Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations

The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.     

Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K, P409A, g10208delAA [two families]). Haplotype analyses indicated that the families with the R112C or g10208delAA mutations were not related. The proband with the D266E lesion had a severe classic phenotype, having developed renal failure at 15 years. In contrast, the patient with the S247C mutation had a variant phenotype, lacking the classic manifestations and having mild renal involvement at 64 years. CONCLUSIONS: These results further define the heterogeneity of alpha-Gal A mutations causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis in these families, and provide additional genotype/phenotype correlations in this lysosomal storage disease.     

Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder. Received: 24 July 1998 / Accepted: 12 September 1998     

Mutation analysis of the Fanconi anemia gene FACC.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by a unique hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C (FACC) has recently been cloned. We have amplified FACC exons with their flanking intron sequences from genomic DNA from 174 racially and ethnically diverse families in the International Fanconi Anemia Registry and have screened for mutations by using SSCP analysis. We identified eight different variants in 32 families; three were detected in exon 1, one in exon 4, one in intron 4, two in exon 6, and one in exon 14. Two of the eight variants, in seven families, did not segregate with the disease allele in multiplex families, suggesting that these variants represented benign polymorphisms. Disease-associated mutations in FACC were detected in a total of 25 (14.4%) of 174 families screened. The most frequent mutations were IVS4 + 4 A-->T (intron 4; 12 families) and 322delG (exon 1; 9 families). Other, less common mutations include Q13X in exon 1, R185X and D195V in exon 6, and L554P in exon 14. The polymorphisms were S26F in exon 1 and G139E in exon 4. All patients in our study with 322delG, Q13X, R185X, and D195V are of northern or eastern European or southern Italian ancestry, and 18 of 19 have a mild form of the disease, while the 2 patients with L554P, both from the same family, have a severe phenotype. All 19 patients with IVS4 + 4 A-->T have Jewish ancestry and have a severe phenotype.     

Analysis of 14 cystic fibrosis mutations in five South European populations

Summary We have analysed five Southern European populations (Albanian, Greek, Italian, Spanish and Yugoslavian) for 14 cystic fibrosis (CF) mutations. The most frequent mutations, apart from F508, were G542X (6.04%), R1162X (3.61%) and N1303K (3.24%). Each of the other analysed mutations were present at a frequency of less than 1% (R347P, R334W, S549RA, S549I, G551D, R553X and W1282X), and four mutations (D110H, I507, S549RT, and S1255X) were not found in this sample. The data presented here allows the use of mutation analysis in 69.5% of Spanish, 58% of Greek, and 56.5% of Italian CF cases.     

Phenylketonuria mutations and their relation to RFLP haplotypes at the PAH locus in Czech PKU families

A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism /variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and I65T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification.     

Nature and Recurrence of AVPR2 Mutations in X-linked Nephrogenic Diabetes Insipidus

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin V₂ receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255del9, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations—R113W, Y128S, R137H, R181C, and R202C—that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methylcytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication.     

Identification of mutation of the phenylalanine hydroxylase gene using an automated DNA sequencer

Mutations were studied in phenylalanine hydroxylase gene of phenylketonuria patients from Kemerovo oblast and Altaiskii krai (15 and 2 families, respectively). The following mutations were identified in exons of this gene: R408W, R261Q, R243Q, Y414C, Y386C, P281L, Y168H, R68S (lead to amino acid substitutions), R243X (leads to stop codon formation), and three splice site mutations (IVS12nt 1g-->a, IVS2nt-13t-->g, IVS7nt 1g-->a).     

Genetic analysis carried out on blood-spots of phenylalanine hydroxylase-deficient newborns detected by northeastern Italian neonatal screening

The aim of this work was to perform genetic analysis on 18 different blood-spot samples collected from neonates detected as hyperphenylalaninemic by Northeastern Italian screening program. DNA was extracted from blood-spots. Exons/introns of PAH gene were amplified by polymerase chain reaction (PCR), and PCR products were purified and sequenced with both forward and reverse primers. The most frequent mutations were IVS12nt1g>a (16.7%) and R408W, P281L and L48S (all together 11.1%). As expected, compound heterozygosity was the usual finding; homozygosity was found only in two patients with R158Q and IVS2nt5g>c mutations. The V230I mutation was reported for the first time in Italy. We found six previously described polymorphisms (V245V, IVS4nt47c>t, IVS2nt19t>c, IVS3nt-22c>t, IVS5nt-54a>g, and E280>Q280). To our knowledge, four genotypes were not previously described: R158Q/V230I present in one patient with classical PKU; and L48S/R408Q, A403V/IVS2nt-13t>g, and G272X/V230I present in patients showing HPA phenotype. Most of the mutations were located in the exons 12 and 7 and in exon/intron 2 (83.3% detection of total mutations in PKU or HPA patients of Northeastern Italy). From a practical viewpoint, the genetic analysis of blood-spots collected on Guthrie cards for neonatal screening for PKU could be a simple method to establish the genotype of neonates. Consequently, the genotype/phenotype correlation could lead to a more accurate diagnosis and prognosis for families.     

Distribution of mutations of acid β-D-glucosidase Gene (GBA) among 68 Russian patients with Gaucher’s disease

Gaucher disease (GD) is the most frequent lysosomal storage disease presenting in all populations. Mutations in the acid β-D-glucosidase gene (GBA) cause development of GD, resulting in a decrease or full loss of activity of this enzyme. We report here the results of the molecular-genetic analysis in 68 Russian GD patients from 65 families with the three types of this disease. The GD genotype has been completely elucidated in 58 patients and in all patients we have found at least one mutant allele (92.6%). Besides frequent mutations (p.N370S, c.1263_1317del (del55), p.L444P, p.R463C, Rec NciI) we have identified rare mutations p.R120W, p.R170C, p.R184W, p.G202R, Rec C (p.R120W; p.W184R; p.N188K; p.V191G; p.S196P; p.G202R; p.F213I), presenting in other populations of GD patients. The mutations p.P236T, p.L249Q, p.L288P, p.P319S, p.V352M, p.W381X, p.A384D identified in this study had not been described before. The GBA mutations identified in Russian patients have been compared with those found in patients of other European countries. Genotype-phenotype correlations in GD are discussed.     

PKU in Slovakia: mutation screening and haplotype analysis

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.     

Mutations in radial spoke head genes and ultrastructural cilia defects in East-European cohort of primary ciliary dyskinesia patients

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2-5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2-3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2-5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes.     

Screening for cystic fibrosis gene mutations by multiplex DNA amplification

Summary We have developed a simple rapid DNA screening test that allows us simultaneously to analyze seven CF mutations (deltaF508, R347P, S549N, G551D, R553X, R334W, 444delA) that together account for about 60% of all CF mutations in the Italian population. It consists of three steps: multiplex polymerase chain reaction (PCR) amplification of exons 4, 7, 10 and 11; restriction endonuclease digestion of the PCR products; and vertical polyacrylamide gel electrophoresis analysis. We have used our multiplex assay for analyzing 15 CF chromosomes (non delta F508) and have found 3 cases of the R553X mutation; the latter have been confirmed by amplification and digestion of exon 11.     

Molecular Diagnosis of 21-Hydroxylase Deficiency: Detection of Four Mutations on a Single Gel

Previous studies of the molecular basis of 21-hydroxylase deficiency have shown four common gene conversion mutations in exons 7 and 8. Current molecular diagnostic protocols use allele-specific oligonucleotide hybridization (ASOH) to individually detect each of these mutations and the corresponding normal alleles. This method is costly, labor intensive, and may not provide quantitative results. To expedite molecular diagnosis in families with 21-hydroxylase deficiency, we have designed and implemented single-strand conformational polymorphism (SSCP) analysis. We applied SSCP analysis to 12 families in whom mutations in exons 7 or 8 had been previously identified by ASOH. Using a single polymerase chain reaction (PCR) amplification, unique conformers can be assigned to three mutations: V281L, Q318X, and R356W. The fourth mutation, T insertion at nucleotide 1761, was detected by heteroduplex analysis of the same PCR product. Thus, we were able to identify all four mutations using a single PCR product on a single gel.     

Two mutations in LDLR gene were found in two Chinese families with familial hypercholesterolemia

Familial hypercholesterolemia (FH) (OMIM 143890) is an autosomal dominantly inherited disease mainly caused by mutations of the gene encoding the low density lipoprotein receptor (LDLR) and Apolipoprotein (Apo) B. First the common mutation R3500Q in ApoB gene was determined using PCR/RFLP method. Then the LDLR gene was screened for mutations using Touch-down PCR, SSCP and sequencing techniques. Furthermore, the secondary structure of the LDLR protein was predicted with ANTHEPROT5.0. The R3500Q mutation was absent in these two families. A heterozygous p.W483X mutation of LDLR gene was identified in family A which caused a premature stop codon, while a homozygous mutation p.A627T was found in family B. The predicted secondary structures of the mutant LDLR were altered. We identified two known mutations (p.W483X, p.A627T) of the LDLR gene in two Chinese FH families respectively.     

Twenty novel mutations in the alpha-galactosidase A gene causing Fabry disease

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A). The nature of the molecular lesions in the alpha-Gal A gene in 30 unrelated families was determined to provide precise heterozygote detection, prenatal diagnosis, and define genotype-phenotype correlations. MATERIALS AND METHODS: Genomic DNA was isolated from affected males and/or carrier females from 30 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and automated sequencing. RESULTS: Twenty new mutations were identified, each in a single family: C142R, G183D, S235C, W236L, D244H, P259L, M267I, I289F, Q321E, C378Y, C52X, W277X, IVS4(+4), IVS6(+2), IVS6(-1), 35del13, 256del1, 892ins1, 1176del4, and 1188del1. In the remaining 10 unrelated Fabry families, 9 previously reported mutations were detected: M42V, R112C, S148R, D165V, N215S (in 2 families), Q99X, C142X, R227X, and 1072del3. Haplotype analysis using markers closely flanking the alpha-Gal A gene indicated that the two patients with the N215S lesion were unrelated. The IVS4(+4) mutation was a rare intronic splice site mutation that causes Fabry disease. CONCLUSIONS: These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis, and help delineate phenotype-genotype correlations in this disease. 相似文献   

Identification of three novel mutations (457 TAT→G,D192G,Q685X) in the Slovenian CF patients

Chromosomes from a cohort of 60 Slovenian families, corresponding to the 121 cystic fibrosis (CF) chromosomes available, were fully scanned for mutations in the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (The 60 families yielded 121 CF alleles because the mother of one patient was also affected). This corresponds to the 27 exons and intron/exon boundaries that have been studied in chromosomes carrying unidentified alleles. As a result of this survey 84% of the alleles are now clearly identified and we describe in this paper three novel mutations (457 TATG, D192G, and Q685X).     

CDKN2A mutations and MC1R variants in Italian patients with single or multiple primary melanoma

We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43-7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C); the novel 5'UTR -21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.     

High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes

We have analyzed 640 Spanish cystic fibrosis (CF) families for mutations in the CFTR gene by direct mutation analysis, microsatellite haplotypes, denaturing gradient gel electrophoresis, single-strand conformation analysis and direct sequencing. Seventy-five mutations account for 90.2% of CF chromosomes. Among these we have detected seven novel CFTR mutations, including four missense (G85V, T582R, R851L and F1074L), two nonsense (E692X and Q1281X) and one splice site mutation (711+3A→T). Three variants, two in intronic regions (406-112A/T and 3850-129T/C) and one in the coding region (741C/T) were also identified. Mutations G85V, T582R, R851L, E692X and Q1281X are severe, with lung and pancreatic involvement; 711+3A→T could be responsible for a pancreatic sufficiency/insufficiency variable phenotype; and F1074L was associated with a mild phenotype. These data demonstrate the highest molecular heterogeneity reported so far in CF, indicating that a wide mutation screening is necessary to characterize 90% of the Spanish CF alleles. Received: 3 July 1997 / Accepted: 20 August 1997     

Spectrum of cystic fibrosis mutations in Serbia and Montenegro and strategy for prenatal diagnosis

We have screened 175 patients for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using nondenaturing polyacrylamide gel electrophoresis (PAGE), denaturing gradient gel electrophoresis (DGGE), and sequencing. Six different mutations (F508del, G542X, 621+1G --> T, 2789+5G --> A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Another 12 mutations (R334W, 2184insA, I507del, 1525-1G --> A, E585X, R75X, M1I, 457TAT --> G, 574delA, 2723delTT, A120T, and 2907delTT) covered an additional 3.36%. A novel mutation (2723delTT) was found in one CF patient (F508del/2723delTT). Thus, a total of 18 mutations cover 82.57% of CF alleles. During our study, 72% of families at risk for having a CF child were found to be fully informative for prenatal diagnosis. Prenatal diagnosis was performed on 56 families; 76 analyses resulting in 16 affected, 38 carriers, and 22 healthy fetuses. These results imply that the molecular basis of CF in Serbia and Montenegro is highly heterogeneous, as is observed in other eastern and southern European populations. Because we detected more then 80% of CFTR alleles, results could be used for planning future screening and appropriate genetic counseling programs in our country.