ESHG Posters 10

M. Omrani¹, S. Salehi Gargari²;
¹Cytogenetic and Molecular Medicine unit, Uromia, IRAN (ISLAMIC REPUBLIC OF), ²Imam Reza hospital, Uromia, IRAN (ISLAMIC REPUBLIC OF).

Despite molecular advances in carrier detection and prenatal diagnosis, in many rural and border area of Iran and Turkey, hemophilia is still a major problem. RFLP/PCR method for factor VIII mutations screening were carried out. Questionnaires for all patients and their families who are receiving clothing factor were obtained. The necessary DNA was gathered using peripheral blood and Protinase K method. PCR and RFLP analysis were carried out according to the literature. Interestingly in many of the villages still it is possible to see affected female with hemophilia A. Even in some families more than 5 members are affected. This happening mostly because of the consanguineous marriage. In more than 70% of the cases the carrier females are marrying with theirs affected first cousin. Therefore their entire child is affected. Despite prenatal diagnosis facilities in the region, still they are not seeking any of them. In more than 60% of the cases family has noted to the disease by accident such as dental operation or general surgery. This makes the duty of the Cytogenesis and counseling centers much heavier. Even it gives an idea to health care organizers that if they going to offer any help in establishing any center in developing countries check many factors like cultural, educational and traditional background of needed people. In this study we are going to present the results of our study in carrier detection as well as sharing our experience with other collogues for working in rural area.

O. Sideleva¹, T. Ivaschenko¹, J. Ostankova¹, M. Petrova², T. Gembitskaya², A. Orlov³, V. Baranov¹;
¹Ott's Institute of Obstetrics and Gynecology, St.Petersburg, RUSSIAN FEDERATION, ²Institute of Pulmology, St.Petersburg, RUSSIAN FEDERATION, ³St. Olga's Childrens Hospital, St.Petersburg, RUSSIAN FEDERATION.

The metabolism of exogenous substances (xenobiotics) via Phase I detoxification enzymes (cytochromes P450) and Phase II detoxification enzymes (glutathione S-transferases; N-acetyltransferases) demonstrates substantial individual variability, thus predisposing to different multifactorial diseases, such as atopy and asthma. Polymorphism analysis of CYP1A1, GSTM1, GSTT1, GSTP1 and NAT2 genes in 109 asthmatic patients and 90 control individuals from the Northern-Western Russia was carried out. Individuals with GSTM10/0, GSTT10/0 genotypes were at approximately 8,5-fold higher risk of developing asthma (OR=8,50; 95%CI: 3,623-10,956). Proportion of GSTM10/0, GSTT10/0, GSTP1 A/A individuals appeared to be significantly increased in the group of asthmatics (19,3%) than in the controls (4,4%, p=0.0007), with an OR of 5,13 (95%CI: 1,849-14,239). The frequency of the Ile-Val polymorphism of CYP1A1 gene and the most common NAT2 polymorphisms was similar in the control group and in asthmatic patients. Null-genotypes of both genes - GSTM1 and GSTT1, combined with GSTP1 A/A genotype might be suggested as genetic factors predisposing to atopic asthma.

A. Kesari, G. S. Pandey, B. Mittal, S. K. Pal;
Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, INDIA.

Telemedicine is an umbrella term that encompasses any medical activities involving an element of distance. It is described as the next frontier in the delivery of health care. Videoconferences and databases facilities are effectively utilized for monitoring public health activities. Telemedicine is creating new way for patients and clinicians to interact and access specialist services which are not locally available. Developing countries have acute shortage of specialist and their services are concentrated in major cities, so in such circumstances, telemedicine can play a crucially role in bridging thegap. A survey was conducted to access the concept of Telemedicine and Telegenetics counseling via Internet among 20 clinicians attending a genetic counseling course, conducted in our department. Participants were from various places of India and most of them were either pediatricians (60%) or gynecologists from peripheral hospitals. A questionnaire was designed to know the general awareness regarding Telemedicine and Telegenetics i.e. the idea of genetic counseling through Internet in India. Cent-percent of the participants agreed that Internet could play a major role in genetic counseling and patient’s management at the peripheral level. Most of clinicians thought that Telegenetics can provide immediate accessibility to experts and will greatly enhance the scope of genetic services in our country. More than 95% of the clinicians agreed to join Tele-consultant group and emphasized the idea of making an interactive websites so that they could be actively connected via net with the tertiary care hospitals after they complete the genetic counseling course.

A Reliable Quantitative Method For Rapid Detection Of Serum Phenylalanine: Application in PKU screening

S. Vallian¹, H. Moeini¹, E. Barahimi²;
¹Isfahan University, Isfahan, IRAN (ISLAMIC REPUBLIC OF), ²Khatam University, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Phenylketonuria (PKU) is an inherited metabolic disease, which is characterized by increased level of serum phenylalanine (Phe). The quantitative measurement of Phe in the patient’s serum is necessary to confirm the disease, and to distinguish PKU from other forms of hyperphenylalaninemia. In this study, we report a novel method for quantitative measurement of Phe in serum. The method was developed using Proteus Rettgeri bacteria, which produce phenylalanine deaminase (transaminase). These bacteria could convert Phe into phenylpyrovate in the culture medium, which can be easily detected by spectrophotometer using ferric chloride reagent. In this method, the standard curve for Phe ranging from 2-30 mg/dL was linear. This method was applied to 33 PKU samples (serum and blood spots collected on filter paper), which their serum Phe had already been tested using the Gutheri bacterial inhibition assay (GBIA) and the HPLC method. The results were essentially similar. The advantage of this method over GBIA is its ability to measure the serum Phe quantitatively without the use of expensive beta-(2-thienyl)-DL-Alanine. This method is now used in our PKU screening program in parallel with the GBIA and HPLC test. So far, no false positive result has been seen using this method, which is occasionally happens with the GBIA. This method provides a fast, simple, cost effective and quantitative assay for PKU screening which is amenable to automation.

Conteporary state of genetic counselling in Czech Republic is on the relatively good level.We have sufficient network of genetic centres (2/1 000 000 inhabitants), number of clinical geneticists -physicians (approx.6/1 000 000) and other graduated in clinical geneticists(8/1 000 000).The level of prenatal diagnoses is on conveniant level: biochemical screening -Triple Test, age indication (35 years), ultrasonic examination (3 times during pregnancy, at least).Due to this secondary prevention 60% of children with inborn chromosomal aberrations are not born.Molecular genetics is quickly developing the number of possible diagnoses is growing.Nevertheless prenatal diagnoses open many ethical, psychological and social problems:The most problematic are the simplified information about pathologic results of biochemical screening, about pathologic karyotype of the fetus (describtion of possible clinical signs of the future child), about possible termination of pathologic pregnancy, about uninformative family with genetic disease caused by rare unusual mutation of well known gene, information about balanced chromosomal translocation of one of the parents etc.Even more tactful information should be to parents who have pregnancy after IVF, waiting a long time for the future child.Even more difficult is refusing of women, who offers donnation of ovum and who for instance balanced translocation or genetic unfavorable family history.The genetic counselling should be everytimes empathetic, fully informative and non- authoritative.

Genetic diseases are unique in that their prevalence varies significantly in different ethnic groups or geographical regions. When designing genetic diagnostic kits, we apply an approach that takes into account these unique properties, to increase detection rate and decrease cost.
Our first EthnixÔ panel is aimed towards Jews of Eastern European origin - the Ashkenazim, which are unique in terms of demographic history and genetic structure. Emerging from a limited number of founders, they have undergone an outstanding expansion of population size and have had the tendency to marry within the religion. As a result, Ashkenazi Jews share a very similar genetic background. This similarity includes some recessive mutations, which, when homozygous, lead to progressive deterioration of one‘s health, life-long disability and death. Relatively few mutations account for most of these diseases.
Our single nucleotide primer extension technology - ProntoÔ, provides a user-friendly, rapid and accurate method for mutation detection. Using ProntoÔ, we developed a testing panel for the most common disease-associated SNPs in Ashkenazim. It tests for mutations which cause Tay-Sachs, CF, Canavan, Gaucher, Fanconi anemia, Bloom syndrome, Familial Dysautonomia, Neimann-Pick, Mucolipidosis type IV and Glycogen Storage disease type I. We apply the same approach in our cancer-predisposition panel for Ashkenazim, which tests for the few cancer-causing mutations in Brca1, Brca2 and APC that are prevalent in this population.
Another panel targets the CFTR gene with mutations that are common throughout Europe as well as mutations that are specific for particular regions.
Future EthnixÔ panels will target additional populations.

Extremely Low Prevalence of Factor V Leiden, FII20210A and FXIIIV34L in Chinese Population

H. Zheng¹, C. C. Tzeng², C. Butt², E. Randell¹^,3, Y. G. Xie¹^,3^,4;
¹Department of Laboratory Medicine, Memorial University of Newfoundland, St. John's, NF, CANADA, ²Department of Pathology, Chi-Mei Foundation Medical Centre, Tainan, TAIWAN REPUBLIC OF CHINA, ³Department of Genetics, Memorial University of Newfoundland, St. John's, NF, CANADA, ⁴Department of Pediatrics, Memorial University of Newfoundland, St. John's, NF, CANADA.

The Factor V Leiden (FVL) and prothrombin G20210A (FIIG20210A) are the two most commonly recognized risk factors for venous thrombosis. In contrast, a gene variant of factor XIII, FXIIIV34L, has been reported to confer protection against arterial thrombosis. The prevalence of these three variants has been determined for most Caucasian populations, however, distribution of these variants in other populations has been poorly studied, especially in Asian populations. To determine the prevalence the three variants in the Chinese population we analyzed 500 unrelated and healthy women from Taiwan area of China. The carrier frequencies, allele frequencies, and the frequencies for all possible genotypes of FVL, FIIG20210A and FXIIIV34L from these samples are given in the Table. The corresponding data for 500 healthy and unrelated individuals from the Newfoundland Caucasian population is included for comparison (Our unpublished data). Our results show that Chinese population has a 9-fold lower prevalence of FIIG20210A, a 24-fold lower prevalence of FVL and more than 250-fold lower prevalence of FXIII34L compared with our Caucasians controls. The present study demonstrates a significant and highly ethnic-dependent distribution of the three variants in the Chinese population compared with Caucasians. The dramatically lower prevalence of FVL and FIIG20210A in the Chinese population suggests that these two gene variants play significantly less role as genetic predisposing factors in Chinese with venous thrombosis. Furthermore, the dramatically lower prevalence of FXIIIV34L in Chinese also suggests that this gene variant might not be a useful candidate allele for further study of arterial thrombosis in Chinese.


	Genotype	Chinese (n = 500)	Newfoundlander (n = 500)
	V/V	499 (99.8%)	259 (51.8%)
FXIIIV34L	V/L	1 (0.2%)	209 (41.8%)
	L/L	0 (0%)	32 (6.4%)
Carrier Frequency		0.2%	48.2%
Allele Frequency of 34L		0.1%	27.3%

	G/G	499 (99.8%)	494 (98.8%)
FIIG20210A	G/A	1 (0.2%)	6 (1.2%)
	A/A	0 (0%)	0 (0%)
Carrier Frequency		0.2%	1.2%
Allele Frequency of 20210A		0.1%	0.6%

	W/W	499 (99.8%)	476 (95.2%)
FVL	W/L	1 (0.2%)	24 (4.8%)
	L/L	0 (0%)	0 (0%)
Carrier Frequency		0.2%	4.8%
Allele Frequency of FVL		0.1%	2.4%

Our first experiences with molecular genetic diagnosis of malignant hyperthermia in the Czech Republic

I. Valaskova, I. Grochova, J. Kadlecova, B. Ravcukova, Z. Lukas;
University of Children´s hospital, Brno, CZECH REPUBLIC.

Malignant hyperthermia (MH) is an autosomal dominant, potentially letal pharmacogenetic predisposition which is considered to be of the main causes of death during anesthesia. Triggering by volatile anesthetics and depolarizing muscle relaxans in susceptible patients leads to an abnormally high release of intracellular Ca²⁺ in sceletal muscle.
In more then 50% of the affected families, MH is caused by mutations in the ryanodine receptor of sceletal muscle( RYR1) encoded by gene on chromosome 19q12.-13.2. Mutation analysis in human RYR1 gene has shown until now about 25 mutations which could be associated with MH predisposition.
To date, MH diagnosis has not been provided in the Czech Republic. We have just started the mutation analysis in RYR1 gene to those patients who either had a documented hyperthermic crisis or positive in vitro muscle contracture test ( IVCT). The most aim our programe is to discover MH families members at risk. This work is supported by grant from the Internal Grant Agency of Ministry of Health in the Czech Republic (IGA MZ ND 6865-3)

L. A. Livshits¹, V. D. Zukin²;
¹Institute of Molecular Biology and Genetics, Kiev, UKRAINE, ²"ISIDA - IVF" clinic, Kiev, UKRAINE.

We have elaborate the pilot genetic testing program based on the information obtained by population screening in different regions of Ukraine. The program involved three categories of populations: 1) members of families with a high risk monogenic hereditary disorders; 2) patients of IVF centers; 3) persons of fertile age from common populations before marriage or before and during pregnancy.
For the first category we have screened the most common mutations and linkage polymorphisms: 320 CF-families, 129 DMD-families, 100 PKU-families and 85 SMA-families. Early prenatal diagnosis was performed based on CVS testing in high-risk families: CF - 66 cases, DMD - 81, PKU - 100, SMA - 85, HD - 2, Fra X - 1, Hemophilia A - 5.
2-nd category. The screening of CFTR gene mutations and long arm chromosome Y microdeletions was performed for 105 infertile man involved in ICSI program. The chromosome Y microdeletions were detected in 5 cases. In two cases we have detected CFTR gene mutation - delF508.
3-rd category. The most common mutations of CFTR, PAH and SMN genes were screened in 175 unrelated persons. 28 persons have been determined as heterozygous carriers of following mutations: delF508 - 5 persons, R408W and Y414C - 2 persons, exon 7 deletion of cenSMN gene - 21 persons. The genetical consulting, partner testing and prenatal diagnosis were recommended for all families at risk.
We have suggested that the such genetic testing programs will be useful for prevention of most common hereditary disorders in Ukraine.

Mutational analyses of potassium channel gene KVLQT1 and identification of a novel long-QT syndrom mutation (T309I)

J. Kadlecova¹, B. Ravcukova¹, I. Valaskova¹, R. Gaillyova¹, T. Novotny², B. Semrad²;
¹University of Children´s Hospital, Brno, CZECH REPUBLIC, ²Dept. of Cardiology, Faculty Hospital, Brno, CZECH REPUBLIC.

Long-QT Syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on ECG and presence of syncope, seizures, and sudden death. Five genes have been implicated in Romano-Ward Syndrome, the autosomal dominant form of LQTS: KVLQT1 (11p15.5), HERG (7q35-36), SCN5A (3p21-24), KCNE1 and KCNE (21q22). Mutations in KVLQT1 and KCNE1 also cause the Jervell and Lang-Nielsen Syndrome, a form of LQTS associated with deafness, a phenotypic abnormality inherited in an autosomal recessive form.
We used mutational analyses to screen LQTS patients from 23 families for mutations in the KVLQT1 gene, a potasium channel subunits that account for most of RWS cases. In six unrelated LQTS patients, singlestrand conformation polymorphism analyses identified aberrant conformers. DNA sequence analyses identified two missense mutations G325R and T309I, localized in the transmembrane domains S6 and pore region. CT substitution resulting in a tyrosine to an isoleucine transition at codon 309 (T309I) was novel. We also identified two single nucleotide polymorphisms in four unrelated LQTS patients, F484F and Y171Y, which have not been previously reported. Further functional studies will be required to determine what effect each of these changes may have on KVLQT1 channel function.
Genetic screening using mutational analysis can improve presymptomatic diagnosis. Familial and sporadic cases affected by mutations in all LQT genes can now be genetically screened to identify individuals at risk of the development of the disorder. This work is supported by the Internal Grant Agency of the Ministry of the Health in the Czech Republic (IGA MZ 5718-3)

Periconceptional use of folic acid in The Netherlands: from science to public health policy

M. C. Cornel¹, L. T. W. de Jong-van den Berg², H. E. K. de Walle³;
¹VU University Medical Center, Amsterdam, NETHERLANDS, ²Social Pharmacy and Pharmacoepidemiology, Groningen Institute for Drug Studies (GIDS), Groningen, NETHERLANDS, ³EUROCAT registration, Medical Genetics, University of Groningen, Groningen, NETHERLANDS.

Introduction:
Neural tube defects (NTD) have a multifactorial etiology. Randomized controlled trials have shown that one exogenous factor, folic acid, reduces the risk of NTD with 50-70%. Dutch health authorities advised in 1993 that women who want to become pregnant should take a 0.4-0.5 mg folic acid tablet daily from 4 weeks before conception till 8 weeks thereafter. A mass media campaign in 1995 informed the public of this advice. In the Netherlands 80-90% of pregnancies are planned. The aim of the campaign was to reach at least 70% of women wishing to conceive and that 65% of these women would use it appropriately.
Awareness and use of folic acid:
At first or second antenatal visit, women filled out a questionnaire. The survey was performed 5 times. The percentages that used folic acid are mentioned in the table. In 2000, 26% of low educated vs. 47% of high educated women used folic acid during the entire advised period. Although an impressive proportion of women complies to the advice, the aim (85%*70%*65%=39% appropriate folic acid use) has not been attained. Yet the percentage of users is higher than other figures reported in the literature so far.
Public health policy:
Although fortification of foods is obligatory in some, and admitted in most countries, the Dutch Health Council advised in 2001 against fortification of regular foods. The Dutch minister of Health stated that the present policy will be continued and attempts to inform women preconception will be intensified.

Percentage of pregnant women that used folic acid
Year	% any time of advised period	% during entire advised period
1994	7.8	0.4
1995	21	4.8
1996	38	15
1998	62	36
2000	61	36

Genetic diagnostic laboratories show a growing interest in the systematic application of quality assurance (QA) in genetic diagnostic laboratories. Many laboratories now participate in external quality assessment (EQA) schemes. In order to evaluate the status of QA implementation in genetic diagnostic laboratories, a survey on QA and accreditation was sent to 206 laboratories from 32 European countries and 5 laboratories in Australia and the USA.
151 participants responded (73 %) to the survey. In order to minimise errors, an appropriate QA system needs to be worked out for the whole procedure of a genetic analysis. Such system is established in 110 of the surveyed laboratories (73%): 45 laboratories follow an international standard, 52 a local or national standard, and 13 laboratories did not specify which guidelines are being used. However, only 82 laboratories are officially inspected: 52 are inspected by a national body, and 30
are accredited by an internationally recognised accreditation body. Thus, it is clear that at present there is no harmonisation on QA among laboratories. The new ISO 17025 standard, which is accepted by most national and international accreditation bodies, could therefore be an important step towards harmonisation of laboratory practice. This new standard also includes requirements for reporting the results. The reporting results of the latest CF EQA schemes illustrate that less than 15% of the laboratories include all the items required by this new standard.

Frequency Of Cx26 Mutations In Deaf Newborns Detected Through A Pilot Universal Neonatal Screening In The Piemonte Region

L. Sbaiz¹, S. Bosso¹, R. Albera², P. Tavormina³, L. Leone¹, G. Restagno¹;
¹Azienda Ospedaliera O.I.R.M.-S.Anna, Torino, ITALY, ²II Clinica ORL, University of Torino, Torino, ITALY, ³ORL Service, O.I.R.M., Torino, ITALY.

Deafness in young infants is commoner than cystic fibrosis, galactosaemia or congenital hypothyroidism, affecting one in 1000 children. Early identification of deaf babies and remedial education is beneficial, with better expressive and receptive language, speech, and social and emotional development.
Pilot screening scheme. The primary screening tool is the measurement of transient evoked otoacustic emissions (TEOAE), administered to all newborns before dismissions from the neonatal center. If the TEOAE test is pathological is repeated three times, then the brain-stem auditory evoked response (BAER) will be measured. When deafness is present, the baby is referred to a specialistic center for audiological assessment and for the application of a rehabilitative protocol and molecular test is performed. Molecular analysis. In developed countries, deafness has an important genetic origin, and at least 70% of genetic cases are autosomal recessive non-syndromic, 80% of which due to mutations in the GJB2 gene. In this pilot screening genetic counselling is offered to parents of deaf babies. In a previous study with direct sequencing of the GJB2 gene, we found mutations in 43 of 74 cases (58%) of severe/profound deafness. The most common mutation is 35delG, accounting for 74% (30/43) of all deafness alleles. The mutations V95M and E47X were detected four and two times, with a relative frequency of 4.6 and 2.3% respectively. Other mutations (L90P, 290-291insA, 333delAA, W24X, E119del, M34T, Q80P) were detected once. We also found four variants of unknown significance (V37I, F83L, R127H, V153I) and the novel alleles M162V, K224Q and R184Q .

Prospective study on Genetic Testing Services quality assurance and harmonization in EU

D. Ibarreta, A. Bock, E. Rodriguez-Cerezo;
IPTS (JRC, European Commission), Seville, SPAIN.

The use of clinically meaningful genetic tests for humans -for diagnostic, confirmatory and predictive purposes - is expanding in all European countries. A few different national regulatory frameworks already exist but there is no harmonization on a European level to ensure a sufficient level of quality, safety and efficacy of genetic testing services in Europe, which is required by society. Genetic testing services are not covered by Council Regulation (EEC) No 2309/93 laying down Community procedures for the authorization and supervision of medicinal products for human and veterinary use or Directive 98/79/EC on in vitro diagnostic medical devices, which applies only to products to be marketed.
There also seems to be a need to set up a European laboratory network to cover rare diseases. Given that research into genetic mutation is so complex, only a few laboratories are in a position to supply an appropriate test for certain diseases, whereas most European countries have at least one laboratory to deal with the more common diseases. To avoid this difficulty, a network of European laboratories, covering the different diseases and genes, could be set up.
JRC (EC) is currently analyzing the potential need and technical options for harmonization and quality assurance of Genetic Testing Services in the EU, taking in consideration already ongoing activities at European level (EQA scheme for HD, Concerted Action for CF, EMQN, etc).

T. K. Kascheeva¹, N. V. Vokhmyanina², I. V. Butomo², O. Romanenko², V. S. Baranov¹;
¹Ott's Institute of Obstetrics & Gynecology, Saint-Petersburg, RUSSIAN FEDERATION, ²Diagnostic Centre (medical genetics), Saint-Petersburg, RUSSIAN FEDERATION.

Prenatal Down's syndrome biochemical screening in St.Petersburg is carried out since 1997, as a double test. Total beta-human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) were studied in 123400 pregnancies dated 1997-2001. Proportion of population covered is more than 84 % in 2000-2001 . About 7 % of screened group had a high risk of Down‘s syndrome (DS) which was calculated by homemade software based on likelyhood ratio and age risk. 74 out of total 191 pregnancies with Down’s fetus were screened . Detection rate in women less 35 was 61 %, in advanced age women - 85 %. According to these findings 15-17 weeks of pregnancy are the most informative period for DS testing . Repetitive biochemical testing carried out after 15-17 weeks usually reduces detection rate and make the decision on invasive karyotyping more ambiguous. These initial results also favor
the use of our homemade test -system produced by “Alkor-Bio” (St.Petersburg) supplemented by new software for Down’s syndrome screening "MedInformatika" (St.Petersburg).

Internal Quality Control: Development of new best practice guidelines for diagnostic molecular genetics laboratories.

S. A. R. Stenhouse¹, S. Patton²;
¹Northern Molecular Genetics Service, Newcastle-upon-Tyne, UNITED KINGDOM, ²EMQN, Manchester, UNITED KINGDOM.

Internal quality control (IQC) refers to all of the policies and procedures which a laboratory puts in place to ensure the error-free processing and analysis of all samples. It covers many areas from staff training to documentation of experiments and reporting and is vital to the provision of a high quality molecular genetics diagnostic service.
A best practice meeting on IQC was held in Edinburgh on 3^rd April 2001 under the auspices of the European Molecular Genetics Quality Network (EMQN) as a satellite meeting of the UK Clinical Molecular Genetics Society‘s spring meeting. From discussion at this event a draft set of best practice guidelines has been developed. The general principles of effective IQC (including the associated problems)will be discussed and illustrated with reference to the draft guidelines.

B. Georgieva¹, A. Todorova¹, I. Tournev², A. Ashikov¹, V. Mitev³, I. Kremensky¹;
¹Laboratory of Molecular Pathology, Hospital of Obstetrics and Gynecology, Medical University, Sofia, BULGARIA, ²Alexandrovska Hospital, Clinic of Neurology, Medical University, Sofia, BULGARIA, ³Department of Chemistry and Biochemistry, Medical University, Sofia, BULGARIA.

Limb-girdle muscular dystrophy type 2C (LGMD2C), a subgroup of sarcoglycanopathies, is caused by mutations in the gamma-sarcoglycan gene, localized on 13q12. Among the described mutations, causing an autosomal recessive muscular dystrophy, a “private” Gipsy mutation C283Y (transition G->A in codon 283) is detected.
The extensive field work in 300 Gypsy living places in Bulgaria revealed about 40 Gypsy patients clinically diagnosed as LGMD. Considering the fact that the Gypsy minority is
an isolated population with high percent of consanguinity and having in mind the autosomal recessive type of inheritance of LGMD2C, a raised carrier frequency of C283Y mutation was expected.
Several screenings for determining the percentage of heterozygosity of C283Y mutation among Gypsy minority were performed. The applied method was direct amplification on dry blood spots from Guthrie cards followed by SSCP. Heterozygotes were confirmed by RsaI restriction digestion.
Screening on 400 Gypsy newborns from Northeast Bulgaria showed high percentage of heterozygosity- 2.25%. Screening on 300 volunteers of a reproductive age from Sliven showed very high percentage of heterozygosity - 7.7%. Investigation on 126 volunteers of a reproductive age from Senovo (Northeast Bulgaria) showed that 22 of them were heterozygotes (17.46%). Screening on 50 Gypsy newborns from Stara Zagora region (Middle Bulgaria) showed no heterozygotes.
The above data show that the disease seemed to be geographically localized to Eastern Bulgaria. It is important to construct a precise map of the regions with high carrier and/or disease frequency. Such regions should be with priority in the Bulgarian healthcare system.

Stable EBV transformed B lymphocyte cell lines derived from residual clinical blood for PE/QA of molecular genetic testing

J. C. Beck¹, S. H. Bernacki², K. Snow³, V. M. Pratt⁴, K. Monaghan⁵, A. K. Stankovic⁶, L. O. Williams⁶, K. Matteson⁷, F. V. Schaefer⁸, M. Friez⁹, A. E. Shrimpton¹⁰, D. H. Farkas¹¹, T. W. Prior¹², L. Wasserman¹³, E. C. Cole¹⁴, T. T. Stenzel²;
¹Coriell Institute for Medical Research, Camden, NJ, ²Duke University Medical Center, Durham, NC, ³Mayo Clinic, Rochester, MN, ⁴LabCorp, Raleigh, NC, ⁵Henry Ford Hospital, Detroit, MI, ⁶Centers for Disease Control and Prevention, Atlanta, GA, ⁷University of Tennesse Medical Center, Knoxville, TN, ⁸H.A. Chapman Institute of Medical Genetics, Tulsa, OK, ⁹Greenwood Genetic Center, Greenwood, SC, ¹⁰SUNY Upstate Medical University, Syracuse, NY, ¹¹Motorola Life Sciences, Pasadena, CA, ¹²Ohio State University, Columbus, OH, ¹³University of California San Diego, San Diego, CA, ¹⁴DynCorp Health Research Services, Durham, NC.

Positive control material for performance evaluation and quality assurance (PE/QA) of diagnostic molecular genetic testing (MGT) is difficult to obtain for many genetic diseases. To determine whether control material could be derived from residual blood collected for routine clinical MGT, 51 bloods were collected for EBV transformation. Eleven cell lines representing 5 genetic disorders were successfully cryopreserved. Results from a logistic regression model indicate that sample age and anti-coagulant (ACD or EDTA) were statistically significant predictors of transformation success; age and sex of the patient were not (p<0.05; samples collected in ACD and stored for fewer days were more likely to transform). Sample volume, hemolysis, whether or not the tube had been opened and prior storage temperature were included in the model. Successful transformation was achieved in samples up to 13 (EDTA) or 14 (ACD) days old and/or with as little as 1 ml blood from both opened and unopened tubes. Acceptable storage conditions were ambient or 4°C for samples <7 days old and 4°C only for samples 8-14 days old. Average time to transformation was 44.0 ±2.5 (SEM) days for ACD samples and 61.3 ±10.8 (SEM) days for EDTA samples. Stability of mutations was verified in ten cell lines after five 10-fold expansions in culture. Each cell line was sent to 5-6 outside MGT laboratories. All mutations were correctly identified by several methods, indicating that samples of this type are likely to be suitable for use as PE/QA material. (Funded by the Centers for Disease Control and Prevention)

Genetic screening of Familial Mediterranean Fever mutations in the Greek population

B. Gkretsi¹, C. Maratheftis¹, M. Vasilakou¹, G. Arlapanos¹, K. Groutidis¹, C. Deltas², C. Yapijakis³, K. Lamnissou¹, K. Groutidis¹;
¹Dept of Biology, University of Athens, Athens, GREECE, ²Dept of Molecular Genetics, The Cyprus Institute of Neurology & Genetics, Nicosia, CYPRUS, ³Dept of Neurology, University of Athens, Medical School, Athens, GREECE.

Familial Mediterranean Fever (FMF) is an autosomal recessive disease that primarily affects populations surrounding the Mediterranean basin e.g. Armenian, Jewish, Arab and Turkish populations. FMF is characterised by recurrent episodes of fever accompanied by abdominal pain, pleuritis and arthritis. The most dangerous complication of FMF is amyloidosis that can lead to end-stage renal failure. The gene for FMF (MEFV) was cloned and missense mutations were found to be responsible for the disease. About 20 mutations have been identified so far, some of them being very freequent. The aim of this study was to investigate the carrier rates of the common MEFV mutations among 250 healthy members of the Greek population. The studied group was consistent only of Greeks whose parents were also of the same ethnicity. Two FMF mutations, V726A and M694V, were considered to be the most common in Greek patients from earlier studies. Our results indicated that none of the studied healthy individuals was carrier of any of these two mutations. We may conclude that the frequency of FMF mutations is extremely low in Greeks. It is possible that the previously identified few FMF Greek patients from others were of different origin.

S. Bräutigam¹, A. Kujat¹, P. Kirst¹, J. Seidel², Ü. Lüleyap³, U. G. Froster¹;
¹University of Leipzig, Leipzig, GERMANY, ²University of Jena, Jena, GERMANY, ³University of Cukurova, Adana, TURKEY.

Phenylketonuria (PKU, OMIM S 261600; McKusick 1986) is one of the most common autosomal recessive disorders in Europe and related to mutations in the PAH gene. The frequency of heterozygotes in Europe is 1:50. The basic defect in PKU is phenylalanine hydroxylase deficiency. The disease is characterised by an accumulation of phenylalanine in blood and nervous system, which leeds to mental retardation. A diagnosis early after birth is very important because the disorder is effectibly treatable by an adequat diet. A disease positiv neonatal screening requires molecular investigations to provide a solid basis for genetic councelling. This is particularly important for adult patients with PKU who are planning their own family. This group of individuums is interested in a rapid and reliable molecular diagnosis of their individual mutation status and the exclusion of a heterozygous mutation in their partners.
Denaturing high-performance liquid chromatography (DHPLC) is a fast and sensitive method for mutation screening which has been applied for mutation detection in various disease related genes. This method has not been previously applied to PKU. Therefore we established DHPLC for PKU mutation screening followd by automated sequencing to analyse rapidly the complete coding sequence of the PAH gene in a total of 125 PKU patients from Saxonia and Turkey. We identified 40 different mutations and polymorphisms in a total of 250 PAH-allels. The mutation detection rate with DHPLC was approximatly 98%. DHPLC has proved to be a fast and reliable method for mutation screening in PKU.

Screening for Cys 282Tyr and His 63Asp mutations of HFE gene in populations of Volga-Ural region of Russia

R. I. Fatkhlislamova, L. Berdina, E. Khusnutdinova;
Institute of Biochemistry and Genetics, Ufa, RUSSIAN FEDERATION.

Hereditary hemochromatosis (HH) is an autosomal recessive disease affecting iron metabolism commonly found in whites. Populations of northern European origin show the highest frequency of HH, with 1 in 300 individuals affected. Two sites of point mutations in the HFE gene - Cys282Tyr and His63Asp - are associated with greater than 90% of HH cases. In presence no information is available on the frequency of HH in Volga-Ural region. To define a carrier frequency we screened for Cys282Tyr and His 63Asp mutations of HFE gene in 6 populations of Volga-Ural region of Russia: Udmurts, Mordvins, Tatars, Bashkirs, Chuvashis and Russian from Bashkortostan. The frequency of the Cys282Tyr mutation is highest in Udmurts and Russians - 13,1% and 10,6% respectively, that corresponds to northwestern European populations. In Mordvins, Chuvashis and Tatars heterozygosity for Cys282Tyr is 7,3%, 7,14% and 6,3% respectively and is absent in Bashkirs. Heterozygosity for His63Asp ranges from 17,7% in Tatars to 31,7% in Mordvins and 31,9% in Russians. The His63Asp mutation is less frequent (occurring in 20,8%-24,6%) in Bashkirs, Chuvashis and Udmurts. A small percentage (0%-3,6%) was found to be homozygous for His63Asp and 12,2% in population of Mordvins. We revealed one case of compound heterozigoty for both mutations in Chuvashis population. Taking into account high frequency for heterozygous Cys282Tyr and His63Asp mutations, we propose that frequency of HH in populations of Volga-Ural region corresponds to European populations, so there is need of screening of subgroups at risk to promote early diagnosis and therapy of this disease.

H. S. Stewart, F. Haydon;
Department of Clinical Genetics, Churchill Hospital, Oxford, UNITED KINGDOM.

Familial fatal insomnia (FFI) is a rare, autosomal dominant progressive prion disease. It is characterised by neuronal degeneration of selected thalamic nuclei and progressive insomnia. Onset ranges from 37-61 years and the average duration of the disease is 13 months. It is associated with a asp178-to-asn mutation of the prion protein gene (PRNP), when the amino acid at position 129 is methionine.
We present the case of a family, in which the father was diagnosed with FFI at the age of 50 years and died 2 years later. Two sons in their second decades attended for genetic counselling, unaware of their father’s diagnosis. The wife of one son was 19 weeks pregnant. Issues surrounding possible predictive and prenatal diagnosis were discussed as for other late-onset neuro-degenerative disorders.
We discuss the issues relating to the possible transmissible nature of this prion disease and the obstetric management of our patient, with reference to the public health implications of prion diseases.

M. Sitska¹, T. Ilus¹, P. Ilisson¹, K. Kuuse¹, P. Tammur¹, T. Reimand¹, A. Ehrenberg²;
¹Medical Genetic Center of the Clinicum of the University of Tartu, Tartu, ESTONIA, ²Women's Clinic of the Clinicum of the University of Tartu, Tartu, ESTONIA.

Basic statistics. Area: 45214 km2. Population: 1,46 million.
Birth rate in 2000: 13 119 newborns. Maternal age at delivery  35….9.5 % (1227 women). Currently about 7% pregnancies undergo invasive prenatal diagnosis (mostly amniocentesis) in Estonia. Incidence of Down Syndrome (SD) before prenatal screening was started (1990-1994) was 1: 700.
Screening .Chromosome anomalies are screened for advanced maternal age ( 35) since 1995. In 2001 , 48% of women  35 had fetal karyotypes.
During the period when prenatal diagnosis has been used (1995-2001) 29 cases (52%) SD of advanced maternal age risk group have been diagnosed prenatally. During the last three years 72% of the SD cases have been detected prenatally.
Maternal serum screening (double test) is not widely used in Estonia: it is routinely offered since autumn 1998 in Tartu, since 2000 in southern Estonia and since 2001 in Tallinn and other part of Estonia. In 2001, 31% pregnant women in Estonia were monitored. In period of 1998 - 2001 altogether 6500 screening tests were done. Positive serum screening was indication for amniocentesis (fetal karyotyping) in 411 (6,4%) cases. Chromosomal abnormalties were detected in 7 (1:59 ) cases, SD in 3 cases.
Conclusion. Incidence of Down Syndrome in Estonia since prenatal screening was started in 1995 is 1: 942. In order to reduce the rate even further, a greater percentage of pregnancies in age group  35 have to be monitored by maternal serum screening. In the future first trimester screening is currently under development in Estonia .

Role of Certified Reference Materials and the Standardisation System in In Vitro Diagnostics

The In Vitro Diagnostica- Medical Device (IVD-MD) directive (Directive 98/79/EC) requires traceability of calibrators and control materials to reference measurement procedures and/or reference material of higher order. According to the VIM [1] traceability is defined as "property of the result of a measurement or the value of a standard whereby it can be related to stated references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties". Hence standards reference materials to support the traceability chain and well understood measurement procedures play an important role in achieving traceability.
It is evident that in the field of clinical chemistry complexities of measurands, the biological variability and commutability have to be taken into account. Otherwise the effect of reference methods and materials on standardization will be limited. Nevertheless the IVD-MD directive is a call to improve comparability of measurement results through more structured and understood approaches for standardization.
In this lecture, the traceability chain will be explained and two approaches towards standardisation using a CRM are discussed and will be compared. The certification process including definition of uncertainty values for both, homogeneity and stability as well as the contribution of characterisation will be presented.
[1] International Vocabulary of Basic and General Terms in Metrology, ISO, 1993

Variations in termination rates in pregnancies diagnosed with Klinefelter syndrome: Data from a cross European study (DADA)

I. Nippert¹, B. Eiben², P. Miny³, J. Horst⁴, F. Louwen¹, T. Marteau⁵, C. Mansfield⁵, M. Reid⁶, D. Kirwan⁷, S. Walkinshaw⁷, M. van Diem⁸, C. Verschuuren-Bemelmans⁸, S. Garcia-Minaur⁹, C. de Vigan¹⁰;
¹Frauengesundheitsforschung, Muenster, GERMANY, ²Institut fuer Klinische Genetik Nordrhein, Oberhausen, GERMANY, ³Universitaetskinderklinik, Basel, SWITZERLAND, ⁴Insitut fuer Humangenetik, Muenster, GERMANY, ⁵Guy's Campus, Kings College, London, UNITED KINGDOM, ⁶University of Glasgow, Glasgow, UNITED KINGDOM, ⁷Liverpool Women's Hospital, Liverpool, UNITED KINGDOM, ⁸Rijksuniversiteit Groningen, Groningen, NETHERLANDS, ⁹Clinica Materno-Infantil, Barakaldo, SPAIN, ¹⁰INSERM, Paris, FRANCE.

Introduction: Klinefelter syndrome (KS) occurs approximately in 1 per 800 male live births. 10%-20% are identified by prenatal diagnosis (PD). Most cases are detected incidentially when PD has been performed because of an increased risk for Down syndrome. Because KS is likely to be unfamiliar to the general population, information given about KS may be of crucial importance for parents' decisions about whether or not to continue with the pregnancy.
Methods: A systematic review of the case notes of all KS diagnosed up to 24 weeks of gestation in 8 European regions in 5 European countries (France, Germany, The Netherlands, Spain and the UK) was conducted. The variables documented included maternal age, parity, gestational age at diagnosis, speciality of health professional providing information before and after PD.
Results: Details of 111 pregnancies and their outcome were obtained. Mean maternal age was 36.9 (±4.6) years, 44.1% pregnancies were terminated. Across the 8 European regions termination rates varied between 76.9% and 0%. Using multivariable logistic regression analysis, the only significant predictor of continuation of the pregnancy was the speciality of the health professionals conducting post diagnosis counseling: the affected pregnancy was more likely to continue when post diagnosis counseling involved only a genetics specialist (RR 2.42 (95% CI 1.14 to 5.92)).
Discussion: There is an association between whether or not a woman terminates a pregnancy affected by an unfamiliar fetal anomaly and the professional background of the health professional providing post-diagnostic counseling. The causal nature of this association remains to be determined.

P. De Marco, M. Calevo, A. Moroni, A. Raso, E. Merello, A. Cama, V. Capra;
G. Gaslini, Genova, ITALY.

Impairment of folate metabolism have been observed in families with Neural Tube defects (NTD). The thermolabile 677CT and 1298AC polymorphisms in MTHFR (methylenetetrahydrofolate reductase) gene have been implicated in the pathogenesis of NTDs, but these mutations can explain only in part the protective effect of folate on NTD. Therefore, other defects in folate metabolism such defective carriers could be involved in the ethiology of NTD. Recently, the human folate carrier (RFC-1) gene has been isolated and characterized. A common polymorphism A80G, changing a histidine with an arginine, in the exon 2, has been reported. In this population-based study, we examined the impact of the RFC-1 A80G variant on NTD risk and the potential interaction between this polymorphism and MTHFR A1298C mutation. We report that the RFC-1 A80G variant is common in the Italian population (0.47). Nevertheless, the allelic frequency was higher among NTD cases and their parents. Heterozygous patients and mothers have OR of 1.72 (95% CI 0.96-3.11) and 1.86 (95% CI 0.68-5.27), respectively. More sensitive risk was calculated for the 80GG genotype of cases (OR=2.35; 95% CI 1.21-4.58). On the contrary, the heterozygous genotype of the mothers and both heterozygous and homozygous genotypes of the fathers did not seem to be significant risk factors. According to multifactorial basis of NTDs, we found that combined genotypes for MTHFR A1298C and RFC-1 A80G polymorphisms of cases (1298AC/80GG and 1298CC/80AG) results in greater NTD risk than heterozygosity and homozygosity for RFC-1 A80G variant alone.

M. Vincent¹, A. Pujol², D. Olivier¹, P. Calvas²;
¹Hôpital Purpan, CHU, Toulouse, FRANCE, ²Service de Génétique Médicale, Hôpital Purpan, CHU, Toulouse, FRANCE.

PAX6, a paired box transcriptor factor, is considered as the master gene control for morphogenesis of the eye. Human PAX6 mutations are associated with a range of ocular abnormalities, including aniridia, various anterior segment defects and foveal hypoplasia.
We carried out a mutational analysis of the PAX 6 gene in 54 unrelated patients with aniridia or one of various closely related syndromes. Despite an association of several methods a deleterious variation was evidenced in only 30 patients : twenty four different mutations, 16 of which are novel, were found.
The spectrum of PAX 6 mutations in our population is highly homogenous with 96% of all mutations leading to premature truncation of the protein (8 nonsense and 4 splice site mutations, 11 insertions and deletions) and only one missense mutation (4%).
Examination of the phenotype did not allow to recognise significant differences whatever the protein was deprived of one or another of its functionnal domains. We present 22 mutations in association with common recognisable aniridia phenotypes and 2 in association with atypical phenotypes : a missense mutation (R19P) in an individual with an unusual microphtalmia-sclerocornea phenotype and a splice site mutation (IVS4+5G>C) in a family presenting a panocular defect associated with a congenital nystagmus.
Our observations support the concept of dosage effects of the PAX 6 mutations as well as presenting evidence for variable expressivity. Genotype-phenotype correlation in ocular defects related to PAX 6 mutations remains difficult despite the increase of observations.

J. Santavy¹, V. Gregor², A. Santava³;
¹Palacky University, Olomouc, CZECH REPUBLIC, ²Dept. of Medical Genetics, Prague, CZECH REPUBLIC, ³Palacky University Hospital, Olomouc, CZECH REPUBLIC.

In our republic, biochemical screening for NTD, AWD, Down’s syndrome and trisomy 18 in the 2nd (AFP, hCG, mE3) or the 1st (AFP, PAPP, free bhCG) trimester of gravidity is available to pregnant women. All of them are examined using specialised ultrasound three times during pregnancy. Women over 37 years are offered fetal karyotyping by cultivation of amniotic or chorionic cells, by FISH or by capillary electrophoresis. Prenatal diagnosis is established in 12 large genetic centres and in several small private clinics. Amniocentesis is done in many gynaecological departments with the experience in fetal medicine. All these investigations are fully paid by Health Insurance.
Main principles of our screening policy are: (1) general availability and (2) voluntarity. Biochemical screening applies the following policies: (3) multimarker screening with computer evaluation including nuchal translucency and (4) cut-off for positivity 1:250 – 1:300.
In 2000 nearly 60,000 specialised sonographical investigations (0,6 % efficiency), 8,000 chromosomal investigations (3 % efficiency) and 1,200 DNA analysis (12 % efficiency) were performed, two last investigations in selected patients only. When we compare effectivity of these three prenatal programs for detection of chromosomal aberration, then only maternal age reasons indicated 1.4 % of them, biochemical screening 4.6 % and specialised ultrasonography 11.6 %. Selectivity for Down’s syndrome was 78 % at FP of 5 % and RT 1:50. These numbers differ in individual departments.
Altogether 425 foetuses with severe types of chromosomal aberrations and congenital defects were found by methods of prenatal diagnosis in 2000.

H. E. K. de Walle¹, L. Henneman², J. B. G. M. Verheij³, F. A. Beemer⁴, J. J. S. Broertjes⁵, C. P. Springer⁶, L. P. ten Kate⁷;
¹EUROCAT, Rijksuniversiteit, Groningen, NETHERLANDS, ²Vrije Universiteit, Amsterdam, NETHERLANDS, ³Dept of Medical Genetics, RUG, Groningen, NETHERLANDS, ⁴Dept Medical Genetics, Utrecht, NETHERLANDS, ⁵Centre for Beta Didactics, RUU, Utrecht, NETHERLANDS, ⁶LUMC, Leiden, NETHERLANDS, ⁷AZVU, Amsterdam, NETHERLANDS.

In 1995, a small group of people in the Netherlands formed the "Initiative Group Community Genetics’. Community Genetics concerns the application of medical genetics to the benefit of as many people as possible and plays an active role in going out to the community. Community genetics involves a wide scope of activities, including genetic education, genetics in primary care, genetic screening, and registration of genetic anomalies and genetics for disadvantaged groups.
Very recently, as a logical consequence, The Dutch Association of Community Genetics (NACG) was founded (2001). Its aim is to stimulate responsible applications of medical genetics in society. Promoting scientific research, encouraging public education and discussion, and organising conferences and workshops pursues this goal.
At this moment the NACG has about 80 members and includes a variety of professions and disciplines, involving both research and application. General practitioners, clinical geneticists, health care workers, midwives, medical psychologists, and epidemiologists are only a few examples.
The NACG just had its first yearly meeting and the intended policy for the nearby future was discussed and approved. A leaflet has been developed, a newsletter (2 times a year) and website are planned. This is done to increase familiarity with community genetics, the NACG and its ideas, and to support the development of community genetic centres at academic universities. Furthermore, the NACG promotes public education and will be programming scientific meetings every year, as they did the last five years.

A. Cambon-Thomsen¹, I. Hirtzlin², C. Dubreuil¹, N. Préaubert³, J. Duchier¹;
¹Inserm U 558, Toulouse, FRANCE, ²Inserm SC11 and U 357, Paris, FRANCE, ³Inserm U 357, Paris, FRANCE.

Biobanks correspond to different situations: research and technological development, medical diagnosis, therapeutic activities. Their status is not clearly defined. The aim of the work was: 1) to make a typology of the different situations encountered and 2) to investigate the way ethical implications were dealt with in various contexts. Data from a survey in 6 EU countries (France, Germany, Netherlands, Portugal, Spain, UK) have been collected in the framework of a European Research Project (EUROGENBANK). A total of 147 structures with biobanking activity were explored through questionnaire and interviews. Results: most of investigated structures belong to public or private non for profit sector, that have a key role for biobanking. This activity is increasing in all countries because few samples are discarded and genetic activity is growing. The size of collections is variable: lots of small collections, few very large ones; purposes of collections are often research or research and healthcare mostly in the context of disease studies. Specific budget is very rarely allocated to biobanking and costs not often evaluated; samples are usually provided free of charges; gift and exchanges are the common rule. Good practice guidelines are generally followed and quality controls performed but quality procedures are not always clearly explained. Associated data are usually computerised (samples traceable or identified). Biobankers generally do not favour centralisation of samples, rather that of data. A European legal and ethical harmonisation is considered likely to facilitate international collaborations. A series of recommendations from the EUROGENBANK project have been issued.

Population Screening, Diagnosis and Care of Mentally Retarded Children with Community Genetic Approach : Indian Experience.

L. N. Mehta, U. P. Dave, N. T. Shetty;
Centre for Research in Mental Retardation (CREMERE), Mumbai, INDIA.

Approach of detection, diagnosis, management and care of the mentally retarded (MR) children in India differs from that of the industrialized countries due to its varied socio-economic, religious, cultural and family structures. The rural, slum and semi-urban population with genetic diversity make the situation more complex by consanguinity. A community-based approach was therefore used for creating awareness about genetic components, and care of the MR children by establishing a "Referral System" with networking of primary health centres.
During 3 years' study, screening of 5.5 lakhs of population was conducted using the existing Primary Health Post infrastructure. Training of medical, paramedical and community health volunteers (N>800), and door-step care by this trained field staff, followed by detection and referral of the MR children to the tertiary Genetic Centre - CREMERE, were the important features. The nature of the disability, causes and recurrence risk were explained through genetic counseling, emphasizing consanguinity and hereditary factors to the parents. Pregnancy monitoring and prevention was stressed in young couples.
The causative factors in total 511 MR children are discussed in view of the present health scenario, genetic expertise and limited laboratory infrastructure in India. Emphasis is given on prevention, when therapy is yet a distant hope. The significant point indicated for health policy planners was the 49% preventable factors (birth asphyxia, infections and low birth weight) and 36% genetic etiology contributing to mental retardation. Integration of genetic health care at the grass root level is functionally demonstrated in this community study.

I. M. Kremensky¹, E. K. Michaylova¹, A. Savov¹, S. Andonova¹, B. Georgieva¹, A. Jordanova¹, V. Dimitrova², T. Chernev², A. Dimitrov², R. Petkova¹, L. Kalayjieva¹;
¹Laboratory of Molecular Pathology, Sofia, BULGARIA, ²Fetal Medicine Department, Sofia, BULGARIA.

Objective: To assess a centralised model for genetic service.
Methodology: All activities related to prenatal screening, diagnosis and prophylactics of inherited disorders are concentrated in a single Centre for Maternal Fetal Medicine in Sofia. The Centre performs: 1) mass neonatal screening for PKU ; 2) selective postnatal biochemical and molecular genetic screening; 3) second and first trimester Down syndrome (DS) screening; 4) prenatal enzyme and DNA diagnosis of the common monogenic disorders and DS . The Centre provides qualified genetic counselling , DNA bank and a genetic register of high-risk families. Results: For 10 years period a total of 1109 families with clinically diagnosed monogenic disorders and DS were referred to the Centre. Prenatal DNA diagnosis were performed in 388 of them - Cystic Fibrosis-94 (24 affected fetuses, 46 carriers), SMA - 31 (5affected fetuses, 18 carriers), b-thalassemia-39 (9 affected fetuses, 20 carriers), PKU-6 (1 affected fetus, 2 carriers), DMD/BMD -24 (4 affected fetuses, 3 carriers), Haemophilia A-13 (2 affected fetuses, 4 carriers), CMT-1 (unaffected), Down syndrome-180 (6 affected fetuses). In 80% of the cases CVS and in 20% - amniocentesis were performed.
Conclusion: For small countries with limited resources the centralised model for genetic services offers a series of advantages. The same infrastructure, communications and genetic register are used for the different screening programs with the same analytic technologies for most of the cases.

Pentaplex X polimorphisms to determine parental origin of X chromosomes in Turner patients.

L. Caenazzo¹, E. Ponzano¹, S. Pelotti², G. Ferri², C. Bini², S. Ceccardi², C. Colalongo², L. Artifoni³, N. A. Greggio³;
¹Department of Medicine and Public Health University of Padua, Padua, ITALY, ²Department of Medicine and Public Health University of Bologna, Bologna, ITALY, ³Department of Paediatric University of Padua, Padua, ITALY.

It have been reported that 40-60% of patients with Turner syndrome (TS) are monosomic for the X chromosome, the remaining cases have a structurally abnormal X or Y or are mosaics with a second cell line with a normal or abnormal sex chromosome. Experimental evidence has demonstrated that 70-80% of 45,X patients retain the maternal X chromosome, while X isochromosomes can be either paternally or maternally derived.
We studied fifthy females with Turner's syndrome, (age ranging from 5 to 32 years) karyotyped and opportunely treated in the Pediatric center twenty-seven out of 50 were mosaics while the other twenty-three were 45,X monosomy.
The 27 females with mosaic pattern were characterised by a large variety of X chromosome with structural abnormalities and different phenotypes.
Furthermore the molecular search for Y chromosomal material on the mosaicism confirmed the cytogenetic findings.
Parental origin of X chromosomes was determined in genomic DNA by PCR using a pentaplex with the five loci amplified dye-labelled in sets of two primer pairs detected in the following size ranges: NED-AME (103-109bp) FAM-DXS101 (179 -233 bp); JOE-DXS10011 (137 -257 bp); FAM-HUMARA (255 -327 bp) and TAMRA-DXS6807 (251 -265 bp). PCR was performed using a Trio-termoblock (Biometra), and separation of the fragments was achieved by capillary electrophoresis using an Applied Biosystems Prism 310 running GENESCAN 2.1 software.
We were able to determine that the parental origin of the single X chromosome was maternal in 90% of the monosomies.

European Molecular Genetics Quality Network (EMQN)- Developing external quality assessment for molecular genetics in Europe

R. Elles¹, S. Patton¹, D. Barton², J. Cassiman³, C. Mueller⁴, B. Bakker⁵, M. Goossens⁶, S. Stenhouse⁷;
¹European Molecular Genetics Quality Network, Manchester, UNITED KINGDOM, ²National Centre for Medical Genetics and University College Dublin, Dublin, IRELAND, ³University of Leuven, Leuven, BELGIUM, ⁴University of Wuerzburg, Wuerzburg, GERMANY, ⁵Leiden University Medical Centre, Leiden, NETHERLANDS, ⁶Hôpital Henri Mondor, Creteil, FRANCE, ⁷Northern Molecular Genetics Diagnostic Service, Newcastle-upon-Tyne, UNITED KINGDOM.

Genetic testing laboratories must answer to the individuals and families who seek their services for the quality and validity of the services they provide. In order to maintain public confidence, it is essential that the highest technical standards are met in public and private sector laboratories; that different standards between European centres are levelled out and that pressure exists to maintain current standards and improve them over time. The European Molecular Genetics Quality Network (EMQN), set up in October 1998 with funding for the European Commission’s framework IV programme, aims to address these issues by raising the standards of quality in molecular genetic testing. The EMQN organises external quality assessment (EQA) schemes and promotes best practice through the organisation of disease-specific workshops. Since 1999, EMQN has provided 28 EQA schemes in ten disease-specific areas and organised 14 disease-specific best practice workshops. The results and guidelines from these schemes and workshops are available on the EMQN website (www.emqn.org). Funding of EMQN by the European Commission finished at the end of March 2002. EMQN is continuing as a not-for-profit organisation linking expert centres in Europe and supported by the subscription of the laboratories for which it provides quality assurance services. Its advisory board will include EQA providers and expert centres.

Y. Krikmann¹, M. Dmitriev¹, A. Tikk¹, A. Metspalu²;
¹Estonian Genome Project Foundation, Tartu, ESTONIA, ²Chair of Biotechnology Insitute and Molecular Cell Biology, Tartu, ESTONIA.

The aim of the Estonian Genome Project is to create a comprehensive database containing the health data and genetic information. This database would be invaluable tool for finding associations between disease phenotypes and particular LD map structures. The database will be established and maintained by the Estonian Genome Project Foundation that has been founded by the Republic of Estonia. The Ethics Committee of the Estonian Genome Project observes all the procedures.
The Human Gene Research Act regulates main aspects of the Estonian Genome Project. In addition to that supportive legal documents (informed consent, material destruction protocols etc.) will be enacted by the Government. The Act is the most comprehensive legal documents in the world regulating population based genome studies. The Act stipulates the rights and duties of gene donors, data protection requirements and other guarantees for the protection of gene donors as well as liability. The Act states administrative and criminal charges for violation of the rights of a gene donor. Every gene donor if she/he has decided to participate have to sign the informed consent form, then description of state of health is prepared by filling the questionnaire together with the family physician and a tissue sample is taken. People can opt out at any moment and they have right to know (if they wish) their own health data and LD map. All research projects on the basis of this database can be performed after the project has been approved by one of the Human Research Ethics Committees.

Introduction of Iranian patients`cell bank in National Research Center for Genetic Engineering and Biotechnology

M. M. Banoei, F. Hormozian, M. Houshmand, M. H. Sanati;
National Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC REPUBLIC OF).

In Iran and many countries in MiddleEast the consanguinous marriage is one of main reason for high prevalance for genetic disorders. In our center we are able to detect someof genetic dieases such as Cystic fibrosis, Galactosaemia,a-1 Antitrypsin deficiency, Myotonic Dystrophy(MD), Huntington disease(HD), Kenedy, Prader- willi, Angel-Man, LHON, MERRF, MELAS, NARP, CPEO, IBM, Leigh Syndroms, Fragile X, Deafness, Acondroplasia, NDT, SMA 1.
We are going to set up molecular methods for other rare genetic disorders.In the other hand,any disorders are very severe and affected individuals die at the first days or month of birth therefore research studies such as DNA analysis of them need more DNA.So we decide to build up cell bank in our center. This bank is a good repository for human cell lines, representing the unique and large samples of rare genetic diseases and ethnic variation of the Iranian population.We concentrate on collecting cells from individuals and family affected by autosomal recessive disorders or other rare diseases.
At present we have restored some cell lines(LCL) from diseases as following:CPEO,Deafness,CAH,Achondroplasia, Huntington`s disease,Cystic Fibrosis,Myotonic Dystrophy,LOHN,MERRF,MELAS, cytogenetic abnormalities and etc.

Rapid scanning of the RET proto-oncogene by denaturing high-performance liquid chromatography (D-HPLC): implications for genetic counselling

I. Torrente¹, A. De Luca¹, C. Conte², E. Flex¹, E. Chiefari³, F. Arturi³, S. Filetti³^,4, G. Novelli², B. Dallapiccola¹^,5;
¹CSS-Mendel Institute, Rome, ITALY, ²Dept. of Biopathology and Imaging Diagnostic, University "Tor Vergata", Rome, ITALY, ³Dept. of Experimental and Clinical Medicine, University of Catanzaro, Catanzaro, ITALY, ⁴Dept. of Clinical Science, University "La Sapienza", Rome, ITALY, ⁵Dept. of Experimental Medicine and Pathology, University "La Sapienza", Rome, ITALY.

Germline activating mutations of the RET proto-oncogene cause three different dominantly inherited cancer syndromes, including multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC). Mutations, involving the somatic cell lineage, are found in about 30% of sporadic medullary thyroid carcinomas (MTC). Early detection of mutations is mandatory for genetic counselling and risk assessment in family members allowing presymptomatic testing and improvement of the disease management. The majority of the activating RET mutations affect exons 10, 11, 13, 14, 15 and 16 and are currently detected by SSCP and restriction enzyme analysis. In order to improve sensitivity, time and cost of the RET mutation analysis, we have developed a Denaturing High Performance Chromatography (DHPLC)-based protocol. In this system mutations can be determined on the basis of the melting behaviour of heteroduplexes, which elute from the column by a combination of temperature and acetonitrile gradient. We performed DHPLC in 141 MTC patients with previously characterized mutations and 35 relatives. Heteroduplex peaks were detected for each mutation tested which produced a distinct and highly reproducible DHPLC elution profile. These results indicated that DHPLC methodology: a) displays a high level of sensitivity, approaching 100% for mutations in the RET proto-oncogene; b) is suitable for rapid genetic testing of members of the MEN2 affected families; c) provides a relatively simple and accurate screening technique by exhibiting advantages over conventional mutation methods, including semi-automated analysis of 96 PCR samples in less than 12 hours and low cost .

E. Girodon¹, C. Férec², M. Audrézet², M. Claustres³, M. Desgeorges³, T. Bienvenu⁴, M. Goossens¹;
¹Service de Biochimie et de Génétique, AP-HP and INSERM U468, hopital Henri-Mondor, Créteil, FRANCE, ²Laboratoire de Biologie Moléculaire et d'Histocompatibilité, CHU Morvan, Brest, FRANCE, ³Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique, Montpellier, FRANCE, ⁴Laboratoire de Biochimie-Génétique, Hopital Cochin, AP-HP, Paris, FRANCE.

Thirty five French molecular genetics laboratories carry out CFTR studies. Each year, around 8300 postnatal studies and 240 classical prenatal diagnoses (risk: ¼) are performed. Almost 50% of the studies concern carrier screening in CF relatives and their partners; 18%, monosymptomatic forms in adults; 17%, atypical forms in children; 12%, suspicion of CF in fetuses displaying echogenic anomalies. Most laboratories screen for the most frequent mutations as a first step and, if necessary, turn to more specialized laboratories to complete CFTR studies. The French CF-network, officially recognized by the Ministry of Health, is composed of three kinds of laboratories, depending on their level of molecular expertise and their implication in the management of the network and in CF research. Those who offer complete CFTR gene analysis are directly funded by the Ministry of Health. The French network works in collaboration with the European CF-Network (coordinated by J.J. Cassiman and E. Dequeker), participating every year in the external quality assessment scheme for CF. Three workshops have been organized in France: at Creteil (April 1997), Lyon (May 1998) and Creteil (December 2001). This workshop, which convened molecular geneticists, clinical geneticists and specialists in charge of CF patients, provided the opportunity to discuss about best practices for CFTR molecular studies, taking into account the European recommendations (Eur J Hum Genet 2000, vol 8, supp 2). The main conclusions of this workshop will be presented.

The Italian national project for standardisation and quality assurance of genetic testing.

D. Taruscio, V. Falbo, G. Floridia, C. Marongiu, C. Pescucci, M. Salvatore;
Istituto Superiore di Sanità, Rome, ITALY.

Genetic services in Italy are characterised by a large number of Laboratories, some performing at outstanding level; however, major problems are lack of homogeneous quality standards as well as of a widespread quality assurance system.
During 1999-2000 the Italian government adopted the National Guidelines on Genetic Testing (http://www.malattierare.iss.it); priority topics include quality standardisation and implementation at both intra- and interlaboratory level. Accordingly, the first national Project for standardisation and quality assurance of genetic tests has been launched on 2000-2002. The project, financially supported by the National Health System, is co-ordinated by D. Taruscio from the Istituto Superiore di Sanità. The main activities of the Project are quality control trials on :
1) cytogenetics (prenatal, postnatal including oncological)
2) molecular genetics (cystic fibrosis, beta-thalassemia, X-fragile syndrome, APC gene).
Eighty Laboratories have been enrolled, covering all Italian Regions; there are 6 inter-regional Working Units (WU). Decisions are discussed by the Steering Committee, including the WU co-ordinators and reference experts. Laboratories participate anonymously, identified by a code number.
Preliminary results either in cytogenetics and molecular genetics trials indicate an overall rate of diagnostic errors approximating 10%. Non-standard nomenclature is used by approximately 15% of Laboratories. The main factors explaining inaccuracies are under evaluation.
Expected results and deliverables include: a) improving quality and homogeneity of Italian genetic services, through, e.g.: reduction of interregional differences; b) improving dissemination of knowledge and quality standards among health operators; c) contributing to harmonisation of protocols; d) elaborating recommendations for a permanent programme on quality assurance.

A. K. Jordanova¹, V. Karcagi², I. Kremensky¹, I. Litvinenko³, M. Uzunova³, I. Tournev⁴, B. Ishpekova⁴, A. Herzegfalvi⁵, I. Simeonova¹, L. Kalaydjieva⁶;
¹Laboratory of Molecular Pathology, Sofia, BULGARIA, ²National Centre for Public Health, Budapest, HUNGARY, ³Clinic of Child Neurology, Sofia Medical University, Sofia, BULGARIA, ⁴Department of Neurology, Sofia Medical University, Sofia, BULGARIA, ⁵Department of Neurology, Budapest, HUNGARY, ⁶Centre for Human Genetics, Edith Cowan University, Perth, AUSTRALIA.

A number of private genetic disorders in Gypsy population have been described and characterized in the recent years. So far, Spinal Muscular Atrophy (SMA) has not been considered specific to Gypsy population. We have studied the molecular characteristics of a total of 38 SMA families - 30 from Bulgaria and 8 from Hungary. The Gypsy SMA patients in Bulgaria represent 35% from all patients analyzed. Our data point to a non-random distribution of the disease in the Roma in Bulgaria. The disease is confined to the Xoroxane Roma, clustered in the South-Eastern part of the country, where an ancestral SMA allele accounts for 98% of SMA chromosomes. We found that this SMA allele has diverged in three closely related conserved haplotypes, carrying different types of SMN gene deletions and conversions. Their combinations determine the varying severity of SMA disease. By contrast, the same ancestral allele is rare in Hungary. It occurs in only 50% of Hungarian SMA chromosomes and is not confined to any specific Romani group.
The high prevalence of SMA in Bulgarian Gypsies, and the existence of a founder SMA allele, raise the question of prevention among the target Xoroxane groups. However, the lack of an inexpensive, reliable, high-throughput method for the detection of deletion carriers makes population-based carrier testing programs unrealistic at this stage. Prenatal diagnosis in known high-risk families still remains the most feasible approach to disease prevention.

4 year experiment of the Centre Médical Jérôme Lejeune to increase the link between clinical medicine and clinical research into genetic intellectual disabilities.

A. Ravel, C. Mircher, H. Blehaut, M. Conte, M. O. Rethore;
Centre Médical Jérôme Lejeune, Paris, FRANCE.

The CMJL is opened since 1997, and offers an annual or biannual follow-up for patients of all age presenting an intellectual disability of genetic origin. These consultations allow a medical, psychological, speech therapy and social follow-up as well as the realization of a biologic checkup, to detect and handle the complications connected to every type of pathology. Since the opening, more than 2000 patients from 0 to 60 years old came regularly in consultation.The diseases are in descending order: Down syndromes (trisomy 21, 75 %), unexplained intellectual disabilities (15 %), other chromosomal abnormalities (4p-, 5p-, 18q-, tri 9p, Willi-Prader, microdeletion; 5 %), and monogenic diseases (fragile X, Rett's syndrome, phenylketonury; 5 %). This important patient cohort of all age allows introducing research programs. The initiative of them is or internal (double blind control clinical trial of folinic acid versus placebo in toddler Down people), or collaborative (epidemiological study on folate enzyme polymorphism; epidemiological study of solid tumor in DS; relation between cataracts, oxidative metabolism and IQ in DS). Our experience exhibits that families are ready to participate to research studies even without direct individual benefice. The therapeutic trial protocols are the most asked by families, but can take place only if the criteria of judgments are very rigorous. The validation of the psychometric or neuropsychological evaluations is possible with the number of subjects of our population and the narrow links existing between the research and the clinical follow-up.

Enabling the Translation of Genetic Research Into Diagnostic Service: A Comparison of Three Cases.

C. S. Albott, L. Van Maldergem;
Institut de Pathologie et Génétique, Loverval, BELGIUM.

Access and availability of diagnosis of very rare inherited diseases (1 in 10 000) presents a real problem to the patient as well as to the health-care provider. Much discussion has occurred concerning the multifarious applications of genetic research and the consequent changes that must occur in basic definitions of treatment, prevention and diagnosis, not to mention etiology. There is, however, a decided lack of clinical evidence concerning the impact of information services which function as intermediaries between centers engaged in research into very rare inherited disorders and the clinicians and patients seeking DNA-based diagnostic analyses. We present here three cases of patients or families in need of diagnosis of a putative genetic disorder. Each case presents a vignette epitomizing a common problem faced in the diagnosis of very rare inherited diseases. In addition, each vignette shows how the problem was overcome through information available on the internet (www.eddnal.com) to researchers and health care professionals. This is the first clinical presentation of the efficacy of a web-based health information service enabling access to DNA-based diagnostic services.

A novel method for human blood DNA purification using automated magnetic particle processor

A. E. Lamberg¹, M. Mehto¹, V. Kymäläinen¹, I. Manger², E. Finne²;
¹Thermo Labsystems, Helsinki, FINLAND, ²Dynal Biotech ASA, Oslo, NORWAY.

KingFisher Blood DNA kit is developed for the purification of genomic DNA from human blood using paramagnetic particles and KingFisher mLÔ magnetic particle processor. The kit is based on two-step process where three different kind of magnetic particles are utilized. In the first step white blood cells are captured from whole blood using leukocyte specific magnetic particles thus leaving behind e.g. red blood cells and serum proteins. In the second step white blood cells still attached to beads are lysed in lysis/binding buffer and released DNA is isolated using DNA binding magnetic particles. Finally the purified DNA is released into elution buffer and is ready for the direct use in PCR or other enzymatic reactions. Fresh human blood (stored maximally 3-4 days at +4ºC) is strongly recommended as starting material. EDTA is recommended as an anticoagulant. Maximal sample volume is 200 µl, which typically gives 4-10 µg of high quality DNA. The approximate processing time for 15 samples is 50 minutes.
The KingFisher Blood DNA kit provides a timesaving and convenient way to purify genomic DNA from human blood samples. The purification process requires no phenol/chloroform extraction or alcohol precipitation, and involves very little handling. As a result, DNA with high quality and yield is obtained for further applications, such as amplifications, digestion with restriction endonucleases and Southern blotting.

Mutation Screening in Lysosomal Enzyme Genes by Real-time Monitoring of Melting Behavior in Oligosaccharides labeled with SYBR Green

E. Paschke¹, M. Czapka¹, K. Paul¹, W. Erwa²;
¹Univ.klinik für Kinder- und Jugendheilkunde, Abteilung für angeborene Stoffwechselerkrankungen, Stoffwechsellabor, Graz, AUSTRIA, ²Klinisches Institut für Medizinische und Chemische Laboratoriumsdiagnostik, Universität Graz, Graz, AUSTRIA.

Lysosomal storage disorders comprise approximately 40 recessive enzyme deficiencies leading to markedly reduced life span and severe deterioration of multiple organ functions. Promising recent results of novel intravenous infusion therapies with recombinant enzyme preparations markedly increase the impact of a complete elucidation of the genotype in affected families. Due to the high number of rare or unknown mutations this includes laborious electrophoretic methods of mutation screening like Single Strand Conformation Polymorphism (SSCP) or Denaturing Gradient Gel Electrophoresis (DGGE).
We evaluated the possibility of using real-time PCR to monitor the melting behavior of fluorescently labeled oligonucleotides for mutation screening. PCR replicons of 100 bp - fragments, overlapping at a length of 12 bp were labeled with the unspecific fluorescent dye SYBR Green and the differences to wild type melting temperature were assesed in a ROCHE Light Cycler. Compared to SSCP the results could be obtained with a considerably shorter time (1,5 vs 8 hours) at a comparable specificity. The increased number of PCR reactions necessary could be reduced by the efficient optimization in real-time PCR. Within two years we were thus able to identify 80-90 % of the affected alleles in 108 patients with 4 rare lysosomal genenzyme deficiencies. 33 (55%) of 60 mutations found were so far unknown. Therefore this approach may be useful for the routine genotyping of patients with rare genetic disorders.