Field validation of experimental challenge models for IPN vaccines.

Atlantic salmon S1/2 pre-smolts from the VESO Vikan hatchery were assigned to study groups, i.p. immunized with commercially available, multivalent oil-adjuvanted vaccines with (Norvax Compact 6 - NC-6) or without (Norvax Compact 4 - NC-4) recombinant infectious pancreatic necrosis virus (IPNV) antigen. A control group received saline solution. When ready for sea, the fish were transported to the VESO Vikan experimental laboratory, where two identical tanks were stocked with 75 fish per group before being transferred to 10 degrees C sea water and exposed by bath to first passage IPNV grown in CHSE-214 cells. The third tank containing 40 fish from each group was challenged by the introduction of 116 fish that had received an i.p injection of IPNV-challenge material. The remaining vaccinated fish were transported to the VESO Vikan marine field trial site and placed in two identical pens, each containing approximately 53 000 fish from the NC-6 group and 9000 fish from the NC-4 group. In the experimental bath challenge trial, the cumulative mortality was 75% and 78% in the control groups, and the relative percentage survival (RPS) of the NC-6-immunized fish vs. the reference vaccine groups was 60% and 82%, respectively. In the cohabitation challenge, the control mortality reached 74% and the IPNV-specific vaccine RPS was 72%. In both models, the reference vaccine lacking IPNV antigen gave a moderate but statistically significant non-specific protection. In the field, a natural outbreak of infectious pancreatic necrosis (IPN) occurred after 7 weeks lasting for approximately 3.5 months before problems due to winter ulcers became dominating. During this outbreak, mortality in the NC-4 groups were 33.5% and 31.6%, respectively, whereas mortality in the NC-6 groups were 6.9% and 5.3%, respectively, amounting to 81% IPNV-specific protection. In conclusion, the IPN protection estimates obtained by experimental challenges were consistent between tanks, and were confirmed by the field results.

Low frequency of Parkin, Tyrosine Hydroxylase, and GTP Cyclohydrolase I gene mutations in a Danish population of early-onset Parkinson's Disease.

Autosomal recessive Parkinson's disease (PD) with early-onset may be caused by mutations in the parkin gene (PARK2). We have ascertained 87 Danish patients with an early-onset form of PD (age at onset < or =40 years, or < or =50 years if family history is positive) in a multicenter study in order to determine the frequency of PARK2 mutations. Analysis of the GTP cyclohydrolase I gene (GCH1) and the tyrosine hydroxylase gene (TH), mutated in dopa-responsive dystonia and juvenile PD, have also been included. Ten different PARK2 mutations were identified in 10 patients. Two of the patients (2.3%) were found to have homozygous or compound heterozygous mutations, and eight of the patients (9.2%) were found to be heterozygous. A mutation has been identified in 10.4% of the sporadic cases and in 15.0% of cases with a positive family history of PD. One patient was found to be heterozygous for both a PARK2 mutation and a missense mutation (A6T) in TH of unknown significance. It cannot be excluded that both mutations contribute to the phenotype. No other putative disease causing TH or GCH1 mutations were found. In conclusion, homozygous, or compound heterozygous PARK2 mutations, and mutations in GCH1 and TH, are rare even in a population of PD patients with early-onset of the disease.

Expanded motor and psychiatric phenotype in autosomal dominant Segawa syndrome due to GTP cyclohydrolase deficiency.

BACKGROUND: Segawa syndrome due to GTP cyclohydrolase deficiency is an autosomal dominant disorder with variable expression, that is clinically characterised by l-dopa responsive, diurnally fluctuating dystonia and parkinsonian symptoms. OBJECTIVE: To delineate the neurological and psychiatric phenotype in all affected individuals of three extended families. METHODS: GTP cyclohydrolase deficiency was documented by biochemical analyses, enzymatic measurements in fibroblasts, and molecular investigations. All affected individuals were examined neurologically, and psychiatric data were systematically reviewed. RESULTS: Eighteen affected patients from three families with proven GTP cyclohydrolase deficiency were identified. Eight patients presenting at less than 20 years of age had typical motor symptoms of dystonia with diurnal variation. Five family members had late-presenting mild dopa-responsive symptoms of rigidity, frequent falls, and tendonitis. Among mutation carriers older than 20 years of age, major depressive disorder, often recurrent, and obsessive-compulsive disorder were strikingly more frequent than observed in the general population. Patients responded well to medication increasing serotonergic neurotransmission and to l-dopa substitution. Sleep disorders including difficulty in sleep onset and maintenance, excessive sleepiness, and frequent disturbing nightmares were present in 55% of patients. CONCLUSION: Physicians should be aware of this expanded phenotype in affected members of families with GTP cyclohydrolase deficiency.

Evaluation of a fetus at risk for dihydropteridine reductase deficiency by direct mutation analysis using denaturing gradient gel electrophoresis.

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH(4)), which is an obligate co-factor of the aromatic amino acid hydroxylases. DHPR deficiency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR-deficient patients are diagnosed by a lack of response to a low phenylalanine diet and by severe neurological symptoms. Final diagnosis is made by measurements of neurotransmitters and pterin metabolites in cerebrospinal fluid (CSF) and urine, in addition to DHPR enzyme activity, which can be assessed in whole red blood cells. Treatment of DHPR deficiency can be difficult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in affected families. Prenatal diagnosis is possible by measuring DHPR activity in different cell types but this is time consuming. More than 25 different mutations have to date been identified in the QDPR gene and direct identification of a mutation in a fetus would be easy and rapid. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and flanking intronic sequences of the QDPR gene. We describe the first prenatal diagnosis conducted using this method.

Treatable neurotransmitter deficiency in mild phenylketonuria.

The authors describe a case of neurologic involvement in mild hyperphenylalaninemia (HPA), not due to tetrahydrobiopterin (BH(4)) deficiency, with low levels of monoamine neurotransmitter metabolites in CSF. The combined BH(4)-Phe loading test suggested a BH(4) response, confirmed by clinical improvement after BH(4) therapy. Molecular study revealed a compound heterozygosity of the phenylalanine hydroxylase alleles: a mild HPA-associated mutation (T380M) and the new mutation D151E. This case demonstrates that even mild HPA, generally considered a benign disorder, may present neurologic impairment.

A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics.

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU). We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis. Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein. Detailed kinetic analyses of PAH expressed in E. coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein. Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000. This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency. Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin.

Molecular analysis of 16 Turkish families with DHPR deficiency using denaturing gradient gel electrophoresis (DGGE).

Dihydropteridine reductase (DHPR) catalyses the conversion of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4), which serves as the obligatory cofactor for the aromatic amino acid hydroxylases. DHPR deficiency, caused by mutations in the QDPR gene, results in hyperphenylalaninemia and deficiency of various neurotransmitters in the central nervous system, with severe neurological symptoms as a consequence. We have studied, at the clinical and molecular levels, 17 patients belonging to 16 Turkish families with DHPR deficiency. The patients were detected at neonatal screening for hyperphenylalaninemia or upon the development of neurological symptoms. To identify the disease causing molecular defects, we developed a sensitive screening method that rapidly scans the entire open reading frame and all splice sites of the QDPR gene. This method combines PCR amplification and "GC-clamping" of each of the seven exonic regions of QDPR, resolution of mutations by denaturing gradient gel electrophoresis (DGGE), and identification of mutations by direct sequence analysis. A total of ten different mutations were identified, of which three are known (G23D, Y150C, R221X) and the remaining are novel (G17R, G18D, W35fs, Q66R, W90X, S97fs and G149R). Six of these mutations are missense variants, two are nonsense mutations, and two are frameshift mutations. All patients had homoallelic genotypes, which allowed the establishment of genotype-phenotype associations. Our findings suggest that DGGE is a fast and efficient method for detection of mutations in the QDPR gene, which may be useful for confirmatory DNA-based diagnosis, genetic counselling and prenatal diagnosis in DHPR deficiency.

Single-step mutation scanning of the 6-pyruvoyltetrahydropterin synthase gene in patients with hyperphenylalaninemia.

BACKGROUND: Deficiency of 6-pyruvoyltetrahydropterin synthase (PTPS) is a recessively inherited disorder that leads to depletion of 5,6,7, 8-tetrahydrobiopterin, the obligatory cofactor for hydroxylation of phenylalanine, tyrosine, and tryptophan. A marker for neonatal detection of PTPS deficiency is hyperphenylalaninemia (HPA). Molecular analysis would provide a simple and reliable means for distinguishing PTPS deficiency from other potential causes of HPA. METHODS: We developed a method based on PCR in combination with denaturing gradient gel electrophoresis (DGGE) that rapidly scans the six coding sequences and all splice sites of the PTPS gene (PTS) for mutations. This method was used to examine the status of the PTS gene in control samples with known PTS mutations and in five patients with PTPS deficiency. RESULTS: Two features of the PTS gene posed particular problems in relation to DGGE analysis: the very high GC content of exon 1, and a 15-bp poly(dT) stretch in the acceptor splice site of intron 1. Both problems were solved by special design of amplification primers. PCR and DGGE conditions were adjusted to allow simultaneous analysis of all six regions of the PTS gene. Using this one-step approach, all control mutations were readily resolved. Among the five PTPS patients, four mutations were identified, including IVS1-3C-->G, IVS2-7T-->A, V57del, and V97M (289G-->A). The IVS1-3C-->G mutation was shown by reverse transcription-PCR analysis to produce multiple splice variants. CONCLUSIONS: We have established a fast and reliable screening method for detection of mutations and small deletions/insertions in the PTS gene. This method should be useful for rapid diagnosis of PTPS deficiency in newborns with HPA.

Relationship among genotype, biochemical phenotype, and cognitive performance in females with phenylalanine hydroxylase deficiency: report from the Maternal Phenylketonuria Collaborative Study.

OBJECTIVE: To examine the relationship of phenylalanine hydroxylase (PAH) genotypes to biochemical phenotype and cognitive development in maternal phenylketonuria (PKU). METHODOLOGY: PAH gene mutations were examined in 222 hyperphenylalaninemic females enrolled in the Maternal PKU Collaborative Study (MPKUCS). A total of 84 different mutations were detected, and complete genotype was obtained in 199 individuals. Based on previous knowledge about mutation-phenotype associations, 78 of the mutations could be assigned to one of four classes of severity (severe PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia [MHP]). Then, 189 MPKUCS subjects were grouped according to the various combinations of mutation classifications. The sample sizes were large enough for statistical testing in four groups with at least one mutation that completely abolishes enzyme activity. These patients are considered functionally hemizygous. RESULTS: The biochemical phenotype predicted from the genotype in functionally hemizygous patients was related significantly to the assigned phenylalanine level. Cognitive performance (IQ) was also significantly related to genotype. The IQ of PAH-deficient mothers with a severe PKU mutation in combination with a MHP mutation or a mild PKU mutation was 99 and 96, respectively, whereas the IQ of PKU mothers with two severe PKU mutations or with one severe and one moderate PKU mutation was 83 and 84, respectively. Of the patients with PKU, 92% had been treated during childhood. Those who were untreated or treated late had lower than average IQ scores for their group of mutation combinations. Females with moderate or mild PKU who were treated early and treated for >6 years showed IQ scores 10 points above average for their group. CONCLUSIONS: The reproductive outcome in maternal phenylketonuria is dependent on prenatal metabolic control and postnatal environmental circumstances. Both factors depend on the intellectual resources of the mother with PKU. The significant relationship among genotype, biochemical phenotype, and cognitive performance observed in the present study is of importance for the development of an optimal strategy for future treatment of females with PKU who plan pregnancy.

Long-term beneficial effects of the phenylalanine-restricted diet in late-diagnosed individuals with phenylketonuria.

The potential benefits to society of treating late-diagnosed mentally retarded persons with phenylketonuria were investigated. In order to ascertain the effects of late dietary intervention, the charts of 124 adults with PKU seen in the metabolic service at the Childrens Hospital of Los Angeles were reviewed. Fifty-nine were diagnosed later than 3 months of age and were over the age of 18 years. They were followed up with medical, psychological, and nutritional assessments. Genotyping was also performed. Twenty-eight have remained on a phenylalanine-restricted diet during the intervening years. All but 3 of the 28 late-diagnosed PKU persons who remained on a restricted diet showed significant intellectual improvement. Seven are able to attend college, 9 are employed, and 12 are attending workshops and/or day care programs. The result of treatment with the phenylalanine-restricted diet was that these individuals could participate in society and were able to arrest the neurodegenerative course characteristic of persons with mutations classified as severe in the phenylalanine hydroxylase gene. We conclude that society could benefit substantially by providing a phenylalanine-restricted diet for late-diagnosed mentally retarded persons with phenylketonuria. Eighteen of 28 such persons who otherwise would have required residential care are living independently.

Necator americanus (Nematoda: Ancylostomatidae) from Africa and Malaysia have different ITS-2 rDNA sequences.

The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. americanus, or that N. americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.

Characterization of Oesophagostomum bifurcum and Necator americanus by PCR-RFLP of rDNA.

Esophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana, where Necator americanus (human hookworm) also exists at high prevalence. Yet, very little is known about the transmission patterns of O. bifurcum, which is in part due to the difficulties in diagnosis and in differentiating some life-cycle stages of O. bifurcum from N. americanus using morphological features. As a first step toward developing a molecular-diagnostic assay, it was evaluated whether ribosomal (r)DNA could provide genetic markers for the identification of O. bifurcum and N. americanus to species. Internal transcribed spacer rDNA (plus flanking and intervening sequences) was analyzed by polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) using several restriction endonucleases. The analysis showed that there was no detectable intraspecific difference in the size of the PCR products among multiple samples, that there was a consistent size difference in the products (of 110 bp or 350 bp, depending on region amplified) between the species, and that there was no significant variation in restriction patterns within each species. These results indicate that the rDNA spanning the internal transcribed spacers provides useful genetic markers for the identification of O. bifurcum and N. americanus to species, which has important implications for developing PCR-based tools to study the epidemiology and population biology of O. bifurcum.

Differentiation of Oesophagostomum bifurcum from Necator americanus by PCR using genetic markers in spacer ribosomal DNA.

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population biology of O. bifurcum.