ESHG Posters 20

Prenatal Diagnosis of Meckel Gruber Syndrome and Dandy-Walker Malformation in four affected consecutive siblings, the fourth one diagnosed at 22 weeks' of gestation

S. Balci¹, F. Teksen², F. Dökmeci³, B. Cengiz³, R. B. Cömert⁴, B. Can⁵, S. Özdamar⁵;
¹Hacettepe University, Faculty of Medicine, Division of Clinical Genetics, Ankara, TURKEY, ²Ankara University, Faculty of Health Education, Department of Basic Health Sciences, Ankara, TURKEY, ³Ankara University, Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, TURKEY, ⁴Hacettepe University , Faculty of Medicine, Department of Radiology, Ankara, TURKEY, ⁵Hacettepe University , Faculty of Medicine, Department of Pathology, Ankara, TURKEY.

Meckel Gruber syndrome (MGS) is a rare autosomal recessive disorder characterized by posterior encephalocele, post-axial polydactyly and dysplastic, polycystic kidneys.
We report a 23 weeks’ old male fetus affected by Meckel Gruber syndrome. Posterior encephalocele, post-axial polydactyly, Dandy-Walker malformation were observed in the ultrasonographic (USG) examination at 22 weeks’ of gestation but lisencephaly and holoprosencephaly were demonstrated with post mortem magnetic resonance (MR) before the autopsy. After the termination of the pregnancy, polycystic dysplastic kidneys were also noted in the post mortem investigation.
The proband was the fourth pregnancy of a consanguineous family which the all three siblings were also affected similarly. Interestingly, the 2 years old affected sister and 23 week’s old male fetus had the same Dandy-Walker malformation.
It was concluded that, family history, sonographic examination, measurement of serum or amniotic fluid alpha-fetoprotein have crucial importance in genetic counselling and prenatal diagnosis of MGS.

Anxiety of mothers participating in second trimester screening for Downs syndrome

S. Vejvalková, M. Simandlova, M. Havlovicová, V. Krutílková, M. Malíková, E. Seemanova, P. Goetz;
UBLG, FN Motol, Prague, CZECH REPUBLIC.

Objective : To test the anxiety during second trimester serum screening for Downs syndrome, we compared 253 mothers with false positive result of second trimester screening and 183 mothers , who underwent invasive prenatal diagnosis because of age .
Methods: We interviewed all mothers 3-8 years after birth of their healthy baby and asked them about their anxiety during the time of waiting on the result of karyotype obtained from amniotic cells. Women were asked to choose one number of 7 point scale (0-6), the individual numbers marked the level of anxiety (stress).We asked them also about their attitudes towards eventual termination of pregnancy (TOP) in the speculative case, that karyotype would be abnormal and we asked them about their opinion of genetic investigation during pregnancy.
Results: Median of anxiety features in the group of mothers with false positive results was 4, median in second group was 3. The result is statistically significant ( p value <0,0005). Statistically significant is also the difference in the number of women with positive attitudes towards eventual TOP (63,2% vs. 75,4%, p value = 0,020 ) and suitability of genetic investigation during pregnancy.( 83% vs. 91,8%, p value =0,018).

The fetal neurology clinic. A multidisciplinary approach for the treatment of the fetus with suspected nervous system pathology.

D. Lev¹^,2, T. Lerman-Sagie¹^,3, N. Zahalkah¹^,4, C. Vinkler¹^,2, M. Yanoov-Sharav¹^,2, L. Ben-Sira⁵, D. Kidron⁶, M. Glezerman¹^,4, G. Malinger¹^,7;
¹Wolfson Medical Center, Holon, ISRAEL, ²Institute of Medical Genetics, Holon, ISRAEL, ³Pediatric Neurology Unit, Holon, ISRAEL, ⁴Department of Obstetrics and Gynecology, Holon, ISRAEL, ⁵Tel Aviv Medical Center, Tel Aviv, ISRAEL, ⁶Pinhas Sapir Medical Center, Kfar-Saba, ISRAEL, ⁷Prenatal Diagnosis Unit, Holon, ISRAEL.

Congenital nervous system anomalies are the second most common congenital anomalies and the most frequent cause of malpractice litigation in the field of prenatal diagnosis. The difficulty in diagnosis resides in various factors: difficult approach to the fetal brain by standard ultrasound, late development of some brain structures and possible lack of neuroanatomic and neuropathologic experience by most sonographers. We reviewed all the cases referred to the Fetal Neurology Clinic at The Edith Wolfson Medical Center during a 2-year period between November 1999 and October 2001.
During this period 128 patients were referred to the Clinic, and 187 examinations were performed (1.46 examinations/patient).The ultrasound findings and the patients’ follow up are summarized. Our team recommended continuing the pregnancy with regular follow up in 59.4% of the patients, in 15.6% the findings were so serious that termination of pregnancy was advised and in 10.6% we advised the patients to consider termination of pregnancy, 7.8% of the patients were referred for MRI and 7% for amniocentesis.
The fertile interaction between the different sub-specialties involved in counseling helped in the differentiation between normal and pathologic cases. In cases with suspected pathology, the team discusses with the parents the possible implications and the meaning of the diagnosis, based on the conjoined clinical experience of the team. This approach enables optimal perinatal management of the pregnancy.

Detection of fetal cell in a minimal amount of maternal blood using HbF staining and polymerase chain reaction

The available fetal cells in maternal blood give a potential source for the detection of fetal abnormalities without risk to the fetus. We attempted to detect fetal cells and fetal DNA in a minimal amount of maternal blood obtained from forearm and fingertip. Fetal cells were isolated from 1 ml of maternal blood by fetal hemoglobin (HbF) staining and microdissection, while DNA was extracted from 200 ml of maternal blood. The isolated fetal cells and DNA were analyzed by polymerase chain reaction using Y chromosome specific primers. These techniques correctly identified the sex of the fetus in all pregnancies tested. Our study showed that fetal DNA could be detected in maternal blood obtained from fingertip at 6 weeks of gestation. The findings suggest that prenatal diagnosis can be carried out as early as 6 weeks of gestation with a small amount of maternal blood using the inexpensive techniques mentioned above.

Trends In Live Birth Prevalence Of Congenital Anomalies. 1979-1999 : The Impact Of Prenatal Diagnosis

C. Stoll, Y. Alembik, M. Roth, B. Dott;
Hôpital de Hautepierre, Strasbourg, FRANCE.

Objectives
To describe the impact of prenatal diagnosis on the birth prevalence of congenital anomalies during 21 years (1979-1999) in a well defined population.
Design
A descriptive population-based study.
Setting
Northeastern France (13,500 births per year).
Methods
Analysis of data from multiple sources on births and terminations of pregnancy after prenatal diagnosis of congenital anomalies in 265,679 consecutive pregnancies of known outcome.
The study period was divided into 3 subgroups 1979-88, 1989-93 and 1994-99.
Results
Between 1979 and 1988 and 1993 and 1999 prenatal detection of congenital anomalies increased from 11.7% to 25.5% and to 31.9%. Termination of pregnancy (TOP) increased in the same proportions during the 3 time periods. However the increase of TOP was much higher for chromosomal anomalies than for non chromosomal congenital anomalies : 21.7, 43.9 and 64.0 vs 4.8, 7.3, and 10.2 respectively. The birth prevalence of Down syndrome fell by 80% from 1979-88 to 1994-99. Sensitivity of prenatal detection of congenital anomalies and TOPs were lower for isolated cases (only one malformation present in the fetus) than for multiple malformations in the same fetus. Sensitivity varied with the type of malformations : it was high for neural tube defect (79.7%) and urinary anomalies (54.8%) and low for congenital heart defects (25.3%) and for oral clefts (27.6%)
Conclusions
The introduction of routine prenatal diagnosis has resulted in a significant fall in the birth prevalence of congenital anomalies. However this fall varied with the types of congenital anomalies.

Congenital deficiency of alpha fetoprotein and associated chromosomal abnormality in the placenta

R. Sharony¹, A. Amiel¹, N. Bouaron¹, D. Kidron², D. Itzhaky³, M. Fejgin¹;
¹The Genetic Institute, Sapir Medical Center - Meir Hospital, Kfar Saba, ISRAEL, ²The Pathology Institute, Sapir Medical Center - Meir Hospital., Kfar Saba, ISRAEL, ³The Immunology Laboratoy, Sapir Medical Center - Meir Hospital, Kfar Saba, ISRAEL.

The role of alpha-fetoprotein (AFP) is unknown for the most part. Several functions of AFP during fetal life have been suggested: regulation of osmotic pressure, mediation of the immune system and growth control. In this report we describe two cases of congenital absence of AFP that were identified by the current methods of detection. The pathological examination results, including an immunohistochemical stain, which refine the levels of AFP detected by the biochemical studies, are enclosed. In order to exclude chromosomal anomalies in the placentae, we preformed complete genomic hybridization analysis on both placentae.Both placentae showed monosomy 16, which was confirmed by FISH. It has been reported that tissue-specific expression of the AFP gene is strongly stimulated by an enhancer present 3.3 to 4.9 kb upstream of the transcription initiation site mapped to 16q22.3-q23.1.An overview of the molecular biology of AFP production is set forth. An explanation is suggested for the lack of symptoms in a newborn of undetected levels of AFP and the mechanism by which this condition might occur. This may shed light on the mechanism by which this rare condition is generated and the lack of any symptoms in affected newborns

The incidence of confined placental mosaicism in non-cultured cells of human spontaneous abortions

I. N. Lebedev, N. V. Ostroverkhova, S. A. Nazarenko;
Institute of Medical Genetics, Tomsk, RUSSIAN FEDERATION.

One of the most important conceptions over the past few years in interpretation of the pathogenesis of abnormal intrauterine human development is the statement about possible discrepancies between karyotype of cells of embryonic and placental origin. During prenatal diagnosis the incidence of confined placental mosaicism is evaluated usually about 1-2%. In order to determine the influence of distribution of cells with chromosomal abnormalities in placental tissues on arrest of embryo development the non-cultured cells from cytotrophoblast and extraembryonic mesoderm of 28 first trimester internal abortions were studied by interphase FISH analysis with centromere-specific DNA probes for each chromosome. 7 discrepancies (25%) between karyotypes of two studied tissues were found. Cell lines with trisomy 7, trisomy 8, monosomy 15 were confined to cytotrophoblast whereas the karyotype of extraembryonic mesoderm cells was normal. One embryo has mosaic monosomy 7 in cytotrophoblast only but in mesoderm the tetraploidy/diploidy mosaicism was detected. Two abortions have chromosome aberrations both in cytotrophoblast and extraembryonic mesoderm but in former the chromosomal abnormalities were in mosaic form with normal cell line whereas in later only the cells with abnormal karyotype were detected. One embryo has 45,X/46,XY/46,XX/47,XXY karyotype in cytotrophoblast and 45,X/46,XY/46,XX in mesoderm. Our data based on analysis of non-cultured cells indicates that the tissue-specific compartmentalization of cell lines with chromosomal abnormalities may have a significant influence on pathogenesis of early embryo development.

Fetal erythroblasts are not the source of cell free fetal DNA in the maternal circulation

X. Zhong¹, W. Holzgreve², S. Hahn³;
¹Prenatal Medicine, Women's Hospital, University Basel, Basel, SWITZERLAND, ²Prenatal Medicine, Women's hospital, University Basel, Basel, SWITZERLAND, ³Prenatal Medicine, Women's Hospital,, Basel, SWITZERLAND.

Fetal cells, specifically fetal erythroblasts, as well as cell free fetal DNA are present in the maternal circulation. Both are currently being investigated as a means for the non-invasive risk free analysis of fetal genetic traits. The origin of cell free fetal DNA is currently unclear. It has been proposed that trafficking fetal erythroblasts may be one source. This is partly due to the apoptotic character that a significant proportion of fetal erythroblasts display, and since elevations in both fetal erythroblast numbers as well as cell free fetal DNA concentrations have been noted in several pregnancy related pathologies. Our examination of fetal cell trafficking and release of cell free fetal DNA in normal and affected pregnancies indicates that no correlation exists between these two fetal molecular and cellular species. This is most evident in pregnancies affected by onset of pre-term labour, where significant elevations in cell free fetal DNA concentrations were detected without any concomitant elevation in fetal erythroblast numbers. Our data therefore suggest that an alternative cell type is the source of cell free fetal DNA. Furthermore, it appears that the release of cell free fetal DNA from this cell type is affected by pathological placental conditions which are not associated with an increase in fetal cell trafficking.

S. Hristoskowa, W. Holzgreve, S. Hahn;
University of Basel, Basel, SWITZERLAND.

Non-invasive risk free prenatal diagnosis can be achieved by the analysis of intact fetal cells and cell free fetal DNA in the maternal circulation. The relationship between these two fetal cellular and molecular analytes is unclear. Recent data have shown that apoptotic fetal cells can be detected in plasma of pregnant women and that many fetal nucleated red blood cells (NRBCs) in the maternal circulation were TUNEL positive. It has, therefore, been suggested that the maternal immune system may clear NRBCs by apoptosis and that this leads to the production of circulatory fetal DNA. On the other hand, this apoptotic phenotype may be associated with erythroid differentiation and enucleation.
For this purpose we have examined whether apoptosis occurs in fetal NRBCs that have crossed into the maternal circulation or rather if this is a physiological phenomenon of NRBC maturation which occurs when these cells are still in the fetal circulation.
Our study, performed on fetal blood samples (n=12), showed that more than 60% of the NRBCs were TUNEL positive. This was true for both cord blood (n=10) samples collected at term as well as those obtained from early stages of gestation (13-16 weeks: n=2). Virtually none of the fetal lymphocytes exhibited such a characteristic.
The apoptotic phenotype displayed by NRBCs therefore does not appear to be due to an interaction with the maternal immune system. This phenotype may, however, help account for the poor analysis of NRBCs by FISH or PCR.

M. Boia¹, V. Botiu², E. Boia³, M. Puiu⁴, C. Ilie⁵, N. Pavel⁶;
¹University of Medicine & Pharmacy, Timisoara, ROMANIA, ²University of Medicine & Pharmacy, Neonatology and Puericulture Clinic, Timisoara, ROMANIA, ³of Medicine & Pharmacy, Pediatric Surgery and Ortopedy Clinic, Timisoara, ROMANIA, ⁴of Medicine & Pharmacy, Department of Medical Genetic, Timisoara, ROMANIA, ⁵of Medicine & Pharmacy, Neonatology and Puericulture Clinic, Timisoara, ROMANIA, ⁶District Hospital, Timisoara, ROMANIA.

Purpose: the diagnostic froming of ultrasound detected lesions, establisment of a correlation between ultrasound and clinical sings, establisment of evolutive stages and terapeutical indications for a selective group of premature new-born.
Material and method: The study was made at Neonatology and Puericulture Clinic for a eight years period (1994-2001). In the studied group was involved 27 patients with central nervous system malformation which was included in this group by anamnestic, clinical, ultrasound, tomographic criterions . Ultrasound was essential method for establish positive diagnosis. The examination was performed with "Sonoage 1500" after a standard protocol of examination wich included coronar and sagital sections.
Results: detected lesions was: cranio-vertebral disrafies 15 cases (55,55%), sindrom Arnold Chiari II - 3 cases (11,11%), sindrom Dandy-Walker - 3 cases (11,11%), corpus calosum agenesis - 5 cases (18,51%), Galen vein malformation - 2 cases (7,40%), arahnoidian cystes - 2 cases (7,40%).
Most lesions was associated: myelomeningocele - corpus calosum agenesis, myelomeningocele - malformation Arnold Chiari II, sd. Dandy- Walker - corpus calosum agenesis.
All cases of central nervous system malformation presented ventriculomegaly in moderate to severe stages - ventriculoperitoneal shunt was performed in 2 cases (7,40%).
Conclusions: The most frequent malformative type was cranio-vertebral disrafies associated with Arnold Chiari malformation II and III, corpus calosum agenesis, sd. Dandy-Walker. By ultrasound, ventriculomegaly was present in all patients of this study.

Y. Li, W. Holzgreve, B. Zimmermann, X. Zhong, S. Hahn;
University of Basel, Basel, SWITZERLAND.

Haemolytic disease of the New-born (HDN), whereby the Rhd mother develops antibodies against her RhD fetus, is still a serious obstetrical problem. In pregnancies at risk for HDN it is useful to know the RhD zygosity of the partner, since there is only a 50% chance that the fetus will at risk for HDN, if he is heterozygous. The determination of this has previously been very difficult and unreliable by conventional antibody assays.
Recent reports have indicated that RhD zygosity can be determined by quantitative fluorescent PCR or Taqman real-time PCR.
We have recently developed a multiplex real-time Taqman assay for the simultaneous analysis of the male SRY locus and RhD gene. Our studies have shown that this assay is very reliable for the analysis of both fetal sex and RhD status using cell free fetal DNA in maternal plasma.
Since one only needs to determine RhD zygosity of the male partner in pregnancies at risk for HDN, we have examined whether our Taqman assay can be used for this purpose.
In our study we examined 39 male blood samples. RhD zygosity was determined both by the ratio of RhD:SRY or RhD:GAPDH loci by Taqman PCR. Our study showed that RhD zygosity could quite clearly be classified as either RhD/RhD and RhD/Rhd by both assays. No discordant results were obtained between the two assays.
Our data, therefore, suggest that real-time PCR can be used to reliably determine the RhD zygosity of male partners in pregnancies at risk for HDN.

E. Boia¹, M. Boia¹, M. Puiu²;
¹University of Medicine & Pharmacy, Timisoara, ROMANIA, ²University of Medicine & Pharmacy, Dep. Genetics, Timisoara, ROMANIA.

The study's specific objectives were: to estabilish the rate of occurancy of the meconium plug syndrome in newborne as a cause of intestinal obstruction; to certifie the role of different clinical presentations of the syndrome in a proper diagnosis; to verifie the main methods used in early differentiation from Hirschprung's disease in neonatal periode.
The authors are studing all newborns hospitalised in our departaments presentind signs and symptoms of intestinal obstruction between 1992-2001.
Sumary of results: In the last decade the authors hospitalised 58 cases of newborns with intestinal obstructions inclouding intestinal atresia and stenosis (19 cases), meconium ileus (6 cases), necrotizing enterocolitis (3 cases), Hirschprung's disease (10 cases), malrotation (4 cases, meconium peritonitis (3 cases) and meconium plug syndrom (13 cases). After exclouding the other causes of intestinal obstruction, the authors corelated meconium plug syndrom with prematurity (84,61%), sweat test significant for cystic fibrosis (15,38%) and hypoglicemia and encreased glucagon prouction (7,69%).
Conclusion:
1. The rate of occurency for meconium plug syndrome in our study was 22,41% inclouding all.
2. Prematurity was registred in 84,61% from all cases with meconium plug syndrome.
3. In two cases (15,38%) the sweat test was significant for cystic fibtosis.
5. Hypermagnesemia was not proved as a cause of hypomotility in any patient in study.
6. The Gastrografin enema exam was also a therapeutic method, all patients begin passing meconium spontaneously after test.
7. Rectal biopsy was capital to exclude Hirschprung's disease.

Fetal Chromosomal Analysis of Pregnancies Following intracytoplasmic Sperm Injection with Amniotic Tissue Culture

H. Samli¹, M. Solak¹, N. Imirzalioglu², Y. Beyatli³, S. Simsek¹, S. Kahraman⁴;
¹Afyon Kocatepe University, Afyon, TURKEY, ²GATA, Ankara, TURKEY, ³Gazi University, Faculty of Science, Dept of Biology, Ankara, TURKEY, ⁴Istanbul Memorial Hospital, Istanbul, TURKEY.

From January 1996 to December 2000, 98 consecutive patients who had become pregnant after Intracytoplasmic Sperm Injection (ICSI) were studied. Hundred and forty-two fetuses of these patients were screened with fetal amniotic tissue culture. Chromosomal anomalies were detected from 6 out of 142 (4.2%) fetuses. Anomalies were as follows, ‘46,XX / 69,XXX / 92,XXXX‘, ‘46,XY / 69, XXY / 92,XXYY‘, ‘47,XY+21‘, ‘47,XY+7‘, ‘47,XXY‘ and ‘45,X0‘. All except one pregnancies were terminated with the consent of the couples. Fetal skin fibroblast cultures were also studied after termination of the pregnancy in order to confirm prenatal diagnosis.
The prevalence of chromosomal anomalies seems to be slightly increased after ICSI, carrying the risk for transmission of chromosomal aberrations of paternal origin and a higher risk of de novo, mainly sex-chromosomal aberrations.

C. Sayar¹, Z. Sahinoglu¹, M. Uludogan¹, B. Türköver¹, N. Elçioglu²;
¹Prenatal Diagnosis Center, Zeynep Kamil Maternity and Children Hospital, Istanbul, TURKEY, ²Dep. of Pediatric Genetics, Marmara University Hospital, Istanbul, TURKEY.

OBJECTIVE: Fetal choroid plexus cysts (CPC) are commonly found at the time of a routine second-trimester scan, but there is much debate as to their clinical significance. The aims of this study were to define the incidence of CPC in an unselected population and describe their association with aneuploidy.
METHODS: 10594 pregnant women that were subjected to second level ultrasonographic analysis at the Department of Perinatology, Zeynep Kamil Maternity and Children Hospital has been studied. 109 cases of CPCs were identified among these group (1.02%) between 16 and 22 weeks' gestation. These patients had genetic counseling, and biochemical testing and amniocentesis were offered to all patients.
RESULTS: The majority of the cases 102/109 presented only isolated CPC and the remaining 7/109 (6.5%) were associated with additional ultrasonographic findings. No relationship was found between the diameter, bilaterality or the complexity of the cyst. Aneuploidies (mostly trisomy 18) were found in 3/102 with isolated CPC (2.94%) and in 3/7 with additional ultrasonographic findings (43%). Advanced maternal age as an additional risk factor was found in only one of isolated CPC, but the remaining two had no other risks so far.
CONCLUSION: The management of isolated choroid plexus cysts remains controversial. Fetus with CPC should be examined carefully by detailed ultrasound assessment to seek for further minor malformations in a specialized center. The review of the literature show that the majority of the authors advocate amniocentesis when the CPC is associated with another ultrasound abnormality.

D. Chu¹, J. Liou², P. Cheng³, S. Chang⁴, C. Sun⁵;
¹Chang Gung University, Tao-Yuan, TAIWAN REPUBLIC OF CHINA, ²Dept OB/GYN, Chang Gung memorial hospital, Taipei, TAIWAN REPUBLIC OF CHINA, ³Dept OB/GYN, Chang Gung memorial hospital, Lin-Kou, TAIWAN REPUBLIC OF CHINA, ⁴Dept. OB/GYN, Chang Gung memorial hospital, Lin-Kou, TAIWAN REPUBLIC OF CHINA, ⁵Dept. Clinical Pathology, Chang Gung memorial hospital, Lin-Kou, TAIWAN REPUBLIC OF CHINA.

The purpose of this study was to determine the number of chromosome 21 present in the fetal cells from pregnancies complicated with Down syndrome by differentiating the chromosome 21s of different parental origins with human chromosome 21-specific DNA marker polymorphism. Forty amniotic fluid samples from pregnancies complicated with fetal Down syndrome were analyzed for D21S11 and interferon-a receptor gene intervening (IFNAR) sequence. Fluorescence-labeled polymerase chain reaction (f-PCR) was performed to amplify these sequences followed by polyacrylamide gel electrophoresis. Data were delineated with automatic DNA sequencer and 35 of 40 (87.5%) fetal Down syndrome samples analyzed for IFNAR showed 3 distinct peaks, each peak represented an individual chromosome 21, while 24 of 30 (80%) cases analyzed for D21S11 showed 3 distinct peaks. There were two Down syndrome samples showed two uneven peaks. By analyzing 98 euploid pregnancies as controls, the ratio of area under the peaks was determined to be 1.31 ± 0.22 and 1.96 ± 0.18 (mean ± SD) for the euploid pregnancies and pregnancies complicated by fetal Down syndrome with two peaks, respectively. Altogether 39 of 40 (97.5%) Down syndrome cases were correctly identified based on either the 3 peaks pattern in at least one of the DNA markers or the relative peak area ratio calculation. It was concluded that polymorphic DNA markers are useful in determining the number of chromosome 21 present in fetal cells. The high sensitivity suggested good prospect of this method in application for prenatal detection of chromosome 21 aneuploidy.

Attitudes of pregnant women towards the risk free prenatal diagnosis of fetal aneuploidies using fetal cells from maternal blood

S. Hahn, R. Müller, C. Troeger, J. Bitzer, W. Holzgreve;
University of Basel, Basel, SWITZERLAND.

A major focus of our group is the development of a risk free non-invasive method for prenatal diagnosis of fetal aneuploidies using fetal cells enriched from the blood of pregnant women. A concern with the introduction of such a new test is that pregnant women may feel coerced to undergo involuntary testing. In order to address this issue we have examined the attitude of 145 pregnant women to the introduction of such a test, consisting of 3 groups:
1.
Women with a normal pregnancy (n=97).
2.
Women judged to have a high risk for a fetal abnormality and who had undergone an invasive prenatal diagnostic procedure (n=24).
3.
Women who had become pregnant following IVF treatment (n=24).
In addition we examined the attitude of 68 non-pregnant women.
Our study showed that:
1.
79,3% would not feel coerced by the introduction of a risk free test.
2.
80% would be interested in first using a non-invasive test as preliminary a screening procedure.
3.
If the test result was found to be normal, 84.4% of pregnant women would not elect to have further tests.
4.
If the result was abnormal, 56% would elect to have the result confirmed by invasive tests.
5.
Women undergoing IVF treatment, would prefer to have the result confirmed by a further non-invasive test (58.3%).
Our data, therefore, indicate that pregnant women would not feel coerced to undergo involuntary testing, and that such a risk free test would be especially welcomed by couples undergoing IVF treatment.

E. Sala¹, N. Villa¹, E. Martinoli², A. Patrizi², S. Mariani³, N. Strobelt³, E. Gautiero¹, R. Solano¹, M. Volonte², L. Dalpra⁴;
¹Genetic Laboratory H S Gerardo, Monza, ITALY, ²Dep. of Biology Genetics for Medical Sciences, University of Milan, Milano, ITALY, ³Dep.of Obstetrics and Gynaecology H S Gerardo, University of Milan-Bicocca, Monza, ITALY, ⁴Dep.of Experimental Environmental Medicine and Medical Biotechnology, University of Milan-Bicocca, Monza, ITALY.

FISH of uncultured amniotic fluid cells and conventional cytogenetic analysis were performed on 83 pregnancies with US abnormalities and 79 on pregnancies with normal US parameters. The AneuVysion^R Assay (Vysis) with specific probes for chromosome 13, 18, 21, X and Y, was used. Amniotic fluid samples were obtained between 12 and 34 weeks’ gestation, 116/162 (72%) being between 15 and 20. In cases with a sole abnormal US finding (n=24) nine aneuploidies were detected (1 tris 13, 8 tris 21). In the group with two or more malformations (n=59) there were 18 aneuploidies (9 tris 18, 3 tris 21, 2 monosomy X,1 tris 13, 2 triploidy, 1 karyotype: 48,XXY,+21). In this group conventional cytogenetic revealed also three chromosomal anomalies not detectable by FISH: a trisomy 16 mosaicism, a 4p- and a r(13). No sex aneuploidies alone were observed. In the group with normal US, 52 cases were analysed because of maternal anxiety and 27 had positive maternal serum screen or advanced maternal age. In this latter group of pregnancies only two trisomies 21 was detected. The complete concordance between FISH data and conventional karyotype analysis observed in our sample, prompt us to consider the interphase FISH as an useful tool in pregnancies with high risk of chromosomal aneuploidies. When ploidy status is known to be normal, the overall risk of chromosomal abnormalities is significantly reduced. Otherwise, the findings of three chromosomal anomalies undetectable by AneuVysion^R Assay confirms that FISH results should be complemented by the conventional chromosomes analysis.

Further experiences with the application of STR analysis for the detection of the most common trisomies by F-PCR

Z. Ban, C. Papp, B. Nagy, L. Lazar, E. Toth-Pal, Z. Papp;
Semmelweis University, Budapest, HUNGARY.

OBJECTIVE: Prenatal cytogenetic analysis for trisomies requires lengthy and work-intensive laboratory procedures. A rapid method for the detection of the most common trisomies is the fluorescent PCR amplification of small tandem repeats (STR) located on the chromosomes 13, 18 and 21. This method is a potential alternative to conventional cytogenetic analysis for the screening of these disorders. Recently the method has been reported to be used as a single screening method for age-related aneuploidies. OBJECTIVE: We compared the results of the routine cytogenetic analysis of amniotic fluid and chorionic villus samples to the results of the fluorescent PCR analysis of the same samples (n=1200). METHODS: The multiplex fluorescent PCR was performed with 3 primer pairs specific for chromosome 21 (D21S11, D21S1411, D21S1312) and 2-2 primer pairs specific for chromosome 18 (D18S851, D18S51) and 13 (D13S631, D13S258). The capillary electrophoresis and fragment analysis was performed on an ABI 310 Genetic Analyser. RESULTS: We found 16 cases of trisomy 21, 10 cases of trisomy 18, 7 cases of trisomy 13 and 3 cases of triploidy. We found the same pathologic samples as the cytogenetic analysis. CONCLUSION: The multiplex fluorescent PCR analysis of STRs proved to be fast and reliable in the diagnosis of the most common trisomies. It is not replacing the cytogenenetic analysis of prenatal samples, but in certain cases like advanced maternal age it is worth considering it as a routine alternative to it.

M. Karimi-Nejad¹, R. Karimi-Nejad¹, H. Najmabadi¹^,2, Y. Shafaghati¹^,2, F. Azimi¹, N. Nabavi-nia¹, F. Afrouzan¹, A. Karimi-nejad¹;
¹Karimi-Nejad Genetics and Pathology Center, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Welfare & Rehabilitation University, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Over 12 years, more than 2000 prenatal tests have been performed with the following indications: 1300 for chromosomal aberrations, 510 hemoglobinopathies, 65 metabolic disorders, 25 muscular dystrophy, 15 fragile X, 6 skin lesions and 1 for ataxia telangiectasia.
The indication, results and outcome of chromosomal study of 1250 amniotic cell cultures mostly at 13-15 weeks are being reported. 662 parous were tested for advanced maternal age; 15 (2.3%) were abnormal, which shows 25 fold increase over general population. The risk is even higher, 2 of 49 (4.4%) parous with advanced maternal age who had previous history of offspring with chromosomal aberration. The highest risk is among parous (15%) where one of the parents bears a balanced translocation; 3/269 (1%) of parous with history of offspring with chromosomal aberrations showed unbalanced karyotypes. There are 2 abnormal karyotypes among 35 parous with low AFP serum, and 1 among 117 pregnancies referred for various reasons including check up. Of the 30 abnormal karyotypes, 11 have tri 21, 6 tri 18, 3 mosaicisms, 2 XXY, and 8 different karyotypes. Besides chromosomal aberrations, one anencephaly, and one full mutation of FMR1 have been detected coincidentally, totalling 32 candidates for therapeutic termination.
Rate of spontaneous abortion of unknown reason, two weeks following the procedure is less that 0.5 %. The final results were reported within 10-14 days. Our experience indicates that early amniocentesis is a safe, acceptable and reliable procedure for detection of fetal chromosomal abnormalitites; we recommend it strongly for at risk pregnancies.

Evalution of balanced and unbalanced chromosomal abnormalities (except classic aneuploidies) in 700 amniocenteses, after birth or termination.

T. Yakut¹, Y. Kimya², U. Egeli¹, D. Demircioglu¹, B. Yigit¹;
¹Department of Medical Biology and Genetics, University of Uludag, Bursa, TURKEY, ²Department of Gynecology, University of Uludag, Bursa, TURKEY.

We wish present our two years experience for prenatal diagnosis since 1999. During this period, we analysed 700 amniocenteses. Amnion fluid of patients were sent to our laboratory due to three main reasons for referral; advenced maternal age, abnormal maternal serum screening test and abnormal ultrasound assesment. None of cases with chromosomal abnormalities do not include chromosomal aneuploidies for chromosomes 13, 18, 21, X, Y and pericentric inversions of chromosome 9 in these evaluation. Three reciprocal, two Robertsonian, one insertional and one de nova translocations as balanced abnormality and 4 unbalanced chromosomal abnormalities were detected in these cases. These unbalanced chromosomal anomalies were as 46,XX,der(1) add(q32-44); 46,XY, 21p+; 46,XX, +mar; 46,XX,(1qh+). Marker chromosome was orginated from chromosome 15 centromeric region which was detected by using flourescence in situ hybridization (FISH). All fetuses were examined after birth or termination. Our finding sugests that 50 % fetuses with unbalanced chromosomal abnormalities (in 2 out of 4) showed some physcial anomalies, such as low air, hydrocephalus, facial and cardiac anomalies, while none of fetuses with balanced translocations had any phenotypical abnormality.

Evaluation Of Histology And Molecular Cytogenetics Of Placenta In 56 I.u.g.r. Cases

N. Villa¹, E. Sala¹, N. Roncaglia², C. Andreotti², C. Colombo³, E. Gautiero¹, R. Solano¹, A. Cappellini⁴, L. Dalpra⁵;
¹Genetic Laboratory, H S Gerardo, Monza, ITALY, ²Dep.of Obstetrics and Gynaecology, H S Gerardo, University of Milan-Bicocca, Monza, ITALY, ³Neonatal Care Unit, H S Gerardo, Monza, ITALY, ⁴Dep.of Pathology, H S Gerardo, Monza, ITALY, ⁵Dep.of Experimental Environmental Medicine and Medical Biotechnology, University of Milan-Bicocca, Monza, ITALY.

The role of confined placental chromosome mosaicism (CPM) in intra-uterine growth retardation (IUGR) has not yet been well established and also the involvement of uniparental disomy (UPD). The aim of the research is to understand if the IUGR, as the only foetal anomaly, is due to CPM and/or UPD. Up to now a total of 56 consecutive foetuses with IUGR (below the 10^th centile) and no major morphological abnormalities were recruited for the study. All foetuses were karyotyped at birth and only two showed an abnormal chromosome constitution (tris 21). Isolated uncultured placental nuclei, prepared from multiple biopsies of placenta at term, were used to perform FISH with Multiprobe-I Kit (Cytocell) specific for each centromere. After FISH a case of tris 15 was detected: 84% of the isolated nuclei showed 3 signals. The standard analysis of 100 blood and 100 skin metaphases revealed a normal karyotype. UPD was excluded using 9 polymorphic loci. The histology of this placenta showed vasculopathy and the cord had 2 vessels. The delivery took place at 28^th week of gestation; the weight of the baby was 540 g (< 10^th centile) and after 4 months 2000 g. He showed severe micropenis, hypospadias, testes not palpable and inguinal hernia. The histology of all the other placentas shows the presence of several features: normal (11/56), chorionitis (5/56), vasculopathy (31/56), cord abnormalities (4/56), hydrops (1) and in 7 cases more than one pathology was present. UPD tests are in progress and results will be discussed.

S. Brankovic-Niksic, S. Nikolic, G. Pilic, T. Kranjac;
Institute of Mental Health, Belgrade, YUGOSLAVIA.

We present our experience in applying cordocentesis as a method of prenatal diagnosis from 1990-1999. In that period we made 674 cordocentesis, for various indications. Gravidity weeks were 18-29, and maternal age distribution was 19-43. The psychosocial characteristics of genetic advice regarding cordocentesis differ comparing to other methods of prenatal diagnosis, because fetal malformation has been already seen by ultrasound and pregnant women is "prepared" for potentially bad outcome. The other reason for different approach in genetic counseling is that termination of pregnancy is performed in high gravidity. In our work we analyzed:
- complication after the punction
- pathological finding in fetal karyotype
- follow up of the babies
- child mortality at deliverycharacteristics of genetic advice in cordocentesis and the psychosocial consequences in terminated pregnancies after cordocentesis.

Molecular-cytogenetic study of human spontaneous abortions by comparative genomic hybridization

D. Azmanov¹, B. Zaharieva¹, V. Dimitrova², Z. Karagiozova², R. Rousseva², T. Chernev², D. Toncheva¹;
¹Department of Medical Genetics, Medical Faculty Sofia, Sofia, BULGARIA, ²Hospital of Obstetrics and Gynecology "Maichin dom", Medical University Sofia, Sofia, BULGARIA.

The frequency of spontaneous abortions is about 6-8% of all recognized pregnancies. Chromosomal aberrations are the main cause for pregnancy loss in more than 50% in the first and 20% in the second trimester of pregnancy. Conventional cytogenetic analysis of spontaneous abortions is usually used for detection of structural and numerical chromosomal aberrations, but disadvantages of the method are difficult tissue culturing and contamination by maternally derived cells. Our previous cytogenetic and molecular genetic studies have indicated that oncogenes and tumor-suppressor genes could play a role in fetal development disturbances.
Comparative genomic hybridization (CGH) is a molecular-cytogenetic technique that allows entire genome screening for numerical and unbalanced structural chromosomal aberrations in a single experiment. CGH analysis was performed on materials from 62 spontaneous abortions with aim to reveal deleted and overexpressed chromosomal regions in miscarriages.
Normal CGH profiles were found in 33 samples (53.23%). The CGH demonstrated trisomies in 20.97 % of cases (trisomies 3, 4, 5, 8, 13, 14, 14, 15, 15, 16, 16, 19, 19), monosomies in 3.23% (monosomy X and 19), hyperdiploidy in 4 cases (6.45 %) and monosomy 9 in combination with gonosomal polysomy in one case (1.61%). Partial chromosomal gains (1p+; 1p+; 1q+; 2q+; 5p+; 5q+; 7p+; 9p+; 9q+; 10p+; 13q+; 16q+; 18p+; 22q+) or losses (16p-) were found in 9 cases (14.52%). In 7 out of 14 amplifications (50%) oncogenic bands were affected which is in agreement with our hypothesis of association between oncogenes and spontaneous abortions.

Mosaic trisomy 13 on chorionic villi in a fetus with body wall complex: fortuitous association or pathogenic hypothesis ?

B. Doray¹, B. Gasser², B. Viville³, F. Girard-Lemaire¹, C. Schluth¹, E. Flori¹;
¹Laboratoire de Cytogénétique, Strasbourg, FRANCE, ²Service d'Anatomie Pathologique, Strasbourg, FRANCE, ³Service de Gynécologie-Obstétrique I, Strasbourg, FRANCE.

Body wall complex (BWC) is a malformative association characterized by the presence of abdominal wall defects associated with limb and visceral anomalies. Two distinguishable phenotypes are delineated: the first one is characterized by craniofacial defects whereas the second one includes urogenital anomalies, abdominal placental attachment and inconstant anomalies of inferior limbs.
We describe here a fetus with BWC detected by ultrasound at 16 weeks’ gestation. Direct chromosome analysis of chorionic villi displayed a trisomy 13 in 20 of 21 cells whereas the cultured cells showed a normal karyotype 46,XY. Foetopathological examination demonstrated a male 17-week-old fetus with an inferior celosomy associated with a short umbilical cord, urogenital anomalies and limb defects. Postmortem karyotype from skin fibroblasts was normal.
Little is known about the pathogenic mechanisms of BWC. At present, the three main theories are early amnion rupture sequence including all phenotypes of BWC, early vascular disruption involved in the craniofacial phenotype and early embryonic maldevelopment with disturbance of the embryonic folding at the origin of the second phenotype. The latter could result of a malfunction of the ectodermal placode or rather of a disturbance of the ectodermal cells deposition into the mesodermal compartment, as suggested by Hartwig (1992).
To our knowledge, chromosome anomalies have not been reported in BWC. Although our results may be fortuitous, it is striking to observe a discrepancy between direct cytogenetic analysis of ectodermal cytotrophoblastic cells and cytogenetic analysis of cultured extra-embryonic mesodermal cells when BWC might result of abnormal ectodermal and mesodermal interactions.

C. Schluth¹, B. Doray¹, F. Lemaire-Girard¹, R. Favre², B. Gasser³, E. Flori¹;
¹Laboratoire de Cytogénétique, Strasbourg, FRANCE, ²Service de Gynécologie Obstétrique SIHCUS-CMCO, Schiltigheim, FRANCE, ³Institut d'Anatomie Pathologique, Strasbourg, FRANCE.

Tetraploidy is characterized by four complete sets of chromosomes (4n = 92). Although it has frequently been reported in early spontaneous abortions, tetraploidy is extremely rare in term pregnancy. Most of late surviving patients are diploid/tetraploid mosaics and present severe mental and physical impairment. To our knowledge, only four tetraploidies were ascertained in the prenatal stage on amniocytes and/or fetal blood lymphocytes.
We report here on the prenatal diagnosis of a diploid/tetraploid fetus: at 11 weeks’ gestation, a karyotype performed on chorionic villi (direct analysis and cultures) for cystic hygroma showed full tetraploidy. Subsequent amniocentesis was considered as normal (despite the presence of a tetraploid clone among the 15 analysed). However a sonographic examination at 18 weeks’ gestation displayed a complex cardiopathy. After termination of pregnancy, chromosome analysis on different tissues confirmed diploid/tetraploid mosaicism.
This observation and the four previously reported in the literature emphasize the difficulty of the prenatal diagnosis of true tetraploidy, first because the features are not specific, often mild or revealed late in pregnancy, second because tetraploidy may be an artifact on in vitro cell cultures or a confined placental mosaicism.
Therefore, in a context of ultrasound abnormalities, when cytogenetic analysis displays tetraploidy, caution is advised and karyotypes on other tissue samples have to be performed and compared with control cultures. Thus the distinction between artifactual and true tetraploidy will be possible.

K. Crkvenac-Gornik, D. Muzinic, D. Begovic, I. Tonkovic-Djurisevic;
Division of Genetics and Metabolism, Department of Pediatrics, University Hospital Centre, Zagreb, CROATIA.

The great majority of chromosomal abnormalities (approximatelly 95%) are due to numerical variation of chromosomes 13, 18, 21, X and Y. For that reason, prenatal diagnostics is necessary to follow up all pregnancies at risk. During 1978-2001, amniocentesis was performed on 17 004 pregnant women. There were following indications for prenatal diagnosis; 72% maternal age of 35 and higher, 4.9% previous child with Down syndrome, 4.74% medicine, X-ray and viruses, 2.12% ultrasound identification of fetal anomaly, 1.81 malformations in children, 1.5% other chromosomal abberations, 1.06% positive maternal serum screening, 1.17% autosomal genopaty, 0.2% history of structural chromosome abnormality in one of the parents.There were found 358 (2.11%) of chromosomal anomalies. Among them 298 (1.75%) are aneuploidies. Results are shown in table.


INDICATION	Trisomy 21	trisomy 18	trisomy 13	sex CHROMO. aneuploidy	TOTAL
Maternal age	150	39	9	37	235
Previous child with Down syndrome	4	1		2	7
Ultrasound identification of fetal anomaly	8	10		6	24
Malformations in children	4		1	5	10
Other chromosomal aberrations	1	2		4?	7
Medicine, X-ray, viruses				7	7
Autosomal genopaty				1	1
Num. abberations in previous pregnancies		1		3	4
Psyche				3	3
TOTAL	167	53	10	68

In conclusion data of this analysis are in concordance with results from other studies. Maternal age is one of the primary indications for prenatal diagnosis of aneuploidy and trisomy 21 is the most common aneuploid condition compatible with survival.

The evaluation the effectiveness of nuchal translucency measurement in screening for congenital heart disease

A. S. Latypov, L. E. Teregoulova, L. R. Samoylova;
Republic Hospital, Kazan, RUSSIAN FEDERATION.

OBJECTIVES: To evaluate possibility first trimester marker, used for Down syndrome screening, named nuchal translucency (NT),for congenital heart defect diagnostics.
METHODS: An unselected group of 3003 pregnant women with a singleton pregnancy underwent first trimester screening at 10-13 weeks' gestation in 2000-2001. Nuchal translucency (NT) was measured by transvaginal sonography. Invasive procedures for fetal karyotyping in cases with NT > or =3 mm were performed and fetuses with abnormal karyotypes were eliminated. A second trimester detailed ultrasound scan was also performed in all cases with nuchal translucency thickness. We tried list of all registered cases of congenital heart defect in newborns and babies from neonatologists, pediatricians, heart surgeons and other sources.
RESULTS: There were 84 cases with NT>3 mm. In this group was found 17 cases of chromosomal anomalies (21+, 45X, 18+). Twelve pregnancies were lost to follow-up .In only 1 case we found two chamber heart. Other outcomes were normal
CONCLUSION: First trimester nuchal translucency measurement is an effective screening test for the prenatal detection of fetuses with chromosome anomalies, including Down’s and Turner syndromes, but effectiveness in congenital heart defect finding requires further study.

I. Tonkovic-Djurisevic, D. Muzinic, D. Begovic, K. Crkvenac-Gornik;
Division of Genetics and Metabolism, Department of Pediatrics, University Hospital Centre, Zagreb, CROATIA.

From 17 004 prenatal cytogenetic investigations, 889 pregnant women have had an indication for early amniocentesis because of infertility. Chromosome analysis of cultured amniocytes with GTG banding showed 13 (1,46%) unbalanced and (2,92%) balanced karyotypes. Thirteen cases of pathological karyotype include 7 trisomies, 4 numerical aberrations of gonosomes and 2 unbalanced translocations , and 10 among them were over the age of 35, that increased the risk.From 26 balanced karyotypes, reciprocal translocation were more common (17; 65.4%) then the robertsonian type (9; 34.6%). A total of 889 infertile couples, in 51 cases one of the parents was already known as a carrier balanced rearrangement.In 20 fetal karyotypes (2.25%) was detected that one of the parents was balanced translocation carrier.It was found one case of de novo robertsonian translocation. The analysis shows high excess of female (20) over male (6) carriers.Prenatal cytogenetic diagnosis should be performed in pregnancies of infertile couples who have had two or more pregnancy losses. They are at risk for carrying a balanced rearrangement since these carriers may lead to offspring with an unbalanced karyotype causing serious congenital anomalies. Many chromosomally imbalanced fetuses are spontaneously aborted before amniocentesis, but partial trisomies of small rearrangements tend to be maintained in pregnancy. Because of the high incidence of chromosome abnormalities in spontaneous abortions at infertile couples, prenatal diagnosis is needed as well.

The significance of congenital anomalies to gestational wastage in a Brazilian University Hospital

D. P. Cavalcanti, A. Aguirre, M. A. Pessoto, M. F. Mello;
UNICAMP, Campinas - SP, BRAZIL.

It is described a prospective investigation at the Women’s Hospital (Caism), to determine the precise causes of gestational wastage. This study included all perinatal deaths (PD) occurring in the obstetrical center at the Hospital between Sept/1999 to April/2001. All PD were analysed with a protocol including genetic-clinical examination, X-rays, clinical photographs, autopsy, and cytogenetic investigation when necessary. From a total of 228 PD, 80 fetus (35%) were malformed. The causes of gestational wastage were classified as maternal (85 cases, 37%), fetal (104 cases, 45%), or unknown (39 cases, 17%). Fetal causes were more commonly found in the group of early neonatal deaths. Besides obstetrical causes, the other main diagnosis found among PD from maternal origin, were arterial hypertension and infectious diseases. Among PD from fetal causes, 24 cases (23%) were twins. The remaining were all malformed foetus distributed as follows: 35 (44%) isolated defects, 15 (19%) foetus with multiple anomalies, and 30 (37%) syndromic fetus. In the isolated group congenital malformations of the CNS (22) followed by uro-genital (12) were the most frequent anomalies. Chromosomal and disruption syndromes, besides skeletal dysplasias were the main syndromes diagnosed. With regard to all the known causes of deaths in the whole group, we observed that secondary prevention is possible in more than half of the deaths with maternal origin. Among deaths from congenital anomalies of the foetus, prevention by genetic counselling and/or prenatal diagnosis is possible in more than 80% of the cases.
Partially Supported by FAPESP, grant 98/16006-6.

Rapid prenatal diagnosis of common autosomal aneuploidies by relative semi-quantitative fluorescence PCR on uncultured amniocytes

C. Philippe¹, H. Rahil¹, J. Solassol², G. Lefort², P. Jonveaux¹;
¹Laboratory of Human Genetics, Vandoeuvre les Nancy, FRANCE, ²Laboratory of Cytogenetics, Montpellier, FRANCE.

Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time consuming, expensive and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance for women with normal results can be performed by molecular analysis of uncultured foetal cells in less than 24 hours. Today, all molecular techniques developed for a fast diagnosis of aneuploidies rely on the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. Our objective was to test a chromosome quantification method based on the analysis of fluorescent PCR products derived from non polymorphic target genes. An easy to set up co-amplification of portions of DSCR1 (Down Syndrome Critical Region 1), DCC(Deleted in Colorectal Carcinoma), and RB1(Retinoblastoma 1) allowed the molecular detection of aneuploidies for chromosomes 21, 18, and 13 respectively. Semi-quantitative analysis was performed in a blind prospective study of 400 amniotic fluids. Four samples (1%) could not be analysed by PCR probably because of a low concentration of foetal DNA. Follow up karyotype analysis was done on all samples and molecular results were in agreement with the cytogenetic data with no false-positive or false-negative results. Our gene based fluorescent PCR approach is an alternative molecular method for a rapid and reliable detection of aneuploidies which can be helpful for the clinical management of high-risk pregnancies.

Deletion 22q11 and conotruncal cardiopathy in four successive pregnancies: contribution of prenatal diagnosis.

CATCH 22, an acronym for cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, and hypocalcemia is associated with a variable deletion on chromosome 22q11 and occurs in patients with dysmorphologic and cardiological syndromes: DiGeorge, velocardiofacial and conotruncal/face syndrome. Estimates suggest that the 22q11.2 deletion occurs in approximately 1/4000 live births, making this disorder a significant health concern, so much so that 22q11.2 deletion studies are becoming part of a standardized diagnostic workup for some isolated heart defects. We report on 4 consecutive 22q11.2 deletion antenatal diagnoses, in an asymptomatic 22q11.2 deletion carrier, ascertained following in utero detection of a conotruncal cardiac defect. Her first pregnancy resulted in a normal female. The second and 3rd pregnancies were terminated following ultrasonographic visualization of conotruncal cardiac defect. FISH analysis of amniotic fluid from the second affected pregnancy confirmed the diagnosis of 22q11.2 deletion, for which the mother herself tested positive. FISH performed on CVS from her 4th and 5th pregnancies were again consistent with the diagnosis of 22q11.2 deletion. Cardiac defects detected on ultrasonograph of the 4th pregnancy were not visualized during the 5th pregnancy, which was terminated on week 15. The ethical issue imposed by termination of what seems to be a normal fetus, albeit carrying 22q11.2 deletion, is underlined. This report highlights the importance of offering 22q11.2 deletion testing to parents of affected probands and couples following in utero detection of a cardiac defect. Antenatal knowledge of the deletion status provides couples with an accurate diagnosis, prognostic information, and recurrence risk.

Y. Rüsing¹, R. Stressig², K. Marquet³, G. Hickmann¹, I. Grund¹, P. Kozlowski¹;
¹Praenatal-Medizin und Genetik Düsseldorf, Düsseldorf, GERMANY, ²Praenatal-Medizin und Genetik Düsseldorf, Düsseldorf, GERMANY, ³Gynaecologist, Aachen, GERMANY.

Abstract
Three cases of deletion 13q
Rüsing Y. 1, Stressig R.1, Marquet K-L.2, Hickmann G.1, Grund I.1, Koslowski P.1
1Prenatal Medicine and Genetic, Duesseldorf, Germany
2 Gynaecologist , Aachen, Germany
We present three cases of deletion in the long arm of chromosome 13 discovered in routine amniocentesis because of maternal age and one case with ultrasonografic indication.
Cases 1 and 2 show a structural aberrant karyotype, with breakpoint in 13q31.
We compare ultrasonografic findings, which showed severe fetal abnormalities at the cerebral features, like holoprosencephalie and exencephalie.
The last one is a special case. Here we present a ring chromosome with deletion in the long arm of chromosome 13. The deleted region of the ring chromosome is established by F-PCR (ABI 310).

Alpha1-antitrypsin deficiency due to maternal uniparental disomy for chromosome 14

M. Blayau¹, S. Odent², C. Dubourg¹, A. Dabadie³, V. David¹;
¹Laboratory of Molecular Genetics and Hormonology, Rennes, FRANCE, ²Medical Genetics, Rennes, FRANCE, ³Pediatry, Rennes, FRANCE.

Alpha1-antitrypsin is a major inhibitor of serine protease and plays a crucial role in protecting pulmonary tissue from degradation by neutrophil elastase. Alpha1-antitrypsin deficiency, an autosomal recessive disorder, predisposes individuals to the development of pulmonary emphysema and is also associated with chronic liver disease. The PI gene is located on the long arm of chromosome 14 at position 14q32.1. Numerous normal or deficient variants of this gene have been described but two major mutations (Z and S) are responsible for the pathology.
We report on a child with developmental delay and alpha1-antitrypsin deficiency (ZZ). His mother is heterozygous for the Z mutation but his father presented with a normal genotype (MM).Exclusion of paternity was discarded and then we began a molecular analysis to explain the genotype discordance and the particular phenotype of the child. Six microsatellites on the long arm of chromosome 14 (D14S264, D14S1057, D14S258, D14S76, D14S67, D14S1162) were tested. Analysis of DNA polymorphisms shows no contribution of the father and two chromosomes 14 from the mother with isodisomy for markers near the PI locus and heterodisomy for the more centromeric markers.
Uniparental disomy as a mecanism of recessive disorders may be evocated when the implicated chromosomic region carries a mutated gene.So, cystic fibrosis due to disomy of chromosome 7 has been reported. We report here the first case of alpha1-antitrypsin deficiency due to disomy of chromosome 14.

Prenatal diagnosis of congenital lipoid adrenal hyperplasia (CLAH) by measuring maternal serum unconjugated estriol(MSuE3) in the 1st trimester of pregnancy.

Z. Ben-Neriah¹, B. Zelikovitch¹, D. Zangen², G. Bach¹;
¹Dpt of Human genetics, Hadassah Medical Center, Jerusalem, ISRAEL, ²Dpt of Pediartrics, Hadassah Medical Center, Jerusalem, ISRAEL.

CLAH is a rare autosomal recessive disease, quite common in the Palestinian population in Israel and is usually diagnosed after birth. The clinical picture is adrenal insufficiency at birth: dark skin, vomiting and failure to thrive. The phenotype is of a female although the karyotype is XY. Unless treated early, death can occur shortly after birth. Treatment with steroids and salts is required for lifetime. The basic defect in this disease is no production of any steroid: cortico- mineralo- and sex steroids. The genetic defect is in the StAR gene, encoding a cholesterol shuttle protein, which transports cholesterol into the mitochondria, where the process of steroidogenesis takes place.
Prenatal diagnosis of CLAH is possible by mutation analysis of the StAR gene, or measuring the MSuE3 during 1^st trimester of pregnancy, between 10-13 weeks. We will present our experience in diagnosing fetuses with CLAH and other types of fetal adrenal insufficiency in the 1^st trimester of pregnancy. We are using a non-invasive method, which, to our best of our knowledge, has not been offered before at such early stage of pregnancy.

Mosaicism (the presence of two or more different cell lines) is reported to occur in approximately 1 % of chorionic villus samples (CVS), although one cell line may be confined to the placenta. Rapid aneuploidy testing is now being applied in many cytogenetic laboratories either to complement or replace conventional karyotype analysis. Assessment of these technologies includes their ability to detect potentially significant abnormalities such as mosaicism.
Quantitative fluorescence-PCR (QF-PCR) detects genomic imbalance by comparing the alleles of amplified STR markers. Routine use of QF-PCR in our centre (>3000 samples to date) has established that this technology can detect low level trisomy mosaicism. However, a previously unreported form of mosaicism at the molecular level was also observed. Nine out of 275 (3.3%) CVS samples showed frond-specific de novo STR alleles. In two of these samples instability was confirmed (one 8bp expansion of D13S742 and one 4bp expansion of D21S1411) by investigation of parental DNA, and in seven by the absence of the novel allele from other fronds and from cultured cells. Mosaicism for a novel allele at a single locus was also detected in uncultured material and cultured cells from one amniotic fluid sample. Eight different STR markers to date have demonstrated novel alleles. Although repeat instability is unlikely to represent an abnormal phenotype, the relatively high frequency of somatic mutation in individual villi is surprising. Case results, possible mechanisms and implications for the interpretation of QF-PCR results will be discussed.

D. Gaillard¹, M. Doco-Fenzy¹, F. Carre-Pigeon¹, H. Sartelet², M. Lorenzato²;
¹Service de Génétique CHU, Reims, FRANCE, ²Laboratoire Pol Bouin, Reims, FRANCE.

Triploidy and tetraploidy are detected in 20% of early spontaneous abortions and a few fœtuses survive beyond midgestation. Polyploid mosaicism is very uncommon. We report seven spontaneous abortions with triploid/tetraploid placental mosaicism, with four empty sacs at seven to sixteen weeks of gestation (WG) and three cases with placenta and fœtus at sixteen, seventeen and twenty-two WG. Karyotyping was not available.
DNA quantification was carried out after pathological investigation suggestive of triploidy The two oldest fœtuses had severe growth retardation (-3SD), syndactyly, adrenal hypopoplasia. The histology of the seven placentas showed hydropic villi with cisterns, vessels, trophoblast hyperplasia and scalloping of the fibrous villi. A few villi exhibited enlarged nuclei. Paraffin embedded placenta sections were used for DNA image cytometry after Feulgen staining, using the CAS 200 image analysis system. At least 200 nuclei were analysed in the mesenchyme of the villi and maternal decidua was used as control (diploid cells with DNA index [DI] near 1). In the seven placentas DI was near 1.5 (triploid nuclei) in 36 to 59% of the cells and near 2 (tetraploid nuclei) in 38 to 51% of the cells. The two oldest fœtuses were triploid (DI near 1.5).
Abnormal fertilization with diandry or digyny are the usual mechanisms reported in triploidy. The seven cases of triploid/tetraploid mosaicism give a new insight with a third mechanism taking place later during segmentation. It could be due to abnormal development of the poles of the spindles during mitosis.

A. V. Gagarina¹, T. V. Kuznetzova¹, T. K. Kascheeva², N. G. Pavlova², I. N. Ogiganova², V. S. Baranov²;
¹Ott’s Institute of Obstetrics and Gynecology RAMS, Saint-Petersburg, RUSSIAN FEDERATION, ²Ott’s Institute of Obstetrics and Gynecology, Saint-Petersburg, RUSSIAN FEDERATION.

Confined placental mosaicism (CPM) is reported to be associated with adverse pregnancy outcomes in form of spontaneous abortions, preterm births and intrauterine growth retardation. Pregnancy progression and perinatal complications in 12 patients with CPM, detected in the first-trimester CVS, were studied. Chromosomes, involved in mosaicism in cytotrophoblast, were different, involving 3,7,14,19,21 and X . Average age of pregnant women was 33,7±7,2 years (M±SD), 7 patients were 35 years and older. The mean value of maternal serum alpha-fetoprotein was 1,33±0,56 MoM and maternal serum chorionic gonadotropin (MShCG) 2,49±1,41 MoM, abnormal high level of MShCG (>2,0 MoM) was noticed in 5 patients. Uterine artery Doppler velocimetry during pregnancy was performed with 4-weeks intervals. Average uterine systolic-diastolic ratio and pulsatility index was found elevated in women with CPM compared with control group at 20 (S/D =2,96±0,23 and 2,13±0,05; p<0,001) and 36 weeks of pregnancy (S/D =2,32±0,17 and 1,71±0,05; p<0,001). Pregnancy complications and adverse outcome were registered in 2/3 of women with CPM. Preeclampsia developed in 3 patients. Stillbirth was registered in 4 cases (1 case of spontaneous abortion at 26 weeks and 3cases of preterm delivery before 37 weeks of pregnancy); intrauterine growth retardation was detected in 3 cases. Morphological signs of the placental insufficiency and villi immaturity were observed in all placentas. Our data reflect a possible negative effect of CPM on the pregnancy outcome, probably due to the placental abnormalities, and supports the previous reports of association between CPM and adverse pregnancy outcome.

Rapid X chromosome dosage by Quantitative fluorescent Polymerase Chain Reaction (QF-PCR) and prenatal diagnosis of Turner syndrome

V. Cirigliano¹^,2, M. Ejarque¹, P. Cañadas¹, C. Fuster², M. Adinolfi³;
¹General Lab, Barcelona, SPAIN, ²Universitat Autonoma, Barcelona, SPAIN, ³University College, London, UNITED KINGDOM.

The quantitative fluorescent polymerase chain reaction (QF-PCR) is an assay designed to perform rapid prenatal detection of common numerical chromosome abnormalities. This method is based on the PCR amplification of highly polymorphic short tandem repeats (STRs). In the course of PCR amplification, a fluorochrome is incorporated into the products, which are then visualised and quantified using an automated DNA sequencer. Several investigations have documented the accuracy of performing rapid prenatal diagnoses of trisomies involving chromosomes 21, 18 and 13.
However, due the unavailability of highly specific STR markers, only in recent times it has been possible to detect X and Y chromosome abnormalities. For the detection of Turner syndrome, several X-linked STRs are included in multiplex PCR assays so that, using up to 4 markers, the likelihood of a normal female to be homozygous for all sequences, is expected to be extremely low (about 2/1000).
Here we report a new method for rapid detection of X chromosome copy number in all prenatal samples. The test is based on QF-PCR amplification of the X-linked HPRT with the autosomal D21S1411 used as internal control for quantification. X chromosome copy number is rapidly and accurately assessed by comparing the ratio between the fluorescent activity of the X specific and autosomal PCR products, thus independently from any calculation of probability.
In its first clinical application this method allowed distinguishing a rare normal female foetus, homozygous for all X chromosome markers used, from a Turner syndrome.

Application of fluorescence in situ hybridization in prenatal diagnosis for identification of rare structural aberration.

B. Pawlowska¹, A. Ilnicka¹, J. Bogdanowicz¹, A. Tomankiewicz-Zawadzka¹, T. Roszkowski², J. Zaremba¹;
¹Institute of Psychiatry and Neurology, Warszawa, POLAND, ²Clinic of Obstetrics and Gynecology, Warszawa, POLAND.

We present results of cytogenetic prenatal investigations in 5 cases of de novo chromosomal rearrangments impossible to diagnose with routine cytogenetic banding techniques. Fluorescense in situ hybridization (FISH) with multiple chromosome specific libraries (chromosome painting) and subtelomeric Chromoprobe Multiprobe T System (Cytocell) were applied to identify or verify these rearrangments.
In two cases translocated fragments were so small, that we had to apply FISH to find out whether the translocations were balanced. In one case we found a small additional fragment on the short arm of chromosome 15. FISH enabled us to identify it as a part of chromosome 2. It coincided with inversion of chromosome 10 in the father. In one case we found de novo pericentric inversion of chromosome 3. In the last case the kariotype was mosaic 45,XO/46,X+mar. In amniocytes the marker was present in 28% of the cells, in leucocytes (obtained by cordocentesis) - in 90 % of the cells. Using painting and subtelomeric probes we found that the marker contained sequences of Y. We also found p-arm subtelomeric region of Y or X chromosome (cohybridization X and Y
p-arm) on both ends of Y. Ultrasonography has shown that the fetal sex was male.

Application of fluorescence in situ hybridization to chromosome analysis in prenatal diagnosis

R. Jankova¹, B. Zaharieva², V. Mazneikova³, V. Dimitrova³, P. Popivanova³, D. Toncheva²;
¹Institute of Immunobiology and human genetics, Institutes, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ²Department of Medical Genetics, Medical Faculty Sofia, Sofia, BULGARIA, ³Univ. Hospital of Obstetrics and Gynecology "Maichin dom", Sofia, BULGARIA.

For over two decades banding has remained the "gold standard" of cytogenetic analysis, providing the first genome-wide screen for abnormalities. However, these analyses are complemented with techniques based on fluorescence in situ hybridization-FISH, which steadily increased the accuracy of cytogenetic diagnosis. The basis of in situ hybridization techniques is the detection of specific nucleic acid sequences on cytogenetic level.
In our laboratory FISH is one of the routine methods of prenatal diagnosis, which previously was introduced and optimized, for molecular cytogenetic diagnostics in oncohematological disorders of over 50 cases. In this study it was applied for detection of trisomy 21 in 11 cases and also for searching of some aberrations of sex chromosomes. Nuclei were prepared from a culture of chorionic villi in three cases and amniotic fluid in the others. Hybridization was performed with specific X/Y and 21q22 probes, and detected with FITC and spectrum orange. In one sample of all analyzed interphase nuclei were found three signals after hybridization with 21q22 probe, indicating that three chromosomes 21 exist (Down syndrome). After using X/Y probe in one case was detected monosomy X, without signal for chromosome Y (Turner Syndrome).
Except for the accuracy of the results, with routine implementation of FISH into laboratories a lot of obstacles, like cases of advanced pregnancy, or problems with cultivating of cells could be overcome. FISH is now the complementary method of choice because of the increased sensitivity and speed with which it can be applied to a variety of cellular targets.

The mutational and haplotype analysis of the phenylalanine hydroxylase gene in families with phenylketonuria from the Bashkortostan.

V. L. Akhmetova, T. V. Viktorova, E. K. Khusnutdinova;
Institute of Biochemistry and Genetics of Ufa Scientific Center of Russian Academy of Sciences, Ufa, RUSSIAN FEDERATION.

Phenylketonuria (PKU) is a common autosomal recessive genetic disorder caused by a large variety of mutations in the phenylalanine hydroxylase (PAH) gene.
This study repots the results of mutational and haplotype analysis in 56 families with phenylketonuria from the Bashkortostan. Our results indicate that the R408W mutation account for over 53% PKU in Bashkortostan. Using SSCP analysis followed by sequencing of 7 and 12 exons of the PAH gene we have identified 5 mutations: R261Q (9,8%), R252W (2,7%), P281L (2,7%), R252P (1%) and 1315+del4 (1%).
We have examined the distribution of haplotypes of four polymorphic alleles of the PAH gene (VNTR, MspI(a), STR and PvuII(a) alleles). Most common PAH haplotype (380-MspI(a)a-240-PvuII(a)A2) appeared in 47% of the PKU chromosomes, but rare occurred on normal chromosomes (4%).
Moreover, we have studied haplotypes associated with PKU mutations. The most prevalent PAH haplotype 380-MspI(a)a-240-PvuII(a)A2 was tightly linked to the most common R408W mutation (67%) and the R261Q mutation (82%). The others mutations detected in this study were associated with various haplotypes.
Thus our investigations demonstrated a strong correlation between the R408W mutation and PAH haplotype 380-MspI(a)a-240-PvuII(a)A2, which correspondents to RFLP-haplotype 2. These dates suggest a common origin for this mutation from Europen populations, where R408W mutation is strongly associated with haplotype 2.
In addition, we have determined the informativity of PAH haplotype for molecular diagnostics of PKU in Bashkortostan that was 95,5%. The data of haplotype and mutational analysis may be used for prenatal diagnostics and carrier screening in PKU families.

Trends in cytogenetic prenatal diagnosis in a reference hospital in Izmir/TURKEY: A comperative study for 4 years

C. Ozkinay;
Ege University Faculty of Medicine,Dept.of Pediatrics,Subdivision of Genetics and Teratology, Izmir, TURKEY.

The aim of the study was to investigate the major changes in the indications, culture success and abnormality rate for conventional cytogenetic prenatal diagnosis for amniotic fluid samples between the period of January 1998 and December 2001 in our area.
Our cytogenetic laboratory provides a prenatal service to obstetrics-gynecology departments of different hospitals in Izmir. Limited number of patients (6-8 per week) is randomly accepted for prenatal cytogenetic study in our center.
Over the 4 years period 1023 prenatal cytogenetic tests were performed in our center. The most common indication was advanced maternal age for each year. But its rate has increased significantly within the years. Culture success rates have improved. When the first two years compared to the last two years the rate of abnormal cytogenetic results were significantly decreased.
The major reason for this observation is probably related to the changes in indications throughout the years.

Seven cases of chromosomal mosaicism detected in amniocentesis and karyotype, phenotype correlations.

C. Gunduz¹, A. Alpman¹, E. Karaca¹, T. Çankaya¹, E. Bora¹, S. Sagol², H. Onay¹, O. Cogulu¹, C. Ozkinay¹, F. Ozkinay¹;
¹Ege University, Faculty of Medicine,Dept.of Pediatrics,Subdivision of Genetics and Teratology, Izmir, TURKEY, ²Ege University, Faculty of Medicine, Dept. of Obstet Gynecol, Izmir, TURKEY.

Cytogenetic test results of 908 amniocentesis performed throughout three years period were evaluated.
Out of 908 amniocentesis, seven cases (0.77%) were detected having the chromosomal abnormalities in more than three cells in total. In all of these cases, different chromosomal variations were observed in abnormal cell lines.
Out of seven cases, four of them were detected with numerical chromosomal abnormalities in mosaic cell line. These were 46,XY[110]/47,XXY[11]; 46,XY[96]/47,XY,+21[4]; 46,XY64]/47,XY,+22[12]; 46,XY[35]/47,XYY[15]. The remaining three cases had structural chromosomal abnormalities in mosaic cell lines. Two of these were balanced translocations (46,XY[150]/46,XY,t(2;12)(q23;q24.2)[5]; 46,XY[65]/46,XY,t(7;8)(q22;p23)[5]) whereas the other one was a rarely seen chromosomal deletion (46,XX[95]/46,XX del 18(q12.1-qter)[5].
In all of the three cases with mosaic structural chromosomal abnormalities fetal cord blood showed normal fetal karyotype and the outcome of these pregnancies were also normal.
Only in one of the four cases with mosaic numerical abnormalities, mosaicism was also detected in the fetal blood and the family chose abortion. The others’ karyotypes in fetal blood were normal (and one of these resulted in spontaneous abortion while the other two resulted in normal outcome).

H. M. Ryu¹, J. R. Han¹, M. Y. Kim¹, J. H. Yang¹, Y. M. Kim², S. Y. Park², K. H. Choi¹;
¹Department of Obstetric and Gynecology, Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA, ²Laboratory of Medical Genetics, Samsung Cheil Hospital and Women's Healthcare Center, Seoul, REPUBLIC OF KOREA.

Objective; With an increasing number of pregnancies being subjected to prenatal karyotyping, large proportions of fetuses with borderline prognosis are detected prenatally as an incidental finding. The couples are faced with a very difficult and personal decision for the pregnancy. This study was designed to evaluate prenatal outcome of borderline prognostic cases in prenatal cytogenetic field. Materials and Methods; We reviewed retrospectively pregnancy outcome of 9,002 prenatal cytogenesis cases during 1990 - 2000. We classified with 4 groups according to prognosis. For precise determination of results, several cytogenetic methods were performed; repeat sampling, C-banding, R-banding, FISH, CGH, NOR banding and so on. Results; Group I (normal); (N=8661, 96.2%), Group II (balanced cases from parental origin); (N=87, 0.97%), Group III (apparently abnormal cases); (N=211, 2.35%, Group IV (borderline cases); (N=43, 0.48%). Through several cytogenetic studies, targeted fetal ultrasound and genetic counseling for borderline cases, pregnancies were continued and delivered healthy babies in 3 cases out of 6 de novo balanced reciprocal translocation, 2 out of 3 de novo robertsonian translocation, 15 out of 25 sex chromosomal aneuploidy, 3 out of 6 marker chromosome, 3 out of 3 discrepant results with prenatal samples. Conclusions; For genetic counseling of borderline cases, molecular cytogenetic study and targeted fetal ultrasound played an important role for option of pregnancy continuation.

Efficiency of Dyna-beads methods for non-invasive prenatal genetic diagnosis using fetal cells

G. Ahangari;
National Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Ghasem Ahangari, Sirous Zeinali and Maryam Ebrahimi.Department of Immunology, National Research Center for Genetic Engineering & Biotechnology, Tehran, Iran. Department of Biotechnology, Pasteur Institute of Iran,Iran.
Prenatal genetic diagnosis has become an essential part of modern obstetric care. Recent advancements in molecular technology and equipments have made fetal diagnosis possible. During the last two decades a number of methods of prenatal genetic diagnosis have become available and have used either in laboratory research or in routine genetic counseling. Non-invasive prenatal genetic diagnosis by circulatory fetal cells in maternal circulation also taking shape. The advantage of non-invasive methods (fetal cells and DNA in maternal blood) is that they provide an opportunity to make a genetic diagnosis without risk therefore, are applicable for use in mass screening programs. We obtained approximately 20 ml blood from 53 women. The mononuclear cells were isolated with ficole. To remove unwanted maternal white blood cells, antibodies against CD45 and CD14 coated with magnetic beads were used as negative selection. Positive selection methods based on using antibody against CD71 and Dynal magnetic to enrich fetal nucleated erythrocytes. DNA was extracted from fetal cells by a standard phenol-chloroform procedure and nested PCR was carried out with appropriate primers for RhD gene and amelogenine gene was used for sex (X and Y) determination. These results suggest that identification of fetal sex and RhD in the maternal blood circulation in 27 or %51 of the case is possible but efficiency of this method compare to others is low.

S. Andonova¹, E. Simeonov², D. Toncheva³, I. Kremensky¹;
¹Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynecology, Medical University, Sofia, BULGARIA, ²Section of Clinical Genetics, Department of Pediatrics, Medical University, Sofia, BULGARIA, ³Department of Medical Genetics, Medical University, Sofia, BULGARIA.

Short tandem repeats (STRs) are highly informative polymorphic genetic markers, widely used in the field of quantitative fluorescent PCR (QF-PCR) aneuploidy testing. With the intention to reduce pipetting errors, cost value and time for analysis, we have developed a multiplex PCR assay in a one-tube reaction for simultaneous analysis of two STRs located on chromosome 21 (D21S11 and D21S1411), one - located on chromosome 18 (D18S535) and one - on sex chromosomes (AMEL). PCR amplification with four Cy-5' fluorescently-labeled primer sets was performed and PCR products were separated on ALFExpress sequencer. Because of inability to use different fluorescent dyes, STRs were selected with different PCR product lenghts, with reported heterozygosity between 0.90 and 0.93. To avoid preferential amplification of shorter fragments and to receive the expected allele ratio the optimal primer amount was adjusted between 2-5 pmol of each primer, number of cylces = 25. The multiplex PCR was optimised with human genomic DNA of normal controls and trisomic 21 samples, no unspecific products were observed. Reported four-plex PCR assay proved to be rapid, accurate and efficient - results were in complete accordance with those obtained after performance of 4 separated single-plex reactions among the same 65 investigated DNA samples.

Applications of two-dimensional electrophoresis and fluorometric enzyme assay for MPS prenatal diagnosis

C. Chuang¹, S. Lin²;
¹Genetics & Metabolism, Dept. of Medical Research, Mackay Memorial Hospital, Taipei, TAIWAN REPUBLIC OF CHINA, ²Dept. of Pediatrics, Mackay Memorial Hospital, Taipei, TAIWAN REPUBLIC OF CHINA.

The mucopolysaccharidoses (MPS), a group of heritable disorders, are transmitted in an autosomal recessive manner, except for MPS II(X-linked). The diagnosis of MPS can be achieved by two-dimensional electrophoresis (2-D EP) for MPS typing determination and enzymatic assay for MPS confirmation. In 2000 - 2001, we had successfully completed prenatal detection by 2-D EP and fluorometric enzyme assay in seven unrelated families having previous affected children; 5 with MPS II, 1 with MPS III and 1 with MPS IV. Amniotic fluids (AF) were taken at 14th ~ 18th weeks of gestation. For 5 AF with high risk of MPS II, the results showed normal EP pattern, in which the chondroitin sulfate (CS) and hyaluronic acid (HA) were demonstrated. The activities of iduronate-2-sulfatase (IDS) were normal in the cultured AF cells. For pregnancy with high risk of MPS III, the 2-D EP result showed a composed pattern of CS, HA, and heparan sulfate (HS). For pregnancy with high risk of MPS IV, the result showed a composed pattern of CS, HA, and keratan sulfate (HS). The enzyme activities of alpha-N-acetyl glucosaminidase and galactosamine 6-sulfatase were all reduced in the cultured AF cells, which were corresponding with the 2-D EP results. On the basis of these results the pregnancies were terminated. By reviewing the 2-D EP method, it is definite, sensitive, and easy to perform for MPS prenatal diagnosis, however, a final confirmation is required by performing an enzymatic assay of that specific enzyme activity.

M. Acquila, M. Pasino, D. Caprino, T. Lanza, F. Bottini, P. G. Mori, M. P. Bicocchi;
Gaslini children's hospital, Genova, ITALY.

Previous studies have shown that haemophilia B (HB) is the result of several mutations, mostly single nucleotide substitutions, in the Factor IX gene. In order to evaluate the impact of mutation analysis on genetic counselling in sporadic and uninformative HB familial pedigrees, we re-analysed 12 patients by dHPLC, who had previously been studied by restriction fragment length polymorphisms (RFLPs). In order to perform mutation analysis, the coding regions, intron/exon splice junctions , 5’ and 3’ untranslated regions, and the promoter and polyadenylation site of Factor IX gene were amplified using the appropriate primers. In all cases unique abnormal dHPLC chromatograms were found, the specific abnormal fragments were sequenced and the mutations were characterised. The distribution detected in our screening studies fits with what is reported in the FIX mutation database. 93% of the mutations occurred in the coding sequences of the gene. They were all independent mutations, mostly (9/12) associated to the severe form of the disease. Point mutations were the most recurrent mutations. Three mutations were novel, unreported in the HB database and none involved CpG dinucleotides. Ten mutations were single base substitutions, one was a base insertion, and one was a four nucleotide deletion. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied. 3/12 de novo events were found in maternal meioses.

Discordant prenatal diagnosis due to a mosaic structural rearrangement of chromosome 21 in two trisomic 21 fetuses.

S. Brisset, A. Aboura, F. Audibert, V. Gautier, R. Frydman, G. Tachdjian;
Hôpital Antoine Béclère, Clamart, FRANCE.

Trisomy 21 mosaicism associated with a structural rearrangement is uncommon. We report on two prenatal diagnoses in which karyotypes showed mosaicism with an aberrant cell line including a rearrangement of chromosome 21. Prenatal diagnoses were performed because of increased nuchal translucency. In the first case, analysis of trophoblast cells revealed an abnormal karyotype: 46,XX/47,XX,+del(21)(q21). The amniocentesis and cordocentesis showed a non-mosaic trisomy 21. In the second case, the trophoblast direct analysis showed a normal male karyotype whereas the long-term culture revealed trisomy 21 mosaicism secondary to a rearrangement of chromosome 21 (either a Robertsonian translocation or an isochromosome): 46,XY/47,XY,rea(21q21q)/45,XY,-21. Amniocentesis confirmed the trisomy 21 in all cells. FISH analysis proved that these rearrangements were derived from chromosome 21. The mechanisms of formation are discussed. The first case could be explained by the deletion of one chromosome 21 in a trisomic zygote with instability of this de novo marker leading to a normal cell line. In the second case different mechanisms are suggested. The rearrangement might occurred postzygotically in a normal embryo with a non-disjunction or a translocation of homologous chromatides of one chromosome 21 leading to a cell line with the rearranged chromosome 21 and another cell line with a monosomy 21. A more complex mechanism of formation is also considered with a cascade of meiotic and subsequent mitotic errors. Our cases underline the importance to combine the short-term and long-term cultures and emphasises the need for confirmatory studies in other tissues when mosaicism is encountered in chorionic villi.

Molecular Diagnostics Of Bulgarian Patients With 17p11.2 Duplication/ Deletion Using Two Sets Of Highly Polymorphic DNA Markers

N. I. Ivanova¹, A. K. Jordanova¹, A. Kantardjieva¹, I. L. Tournev², B. Ishpekova², V. Gergelcheva², M. Daskalov², I. O. Litvinenko³, S. Veleva², V. Mitev⁴, I. M. Kremensky¹;
¹Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynecology, Sofia Medical University, Sofia, BULGARIA, ²Department of Neurology, Sofia Medical University, Sofia, BULGARIA, ³University Pediatric's Hospital, Sofia Medical University, Sofia, BULGARIA, ⁴Department of Biochemistry, Sofia Medical University, Sofia, BULGARIA.

Charcot-Marie-Tooth (CMT) disease is the most common inherited disorder of the peripheral nervous system. The majority of patients with demyelinating neuropathy (CMT1) have 1,5Mb duplication/deletion in 17p11.2 chromosome region. It is responsible for nearly 80% of authosomal-dominant CMT cases.
We have analyzed a total of 28 CMT1 families with clinical data for authosomal-dominant inheritance for 6 highly polymorphic markers closely linked to 17p11.2 region: D17S921, D17S122, D17S1357, D17S1358, D17S2226 and D17S2227. We have set up analytical conditions on ALF fragment analyzer to receive reliable and reproducible gene dosage differences in affected subjects and controls. We have detected 11 families with duplication and 1 with deletion. The combination of markers D17S921 and D17S122 was informative in 9 cases. Additionally testing for D17S1357, D17S2227 and D17S2226 revealed another 3 cases of 17p11.2 duplication. With these five markers we were able to identify duplicated/deleted and normal alleles in 100% of CMT1A and HNPP families. Polymorphic markers have been arranged in two sets based on their informativeness and length of repeats. The first one included D17S921, D17S122, D17S2227 and allowed correct and fast molecular diagnosis of 17p11.2 duplication/deletion in 90% of cases. The second one comprising D17S1357and D17S2226, we used in non-informative cases and as a verification test. Marker D17S1358 was informative in only 2 cases and therefore it is not currently used in our laboratory diagnostic practice. The described two-step procedure is fast, efficient and inexpensive, which makes it suitable for routine postnatal and prenatal diagnostics of 17p11.2 duplication/deletion in Bulgarian CMT1 families.

Recurrence of achondrogenesis type II within the same family: evidence for germline mosaicism

G. Mortier¹, M. Le Merrer², A. Delezoide³, N. Laurent⁴, C. Thauvin-Robinet⁵, T. Rousseau⁶, P. Coucke¹, L. Faivre⁷;
¹Department of Medical Genetics, Ghent University Hospital, Ghent, BELGIUM, ²Département de Génétique, Hôpital Necker Enfants Malades, Paris, FRANCE, ³Service de Foetopathologie, Hôpital Robert Debré, Paris, FRANCE, ⁴Service d'anatomo-pathologie, Dijon, FRANCE, ⁵Centre de Génétique, Hôpital d'Enfants, Dijon, FRANCE, ⁶Centre de Médecine foetale, Maternité, Dijon, FRANCE, ⁷Centre de Génétique, Dijon, FRANCE.

Achondrogenesis type II is a lethal skeletal dysplasia, characterized i) clinically by short-limbed dwarfism with short trunk, anasarca, and disproportionately large cranium, and ii) radiologically by lack of complete ossification of the vertebral bodies. New dominant mutations within COL2A1 have been identified in this disorder. Here we report on two pregnancies of a healthy, nonconsanguineous young couple. In the first pregnancy, severe micromelia and generalized edema were noted on ultrasound at 21 WG. Clinical evaluation of the fetus after termination of the pregnancy revealed short-limbed dwarfism with short trunk, narrow thorax, large head, generalized edema, flat face, short nose with anteverted nostrils, micrognathia and short neck. Radiographs showed typical findings of achondrogenesis type II, including very short and squared tubular bones, with irregular metaphyseal spurring, short iliac wings and absence of ossification of ischiatic and pubic bones. The vertebral bodies were very poorly ossified and bipartite in the dorsal region. Metacarpals and phalanges were small, with punctiform first metacarpals. Histologic studies confirmed the diagnosis. In the second pregnancy, fetal hygroma was noted at 11 WG and similar clinical, radiographic and histologic findings were observed. Molecular analysis of gDNA extracted from amniotic cells of the second fetus revealed heterozygosity for a G316D mutation in the COL2A1 gene. This mutation was absent in gDNA extracted from the parental blood lymphocytes. Although we could not prove the presence of the mutation in the first fetus because of lack of appropriate materials, we strongly believe that our data are in favour of germline mosaicism.

Prenatal diagnosis of partial trisomy 18 by quantitative fluorescent PCR (QFPCR)

M. Macek¹, M. Matejckova¹, M. Brouckova¹, J. Marek², M. Simandlova¹, V. Krutilkova¹, M. Malikova¹, D. Chudoba¹, D. Novotna¹, E. Kulovany³, V. Povysilova⁴, M. Havlovicova¹;
¹Institute of Biology and Medical Genetics, Prague 5, CZECH REPUBLIC, ²Kardiocenter of University Hospital Motol, Prague 5, CZECH REPUBLIC, ³Clinic of Obstetrics and Gynaecology of University Hospital Motol and of 2nd Medical School of Charles University, Prague 5, CZECH REPUBLIC, ⁴Institute of Pathologic Anatomy of 2nd Medical School of Charles University, Prague 5, CZECH REPUBLIC.

The aim of this report is to document the importance of the QFPCR for rapid prenatal diagnosis. This pregnancy was referred in 24^th week because of decreased maternal serum hCG (0,23 MoM) and abnormal ultrasound examination. It revealed ren arcuatus, cardiac anomaly. Karyotype from amniocytes was 46,XX. QFPCR analysis confirmed normal STR pattern for chromosomes 13,21,X and Y using D13S258,D13S631,D21S11, D21S1414,X22,XHPRT STR markers. Female sex of the fetus was confirmed. D18S535 STR marker (18q12.2-q12.3) characteristics brought evidence of normal diallelic pattern with peak ratio 1,2. Repeated examinations of STR D18S51 marker disclosed diallelic type of partial trisomy 18 in 18q21.3 region with peak ratio 2,41 (2,31-2,60). The mother decided for the abortion of this fetus. In aborted fetus ventricular septal defect, open foramen ovale and ductus arteriosus, pericardial exudate, aplasia of umbilical artery, ren arcuatus with cystic dysplasia of the caudal kidney pole were found. These anomalies correspond to the described features of partial trisomy 18q21.1. The abnormal chromosome was inherited from the mother as follows from the comparison from parental and fetal electrophoretic patterns. These STR markers did not allow to ascertain origin in first or second meiosis, because the mother was homozygous for D18S51 STR marker. QFPCR provides exact, rapid diagnosis from microquantity of cells, determination of parental origin also in partial trisomies, non detectable by the current cytogenetic methods in the range of 270-310 bp as was in this case. Supported by grants IGA 6462-3,6411-3,LN-00A079,11130003,00000064203

Placenta/fetus discrepancies involving structural abnormalities of chromosome 8 detected in a prenatal diagnosis.

A. Soler¹, A. Sánchez¹, A. Carrió¹, C. Badenas¹, M. Milà¹, A. Borrell²;
¹Servei de Genètica. Hospital Clínic, Barcelona, SPAIN, ²Servei d'Obstetrícia. Hospital Clínic, Barcelona, SPAIN.

Confined placental mosaicism is detected in 1-2% of pregnancies, but most of them involve aneuploidies. Structural mosaicism is a very rare event and difficult to interpret. We describe the case of a pregnant woman referred for prenatal diagnosis due to advanced maternal age. Semidirect cytogenetic analysis performed on chorionic villi showed a mosaic 46,XX,i(8q)/46,XX,del(8)(p11.2) karyotype, demonstrated by FISH. Karyotypes of the parents were normal. An amniocentesis was performed at 15 weeks, when ultrasonographical examination showed comunicant hydrocephaly. Cytogenetic analysis of cultured amniocytes showed a 46,XX,dup(8)(p23p11.2) karyotype. The pregnancy was terminated; pathologic findings included club feet, clenched left hand, subcutaneous edema and bilateral hydrocephaly. Molecular studies using chromosome 8 microsatellites performed on parents’ blood and fetal tissues revealed a maternal meiotic origin of the inv dup(8p) with deletion of distal p23 region and duplication of remaining 8p, in agreement with other published cases (Floridia et al. 1996). We propose a model to explain the cytogenetic findings, which includes a first maternal meiotic error giving rise to a large dicentric isochromosome 8 present in the ovum, a second error in one of the first zygote divisions with misdivision of the dicentric 8 giving rise to a cell line with del(8p) confined to trophoblast and the othe cell line with inv dup(8) confined to fetal tissues, and a third error in trophoblast giving rise to a new cell line with isochromosome 8q.

Tetrasomy 12p or Syndrome de Pallister-Killian. Interest of the diagnostic on buccal smear

J. Vigneron¹, M. J. Gregoire², M. Andre³, C. Moret⁴, J. M. Hascoet³;
¹Unité de Fonctionnelle de Génétique, Maternité Regionale, Nancy, FRANCE, ²Laboratoire de Génétique, CHRU, Vandoeuvre les Nancy, FRANCE, ³Néonatologie, Maternité Regionale, Nancy, FRANCE, ⁴Neuroradiologie, Hôpital Central, Nancy, FRANCE.

We present a new case of 12p Tetrasomy diagnosed on buccal smear in the neonatal period.
A macrosomia was diagnosed during the gestation. A maternal diabetes was excluded.
At birth, the child was referred to NICU for hypotonia and neonatal distress.
Clinical evaluation showed a coarse and oedematous face, nuchal skinfolds, a mouth with downturned corners, dysplastic ears, a posterior cleft palate, an hypopigmented area of the scalp leading to the hypothesis of Pallister-Killian.
The neuroradiologic work-up showed a callosal dysgenesis with abnormal gyration pattern.
The diagnosis was prooved by direct FISH of buccal smear and controlled by fibroblast caryotype.
This case report illustrates the importance of perinatal US manifestations of PKS, the importance of caryotyping any foetus with unexplained macrosomia justified by the severe psychomotor retardation and the unfavourable pronostic.
Buccal smear allows easy and reliable diagnosis of this condition in a fraction of the time necessary for conventional caryotype on cultured fibroblasts.

Detection of trisomy 21 in a fetus during the investigation for Tay Sachs disease; prenatal cytogenetic study should be performed associated with molecular or enzymatic studies

A. Alpman¹, G. Sapmaz¹, E. Karaca¹, T. Cankaya¹, E. Bora¹, O. Cogulu¹, C. Gunduz¹, W. J. Kleijer², F. Ozkinay¹;
¹Ege University Faculty of Medicine,Dept.of Pediatrics,Subdivision of Genetics and Teratology, Izmir, TURKEY, ²Erasmus Medical Centre Rotterdam Department Clinical Genetics, Rotterdam, NETHERLANDS.

A family whose previous 2 children died from Tay Sachs disease applied for genetic counseling. Tay Sachs disease had been confirmed by enzymatic study in the second child and the mother was 6 weeks pregnant. They were told that prenatal diagnosis for Tay Sachs and cytogenetic study for chromosomal abnormalities in chorionic villus sampling (CVS) would be available at the 12th week of gestation. CVS was performed at the week of 12th gestation. Enzymatic study showed a normal fetus for Tay Sachs but cytogenetic study revealed a fetal karyotype of trisomy 21. Genetic counseling was given to the family and pregnancy was terminated according to the family’s willing and ethic committee proposal at the 14th week of pregnancy.
Cytogenetic study should be offered in all pregnancies, which prenatal molecular or enzymatic studies are performed.

The informative analysis and prenatal diagnosis in the families with Duchenne muscular dystrophy in Moldova.

P. Stratulat, V. C. Sacara;
Scientific Research Institute of Mother and Child Health Service, Chisinau, REPUBLIC OF MOLDOVA.

Introduction: Duchenne muscular dystrophy (DMD) is a severe X-linked disease with an incidence of 1 in 3,500 males. Until recently, the most accurate diagnostic tests for DMD were the determination of serum creatine-kinase levels, muscle biopsy, and EMG. However, the application of recombinant DNA technology to the diagnosis of DMD has resulted in the development of more accurate tests.
Methods: 81 families with increased risk of DMD passed clinico-neurological, CK test, muscle ultrasonography investigations and molecular study. MPCR's were performed for deletion detection (18 different exons of dystrophin gene were tested in patients DNA) and RFLP-analysis (pERT87-8/TaqI, pERT87-15/BamH1 and 16 intron/TaqI polymorphisms).
Results and Discussion: About 76 % of probands were proved to be carriers of dystrophin gene deletion by MPCR. The given test highly is informative at the patients from Moldova. The analysis of informative families with MDD/B on the three intragenic polymorphisms pERT87-8/Tag1, pERT87-15/BamH1 and 16intron/Tag1 has revealed high of polymorphism 16intron/Tag1 (47,36 %) and low percent of informative was determined at the pERT 87-15/BamH1 polymorphism (21%). Smaller percent of informative has given polymorphism pERT87-8/Tag1 (41,77 %). These dates coincides with theoretical accounts of populations frequencies of the pERT87-8/Tag1, pERT87-15/BamH1 and 16 intron/Tag1 alleles which were counted by Hardy-Weinberg equilibrium.
Conclusion: The algorithm of molecular researches, selected by us, allows to define informative in 76 families (93% cases) and, accordingly, to conduct clinical, preclinical and prenatal diagnosis in DMD families.

Systematic follow up of 151 infants after diagnosis of nuchal anomalies at the 1st trimester

C. Baumann¹, R. Delagarde², E. Vuillard³^,4, J. Oury⁵;
¹Clinical Genetics Unit, Hôpital Robert Debré, Paris, FRANCE, ²Clinical Genetics unit,hopital Robert Debre, Paris, FRANCE, ³prenatal diagnosis unit, Paris, FRANCE, ⁴Hopital Robert Debre, France, FRANCE, ⁵prenatal diagnosis unit ,Hopital Robert Debre, Paris, FRANCE.

AIMS: to assess developmental outcome of infants with nuchal anomalies at first trimester scanning and normal conventional karyotype.
DESIGN : Between 1994 and 2001, abnormal nuchal thickness was observed in 360 pregnancies. All measurement were performed by the same observer (EV). Nuchal anomalies were subdivided in nuchal thickening, nuchal translucency, and cystic hygroma. The fetuses were karyotyped, other anomalies were carefully sought and prenatal genetic counselling given accordingly.
RESULTS : For 184 fetuses, the parents finally elicited to terminate the pregnancy. 176 children were delivered (18 of them with scanning anomalies). All delivered babies were examined by one examiner (CB), and clinical follow up at 3, 6, 12 and 24 months was offered. 21 were lost to follow up. Among the remaining 151 newborns, 136 (90%) were considered to have normal psychomotor development at age 2. Among these, 21 (15%) had malformations (prenatally detected in 17 cases) : 12 isolated (8.8%) and 9 multiple (5.3%). In 15 cases, (10%) delayed psychomotor development was shown, either isolated (7 cases) or associated with identified (cyto)genetic syndromes (8 cases, 7 of them diagnosed postnatally). Ultrasonographic anomalies most predictive of an abnormal neurodevelopmental outcome were : nuchal hygroma, persistence of US anomalies in 2nd trimester, and presence of associated CHD.
CONCLUSIONS : Neonates presenting a nuchal anomaly during pregnancy are at high-risk for psychomotor developmental delay, even when neonatal evaluation appears “normal”.

Fraser syndrome : Report of 5 additional fetal cases and review of the literature

J. Martinovic¹, N. Laurent², A. L. Delezoide¹, T. Rousseau³, C. Esculpavit¹, L. Faivre⁴, N. Morichon-Delvalez¹, M. Vekemans¹, E. Justarbo², P. Sagot³;
¹Service d'Histo-Embryologie et Cytogénétique, Departement de Génétique, Hopital Necker-Enfants Malades, Paris, FRANCE, ²Service d'Anatomie et de Cytologie Pathologique, Centre Hospitalier Universitaire, Dijon, FRANCE, ³Clinique Gynécologique et Obstétricale, Centre Hospitalier Universitaire, Dijon, FRANCE, ⁴Centre de Génétique, Centre Hospitalier Universitaire, Dijon, FRANCE.

The aim of this paper is a review of the diagnostic criteria of the Fraser multiple malformation syndrome (MIM 219000) in its severe fetal form. We based our study on the review of the literature, and report on the five additional fetal cases from two non-consanguineous sibships.
We reviewed 21 detailed fetal descriptions of Fraser syndrome from the literature (Thomas et al., Boyd et al., Ramsing et al.), and compared total of 26 fetal cases to 68 pediatric cases reviewed by Gattuso et al., the last representing viable milder form of the Fraser syndrome.
A quantitative estimate of the frequency of the principle clinical manifestations in the fetal versus pediatric population was obtained. The sensitivities of major criteria in fetuses varied between 77% (cryptophtalmos) and 96% (syndactyly), and of minor criteria varied between 81% (renal agenesis/laryngeal anomalies) and 23% (cleft lip/palate). Different renal anomalies were present in 96% of fetuses. Severe form of the syndrome should be more frequently diagnosed in-utero regarding a very poor prognosis and high perinatal lethality. Furthermore, a high recurrence risk of 25% among sibs warrants diagnosis for appropriate genetic counselling, the prenatal diagnosis being also much facilitated in further pregnancies in the cases with positive family history. Therefore, we are emphasizing the frequency of renal agenesis and laryngeal anomalies among fetal population and suggesting these two features should integrate major diagnostic criteria of Fraser syndrome in fetuses.