ESHG Posters 4

E. Obersztyn, Z. Helias-Rodzewicz, A. Sobczynska-Tomaszewska, E. Bocian, T. Mazurczak;
National Institute of Mother and Child, Warsaw, POLAND.

Uniparental Disomy (UPD) is the inheritance of two homologous chromosomes only from one parent in a euploid offspring. Loss of heterozygosity in the uniparental isodisomy may be a cause of clinical expression of autosomally recessive disorders, when only one of the parents is the carrier of gene mutation. In approximately 7-10% patients with Silver-Russell syndrome maternal UPD7 is observed.
Clinical, cytogenetic and molecular studies carried out in the case of a 2 years old girl affected with cystic fibrosis (CF) as a result of maternal isodisomy of chromosome 7 are presented. Clinical diagnosis of CF was confirmed by mutation analysis in the CFTR gene which revealed that patient is the homozygote of delF508 mutation. Severe intrauterine and postnatal growth retardation (body weight -4SD, body length -6SD), relative macrocephaly, clinodactyly of 5th finger, feeding difficulties, hypoglycaemic episodes in the neonatal period and dysmorphic features suggested Silver-Russell syndrome. Psychomotor development was within the normal range. Karyotype in the lymphocytes was normal - 46,XX. Analysis of polymorphic microsatellites markers at loci: D7S507, D7S2422, D7S460, D7S1517 and GTNOS showed maternal UPD7. Analysis of delF508 mutation revealed that only mother was the heterozygote of this mutation.
Results of our studies explain the cause of severe somatic retardation in the patient.The diagnosis was the basis for verified genetic counselling concerning recurrence risk of cystic fibrosis in that family.

The case of cystic fibrosis in 46,XY phenotypic newborn girl with Smith-Lemli-Opitz syndrome

E. A. Grigori, P. M. Stratulat, E. P. Tabac, L. Sinitsina;
Institute of Childhood and Motherhood Health Care, Chisinau, REPUBLIC OF MOLDOVA.

In presented study we are describing a phenotype of newborn girl consulted concerning congenital abnormalities. Her mother was 20 years old, father was 25 years old, apparently healthy. The parental consanguinity wasn't discovered. Child was born from fifth pregnancy, complicated with toxemia, at 38 week of gestation, birth weight was equal to 2700g, head circumference of 33 cm. The first four pregnancies terminated with spontaneous abortion. There were the following phenotypic abnormalities: broad nasal bridge with broad nose and anteverted nostrils epicanthal folds, low set ears, increased nasolabial distance, broad maxillary alveolar ridges, micrognatia, cleft palate, short neck with pterygium colli, postaxial polydactyly of hands and feet, hypoplastic of thumbs, proximal cutaneous syndactyly of toes II-III, congenital heart defect, external genitals feminine. The karyotype of child was 46,XY. The karyotypes of both parents were normal. The child died at the age of 15 days. It was found the following malformations at autopsy: microghiry, heart trilocalar with common ductus arteriosus, fibrosis interstitial of pancreas with dilatation cystic of sinuses, cystic dysplastic changes of both kidneys, bronchitis and broncholith with mucus obstruction. Thus we suggest that above mentioned case is presented by association of Smith- Lemli-Opitz syndrome in phenotypic female a 46,XY karyotype and cystic fibrosis caused by different recessive genes.

S. D. Ghimbovschi¹, T. E. Ivaschenco², T. K. Kascheeva², S. A. Groppa¹, V. S. Baranov²;
¹Institute of Childhood and Motherhood Health Care, Chishinau, REPUBLIC OF MOLDOVA, ²Scientific Institute of Midwifery and Gynecology, S-Petersburg, RUSSIAN FEDERATION.

Introduction: Taken into consideration that there are 72% of mutant chromosomes with non-identified mutations in moldavian patients with Cystic Fibrosis (CF), we performed prenatal diagnosis (PD) using both molecular-genetic and biochemical methods. Using PCR-RFLP analysis and investigation of the level of the some enzymes in liquor amnii (aminopeptidaze, g-glutamiltranspeptidaze and alkaline fosfotaze) have been applied PD in 12 CF-families.
Results: In all 5 cases of PD in first trimester of gestation we used only molecular-genetic methods. In 2 cases we based only on the availability of the DF508; in 1 case - on the availability of DF508 and R347P; in 2 cases - on the availability of DF508 and allelic distribution in CS7/Hin6.I and Km19/PstI systems (in both allele 2 was linked with unknown mutations). In all of these cases were established healthy fetuses. In 4 (from 7) cases of PD in the second trimester of gestation we used both the molecular-genetic (availability of DF508) and biochemical methods. Due to absence of information about "guilty" mutations in 3 CF-families we carried out the PD in the second trimester of gestation based only on the data of the enzyme’s level in liquor amnii. As a result of our PD investigations we confirmed cystic fibrosis in 4 fetuses.
Conclusion: Using both molecular-genetic and biochemical approaches of investigation allows performing of PD in total number of pregnancies with major risc in CF in situation when there are not sufficiently information about molecular-genetic structure of mutations.

Preimplantation Genetic Diagnosis for cystic fibrosis in populations with molecular heterogeneity, based on multiplex sequence variation detection throughout the CFTR gene

C. Vrettou¹, M. Tzetis¹, J. R. Traeger-Synodinos¹, G. Palmer², E. Kanavakis¹;
¹University of Athens, Medical Genetics, Athens, GREECE, ²Center for Reproductive Medicine, Alpha Lab, Athens, GREECE.

Cystic fibrosis (CF) is targeted as one of the priority genetic diseases for prevention programmes. Preimplantation Genetic Diagnosis (PGD) represents an alternative approach to prenatal diagnosis, especially for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction for male infertility who have the additional risk of transmitting CF. The clinical application requires optimization of single cell genotyping protocols to minimize PCR failure, allelic drop out (ADO) and contamination. In addition the protocol should allow detection of the wide spectrum of potential CF-affected genotypes, especially relevant for Southern European populations. To this end we developed a flexible multiplex PCR protocol allowing analysis of sequence variations in any combination amongst 7 CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested-PCR and DGGE analysis, along with the intragenic dinucleotide microsatellite IVS8CA. The experiments were carried out on 390 single lymphocytes from 3 compound heterozygous CF patients, one heterozygote and one non-CF individual. PCR efficiency between exons varied from 90% to 100%, and ADO from 0% to 3.8%. IVS8CA was co-amplified with PCR efficiency of 92.4% and 10.8% ADO (evaluated by sizing the fluorescent PCR product). No contamination was observed in any set of experiments. The present method overcomes the need of separate assays for each CF mutation, and additionally allows analysis of linked polymorphic sequence variations (when informative), useful for minimizing misdiagnosis and/or indirect diagnosis. This method proved robust and flexible for diagnosing diverse CF genotype combinations in single cells.

Preimplantation genetic diagnosis (PGD) of cystic fibrosis by multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene.

C. Moutou¹, N. Gardes¹, S. Viville¹^,2;
¹Hôpitaux Universitaires de Strasbourg SIHCUS-CMCO, Schiltigheim, FRANCE, ²Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, FRANCE.

One major limitation of pre-implantation genetic diagnosis (PGD) practice comes from the need to develop single cell PCR protocols. For Cystic fibrosis (CF), for which almost 1000 mutations have been identified, the development of a mutation-based PGD protocol is impracticable. An 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 a new PGD protocol for CF, 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. A PCR signal was obtained in 97% of the single lymphoblasts (151/155) tested. A complete haplotyping was achieved in 137/151 (91%) lymphoblasts and a 6% rate of allele drop-out (ADO) was observed. During clinical application, 94% of blastomeres gave PCR signals and a complete haplotype could be assigned to 84% of them. With the degree of polymorphism of the markers (48 and 39%) and their co-amplification with 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 confirmation of the diagnosis, detection of contamination and could give an indication on the ploidy of the embryos tested.

Non-visualization of the fetal gallbladder: a predictive sign of cystic fibrosis?

D. Demarsy¹, M. C. Malinge¹, A. Guichet¹, C. Lépinard¹, D. Bonneau²;
¹Service de Génétique Médicale, CHU Angers, FRANCE, ²Centre Hospitalier Universitaire, Angers, FRANCE.

The non-visualization of the gallbladder without additional malformations has been described in fetuses with trisomy 21, biliary atresia and gallbladder agenesia. On another hand, a number of fetuses with an isolated absence of the gallbladder have a normal outcome. It is well known, however, that cystic fibrosis must be suspected when the absence of the gallbladder is associated with hyperechogenic bowel. We report here 5 cases where the isolated non-visualization of the gallbladder led us to diagnose cystic fibrosis by DNA analysis at 20-24 weeks of gestations. Four fetuses were homozygous DF508/DF508 and 1 was coumpound heterozygote DF508/G551D. All pregnancies were terminated and the gallbladder was found to be present but hypoplastic on pathological examination in all 5 fetuses. Several hypothese may be proposed to explain this finding. 1) a cystic duct obstruction from inspissated bile or mucus. Pathological examinations of the biliary tract were however normal in all fetuses.2) a contracted gallbladder. 3) an hyperechogenic bowel might have been associated earlier in pregancy and might have disappeared when ultrasound examinations were performed. In agreement with this hypothesis, one of the fetuses reported her was found to have an absent gallbladder associated with hyperechogenic bowel at 14 weeks of gestation whereas the anomaly of the gallbladder was isolated at 15 weeks of gestation. We suggest therefore to performe chromosomal and DNA analyses on amniotic fluid to exclude trisomy 21 and cystic fibrosis respectiviely when the gallbladder is not visualized at 22 weeks of gestation.

Non-invasive Prenatal Detection Of A Paternal Inherited Cystic Fibrosis Mutation In Maternal Plasma

C. González-González, M. García-Hoyos, M. J. Trujillo, M. Rodríguez de Alba, J. Gallego, I. Lorda-Sánchez, C. Ayuso, C. Ramos;
Department of Genetics. Fundación Jiménez Díaz, Madrid, SPAIN.

The discovery of the presence of circulating fetal DNA in maternal plasma and serum opens up new strategies to perform non-invasive procedures to obtain a reliable diagnosis without any risk for the fetus. In this way, it has been used for non-invasive prenatal diagnosis of fetal gender, Rh factor, and paternally inherited disorders like myotonic dystrophy.
The big potential of the detection of fetal DNA from maternal plasma by PCR lies in the possibility to avoid any risk for the fetus in the invasive procedure and its feasibility for a clinical application. Because of the maternal DNA contamination this kind of analysis is limited to those sequences of paternal origin and thus we attempted to detect only the paternal mutation.
In our study we have used the PCR method to demonstrate a non-invasive prenatal analysis, in maternal plasma, of an autosomal-recessive disorder like Cystic Fibrosis in which both parents have different mutations, having successfully detected a paternally inherited CF mutation in heterozygosis.
The interference of the maternal DNA during the PCR amplification represents the main disadvantage. At this point it is essential to find new strategies in order to obtain a non-invasive diagnosis with results as precise as the ones obtained by invasive procedures.

Microscopic fetal pathological examination can lead to unexpected diagnosis of cystic fibrosis

P. Y. J. Marcorelles¹, M. P. Audrezet², J. J. Chabaud³, P. Parent⁴, M. Le Bris⁵, M. De Braekeeler⁵, C. Ferec², N. Lagarde¹;
¹Service d'Anatomie Pathologique, CHU, Brest, FRANCE, ²Laboratoire de Génétique Moléculaire, CHU, Brest, FRANCE, ³Clinique Pasteur, Brest, FRANCE, ⁴Service de Génétique Médicale, CHU, Brest, FRANCE, ⁵Service de Cytogénétique CHU, Brest, FRANCE.

We are reporting a case of a 19 weeks gestational age fetus terminated for myelomeningocele discovered on early ultrasound examination . Parents were non related with no story of familial genetic disease. On microscopic examination of fetal pancreas, slight cystic dilatations of small pancreatic ducts were consistent with cystic fibrosis as these dilatations are known as the earliest histologic change described in the pancreas. Molecular analysis of the CFTR gene by DHPLC on the genomic DNA of the parents revealed the presence of the mutations deltaF508 and E60X, thus confirming this diagnosis.
Normal pancreatic development is caracterised by the increase of acinar number during pregnancy. In case of cystic fibrosis, acini become scarce with extensive diffuse fibrosis and excretory ducts may be distented with eosinophilic plugs in their lumina.. Others additional findings can be meconium ileus, or meconial peritonitis and on microscopic view widespread obstruction of exocrine glands ducts. However none of these morphological findings can be found in every fetal or perinatal case but becomes usual in older children.
To conclude, this case report points out the need of thorough fetal examination which can discover others abnormalities than the expected ones and allows a better pre-natal genetic counselling.

Screening for CFTR Gene Mutations and Polymorphisms in Patients with Chronic Pancreatitis

A. Nikolic¹, J. Kusic¹, D. Radojkovic¹, S. Lukic², T. Milosavljevic², A. Savic¹;
¹Institute of Molecular Genetics and Genetic Engineering, Belgrade, YUGOSLAVIA, ²Clinical Center of Serbia, Department of Gastroenterology, Belgrade, YUGOSLAVIA.

Chronic pancreatitis is an inflammatory disorder in which progressive and irreversible structural changes of the pancreas result in a permanent impairment of both exocrine and endocrine function. Both environmental and genetic factors are known to induce chronic pancreatitis. Recent reports have revealed genetic basis of chronic pancreatitis showing that mutations in Cationic Trypsinogen, CFTR and SPINK1 genes are associated with this disorder.
This study was undertaken to investigate the association of mutations and polymorphisms in CFTR gene with chronic pancreatitis in Yugoslav patients. Thirty-nine patients (alcohol-related pancreatitis in 29 and idiopathic pancreatitis in 10) were examined using the combination of PCR and subsequent direct (HA, PSM) and indirect (SSCP, DGGE) mutation detection methods.
F508del mutation was found in two patients (5.1%). 5T allele at Tn polymorphic site was not found in any of the analyzed patients, while frequencies of 7T and 9T allele were 85.9% and 14.1%, respectively. Eighteen patients (46.1%) had 2694T→G polymorphism in exon 14a, while three (7.7%) had 4002A→G polymorphism in exon 20. One patient showed also, in combination with 2694T→G polymorphism, the change in exon 6a, but this change should be defined through sequencing.
Obtained frequency of 2694T→G polymorphism in Yugoslav patients with chronic pancreatitis (25.6%) closely resembles its frequency in healthy Yugoslav population (29%). Still, obtained frequency of CFTR mutations is slightly increased in these patients which indicates possible association of mutations in CFTR gene with chronic pancreatitis, but further investigations on larger cohort of patients are required in order to confirm these findings.

Molecular epidemiology of cystic fibrosis in Brittany, France: a retrospective study from 1960

V. Scotet¹, D. Gillet², I. Duguépéroux², M. P. Audrézet¹, G. Bellis³, B. Garnier³, M. De Braekeleer¹^,2^,3, C. Férec¹;
¹INSERM EMI 01-15, Laboratoire de Génétique Moléculaire, Brest, FRANCE, ²Laboratoire de Cytogénétique, Brest, FRANCE, ³Ined, Paris, FRANCE.

Cystic fibrosis (CF) is the most common severe autosomal recessive disease that affects children in Caucasian populations. Characterized by pulmonary and digestive disorders, CF is caused by mutations in the CFTR gene. Near 1000 mutations have been identified worldwide. The aim of this study was to define the spatial and temporal distribution of CF and of its mutations in Brittany (western France) where the disease is frequent.
We retrospectively registered all the patients born in Brittany since 1960, by crosschecking different data sources. We contacted councils to obtain patients' residence place.
A total of 520 patients were registered. We assessed CF incidence according to administrative and ecclesiastic divisions and its evolution over decades. Incidence was 1/2630, with a west/east gradient which was confirmed over time (Finistère: 1/2071 vs. Ille-et-Vilaine: 1/3286). This high frequency may result from founder effects and genetic drift. Currently, incidence is decreasing mainly because of prenatal diagnosis. Moreover, we determined the mutations spectrum and their spatial distribution. We obtained an excellent detection rate (99.7%). Western Brittany presented a specific spectrum (1078delT, G551D, W846X, 4005+1G>A), whereas the eastern part show a spectrum more similar to the French one.
This study relates the regional specificities of the CFTR gene and highlights the disparities that existed in Brittany. This results from different isolation degrees and population movements. It is the first time that a so detailed study is performed in a large population. This better knowledge of CF epidemiology allows to improve diagnostic strategies and to refine genetic counselling.

Notification of Cystic Fibrosis as primary cause of death in Rio de Janeiro State, Brazil, from 1979 to 1998

C. L. A. Paiva¹, L. Janotti², C. Naurath³, S. R. Middleton⁴, R. M. Lugarinho⁵, S. R. dos Santos⁴;
¹Genetics and Molecular Biology Unit, Universidade do Rio de Janeiro(UNIRIO), Rio de Janeiro, BRAZIL, ²Universidade do Rio de Janeiro (UNIRIO), Rio de Janeiro, BRAZIL, ³Universidade do rio de Janeiro (UNIRIO), Rio de Janeiro (RJ), BRAZIL, ⁴Universidade do Rio de Janeiro (RJ), Rio de Janeiro (RJ), BRAZIL, ⁵Universidade do Rio de Janeiro, Rio de Janeiro (RJ), BRAZIL.

Cystic Fibrosis (CF) is an autossomal recessive disease mapped to 7q31-q32. Lung disease accounts for approximately 95% of its morbidity and mortality, with an incidence of 1 in 2,500 Caucasians. The aim of this work was to investigate the notification of CF as primary cause of death in Rio de Janeiro State (RJ) for 20 years. Our data were extracted from the Brazilian Data-SUS CD-rom entitled "Sistema de informacão sobre mortalidade/1979-1998". Our results show that 27 infants, younger than 1 year, died from CF in RJ from 1979 to 1988, and 13 infants died from CF from 1989 to 1998. The same sorts of results were observed throughout Brazil (252 cases from 1979 to 1988 and 178 cases from 1989 to 1998). For Brazil, Qui-square test indicates that these differences are significant (p<0.01), as well as for Rio de Janeiro State (p<0.05). For the age group 15 to 24 years the numbers of death notifications have increased. These results indicate that there was an improvement in the quality of life of CF patients. Eighty people out of 93 died from CF in Rio de Janeiro City and 13 deaths occurred in five smaller cities, although their permanent addresses were distributed among 12 different cities out of 31. It is worth mentioning that notification of CF as primary cause of death occurred in cities, but one, where there was at least one Faculty of Medicine. This may account for a better acknowledgement of CF and for a more accurate death notification.

Molecular-genetic analysis of polymorphic variants of GSTM1, CYP1A1 genes and CFTR gene mutations in patients with chronic pulmonary diseases from Bashkortostan

D. Yanbaeva, G. Korytina, O. Macarova, E. Khusnutdinova, T. Viktorova;
Institute of Biochemistry and Genetics, Ufa, RUSSIAN FEDERATION.

Chronic pulmonary diseases are one of the major widespread causes of illness. The search for pulmonary disease susceptibility genes is a complex problem, connecting with the influence of environmental factors in the pathogenesis of these disorders. Nevertheless, a large number of epidemiological studies suggest that genetic factors play a role. One possibility to account for a susceptibility to the effects of environmental pollutants may be genetic variations in the xenobiotic metabolizing enzymes such as glutathione-S-transferase M1 (GSTM1) and cytochrome P450 1A1 (CYP1A1). In addition, heterozygosity for predominant delF508 mutation of CFTR gene is associated with pulmonary disease such as chronic bronchitis and asthma.
In order to determine the possible role of detoxifying enzymes gene mutations and mutation delF508 of CFTR gene in the pathogenesis of pulmonary diseases we have studied 45 patients (22 with bronchopneumonia, 12 with chronic bronchitis, 7 with asthma and per 2 with bronchiectasis and bronchoobstructive syndrome) and compared the results with 68 subjects from control group. The GSTM1 0/0 genotype was found in 37% of affected individuals and in 49% in control subjects. There were no significant differences (p>0,05). For CYP1A1 locus the Val allele was detected in 7% of affected individuals and only in 3% in control, but the differences are not significant (p>0,05). CYP1A1 Ile/Val genotype increased approximately twofold in affected individuals (15%). We have not found an individual with delF508 mutation of CFTR gene. In future studies of multiple gene polymorphisms we should include other candidate genes and will take more affected patients.

Possibilities and barriers in the implementation of a preconceptional screening programme for cystic fibrosis carriers: a focus group study

F. Poppelaars¹, G. van der Wal¹, J. Braspenning², M. Cornel¹, L. Henneman¹, M. Langendam¹, L. ten Kate¹;
¹VU University Medical Center, Amsterdam, NETHERLANDS, ²University of Nijmegen, Nijmegen, NETHERLANDS.

Background. Since the identification of the cystic fibrosis (CF) gene it has become possible to perform CF carrier screening. Despite the positive results of pilot studies in various countries, carrier screening is not yet standard practice. The question arises as to whether this might be due to barriers in implementation.
Objective. The objective was to explore possibilities and barriers in the implementation of a nation-wide preconceptional CF carrier screening programme.
Methods. Sessions with two focus groups of CF patients and CF relatives, one focus group of people from the target population (couples planning to have children), and two focus groups of care-providers (general practitioners, and health care workers in the Municipal Health Services).
Results. In general, there is a positive attitude among CF patients and their relatives, the target population, and care-providers towards preconceptional CF carrier screening. The most important barriers in implementation are the problem of reaching the target population, the heavy workload of GPs, the limited knowledge about CF in general, and the absence of a preconceptional consultation setting.
Conclusion. Different intervention strategies will be necessary to overcome the various barriers in the organisation and execution of the screening. The positive attitude towards preconceptional CF carrier screening, in combination with the willingness of the care-providers to participate in providing (a part of) the screening programme, will make it easier to overcome the barriers.

Evaluation of the probe specificity used in INNO-LiPA CFTR: No false positives or cross-reactions with benign variants or rare mutations.

INNO-LiPA CFTR12 and INNO-LiPA CFTR17 + Tn respectively identify 12 and 17 CF-related mutations and their wild-type sequences, as well as the CBAVD-related polymorphism Tn. The technology used is a simple reverse hybridisation of amplified product on a nitro-cellulose strip carrying specific oligonucleotide probes as parallel lines. The amplified product is the result of an optimised multiplex amplification. Hybridisation and stringent wash occur at the same temperature and can be performed either manually or using Auto-LiPA. Both assays were successfully validated in a European multicenter study.
Individuals, homozygous for a mutant or a wild-type allele, will only hybridise with the corresponding probe, whereas individuals heterozygous for a particular mutation will hybridise with both the mutant and wild type probe for this mutation. We undertook to test the INNO-LiPA CFTR assay specificity for samples containing benign variants or rare CF-related mutations, of which the sequences are, covered by the INNO-LiPA CFTR mutant and wild type probes. The benign sequence variants were introduced by mutagenesis and controlled by sequence analysis. In addition, three clinical samples with proven presence of F508C and linked with the S1251N mutation were included. For the rare mutations, clinical samples were available. None of the benign variant sequences tested showed any false-positive reaction with the corresponding mutant probe on the strip; in all these cases, the wild-type probe remained positive. For the rare mutations, no cross-reactivity with the mutant probes was observed. We could conclude that in all these samples, the INNO-LiPA probe reactivities allowed correct clinical interpretation.

Prevalence Of Cftr Mutations In Newborns With Increased Irt Detected Through A Pilot Neonatal Screening For Cystic Fibrosis In The Piemonte Region

G. Restagno¹, L. Sbaiz¹, A. Gomez¹, E. Cocco¹, S. Bosso¹, A. Sedita², E. Bignamini³, L. Anfossi³, L. Leone⁴;
¹Molecular Genetics Service, Azienda Ospedaliera O.I.R.M.-S.Anna, Torino, ITALY, ²Molecular Genetics Service, Azienda Ospedaliera O.I.R.M.-S.Anna, Torino, ITALY, ³Cystic Fibrosis Center, A.O. O.I.R.M.-S.Anna, Torino, ITALY, ⁴Department of Clinical Pathology, Azienda Ospedaliera O.I.R.M.-S.Anna, Torino, ITALY.

Neonatal screening strategy. A two-step protocol combining the assay for immunoreactive trypsinogen (IRT) with the analysis of 30 mutations in the CFTR gene using the OLA-PCR-SCS. Neonates with two mutations are referred directly for clinical assessment and confirmatory sweat test; infants with one mutation are recalled for sweat test at age 4-5 weeks. A genetic counselling benefit is offered to parents when trypsinogen/DNA screening is performed. This strategy results in early and accurate diagnosis of cystic fibrosis but an excess of heterozygotes among neonates with hypertrypsinaemia has been reported.
Prevalence of heterozygosity in hypertrypsinaemic newborns. We have assessed the heterozygosity frequency among 50.956 children born from July 2000 to December 2001 and screened for CF. 996 (1.9%, i.e. 1/51) of those tested had an IRT level greater than the decisional threshold and were analysed for mutations. 19 were CF with a positive sweat test and 80 were carriers. Incidence of CF was 1/2682, leading to a carrier frequency of 1/26 while the estimated frequency of heterozygtes in children with hypertrypsinaemia was 1/9, three times greater than the general population. The number of different mutations in carriers is greater than in CF children (13 versus 10) and than in a cohort of 1574 newborns from our region, not selected by IRT, analysed in a previous pilot screening (10 different mutations). The identification of an excess of heterozygotes in hypertrypsinaemic newborns remains an important matter that can be managed only with effective counseling strategies in the context of newborn screening.

A. Schiller, J. Dunker, U. Larsson, M. Storgärds, A. Alderborn;
Pyrosequencing AB, Uppsala, SWEDEN.

Cystic fibrosis is a lethal recessive genetic disorder in Northern European populations affecting 1 live birth in 1600-2500. In contrast to many other recessive disorders, the carriers of CF mutations (1 in 25 Caucasians) have no biochemical or physiological alterations by which they could readily be identified. As a consequence the search for genetic markers became a matter of decisive importance. Thirteen years after the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and the characterization of over 990 mutations, CF is now to become the first disease targeted for population-wide genetic screening.
Pyrosequencing AB (Sweden) offers a PSQÔ 96 System well suited to meet the demand for research and routine testing of mutations in the CFTR gene, providing simple, rapid, cost efficient and extremely accurate detection, of all relevant mutations, including internal controls for each analyzed position. PyrosequencingÔ is a DNA sequencing technology based on real-time detection of pyrophosphate released upon nucleotide incorporation. Highly reliable CF assays have been designed and developed for all CFTR mutations with a population frequency of ³ 0.1% including point mutations as well as insertions and deletions. The assays are greatly condensed including multiplex design of both PCR and sequencing reactions. The assays have also been optimized to even include less common mutations in the same analysis simply by extending the number of nucleotide dispensations in each pyrosequencing reaction. In addition to these two approaches, the same assays have been used for population-based determination of carrier frequencies using allele quantification.

An automated high throughput screening system for detection of 33 clinical mutations and 7 SNPs in the cystic fibrosis gene

E. H. Schreiber¹, R. Tam¹, I. McLaughlin², S. J. Scharf²;
¹Celera Diagnostics, Alameda, CA, ²Applied Biosystems, Foster City, CA.

Cystic Fibrosis (CF) is an inherited disorder that affects children and young adults. Causative for respiratory disease and pancreatic dysfunction are mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We have previously offered an assay (for research use only) that detects 31 mutations in the CFTR gene. This single-tube assay is based on multiplex PCR amplification and subsequent detection of the alleles by oligonucleotide ligation assay (OLA). To meet both the list of essential mutations in the recently issued ACMG guidelines for population screening of CF carriers, and the need for high throughput screening, we revised the design of the PCR/OLA-based Cystic Fibrosis assay by including all required mutations and enabled detection on the Applied Biosystems 3100 Genetic Analyzer ®.
The new assay detects the following 33 mutant and normal alleles:
S549R, S549N, R553X, G551D, V520F, del I507, delF508, 3876delA, 1717-1 G->A, G542X, R560T, 3120+1G->T,R347P,R347H,I148T, W1282X,R334W,1078delT,3849+10 kbC->T, R1162X,N1303K,3659delC,3905insT,A455E, R117H,394delTT,2184delA,2789+5G->A, 1898+1G->A,621+1G->T,711+1G->T,G85E. Further, the assay detects these polymorphisms recommended for reflex testing: F508C,I506V,I507V and polyT in intron 8.
The instrument enables automated DNA sequencing and fragment analysis applications using an array of 16 capillaries. Up to 192 samples can be processed on two 96-well plates within 8 hours of unattended operation.The data are analyzed with Genotyper® software in an automated fashion for final review by the investigator. The assay works with purified genomic DNA and blood samples (Guthrie cards). We have tested a reference panel of 25 CF samples from Coriell laboratories and found that all samples were genotyped correctly.

Psychological impact of the introduction of a pilot for newborn screening of Cystic Fibrosis

T. Pàmpols¹, A. Maya¹, M. Puliol¹, F. Borja¹, T. Casals², J. Bellón³, R. Prats⁴;
¹Institut de Bioquímica Clínica.CDB.Corporació Sanitària Clínic, Barcelona, SPAIN, ²Institut de Recerca Oncoloógica, Barcelona, SPAIN, ³Consellería de Sanitat i Seguretat Social.Govern Balear, Palma de Mallorca, SPAIN, ⁴Departament de Sanitat i Seguretat Social.Generalitat de Catalunya, Barcelona, SPAIN.

The Institut de Bioqufmica Clfnica currently analyses 74,000 newborn/year for PKU , CH and since September 1999 CF as a pilot . The CF protocol is based in a three-tier system: a) inmunoreactive trypsine test at 2-5 days of life ,b) in testing positives a retest at 20-40 days, c) in retesting positives a DNA test for 31 mutations , sweat test and clinical examination .
The 171,693 newborns screened for CF have generated 2,500 consultations revealing anxiety or adverse emotional reactions :
-The request of the sample for the retest . In spite of the text of the letter has been carefully reviewed. Number of recalls 1,957 (1.14 % ).
-The communication of a positive retest . N= 480 (0.28 %) . At this point baby still can be heterozygous or normal , in fact only a 6.6 % of them will be CF. Parents began to be aware of the severity of the disease as well as of the limitations of treatment . Benefits of genetic counselling are hardly perceived at this time.
- The finding of the mutation in only one allele. It still raises two possibilities a CF genetic compound or an heterozygous . Results of sweat test and medical exams are very important . But the screening of the whole gene takes a long time and the anxiety persist.
- The access of the parents to Internet is specially traumatic.
In conclusion all this psychosocial components will be considered in the final pilot evaluation.

DNAH3: Characterization ofthe sequence and mutation search in patients with Primary Ciliary Dyskinesia

J. L. Blouin¹, C. Gehrig¹, M. Armengot², M. Rutishauser³, M. Jorissen⁴, D. Jeganathan⁵, L. Bartoloni¹, C. Rossier¹, G. Duriaux-Sail¹, N. Scamuffa¹, H. Mitchison⁵, C. D. DeLozier-Blanchet¹, S. E. Antonarakis¹;
¹Medical Genetics, University Hospital and School of Medicine, Geneva, SWITZERLAND, ²Otorhinolaryngology Service, University Hospital, Valencia, SPAIN, ³Universitätskinderklinik, Basel, SWITZERLAND, ⁴Human Genetics, University Hospital, Leuven, BELGIUM, ⁵Royal Free and University College Medical School, University College, London, UNITED KINGDOM.

Primary Ciliary Dyskinesia (PCD) is an autosomal-recessive disorder with an incidence of 1/20’000, characterized by dysmotility of cilia/flagella. In addition to upper respiratory tract infections, bronchiectasis and male subfertility, 50% of cases show situs inversus (Kartagener syndrome, KS). Our linkage analysis (Blouin et al. EurJHumGenet 8:109-118) indicated extensive locus heterogeneity, in concordance with the variety of ultrastructural defects of cilia/flagella. We identified potential loci on chromosomes 3p, 5p, 8q, 11p, 15q, 16p, 17q and 19q, colocalizing with genes for dyneins, the major proteins of dynein arms defective in 50% of patients and therefore strong candidates for PCD. Our most suggestive/nearly significant linkage interval on chr.16p near marker D16S748 (NPL score =2.96 on families with dynein arm deficiency) contains the gene for axonemal dynein heavy chain DNAH3. We report here characterization of the DNAH3 gene and mutation search by sequencing all exons in patients with PCD. Genomic and transcriptional organization were determined by RT-PCR and searches of genomic sequences and ESTs. The gene spans 200 Kbp and is composed of 62 exons coding for a 4410 residues protein, highly homologous to paralogues and orthologues. Mutation search in 7 patients with PCD showing allele segregation compatible with linkage to DNAH3 revealed 9 exonic variants. Screening of the translated variants in more than 100 control population chromosomes of same ethnic origin suggests that amino-acid substitutions P1197L and A3529D observed in patients with KS and ciliary ultrastructural deficiency on either inner dynein arm/stroke or both dynein arms might be pathogenic mutations.

Primary Ciliary Dyskinesia: Mutation analysis in Dynein light chain genes mapping to chromosomes 1 (Hp28) and 22 (DNAL4)

C. Gehrig, C. Albrecht, G. Duriaux Saïl, C. Rossier, N. Scamuffa, C. DeLozier-Blanchet, S. E. Antonarakis, J. L. Blouin;
Medical Genetics, University Hospitals and School of Medicine, Geneva, SWITZERLAND.

Primary Ciliary Dyskinesia (PCD) is an autosomal recessive disorder affecting the ultrastucture of respiratory tract cilia and spermatozoa flagella, causing dysmotility to immobility and male sterility. When respiratory infections and bronchiectasis are associated with situs inversus (half of cases), the disease is referred to as Kartagener Syndrome. PCD is genetically heterogeneous, with only a few mutations having been described to date. Our linkage analysis in 31 families (Blouin et al. 2000, EurJHumGenet 8:109-118) failed to reveal a single major locus, but suggested linkage to several regions containing candidate genes for PCD. These candidates include dynein genes, the major elements of the dynein arms, such as the novel human light chain genes Hp28 (chromosome1p35) and DNAL4 (22q12-13). For Hp28, there is suggestive evidence for linkage in the appropriate genomic interval on chr.1p (NPL=1.37). We screened 54 unrelated patients for mutations in these two genes with SSCA; electrophoretic variants were sequenced. No obvious pathogenic mutations were revealed, but nucleotide variations were observed at both loci. In Hp28 (previously screened in 7 patients: Pennarun et al., 2000, EurJHumGenet 9:P1584), two nucleotide changes were detected: A65V in exon 2 was later confirmed as a frequent polymorphism in a control population (CEPH), whereas the second variant was located in an intron (IVS3-10). In DNAL4, variants were observed in intron1 (IVS1+31) and in intron2 (IVS2+20). These studies suggest that neither Hp28 nor DNAL4 are frequently mutated in PCD.
Study supported by the Swiss National Funds and the Carvajal Foundation.
Authors CG and CA contributed equally.

Genotype-phenotype correlations in a group of Yugoslavian adult cystic fibrosis patients

D. Radivojevic¹, M. Djurisic², P. Minic², M. Guc-Scekic², T. Lalic²;
¹Mother and Child Health Institute of Serbia, Belgrade, YUGOSLAVIA, ²Mother and Child Health Institute of Serbia "Dr Vukan Cupic", Belgrade, YUGOSLAVIA.

Cystic fibrosis (CF) is the most common serious genetic disorder among Caucasian populations, mostly diagnosed in childhood. Since the number of adult CF patients is significant, usually two groups of patients are defined: first, with late diagnosis (at the age of 16 or older) and second, patients diagnosed before their 16th birthday (early diagnosis). In a group of 164 Yugoslavian (YU) CF patients whose DNA samples were analyzed for the presence of CFTR mutations, 9,76% (16/164) were adults. Three of 19 CFTR mutations identified in YU CF patients were found in adult patients: dF508, R334W and A120T. Complete genotypes were determined in 43,75% (7/16) of adult CF patients, while 9 of them (56,25%) had at least one unidentified CF allele. Genotype dF508/dF508 was found in 31,25% (5/16) of adult patients, another 5 of them (31,25%) had dF508/non-dF508 genotype, and the group of 6 adults (37,50%) had non-dF508/non-dF508 genotype. Patients were compared considering their clinical data (sex, age at diagnosis, age, lung function tests, sweat test, pancreatic status) and determined genotypes.In this work, authors will discuss possible connection between genetic features and manifestations and prognosis of the disease in analyzed group of YU adult CF patients.

N. Mosse, K. Mosse, G. Tsukerman;
Institute for Hereditary Diseases, Minsk, BELARUS.

Several years ago the prevalence of delta-F508 mutation has been studied in 2,598 newborns chromosomes from Belarus population. The frequency of delta-F508 mutation heterozygote carriers was 0.014 or 1:72. As this mutation accounted for 62% of CF alleles in our patients, so CF incidence had to be 1:8000. Then a whole population pilot screening of 146 701 newborns, based on initial estimation of IRT at cut-off 70 ng/ml followed by direct CF gene analysis of positive samples, confirmed the expected frequency, which is much less then 1:2500. Examination of 232 CF chromosomes from Belarus patients for the presence of the mutations, originally identified in European population, has shown that delta-F508 mutation covered 61,2% of CF chromosomes, N1303K - 2.5%, G542X - 1.3%, W1282X - 0.9%, R334W - 0.4%, R347P - 0.4%, S549N - 0.4%, R553X - 0.4%. Mutations G551D, R560T, 621+1GT, 520F, DI507, 1717-1GA, R117H, A455E, 3849+10kb were not found. Recently a large genomic deletion, spanning introns 1-3 of CFTR gene, was identified and termed CFTRdel2,3(21kb). Our data show that this mutation, frequently observed in Central and Eastern Europe, is particularly common in Belarus - 6% of all CF chromosomes. Thus the total detection rate of the found mutations is 73,7%. The first trimester prenatal diagnosis has been performed in 34 CF-families by means of direct mutation analysis and combined analysis using the intragenic polymorphism IVS6a-GATT in the CFTR gene. Eight affected fetuses and 15 carriers were detected.

Analysis of candidate genes in the region of the cystic fibrosis modifier 1 (CFM1) locus.

D. Markiewicz¹, X. Yuan¹, M. Corey¹, R. Rozmahel², P. Durie¹, J. Zielenski¹, L. Tsui¹, &. The CF Modifier Collaborative Group³;
¹The Hospital for Sick Children, Toronto, ON, CANADA, ²University of Toronto, Toronto, ON, CANADA, ³., European and North American CF Centers, ON, CANADA.

Cystic Fibrosis (CF) is an autosomal recessive disease, most common among Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. There is considerable genotype-phenotype association but clinical variation observed in CF is also determined by additional, secondary genetic modifiers and environment factors. We previously demonstrated the presence of a CF modifier locus (CFM1), contributing to the predisposition of meconium ileus (MI). Association studies (transmission disequilibrium tests, TDT) using MI and non-MI CF families with at least one affected child led to refinement of the region on human chromosome 19, region q13.2. Of 18 local markers tested, the strongest association with MI was detected for microsatellite in intron one of the KCNN4 gene. While detailed analysis of KCNN4 is in progress, we have extended our analysis of genes in its immediate proximity. A gene (NM019108) immediately distal to KCNN4 has been analyzed as candidate for CFM1. This predicted gene consists of 14 exons, spanning 23 kb. It is partially confirmed by EST data. Sequencing analysis of the exons of this gene from MI-discordant sibpairs revealed 3 non-coding sequence variants. We have performed preliminary studies one of these variants (648+46T/G) and an intragenic, complex microsatellite marker in MI and non-MI CF patients and families, respectively. No significant allelic association could be detected with these two markers but studies will continue with the remaining ones.

Rare Mutations and Polymorphisms of the Cystic Fibrosis Gene in Patients with Alcoholic and Early-Onset Idiopathic Chronic Pancreatitis

B. Steiner¹, K. Truninger²^,3, H. E. Blum³, B. Müllhaupt², B. Seifert⁴, S. Gallati¹;
¹Human Molecular Genetics, Bern, SWITZERLAND, ²Department of Internal Medicine, Zürich, SWITZERLAND, ³Department of Medicine II, Freiburg, GERMANY, ⁴Dept. of Biostatistics, Zürich, SWITZERLAND.

The pathogenesis of chronic pancreatitis (CP) is poorly understood. Genetic studies identified mutations in the cationic trypsinogen gene, the serine protease inhibitor, Kazal type 1 gene, and the cystic fibrosis (CFTR) gene in patients with CP of different etiologies. The aim of the present study was to perform a comprehensive DNA testing of the CFTR gene in patients with early-onset ICP (ICP) and alcoholic CP (ACP). Furthermore, single nucleotide polymorphisms (SNP‘s) induced alterations of motif scores of the serine/arginine-rich (SR) proteins SF2/ASF, SRp40, SRp55, and SC35 were analyzed using score matrices. CF causing mutations were found in 4/14 ACP patients (29%; 6.5 times the expected frequency, p < 0.05) and in 5/13 ICP patients (39%; 8.7 times the expected frequency, p < 0.05). 2 (14%) ACP and 4 (31%) ICP patients were compound heterozygous. The frequency of SNP’s was increased in patients with ACP and ICP. The SNP nt2694T/G in exon 14a reduces a SRp40 score motif and generates a new SRp55 high score motif. The SNP nt4521 in exon 24 eliminates a SC35 motif. The intronic SNP nt3041-92G/A eliminates a SRp55 and a SC35 motif and SNP nt3601-65C/A eliminates a SRp55 motif but increases a SC35 motif score. Mutations and SNP’s of the CFTR gene are associated with ACP and ICP. The most frequently identified SNP‘s clearly change the motif scores of different SR proteins, supporting the idea that the combination of mutations and SNP’s may be important in the pathogenesis of CP by affecting splicing efficiency.

Routine analysis of the CFTR IVS(8)T polymorphism discloses two pathogenic mutations

C. Kraus¹, L. Naehrlich², S. Mattes³, A. Reis¹;
¹Institute of Human Genetics of the Friedrich-Alexander University, Erlangen, GERMANY, ²Pediatric Hospital of the Friedrich-Alexander University, Erlangen, GERMANY, ³Pediatric Hospital Thueringen-Clinic, Saalfeld, GERMANY.

Cystic fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, caused by hundreds of mutations in the cystic fibrosis conductance transmembrane regulator (CFTR) gene. Routinely, we use an Oligonucleotide ligation assay (Applied Biosystems) to screen for 31 mutations in the CFTR gene, including 24 of the most common. Additionally, CFTRdele2,3(21kb), which occurs with a frequency of 1% in our population is analyzed by multiplex PCR. With regard to CBAVD the polymorphism IVS(8)T were examined by PCR/restriction anaylsis and subsequent PAGE. Generally, the poly-T tract in intron 8 exists in three variants, 5T, 7T, and 9T. The 7T and 9T variants generate a predominantly normal transcript, whereas the 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males (CBAVD). The existence of further allelic variants (3T, 6T and 8T) was shortly mentioned in an abstract last year. From our CF patients two out of 125 exhibited an abnormal migration pattern in the PAGE of IVS(8)T. In one patient PAGE results suggested in addition to a 9T allele a 6T allele. Sequencing, however, disclosed the 6T allele as a 7T allele carrying a splice site deletion (IVS-1delG). In the other patient PAGE displayed a 7T and 9T allele with an abnormal heteroduplex formation. In this case a IVS8-2A->C transversion was discovered in cis with the 7T allele. We therefor conclude that any atypical T variant has to be carefully analyzed for the underlying mechanism.

Prevalance of MEFV gene mutations in FMF phenotype II patients with renal amyloidosis

B. Balci¹, E. Yilmaz¹, S. Gucer², N. Besbas³, K. Tinaztepe², A. Bakkaloglu³;
¹Hacettepe University, Faculty of Medicine, Dept. of Medical Biology, Ankara, TURKEY, ²Hacettepe University, Fac. of Medicine, Dept. of Pediatric Pathology, Ankara, TURKEY, ³Hacettepe University, Fac. of Medicine, Dept. of Pediatric Rheumatology and Nephrology, Ankara, TURKEY.

Familial Mediterranean Fever (FMF) is an inherited inflammatory disease that is principally recognized in Jewish, Armenian, Turkish and Middle Eastern Arab populations. The disease is characterised by recurrent febrile episodes of fever and serosal inflammation manifested by sterile peritonitis, pleuritis and synovitis. Amyloidosis of the AA type is the most severe manifestation of the disease.
Individuals with two MEFV mutations may be divided into three clinical categories; in the group of phenotype I FMF patients; the amyloid development appears after the beginning of symptoms like, fever, abdominal pain and inflammatory attacks. In some cases, renal amyloidosis may develop before other manifestations of FMF. In this group of patients which are named as phenotype II, the disease is asymptomatic until the development of renal amyloidosis. The last category, phenotype III, includes clinically unaffected gene carriers.
The aim of the present study is to confirm the presence of MEFV mutations in phenotype II patients. 25 paraffin blocks from patients with renal amyloidosis and who were deceased which are thought to be phenotype II were analyzed for MEFV gene mutations. The distribution of the four most common mutations among phenotype II patients was; 38 % for M694V, 8 % for M680I, 4 % for V726A and 4 % for E148Q. The distribution of the four common mutations among FMF phenotype I patients (M694V 51.55 %, M680I 9 %, V726A 2,88 % and E148Q 3,55 %) was not significantly different from that found in phenotype II patients.

The differential contribution of MEFV mutant alleles to the clinical profile of familial Mediterranean fever

R. Gershoni-Baruch¹, R. Brik¹, M. Shinawi¹, A. Livneh²;
¹Rambam Medical Center, Haifa, ISRAEL, ²Sheba Medical Center, Tel-Aviv, ISRAEL.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. Five sequence alterations (M694V, V726A, M680I, M694I and E148Q), in the MEFV gene, account for the majority of FMF chromosomes. The wide clinical variability of the disease has been related to MEFV allelic heterogeneity. M694V homozygotes have a severe form of the disease. Mutations E148Q and V726A have reduced penetrance. The clinical features, associated with the M680I and the complex V726A-E148Q allele, are not well defined. This study further characterizes the phenotypic profile associated with the major MEFV mutations. We investigated 220 FMF patients, in whom both FMF alleles have been identified, and found that different genotypes are characterized by a specific allelic related clinical profile and penetrance. Homozygotes for the M694V mutation and the complex V726A-E148Q allele are the most severely affected and often endure renal amyloidosis. Homozygotes for the M680I and V726A alleles and compound heterozygotes for either the M694V or the V726A-E148Q alleles in combination with either the E148Q, the V726A or the M680I alleles are significantly less severely affected. The morbididity associated with the complex V726A-E148Q allele by far outweighs that associated with the V726A allele, bearing evidence to the fact that the E148Q mutation is not a benign polymorphism. These findings increase our understanding of the role of allelic variability in disease expression.

Male gender increases susceptibility to amyloidosis in FMF patients homozygous for the M694V-MEFV mutation.

R. Gershoni-Baruch¹, R. Brik¹, M. Shinawi¹, A. Livneh²;
¹Rambam Medical Center, Haifa, ISRAEL, ²Sheba Medical Center, Tel-Aviv, ISRAEL.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and serositis and predisposition to renal amyloidosis. Five sequence alterations (M694V, V726A, M680I, M694I and E148Q), in the MEFV gene, account for the majority of FMF chromosomes. Differences in the clinical expression have been partly attributed to MEFV allelic heterogeneity. The M694V/M694V genotype is associated with a severe form of disease. Otherwise, a role for additional genetic and/or environmental modifiers has been proposed. Of these, male gender was found to influence disease penetrance and susceptibility to renal amyloidosis. The aim of this study was to further investigate the contribution of sex to the phenotypic profile, in FMF. We thus performed a sex-phenotype correlation analysis on a large cohort consisting of 124 FMF patients who were all homozygous for the M694V mutation, thus precluding the weight of allelic heterogeneity. Although a preponderance of male patients was documented (73:51; 1.4), the overall male:female ratio was significantly higher among patients with amyloidosis (32:15; 2,1) than among patients without amyloidosis (41:36; 1.1). The calculated FMF severity scores were equally high among male and female patients (9.5±3.0 and 9.7±2.8, respectively). Male gender acts as an MEFV-independent factor increasing susceptibility to renal amyloidosis (OR = 2.37; 95% CI = 1.06-5.26).

Familial Mediterranean fever - Results of MEFV gene analysis and detection of two novel mutations

J. Trübenbach¹, J. Decker¹, B. Zabel², G. Wildhardt¹;
¹Bioscientia, Ingelheim, GERMANY, ²University Children´s Hospital, Mainz, GERMANY.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder (MIM 249100) characterized by recurrent episodes of fever and serosal inflammation with peritonitis, arthritis, erythema. Amyloidosis causing renal failure is the most severe complication of the disease which primarily affects populations of Mediterranean extraction.
In 1997, a gene for FMF (MEFV) was identified. The MEFV gene product (pyrin/marenostrin) consists of 781 amino acids encoded by 10 exons. The specific protein function remains unknown but the expression in polynuclear leukocytes suggests an essential role in inflammation processes.
Several FMF studies helped to identify MEFV mutation hot spots. Based on these data, a stepwise procedure was applied for FMF routine diagnostics. Stage 1 consisted in sequencing of exon 10 where most MEFV mutations are found. Stage 2 meant was analysis of exons 2, 3 and 5, the location of several additional FMF-associated mutations. In Stage 3 the remaining exons were sequenced.
Our diagnostic strategy applied to 94 cases resulted in the identification of one MEFV-mutation in at least 50 cases. In 31 patients the genetic cause of the disease could be verified. In addition, two novel mutations associated with FMF were found. The mutation E148V (in exon 2) affected one patient, the mutation I591T (in exon 9) was detected in two unrelated patients.
Our three-level FMF diagnostic study proved to be efficient without missing the identification of novel mutations. The availability of the molecular test has major clinical implications considering the prognostic value of some MEFV-mutations and the variability of the disease phenotype.