ESHG Posters 13

S. Vallian¹, E. Barahimi², M. Shamoradgoli¹, M. Hosseini³, H. Moeini¹;
¹Isfahan University, Isfahan, IRAN (ISLAMIC REPUBLIC OF), ²Khatam University, Tehran, IRAN (ISLAMIC REPUBLIC OF), ³NRCGEB, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Phenylketonuria (PKU) is a metabolic genetic disease, in which the phenylalanine hydroxylase (PAH) gene is mutated, resulting in the elevation of blood phenylalanine (Phe). The disease is associated with a severe irreversible mental retardation. However, early detection of the disease and the elimination of Phe from diet could prevent the PKU symptoms. In a screening program to analyze the PKU mutations in Isfahan, we examined 1611 institutionalized mentally retarded patients, ranging from 7-27 years old. Of the patients examined, 36 (2%) were positive using the Guthrie bacterial inhibition assay (GBIA). Quantitative measurement of the serum Phe showed that among the patients tested, 33 had increased level of Phe (above 10 mg/dL). Preliminary studies on the PAH mutations in these patients resulted in the detection of several mutations including the delL364, R261X, K341T, G272X and S273F. The delL364 mutation is, so far, the predominant one (about 3%). Furthermore, our analysis showed that 66% of the patient are the results of consanguineous marriages, in which the parents were first cousin, indicating the role of this kind of marriages in the prevalence of the disease in Isfahan.

Congenital Disorder of Glycosilation type Ia in a patient with Jouber syndrome and arachnodactyly

B. Cser¹, É. Morava¹, J. Karteszi¹, K. Huyben², L. Szonyi³, R. Wevers², G. Kosztolanyi¹;
¹Dep.Med.Genetics and Child Development, PTE, Pécs, HUNGARY, ²Lab.of Pediatrics and Neurology, Nijmegen, NETHERLANDS, ³Dep.of Pediatrics I., Budapest, HUNGARY.

Many forms of CDG are currently known. Serum transferrin isofocusing is the method of choice for screening however, most metabolic laboratories do not routinely use this technique. The method should be specifically requested in any child with an undefined multi-system disorder. As part of the EUROGLYCAN project we started a systemic screening for CDG in hypotonic patients. In the first hundred individuals studied we found one positive case. The two year-old patient was examined with mental retardation, profound hypotonia, random eye movements, ptosis, flat face, arachnodactyly, generalised joint hyperflexibility and lipid hepatopathy. Dysmorphic facial features, mental retardation, hypotonia, ophthalmic symptoms and cerebellar vermis hypoplasia suggested Joubert syndrome. Karyotype analysis was normal. Persistent hepatomegaly and increased hepatic enzymes raised the possibility of a metabolic disease. Serum transferrin isoelectric focusing and leukocyte enzyme studies established the diagnosis of CDG Ia (Congenital Disorder of Glycosylation). Characteristic features of CDG Ia include severe muscle hypotonia, mental retardation, ataxia, ophthalmologic involvement, failure to thrive, abnormal fat distribution, cerebellar atrophy and hepatopathy. Our patient had normal growth, normal fat distribution and no inverted nipples or strabismus, however, arachnodactyly and joint hyperlaxity were pronounced. Our case who is the first patient diagnosed with CDG-Ia in Hungary contributes to the phenotypic spectrum of this recently identifiable metabolic disorder.

Necrosis-like cell death in Prolidase Deficiency (PD) fibroblasts: molecular, biochemical and morphological characterization of five new PD cases.

A. Forlino¹, A. Lupi¹, P. Vaghi², A. Icaro Cornaglia³, E. Campari¹, A. Calligaro³, G. Cetta¹;
¹Dipartimento di Biochimica, University of Pavia, Pavia, ITALY, ²Centro Grandi Strumenti, University of Pavia, Pavia, ITALY, ³Dipartimento di Medicina Sperimentale, University of Pavia, Pavia, ITALY.

Prolidase Deficiency (PD) is a rare recessive disorder caused by mutations in the prolidase gene. It is characterized mainly by skin lesions, mental retardation and recurrent infectious. We identified the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a G to A transversion in two siblings leading to a G448R substitution. A G+1 to C transition in one allele of intron 11, causing the skipping of exon 11, was detected in a third proband and this is the first report of a mutation in a splicing donor site of the prolidase gene. A G-1 to A transversion in intron 7 was identified in two unrelated probands and shown to cause multiple alternative spliced transcripts. Long term cultured fibroblasts from these PD patients were used to investigate the biochemical, microscopical and ultrastructural changes in affected cells. Light and electron microscopy revealed that patients’ cells were more round and branched than controls. We also detected increased cytosolic vacuolization, plasma membrane interruptions, mitochondria swelling and mitochondrial matrix and cristae modifications. JC-1 labelling showed decreased mitochondrial membrane potential. An intracellular accumulation of the Gly-Pro dipeptide was revealed by capillary electrophoresis analysis. The composite data provide new insights into PD physiopathology, suggesting the activation in skin fibroblasts of a necrosis-like cellular death, which could be responsible for the typical skin lesions in this disease.

Five years of molecular diagnosis of hypophosphatasia : benefits for genetic counseling and for understanding the molecular basis of the disease

E. Mornet;
Laboratoire SESEP, Université de Versailles-Saint Quentin en Yvelines, Versailles, FRANCE.

Hypophosphatasia is an inherited disorder caused by a deficiency of bone alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The disease results in low or nil bone and dental mineralization. It is highly variable in its clinical expression, due to the strong allelic heterogeneity in the TNSALP gene. Since 1997, we studied 127 patients or families of patients from European, North-American and Australian origins and identified by sequencing 101 mutations among which 91 were not previously described. Twenty-six molecular prenatal diagnoses were performed in 22 families affected by the lethal form of the disease. The great number of mutations collected by our laboratory constituted a powerful tool for studying the correlations of genotype and phenotype, for understanding the cases of dominant transmission, and for elucidating the role of alkaline phosphatase in bone mineralization. For that, we used a panel of methodologies such as site-directed mutagenesis, 3D modeling and monoclonal antibodies directed against the protein. Our results allowed us to distinguish severe and moderate alleles, to discriminate recessive and dominant mutations, to show that the dominant effect of some mutations result from an inhibitory mechanism and to identify major functional regions of the enzyme, a result of significant importance in understanding the role of this enzyme in bone mineralization. These results have been immediately used to improve genetic counseling and molecular diagnosis of the disease and in the long term, could help to discover drugs for treatment of the disease.

R. Petrovic¹, J. Chandoga¹, J. Futas¹, L. Dvorakova², M. Hrebicek²;
¹Centre of Medical Genetics, University Hospital, Bratislava, SLOVAKIA, ²Institute of Inherited Metabolic Disorders, Prague, CZECH REPUBLIC.

Introduction: Up to now about twenty inherited peroxisomal diseases has been described. Except of X-linked adrenoleukodystrophy (X-ALD), all diseases are based on autosomal recessive type of inheritance. This defect is based on peroxisome biogenesis or deficiency in individual peroxisomal protein.
Methods: Gas Chromatography/Mass Spectrometry (GC/MS) analysis of very long chain fatty acids (VLCFA), phytanic acid, pipecolic acid and urinary organic acids were used for diagnostics of peroxisomal diseases with defect in b-oxidation route, primary hyperoxaluria I and mevalonic aciduria. In some cases digitonin permeabilisation of cultured fibroblasts was realised. For X-ALD mutations analysis cDNA sequencing was used.
Results: One case of the peroxisome biogenesis disorder and three cases of single peroxisomal enzyme deficiencies with fatal course to 3 year were diagnosed. X-ALD with variable phenotypes was found in five male patients. In two patients (brothers) of age about 30 this disease was presented as adrenomyeloneuropathy associated with adrenal insufficiency and Addison crises. In the other sibling X-ALD as childhood cerebral form and until now asymptomatic form was manifested. Among the X-ALD patients there is also a 12-year old boy with 5 year continual therapy with Lorenzo’s oil. He has a slow progression of disease and nearly physiological VLCFA serum values. One case of primary hyperoxaluria I with kidney stones at 4 year and mevalonic aciduria were also diagnosed.
Conclusions: During the last five year inherited disease of peroxisomal compartment has been definitively proved for 11 patients. Two novel mutations of ALD gene were identified c.1898G>T (S6331I) and c.1979G>C (R660P).

Rapid Genetic Testing for Gaucher Disease: From Restriction Fragment Length Polymorphism (RFLP) to Reverse-Hybridization

G. Kriegshäuser¹, D. Halsall², A. Moritz¹, F. Kury¹, C. Oberkanins¹;
¹ViennaLab Labordiagnostika GmbH, Vienna, AUSTRIA, ²Dept. Clinical Biochemistry, Addenbrook´s Hospital, Cambridge, UNITED KINGDOM.

Gaucher disease (GD), the most frequent lysosomal storage disorder, is an autosomal recessive disease characterized by deficiency of glucocerebrosidase (GBA). Mutations in the GBA gene cause the disease and enzyme deficiency results in accumulation of glucocerebroside, mainly within cells of the monocyte/ macrophage lineage, which may lead to splenomegaly, hepatomegaly, thrombocytopenia, bone marrow suppression, and bone lesions. The disease is panethnic and has been divided into three major types on the basis of the absence (type 1) or the presence and severity of neurologic manifestations (type 2 and type 3). The most common variant of the disease is type 1, which is particularly frequent in the Ashkenazi Jewish population with an estimated disease frequency of 1 in 850 and a carrier rate to approximate 1 in 15. We have developed a reverse-hybridization assay (Gaucher Disease StripAssay) for the simultaneous detection of eight point mutations (84GG, IVS2(+1), 1226G, 1297T, 1342C, 1448C, 1504T and 1604A) and two multiply mutated alleles derived from rearrangements between the structural gene and the 16-kb downstream pseudogene (RecNciI, RecTL). The test is based on a single, multiplex DNA amplification reaction and ready-to-use test strips presenting a parallel array of oligonucleotide probes for each wild-type and mutated allele. The Gaucher Disease StripAssay was used to screen a cohort of 91 English Gaucher Disease patients previously genotyped by RFLP. Data obtained with both assays will be presented and discussed with respect to specificity, sensitivity, design and throughput.

S. Opitz¹, M. Kaufmann¹, E. Schwinger¹, C. Zühlke¹, B. Käsmann-Kellner²;
¹Institut für Humangenetik, UKL, Lübeck, GERMANY, ²Augenklinik d. Universitätskliniken des Saarlandes, Homburg/ Saar, GERMANY.

Oculocutaneous albinism (OCA) is an autosomal recessive disorder with heterogeneous genetic background. After extended clinical investigations of 176 unrelated patients [BKK; standardised examination protocol] with albinism we extracted DNA from blood samples for molecular genetic analyses of the genes responsible for OCA1 and OCA2. OCA1 is caused by mutations in the tyrosinase gene (TYR) encoding the key enzyme in melanin biosynthesis. We investigated the 5 exons of the TYR gene by PCR and SSCP and found variations in 66 individuals (1 conformational polymorphism in 46 patients, 2 in 17 patients, and 3 in 3 patients). OCA2 resulting from mutations in the human homologue of the mouse pink eye gene is the most common type of albinism world-wide. Analysing exons 3 to 20 of the P gene (consisting of 25 exons) by PCR and SSCP aberrant signals could be detected in 22 persons (preliminary results: for 21 patients 1 difference to the normal allele was found, one patient showed abnormalities in 2 exons). In 8 patients conformational deviations were detected by SSCP in both the TYR and the P gene. In our sample of German origin about 37% of the patients show SSCP changes in the TYR gene and 12.5% in the P gene (exons 3-20) suggesting that mutations at this locus are possibly underrepresented in Caucasians.
Here, we present SSCP data of the complete TYR and P gene. Differentiation between polymorphisms and relevant mutations was carried out using sequence analysis.

An Automated Screening Test for Multiple Mutations Associated with Hereditary Iron Overload

A. Moritz, G. Kriegshäuser, F. Kury, C. Oberkanins;
Viennalab Labordiagnostika, Vienna, AUSTRIA.

Inherited iron overload is a heterogenous disorder, including "classic" autosomal recessive hereditary haemochromatosis (HH), as well as juvenile and autosomal dominant forms of the disease. The most prevalent variant among Caucasians is autosomal recessive HH due to mutations in the HFE and transferrin receptor-2 (TFR2) genes. More recently, mutations in the genes for ferroportin (FPN1/SLC11A3/IREG1) and ferritin heavy chain (FTH1) were found to be associated with autosomal dominant iron overload. In most cases therapeutic phlebotomy provides an effective and inexpensive lifelong treatment. DNA testing is now routinely used to support the diagnosis in patients with abnormal iron parameters, for the presymptomatic identification of individuals at risk, and its potential for population screening programs is currently under discussion.
We have developed a reverse-hybridization assay (Haemochromatosis StripAssay) for the rapid and simultaneous detection of 18 known mutations in the HFE, TFR2, FPN1 and FTH1 genes. The test is based on multiplex DNA amplification and ready-to-use test strips containing oligonucleotide probes for each wild-type and mutated allele immobilized as parallel lines. The entire procedure from blood sampling to the identification of mutations requires less than 6 hours, and may be carried out manually or essentially automated using existing instrumentation (e.g. TECAN profiBlot). (oberkanins@viennalab.co.at)

S. K. Heath¹, G. Gray¹, A. Harper¹, P. J. McKiernan², M. Preece¹, A. Green¹;
¹W. Midlands Regional Laboratory for Inherited Metabolic Disorders, Birmingham Children's Hospital, Birmingham, UNITED KINGDOM, ²Liver Unit, Birmingham Children's Hospital, Birmingham, UNITED KINGDOM.

Tyrosinaemia Type I is caused by a deficiency of the enzyme fumarylacetoacetase (FAA), the last enzyme in the catabolic pathway of tyrosine. As with most other inborn errors of metabolism, carrier status cannot be reliably excluded using biochemical markers (ie metabolites or enzymes), and is a particular issue for consanguineous families. Prenatal diagnosis by biochemical methods may also be problematic for some families.
The FAA gene maps to 15q23-25, has 14 exons, and more than 34 different mutations have been reported. There are four common mutations associated with this disorder, most of the remainder are private mutations. Of the 37 patients (from 35 families) at Birmingham Children’s Hospital, 62% of disease alleles were accounted for by these four mutations. 27% were G192T, 27% IVS12+5 G to A, 6.8% IVS6-1 G to T, and 1.4% G1009A. 68% of these patients are of Asian origin: within this group G192T is exclusive, and IVS12+5 G to A is more common than in the whole group.
Mutation screening by single-stranded conformational polymorphism (SSCP) analysis was developed to identify the remaining disease-causing mutations. 8 patients had further testing (14 untyped alleles). From this group of patients, 9 additional mutations were identified of which 6 were novel, and together accounted for 13 of the 14 disease-causing alleles. Five of the mutations were mis-sense mutations (S23P, H133R, P156Q, T325M and S352R) and one splice site mutation (IVS9-2 A to G). These findings have led to an improved approach to diagnosis of tyrosinaemia type I for clinical practice.

R. Farrugia¹, R. Naudi¹, S. Attard Montalto², R. Parascandolo², C. A. Scerri¹, C. Bartolo³, A. E. Felice¹;
¹Laboratory of Molecular Genetics University of Malta, Msida, MALTA, ²Department of Paediatrics St Luke's Hospital, G'Mangia, MALTA, ³Synergene Technologies Ltd, Attard, MALTA.

A higher than usual frequency of hyperphenylalaninaemia due to tetrahydrobiopterin (BH₄) deficiencies, specifically Dihydropteridine Reductase (DHPR) deficiency, is present in the Maltese population. Classical Phenylketonuria due to Phenylalanine Hydroxylase deficiency has not been identified to date.
Molecular analysis of the DHPR gene in 3 families (4 probands born to unrelated parents over a span of 4 years) has identified the G23D mutation, a previously identified mutation in 2 Italian and 1 other Maltese patient. This glycine to aspartic acid change at the 23^rd amino acid in the protein alters a highly conserved amino acid in the NADH binding domain of the DHPR gene.
Population studies have shown this mutation to be abnormally frequent in the Maltese population. A heterozygote carrier rate of 2% has been established in a cohort of 400 random Maltese neonatal DNA samples. This mutation is of Mediterranean origin and is a clear example of a founder effect.
A neutral polymorphism, L132L, in the DHPR gene had previously been identified in a patient carrying the G23D mutation. This polymorphism was not present in our DHPR patients, however 6 out of 7 patients manifesting clinical symptoms typical of Dopa Responsive Dystonia (DRD) also had the L132L polymorphism in either homozygosity or heterozygosity. DRD in these 7 patients, from 4 unrelated families, is believed to be due to GTP Cyclohydrolase I deficiency - the first enzyme in the biosynthesis pathway of BH₄. Molecular and biochemical analysis are currently being carried out to identify the causative mutation in these patients.

Detection of two novel large mutations in SLC7A9 by semi-quantitative fluorescent multiplex PCR

M. Font-Llitjós¹, M. Palacín², V. Nunes¹;
¹Institut de Recerca Oncològica, L'Hospitalet de Llobregat, SPAIN, ²Universitat de Barcelona, Barcelona, SPAIN.

Cystinuria is an autosomal recessive aminoaciduria in which two clinical types have been described: I and nonI. Mutations in the cystine and dibasic amino acid transporter cause cystinuria: mutations in the heavy subunit, rBAT, coded by SLC3A1 gene, cause type I cystinuria while mutations in the light subunit bo,+AT, coded by SLC7A9 gene, cause non-type I cystinuria. Using multiplex semi-quantitative fluorescent PCR we have amplified the 13 exons of SLC7A9 together with exon 5 of DSCR1 (located on chromosome 21) as a double dose control gene. The PCR products were loaded in a 48 well acrylamide gel together with an external fluorescent size standard and run in an ABI PRISM 377 DNA sequencer. The results were processed by GENESCAN™ software. With this technique we have detected two novel large mutations in 2 Spanish families: a 5kb deletion and a 5kb duplication, both affecting exon 12, originated by the crossing over of two 195 bp sequences, which difer by 1 nucleotide, separated by 4778 bp. This method is able to detect size differences from a single base to whole exons missing, which makes it useful for scaning genes with a small to medium number of exons. This technique requires 40-60 times less DNA than Southern blot, is reproducible and can be very useful to rapidly scan a large number of samples.

Three Novel Mutations In The Cyp21 Gene In Patients With The Classical Form Of Congenital 21-hydroxylase Deficiency

M. P. De Mello¹, O. V. Carvalho-Netto¹, F. C. Soardi¹, S. H. V. Lemos-Marini², G. Guerra-Junior², M. T. M. Baptista³;
¹Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, BRAZIL, ²Dept. de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, BRAZIL, ³Dept. de Clínica Médica, Disciplina de Endocrinologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, BRAZIL.

Deficiency of 21-hidroxilase is the most frequent cause of congenital adrenal hyperplasia. Usually, CYP21 affected genes bear one of the eight mutations also present in the pseudogene CYP21P. The objective of this work was to determine new mutations in alleles that did not present pseudogene-originated mutations and in alleles of a patient with phenotype-genotype conflicting clinical form. The occurrence of three new mutant alleles is described. A mutation resulting from the insertion of an adenine between nucleotide 992 and 993 in the exon 4 was found in two heterozygous siblings. It was inherited from the father whereas the maternal allele bears the R356W. The reading frameshift changes the Lysine170 to a Serine; from this point on every amino acid changes and it creates a stop codon at the position 394. Correct segregation was confirmed by digestion with Pst I. A guanine to a adenine change in the nucleotide 166 caused G56R mutation. It is inherited from the mother and the paternal allele bears the IVS2AS,A/C-G,-12 microconversion. Segregation was confirmed by digestion with Apa I. A salt-losing patient was previously genotyped as having I172N from father and V281L from the mother, a genotype compatible with the non-classical form. CYP21 sequencing reveled that the maternal allele also bears a novel mutation. It is a G to A change at the position 391 within intron 2 splice donor consensus sequence. It is at +4 nucleotides from the GT splice donor site. An in silico analysis reveled that it completely suppressed the splice site.

Somatic and germinal mosaicism for the steroid sulfatase gene in an X-linked ichthyosis carrier

S. Cuevas, L. Gonzalez, A. Jimenez, M. Rivera, S. Kofman;
Hospital General de Mexico, Mexico, D.F., MEXICO.

Steroid sulfatase (STS) deficiency results in X-linked ichthyosis (XLI). It is characterized by dark, adhesive and regular skin scales and has a similar frequency in different geographic areas. A large majority of XLI patients present complete deletion of the STS gene which is located on Xp22.3. Mosaicism for the STS gene has not yet been reported in XLI. In the present study, we describe an XLI patient with complete deletion of the STS gene and his mother who harbored somatic and germinal mosaicism for this molecular defect. The family (XLI patient, grandmother, mother and sister) was analyzed through STS enzyme assay and PCR, DNA markers analysis and FISH of the STS gene. STS activity was undetectable in the XLI patient, very low in the mother and normal in the grandmother and sister. PCR analysis of the XLI patient showed a deletion from regions DXS1139 to DXF22S1 including the STS gene. FISH analysis performed in maternal oral cells and leukocytes showed one copy of the STS gene in 80% of the cells and two copies in the rest. The grandmother and sister showed two copies of the STS gene. DNA markers analysis allowed to identify that the origin of the X chromosome with the deletion of the STS gene corresponded to the grandfather. We report the first case in which the XLI is caused by the presence of a somatic and germinal mosacism of the STS gene

M. Tesarova¹, J. A. Mayr², L. Wenchich¹, H. Hansikova¹, M. Eleder¹, K. Blahova¹, W. Sperl², J. Zeman¹;
¹Dept. of Pediatrics and Center for Integrated Genomics, Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, ²Dept. of Pediatrics, University in Salzburg, Salzburg, AUSTRIA.

Alpers progressive infantile poliodystrophy, a neurodegenerative disease with liver dysfunction may be associated with impairment of function of mitochondrial enzymes and/or depletion of mitochondrial DNA (mtDNA). MtDNA depletion is a quantitative disturbance of mtDNA, characterised by tissue-specific reductions in mtDNA copy number. We describe two infants with liver impairment and progressive neuromuscular disease characterised by hypotonia, visual disturbances, refractory epilepsy, psychomotor retardation and profound brain atrophy. Progressive course of the disease with increased level of lactate in blood and cerebrospinal fluid suggested a disturbance in mitochondrial energy generating systems. In both patients, the analysis of hepatocyte ultrastructure revealed enormous multiplication of mitochondria of various size and microvesicular steatosis, histochemical investigations demonstrated cytochrome c oxidase deficiency. Southern blot analyses revealed markedly reduced content of mtDNA in liver (11% and 10% of the mean values in controls), brain cortex (15% and 30% of the mean values in controls) in both patients and in cultured fibroblasts (25% of the mean values in controls) of one patient. MtDNA content in muscle and heart was normal. The activities and protein amount of respiratory chain complexes were mildly decreased in isolated liver mitochondria in comparison with controls but they were normal in muscle and heart mitochondria and in fibroblasts. In one patient, mild decrease of mtDNA polymerase gamma activity was found, but its ratio to citrate synthase was normal.
Supported by IGA-MZ NE 6555-3.

A novel approach for reliable identification of cytochrome P450 alleles by multiplex assays on PyrosequencingÔ

S. Eriksson¹, M. Wadelius², G. Frenne², A. Alderborn¹;
¹Pyrosequencing AB, Uppsala, SWEDEN, ²Dept. of Clinical Pharmacology, Uppsala, SWEDEN.

The P450 cytochromes are important metabolisers of a large number of pharmaceutical substances. Polymorphic variation in these genes defines different alleles, often associated with decreased catalytic activity of the gene product. The aim of this study was to set up rapid and reliable assays to identify alleles of CYP2D6, CYP2C9 and CYP2C19 by PyrosequencingÔ.
PyrosequencingÔ, or real-time sequencing, is a fast and accurate method for SNP analysis. Pyrosequencing AB (Sweden) manufactures the PSQÔ 96 System for analysis of up to 96 SNP assays in 10 min, and the PTP system for high throughput SNP scoring. Dedicated softwares automatically delivers genotype and quality assessment for each sample. A major advantage with Pyrosequencing is its combination of accuracy, speed and ease-of-use.
Pyrosequencing assays for determination of four single nucleotide polymorphisms (SNPs) and two deletion polymorphisms were developed to identify the functional allele *2 and five of the non-functional CYP2D6 alleles (*3, *4, *6, *7 and *8). The establishing of a multiplex genotyping procedure enabled simultaneous scoring of these polymorphisms in only two Pyrosequencing reactions. 130 patient samples were analysed and the results compared to genotyping by RFLP. Pyrosequencing correctly identified 100% of the alleles identified by RFLP and, in addition, scored two alleles that had not been assessed in the RFLP method. Furthermore, multiplex assays for identification of the alleles CYP2C9*2 and *3 and CYP2C19 alleles *2, *3 and *4 were successfully set up and verified.
In summary, the Pyrosequencing approach enables reliable, cost efficient, and non-labour-intensive identification of CYP alleles.

Mapping of the gene for multiple sulfatase deficiency (MSD) by functional complementation using microcell-mediated chromosome transfer

M. P. Cosma¹, S. Pepe¹, G. Parenti², A. Belli¹, A. Mankad³, M. Grompe³, D. A. Trott⁴, R. F. Newbold⁴, A. Ballabio¹^,5;
¹TIGEM Telethon Institute of Genetics and Medicine, Naples, ITALY, ²Department of Pediatrics, Federico II University, Naples, ITALY, ³Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR, ⁴Department of Biological Sciences, Brunel University, Uxbridge, UNITED KINGDOM, ⁵Medical Genetics, II University of Naples, Naples, ITALY.

Multiple sulfatase disease (MSD) is a rare autosomal recessive disorder characterized by the deficiency of all lysosomal sulfatases. This causes a severe phenotype resulting from the association of features of all single sulfatase deficiencies. Cloning of the MSD gene has been hampered by absence of familial cases suitable for linkage mapping. A recent study demonstrated that the biochemical basis of this disease is a defect of a post-translational modification, which appears necessary for sulfatase catalytic function. However, no information is available on the protein performing such modification, and consequently of the gene mutated in this disease. We have used microcell mediated chromosome transfer to clone by complementation the gene associated with multiple sulfatase deficiency (MSD). Briefly, a panel of human/mouse monochromosomal hybrids containing single human chromosomes tagged with a selectable marker was used as a source of normal human donor chromosomes. All 22 autosomes human chromosomes were serially transferred from the hybrids into an MSD cell line by microcell-mediated chromosome transfer. After selection, we have isolated resistant clones and measured ARSA, ARSB, and ARSC activities. The results obtained so far clearly indicate functional complementation for all three sulfatases tested in the presence of a specific human chromosome derived from the donor cells. These results will be presented at the meeting together with submapping data which are being obtained by cytogenetic techniques and microsatellite analysis on hybrids containing radiation-fragmented chromosomes. Ultimately, we hope to identify the MSD gene by testing candidate genes from the smallest complementing chromosomal region.

U. Caruso, M. C. Schiaffino, G. Migliaccio, D. Campanella, R. Lorini;
University Department of Pediatrics, G. Gaslini Institute, Genova, ITALY.

Peroxisomal disorders are an extremely heterogeneous group of genetic disorders without strict correlation between clinical picture and biochemical abnormalities. The definitive diagnosis is a complex procedure which includes metabolite analysis and enzymatic, molecular and genetic studies. VLCFA in plasma has been largely indicated as the first screening test. However the information allowed by this assay is limited to the VLCFA ß-oxidation and in many cases it is unsuitable to indicate the right diagnosis. We propose to use a diagnostic flowchart based on two simultaneous investigations: VLCFA, pristanic and phytanic acids in plasma [1] and plasmalogens in erythrocytes [2]. These analyses can be carried out at the same time on the same EDTA blood sample by GC-MS and stable isotope dilution in less of 24 hours. By the combination of the results (VLCFA profile, pristanic/phytanic ratio, plasmalogen concentration) it is possible to propose a differential diagnosis among almost all peroxisomal disorders: generalised defects, ß-oxidation defects (bifunctional protein or thiolase), X-ALD, acyl-CoA oxidase deficiency, Refsum disease, classical RCDP, DHAP-AT or alkyl-DHAP synthase deficiency. This procedure can be easily applied by a laboratory with experience in metabolite analysis without any special facility (but a GC-MS) and it has been successfully used in our laboratory from five years leading to 19 new diagnosis, all confirmed by enzymatic and molecular studies.
[1] U. Caruso et al.: J Inher Metab Dis 19 (Suppl 1):83, 1996.
[2] U. Caruso: Rapid Communications in Mass Spectrometry 10:1283-85, 1996.

Genetic contribution of thrombophilic mutations to pregnancy complications: Fact or fiction?

C. Makatsoris¹, T. Hatzis², A. Hatzaki¹, T. Antoniadi¹, M. B. Petersen¹;
¹Genetics and Molecular Biology Laboratory, "MITERA" Maternity and Surgical Centre, Athens, GREECE, ²Haematology Department, "MITERA" Maternity and Surgical Centre, Athens, GREECE.

Over a period of 3.5 years, 406 patients (in the majority of the cases pregnant women) have been referred to our laboratory setting. All women were tested for the Factor V Leiden G1691A mutation, prothrombin G20210A mutation, and the MTHFR C677T polymorphism. We have categorised the patients into 9 groups to facilitate the statistical analysis, and compared them with a control group of 160 blood donors (FV Leiden mutation-allele frequency 2.5%, prothrombin mutation-allele frequency 2.2%, and MTHFR polymorphism -allele frequency 35.3%). Results are summarised in the table below. The chi squared statistical analysis (genotype frequencies) revealed statistical significance for the FV Leiden mutation in groups G (P<0.001), H (P<0.001), and I (P<0.01); for the prothrombin mutation in groups D (P=0.01), and H (P<0.001); and for the MTHFR polymorphism in groups B (P=0.01), H (P<0.05), and I (P<0.001). Our data suggest that pregnancy related thromboembolic complications (pulmonary embolism, deep vein thrombosis either prepartum or postpartum) are exacerbated by thrombophilic mutations such as FV Leiden and prothrombin, because pregnancy itself is an additional thrombotic factor for these women.


Patient group	N (number of patients)
A (1 miscarriage)	76
B (>2 miscarriages)	169
C (stillbirth)	18
D (pre-eclampsia)	16
E (increased resistance of uterine veins)	12
F (unsuccessful IVF attempts)	21
G (placental abruption)	4
H (thromboembolism)	24
I (>1 complications)	66
TOTAL	406

R. Gjergja¹, I. Barisic¹, O. Jadresin¹, K. Fumic²;
¹Children's University Hospital Zagreb, Zagreb, CROATIA, ²Clinical Hospital Center Rebro, Zagreb, CROATIA.

Menkes disease is an X-linked recessive disorder affecting the metabolism of copper. The Menkes gene product (MNK) is a transmembrane copper-transporting P-type ATP-ase considered to be the main efflux protein in human tissues. Although many patients have a severe clinical course characterised by progressive neurodegeneration, connective tissue disturbances, distinctive facial appearance, hair abnormalities, and poor outcome, variable allelic forms presenting as mild Menkes disease or occipital horn syndrome can be distinguished. Neuroimaging usually shows cortical cerebral and cerebellar atrophy as a result of progressive and extensive degeneration of grey matter, secondary demyelination, subdural accumulation of fluid, or multifocal areas of ischemic infraction. We report two infants with remarkable early diffuse white matter involvement on neuroimaging suggesting at first Krabbe disease. Diagnostic evaluation yielded low levels of serum copper and ceruloplasmin, high 64CU uptake in fibroblasts and DNA analysis ultimately confirmed the diagnosis of Menkes disease. It is concluded that Menkes disease should be considered in any male infant who presents with white matter changes, even in the absence of other distinctive features of the Menkes disease spectrum. As early diagnosis and treatment can significantly improve the outcome, Menkes disease should be included in the differential diagnosis of leukoencephalopathies.

E. Y. Voskoboeva, T. M. Boukina, A. M. Boukina, E. Y. Zakharova, V. S. Akhunov;
Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION.

A special programme for the diagnosis and prevention of inherited metabolic diseases was developed in Russia. During 20 years more than 600 patients with different types of lysosomal storage diseases (LSD) were diagnosed in our Department. Since 1993 year the DNA-diagnostic for some LSD has been started. The molecular and mutation analysis has been performed in a total of 37 MPS II patients, 8 MPS VI patients, 35 MPS I patients, 8 patients with a-mannosidosis, 42 patients with Gaucher disease, 8 patients with NCL type 2, 1 patient with NCL type 3, 1 patient with Tay-Sachs disease and 1 patient with Wolman disease. The results are shown on the Table. Based on the results obtained 4 prenatal diagnostic and 5 carrier detection have been performed.

Tabl.
Disease	Point mutations	Del\ins	Structural alterations	Affecting splicing	Frequent mutations	Novel mutations
Gaucher disease	56	1	-	-	N370S 36 alleles (42,9%), L444P 16 alleles (19%)	A384N
a-mannosidosis	16	-	-	-	R750W 16 alleles (100%)	-
MPSI	37	2	-	-	Q70X 26 alleles (37%)	Q63X, A75P, P533L, Y343X, W487R
MPSII	25	3	3	6	-	DelT72,delT305,A79E,Y54D,L102R, H159P, D198G,G224E,G340D,C432Y,D478Y,R443X, int1/ex2 a->g, ex4/int4 g->a, ex6/int6 g->a, complete IDS deletion, ex5-6 deletion.
MPSVI	14	2	-	-	R152W 6 alleles (37,5%)	L98P, del 7 bp 238-243, del T245, R152W Q160R,Q160X,R315X, L360P, Y513X
NCL type 2	13	-	-	-	R208X 12 alleles (69%)	R206H
NCL type 3	-	2	-	-	-	FsA349
Tay-Sachs disease	-	2	-	-	-	-
Wolman disease	1	1	-	-	-	-

E. Z. Lomonosova¹, A. M. Boukina¹, O. V. Shehter¹, F. K. Lagkueva², G. E. Rudenskaya¹, E. L. Dadaly¹;
¹Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION, ²Republic hospital for children, Alania, Vladikavkas, RUSSIAN FEDERATION.

X-linked adrenoleukodistrophy (X-ALD: McKusick 300100) is severe neurological disease associated with elevated levels of very long chain fatty acids (VLCFA) resulting from a deficiency in peroxisomal VLCFA b-oxidation. X-ALD gene has been mapped to Xq28. The gene encodes a peroxisomal membrane transporter protein of unknown function. More than 360 different mutations of the gene have been reported and most of them are unique.
Eleven patients were diagnosed by using VLCFA analysis. The mutation analysis has been performed in 8 Russian patients with childhood cerebral form X-ALD. 6 different mutations have been identified using such methods as PCR-SSCP analysis and direct nonradioactive sequencing. Three of them (R152L, Y296C, and S606L) have previously been reported. Three mutations detected were novels ones: fs164 (c494delG), L138del or L139del in exon 1 and D555N in exon 7 . In one family with two affected boys the mutation fs164 (c494delG) was found in both sibs. But the c494delG has not been detected in DNA sample extracted from leukocytes of the mother. We suggest that the mother has germline mosaicism. The same situation was previously described for other X-linked disease but not for X-ALD (J. G. Gleeson, Am. J. Hum. Genet. 67:574-581, 2000).

Expression and characterization of several glucocerebrosidase mutations causing Gaucher disease in Spanish patients.

M. Montfort¹, L. Vilageliu¹, A. Chabás², D. Grinberg¹;
¹Departament de Genètica, Universitat de Barcelona, Barcelona, SPAIN, ²Institut de Bioquímica Clínica, Corporació Sanitària, Barcelona, SPAIN.

Gaucher disease is a lysosomal storage disorder characterized by the accumulation of glucosylceramide. This accumulation is a consequence of the reduced activity of the lysosomal enzyme glucocerebrosidase. Several different mutations in the glucocerebrosidase gene were identified in the Spanish Gaucher disease patients, some of which were new. In order to characterize the resulting enzyme, these mutant alleles were expressed in an improved baculovirus system. This novel gene expression system allows rapid and efficient generation of recombinant baculovirus DNAs by site-specific transposition in E. Coli. In particular cDNAs corresponding to the following mutated alleles were studied: P182L, N188S, R257X, Y313H, E326K, N370S, P391L, N392I, I402T, D409H, L444P, N188S+E326K and E326K+L444P.
Expression of wild type cDNA results in an overexpression of acid b-glucosidase activity (mesured with 4MU-b-glucoside as substrate) of 6 to 10-fold the value of control fibroblasts. In the case of I402T, D409H, N370S, L444P, N188S+E326K and E326K+L444P mutations, expression studies revealed an activity lower than 30% of the wild-type values. Mutations N188S and E326K expressed separately show an activity greater than 30% of the wild-type values. In contrast, P182L, R257X, Y313H, P391L and N392I mutant alleles present no significant activities. Some kinetic properties were also analysed and western blots were carried out.

S. P. Lin¹, G. J. Lee-Chen², C. K. Chuang¹, W. L. Hwu³, M. T. Kuo¹, C. Yang⁴;
¹MacKay Memorial Hospital, Taipei, TAIWAN REPUBLIC OF CHINA, ²National Taiwan Normal University, Taipei, TAIWAN REPUBLIC OF CHINA, ³National Taiwan University Hospital, Taipei, TAIWAN REPUBLIC OF CHINA, ⁴Taipei Medical University Hospital, Taipei, TAIWAN REPUBLIC OF CHINA.

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder caused by deficiency of the lysosomal enzyme, a-L-iduronidase. It represents the prototype of all MPS. The spectrum of its phenotypes ranges from severe (IH) to intermediate (IH/S) to mild (IS). We have identified 9 MPS I patients, 6 males and 3 females with age from 5 to 37 years old, and 6 of them are IH/S and 3 are IH. The diagnosis is achieved by physical examination and 3 sequential laboratory tests, which are the quantitative detection of excessive excretion of glycosaminoglycans (GAGs) in the urine, urine GAG qualitative 2-D electrophoresis, and finally demonstration of the specific enzyme defect in leukocytes and/or cultured fibroblasts. Molecular investigation was also performed on 7 patients. And 11 mutations, Ml1 (G to A transition in the initiation codon ATG), A79V, Y343X, L346R, T364M, Q584X, R619G, 388-3c-->g, 1447del 27 and 1474ins15, were detected from 14 alleles. Mutation detection of the 2 newly diagnosed patients from one family is to be done later. Bone marrow transplantation has been suggested, but none of these patients received the treatment. The clinical findings of the 9 MPS I patients in detail will be presented in the meeting.

L. Feliubadaló¹, M. Arbonès¹, M. Palacín², V. Nunes¹;
¹Institut de Recerca Oncològica-IRO, L'Hospitalet de Llobregat, SPAIN, ²Universitat de Barcelona, Barcelona, SPAIN.

Cystinuria (OMIM 220100) is an autosomal recessive disease with an average prevalence of 1/7000. It is due to an impairment in the renal and intestinal absorption of cystine and dibasic amino acids. Mutations in SLC3A1 cause type I cystinuria, whereas mutations in SLC7A9 cause non-type I cystinuria.
Here we describe the identification of the murine ortholog of SLC7A9 and the generation of a knock out model for non-type I cystinuria.
The complete open reading frame of the mouse Slc7a9 was obtained with overlapping IMAGE clones. After screening a genomic library from the 129 murine strain, a clone with a15.7 Kb insert was identified, containing the first 11 exons (out of 13) of Slc7a9. Homologous recombination was performed in E14.1 ES cells (derived from 129 embryos) and two clones were isolated, which were heterozygous for a mutation in Slc7a9. The mutation substitutes exons 3 to 9 for the neomycin resistance gene. The resulting gene encodes a protein that is truncated before the first of the 12 predicted transmembrane segments. The recombinant cells where microinjected into C57BL/6 blastocysts, and several chimeric mice were obtained. They were mated to C57BL/6 mice, and the resulting heterozygotes were intercrossed to give a F2 with the expected mendelian genetic ratios. Mice homozygous for the mutation are fertile and do not present any apparent external problem at leastuntil their current age (11 weeks). The biochemical and morphological characterization of this mouse model is in progress.

Novel mutation of human OCTN2 carnitine transporter in a patient with severe ischaemic heart disease

J. Bene, K. Komlósi, V. Havasi, B. Melegh;
Dept. of Medical Genetics and Child Development, University of Pécs, Pécs, HUNGARY.

Novel mutation of human OCTN2 carnitine transporter in a patient with severe ischaemic heart disease
Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defect of the tissue carnitine transport. Impaired carnitine transport can be caused by mutations of the carnitine transporter gene SLC22A5 encoding the organic cation transporter (OCTN2), a Na⁺ dependent carnitine transporter. It has been already recognized that the OCTN2 mutations are associated with different phenotypic presentations, and differences can be observed even in patients with identical mutations. However, cardiac symptoms are always present in the affected individuals, and there are indications that certain mutations can also be associated with heart problems even in heterozygotes. In a female patient with moderate cardiomyopathy myocardial infarction developed at the age of 45, and due to the persisting coronary insufficiency, later an artery bypass grafting was necessary by open-heart surgery. At the age of 70 myocardial infarction developed again. Examination of the SLC22A5 gene revealed a C-T transition at the 15 np of the exon V (in a heterozygote form) which is associated with serine phenylalanine replacement at the amino acid position 280, which has not been reported elsewhere. This amino acid exchange means a replacement of an aminoacid with potential functional residue. Whether this mutation plays a role in or contributes to the pathology or can be at least regarded as a mutation which generates an additional susceptibility, remains to be elucidated.

J. Zschocke¹^,2, P. Guldberg³, R. A. Wevers⁴, R. Santer⁵, J. V. Leonard⁶, G. F. Hoffmann², D. Kohlmüller², E. Mayatepek²;
¹Inst. of Human Genetics, Univ. of Heidelberg, Heidelberg, GERMANY, ²Div. of Metabolic and Endocrine Diseases, Dept. of Pediatrics, Heidelberg, GERMANY, ³Dept. of Tumor Cell Biology, Inst. of Cancer Biology, Copenhagen, DENMARK, ⁴Lab. of Neurology, Univ. Hospital Nijmegen, Nijmegen, NETHERLANDS, ⁵Univ. Children’s Hospital, Kiel, GERMANY, ⁶Inst. of Child Health, London, GERMANY.

Flavin-containing mono-oxygenase 3 (FMO3) is required for N-oxidation and detoxification of many endogenous and exogenous compounds including biogenic amines and several common drugs. Individuals with severe FMO3 deficiency have a constant fish-like body odour (fish odour syndrome) due to insufficient N-oxidation of trimethylamine (trimethylaminuria), whilst mild deficiency is associated with transient or intermittent malodour. So far, seventeen mutations or amino acid variants of FMO3 have been identified. A common FMO3 gene allele [E158K,E308G] (homozygous in 4 % of the German population) was shown to be associated with reduced FMO3 function. We now report the result of more extensive biochemical and molecular studies, using a DGGE mutation scanning method, in individuals with constant or intermittent malodour. Malodour in two patients was related to carnitine treatment, a well-known but previously unexplained side effect. Nine novel mutations in the FMO3 gene were identified; all but one are missense mutations that may leave residual enzyme activity. Mild mutations were identified in combination with severe mutations, or in homozygous state, in patients with mild variants of trimethylaminuria as well as patients with fish-like malodour under carnitine treatment. In summary, mutations in the FMO3 gene cause a broad spectrum of phenotypes ranging from severe fish odour syndrome to mild enzyme deficiency that is common in the general population but only intermittently associated with malodour. In view of the important metabolic functions of FMO3, its mild deficiency may play a role as susceptibility factor in various pharmacological and other pathophysiological conditions.

Mitochondrial DNA mutations in Russian patients with different forms of mitochondrial diseases

E. Y. Zakharova, E. Y. Voskoboeva;
Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION.

During last 4 years 34 patients with mitochondrial diseases were investigated in our Department. Based on clinical data the patients were divided on four different groups: 10 patients with MELAS syndrome; 11 patients with KSS; 4 patients with MERRF syndrome; 8 patients with LHON.
Molecular-genetic analysis revealed that the patients with MELAS have A3243G mutations (n= 6), del 4977 (n=2) and other deletions mtDNA (n=2). In group of patients with MERRF syndrome mutations A8344G (n=1), del 4977 (n=2) and other deletions mtDNA(n=2 ) were found. In group of patients with LHON mutations G3460A (n=1), G11778A (n=6), T14484C (n=1) were found. Different size deletions of mtDNA were detected in patients with KSS (n=5), 6 patients with deletions detected had “common deletion” 4977bp. In two patients from this group the point mutation A3243G was also found.
The phenotypes of 8 patients did not correspond to the genotypes detected: 3 patients with MELAS and 3 patients with MERRF had different deletions, 2 patients with KSS had point mutation A3243G.

Determination of DL-pipecolic acid in body fluids by gas chromatography-mass spectrometry

J. Futas, R. Petrovic, J. Chandoga;
Centre of Medical Genetics, University Hospital, Bratislava, SLOVAKIA.

Background: Pipecolic acid (PIPA) is a metabolite of lysine degradative route. PIPA pathway accompanies the major saccharopine pathway of L-lysine degradation, being prominent only in brain, while it is obligate for D-lysine. Hyperpipecolatemia is found as an overflow effect in familial hyperlysinemia. PIPA accumulates in body fluids in some disorders of peroxisome biogenesis leading to deficient activity of L-pipecolic acid oxidase, too.
Objective: Simple quantification of PIPA in body fluids as a tool for biochemical diagnostics of inherited metabolic diseases.
Methods: Gas chromatography- mass spectrometry determination of PIPA as a N-ethoxycarbonyl ethyl ester prepared by reaction with ethylchloroformate in the presence of ethanol and pyridine. Reaction was performed in the matrix of deproteinized (acetonitrile-ethanol) biologic sample after extraction of neutral lipids (hexane) without preliminary isolation of amino acids. A capillary column with non-chiral phase was used.
Results: N-ethoxycarbonyl ethyl ester of PIPA was easily noticeable in the vacant area of chromatogram. Detection limit for L-pipecolic acid standard solution was 0,1 mmol.l^-1 under used conditions. Physiological values for PIPA concentration in serum and urine of patients without peroxisomal disorder were determined. In a male child (6 months) suspected for generalised peroxisomal disorder the serum PIPA level reached 101 mmol.l^-1, with urine PIPA level of 55,1 mmol/mol creatinine.
Conclusion: A simple and reliable method for determination of DL-pipecolic acid in body fluids is presented. It has the advantage of simultaneous analysis with other amino acids in one run and so it may be a component of a screening program.

A. Tabasi¹, F. Sharifpanah², D. Fathi³, M. Meftahpour³, A. Latifi-Sofi³, M. Houshmand²;
¹Farabi hospital, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²National Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC REPUBLIC OF), ³Shariati Hospital, Tehran, IRAN (ISLAMIC REPUBLIC OF).

We report herein on 9 Iranian patients with mitochondrial DNA (mtDNA) deletions, found among 11 patients with chronic progressive external ophthalmoplegia (CPEO). Of the 11 patients, who reffered to our lab, 6 had CPEO, one had Kearns-Sayre syndrome and 3 had IBM and one reffered for mitochondrial myopathy. The authors investigated DNA extraction from blood samples for occurrence of mtDNA deletions by multiplex PCR analyses. Four patients had multiple deletions. Five patients showed single deletions, 7 or 5 kb deletions; 3 of them had the same 'common deletion' of 4977 bp. There was no correlation between clinical severity, and the size of the mutated mtDNA, suggesting that there are still unknown factors influencing the disease phenotype.An etiologic association between the somatic multiple mtDNA deletions in CPEO and clinical manifestations other than the myopathy has so far not been demonstrated. For more investigation muscle biopsy took from patient's. Because of the mtDNA deletions reported mostly in the patient's muscles than blood samples, we are going to analyses muscle biopsy from patients by more sensetive methods such as southern blot and expand long PCR.

Statistical Evaluation of the Aminoacidopathies Diagnosed in the Laboratory of Human Genetics of Cluj County Hospital, Integrated in the Department of Cell and Molecular Biology of "Iuliu Hatieganu" University of Medicine and Pharmacy from Cluj-Napoca, Romania, Between 1981-2001

R. C. Vulturar¹, V. Pascu², E. Opris², L. Budisan², I. Benga³, G. Benga⁴;
¹Department of Cell and Molecular Biology, "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, ROMANIA, ²Laboratory of Human Genetics of Cluj County Hospital, Cluj-Napoca, ROMANIA, ³Neuro-Psychiatry Clinic of Cluj Children Hospital, Department of Paediatric Neurology, "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, ROMANIA, ⁴Department of Cell and Molecular Biology of "Iuliu Hatieganu" University of Medicine and Pharmacy, Laboratory of Human Genetics of Cluj County Hospital, Cluj-Napoca, ROMANIA.

The exact incidence of many inborn errors of metabolic diseases (IEMD) are unknown in many parts of the world. The normal pathways of amino acids could be affected by deficiences of enzymes with accumulation of toxic substances or by the deficiences in transport systems, because several specific transport systems ensure virtually complete (re-)absorbtion of amino acids in gut and kidney. These defects of transport systems are often detected only through elevation of the respective amino acids in urine with normal (or low) values in plasma. In the Laboratory of Human Genetics of Cluj County Hospital were performed plasma and/or urine amino acid analyses by two-dimensional thin layer chromatography procedure (Wadman & al. 1981) in children suspected for having perturbances of amino acid metabolism. Thus, a number of over 1800 samples from patients reffered by the Neuro- Psychiatry Clinic and other Paediatric Services from Cluj-Napoca were analysed between 1980-2001. Most of the samples (74.2%) were reffered by the Department of Paediatric Neurology of Cluj County Hospital. Out of the total samples, 56 cases were diagnosed as hyperphenylalaninemias/ phenylketonurias, one case of oculocerebrorenal Lowe syndrome, 4 cases of cystinurias, 28 cases of unspecified generalized hyperaminoacidurias. We also include a case of sarcosinemia, two cases of histidinuria without histidinemia, rare and still controversial genetic disorders concerning neurologic abnormalities determination. In our statistic over 26% of total samples had presented alteration of the normal pattern of amino acids chromatograms, without significance of aminoacidopathies; besides, 8.33% of total urinary samples shows excretion of beta-aminoisobutyric acid.

Carnitine Palmitoyltransferase 2 Deficiency: Attempt at Correlation between Genotype and Clinical presentation

L. Thuillier¹, H. Rostane², S. Gobin¹, F. Demaugre³, M. Brivet⁴, C. Prip-Buus⁵, J. M. Saudubray⁶, J. P. Bonnefont¹;
¹INSERM U393, Paris, FRANCE, ²Laboratoire de Biochimie, Hôpital Necker-Enfants Malades, Paris, FRANCE, ³INSERM U370, Paris, FRANCE, ⁴Service de Biochimie, CHU Bicètre, Le Kremlin Bicètre, FRANCE, ⁵CNRS UPR 1524, CHU Cochin, Paris, FRANCE, ⁶Département de Pédiatrie, Hôpital Necker-Enfants Malades, Paris, FRANCE.

Carnitine Palmitoyltransferase 2 (CPT2) deficiency, a common autosomal recessive disease of the mitochondrial long-chain fatty acid (LCFA) oxidation, may result in distinct clinical phenotypes, namely a mild adult muscular form, and a severe hepatocardiomuscular infantile/neonatal form. This phenotypic heterogeneity remains poorly understood. We therefore undertook the analysis of twenty CPT2-deficient patients ("adult form": n=13, "infantile form": n=7) on a molecular, enzymatic and functional point of view. Analysis of the CPT2 gene by DGGE and/or direct sequencing enabled us to detect thirteen mutations including five novel ones: 371G>A (R124Q), 437A>C (N146T), 481C>T (R161W), 983A>G (D328G), and 1823G>C (D608H). Taking into account our results and the literature data (33 mutations) it appears that: 1/the combinations of allelic mutations identified in the muscular form have never been detected in the hepatocardiomuscular one, 2/ in adult CPT2-deficient patients, at least one of the two mutations constantly lies in exons 1-3, while 3/ all "severe" missense mutations observed in the infantile form of the disease are constantly located in exons 4-5 of the CPT2 gene.
Moreover, it appears that the difference in severity between the adult and the infantile phenotypes is clearly related to a difference in their respective levels of mitochondrial LCFA oxidation, and in a less extent, to their residual CPT2 activity in lymphocytes and/or fibroblasts. Understanding the relationship between the type of CPT2 mutation, the residual CPT2 activity, and the resulting LCFA oxidation level remains the key point for unravelling the genotype-phenotype correlation.

Identification Of 14 Novel Mutations In The Slc3a1 Gene In Spanish Cystinuria Patients And Functional Analysis Of The Mutation L89p

M. Jiménez-Vidal¹^,2, M. Palacín², V. Nunes¹;
¹Institut de Recerca Oncològica, L'Hospitalet de Llobregat (Barcelona), SPAIN, ²Departament de Bioquímica i Biologia Molecular. Facultat de Biologia. Universitat de Barcelona, Barcelona, SPAIN.

Cystinuria is a heritable disorder of amino acid transport characterized by the defective transport of cystine and the dibasic amino acids through the brush border epithelial cells of the renal tubule and intestine tract. Initially, three types of cystinuria (I, II, and III) were described based on the urinary excretion of cystine and dibasic amino acids in obligate heterozygotes. The SLC3A1 gene -coding for a high subunit of the heteromeric amino acid transporters (HATs), rBAT- is responsible for type I cystinuria, whereas the SLC7A9 gene -coding for a light subunit of the HATs, b0,+AT- is involved in determining non type I (before types II and III) cystinuria.
We have screened the entire coding sequence and the intron/exon boundaries of the SLC3A1 gene in 50 spanish cystinuria patients by means of single strand conformation polymorphism (SSCP) and DNA sequencing.
We identified 14 new mutations in SLC3A1 that increases the number of mutated alleles so far characterized in this gene to 88, and accounts for 83% of the type I chromosomes studied.
Functional analysis of the mutation L89P -located at the unique putative transmembrane domain of rBAT and affecting a conserved amino acid residue- in Xenopus oocytes and after coexpression with b0,+AT in HeLa cells, revealed significant residual transport activity and impaired maturation and transport to the plasma membrane, suggesting a trafficking defect.

The clinical and genetic characteristics of congenital plasma dopamine beta-hydroxylase deficiency: a severe orthostatic syndrome

R. A. Wevers¹, J. Deinum², G. C. H. Steenbergen-Spanjers¹, J. Lenders¹^,2, M. Jansen¹, L. P. W. J. van den Heuvel¹, F. Boomsma²;
¹University Medical Center, Nijmegen, NETHERLANDS, ²Erasmus Medical Center, Rotterdam, NETHERLANDS.

Introduction: Dopamine ß-hydroxylase (DßH) catalyses the conversion of dopamine into norepinephrine. Plasma DßH deficiency has been associated with a severe congenital orthostatic syndrome with absent plasma (nor)epinephrine. Low plasma DßH, however, also occurs in 5% of normal subjects having normal (nor)epinephrine. The genetic basis of symptomatic DßH deficiency has never been investigated.
Methods: We sequenced the DßH gene in two unrelated patients with DßH deficiency and an orthostatic syndrome. We determined plasma DßH in 49 healthy blood donors to identify asymptomatic individuals with low plasma DßH activity to sequence their DßH gene.
Results: Two mutations uniquely associated with plasma DßH deficiency and absent plasma (nor)epinephrine were found in the patients with the orthostatic syndrome. One patient was homozygous for a splice site mutation (IVS1+2 T>C), and the other was compound heterozygote for this splice site variant and a deletion of base 575. In blood donors with low plasma DßH activity we found a mutation at -1021, immediately upstream of the transcription initiation site. Individuals homozygous for
-1021T had almost absent plasma DßH activity.
Conclusions: Our study is the first to describe pathogenic mutations in the DßH gene. It defines the genetic basis of DßH deficiency with absent plasma (nor)epinephrine leading to an orthostatic syndrome. It also explains the genetic background of asymptomatic DßH deficiency. The concurrence of homozygosity for the DßH -1021 T-allele with low plasma DßH in healthy individuals suggests that the -1021-locus determines secretion of DßH into the blood without interfering with normal catecholamine synthesis.

Genetic Screening for Simultaneous Diagnosis of 101 Inborn Errors of Metabolism (IEM) in Critical Neonates by Gas Chromography / Mass Spectrometry (GC/MS).

U. P. Dave¹, I. Matsumoto², C. Zhang²;
¹Centre for Research in Mental Retardation (CREMERE), Mumbai, INDIA, ²Matsumoto Institute of Life Science (MILS), Kanazawa, JAPAN.

Newborn screening, recommended by WHO, is a preventive public health strategy to decrease morbidity and mortality in infants. India lags behind in introducing such health programs due to different national health priorities, and genetic screening is also perceived as expensive and concerns rare disorders.
GC/MS was first applied in 1966 worldwide in the diagnosis of IEM because of its accuracy, sensitivity and power of analyzing multiple compounds simultaneously, but was introduced in India through the present study in 1998. Matsumoto's method of GC/MS analysis for 101 metabolic disorders of amino acids, organic acids, sugars, sugar alcohols, sugar acids, nucleic acids and nucleic acid bases was used. The air dried urine filter paper offered an easy mode of transport from small towns and rural areas having limited health and laboratory infrastructure.
We report 16.5% (93 of 565 referrals) metabolic abnormalities in children, while 29.7% (19 of 64) in high-risk neonates. The genetic factors in neonates were consanguinity (26%), death of earlier sibs (21%) and h/o mental retardation (5-8%). Convulsions (37%), respiratory distress (26%), lethargy (21%) and refusal to feed (21%) were the predominant clinical manifestations.
Genetic counseling to the family was done explaining the role of hereditary, consanguinity, recurrence risk, scope of prenatal diagnosis to prevent IEM in their families. India, being diverse in its socio-cultural, religious and racial background, the importance of GC/MS is indicated in establishing its genetic epidemiology, which is currently lacking. The few cases highlighting the application of biomedical mass spectrometry in genetic screening will be illustrated.

Determination of HLCS genomic structure and mutation identification in four patients with HLCS deficiency

S. Malvagia¹, A. Morrone¹, M. A. Donati¹, E. Pasquini¹, S. Funghini¹, A. Boneh², H. Peters², F. Ciani¹, E. Zammarchi¹;
¹Dpt of Pediatrics, University of Florence, Florence, ITALY, ²Murdoch-Childrens Research Institute, Melbourne, AUSTRALIA.

Holocarboxylase synthetase (HLCS) deficiency is a rare AR biotin metabolism disorder caused by absent or reduced HLCS enzymatic activity. This enzyme catalyses the biotinylation of the four human biotin dependent carboxylases. Its deficiency leads to biotin-responsive multiple carboxylase deficiency (MCD).
The human HLCS gene has been mapped to chromosome 21q22.1. Up to now, sequencing analyses in the HLCS gene have been performed on its 2466 bp cDNA, making the confirmation of the genetic lesions at the genomic level difficult. We report on the map of HLCS genomic structure, deduced from the DNA sequence of human chromosome 21 published by the gene card web sites http://hgp.gsc.riken.go.jp; http://bioinfo.weizmann.ac.il/cards-bin/carddisp?HLCS. All Exon/Intron boundaries were tested using genomic primer. The putative deduced 9 exons, encompassing the coding region, were confirmed and PCR conditions for amplification of all genomic fragments were set up. These data confirm that the HLCS gene contains 9 exons plus two additional exons upstream of the ATG codon.
The newly available HLCS genomic characterization allowed us to identify of 4 known and 2 new genetic lesions in four patients with HLCS deficiency. The known G581S mutation was reconfirmed with new intronic primers since an undetected intron was contained in the fragment amplified by published exonic primers which were wrongly used for enzymatic analysis at genomic level.

Mutation detection of Galactose-1 phosphate uridyltransferase(GALT) gene in Iranian Galactosemia patients.

N. Naghibzadeh Tabatabai¹, F. Mirzajani², S. Zare Karizi¹, S. Ghandili², M. Falahian³, J. Jalali⁴, M. Nakhaie⁴, M. Houshmand²;
¹Azad University, Tehran, IRAN (ISLAMIC REPUBLIC OF), ²National Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC REPUBLIC OF), ³Tehran Medical University, Tehran, IRAN (ISLAMIC REPUBLIC OF), ⁴Iran Medical University, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Galactosemia is a clinically sever and heterogenous autosomal recessiv disorder caused by deficiency of galactose-1-phosphate uridyltransferase (GALT)activity.The numerous point mutations have been identified in the GALT gene and the prevalence of the mutations in some ethnic groups have been reported indicating the differences in the frequency of the mutations amongst patients belonging to the different ethnic groups. Eleven unrelated Galactosemia families ( 27 DNA
samples ) were tested for the presence or absence of the two common mutations of GALT gene i.e. Q188R and K285N mutant alleles, using PCR-RFLP protocol. The frequency of the mutated alleles were found to be 36% and 9 % respectively. More study is being under taken on one patient with confirmed Galactosemia who is heterozygote for K285N mutation and most probably demonstrating a case of compound heterozygote.
More investigation will be carried out on Iranian patients with biochemically confirmed galactosemia, concerning detemination of eight common alleles
including Q188R, K285N, S135L, L195P, Y209S, F171S, Q169K and X380R alleles in the collected samples and the frequency of the mutations in Iranian galactosemia patients will be reported.

E. Malonova¹, A. M. Robreau², J. Zeman¹, H. Puy², V. Silva², R. Rosipal¹, J. C. Deybach², P. Martasek¹;
¹Center of Integrated Genomics, Department of Pediatrics, Ist Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, ²Centre Francais des Porphyries, INSERM U490, Hopital Louis Mourier, Colombes, FRANCE.

Porphyria variegata (PV) is an autosomal dominant acute hepatic porphyria caused by a partial defect in heme synthetic enzyme protoporphyrinogen oxidase (PPO). PV is clinically characterized by acute attacks of neurovisceral crises and/or dermatologic manifestation. The clinically manifest patients show typical plasma fluorescence at 626 nm, have increased fecal porphyrins and, during acute attacks, have increased porphyrin precursors in urine. The activity of PPO in heterozygous patients is about 50%. The human PPO cDNA and gene has been cloned and mapped to chromosome 1q22-23. The gene spans about 5 kb, contains 13 exons, the cDNA encodes a protein of 477 amino acids.
We have studied 6 unrelated Czech families with clinically and biochemically diagnosed PV. Four new mutations were detected: one deletion (1393 del 8 bp), two missense mutations (W227G, C459Y) and one splicing mutation (IVS 6+1 G®A). Although the relationship is not known, the deletion 1393 del 8 bp was identified in two of these families. In the last family the already described deletion 1177 del G was found. The pathological significance of missense mutations was determined by their expression in prokaryotic system. Both these missense mutations resulted in the absence of PPO activity in E. coli which is in good agreement with an expected 50% activity in the heterozygous state. Availability of screening for all PV families will ensure correct diagnoses in all gene carriers and prevents life-threatening porphyric attacks. (Supported by Charles University grant GAUK 5/200/c and LN00A079 from MŠMT of Czech Republic)

S. Bernabini¹, B. Minuti¹, E. Pelo¹, F. Martinelli², W. Borsini³, M. R. Scordo³, A. Sodi⁴, R. Mignani⁵, M. L. Battini⁶, A. Sessa⁷, F. Torricelli¹;
¹Cytogenetic and Genetic Unit, Careggi Hospital, Florence, ITALY, ²Nephrology and Transplant Unit, Careggi Hospital, Florence, ITALY, ³Departiment of Neurosciences, University of Florence, Florence, ITALY, ⁴Departiment of Ophtalmology, University of Florence, Florence, ITALY, ⁵Nephrology and Dialysis Unit, Infermi Hospital, Rimini, ITALY, ⁶Departiment of Dermatology, University of Florence, Florence, ITALY, ⁷Nephrology Unit, Vimercate, Vimercate, ITALY.

Fabry disease is an X-linked recessive disorder of glycolipid metabolism resulting from a deficiency of the lysosomial enzyme a -galactosidaseA(GLA). The reduced enzyme activity leads to the progressive accumulation of glycosphingolipids throughout the body, particularly in: skin, kidney, nervous system, eye, heart. The a-galactosidaseA gene is located at Xq22 and consists of seven exons; more than 150 mutations have been identified in unrelated Fabry subjects. Our work was conducted to detect mutations in ten unrelated Italian families. All the subjects have been estimated clinical to renal, cutaneous, cardiac and neurological level; in all it has been executed the dosage on blood of a -GLA-A. Up until today, we identified seven affected males and eleven female carriers. In two families the study is still in progress, in one family we identified no mutations. All of the exons are small enough to allow amplification by the polymerase chain reaction (PCR) and investigation of sequence changes by Conformation Sensitive Gel Electrophoresis (CSGE) analysis and sequencing. We have detected 9 mutations: 7 single base substitutions (6 missense and 1 nonsense), 1 small deletion (delCT ex7) and one splice site alteration (IVS4-16 G ->A).The missense mutations identified are G35R,R112C,R227Q,R301P and D313Y.In a family it has been characterized in all the males affected and the females carriers the mutation missense in exon 6 (R301P) and one mutation of splicing in intron 4 (IVS4).This study confirm the elevated heterogeneity of the mutations in a-GLA-A gene in patients with Anderson-Fabry disease that accompanies to one extreme clinical variability.

Molecular analysis of three Czech patients with 3-hydroxy-3-methylglutaryl CoA lyase deficiency

L. Mrázová¹, E. Pospíšilová², J. Hrdá²;
¹Charles University 1st Faculty of Medicine, Prague, CZECH REPUBLIC, ²General Faculty Hospital, Prague, CZECH REPUBLIC.

Mitochondrial 3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the last step of leucine catabolism and the ketogenic pathway. Deficiency of HL caused by mutations in HL gene leads to 3-hydroxy-3-methylglutaric aciduria (HA) an autosomal recessive metabolic disorder. Three unrelated patients with HA were diagnosed in our Institute. The diagnosis was confirmed by measurement of enzyme activity HL in lymphocytes and/or fibroblasts at all three patients and their family members.
To confirm the diagnosis at molecular level we have directly sequenced PCR or RT/PCR products amplified from probands gDNA or cDNA. In two cases we have found known homozygous missense point mutations 698A→G (H233R) and 122G→A (R41Q), respectively. In the last case, 3-years old girl, we have found heterozygous mutations H233R/del41G. Del41G (Pro9fs(-1)) is a novel one base deletion in exon1, which leads to a frameshift and premature stop codon after 32 aminoacids. Subsequently we have set up PCR-RFLP assays for mutations H233R and del41G to verify results and to determine mutations in family members. The results of molecular analysis correlate with measured enzyme activity.

DNA analysis of the phenylalanine hydroxylase gene in Puerto Rican phenylketonuria patients

A. Sanchez-Valle¹, D. Pizarro¹, R. Valle¹, J. Y. Renta¹, M. González², C. L. Cadilla¹;
¹University of Puerto Rico School of Medicine Dept. of Biochemistry, San Juan, PR, ²Dept. of Pediatrics, San Juan, PR.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. The PAH gene harbours many mutations (~ 400 variant alleles), which cause various degrees of phenylalanine hydroxylase deficiency. Missense mutations, represent ~ 60% of the mutations so far recorded. The Puerto Rico Newborn Screening Program has found that the incidence of PKU in the island of Puerto Rico (PR) is 1:18,000 on average. The most frequent hyperphenylalanemia in PR is classic PKU. In the present study we screened 15 classical PKU Puerto Rican patients for mutations by PCR amplification of all exons followed by DGGE and/or SSCP analysis and direct DNA sequencing. We found several polymorphic sites in exons 4, 7, 11, and 12 and a novel frameshift mutation in exon 4. The novel mutation was found in one patient with a homozygous insertion of an A nucleotide at codon 120. Two patients were homozygous for the E280K mutation and another patient was homozygous for the R176X mutation.. The E280K mutation has been found in high frequency among Cuban PKU patients, whereas the R176X mutation was found previously in Southern Europe PKU patients. Some patients carried missense mutations in addition to another homozygous mutation in the PAH gene. NIH-NIGMS grant R25-GM61838 and RCMI grants G12RR03051 and 1P20RR11126 from NIH-NCRR supported this study.