ANALYSIS OF EXTRA- AND INTRAGENIC MARKER HAPLOTYPES AS PART OF MOLECULAR DIAGNOSIS OF CYSTIC FIBROSIS IN PATIENTS FROM SERBIA

Cystic fibrosis (CF) is the most common fatal autosomal recessive disease in Caucasians. since characterization of F508del, the predominant mutation in different countries, more than 1500 mutations have been discovered in the CFTR gene, including a large number of polymorphisms. After molecular screening of 222 CF patients from serbia, we detected 21 different CFTR mutations, F508del being the most frequent (69.59% of CF alleles). A total of 21 mutations cover almost 80% of CF alleles in this group. since the molecular basis of CF is highly heterogeneous in our population, studying thye haplotype association with normal and CF chromosomes could be very helpful in all cases where one or both mutations remain unidentified. haplotype analysis was done using six diallelic sites and one tetranucleotide repeat (Xv2C-KM19-MP6D9-j44-Ivs6a(GATT)-M470v-T854T) on 99 F508del, 90 non-F508del, and 105 normal chromosomes. strong linkage disequilibrium was observed for CFTR mutations and one haplotype (1-2-2-1-6(2)-1-1), while normal chromosomes were mostly associated with another (1-1-2-1-6(2)-1-2). The obtained results show that in most cases it would be possible with this group of polymorphisms to separate normal chromosomes from chromosomes which carry the CFTR mutation. As far as prenatal diagnosis of cystic fibrosis is concerned, haplotype analysis can be used as a helpful method of indirect diagnosis in families at risk with one or both CF alleles uncharacterized.

The Ivs6aGATT polymorphism was detected by difference in mobility of the PCR product on 8% PAGe gel.Allele 1 showed seven repeats, allele 2 six, and allele 3 eight repeats (D o r k et al., 1992).
Other haplotypes were determined after restric-

ResUlTs
After screening of 222 CF patients from serbia, we detected 21 different CFTR mutations, F508del being the most frequent (69.59% of CF alleles).A total of 21 mutations cover 80% of CF alleles in this group.Mutation analysis showed the presence of 27 different genotypes; 22 patients (9.90%) had both CF alleles remaining uncharacterized.
Further haplotype analysis was done for 147 members of 44 families at risk.Four diallelic extragenic RFlPs were analyzed: j44 (located less then 10 kb away from the 5' end of the CFTR gene); and Xv-2c, KM.19, and MP6-D9 (located 175 kb, 125 kb, and 40 kb, respectively, ahead of exon 1 of the gene (Fig. 1).DNA samples were also analyzed for the presence of two intragenic diallelic single nucleotide polymorphisms (sNPs): M470v (located in exon 10) and T854T (located in exon 14a).Finally, the last polymorphism analyzed was an intragenic tetranucleotide repeat, microsatellite (sTR) Ivs6aGATT, located at the junction of intron Ivs6a and exon 6b (Fig. 2).The allele frequencies are shown in Table 1.All polymorphisms exhibit strong linkage disequilibrium with the F508del mutation, which was the most frequent one in the analyzed group of patients (Fig. 3).
haplotypes were assigned where the phase could be established by typing both parents and affected child in each CF pedigree.The results showed the presence of 14 different haplotypes, six of them being the most common (Table 2).One (1-2-2-1-6(2)-1-1) was found mostly associated with the F508del mutation (91%) because of linkage disequilibrium.The most frequent (36%) haplotype in N chromosomes was (1-1-2-1-6(2)-1-2), while all six of the most common haplotypes were associated with non-F508del CF chromosomes (with frequencies in the range of 11-25%).

DIsCUssION
Polymorphism analysis is often used as a valid procedure to assess the genetic heterogeneity of a population or to evaluate the origin of a mutation  , 2003).since the main goal of diagnostic analysis is to allow families at risk to diagnosis, studying of haplotype association with normal and CF chromosomes could be very helpful in all cases where one or both CF alleles remain uncharacterized.
Analysis of six diallelic sites and one sTR showed that all of them were in linkage disequilibrium with the most frequent CFTR mutation worldwide, F508del.Almost exclusively, allele 1 of Xv-2c, j44, M470v, and T854T, and allele 2 of KM.19, MP6-D9, and Ivs6aGATT repeats were found in association with this mutation.This specific diallelic haplotype (1-2-2-1-6(2)-1-1) is commonly associated with F508del in other populations too, supporting the theory of an ancient origin of this mutation as one that arose after one mutational event, and further spreading of it from the south through other parts of the europe (D o r k et al., 1992; M o r r a l et al., 1994; K a n a v a k i s et al., 2003).The same haplotypes were also found in normal and non-F508del CF chromosomes, but in much lower frequencies (7.6% of N and 15% of non-F508del mutations).
The most common (36%) and probably the oldest haplotype associated with normal chromosomes was 1-1-2-1-6(2)-1-2, also found in 21% of CF chromosomes which did not carry the F508del mutation.however, it can be expected that normal chromosomes can be separated from the CF chromosome, mostly due to high frequency of allele 1 of the Ivs6aGATT repeat (76% of normal chromosomes carried allele 1, compared to only 4% of F508del chromosomes and half of non-F508del chromosomes).All six common haplotypes were found in non-F508del chromosomes (11/25%), suggesting heterogeneity and different origin of those mutations.The high mutational heterogeneity and haplotype variability shown in the serbian population is similar to those found in other populations in our region.This is also consistent with an earlier appearance of CFTR mutations in southeast europe, which would allow time for haplotype recombination (D o r k et al., 1992; K a n a v a k i s et al., 2003).
Knowledge of the population genetics and chromosomal haplotype of various CFTR mutations would significantly facilitate mutation diagnosis, especially in populations with high mutational heterogeneity, like ours.With respect to prenatal diagnosis, especially in cases where one or even both CF alleles were not identified using direct methods for mutation screening, a combination of methods for detection of CFTR mutations and haplotype analysis could provide rapid, accurate, and reliable prenatal diagnosis for almost all couples at risk of having a child affected with cystic fibrosis in our country.
cedure (M i l l e r et al., 1988).DNA samples were screened for the presence of CFTR mutations by heteroduplex analysis on PAGe for the presence of F508del (K e r e m et al., 1989; R o m m e n s et al., 1990), simultaneous in vitro qualitative detection of 29 CFTR mutations frequent in europe (elucigene tm CF 29 kit, Orchid), DGGe analysis of PCR amplified exons 1-24 (F a n e n et al., 1992; G h a n e m et al., 1992; C o s t e s et al., 1993), and sequencing (s a n g e r et al.

Fig. 3 .
Fig. 3. Allele frequencies of polymorphisms associated with the F508del mutation (M o r r a l et al., 1994; C l a u s t r e s et al., 1996, M o r r a l et al., 1996; A n g e l i c h e v a et al., 1997; P e t r e s k a et al., 1998; K a n a v a k i s et al.

Table 1 .
Table 1.Allele frequencies of polymorphisms at the CF locus in a group of Serbian normal (N) and CF chromosomes.

Table 1 .
Frequencies of the most common marker haplotypes in a group of analyzed N (normal) and CF chromosomes from Serbia.