ESHG Posters 17

Genotype Analysis of the NF1 Gene in the French Canadians From the Québec Population

J. Thirion¹, L. Fang¹, N. Chalhoub¹, W. Li², J. Feingold³, J. Ortenberg⁴, B. Lemieux¹;
¹Faculté de Médecine CHUS, Sherbrooke, PQ, CANADA, ²Rockefeller University, New York, NY, ³Inserm 393, Hôpital Necker, Paris, FRANCE, ⁴McGill University, Montreal, PQ, CANADA.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that afflicts about 1 in 3,500 individuals. The NF1 gene spans about 350 kb at 17q11.2 with 60 exons and has a high mutation rate leading to 50% sporadic cases. We genotyped 19 NF1 families including 85 individuals (45 affected and 40 unaffected) from the French Canadians of the Québec population and investigated deletion mutations, allele frequency distribution, linkage and linkage disequilibrium (LD) by using six intragenic polymorphic markers including 2 RFLPs (EcoRI and RsaI) and 4 microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0) which are distributed along an approximately 65kb of the gene. Genotype analysis indicated families 7610 and 7473 bear unusual deletions. In Family 7610 the deletion removed the entire NF1 gene except exons1 to 4b. The breakpoint of the deletion is located between exons 4a and 4b. The deletion 7473 was derived from the maternal chromosome and exons 1 to 5 were deleted. The breakpoint of the deletion is located between exons 7 and 13. Clinical manifestations were mild suggesting that deleting either upstream or downstream contiguous sequence may not lead to a severe phenotype. Deletion of both upstream and downstream sequences on both sides of the NF1 gene may be needed to cause severe clinical features. The allele frequencies of microsatellites IVS27AC28.4 and IVS38GT 53.0 are compared to previously reported data from Caucasians, including Spanish and Italians. The difference is statistically significant (P<0.0036) for marker IVS27AC 28.4 between the Québec French Canadian and the Italian population.

Mutation Analysis of the EXT Genes in Taiwanese Patients with Hereditary Multiple Exostoses

F. Tsai, Y. Shi, J. Wu, C. Tsai;
China Medical College Hospital, Taichung, TAIWAN REPUBLIC OF CHINA.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by short stature , cartilaginous excrescences near the ends of the diaphyses of the bones of the extremities,and increased risk of chondrosarcoma . Three chromosomal loci have been comprised in this genetically heterozygous disease: EXT1 gene on chromosome 8q23-q24, EXT2 on 11p11-p13, and EXT3 on 19p. Both the EXT1 and EXT2 genes had been cloned and been defined as a new family of potential tumor suppressor genes. We had analyzed five patients with clinical features of multiple exostoses, of which one is sporadic and four are familial cases. For determining the mutant spectrum of disease attributable to abnormalities in the three EXT loci, linkage studies were performed before the mutation analysis. The results showed that one family linked to EXT1 locus and three linked to EXT2 locus. Four novel mutations were identified: a frameshift mutation (K218fsX247) and a nonsense mutation (Y468X) in EXT1 gene; a missense mutation (R223P) and a nonsense mutation (Y394X) in EXT2 gene. Moreover, according to the definite disease-causing allele, linkage analysis provides a reliable clue and could be utilized as an alternative approach for clinical and prenatal diagnosis.

R. Combi¹, S. Duga¹, R. Asselta¹, S. Boi¹, A. Oldani², M. Zucconi², L. Ferini-Strambi², M. Malcovati¹, L. Dalprà³, M. L. Tenchini¹;
¹Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, ITALY, ²Sleep Disorders Centre, University of Milan, Medical Faculty, Scientific Institute H San Raffaele, Milan, ITALY, ³Department of Experimental, Environmental Medicine and Medical Biotechnologies, University of Milan-Bicocca, Monza, ITALY.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), characterised by clusters of nocturnal seizures with a frontal lobe semiology, mostly occurring during non-REM sleep, follows autosomal dominant inheritance with incomplete penetrance (70-80%). Three loci have been associated to this syndrome: ENFL1 (20q13.2), ENFL2 (15q24) and ENFL3 (1q21). Three mutations responsible for ADNFLE have been reported in the CHRNA4 gene (ENFL1 locus), coding for the alpha4 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) and two mutations have been found in the CHRNB2 gene (ENFL3 locus), coding for the beta2 subunit of the same receptor. However, the identified mutations account for a minority of ADNFLE cases. Additional brain-expressed nAChR subunit genes (alpha2-7 and beta2-4) are candidates for ADNFLE. We performed linkage analyses to evaluate the association between ADNFLE and alpha2-7 and beta2-4 subunits in four families. Six chromosome regions were analysed: 1q21 (CHRNB2), 8p21 (CHRNA2), 8p11.2 (CHRNA6 and CHRNB3), 15q14 (CHRNA7), 15q24 (CHRNA5/A3/B4) and 20q13.2 (CHRNA4). Significantly negative LOD score values were obtained in each family, except in two cases (CHRNA4, family 32; CHRNA2, family 10), where, however, no mutations were detected by sequence analysis.
Besides further supporting locus heterogeneity of the disease, these findings exclude the involvement of all known neuronal brain-expressed nAChR subunits in the etiopathogenesis of ADNFLE in the analysed families and demonstrate the existence of at least a fourth locus, probably not belonging to the nAChR gene family, involved in this syndrome.
This project is supported by the Italian Telethon Foundation, grant E.C0839.

M. V. Nechyporenko, L. A. Livshits;
Institute of Molecular Biology and Genetics, Kiev, UKRAINE.

We carrier out genetic investigation of 100 patients with clinically diagnosed phenylketonuria in order to identify PAH gene mutations. Using DGGE and dHPLC techniques we have screened mutant alleles in exons 1, 3, 7, 11, 12 of PAH gene. Identification of mutations were performed by RFLP and direct DNA sequencing. The most frequent mutation found according to our study was R408W (57 %). The frequency of other mutations were: R158Q – 3,5%, R252W – 2,9%, P281L – 2,3%, Y414C – 1,5%, Ivs10nt546, Ivs12nt1, R261Q, G272X, S273F, R413P – 0,6-1%. PAH gene STR and VNTR polymorphisms analysis were performed in this group of patients too.
PKU is a highly heterogeneous trait showing a broad continuum of phenotypes. On the basis of individual data on phenylalanine tolerance and pretreatment phenylalanine serum the 24 patients were assigned to one of the four arbitrary phenotype categories: classic PKU, moderate PKU, mild PKU and MNH. 19 patients with genotypes R408W/R408W (7 patients), R408W/R158Q (2), R408W/R261Q (1), R408W/Ivs10nt546 (1), R408W/Ivs12nt1 (1), S273F/R413P (1), R252W/x (1), R408W/x (1), x/x (4) had classic PKU. 3 patients with genotypes R408W/P281L, R261Q/x, x/x were diagnosed as moderate PKU and 2 patients (genotypes R158Q/x and x/x) had mild PKU. It is interesting to note that two untreated patients with genotype R408W/Y414C were diagnosed as classic PKU and MHP basing on clinical date (they are 11 and 7 years old). This date confirm the necessity of treatment for PKU patients with mild PAH gene mutations.

M. C. Addor¹, V. Kaltenrieder¹, F. Gudinchet², D. F. Schorderet¹;
¹Division of Medical Genetics, CHUV - 1011 Lausanne, SWITZERLAND, ²Department of Radiology, CHUV - 1011 Lausanne, SWITZERLAND.

Inherited isolated brachydactyly type C (OMIM 113100) is an autosomal dominant disorder with marked variability. Type C consists of shortness of 1st metacarpal, 2nd, 3rd and 5th middle phalanges, hypersegmentation of the proximal phalanges of the 2nd and 3rd digits and ulnar deflection of the index. In most families, the anomalies are restricted to the hands. Associated findings such as short stature, radio-ulnar and humero-radial abnormalities, wedging of vertebrae, hip dysplasia, epiphyseal changes, foot anomalies and cupped ears have also been reported.
Locus heterogeneity for brachydactyly type C has been demonstrated. In a kindred with anomalies of upper and lower limbs reported by Haws (1963), a gene has been localized in 12q24 (Polymeropoulos et al., 1996); brachydactyly limited to the hands was mapped to chromosome 20q11.2 (Lin et al., 1996). Several mutations in the morphogen CDMP1, a member of TGF-b superfamily mapping to 20q, were predicted to cause haploinsufficiency (Polinkovsky et al., 1997). We present the clinical findings of 4 affected members of a 3-generation family with involvement of upper limbs only. Blood was obtained from 8 members of the family. Haplotype analysis showed segregation of the disease phenotype with markers on chromosome 20q11.2, but not on chromosome 12q24. Subsequently, mutation analysis by sequencing showed a t1380c transition resulting in a F354S mutation in CDMP1. This mutation segregated in the 4 affected patients and was never observed in normal members of the family, nor in 50 controls. As far as we know, this mutation has never been described before.

DGGE analysis of the low density lipoprotein receptor gene mutations in patients with familial hypercholesterolemia in Greece

G. V. Z. Dedoussis¹, C. Pitsavos², G. Sokari¹, M. Prassa¹, J. Skoumas², P. Toutouzas²;
¹Department of Science Dietetics-Nutrition, Harokopio University of Athens, Athens, GREECE, ²Ippocratio Hospital, Department of Cardiology, Medical School of Athens, Athens, GREECE.

We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 45 Greek patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Eight aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. These mutations were located in 6 different exons (exons 2, 6, 8, 9, 12, 14). Among them 8 were missense mutations (C6W, S265R, A370T, Q363P, Q363X, D365E, V408M, G571E) and 1 was a splice defect (2140+5G>A). The splice site mutation and the Q363P are detected for the first time in the Greek population. The prepositus for the splice defect was also double mutant for the mutations Q363X and D365E previously found in Greek-Cypriot subjects. These identified mutations co-segregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype since these were the only molecular defects identified in the entire region and splice site consensus sequences. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Greek population

Mutation and haplotype analysis of ABCA4 in mixed Spanish families and implication of this gene in a pattern dystrophy phenotype.

R. Gonzàlez-Duarte¹, E. Paloma¹, A. Martínez-Mir¹, R. Coco², L. Vilageliu¹, S. Balcells¹;
¹Departament de Genètica, Universitat de Barcelona, Barcelona, SPAIN, ²IOBA, Universidad de Valladolid, Valladolid, SPAIN.

Studies of genotype-phenotype correlations highlighted the function of ABCA4 in retinitis pigmentosa (RP), cone-rod dystrophy (CRD) and Stargardt disease (STGD). Initial screening of ABCA4 variants showed a correlation between the type of mutation and the severity of the disease. Later, in vitro studies of several recombinant ABCR mutants revealed a wide range of severity of biochemical defects [Sun et al., 2000]. In the present study we have undertaken mutational and haplotype analysis of ABCA4 in three mixed pedigrees segregating different retinal dystrophies to identify the combination and type of mutations causing the diseases. In family I, we have shown cosegregation of different ABCA4 alleles with STGD, FFM, CRD and even pattern dystrophy simulating FFM (FFM-like PD). To our knowledge, this is the first report of a PD phenotype explained by mutations in ABCA4. Based on the fact that this disease is milder than STGD, it is tempting to speculate that it may be explained by a combination of two mild alleles. On the other hand, in family II, segregating STGD and RP phenotypes, the involvement of ABCA4 in STGD is clear, but this is not the case for RP. Finally, in family III, also segregating STGD and RP, ABCA4 fails to explain either phenotype. Our data highlight the wide allelic heterogeneity involving this gene and support the genetic heterogeneity (beyond ABCA4) of mixed STGD/RP pedigrees.

C. Fey¹, A. R. J. Curtis¹, P. F. Chinnery², A. Lombès³, C. M. Morris¹, A. Curtis¹, J. Burn¹;
¹Institute of Human Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, UNITED KINGDOM, ²Department of Neurology, University of Newcastle upon Tyne, Newcastle upon Tyne, UNITED KINGDOM, ³Institut de Myologie, INSERM U523, Paris, FRANCE.

We have identified a new dominant neurodegenerative disease, neuroferritinopathy, which results from a pathological mutation in the ferritin light chain gene on chromosome 19 (OMIM: 606159). A single adenine insertion in exon 4 disrupts the DE loop and E helix at the carboxy-terminal end of the subunit and leads to brain iron accumulation and a variable clinical phenotype that can mimic Huntington’s disease, parkinsonism and dystonia. Several cases have now been recognised in the UK and share a common haplotype. We have now identified the same mutation in a French family, previously described in the literature with an atypical dystonia, MRI evidence of basal ganglia degeneration and reduced activities of several mitochondrial respiratory enzymes (Caparros-Lefebvre et al 1997 J Neurol Neurosurg Psychiatry, 63: 196-203). We show that this family shares the closest marker in the disease haplotype found in north England. Genealogical research has linked some of the English families to the 18th century Coulthard family of North Cumbria. The surname has been traced to a Norman extraction based on the town of Coudehard raising the possibility of a much more ancient origin for the mutation. Further genealogical and haplotype analysis will clarify the degree to which this mutation should be sought in French people with atypical late onset neurological basal ganglia dysfunction.

M. C. Lemos¹^,2, D. Rodrigues¹, L. Gomes¹, P. T. Christie², R. V. Thakker², M. Carvalheiro¹;
¹Serviço de Endocrinologia, Diabetes e Metabolismo, Hospitais da Universidade de Coimbra, Coimbra, PORTUGAL, ²Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UNITED KINGDOM.

Nephrogenic diabetes insipidus (NDI) is a congenital disorder characterised by the excretion of large volumes of diluted urine due to impaired renal concentration in response to the hormone arginine vasopressin (AVP). Most cases are inherited in an X-linked recessive manner and present mutations in the V2 vasopressin receptor (AVPR2) gene. A few cases are autosomal recessive, due to mutations in the AVP-sensitive water channel gene, aquaporin-2 (AQP2).
A female index case presented with an average fluid intake of 12L/day. An additional four male subjects were affected, presenting an inheritance suggestive of an X-linked disorder. Clinical and biochemical analysis of the index case showed incomplete responses to the water deprivation and vasopressin loading tests. Partial hemodynamic and coagulation responses to the synthetic V2-specific agonist dDAVP were observed. Genetic analysis was performed by PCR amplification of the coding regions and exon/intron boundaries of the AVPR2 gene, followed by automated DNA sequencing.
A 493G→C transversion in exon 2 was observed, leading to the substitution of an alanine by a proline at codon 165. Restriction enzyme analysis allowed confirmation of the mutation and its cosegregation with the disease. In addition, analysis of 105 alleles from 70 unrelated individuals revealed an absence of this abnormality.
Our study has, therefore, identified a previously unreported mutation of the AVPR2 gene, affecting a transmembrane domain of the receptor. The marked degree of NDI symptoms exhibited by the heterozygote female patient, in spite of her partial responses to stimulation tests, could be explained by a mechanism of skewed X-inactivation.

Mutation in the gene for protein tyrosine phosphatase SHP-2 (PTPN11) in a large family with Noonan/cardio-facio-cutaneous syndrome

E. Schollen, G. Matthijs, E. Legius, J. Fryns;
Center for Human Genetics, Leuven, BELGIUM.

Noonan syndrome (NS, MIM 163950) is an autosomal dominant condition characterised by facial dysmorphy, congenital cardiac defects and short stature. Most of the clinical features of Noonan syndrome overlap with cardio-facio-cutaneous (CFC) syndrome. In CFC syndrome patients are more severely affected with specific skin abnormalities and moderate mental retardation. Recently, a gene responsible for Noonan syndrome in some families has been cloned. Missense mutations in PTPN11, the gene encoding the non-receptor protein tyrosine phosphatase SHP-2, is responsible for at least 50% of the Noonan syndrome cases.
A large, four generation Belgian family with NS in some and CFC syndrome in other family members, was previously used to fine map the Noonan syndrome candidate region in 12q. We now report the identification of a mutation (Gln79Arg) in the PTPN11 gene in this large family. The mutation was found in both the CFC and NS individuals from this family. We believe that CFC syndrome is a heterogeneous condition (as well as NS) and that some CFC syndrome cases may be the result of variable expression of an SHP-2 mutation. Screening of a larger group of Noonan syndrome and CFC syndrome patients is in progress.

H. M. Fodstad¹, P. J. Laitinen¹, H. Swan², L. Toivonen², M. Viitasalo², K. Kontula¹^,2;
¹University of Helsinki, Helsinki, FINLAND, ²Helsinki University Central Hospital, Helsinki, FINLAND.

Long QT syndrome (LQTS) manifests as prolonged QT interval on ECG, ventricular arrhythmias and risk of sudden death. The prevalence of inherited LQTS is estimated as 1:10 000. Mutations of five ion channel genes (KCNQ1, HERG, KCNE1, KCNE2 or SCN5A) may cause LQTS. We have identified two founder mutations (KCNQ1-Fin and HERG-Fin) that together account for 35-40% of LQTS cases in the genetically isolated Finnish population.
This study was conducted to approximate the prevalence of inherited LQTS in the Finns and, in particular, to screen for mutations of SCN5A among LQTS probands. The causal mutation was documented in 86/236 (36%) families studied, and 631 mutation carriers were identified corresponding to a population prevalence of 1:8000 (Table 1). Case detection rate suggests that the actual prevalence is at least 1:5000. Simple PCR assays for the KCNQ1-Fin and HERG-Fin mutations alone detect 35% of cases. Samples from 150 LQTS probands without prior mutation detection were screened for SCN5A mutations. One amino acid change (G5851T corresponding to V1951L) and two silent polymorphisms (G4218A and C5457T) were detected.
In conclusion, DNA analyses show great promise in establishment of the LQTS diagnosis and may identify variants showing a disease-modifying role. Inherited LQTS may be more prevalent in Finland than many other Western populations. The LQT1, LQT2 and LQT3 subtypes of LQTS occur in relative frequencies of 75%:21%:4%, respectively.

Table 1.
LQTS type	Mutation carriers	Families	Mutation
KCNQ1	475	66	6
KCNQ1-Fin	428	59
HERG	133	19	9
HERG-Fin	71	9
SCN5A	23	1	1
All LQTS patients	631	86	16

J. Kasnauskiene, L. Cimbalistiene, V. Kucinskas;
Human Genetics Centre of Vilnius University, Vilnius, LITHUANIA.

We report the spectrum of the PAH gene mutations in patients with phenylketonuria residing in Lithuania. A total of 184 independent PAH chromosomes were investigated. All 13 exons of the PAH gene of all PKU probands were scanned for DNA alterations by denaturing gradient gel electrophoresis (DGGE). In the cases of a specific DGGE pattern was recognized, mutations were identified by direct fluorescent automated sequencing or by restriction enzyme digestion analysis. 19 different PAH gene mutations were identified on 173 PKU chromosomes (95%). The most common ones were R408W (73.6% chromosomes) and R158Q (6.6% chromosomes) whereas the remaining mutations appeared to be rare (relative frequencies were from 0.55% to 2.2%.). In 11 PKU chromosomes for 9 patients repeated DGGE scanning of the whole coding region of the PAH gene and sequencing exons if specific pattern was recognized revealed no mutations. Most likely, in these cases mutant chromosomes may harbor large deletions or intronic splice mutations in the PAH locus.
52 individuals with PKU were found to be homozygous for the PAH gene mutation. The vast majority of such patients (51 or 57%) appeared to be homozygous for R408W (PAH genotype R408W/R408W), while the proband in one family was homozygous for R158Q (PAH genotype R158Q/R158Q). 33 (36%) patients with PKU were compound heterozygous: in 31 (33%) cases R408W and a rare mutation were identified and in two cases both mutations were rare.

Mutations of the CYP11A gene could not explain all cases of congenital lipoid adrenal hyperplasia without StAR gene mutation in our Caucasian population.

D. Mallet¹, S. Portrat-Doyen¹, A. Lecoq¹, R. Brauner², B. Leheup³, J. Carel⁴, Y. Morel¹;
¹Laboratoire de Biochimie Endocrinienne et Moléculaire, INSERM U329, Hôpital Debrousse, Lyon, FRANCE, ²Service d'Endocrinologie Pédiatrique, Hôpital Necker enfants malades, Paris, FRANCE, ³Service de Pédiatrie, CHU de Nancy, Nancy, FRANCE, ⁴Service d'Endocrinologie Pédiatrique, Hôpital St Vincent de Paul, Paris, FRANCE.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is a rare autosomic recessive disorder affecting the first step of steroid biosynthesis, leading to the absence of glucocorticoids, mineralocorticoids and sex steroids. Affected individuals present with salt loss, dehydration and patients with XY genotype are phenotypically females. In contrast with other CAH, this disease is not caused by a defect in the enzyme (P450scc), but in the Steroidogenic Acute regulatory protein (StAR), implicated in the transport of cholesterol from the outer to the inner mitochondrial membrane where P450scc is localized. Almost all lipoid CAH patients (41/42) who are not of Caucasian origin are homozygous or compound heterozygous for mutations of the StAR gene. By contrast, in our 11 unrelated Caucasian families, we have found only 6 families with mutations of the StAR gene. In the 5 other ones, StAR mRNA and Dax-1 and SF-1 genes, of which mutations cause adrenal insufficiency, have been sequenced and appear to be normal. As a de novo heterozygous mutation of the CYP11A gene encoding P450scc has recently been reported to cause lipoid CAH, we have sequenced this gene and found mutations in two patients.
This study shows that mutations of the StAR gene are responsible for lipoid CAH in only 60% of patients of Caucasian origin. Compound heterozygous mutations of the CYP11A gene can also be responsible if residual P450scc enzymatic activity produces enough progesterone to avoid spontaneous abortion. Nevertheless, other genes should be involved to explain the genetic lesion in our 3 remaining families.

L. Martorell¹, D. Monckton², M. Baiget¹;
¹Genetics Unit. Hospital Sant Pau, Barcelona, SPAIN, ²Division of Molecular Genetics. Institute of Biomedical and Life Sciences,University of Glasgow, Glasgow, UNITED KINGDOM.

Myotonic dystrophy (DM1) is caused by the expansion of an unstable CTG repeat located in the 3'-untranslated region of the DM gene (DMPK). The number of CTG repeats is polymorphic in the general population a ranging from 5 to 37 repeats. DM1 patients have expansions of greater than 50 repeats and up to many thousands. The size of the repeat is positively correlated with the severity of the disease and inversely correlated with the age of onset of symptoms.Dramatic instability with very large intergenerational increases and contractions is observed in DM1 patients.Detailed studies of somatic mosaicism have revealed that it is tissue specific, biased toward further expansion and continuous throughout the life of an individual.The trinucleotide repeats instability mechanisms involved in DM1 are unknown.In order to gain a better understanding of the dynamics of repeat instability in the male germline, we have used sensitive small pool-PCR analyses (SP-PCR) to compare blood and sperm DNA from 23 males, with different age, CTG repeat expansion and clinical form.Analysis of sperm DNA from control individuals showed that small normal alleles were stable. Sperm samples of DM1 patients revealed both different levels of mosaicism and patterns of distribution for the expanded allele, designing a characteristic pattern in each clinical group. The comparison of these results with those obtained from peripheral blood will allow us to define, in accordance with age and clinical form, specific patterns of mosaicism in somatic and germline cells for DM1 individuals.

N. Pronina, R. Lugovska, P. Vevere, A. Kornejeva;
Latvian State Medical Genetics centre, Childrens` Hospital "Gailezers", Riga, LATVIA.

Phenylketonuria (PKU), an autosomal recessive genetic disorder, is caused by a deficiency of the hepatic phenylalanine hydroxylase enzyme (PAH). Sixty patients were detected from 1980 to 2001; the approximate incidence of PKU is 1 of 8700 new-borns in Latvia.
Fifty patients from 48 unrelated families were screened for the presence of six PAH gene mutations: R408W, R261Q, R252W, G272X R158Q and IVS10nt546 using ASO and PCR/RED methods. The mutation detection rate was 86,0% among the studied alleles. Out of 100 alleles under study, 77 (77%) were identified as defective due to R408W. Twenty-eight patients were homozygotes for R408W, 21 were compound heterozygotes for R408W. Other five mutations had a very low incidence of PKU alleles: R158Q -4%, R261Q - 2%, G272X - 1%, R252W - 1%, IVS10nt - 1%. In 14 cases (14%) probands were compound heterozygotes for different PAH locus mutations, one of them was unidentified. The location of unknown mutations was found using the DGGE method. DNA sequence analysis of the exons which showed positive DGGE signals found out 4 different mutations: E280K (5%), A104D (2%), E178G (1%), P281L (1%). The sequence analysis of second allele for other chromosomes is in progress.
Presence of severe R408W mutation in 77% of PKU alleles explains the high rate of PKU patients with severe phenotype.

Highly skewed X-inactivation pattern in a female with unique presentation of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency.

L. Dvorakova¹, J. Hujova¹, R. Dobrovolny¹, L. Stolnaja¹, E. Tietzeova¹, M. Hrebicek¹, M. Kumsta², I. Sebesta¹^,2;
¹Inst. of Inherited Metabolic Disorders, Prague, CZECH REPUBLIC, ²Inst.Clin.Biochem., Prague, CZECH REPUBLIC.

Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is an X-linked recessive disorder of purine metabolism characterised with onset of gout and/or urolitiasis often in adolescence. This phenotype occurs almost exclusively in males. We follow a girl who presents gouty arthritis of big toe and hyperuricaemia from the age of 9 years. Normal activity of phosphoribosylpyrophosphate synthetase, loss of HPRT activity, raised adenine phosphoribosyltransferase activity and raised nicotinamide adenine dinucleotide concentration in erythrocytes revealed partial HPRT deficiency. The loss of HPRT activity was found also in the patient’s father, who presented with renal colics and gout since the age of 18 years. The sister of our female patient is asymptomatic and her HPRT activity is within normal limits.
Mutation analysis revealed that both sisters inherited from their father a previously described mutation in the 3rd exon of HPRT gene, c.158T>C (V53A). X-inactivation study performed in peripheral blood leukocytes showed that the X-inactivation is highly skewed in both sisters leaving the paternal mutated chromosome predominantly active in the symptomatic girl, while the second asymptomatic girl has almost exclusively active maternal non-affected chromosome.
Conclusions: 1. The unusual description of phenotype shows a possibility of presence of partial HPRT deficiency in girls with unexplained hyperuriceamia.
2. Results of biochemical tests performed in heterozygotes may fail because of X-inactivation status. Mutation analysis is necessary for a reliable identification of carriers of mutated gene.
(Supported by grants NE 6557-3-01 of the Czech Ministry of Health and MSM-111100005 of the Czech Ministry of Education)

M. Schwartz¹, J. Vissing²;
¹Department of Clinical genetics, Rigshospitalet, Copenhagen, DENMARK, ²Department of Neurology, Rigshospitalet, Copenhagen, DENMARK.

Mitochondrial DNA (mtDNA) is thought to be strictly maternally inherited in mammalian species: Sperm mitochondria disappear in early embryogenesis, either through selective destruction, inactivation or by simple dilution due to the surplus of oocyte mitochondria. A very small amount of paternally inherited mtDNA has been detected by PCR, identified after several generations of interspecific backcrosses. However, recent studies using microinjected sperm into mouse oocytes support the hypothesis that sperm mitochondria are targeted for destruction by nuclear-encoded proteins, but the underlying mechanism remains a mystery. In this report we show for the first time that paternally derived mtDNA can indeed survive and contribute substantially to the mtDNA pool in man. In a patient with mitochondrial myopathy due to a novel two base pair (bp) deletion in the ND2 gene, 90% of the muscle mtDNA was paternal in origin. The two mtDNA haplotypes were different at 18 positions. The mtDNA haplotype in muscle was identical to the haplotype found in the father, while that of blood was identical to the mtDNA haplotype in the mother. This phenomenon may be more common than generally believed because mitochondrial haplotypes are often not investigated and because substantial differences are required in order to distuguise the haplotypes in a routine analysis.

Haploinsufficiency of DYRK1A on chromosome 21q22.2 is associated with microcephaly

M. Hoeltzenbein¹, A. Schröer¹, J. Wirth¹, B. Heye², S. Stengel-Rutkowski², N. Tommerup³, H. H. Ropers¹, V. M. Kalscheuer¹;
¹Max-Planck-Institute for Molecular Genetics, Berlin, GERMANY, ²Kinderzentrum, München, GERMANY, ³Wilhelm Johannsen Centre for Functional Genome Research, IMBG, The Panum Institute, University of Copenhagen, Copenhagen, DENMARK.

Recent studies of primary microcephaly are frequently based on homozygosity mapping in consanguineous families and have revealed 5 loci for recessive forms of microcephaly, but so far no gene has been identified by this approach. Examination of balanced translocations is an alternative approach to identify genes involved in microcephaly. We report on a 12-year old girl with prenatal onset of microcephaly, a severe developmental delay including speech defect and seizures. Structural brain malformations were excluded by MRI. Chromosome analysis revealed an apparently balanced translocation t(2;21)(q22;q22). Molecular cytogenetic techniques were used to find breakpoint-spanning clones. Subsequent breakpoint analysis showed that the chromosome rearrangement led to disruption of the DYRK1A gene. Semiquantitative RT-PCR experiments revealed a 50% reduction of DYRK1A expression in the patient's lymphoblastoid cell line compared to a control cell line. The chromosome 2 breakpoint maps within the putative tumor suppressor LRP1B, a member of the low density lipoprotein receptor family.
Dyrk-related kinases belong to a new family of protein kinases that have been suggested to be involved in the regulation of cellular growth and development. The Drosophila homolog minibrain (mnb) is required for normal postembryonic neurogenesis and mutations in this gene lead to size reduction of the brain. We could show that haploinsufficiency of DYRK1A is associated with microcephaly. This is supported by the finding of microcephaly as a main feature in patients with monosomy 21q22. We would suggest that other patients with primary microcephaly should be screened for mutations in the DYRK1A gene.

Current models of mutagenesis applied to missense single-nucleotide substitutions in the human lamin A/C gene

A. Todorova¹, M. Dabauvalle², C. R. Müller¹;
¹Department of Human Genetics, Biozentrum of the University of Würzburg, Würzburg, GERMANY, ²Department of Cell and Developmental Biology, Biozentrum of the University of Würzburg, Würzburg, GERMANY.

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the gene, coding the lamin A/C nuclear lamina proteins. Surprisingly, mutations in the nuclear envelope proteins lead to muscular dystrophy. The pathological mutations in that case are mainly missense, which are most informative for understanding mechanisms of mutagenesis.
We analysed 27 missense substitutions of the lamin A/C gene, regarding their possible origin. Transitions account for 59.3% versus transversions 40.7%. Nine transitions (33.3%) in CpG islands could be explained by methylation-mediated deamination. We analysed the mutability at dinucleotide level. As expected the CG dinucleotide was most frequently affected. Single-base mutability was assessed on non-CpG mutations and the obtained order was: G=C>T>A. Other mechanisms of mutagenesis (e.g. slipped-mispairing during replication) may require specific flanking sequences (arrest sites for polymerase a, direct repeats, palindromes and symmetric elements). We analysed 15 nucleotides upstream and downstream the mutation. Slipped-mispairing hypothesis is applicable in 37% of the cases. The arrest site for polymerase a was associated with 25.9% of mutations. Direct repeats, palindromes and/or symmetric elements were almost invariably present in all the analysed areas.
In conclusion, there is no a single mechanism, which can explain all the cases. Among the investigated mechanisms, the "environment", i.e. the presence of repeated motifs seems to play a major role.

Outcome of three donor splice site mutations accounting for congenital afibrinogenemia and order of intron removal in the fibrinogen alpha gene (FGA).

M. Neerman-Arbez, A. David, C. Attanasio;
Division of Medical Genetics, University Medical Centre, Geneva, SWITZERLAND.

Congenital afibrinogenemia (MIM # 202400) is a rare, autosomal recessive disorder characterised by the complete absence of circulating fibrinogen. Our studies on the molecular basis of the disease showed that the most common genetic defect is a donor splice mutation in FGA intron 4, IVS4+1G>T. Two other FGA donor splice mutations, in intron 1 (IVS1+3A>G) and in intron 3 (IVS3+1_+4delGTAA) were identified in afibrinogenemia patients. Because it was impossible to directly study the effect of these mutations on mRNA splicing in patient hepatocytes we designed a transfected cell approach. For the common IVS4 mutation, multiple cryptic donor splice sites in exon 4 and intron 4 were found to be utilised. One of these, situated 4 bp downstream of the normal site was used in 85% of transcripts resulting in a 4 bp insertion-frameshift leading to premature truncation of FGA. Analysis of the IVS1+3 mutation showed intron 1 inclusion in the majority of transcripts, while the IVS3delGTAA mutation caused exon 3 skipping. The different outcomes of these donor splice mutations appear to follow the model proposed by Byers et al.(EJHG 2001, vol 9 suppl 1 p.80) in a study of fibrillar collagen genes, where donor splice mutations occurring in a rapidly-spliced intron with respect to upstream introns lead preferentially to exon skipping, while mutations in later-spliced introns lead to intron inclusion or cryptic splice site utilisation. Indeed, we found that in FGA introns 2 and 3 were spliced first, followed by intron 4 and finally intron 1.

DQA1 polimorphism analysis in Congenital Adrenal Hyperplasia (CAH) patients from Russia.

N. Osinovskaya, T. Ivaschenko;
Institute of Obstetrics & Gynecology, St.Petersburg, RUSSIAN FEDERATION.

Virilizing congenital adrenal hyperplasia (CAH) is the most common cause of genital ambiguity. 90-95% of CAH cases are caused by 21-hydroxylase deficiency. Particular forms of 21-hydroxylase deficiency are associated with particular combinations of HLA-antigens or haplotypes. Typing of DQA1 alleles (0101:0102, 0103, 0201:0601, 0301, 0401:0501) was carried out in DNA samples of 86 CAH patients (23 with salt wasting (SW) form , 13 with simple virilizing form (SV) , 50 with nonclassic (NC) form and of 50 unrelated healthy donors. Significant decrease of DQA1 0401:0501 alleles was registered in SW patients compared to SV , NC patients and controls (15%, 25%, 40% and 36% respectively; p<0.05). About 39% of the chromosomes in SW patients and 16% in SV patients had either major deletion or large conversions modifying the CYP21B gene. These mutations were predominantly identified in chromosomes with DQA1 0101:0102/0201:0601 alleles. Another major mutation 656A -G was registered in 22% of SW and 16% of SV groups. This nucleotide change corresponds to DQA1 0103/0401:0501 alleles. The frequency of DQA1 alleles in NC group of CAH patients is not different from this one in the control group. DQA1 alleles typing may be used, as additional method in prenatal diagnosis of CAH.

S. Ennis¹^,2, J. Ng Wanjing², G. Kearney³, M. Fitzgerald³, R. Stallings¹^,2, D. E. Barton¹^,2, A. J. Green¹^,2, L. Gallagher³, M. Gill³;
¹Department of Medical Genetics, University College Dublin, Dublin, IRELAND, ²National Centre for Medical Genetics, Our Lady’s Hospital for Sick Children, Crumlin, Dublin, IRELAND, ³Department of Psychiatry, Trinity Centre for Health Sciences, St. James's Hospital, Dublin, IRELAND.

Autism is a relatively common primary developmental disorder, with a significant genetic component. Routine investigations for the cause of autism, including chromosome and fragile X analysis, almost always are normal. There is a polygenic component to autism, and large sib-pair studies have been carried out by a number of groups worldwide. Several groups have identified associations between autism and a wide area of chromosome 2q, flanked by markers D2S364 and D2S2188 (Buxbaum et al & the IMGSAC respectively) which shows the strongest evidence for linkage. These results strongly suggest a predisposing gene(s) to autism within the 2q region.
We have recently identified a patient with high-functioning autism, who has a small but cytogenetically visible de novo deletion of chromosome 2q32 which falls within these linkage findings. This would appear to be the smallest known deletion of this part of chromosome 2, suggesting that the deletion includes a predisposing gene(s) for autism.
Fine mapping of the deleted region was carried out using markers from 2q32, to map the exact size of the deleted region. This reduces the very large linkage region by >75% to approximately 8.7 megabases. The region contains approximately 16 known genes and 16 ESTs, a number of which are potential candidates for autism.
We present results of a wider study for the presence of submicroscopic microdeletions, and of candidate genes, incorporating both linkage disequilibrium and mutational screening approaches in a panel of 77 Irish families with autism within the mapped region.
Am J Hum Genet 2001 Jun;68(6):1514-20, Sep;69(3):570-81

Mutations of the PKD2 gene in families with autosomal dominant polycystic kidney disease in Czech Republic

J. Reiterová¹, J. Stekrová², V. Kebrlová², M. Kohoutová², M. Merta¹, J. Zidovska²;
¹Dept.of Nephrology, 1st Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, ²Institute of Biology and Medical Genetics, Prague, CZECH REPUBLIC.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease.The disease is caused by mutations of the PKD1 and PKD2 genes. DNA presymptomatic diagnosis is performed in our referential laboratory, using highly polymorphic microsatellite markers for DNA linkage analysis of both genes. Presymptomatic DNA diagnosis was performed in 186 unrelated ADPKD families. Detection of PKD2 mutations was established in 39 families (in 9 families the disease was clearly linked to PKD2 gene and in 30 families with mild clinical course was not possible to exclude the linkage to PKD1 gene) and in 27 patients with end stage renal failure later than in 63 years.An affected member from each family was analyzed by heteroduplex analysis (HA). Samples which exhibited shifted bands on HA were amplified and after purification sequenced in both directions. Twelve mutations were identified, nine mutations unique for Czech population and four mutations from unique mutations were not presented. Four new unique mutations are the following: 1.nonsense mutation in exon 1, 145 C>T (Q49X), 2.missense mutation in exon 4, 917 G>A (R306Q), 3.frameshift mutation in exon 4, 1078-1081 del C, 4.missense mutation in exon 5, 1258 A>G (R420G).Segregation of the mutation with the disease in each family was tested by HA or sequencing. Establishment of localization and type of mutations and genotype/phenotype correlation in affected families will improve presymptomatic DNA diagnosis and could help to assess the clinical prognosis of ADPKD patients.
Supported by the grant project IGA MZ CR NE/5996-3.

Type VII collagen gene (COL7A1) mutations survey in Italian patients affected by epidermolysis bullosa dystrophica

M. Colombi¹, R. Gardella¹, D. Castiglia², P. Posteraro², N. Zoppi¹, E. Nebuloso², M. Paradisi², G. Tadini³, J. A. McGrath⁴, G. Zambruno², S. Barlati¹;
¹University, Brescia, ITALY, ²Istituto Dermopatico dell'Immacolata,IRCCS, Roma, ITALY, ³Policlinico, IRCCS, Milano, ITALY, ⁴Guy's King's College and St Thomas'Hospital, London, UNITED KINGDOM.

Dystrophic epidermolysis bullosa (DEB) is a rare skin disorder showing clinical heterogeneity and transmitted either in dominant (DDEB) or recessive (RDEB) mode. All variants of DEB have been associated to mutations in type VII collagen gene (COL7A1). More than 200 mutations, often specific to individual families, have been disclosed in this gene and some of them have been shown to be characteristic of certain ethnic populations.
We report the survey of COL7A1 mutations in Italian DEB patients. From the analysis of 51 DEB families 42 mutations were identified, 18 of which are novel. Genotype-phenotype correlations were in line with the general rules already drawn in DEB. In the characterised families, we performed 70 analyses of the carriers of mutations and 3 prenatal diagnoses.
Six frequent mutations were identified in Italian RDEB patients, i.e. the 497insA, the 4783-1G->A, the 7344G->A, the 425A->G, the G1664A, and the 8441-14del 21. While the 7344G->A and the 425A->G mutations have been found in different populations, the remaining have been identified prevalently or only in Italian patients. The haplotype analysis, together with the common geographic origin of the patients carrying the frequent Italian COL7A1 mutations, has evidenced an ancestral common origin of the mutated alleles. Altogether the 6 frequent mutations cover about 43% of RDEB alleles in Italian patients and therefore they should be screened firstly in the patients not yet characterised at molecular level.

Albright’s Hereditary Osteodystrophy: Screening for GNAS1 mutations by DHPLC and expansion of the mutation and polymorphism spectrum.

S. J. Rickard¹, L. C. Wilson²;
¹Institute of Child Health, London, UNITED KINGDOM, ²Institute of Child Health & Great Ormond Street Hospital, London, UNITED KINGDOM.

Albright’s Hereditary Osteodystrophy (AHO) results from heterozygous deactivating mutations in the GNAS1 gene and is associated with short stature, obesity, brachymetaphalangia, ectopic ossifications and learning disability. The inheritance is autosomal dominant but modified by genomic imprinting. Additional endocrine abnormalities, known as pseudohypoparathyroidism (PHP1a), are associated with mutations of the maternal but not the paternally derived GNAS1 allele. The AHO phenotype is heterogeneous and the diagnosis is difficult to confirm in the absence of PHPIa without access to GNAS1 mutation screening.
GNAS1 lies within a complex imprinting cluster at chromosome 20q13. It comprises 13 exons spanning 20kb and encodes the 394 amino-acid alpha subunit of the adenylyl cyclase stimulatory protein, Gs.
We have screened over 80 patients with features of AHO for GNAS1 mutations using DHPLC for exons 2-13, and sequencing for exon 1. To date we have identified fourteen mutations spread throughout the gene and two novel exonic variants, V36V and S54G which are likely to be polymorphic. In addition, we have identified a small GCC expansion in the 5’UTR of an affected patient, a common intronic deletion polymorphism close to a splice site, and five further intronic variants. These findings together with evidence for and against pathogenicity will be presented in more detail.

T. Antoniadi¹, A. Hatzaki¹, A. Pampanos², T. Iliades³, N. Voyatzis³, J. Economides⁴, P. Leotsakos⁴, P. Neou⁵, M. Tsakanikos⁵, M. Grigoriadou², A. Skevas⁶, M. B. Petersen¹^,2;
¹"MITERA" Maternity &Surgical Center, Athens, GREECE, ²Institute of Child Health, Athens, GREECE, ³Aristotle University of Thessaloniki, Thessaloniki, GREECE, ⁴"Aghia Sophia" Children's Hospital, Athens, GREECE, ⁵"P.&A. Kyriakou" Children's Hospital, Athens, GREECE, ⁶University of Ioannina, Ioannina, GREECE.

Mutations in the GJB2 gene encoding the gap-junction protein connexin-26 (Cx26) on chromosome 13q11 have been shown as a major contributor to prelingual, non-syndromic recessive deafness. A variety of techniques has been developed for screening the GJB2 gene for known and unknown mutations, especially the most frequent mutation in the Caucasian population, the 35delG. However, as there are almost a hundred sequence alterations identified so far, and their geographic distribution differs a lot, there is great interest for a rapid and easy method of identifying other mutations. Here we present easy screening techniques for the 3 recurrent GJB2 mutations in the Greek population, besides the 35delG. These mutations, K224Q, W24X and L90P, were originally identified by DGGE and/or direct genomic sequencing of the coding region. We developed ARMS-PCR for detecting the L90P and K224Q mutations and PCR-RFLP for the W24X mutation, using 6 previously genotyped samples. Additionally, 25 unrelated Greek patients (21 familial and 4 sporadic cases) with non-syndromic hearing impairment (NSHI), previously screened for the 35delG mutation and found to be negative (20 familial cases) or heterozygotes (1 familial and 4 sporadic cases), were screened for the 3 mutations. We found the L90P mutation in compound heterozygosity with the 35delG in a sporadic case using the newly standardized method.
The fast and easy detection of recurrent mutations can significantly contribute to the diagnosis of deafness, carrier detection and genetic counseling.

A. L. Jimenez¹, M. Valdes², M. Rivera³, S. Kofman-Alfaro³, S. Cuevas³;
¹Hospital General de Mexico, México DF, MEXICO, ²Centro Nacional de Rehabilitacion (INO), Mexico DF, MEXICO, ³Hospital General de Mexico, Mexico DF, MEXICO.

Servicio de Genetica, Hospital General de Mexico, Facultad de Medicina, UNAM and *Centro Nacional de Rehabilitacion (INO). Mexico D.F., MEXICO.
X-linked ichthyosis (XLI) is an inherited disease characterized by dark, adhesive and regular skin scales present at birth or early after birth. The primary defect of XLI is the deficiency of steroid sulfatase enzyme (STS), which hydrolyzes 3-beta-hydroxysteroid sulfates. The STS gene locus is located on Xp22.3. Reports in the literature indicate a complete deletion of STS gene and flanking sequences in 85-90% of XLI patients, while only 8 intragenic deletions and 11 point mutations have been reported. This study reports a Mexican patient with XLI and a novel partial deletion of the STS gene. XLI diagnosis was confirmed through STS assay in leukocytes using 7-[3H]-dehydroepiandrosterone sulfate as a substrate. The STS gene was analyzed by PCR. STS activity was undetectable in the XLI patient (0.0 pmol/mg protein/h) and very low in his mother (0.32 pmol/mg protein/h vs 0.84 pmol/mg protein/h of normal control). PCR analysis showed no amplification of exon 9 and normal amplification of exons 1-8 and 10 of the STS gene. We have analyzed more than 120 XLI patients and this is the first deletion in exon 9 of the STS gene reported in the literature.

M. R. Rivera¹, A. L. Jimenez², S. Chiñas¹, M. Arenas³, S. A. Cuevas¹, S. H. Kofman-Alfaro¹;
¹Hospital General de Mexico, Mexico DF, MEXICO, ²Hospital General de Mexico servicio de Genetica, Mexico DF, MEXICO, ³Centro nacional de Rehabilitacion (INO), Mexico DF, MEXICO.

Norrie disease (ND) is an uncommon X-linked recessive neurodevelopmental disorder characterized by bilateral congenital blindness, one half of the cases present mental retardation and one third have sensosorineural deafness.
The ND gene is localized in Xp11.3 and has three exons, one untranslated and it encodes a polypeptide of 131 a.a. called norrina, that has an important function in vascularization and differentiation of the inner retina.
More than 70 nonsense and missense mutations, a translocation, an inversion, and several deletions in the spectrum of ND have been reported. The high number of pathological changes in this small gene suggests that norrina conformation is extremelly prone to disruption from changes ocurring anywhere in its structure.
We describe 2 novel mutations in the ND gene in two mexican families. The affected individuals, showed typical ocular features of ND with deafness and mental retardation. Exons 2-3 of the Norrie gene were analyzed through PCR and DNA sequencing. Family 1, showed a missense mutation (A97P) within exon 3 in the patien and heterozygocity in the mother and sister. Family 2, present a deletion of 246 pb within exon 3 observed only in the patient.

Clinical and molecular characterisation of Osteogenesis imperfecta in patients from Lithuania

E. Benusiene, V. Kucinskas, R. Slibinskas, D. Kasperaviciute;
Human Genetic Centre, Vilnius University, Vilnius, LITHUANIA.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder caused in >90% cases by dominant mutations in the genes COL1A1 and COL1A2, which encode the proa1(I) and proa2(I) chains of type I procollagen respectively. The severity of the OI phenotype is strongly associated with the polypeptide chain type, mutation site, flanking sequences, and residue substituted for glycine. The aim of the study was to evaluate clinical manifestation of OI on the molecular basis of disease.
Probands from 16 OI families (type I) and 22 sporadic patients with OI type I (11), type II (3) and type III (8) were screened for mutations in 40 exons of the COL1A1 gene using DNA heteroduplex analysis. Direct DNA sequencing revealed 10 OI (type I, III) causing mutations in 12 unrelated patients. Out of them, 8 (E500X, R183X, c.2165-2166insCTCTCTAG, c.1787delT, c.1786-1787insC, IVS19+1G>A, IVS20-2A>G, IVS22-1G>T) were null mutations (due to a premature stop codon arising either directly from a point mutation or indirectly from a frameshift mutation, or from a mutation causing an abnormality in mRNA splicing) leading to mild OI phenotype. OI was differently manifested in related patients with identical genotype and ranged from mild to severe phenotype in two families. This finding suggests that the phenotypic expression of the disease may be influenced by other factors (genetic or epigenetic), which may be important in the process of bone formation. The relation between location and nature of the glycine substitution and severity of OI was observed for expressed point mutations (G79R, G481A).

Ferroportin 1 (FP1) is a human iron exporter expressed in enterocytes, macrophages, hepatocytes and placental syncytiotrophoblasts. FP1 coding gene, SCL11A3, consists of 8 exons and maps to 2q32.
Heterozygous mutations of FP1 cause hemochromatosis type 4 (HFE4). At variance with classic hemochromatosis HFE4 shows dominant inheritance, high serum ferritin before increased transferrin saturation and iron accumulation both in hepatocytes and macrophages. Two mutations of FP1 have been reported: (N144H) in exon 5 and (A77D) in exon 3.
We report the finding of a new FP1 mutation in 2 female patients (mother and daughter) with high serum ferritin, normal transferrrin saturation and signs of hepatic iron overload at liver biopsy and SQUID. Direct sequencing of the 8 exons of FP1 gene in both patients identified an heterozygous GTT deletion in exon 5, corresponding to a valine deletion in a valine triplet at position 160-162 (V162del) in the protein. The same mutation was not present in 50 normal controls.
The deletion occurs close to N144H in the highly conserved putative transmembrane domain involved in iron binding/transport. The finding of two different mutations in this domain provides further evidence of its relevance for FP1 function and suggests a loss of function as the mechanism of the disease. Our results add further support to the heterogeneity of hemochromatosis in Italy.

A. Moschik¹, A. Kujat¹, S. Spranger², C. Grünauer-Kloevekorn¹, U. G. Froster¹;
¹University of Leipzig, Leipzig, GERMANY, ²Praxis für Humangenetik Dr.S.Spranger/PD Dr.B. Kazmierczak, Bremen, GERMANY.

Holt-Oram syndrome (OMIM #142900, McKusick 1986, syn: Heart-Hand syndrome) is a rare disorder involving developmental defects of heart and upper limbs. Main symptoms include ASD, VSD and defects of the thumb and radius. Malformations of the lower limbs never were described. The syndrome follows an autosomal dominant pattern of inheritance with complete penetrance and variable expression. This developmental disorder is associated with mutations in theTBX5 gene which plays an important role in the morphogenesis of heart and limbs in vertebrates. The TBX5 gene contains a highly conserved T-box DNA binding domain. The Holt-Oram phenotype results from haploinsufficiency of TBX5.
We performed the mutation analysis of the TBX5 gene by direct automated sequencing of the coding region (exons 2 to 9) and the exon-intron boundaries in 20 unrelated patients with various malformations of the upper limbs and heart, clinically diagnosed as Holt-Oram syndrome. In three cases a familiarity of the heart-hand defects occured. This analysis of the TBX5 gene identified four different mutations in five unrelated families. Three mutations were identified for the first time, only one mutation was described previously. All patients with disease related mutations in the TBX5 gene presented variable defects of the heart and upper limbs. Further studies involving clinical and genetic investigations are necessary to correlate specific mutations in the TBX5 gene with phenotype expression of the heart and hand defects.

Paternal 11p15.1-pter heterodisomy associated with the Beckwith-Wiedemann phenotype resulting from malsegregation of criptic familial translocation

S. Russo¹, B. Dossena², M. Mencarelli¹, A. Bergo¹, F. Cogliati¹, F. Cavalleri¹, M. Tibiletti², L. Larizza³^,1;
¹Istituto Auxologico Italiano, Milano, ITALY, ²Ospedale Circolo, Insubria University, Varese, ITALY, ³Department of Biology and Genetics, University of Milan, Milano, ITALY.

Beckwith-Wiedemann (MIM#130650) is a developmental disorder with variable phenotype and genetic heterogeneity. Pre and postnatal overgrowth, macroglossia, and anterior abdominal wall defects represent the main clinical signs of the syndrome associated with an increased susceptibility (7.5%) to a variety of embryogenetic tumors such as Wilms' tumors, adrenocortical carcinoma and hepatoblastoma. The BWS locus is subjected to genomic imprinting, with lesions involving a cluster of genes on 11p15.5. A small fraction (2%) of patients carry chromosome 11p15.5 abnormalities, duplications of paternal origin and balanced translocations or inversion with breakpoints on the maternal chromosome. Paternal uniparental disomy (pUPD) (20%), point mutations in the p57 gene (5-20%) and alteration in the methylation pattern of H19 and IGF2 imprinted genes are the most common pathogenetic mechanisms.
We refer on a patient displaying features such as gigantism, macroglossia, anterior abdominal wall defects and nephromegaly, consistent with BWS. The family records signaled two spontaneous abortions and a brother presenting with BWS features and nephroblastoma who deceased at four weeks. Standard karyotyping did not evidence chromosomal abnormalities. Microsatellite segregation analysis carried on to assess 11pUPD revealed paternal heterodisomy within 11p15.4-pter. By using the multiprobe telomeric FISH method the suspicion of a criptic translocation involving 11p15 was confirmed by the finding of an unbalanced translocation with derivative 21/11 in the proband and a balanced t(11;21)(11p15.4;21q22.3) in proband's normal brother and father. Microsatellite analysis of 21q22.3-qter region allowed to map the 21q monosomy between D21S890 and qter. The contribution of 21q monosomy to the proband phenotype is under study.

ALK1 gene and Hereditary Hemorrhagic Telangiectasia (HHT): preliminary results of a screeening on 52 Italian families identify 13 new mutations.

C. Olivieri¹, E. Buscarini², G. Delù³, L. Malvezzi³, F. Pagella³, E. Mira³, C. Danesino¹;
¹Medical Genetics, University Of Pavia, Pavia, ITALY, ²Gastroenterology, "Ospedale Civile di Crema", Crema, ITALY, ³Otorinolaringoiatric Clinic, IRCCS "S.Matteo", Pavia, ITALY.

Hereditary Hemorrhagic Telangiectasia (HHT) (OMIM 187300) is an autosomal dominant disorder caused by mutations in either one of two genes: Endoglin (ENG, OMIM #131195) (HHT1) and Activin Like Kinase1 (ALK1, OMIM #601284 ) (HHT2). Evidence for a third locus has also been reported.
The clinical presentation typically includes epistaxis and telangiectasies; a diagnosis can be held for confirmed if 3 of the 4 suggested diagnostic criteria (epistaxes, telangiectasies, visceral lesions, positive family history) are present. The phenotype is highly variable and penetrance is complete by the age of 40 years. Arterovenous fistulae are frequently observed in liver, lungs and brain and may cause severe life threatening complication.
The number of mutations identified so far is limited; in particular for ALK1 only 24 mutations have been reported, studied in single patients or in very small samples. Here we report the preliminary results of the first screening conducted on 52 Italian families in which at least one subject results clinically affected by HHT. We analysed exons 3, 7 and 8 of the ALK1 gene by either SSCP or dHPLC techniques. We were able to identify 16 mutations, 13 of them are previously unrepoeted; 2 of them were found in 2 unrelated families with the same geographic origin. 6 mutations have been found in exon 3, which codifies for the extracellular receptor domain; 4 mutations in exon 7 and 4 in exon 8 which codify for the intracellular Tyrosin-kinase domain. Six of these patients (37,5%) present liver involvement.

Novel SCN5A Mutation Leading Either to Isolated Cardiac Conduction Defect or Brugada Syndrome in a Large Family

J. Schott¹, F. Kyndt¹, V. Probst², F. Potet¹, J. Chevallier², S. Demolombe¹, I. Baro¹, J. Moisan², D. Escande¹, H. Le Marec²;
¹INSERM U533, Nantes, FRANCE, ²CHU de Nantes, Nantes, FRANCE.

The SCN5A gene encoding the human cardiac sodium channel a subunit plays a major role in cardiac electrophysiology. Mutations in SCN5A lead to a large spectrum of phenotypes including the long-QT syndrome, the Brugada syndrome and isolated progressive cardiac conduction defect (ICCD or Lenègre disease). In the present study, we report the identification of a novel SCN5A missense mutation causing either Brugada syndrome or ICCD in the same pedigree. In a large French family, we identified a G-to-A mutation at position 4372 that was predicted to change a glycine for an arginine (G1408R) between the DIII-S5 and DIII-S6 domains of the sodium channel protein. Among 45 family members, 13 were carrying the G1408R mutation. Four patients from 2 family collateral branches showed typical Brugada phenotype including ST segment elevation in the right precordial leads and right bundle branch block. One symptomatic patient with Brugada phenotype required implantation of a cardioverter-defibrillator. Seven patients from 3 other family collateral branches had ICCD but no Brugada phenotype (negative flecainide test). One patient with ICCD had episodes of syncope and required a pacemaker implantation. Expression study of the G1408R mutated SCN5A showed no detectable Na⁺ current but a normal protein trafficking. We conclude that the same mutation in the SCN5A gene can lead either to Brugada syndrome or to ICCD. Our findings suggest that the consequence of the same SCN5A mutation may be individual or branch specific. Cosegregation of a modifier gene could explain these branch-specific phenotypic differences.

Mitochondrial mutations in non syndromic sensorineural hearing impairement : A large spectrum of mutations.

S. Marlin¹, D. Feldmann², E. Chapiro², F. Denoyelle³, D. Sternberg⁴, D. Weil⁵, C. Petit⁵, E. N. Garabédian³, R. Couderc²;
¹Genet Dept, A.Trousseau Hosp, APHP, Paris, FRANCE, ²Biochemistry and molecular biology Dept, A. Trousseau Hosp , APHP, Paris, FRANCE, ³ORL Dept, A.Trousseau Hosp, APHP, Paris, FRANCE, ⁴Biochemistry Dept, Pitié-Salpétriêre Hosp , APHP, Paris, FRANCE, ⁵Unit Génétique des déficits sensoriels, CNRS URA 1968, Institut Pasteur, Paris, FRANCE.

Mitochondrial (mt) DNA mutations identified in maternally inherited hearing loss (MIHL) suggest an important role for mitochondria in the function of the inner ear. Fifty five families with non syndromic MIHL, eight sporadic patients with non syndromic hearing loss (NSHL) after aminoglycoside treatments and 29 sporadic patients with NSHL have been collected. For each proband the deafness was documented : age of onset, audiometry, temporal bones CT scan. ARNr 12S, ARNt leucine and ARNt serine were analysed by denaturing gradient gel electrophoresis and sequencing. The A1555G mutation was present and homoplasmic in a large family (19 affected patients).The affected subjects presented a congenital bilateral and sensorineural hearing loss. The deafness was severe to profound and age-stable. T7511C mutation was observed homoplasmic or heteroplasmic in two large french families.The age at onset of deafness was variable. The bilateral and sensorineural hearing loss was stable or progessive. The A3243G mutation, usually observed in syndromic deafness (MELAS and MIDD), was founded in one family with an isolated sensorineural hearing loss (4 affected patients). The hearing impairement was postlingual and evolutive with variable severity (mild to severe). No diabetes mellitus, cardiomyopathy or neurologic symptoms were associated. No mt mutation was observed in sporadic cases. In conclusion, we have observed a mitochondrial mutation in 7.2% of non syndromic MIHL patients with a low prevalence of A1555G (1.8%). A3243G was observed for the first time in a family with NSHL. These results suggest that many different mitochondrial mutations could be involved in non syndromic MIHL.

Brain Asymmetry In Beckwith-wiedemann Syndrome, A Marker For Paternal Isodisomy ?

P. Bitoun¹, F. Rigaudiere'², C. Junien³, I. Henri³, J. Gaudelus⁴;
¹C.H.U. Paris-Nord,Hopital Jean Verdier, Genetique Medicale, Bondy, FRANCE, ²Hopital Lariboisiere, Paris, FRANCE, ³Inserm U 383, Paris, FRANCE, ⁴Hopital Jean Verdier; Pediatrie, Bondy, FRANCE.

Hemihypertrophy is acommon finding of the Beckwith-Wiedemann syndrome (BWS) particularly in limbs and occasionally in the face or jaw. Brain hemihypertrophy is not commonly reported.
We describe a female patient with right hemihypertrophy noted at birth by right facial hemihypertrophy and jaw asymmetry. Limb involvement was also noted in lower more than in upper limbs. Right brain hemihypertrophy was found using visual evoked potentials (VEP) and persists after 14 years of follow-up.
Paternal isodisomy was demonstrated using molecular markers for the BWS critical region as published in 1993*. This patient has developed normally albeit with recurrent ear infections , right leg hypertrophy requiring 1cm sole on left leg and slight scoliosis. She has otherwise normal growth and development.
The question raised is whether this brain asymmetry reflected by the asymmetric VEP is simply a marker for the BWS or a marker for the isodisomy. The evidence on this issue will be discussed and recommendations made to routinely study the VEP in BWS patients.
*Henry I, Puech A, Riesewijk A, Ahnine L, Mannens M, Beldjord C, Bitoun P, Tournade MF, Landrieu P, Junien C. Somatic mosaicism for partial paternal isodisomy in Wiedemann-Beckwith syndrome: a post-fertilization event. Eur J Hum Genet. 1993;1(1):19-29.

Analysis of large structural changes of the factor VIII gene involving intron 22 in severe haemophilia A

A. Bors¹, H. Andrikovics¹, L. Kalmar¹, I. Klein², A. Varadi², L. Nemes¹, A. Tordai¹;
¹Institute of Hematology and Immunology, National Medical Center, Budapest, HUNGARY, ²Institute of Enzymology of the Hungarian Academy of Sciences, Budapest, HUNGARY.

Haemophilia A (HA), the deficiency of the coagulation factor VIII (fVIII), is the most common severe, sex-linked inherited bleeding disorder. In approximately 50% of the patients with severe HA (fVIII level<1%), the disease is caused by fVIII gene intron 22 (IVS22) inversions, generally occuring as a result of an intrachromosomal recombination between the F8A gene located in IVS22 and one of its two extragenic homologous copies located telomeric from the fVIII gene. We studied 101 unrelated, severe HA-patients or obligate carriers by Southern blotting. We found known, altered inversion patterns in 54 (53%) cases: 38 (70%) distal, 12 (22%) proximal types and 4 (8%) unusual patterns. We observed loss or exclusively altered intronic bands in two cases of the latter group. We proved the existence of large deletions involving IVS22 and exon 22 or 23 by exon-specific amplification. The remaining two patients showed extra homologous F8A copies (4 bands in affected males) on Southern analysis that were not corresponding to type 3A or B inversions. To further analyse these two cases, "long-distance" PCR (LD-PCR) was performed for separate amplifications of the intronic and extragenic copies of F8A. In both cases, LD-PCR showed a normal and an inversion-affected intronic copy of F8A and normal extragenic copies, suggesting that an extra (possibly inserted) intronic fragment participated in the inversion process. The present cases further support the theory that, the structure of fVIII IVS22 represents a hot spot for large gene rearrengements and emphasize the importance of alternative mutation mechanisms.

Results of mutation testing in >400 families affected by Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency

M. M. Coleman¹, A. Wallace¹, S. Ramsden¹, D. A. Price², P. E. Clayton², R. Elles¹;
¹NW Regional Molecular Genetics Lab, St Mary's Hospital, Manchester, UNITED KINGDOM, ²Endocrinology Dept, Royal Manchester Children's Hospital, Manchester, UNITED KINGDOM.

21-hydroxylase deficiency is an autosomal recessive disease that presents with a range of symptoms. The late onset form is often so mild that it may be undiagnosed in affected individuals (cryptic CAH). Symptoms of classical CAH ranges from simple virilisation to a potentially life threatening inability to retain salt. There is a strong demand for prenatal diagnosis from couples who have already had an affected child. Prenatal diagnosis is usually carried out by linkage analysis with microsatellite markers closely flanking the functional 21-hydroxylase (CYP21) gene. Couples may opt for an in-utero treatment regime for affected female pregnancies.
The majority of the mutations causing 21-hydroxylase deficiency are known but testing for them is complicated by the complex arrangement of the genes responsible. We currently carry out testing for large scale deletions/conversions of the CYP21 gene by Southern blot analysis. We also screen for a common form of deletion and eight other mutations using direct ARMS tests. The CYP21 genes of affected individuals with one or more mutations undetected following these tests are sequenced in four overlapping fragments.
This strategy has identified both mutations in approximately 95% of >400 families tested and enabled us to assess the frequency of each mutation in British patients with 21-hydroxylase deficiency. In addition, this study has highlighted a number of interesting cases where for example there is evidence of mosaicism, de novo mutational events and parents with cryptic CAH. These observations indicate the need for mutation screening prior to prenatal diagnosis by linkage analysis wherever possible.

B. L. Loeys¹, J. De Backer¹, S. Symoens¹, P. Coucke¹, G. Pals², A. De Paepe¹;
¹Ghent University Hospital, Ghent, BELGIUM, ²Vrije Universiteit Medisch Centrum, Amsterdam, NETHERLANDS.

The clinical diagnosis of MFS, an autosomal dominant multisystemic disorder, is based on a set of major and minor criteria, known as the "Ghent nosology". MFS is caused by mutations in the fibrilline-1 gene (FBN1). More than 200 FBN1-mutations have been identified. However, due to incomplete mutation uptake and the suggestion of a second locus on chromosome 3p, the issue of locus heterogeneity in MFS remains under debate.
We present results on a cohort of 103 patients fulfilling the clinical diagnosis of MFS according to the Ghent nosology, in which a thorough mutationanalysis was done by CSGE/SSCP followed by DHPLC or sequencing. Initial mutationscreening allowed to identify an FBN1-mutation in 74 patients. Next, sequencing of all FBN1-exons was performed in 16 patients and screening by DHPLC in 13 patients, identifying respectively 7 and 5 additional mutations. In 5 more patients with a positive family history of MFS, but no mutation identified, segregation analysis showed linkage to the FBN1-locus.
In 12 patients (~10%) the involvement of FBN1 could not be proven by this approach. The phenotype of these patients did not differ from the others with respect to the distribution of major clinical manifestations. Southern blot analysis to exclude large deletions in the FBN1-gene is in progress. Most likely, a portion of FBN1-mutations remains undetected because of technical limitations.
In conclusion, the involvement of the FBN1-gene could be demonstrated in ~90% of all MFS patients, which strongly suggests that this gene is the predominant if not the sole locus for MFS.

Identification Of A Novel Missense Mutation (q283p) In A C282y Heterozygote Hemochromatosis Proband From Brittany (western France)

G. Le Gac¹^,2^,3, C. Mura²^,3, S. Jacolot²^,3, G. Esnault⁴, V. Scotet¹^,3, M. C. Merour¹, F. Y. Dupradeau⁵, J. Rochette⁵, C. Férec¹^,3;
¹Etablissement Français du Sang - Bretagne, Brest, FRANCE, ²Université de Bretagne Occidentale, Brest, FRANCE, ³Inserm (emi 01-15), Brest, FRANCE, ⁴Université de Nantes, Nantes, FRANCE, ⁵Université de Picardie Jules Verne, Amiens, FRANCE.

Introduction : Depending on the population studied, between 4 and 35% of hemochromatosis probands have a least on chromosome without an assigned HFE mutation (i.e. C282Y, H63D and S65C). In the present study, we have performed a D-HPLC scanning of the HFE coding region in twenty HC probands from Brittany to define if they carry uncommon mutations liable to explain their iron overload phenotype. Material : The 20 HC probands were selected because a transferrin saturation above the threshold (³ 60% in men ³ 50% in women) in absence of others known cause of iron overload. Results : D-HPLC scanning had allowed us to detect a novel missense mutation in exon 4 of the HFE gene from one proband who was also C282Y heterozygous. Subsequent sequencing analysis identified an adenine to cytosine transversion at position 848 which changes amino acid 283 from glutamine to proline (Q283). Another family member (sister) was also found C282Y/Q283P compound heterozygote , presenting with a transferrin saturation level of 74%. Theoritical structural analysis shows that folding of HFE is blocked by a proline in position 283 which cannot complete the H-bonding network and could have a deleterious effect on the key disulphide bridge in the a3 domain. Conclusion : With the description of ten other rare or private causative mutations, our study confirms that it may be of particular interest to perform a complete analysis of the HFE coding region in HC probands with at least one chromosome lacking the C282Y, H63D and S65C mutations.

Fabry disease: Exclusion of D313Y as a disease causing mutation of the alpha-Galactosidase A (GLA) gene.

T. Neumann, A. Eigel, M. Herrmann, N. Bogdanova, J. Horst;
Institut für Humangenetik, Westf. Wilhelms-Universität, Münster, GERMANY.

Fabry disease is a X-linked recessive metabolic disorder caused by mutations in the alpha-Galactosidase A (GLA) gene. The identification of the disease causing mutation in a given Fabry family permits precise genetic counselling, heterozygote detection of female family members and prenatal diagnosis.
We present a large pedigree of a German Fabry family with mutation analysis in more than thirteen individuals. All affected males had died. Mutation analysis in a putative female carrier (suspected by biochemical GLA measuring) showed the sequence variant D313Y of the GLA gene. D313Y was previously described as a disease causing mutation by Eng et al. in 1993. In a prenatal diagnosis (CVS) of a male pregnancy we detected the D313Y sequence variant. In order to confirm our results, we performed a biochemical GLA measurement. Unexpectedly the CVS cells showed an alpha-Galactosidase A activity in the normal range.
Molecular genetic investigations in further family members (i) identified the D313Y variant in a healthy 49 year old male and (ii) excluded this sequence variant in an obligate female carrier, whose DNA became meanwhile avaible for genetic analysis. Genomic DNA sequencing in this individual revealed the nonsense mutation W348X in exon 7 of the GLA gene. Pedigree analysis confirmed that W348X, as distinct from D313Y, cosegregates with the disease in the family.
Our results show that the D313Y sequence variant of the GLA gene is an amino acid substitution, whose influence -if at all- on the severity of the Fabry phenotype remains to be clarified.

L. Musante¹, H. G. Kehl², F. Majewski³, P. Meinecke⁴, S. Tinschert⁵, G. K. Hinkel⁶, S. Schweiger¹, G. Gillessen-Kaesbach⁷, D. Wieczorek⁷, M. Hoeltzenbein¹, H. H. Ropers¹, V. M. Kalscheuer¹;
¹Max-Planck-Institute for Molecular Genetics, Berlin, GERMANY, ²Klinik und Poliklinik für Kinderheilkunde-Kardiologie, Universitätsklinikum Münster, Münster, GERMANY, ³Institut für Humangenetik und Anthropologie, Universität Düsseldorf, Düsseldorf, GERMANY, ⁴Abteilung für Medizinische Genetik, Altonaer Kinderkrankenhaus, Hamburg, GERMANY, ⁵Institut für Medizinische Genetik, Charite, Humboldt-Universität, Berlin, GERMANY, ⁶Institut für Klinische Genetik, Technische Universität, Dresden, GERMANY, ⁷Institut für Humangenetik, Universitätsklinikum Essen, Essen, GERMANY.

†Deceased.
Noonan-syndrome (NS, MIM 163950) is a well recognized autosomal dominant multiple malformation syndrome with an estimated incidence of 1 in 1,000 to 2,500 live births. Affected individuals have proportionate short stature and a characteristic facial appearance with hypertelorism, ptosis, downward slanting palpebral fissures, and low set posteriorly rotated ears. In addition, cardiac involvement, most commonly pulmonary valve stenosis and hypertrophic cardiomyopathy, is frequently seen. NS is genetically heterogenenous. Linkage to a 5 cM region on chromosome 12q24 has been reported previously and only recently, mutations in the protein tyrosine kinase gene PTPN11, have been described. In a small sample, missense mutations were found in more than 50% of NS cases (Tartaglia, M et al., Nat. Genet. 29: 465-468, 2001). We have screened PTPN11 for mutations in 86 clinically well characterized familial and sporadic NS cases and identified 14 different missense mutations in 29 (34%). Seven mutations, 5 in the N-SH2 domain, 1 in the C-SH2 domain and 1 in the PTP domain, are novel and have not been described before. In addition, we found 4 of 7 mutations described by Tartaglia et al (Asp61Gly, Tyr63Cys, Ala72Ser, Asn308Asp) repeatedly in apparently unrelated cases. Most mutations cluster in the SH2 domain at the N-terminus (N-SH2), which acts as a molecular switch between the inactive and active protein form. No PTPN11 mutations were detected in 4 patients with cardio-facio-cutaneous syndrome (CFC), which shares many phenotypic similarities with NS.

N. S. Udar¹, C. Kenney², M. Chalukya¹, T. Anderson¹, V. Yellore¹, S. Yelchits¹, D. Brown², A. B. Nesburn², K. W. Small¹;
¹University of California Los Angeles, Los Angeles, CA, ²Ophthalmology Research Laboratories Cedars-Sinai Medical Center, Los Angeles, CA.

Keratoconus has an approximate incidence of 50-230/100,000 in the general population. Progressive non inflammatory corneal thinning is a characteristic feature of this disease. The age of onset is at puberty and the disorder is progressive until the third to fourth decade of life when it usually arrests. It is a major cause of cornea transplantation in developed countries. Genetic factors have been suggested as a cause of keratoconus. Both autosomal dominant and autosomal recessives forms of vertical transmission/inheritance have been suggested. There are many reports suggesting involvement of BIGH3. The BIGH3 gene is expressed in the cornea and localizes to human chromosome 5. Mutations in this gene are responsible for causing corneal dystrophies. In addition, the protein levels of BIGH3 are reported to be altered in keratoconus tissues. We screened 16 individuals, representing different families with keratoconus and for mutations within the BIGH3 gene. Although we found more than 6 sequence variations we did not find any protein altering changes. We concluded that the BIGH3 gene is not responsible for causing keratoconus in this patient population.

M. Houshmand¹, E. Sharifpanah¹, M. Sanati¹, A. Tabasi², Y. Shafeghati¹;
¹National Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²Farabi hospital, Tehran, IRAN (ISLAMIC REPUBLIC OF).

PURPOSE: To define the prevalence of a panel of mitochondrial DNA (mtDNA) mutations associated with Leber's hereditary optic neuropathy (LHON) in the Iranian LHON population. LHON-associated mtDNA mutations have been found in LHON patients from around the world, but the Iranian LHON population has not been studied. METHODS: 23 Iranian patients were defined clinically as having LHON on the basis of painless, subacute, bilateral optic neuropathy and the exclusion of other causes of subacute optic neuropathy. MtDNA was extracted from blood of the probands and healthy members of their families and assayed for a panel of primary LHON-associated mtDNA mutations by polymerase chain reaction (PCR)-based methods. We studied four well-known LHON-associated primary mutations (at nucleotide positions 11778, 3460, 14484, and 14459) in all 23 probands. RESULTS: Among the 23 probands tested for four common LHON mutations, 3 carried the 11778 mutation, 1 carried the 14459 mutation, 1 carried the 3460 mutation and no one carried the 14484 mutation. The phenotype of female of members of family who carried 11778 mutation showed the normal variant even they had the same level of this mutation. Beside one patient who carried 3460 mutation all of our patients were male. We could not find any new point mutation in ND6 gene of other patient even this gene is hot spot for LHON. CONCLUSION: The results of mtDNA analysis of the Iranian LHON patients appear to be the same from those of previous reports.

Mutations in the GJB-2 gene in patients with non-syndromic hearing loss from endogamous population of Roms (Gypsies)

G. Minarik¹, V. Ferak¹, E. Ferakova¹, A. Ficek¹, H. Polakova², A. Zatkova², L. Kadasi²;
¹Faculty of Natural Sciences Comenius University, Bratislava, SLOVAKIA, ²Institute of Molecular Physiology and Genetics SAS, Bratislava, SLOVAKIA.

Mutations in the connexin 26 gene (GJB-2) represent a major cause of autosomal recessive non-syndromic hearing loss (NSHL) worldwide. In most Caucasian populations, the 35delG mutation in this gene was found to account for up to half of the genetic non-syndromic childhood deafness. In populations of non-European ethnic background, other GJB-2 gene mutations are occasionally common, e.g. 167delT in Ashkenazi Jews, R143W in Africans, 235delC in Koreans.
DNA samples from 54 unrelated NSHL patients from endogamous and inbred population of Roms (Gypsies) from Eastern Slovakia were screened for two GJB-2 mutations: 35delG and W24X. A single patient was found homozygous for 35delG, 8 were homozygous for W24X, 5 compound heterozygotes 35delG/W24X, 3 heterozygous for W24X (and an unidentified mutation), and one heterozygous for 35delG (and an unidentified mutation). In remaining 36 patients, no GJB-2 mutation was found. Thus, in Slovak Roms, W24X accounts for 23,52 %, whereas 35delG for only 5,88 % of all GJB-2 gene mutations in NSHL patients. So far, the W24X mutation was observed in two Pakistani NSHL families, which is in accordance with the hypothesis of Indian origin of European Roms.

Analysis of polymorphism for UGT1*1 exon 1 promotor in neonates with pathologic and prolonged jaundice

A. Ulgenalp, .¹, N. Duman¹, F. Schaefer², L. Whetsell², E. Bora¹, H. Gülcan¹, A. Kumral¹, H. Ören¹, Ö. Giray¹, D. M. Erçal¹, H. Özkan¹;
¹Dokuz Eylül University, Izmir, TURKEY, ²H. A. Chapman Institute of Medical Genetics, Tulsa, OK.

The role of Gilbert's syndrome, which is associated with unconjugated hyperbilirubinemia and decreased bilirubin UDP-glucuronosyltransferase activity, in neonatal jaundice is still not well known. We aimed to investigate whether a TATA box polymorphism in the promoter of the UGT1*1 exon I, the most common detected DNA polymorphism in Gilbert's syndrome, is a contributory factor in unexplained pathologic or prolonged jaundice. 38 neonates who had unexplained pathologic jaundice, 37 neonates who had unexplained prolonged jaundice, and 35 healthy, nonjaundiced neonates were enrolled in the study. Genomic DNA was isolated from blood and polymerase chain reaction amplification was used to examine sequence variation of the promoter upstream of UGT1*1 exon I. Genotypes were assigned as: 6/6 (homozygous for a normal allele bearing the sequence (TA)6TAA), 7/7 (homozygous for an abnormal allele with the sequence (TA)7TAA), and 6/7 (heterozygous with one of each allele). Of the 110 infants, 10 (9%) had 7/7, 51 (46%) had 6/7, and 49 (45%) had 6/6 genotype. Although the percentage of 7/7 was higher in the group with pathologic jaundice (13%) than in the group with prolonged jaundice (8%) and in the control group (6%), the difference between three groups were not statistically significant. Also no differences were observed among different genotypes and mean serum total bilirubin concentrations. In conclusion, we showed that TA 7/7 and TA 6/7 genotypes are not rare in our population and the presence of those polymorphisms alone does not play a significant role in the etiology of unexplained pathologic or prolonged neonatal hyperbilirubinemia

An efficient strategy for molecular diagnosis of Wilson disease in the Sardinian population.

M. Lovicu¹, A. Cao¹, G. Loudianos²;
¹IRTAM-CNR, Cagliari, ITALY, ²Ospedale Regionale Microcitemie, Cagliari, ITALY.

Wilson disease (WD) is an autosomal recessive disorder of copper transport resulting from mutations in the ATP7B gene. Mutation analysis of the ATP7B gene carried out in 76 unrelated WD Sardinian families allowed the characterization of 94% of the chromosomes analyzed and led to the identification of 16 WD causing-mutations ( Hum Mut .14: 294-303, 1999). Six of the mutations identified are frequent and account for the molecular defect 85% of WD chromosomes. In an effort to increase our capability of molecular diagnosis of WD in Sardinians we have set up a multiplex PCR method for the detection of the 6 most frequent mutations, coupled with reverse dot blot analysis. Primers and ASO probes sequences were designed to permit common experimental conditions during the multiplex PCR and reverse dot blot analysis respectively.
Using this procedure, we confirmed the results obtained in the patients previously characterized by SSCP analysis. Molecular diagnosis of an additional group of 51 DNA samples of Sardinian origin revealed again concordant results using both reverse dot blot and SSCP methods. On the basis of these data we conclude that the most efficient strategy for molecular diagnosis of WD in Sardinian population consists in a preliminary mutation screening for the six most common mutations using multiplex PCR followed by reverse dot blot analysis. Samples not characterized by this first step of analysis will be subsequently analyzed using SSCP analysis for all the exons and the promoter region of ATP7B gene.

C. Dubourg¹^,2, L. Pasquier³, M. Blayau¹, M. Durou¹, L. Lazaro³, S. Odent³, V. David¹^,2;
¹Laboratoire de Génétique Moléculaire, CHU Pontchaillou, Rennes, FRANCE, ²Umr 6061 cnrs, Rennes, FRANCE, ³Service de Génétique Médicale, CHU Pontchaillou, Rennes, FRANCE.

Holoprosencephaly (HPE ; 1,2/10.000 live births ; 1/250 conceptuses) is a common development defect affecting both the forebrain and the face. Clinical expressivity is variable, ranging from a single cerebral ventricule and cyclopia to clinically unaffected obligated carriers in familial HPE. The disease is genetically heterogeneous but additional environmental agents also contribute to the aetiology of HPE. This study includes 126 unrelated nonchromosomal HPE cases (76 typical HPE, 25 atypical cases, 25 polymalformative cases). We provide clinical data regarding the subgroup of typical HPE and report 21 novel heterozygous mutations (16% for all the cases, 25% for typical HPE), 12 in Sonic hedgehog gene (SHH), 5 in ZIC2, 3 in SIX3, and 1 in TGIF. Ten mutations were found in familial cases whereas 11 mutations were identified in apparently sporadic cases. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alteration in the coding sequence, genetic screening has revealed missense codons with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HPE where the opportunity for prenatal diagnosis may be considered. We report here a functional test where the significance of SHH aminoacids replacements observed in the human population is tested by the C3H10T1/2 osteoblast transformation and phosphatase alcaline production under the Sonic Hedgehog action.

Mutations in the gene for SLURP-1 in patients with Mal de Meleda (MDM) with recessive and pseudo-dominant inheritance

H. C. Hennies¹, K. M. Eckl¹^,2, H. P. Stevens³, G. G. Lestringant⁴, M. Westenberger-Treumann⁵, H. Traupe⁶, P. M. Frossard⁴, R. Stadler⁵, I. M. Leigh³, A. Reis⁷, P. Nürnberg¹;
¹Max-Delbrück-Centrum, Berlin, GERMANY, ²Free University, Berlin, GERMANY, ³St Bartholomew's and the Royal London School of Medicine, London, UNITED KINGDOM, ⁴Tawam Hospital and Faculty of Medicine and Health Sciences, Al Ain, UNITED ARAB EMIRATES, ⁵Klinikum, Minden, GERMANY, ⁶University, Münster, GERMANY, ⁷University, Erlangen, GERMANY.

Mal de Meleda (MDM), or keratosis palmoplantaris transgrediens of Siemens, is a hereditary skin disorder characterised by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis. There is no associated involvement of other organs, however, a rather broad spectrum of clinical presentations with other variable features is characteristic. MDM was first described in patients from the isle of Mljet (Meleda) in Croatia. Recently, mutations in the ARS (component B)-81/s gene on chromosome 8q24-qter were identified in patients with MDM. We have shown lately that a very similar phenotype of transgressive PPK is not linked to chromosome 8q24-qter in several families from the United Arab Emirates. Here we present further families with transgressive PPK. Three novel mutations in ARS (component B)-81/s were identified in consanguineous families from Turkey, Palestine, and the United Arab Emirates: two different mutations affecting the same codon, both resulting in the amino acid change G86R, and a mutation that alters the translation initiation codon. In a German family without known consanguinity, which was originally supposed to have a dominant form of transgressive PPK, we demonstrated a pseudo-dominant inheritance. Three children and their affected mother were homozygous for the mutation W15R while the unaffected father was heterozygous. Pseudo-dominance was confirmed by the analysis of several neighbouring microsatellites. Our findings show that the MDM type of transgressive PPK may be caused by SLURP-1 mutations in patients from various origins. A founder effect is supposed to be responsible for MDM on Mljet, however, here we demonstrate allelic heterogeneity for mutations in SLURP-1.

N. Nekrasova, I. Shagina, A. Petrin, A. Polyakov;
Research center for Medical Genetics, Moscow, RUSSIAN FEDERATION.

Mutations in the gene for connexin 26 are the major cause of autosomal recessive inherited non-syndromic congenital hearing loss. A single mutation 35delG is responsible for the majority of affected alleles in different populations. In several Russian populations average carrier frequency of 35delG mutation is 1/46.7. We have analyzed 109 unrelated patients affected by sensoneural hearing loss for 35delG mutation. All of ours patient had early onset deafness with various degree of hearing loss. First group consisted 87 patients with normal hearing parents, in second group19 patients had both parents with hearing loss, in 3 patients only one parent had hearing loss (third group). In the first group we have found 33 families (38%) with mutation 35delG. In 22 patients mut 35delG was in homozygous state, in 11 patients it was in heterozygous state. In the second group all the patients had mut 35delG. Sixteen patients (84%) had mut 35delG in homozygous state; others had mut 35delG in heterozygous state. In the third group mut 35delG have been found in one patient in heterozygous state. We have accounted these results on a percent of chromosomes affected by mut 35delG. In the group of patient with normal hearing parents quota of injured chromosomes was 31.6%, in group of “deafness” families it was 92%. Summer percent of chromosomes with mut 35delG was 41.7.

S. Tartaglia¹, R. Doliana², D. Volpin³, S. Mason¹, R. Tenconi¹, M. Clementi¹;
¹Genetica Clinica Epidemiologica, Padova, ITALY, ²Centro Oncologico, Aviano, ITALY, ³Diaprtimento Istologia, Padova, ITALY.

Elastic fibers are major constituents of the extracellular matrix and confer to connective tissues the properties of resilience and elastic recoil. Recently EMILIN1 and EMILIN2, largely distributed in connective tissues, have been isolated. The structural components of elastic fibers have been found to be defective in some heritable human diseases, such as Williams syndrome and familial supravalvular aortic stenosis for elastin and Marfan's syndrome and congenital contractural aracnodactyly for fibrillin. . However, no mutations responsible for many diseases due to anomalies of elastic fibers/connective tissue have been detected. Since EMILIN1 and EMILIN2 are found only within elastic fibers, and they co-react, mutations of these gene family are expected to give rise to alterations of this extracellular matrix component. We have searched for mutations of EMILIN1 and EMILIN2in 8 families with Marfan-like phenotype.
For each family a three-generation pedigree, physical examination of all probands and their first degree relatives has been performed. DNA was obtained after informed consent of probands and first degree relatives.
A mutation analysis has been performed using SSCP and CSGE techniques.
At present no mutations have been identified in the families. Although the low number does not allow us to make final conclusions, EMILIN family genes seem not to be involved in Marfan-like phenotypes.

Four new mutation and neutral polymorphisms of the low density lipoprotein receptor gene in St. Petersburg familial hypercholesterolemia.

Y. A. Tatishcheva;
Institute for Experimental Medicine, St. Petersburg, RUSSIAN FEDERATION.

Familial hypercholesterolemia (FH) is an inherited metabolic disease with a dominant mode of inheritance. It is quite common in most human populations (1:500) and results in reduction of low-density lipoprotein (LDL) catabolism followed by premature coronary heart disease. The disease is caused by mutations in the LDL receptor gene.
In order to develop presymptomatic diagnostic tools for management of the FH we aimed to study the LDL receptor gene mutation spectrum in St.-Petersburg (Russia). We have created a DNA bank from 100 unrelated patients with clinical picture of FH. Earlier, seven mutations in the LDL receptor gene were found in some of the probands. In view of the growth of the collection of DNA samples, we continued the search for genetic abnormalities in exons 4 and 10 of this gene. This exons were amplified by polymerase chain reaction (PCR) and screened for presence of the mutations via combined single-strand conformation polymorphism -heteroduplex analysis (SSCP-HA). The fragments showing shifted mobility in polyacrylamide gel electrophoresis were sequenced by method of Sanger. Up-to-date, we have identified four new mutations - A130P, G128G, C146R, C188Y. Rapid methods for mutation detection were developed. Cosegregation of mutations and high cholesterol levels proves the role of mutations in disease development. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.

First description of a recessive form of Central Core Disease, transitorily presenting as Multi-minicore Disease and associated with a homozygous mutation in RYR1.

A. Ferreiro¹, N. Monnier², N. B. Romero¹, J. P. Leroy³, C. Bönneman⁴^,5, V. Straub⁶, C. A. Haenggeli⁷, T. Voit⁶, J. Lunardi², M. Fardeau¹, P. Guicheney¹;
¹INSERM U523/Institut de Myologie, Paris, FRANCE, ²Laboratoire Biochimie de l'ADN, CHU Grenoble, Grenoble, FRANCE, ³Service d'Anatomie Pathologique, CHU Brest, Brest, FRANCE, ⁴The Children's Hospital, Philadelphia, PA, ⁵Georg August Universitat, Göttingen, GERMANY, ⁶Department of Pediatrics, University of Essen, Essen, GERMANY, ⁷Hôpital des Enfants, Geneva, SWITZERLAND.

Multi-minicore Disease (MmD) is an autosomal recessive congenital myopathy characterized by the presence of multiple small zones of sarcomeric disorganization and mitochondria depletion ("minicores") in muscle fibers. Its clinical phenotype is heterogeneous. To identify the genetic basis of the disease, we performed a genome-wide screening in a consanguineous Algerian family in which 3 children presented in infancy with generalized muscle weakness, more evident in pelvic girdle and hands, and joint hyperlaxity (“distal involvement” phenotype). Their first muscle biopsies (at 2 and 4 years of age) showed multiple minicores. By homozygosity mapping, linkage of the disease to the 19q13 region was identified in this family and, subsequently, in 3 additional MmD families showing a similar phenotype (LS=5.19 for D19S570 at q=0.00).
A gene encoding the skeletal muscle ryanodine receptor, RYR1, is located in this region. Heterozygous RYR1 mutations cause 2 autosomal dominant entities: the congenital myopathy Central Core Disease (CCD) (C-terminal domain mutations) and Malignant Hyperthermia Susceptibility (N-terminal domain mutations). In the Algerian family, we identified the first homozygous missense mutation (P3527S) in the central part of RYR1. New muscle biopsies at adulthood revealed typical CCD with rods in the 3 patients and absence of cores in the healthy parents.
This subgroup of families linked to 19q13 constitute the first variant of CCD with genetically proven autosomal recessive inheritance and transient presentation as MmD. This work also illustrates the age-related modification of the morphological lesions in congenital myopathies, and represents the first description of a genetic defect underlying the MmD phenotype.

Mutation analysis in Crouzon and Pfeiffer syndromes identifies novel substitutions in the tyrosine kinase regions of the fibroblast growth factor receptor-2 (FGFR-2)

V. El Ghouzzi¹, S. Heuertz¹, J. Bonaventure¹, M. Le Merrer¹, A. Munnich¹, D. Renier², E. Lajeunie¹^,2;
¹INSERM U393, Paris, FRANCE, ²Dept of neurosurgery, Necker hospital, Paris, FRANCE.

Crouzon and Pfeiffer syndromes are autosomal dominant craniosynostoses characterised by premature fusion of one or more cranial sutures and dysmorphic facial features with or without limbs abnormalities. These conditions are associated with heterozygous mutations in three members of the fibroblast growth factor-receptor family (FGFR1-3) but the great majority of them are missense substitutions exclusively located in the extracellular domain of FGFR2. Studying a large series of 73 unrelated patients, we found 28 different heterozygous mutations in 66 cases (90%). This included 22 "conventional" mutations in 59 individuals (the P252R mutation in FGFR1, the A391E mutation in FGFR3, and 20 different mutations in exons IIIa and IIIc of FGFR2) and 6 novel FGFR2 substitutions in 7 patients. Interestingly, two of these (N549H and K641R) were found in the intracellular domain of FGFR2, within the two tyrosine kinase regions of the receptor and were associated with Crouzon syndrome (2 cases) and Pfeiffer syndrome (1 case), respectively. Sequence alignments of FGFR2 and FGFR3 showed that asparagin 549 in FGFR2 corresponds to asparagin 540 in FGFR3 which is mutated in hypochondroplasia (N540K) suggesting that the N549H substitution may be an activating mutation of FGFR2. Extensive screening of intracellular domains of FGFR1 and FGFR2 are now being investigated for the 10% remaining cases of our series. These results show that FGFR2 mutational spectrum is not restricted to the Ig-loops of the extracellular region as previously thought and suggest that all Crouzon and Pfeiffer cases are associated with FGFRs mutations.

E. V. Vasil'ev¹, E. Y. Polyakova², V. A. Peterkova², P. O. Rumyantsev³, D. V. Zaletayev¹;
¹Research Centre for Medical Genetics, RAMS, Moscow, RUSSIAN FEDERATION, ²Research Centre for Endocrinology, RAMS, Moscow, RUSSIAN FEDERATION, ³Research Centre for Medical Radiology, RAMS, Obninsk, RUSSIAN FEDERATION.

Multiple endocrine neoplasia type II syndromes (MEN IIA and IIB ) are an autosomal, dominantly inherited disorders characterized by the occurrence of medullary carcinoma of the thyroid (MTC), hyperparathyroidism and pheochromocytoma. These syndromes result from mutations of the RET proto-oncogene on chromosome 10. Germ line mutations are also seen in patients with familial medullary carcinoma of the thyroid (FMTC). MTC is associated with high morbidity and mortality. The use of direct DNA testing is an attempt to improve the cure rate because in more than 90% of individuals with a RET proto-oncogene mutation, MTC will develop.
The aim was detection of RET protooncogene molecular anomalies causing MEN II syndromes. We have studied 5 familial and 7 sporadic cases of the MEN IIA syndrome and 19 sporadic MTC cases. In all of these familial, 5 sporadic cases of MEN II A and 6 MTC cases direct sequence and restriction analysis of the exon 11 revealed the most common cysteine-to-arginine change in the codon 634 (TGC→CGC). Results of SSCP and heteroduplex analysis of the 10 and 11 exons in 2 remaining patients with sporadic MEN II A and 13 individuals with sporadic MTC were negative, but some of them have shown mobility shifts at 12-14 exons. Anomalies are characterized. In 2 families with MEN IIB syndrome a common germ line mutation of codon 918 was detected.
Early diagnosis by direct DNA testing led to early treatment with curative total thyroidectomy in two children from MEN IIA families and make possible prenatal diagnostics.

Molecular characterisation of X-linked properdin deficiency in a large French family

E. Bieth¹, F. Fortenfant¹, J. Enjalran¹, A. Sanchez², P. Calvas¹, A. Blancher¹;
¹Hôpital Purpan, Toulouse, FRANCE, ²Hôpital, Albi, FRANCE.

Properdin deficiency type I, is a rare X-linked disorder (MIM 312060) strongly predisposing to meningococcal disease. Properdin stabilises the labile C3 convertase of the alternative pathway of the complement system. We report a large French family with four properdin deficient males in three generations: an affected grand-father with four daughters and three diseased grand-sons. All but one developed meningitis caused by Neisseria meningitidis serogroup Y or serogroup B. No functional activity of properdin was detected in the affected males. The properdin level was bellow the detection limit in two of them and deeply decreased in the others. In three obligate carriers, the properdin level was reduced near half of the standard value (14, 15, 17 mg/l; controls = 22-38 mg/l). One expressed a normal rate (25,3 mg/l). By sequencing long-PCR products of the properdin gene (PFC gene) we found a base change at the 3’end of the consensus sequence of intron 8 (namely 1205-1G>T). This mutation inactivates a splice site in the PCF gene as demonstrated by analysis of the leukocytes transcription products in affected patients. It is to date the first splice mutation demonstrated in this gene. Interestingly, one among the four female carriers developed meningitis, and shared with her two carrier sisters, chronic rheumatic diseases associated to marker HLA-B27. HLA-B locus and properdin factor B (C3 proaccelerator) being closely linked on chromosome 6, relationship between properdin deficiency and chronic rheumatic diseases will be discussed.

A non-glycine mutation in the C-propeptide of the alpha1(I) collagen chain causes mild Osteogenesis imperfecta and EDS-like features

S. Symoens¹, L. Nuytinck², P. J. Coucke¹, B. Loeys¹, E. Legius³, A. De Paepe¹;
¹University Hospital - Center of Medical Genetics, Ghent, BELGIUM, ²Innogenetics, Ghent, BELGIUM, ³K.U.L. - Center for Human Genetics, Leuven, BELGIUM.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by a wide range of mutations occurring in the genes encoding type I collagen (COL1A1-COL1A2). These mutations mainly represent single-base changes resulting in the substitution of critical glycine residues but also deletions and insertions are reported.
We studied a 10-year-old girl with mild type I OI. The proband’s delivery was complicated by fracture of the clavicle and pneumothorax. Postnatally, no further fractures occurred. To date, the patient has blue sclera and suffers from mild hyperlaxity of skin and joints.
We analyzed type I collagen production at the protein and molecular level. Although no abnormalities were observed on protein analysis, we identified a de-novo c3890A>G transition within exon 49 of the COL1A1 gene, resulting in a M1264V substitution in the C-propeptide. This transition causes the activation of a cryptic splice site inside the exon. As a result, the last 25 bp of exon 49 are spliced out, and due to a frameshift, a premature stopcodon is created in exon 50. The normal and both aberrant cDNA transcripts (11/ 20 clones carry the 25-bp deletion, 2/20 clones M1264V substitution) are detected.
At the protein level, the frameshift mutation results in the removal of 5 of the 6 critical cysteine residues in the C-propeptide, necessary for the incorporation of the chain in collagen type I molecules. The mild phenotype of this patient suggests that this latter protein defect is predominant and causes haploinsufficiency. Further biochemical characterization will be performed.

L. Renard¹, C. Godfraind², L. Boon³, M. Vikkula⁴;
¹Department of Radiotherapy, Cliniques universitaires St-Luc, Université catholique de Louvain, Brussels, BELGIUM, ²Laboratory of Neuropathology, Cliniques universitaires St-Luc, Université catholique de Louvain, Brussels, BELGIUM, ³Division of Plastic Surgery, Cliniques universitaires St-Luc, Université catholique de Louvain, Brussels, BELGIUM, ⁴Laboratory of Human Molecular Genetics, Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, Brussels, BELGIUM.

Paragangliomas (OMIM# 168000), (carotid body tumours) are vascularized tumors of the head and neck. They may be unilateral, although with inherited predisposition, they tend to be bilateral. It has been observed that paragangliomas are often inherited via the paternal line; thus maternal imprinting occurs. This helped in finding linkage to 11q23-qter (Heutink et al. 1992), which led to the identification of a mutated gene, SDHD, which encodes the small subunit of cytochrome b in the succinate-ubiquinone oxidoreductase complex (PGL1) (Baysal et al. 2000). This enzyme complex, constituted from SDHA, B, C and D, is important for the tricarboxylic acid cycle and the aerobic respiratory chains. Thus, loss of SDHD may lead to chronic hypoxic stimulation of cellular proliferation that leads to tumorous growth. Mutations in the SDHB and C genes (PGL3 and PGL4) have also been identified, but in families with non-imprinted inheritance of paragangliomas (Niemann, and Muller, 2000; Astuti et al., 2001). Interestingly, another PGL locus was mapped more telomerically on 11q (PGL2).
We report a family with three generations affected with paragangliomas. Generation skipping of phenotype was noted once, when the disorder seemed to be inherited from the mother. Thus, the SDHD gene became the most likely candidate. In fact, we identified a novel splice site mutation that co-segregated with the phenotype. Interestingly, six non-affected carriers were observed, all of which had inherited the mutant allele from their mother. The identification of carriers enables genetic counseling, an important aspect for these usually treatable vascular tumors. (vikkula@bchm.ucl.ac.be)

N. Boutet¹, G. Chartier², D. Lafon², V. Drouin-Garraud³, P. Sarda⁴, D. Griffiths², M. Longy⁵, D. Lacombe⁶, P. Gorry⁵^,6;
¹Institut Bergonie, Bordeaux, FRANCE, ²Institut Bergonié, Bordeaux, FRANCE, ³Unité de Génétique Clinique, Hôpital. Ch. Nicolle, Rouen, FRANCE, ⁴Unité Génétique Médicale, CHU de Montpellier, Montpellier, FRANCE, ⁵Laboratoire de Génétique Oncologique, Institut Bergonié, Bordeaux, FRANCE, ⁶Service de Génétique Médicale, CHU Pellegrin-Enfants, Bordeaux, FRANCE.

"Hedgehogopathies" is a term which designates a group of hereditary dysmorphic syndromes where an alteration of the Hedgehog signalling pathway in vertebrates occurs. Many actors in this transmission pathway have been identified by genetic studies in Drosophila ; their homologues in man are candidate genes for dysmorphic syndromes.
In humans, Sonic Hedgehog is mutated in Holoprosencephaly, Patched in Gorlin syndrome, and GLI-3, the homologue of the Cubitus interruptus gene in Drosophila, is implicated in Greig syndrome and in Pallister-Hall syndrome. Its co-activator, the transcription factor CBP is mutated in Rubinstein-Taybi syndrome. Finally, one of the target genes of SHH, Pitx2, is responsible for Rieger syndrome.
We have implemented genetic diagnosis of Gorlin syndrome (OMIM 180500), of Greig (OMIM 175700) and Pallister-Hall syndromes and also of Rieger syndrome (OMIM 180500).This work allowed us to consider phenotype-genotype relationships of these different hereditary diseases. We identified 9 novel mutations of the Patched gene, 4 novel mutations of the Gli3 gene and one new mutation of the Pitx2 gene. While a correlation exists between the mutation spectrum and Greig and Pallister-Hall syndromes, no correlation is found in Gorlin syndrome. Finally, the Reiger syndrome is characterized by major genetic heterogeneity. Clinical data and molecular results are discussed in the light of our understanding and knowledge of the Hedgehog signaling pathway in vertebrates.