Cystic fibrosis screening: lessons learned from the first 320,000 patients.

PURPOSE: To examine the data from > 335,000 Cystic fibrosis (CF) tests to detect unsuspected findings and obtain clinical data when indicated to optimize genetic counseling. METHODS: A proprietary database containing 335,204 consecutive CF DNA tests and 445 CF prenatal diagnostic tests was queried. Clinical information was obtained for prenatal and selected nonprenatal cases by telephone contact with physician offices. RESULTS: The mutation 1078delT was found in much lower frequency than expected with rates of only 1:55,867 tests and 0.06% of CF mutations. This level is below the threshold set by the American College of Medical Genetics. Homozygosity was observed for 2789+5G>A in a 29-year-old women and compound heterozygosity with delta F408 in a 40-year-old woman with isolated chronic sinusitis. Many patients elected prenatal diagnosis when not at a 1:4 risk due to echogenic bowel or IVS-8 5T issues. CONCLUSIONS: With the exception of 1078delT, all CF mutations in the ACMG panel were detected with a frequency of > 0.1% of CF chromosomes. When ACMG guidelines are strictly adhered to, population-based CF carrier screening will accurately identify couples at risk for having children with CF.     

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.     

Comprehensive Cystic Fibrosis Mutation Epidemiology and Haplotype Characterization in a Southern Italian Population

We screened the whole coding region of the cystic fibrosis transmembrane regulator (CFTR) gene in 371 unrelated cystic fibrosis (CF) patients from three regions of southern Italy. Forty‐three mutations detected 91.5% of CF mutated chromosomes by denaturing gradient gel electrophoresis analysis, and three intragenic CFTR polymorphisms predicted a myriad of rare mutations in uncharacterized CF chromosomes. Twelve mutations are peculiar to CF chromosomes from southern Italy: R1158X, 4016insT, L1065P and 711+1G>T are present in 6.3% of CF chromosomes in Campania; G1244E and 852del22 are present in 9.6% of CF chromosomes in Basilicata and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D are frequent in Puglia (19.6% of CF alleles). Several mutations frequently found in northern Italy (e.g., R1162X, 711+5G>T) and northern Europe (e.g., G551D, I507del and 621+1G>T) are absent from the studied population. The I148T‐3195del6 complex allele was present in two CF chromosomes, whereas I148T was present in both alleles (as a single mutation) in another CF patient and in five CF carriers; this could result from crossover events. The haplotype analysis of three intragenic polymorphisms (IVS8CA, IVS17bTA and IVS17bCA) compared with data from other studies revealed that several mutations (3849+10kbC>T, 1717‐1G>A, E585X, 3272‐26G>A, L558S, 2184insA and R347P) originated from multiple events, whereas others (R1158X and S549R) could be associated with one or more intragenic recombinant events. Given the large population migration from southern Italy, knowledge of the CF molecular epidemiology in this area is an important contribution to diagnosis, counselling and interlaboratory quality control for molecular laboratories worldwide.     

Premarital and prenatal screening for cystic fibrosis: experience in the Ashkenazi Jewish population.

PURPOSE: Since the early 1990s, Dor Yeshorim (DY) and the Mount Sinai School of Medicine (MSSM) have conducted premarital and prenatal carrier screening for cystic fibrosis (CF) in the Ashkenazi Jewish (AJ) population as part of their genetic testing programs, respectively. Together, over 170,000 screenees have been tested. In this study, we report the CF mutation frequencies in over 110,000 screenees who reportedly were of 100% AJ descent from the DY program and MSSM. In addition, the CF mutation frequencies in a group of > 7,000 screenees for AJ diseases who were of < 100% AJ descent are reported. METHODS: Testing for CF mutations was performed by either PCR and restriction digestion or ASO hybridization analyses at MSSM or sent to various academic and commercial laboratories by DY. RESULTS: The overall (and individual) carrier frequency for the five common AJ mutations, W1282X (0.020), DeltaF508 (0.012), G542X (0.0024), 3849+10kb C>T (0.0020), and N1303K (0.0016), among screenees who were 100% AJ was 1 in 26; when D1152H and the rare 1717-1G>A were included, the overall carrier frequency increased to approximately 1 in 23. In four families with D1152H, five compound heterozygotes for D1152H and W1282X (n = 2), DeltaF508 (1) or 3849+10kb C>T (1) were identified. In contrast, the carrier frequency for screenees reporting < 100% AJ descent was approximately 1 in 30 for the seven mutations. CONCLUSIONS: The carrier frequency for five common CF mutations in a large 100% AJ sample increased from 1 in 26 to 1 in 23 when D1152H was included in the panel. Addition of D1152H to mutation panels when screening the AJ population should be considered because compound heterozygosity is associated with a variable disease phenotype. Further studies to delineate the phenotype of CF patients with this mutation are needed.     

Cystic fibrosis screening: a fetus with hyperechogenic bowel may be the index case.

BACKGROUND: The potential of hyperechogenic fetal bowel to act as a hallmark for prenatal cystic fibrosis screening in the general population is controversial. METHODS: Our goal was to evaluate the incidence of cystic fibrosis in 209 fetuses with hyperechogenic bowel diagnosed at routine ultrasonography and with no family history of cystic fibrosis. The diagnosis of cystic fibrosis was based on prenatal screening for the eight mutations most frequently observed in France (deltaF508, deltaI507, 1717-1G-->A, G542X, G551D, R553X, W1282X, N1303K) and at postnatal follow up. RESULTS: The overall incidence of cystic fibrosis was 7/209 (3.3%) which is 84 times the estimated risk of CF in the general population (112500). Of these seven cases, six were diagnosed prenatally based on DNA analysis (deltaF508/deltaF508, n=5; deltaF508/G542X, n=1). One case in which only one mutation had been recognised was diagnosed clinically after birth (deltaF508/unidentified mutation). Of the seven cases, none was diagnosed at 16-19 weeks, four at 16-24 weeks, and three after this. The incidence of heterozygous fetuses (15/209, 7%) was not significantly higher than the 5% expected in the general population. The mutations involved in these heterozygous cases were deltaF508 (n=13), G542X (n=1), and G551D (n=1). CONCLUSIONS: Screening for cystic fibrosis should be offered to families in which fetal hyperechogenic bowel is diagnosed at routine ultrasonography. This underlines the need to review genetic counselling in this situation where the fetus is the index case for a genetic disease.     

Mucopolysaccharidosis I mutations in Chinese patients: identification of 27 novel mutations and 6 cases involving prenatal diagnosis

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease that results from the deficiency of α-l-iduronidase and is transmitted in an autosomally recessive manner. This report describes the first systematic screening for mutations in Chinese MPS I patients from mainland China, wherein we have summarized the phenotype/genotype correlation of the individuals in Chinese MPS I patients. Mutational analyses were performed in 57 unrelated Chinese MPS I patients. Overall, 105 mutant alleles were identified from a set of 41 different mutations. Notably, of these 41 mutations, 27 were novel mutations that consisted of 8 splicing mutations (c.1-2C>G, c.296+4G>A, c.300-1G>C, c.792+1G>C, c.973-4G>A, c.1189+5G>T, c.1402+1G>T and c.1402+2T>G), 1 nonsense mutation (p.W41X), 1 insertion (c.668-670ins GCG), 5 duplications (c.531dupT, c.657dupG, c.883dupC, c.1147dupG and c.1225dupG), 3 deletions (c.349delT, c.1593delG and c.1244-1271del27),1 nucleotide substitution c.2T>C and 8 missense mutations (p.H33P,p.F52L, p.G168V,p.T179R,p. E182D, p.L237R, p.L238R and p.L421P). The missense mutations p.A79V and p.L346R, which accounted for 16.7% (19/114) and 12.3% (14/114) respectively, were the common mutations in Chinese patients but were rare in the mutational profiles reported for other populations. These results indicate that Chinese MPS I patients may have a different mutational spectrum compared to those of other populations. Moreover, for the first time in China, molecular genetic methods were used for prenatal diagnosis of six cases in five families.     

Prenatal diagnosis in 3,000 women for chromosome,X-linked,and metabolic disorders

In 3,000 women referred for prenatal diagnosis, 110 (3.7%) abnormal fetuses were detected and 85 therapeutic terminations were performed. There were five main reasons for referral. Among the 2,227 women referred because of maternal age 35 years and older, there were 51 (2.3%) who had aneuploid fetuses. In the 297 women referred because of a previous child with Down syndrome, 3 aneuploid fetuses (1.0%) were detected. Of the 55 couples where one spouse was a carrier of a balanced chromosome rearrangement, 6 chromosomally abnormal fetuses were found (10.9%) (all the offspring of maternal carriers). In the latter group, five of the six heterozygotes with abnormal findings were carriers of tdic (13;21) translocations. In the 82 cases with a history of X-linked disorders, there were 40 males (49%). Thirty-five women were referred because of inborn errors of metabolism: 10 affected fetuses were found (28%). There was a greater proportion of sexchromosome aneuploids as compared to trisomy 21 fetuses in the 35 to 39 year maternal age group. This was reversed in the group of women 40 years old and older. Of the 25 abnormal fetuses not terminated, 6 were sex chromosome aneuploids and 10 involved X-linked conditions where the progeny could be further prenatally monitored (eg, X-linked hydrocephalus) or treated (eg, hemophilia). In the remaining 9 the parents expressed diverse reasons for their choice. Repeat amniocentesis was required in 2.9% of cases. One case of maternal cell contamination and one case of unconfirmed mosaicism were the only diagnostic errors found in the study. In the last 1,000 specimens referred, the average time necessary for a karyotypic result was 15.6 ± 5.6 days after amniocentesis.     

Mutation analysis in 24 French patients with glycogen storage disease type 1a.

Both alleles of 24 French glycogen storage disease type 1a patients were sequenced: 14 different mutations allowed the identification of complete genotypes for all the patients. Nine new gene alterations are reported. Five mutations, Q347X, R83C, D38V, G188R, and 158 del C, account for 75% of the mutated alleles. These data show that the molecular pathology of the glucose-6-phosphatase gene is heterogeneous in this population. Complete genotyping of the index case by systematic sequencing is necessary to allow prenatal diagnosis in chorionic villi for at risk couples.     

Genetic testing for cystic fibrosis: evaluation of the Elucigene CF20 kit in blood and buccal cells

Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-cost methods, permitting the simultaneous detection of multiple mutations. The Elucigene CF20 kit developped by Cellmark Diagnostics, uses multiplex ARMS, which allows the screening for 20 CFTR gene mutations (deltaF508, G542X, N1303K, 1717-1G>A, G551D, W1282X, R553X, deltaI507, 1078delT, 2183AA>G, 3849+10kbC>T, R1162X, 621+1G>T, R334W, R347P, 3659delC, R117H, S1251N, E60X, A455E ) in a work day without specific instrumentation. The kit distinguishes between homozygotes and heterozygotes for deltaF508, but not for rare mutations. The kit detects from 68 to 92% of defective alleles in Caucasians. We evaluate the kit in a blind study in two independent laboratories. Thirty blood samples and thirty mouthwash samples from CF patients, carriers and unaffected individuals were analysed by the Elucigene CF20 kit. All the samples were previously analysed by denaturing gradient gel electrophoresis and sequencing. The Elucigene CF20 kit consists of three multiplexes. Each mutiplex contains ARMS specific primers for six to eight mutations and two control reactions. The absence of the upper control fragment indicates that a repeat test is required. We demonstrated a first time amplification rate of 98.3%: of the 60 samples tested, one required a reamplification. Results compared with the reference method demonstrated that in all cases where one or more of the 20 mutations detected by the kit were present in the test set, the kit accurately identified them. Reproducibility was assessed by repeating the analysis of a blood and mouthwash sample five times. Cross reactivity between R117C and R117H, R117P and R117H, R347P and R347H, deltaI507 and deltaF508, G551D and R553X were evaluated. Only a cross reactivity between R347P and R347H was observed. The kit is specially useful for first line study of patients and carrier identification.     

Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis

Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application.     

Type 1 Gaucher disease: null and hypomorphic novel chitotriosidase mutations-implications for diagnosis and therapeutic monitoring

Human plasma chitotriosidase (Chito) is a useful diagnostic and therapeutic biomarker for Type 1 Gaucher disease (GD). However, approximately 40% of Caucasians are heterozygous or homozygous for a common null mutation, c.1049_1072dup24 (dup24) in the chitotriosidase gene (chitinase 1, CHIT1), that complicates interpretation for heterozygotes and precludes use for null homozygotes. 320 Type 1 GD patients were screened for CHIT1 genotype and plasma Chito enzyme levels; 37% were heterozygous and 4% were homozygous for the CHIT1 dup24 allele. Four patients who had no or very low plasma Chito activities had wild-type (wt)/dup24 or wt/wt CHIT1 genotypes, suggesting the presence of other mutations. Sequencing their CHIT1 genes revealed three novel mutations: p.E74K (E74K), p.G102S (G102S), and a complex exon 10 lesion (c.[1060G>A; 1155G>A; 1156+5_1156+8delGTAA], p.[G354R; L385L; missplicing], designated "complex E/I-10"). The G102S mutation was common in Type 1 GD patients and controls ( approximately 30% of alleles). In contrast, the E74K mutation was rare, present only in three Type 1 GD patients ( approximately 1% of alleles), all of Ashkenazi Jewish (AJ) descent, but it was not found in normal controls. The complex E/I-10 mutation occurred in two Caribbean Hispanic/African Type 1 GD patients and was present in 0 to 6% of alleles among normal controls from different populations. In vitro expression demonstrated that the E74K and G102S alleles had approximately 51% and approximately 23% of wild-type Chito catalytic efficiency, respectively. Expression of the G354R allele alone or with the L385L silent substitution did not produce detectable Chito activity or protein. RNA studies indicated that the complex E/I-10 allele also caused missplicing. Recognition of these mutations, particularly G102S, will facilitate the use and interpretation of plasma Chito activities for disease diagnosis, estimating disease severity, and monitoring therapeutic efficacy in GD.     

四个遗传性耳聋家系的TMC1基因变异分析及产前诊断

目的对4个耳聋家系进行遗传性耳聋基因变异的筛查,为家系遗传咨询与产前诊断提供依据。方法应用二代测序技术对家系先证者进行耳聋基因检测,并对可疑基因变异采用Sanger双向测序对先证者及家系成员进行验证,确定可疑致病变异后,对3个家系高危胎儿进行产前诊断。结果家系1先证者检测到TMC1基因c.100C>T(p.R34X)和c.642+4A>C复合杂合变异,家系2先证者检测到TMC1基因c.582G>A(p.W194X)和c.589G>A(p.G197R)复合杂合变异,家系3先证者检测到TMC1基因c.1396_1398delAAC和c.1571T>C(p.F524S)复合杂合变异,家系4先证者检测到TMC1基因c.2050G>C(p.D684H)纯合变异,4个家系先证者父母均为携带者,其中c.642+4A>C、c.1571T>C(p.F524S)变异位点既往未见报道;产前诊断结果显示3个家系胎儿均不是患者,出生后随访至2019年9月,听力未见异常。结论TMC1基因变异是4个耳聋家系的可能致病原因,分子生物学的发现增加了对TMC1基因功能的认识并丰富了人类基因变异数据库,为家系遗传咨询和产前诊断提供了依据。     

CYP21A2 mutations in Portuguese patients with congenital adrenal hyperplasia: identification of two novel mutations and characterization of four different partial gene conversions

More than 90% of congenital adrenal hyperplasia (CAH) cases are caused by 21-hydroxylase deficiency. In this study, the CYP21 gene was genotyped in 56 Portuguese unrelated patients with clinical symptoms of 21-hydroxylase deficiency, in a total of 112 independent alleles. CYP21A2 mutations were identified in 99.1% of the alleles. The most common point mutation was 1688G>T (25.9%). A previously unreported partial gene conversion, extending from exon 1 to 7, was found in 16.1% of the alleles, in most cases associated to the mutation 1688G>T in the other chromosome, and in patients with nonclassical CAH. Other three distinct partial gene conversions were also identified, with lower frequencies: one extends from exon 1 to 3 and the others from exons 3 to 7 and 3 to 8. Two novel mutations were identified in two salt-wasting patients: a putative splicing mutation, IVS2+5G>A, and the transition 2557C>T, that gives rise to the nonsense mutation R445X. Seven point mutations and a partial gene conversion were responsible for 88 of the studied disease causing alleles, and the overall concordance between genotype and phenotype was 92.9%. With this study the molecular basis of CAH was characterized, for the first time, in Portuguese patients, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling.     

泰国缺失型α地中海贫血1的产前基因诊断

目的对携带泰国缺失型α地中海贫血1,可能生育α地中海贫血高危胎儿的夫妇进行产前基因诊断。方法应用聚合酶链反应、DNA测序等方法对夫妇及胎儿DNA进行基因分析。结果4个家系的孕妇均为泰国缺失型α地中海贫血1复合α地中海贫血2的Hb H病患者,5个家系的孕妇或丈夫为泰国缺失型α地中海贫血1杂合子;其配偶为东南亚缺失型α地中海贫血1杂合子。他们的胎儿检出2例Hb Bart’s胎儿水肿综合征,1例Hb H病,4例α地中海贫血杂合子,2名正常。DNA测序分析证实PCR的检测结果。结论泰国缺失型α地中海贫血1的产前基因诊断研究,对遗传咨询和地中海贫血产前基因诊断具有重要意义。     

葡萄糖6磷酸酶基因热点突变检测结合1176多态位点连锁分析快速产前诊断Ia型糖原累积病

目的探讨中国人Ⅰa型糖原累积病简便、快速、准确的产前诊断方法。方法通过限制性内切酶图谱分析了葡萄糖6磷酸酶(glucose-6-phosphatase，G6Pase)基因727G→T和R83H的突变，并结合1176位点单核苷酸多态性连锁分析，对3个Ⅰa型糖原累积病家系进行了基因诊断和产前诊断。对发现的突变及1176位点多态性用DNA测序证实。结果3个家系先证者G6Pase基因的2个等位基因均携带727G→T突变，分别来自其父母。家系1和3胎儿为727G→T突变杂合子；家系2胎儿不携带该突变。1176位点单核苷酸多态性分析显示，3名胎儿1176位点单核苷酸多态性与3名先证者不同。DNA直接测序结果与限制性内切酶图谱分析结果相符。家系1和家系2胎儿已出生，井证实与产前诊断结果相符。结论通过限制性内切酶酶切法筛查727G→T和R83H突变结合1176位点单核苷酸多态性连锁分析可简便、快速、准确地诊断和产前诊断Ⅰa型糖原累积病。     

Clinical, Functional and Genetic Analysis of Twenty-Four Patients with Chronic Granulomatous Disease - Identification of Eight Novel Mutations in CYBB and NCF2 Genes

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and cannot produce superoxide anions. The most common form is caused by mutations in CYBB encoding gp91phox. We investigated 24 CGD patients and their families. Twenty-one mutations in CYBB were classified as X91(0), X91(+) or X91(-) variants according to cytochrome b (558) expression. Point mutations in encoding regions represented 50?% of the mutations found in CYBB, splice site mutations 27?%, deletions and insertions 23?%. Eight mutations in CYBB were novel leading to X91(0)CGD cases. Two of these were point mutations: c493G>T and a double mutation c625C>G in exon 6 and c1510C>T in exon 12 leading to a premature stop codon at Gly165 in gp91phox and missense mutations His209Arg/Thr503Ile respectively. Two novel splice mutations in 5'intronic regions of introns 1 and 6 were found. A novel deletion/insertion c1024_1026delCTG/insT results in a frameshift introducing a stop codon at position 346 in gp91phox. The last novel mutation was the insertion of a T at c1373 leading to a frameshift and a premature stop codon at position 484 in gp91phox. For the first time the precise size of two large mutations in CYBB was determined by array-comparative genomic hybridization and carriers' status were evaluated by multiplex ligation-dependent probe amplification assay. No clear correlation between clinical severity and CYBB mutations could be established. Of three mutations in CYBA, NCF1 and NCF2 leading to rare autosomal recessive CGD, one nonsense mutation c29G>A in exon 1 of NCF2 was new.     

Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosis

One major limitation of pre-implantation genetic diagnosis (PGD) practice comes from the need to develop single cell PCR protocols. For a disease such as cystic fibrosis (CF), for which almost 1000 mutations have been identified, the development of a mutation based PGD protocol is impracticable. An elegant way to overcome this problem is to set up an indirect diagnosis using polymorphic markers allowing the identification of the pathogenic haplotype instead of the mutation. We present here a new PGD protocol for CF. Our strategy is based on a multiplex fluorescent PCR co-amplifying the DeltaF508 mutation and two CFTR intragenic polymorphic microsatellites (IVS8CA and IVS17bCA). Such an approach is justified since in 91% of the cases at least one partner of the couple carries the DeltaF508 mutation. The use of intragenic markers reduces the risk of misdiagnosis due to meiotic recombination. In 97% of the single lymphoblasts (151/155) tested a PCR signal was obtained. A complete haplotyping was achieved in 137/151 (91%) lymphoblasts and a 6% rate of allele drop out (ADO) was observed. Three cases were performed. Case one was at risk of transmitting mutations DeltaF508 and R1162X, case 2 DeltaF508 and R1066C and case 3 DeltaF508 and 1341+1A. Considering these three cases and the re-analysis of the affected embryos, we have analysed 62 blastomeres from which we had PCR signal for 58 (94%) and a complete haplotype for 49 (84%). With the degree of polymorphism of the markers used in this work (48 and 39%) and the fact that we co-amplified the F508 locus our test should be suitable for nearly 80% of the couples requesting PGD for CF. This fluorescent multiplex PCR indirect diagnosis provides also a safer test since it allows the confirmation of the diagnosis, the detection of contamination and could give an indication on the ploidy of the embryos tested.     

Cystic fibrosis mutations in Israeli Arab patients

Mutation analysis was performed on 42 unrelated Israeli Arab CF patients. The previously known mutations in this population, DF508, N1303K, G542X, 4010delTATT, and S549R(T>G), were identified in 57 CF alleles, leaving 28 CF alleles with unknown mutations. Screening of the coding sequence of the CFTR gene by a single strand conformation analysis (SSCA) and direct sequencing revealed three point mutations and two intragenic deletions, including 2183AA>G, R75X, S549R (A>C), 3120+1Kbdel8.6Kb and del(exon2). In the present sample of Israeli Arab patients, 12 mutations account for 92% of the CF alleles. The mutations DF508, N1303K, W1282X and 3120+1Kbdel8.6Kb were found in all Arab ethnic subgroups. The mutations G85E, R75X, 2183AA>G, and del(exon2) were confined to Muslim Arabs, and the mutations 4010delTATT, S549R(A>C) and G542X were confined to Christian Arabs. Hum Mutat 14:543, 1999.     

The incidence of different cystic fibrosis mutations in the Scottish population: effects on prenatal diagnosis and genetic counselling.

We present an analysis of the frequency of 16 different cystic fibrosis (CF) mutant alleles in the Scottish population. Each allele was detected in DNA amplified by the polymerase chain reaction (PCR) either directly on polyacrylamide gels, on agarose gels after restriction enzyme digestion, or by using allele specific oligonucleotides. Among 506 CF chromosomes, of predominantly Scottish origin, the frequencies of the different mutations were delta F508 0.71, G551D 0.05, G542X 0.04, R117H 0.01, 1717-1G----A 0.01, A455E + delta I507 + R553X + R560T + W1282X + 621 + 1G----T combined 0.03, unpublished 0.01, and unknown 0.13. No examples of D110H, R347P, S549N, S549I, or 2566ins AT mutations were found. The relevance of this type of analysis for both prenatal diagnosis and heterozygote screening is discussed.     

Comparison of the CFTR mutation spectrum in three cohorts of patients of Celtic origin from Brittany (France) and Ireland

This study aims to compare the spectrum of the mutations identified in the gene responsible for cystic fibrosis in three cohorts of patients of Celtic origin from Brittany and Ireland. It included 389 patients from Brittany, 631 from Dublin and 139 from Cork. The CFTR gene analysis relied on the detection of the most common mutations, followed by a complete gene scanning using DGGE or D-HPLC. High mutation detection rates were obtained in each cohort: 99.6%, 96.8%, and 96.0% respectively. A high frequency of the c.1652_1655 del3 mutation (F508del: 74.8% to 81.3%) and of the "Celtic" mutation (c.1784G>A (G551D): 3.7% to 9.7%) was observed in each population. Apart from this, the mutation spectrums differed. In Brittany, the most common abnormalities were: c.1078delT (3.6%), c.4041C>G (N1303K: 1.4%), c.2670G>A (W846X(2): 1.0%) and c.1717-1G>A (1.0%), whereas in the cohort of Dublin, the main mutations were: c.482G>A (R117H: 3.0%), c.1811G>C (R560T: 2.4%) and c.621+1G>T (1.7%). Finally, in the Cork area, only the c.482G>A mutation (R117H) reached a frequency of 1%. Two previously-unreported mutations were identified in the Dublin cohort: c.2623-2A>G and c.3446T>G (M1105R). This collaborative study highlights the similarities of the CFTR alleles in the Breton and Irish populations, but also the disparities that exist between these populations, despite their common origin. Each population has its own history, with its mixture of founder effects and genetic drifts, which are at the origin of the current mutation distribution. The molecular study of the CFTR gene provides new tools for retracing European populations' histories.