OUDIANOS G, CAO A, PIRKIX M, PICA A: Deletion &Thalassemia: The 7.2 kb Deletion of Corfu So-Thalassemia in a Non-P-Thalassemia Chromosome. Blood 1990, 75:1747-1748. .. . ^ Descntxs a &rnily with b-thalassemia caused by the same deletion found in the Corfu GP-thalasremia. 10.

Thalassemia with 1985. i:241-243.

1984,

3.

genetic

A,

MT, DI of Beta-

20.

WONG K6w.w Using DNA.

C, DOWLING CE, SNIU RK, HIGUCHI RG, ERUCH m JR HH: Characterization of p-Thaksemia Mutations Direct Genomic Sequencing of Amptied Single Copy Nature 1987, 330:3&i-386.

21. .

MONNI G, OUA G, RosATE~L~ C, CAO h Second Trimester Placental Biopsy Versus Amniocentesis for Prenatal Diagnosis of P-Thalassemia N EnglJ &fed 1990, 322:&l. According to the results of this paper, second trimester placental biopsy appears to be a safe and ekent procedure for the prenatal diagnosis of P-thalassemia. 22. ..

ROSATEUJ C, SAIW R, TUWRI T, SW MT, DI TUCCI 4 DE MURTAS IM, LOUD~ANOS G, MONNI G, CAO A: Reliability of Renatal Diagnosis of Genetic Diseases by Analysis of Amplified Trophoblast DNA / IWed Gene/ 1990. 27:249-251. Description of a large set of experiences of prenatal diagnosis of pthalassemia by dot-blot analysis on amplikd trophoblast DNA with allelic-specific oligonucleotide probes. The result indicates that when careful dissection of trophoblast tissues from maternal decidua is car. ried out, amplification of chorionic villi DNA is not associated with amplification of maternal DNA sequences, indicating a high reliability of prenatal diagnosis based on DNA amplification. 23.

WORLD HEALTH Hemoglobinopathies

24.

WR!&I BS. ROM~II~NS JM, BKHANAN JA, MAWEWICZ D. Cox TK, CHAKIwWrn A, BUC~WUD M. TSUI L-C: ldentitication of the Cystic Fibrosis Gene: Genetic Analysis. Science 1989, 24.5: 10731080. -

A Cao, MC Rosatelli and Eti Evolutiva, Universiti

ORGANIZXION: Control.

Update of the Bull World Heal&

R Galanello, Institute Cagliari, Via Jenner

di Clinica s/n I-09100

Progress of Organ 1985.

e Biologia Cagliari,

dell’ Italy.

’ .