Mutation profiles of phenylketonuria in Quebec populations: evidence of stratification and novel mutations.

Independent phenylketonuria (PKU) chromosomes (n = 109) representing 80% of a proband cohort in Quebec province carry 18 different identified mutations in 20 different mutation/haplotype combinations. The study reported here, the third in a series on Quebec populations, was done in the Montreal region and predominantly on French Canadians. It has identified three novel mutations (A309D, D338Y, and 1054/1055delG[352fs]) and one unusual mutation/RFLP haplotype combination (E280K on Hp 2). The relative frequencies and distribution of PKU mutations were then compared in three regions and population subsets (eastern Quebec, French Canadian; western Quebec, French Canadian; and Montreal, non-French Canadian). The distributions of the prevalent and rare mutations are nonrandom and provide evidence for genetic stratification. The latter and the presence of eight unusual mutation/haplotype combinations in Quebec families with European ancestries (the aforementioned four and M1V, I65T, S349P, and R408W on Hp 1) corroborate demographic and anthropologic evidence, from elsewhere, for different origins of French Canadians in eastern and western Quebec.     

Carrier screening for phenylketonuria: comparison of two discriminant analysis procedures

Absence of a convenient, direct enzyme assay for detecting phenylketonuria (PKU) heterozygotes has resulted in continued effort to develop an accurate and reliable procedure to discriminate the heterozygous individual from the homozygous normal. Our study compares two statistical procedures that combine the semifasting plasma phenylalanine and tyrosine concentrations with the individuals' prior probability of being a heterozygous carrier in order to discriminate carriers from noncarriers. The results of this comparison indicate that the quadratic discriminant function is superior to the linear discriminant function as a method of carrier testing both in theory and in practice. An interactive computer system is described that facilitates the clinical utilization of the quadratic discriminant function.     

Nitrogen immobilization in leaf litter at two Mediterranean ecosystems of SW Spain

Nitrogen immobilization in relation to the dynamics of lignin and tannins in nine different types of leaf litter was investigated during a 2-yr study at two Mediterranean ecosystems of SW Spain. Net nitrogen immobilization for all the species was higher in a forest than in the more nutrient-poor soil of a shrubland. Absolute amount of lignin increased in both ecosystems in the first 2–4 months whereas tannin rapidly decreased in the same time period. Increases in lignin were significantly correlated to losses of tannins during decomposition. Initial tannin content was the best predictor of the maximum amount of immobilized nitrogen in litter in both ecosystems. Mechanisms that could explain the immobilization of nitrogen in litter are discussed.     

Climax epiphytic communities in Mediterranean Spain

The Antitrichetalia curtipendulae epiphytic climax communities of Šmarda & Hadàč from the Spanish Mediterranean area have been classified by numerical and traditional floristic methods. Ninety-eight phytosociological relevés have been recorded from a number of Iberian forests. Our results indicate two clusters that can be easily related to two different alliances: Lobarion pulmonariae (Nephrometum resupinati) and Pterogonio gracile-Antitrichion californicae (Antitrichielum californicae). A phytogeographical and syntaxonomical discussion is followed by a suggested Red List of threatened lichens and bryophytes occurring in these communities in the Spanish Mediterranean area with the aim of advancing conservation of endangered and vulnerable species.     

The identification of two mis-sense mutations at the PAH gene locus in a Turkish patient with phenylketonuria

Summary DNA sequence analysis of the 13 exons and intron/exon boundaries of the phenylalanine hydroxylase (PAH) gene has detected two base transitions, resulting in mis-sense mutations, in the genomic DNA of a Turkish patient (E1) with phenylketonuria (PKU). The Leu⁴⁸Ser amino acid substitution was associated with the mutant haplotype 3 allele and the Glu²²¹Gly amino acid substitution with the mutant haplotype 4 allele of this family. Allele-specific oligonucleotide (ASO) dotblot analysis subsequently detected the Leu⁴⁸Ser mutation in the haplotype 4 PKU alleles of nine (18.8%) of the 48 unrelated Caucasian PKU families investigated. This mutation results in mild PKU in the homozygous state. The Glu²²¹Gly mutation has only been detected within patient E1 and his father.     

Predation upon reptiles in Mediterranean habitats of Chile,Spain and California: A comparative analysis

Summary Pradation on reptiles at three Mediterranean-type habitat sites was assessed by computing the incidence of reptiles as a percentage of vertebrates in the diet of each predator species and the incidence of each reptile species as prey for the entire assemblage of predators at each locality. The overall importance of reptiles is lowest in Chile, intermediate in California, and highest in Spain. These differences do not appear to result from interlocality variation in the size distributions of predators or of prey. The incidence of particular reptile species as prey is correlated with their relative abundances in Spain and California, but not in Chile. Behavioral and morphological attributes evidently make some species more vulnerable to predation and others less so than their abundances would predict. Predation on the speciose lizard genus Liolaemus in Chile is sufficient to promote behavioral responses but not major morphological divergence.     

Mutation analysis of genes related to methylmalonic acidemia: identification of eight novel mutations

Molecular Biology Reports - Methylmalonic acidemia (MMA), an inherited metabolic disease, results from genetic defects in methylmalonyl-CoA mutase or any of the proteins involved in...     

Compound heterozygosity in nonphenylketonuria hyperphenylalanemia: the contribution of mutations for classical phenylketonuria

Hyperphenylalaninemia (HPA) results from defective hydroxylation of phenylalanine in the liver, in most cases because of defective phenylalanine hydroxylase. HPA is highly variable, ranging from moderate elevation of plasma phenylalanine with no clinical consequences to a severe disease, classical phenylketonuria (PKU). Non-PKU HPA was found in excess of PKU in Israel, while the opposite is true in Europe. To study the genetic basis of non-PKU HPA, we performed haplotype analysis at the phenylalanine hydroxylase locus in 27 families with non-PKU HPA. All individuals with this condition were compound heterozygotes. In six of these families, in which both PKU and non-PKU HPA were segregating, haplotype analysis showed that non-PKU HPA resulted from compound heterozygosity for a PKU mutation and a second mutation, with milder effect, which is probably expressed only when it interacts with the severe mutation. The involvement of PKU mutations in non-PKU HPA was further demonstrated in Jewish Yemenite families with non-PKU HPA, in which the individuals with this condition were carriers of the single PKU allele which exists in this community. In addition, two previously known PKU point mutations (R261Q and R408W) were found in individuals with non-PKU HPA. These mutations are associated, in our population, with the same haplotypes as those with which it is associated in Europe. Based on the above-mentioned genetic model for non-PKU HPA, successful prenatal diagnosis of this condition was performed in one family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenylketonuria in Spain: RFLP haplotypes and linked mutations

In order to investigate the molecular basis of phenylketonuria (PKU) in Spain, we analyzed the restriction fragment length polymorphism (RFLP) haplotypes and common mutations in the phenylalanine hydroxylase (PAH) gene in 32 unrelated Spanish PKU families. The distribution of RFLP haplotypes differs from that of northern Europe. Mutant haplotypes 2 and 3 were completely absent in our sample. Approximately 65% of the mutant alleles are confined to three RFLP haplotypes, namely haplotypes 1, 6 and 9, also frequently found in other Mediterranean populations. We screened for previously described PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides, and found IVS10,165T, E280K and P281L as the major mutations, representing 41% of the PKU alleles. Other mutations found were Y414C, and a new one, P244L. Mutations R408W and IVS12, prevalent in northern Europe, as well as others present in southern European populations (R252W, R261Q, L249F) were not detected in our sample. Our results reveal the genetic heterogeneity present in the Spanish PKU population, which shows similarities to others of Mediterranean origin.     

Dairy goat grazing systems in Mediterranean regions: A comparative analysis in Spain, France and Italy

In order to maintain small ruminant grazing systems it is necessary to know precisely how they work and what actions can be taken to improve them. The objective of this paper was to characterize, classify, analyse and propose improvements for the dairy goat grazing systems in three countries from the western Mediterranean area: Spain, France and Italy. A multivariate analysis was conducted with 21 indicators obtained from the data collected from 45 farms. As a result of the multivariate analysis, 82.5% of variance was explained by two principal components. The first component included proportion of cultivated pasture area and cultivated pasture area per goat. The second included goats present and forage supply per goat. After conducting a cluster analysis based on these two principal components, farms were classified into four groups. Group 1 was made up of French and Italian farms, which had a smaller territorial base but a high cultivated pasture area per goat, contributing to greater self-sufficiency in feed. However the concentrate per goat was too high in relation to milk production, which was medium. The difference between milk income and feed cost is also medium. Group 2 was basically made up of Italian farms. The farms in this group had the most extensive management systems, with a low use of inputs. However, the milk production was low, meaning that the difference between milk income and feed cost was also low. Group 3 was mainly made up of Spanish farms. On these farms the concentrate supply was excessive and the forage supply was low. The milk production per goat was medium and the difference between milk income and feed cost was small. Group 4 was made up mainly of French farms. The farms of this group had a moderate supply of concentrate although the forage supply was very high. They had a high milk production, which lead to a large difference between milk income and feed cost per goat.The main weaknesses observed are related to feeding management, particularly grazing, and to the goat productivity. In the former case research on the nutritional utilization of rangelands and pastures and correct feed supplementation is to be encouraged. In the latter, the production capacity of the goats present in these systems should be improved, without forgetting the balance between hardiness and general productivity.     

Missense mutations prevalent in Orientals with phenylketonuria: molecular characterization and clinical implications

Two missense mutations in the phenylalanine hydroxylase (PAH) genes of Orientals with phenylketonuria (PKU) have been identified. A G-to-A transition in exon 7 of the gene results in the substitution of Gln243 for Arg243 (R243Q) and accounts for 18% of all PKU chromosomes among Chinese. An A-to-G transition in exon 6 of the gene results in the substitution of Cys204 for Tyr204 (Y204C) and identifies about 13 and 5% of all PKU chromosomes in the Chinese and Japanese populations, respectively. The R243Q construct produced less than 10% of normal PAH activity in in vitro expression analysis in a eukaryotic cell system, and patients homozygous for this substitution exhibit a severe clinical phenotype. These results are consistent with previous findings in this expression system. The Y204C construct, however, produced near normal levels of PAH enzyme activity and immunoreactivity in this in vitro expression system. Because this substitution is present only on PKU chromosomes, it is a valuable marker for identifying the corresponding mutant allele for carrier screening of PKU. With the characterization of these two substitutions, about 60% of PKU alleles in China can now be identified. The continuing search for additional PKU mutations will permit effective carrier screening and prenatal gene diagnosis of PKU in East Asia.     

Planktonic chlorophyll a and eutrophication in two Mediterranean littoral systems (Mallorca Island,Spain)

Two Mediterranean littoral systems of Mallorca Island have been studied using planktonic chlorophyll a (total concentration and seasonal variations) as trophic indicator. The studied systems are quite different in geomorphology, physicochemical trends and anthropic incidence. S'Albufera-Alcudia Bay is the drainage outlet of a coastal marsh, with a salt wedge estuarine regime. Andratx Harbour is a narrow bay affected by a high anthropic incidence due to the presence of a small fishing port and mainly a large leisure harbour. Both systems are affected by a typical Mediterranean climate and a negligible tide incidence. The present work is based on several year monitoring of physical, chemical and biological water parameters. In S'Albufera-Alcudia Bay, Chl a concentration ranged from 0.01 to 17.10 mg m^–3, the values dropping from the inland site to the sea. The phytoplankton biomass gradient takes its origin in physical and chemical differences. The variance is mainly related to the inland-sea water axis. Nitrogen and silica enriched inland waters are the main source of eutrophication, hence their relation with Chl a. These nutrients entering the Albufera are mainly derived from leaching from farmed soils. The transition from an inland eutrophic system to an oligotrophic marine one show the filter character of these coastal marshes. In Andratx Harbour, Chl a ranged from a non detectable concentration to 6.36 mg m^–3. The ultraplankton (<5 m) was the most important contributor to total concentration. The ultraplankton in this system is related to influence of more oligotrophic open seawater. A greater proportion of microplankton (>20 m) is related both to higher nutrient concentrations and a higher water column stability. In Andratx Harbour, the enclosure state seems very important for development of phytoplankton blooms.     

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.     

Epidemiology of Mediterranean theileriosis in Extremadura region, Spain

Extremadura is an extensive region (41,000 km2) located in the Southwest of Spain, where animal husbandry is a very important economical resource. Livestock parasitism of ticks is common in the region and it is favoured by the environmental conditions and management systems. Mediterranean theileriosis (MT) is perhaps the most important tick-borne disease of cattle. Although Theileria annulata has been identified in Extremadura since 1933, little epidemiological information is available. During the last decade, we have conducted different studies with the aim to clarify the epidemiological situation. Laboratory diagnosis, identification of tick species and studies on their seasonal dynamics have been carried out. Moreover, T. annulata was isolated and cultivated and experimental transmission of the parasite was made. Finally, several seroepidemiological tests in livestock from different origins have been performed. From 1991 to 1995 a total of 236 of MT clinical cases have been diagnosed. The following tick species were identified: Hyalomma marginatum marginatum (64.0%), Rhipicephalus bursa (25.6%), Hyalomma lusitanicum (9.6%) and Rhipicephalus sanguineus (0.5%). Clinical cases are detected from May until November with peaks in May-June and October that correspond to the periods of maximum activity of adults of H. m. marginatum and H. lusitanicum, respectively. From our results of seroepidemiological studies the Extremadura region could be considered as endemic for MT. The disease is unstable in semi-extensive systems (dairy cows) and tends to stabilisation on extensive farms for meat and fighting bulls.     

Aeropalynological and phenological study in two different Mediterranean olive areas: Cordoba (Spain) and Perugia (Italy)

ABSTRACT

An aerobiological and phenological investigation on the olive tree was carried out during three years in two areas: Cordoba (Spain) and Perugia (Italy). In these countries, this species is economically important and those areas were chosen because of the long series of aeropalynological data (1982–1998) available, obtained by means of identical volumetric pollen traps. The aim of this study was to use phenological observations to prove the real contribution to the pollen curves in different cultivated areas. Results show that in Cordoba province (302.152 ha) the pollen curve is characterised by different peaks because of the pollination of different cultivated crops. In some cases, these crops are located far from the pollen trap (50 km) but pollen is transported thanks to favourable winds during the flowering period. In Perugia (750 ha) the pollen curve is characterised by only one peak; it is very concentrated because of the proximity of the investigated crops. The objective of this research was to obtain information on this species in order to elaborate statistical models aimed at forecasting the potential fruit production based on the amount of pollen released into the atmosphere.     

Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene

Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II–III) predominated in males (p < 0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1–SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN–SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.     

Molecular genetics of phenylketonuria in Mediterranean countries: a mutation associated with partial phenylalanine hydroxylase deficiency.

We report the characterization of a mutation in the phenylalanine hydroxylase (PAH) gene associated with partial residual activity of the enzyme. This point mutation (280glu----lys) was found by sequencing a mutant cDNA clone derived from a needle biopsy of the liver in a child with variant form of phenylketonuria. There is a strict concordance between homozygosity for the mutation and this particular phenotype. The (280glu----lys) mutation is linked to an original and rare RFLP haplotype at the PAH locus found in south Europe and North Africa. So far, this genotype-haplotype association is both inclusive and exclusive. Thirty-three PAH-deficient patients were screened for the mutation by using polymerase chain-reaction amplification of their genomic DNA extracted from Guthrie cards. Since a large number of patients can be screened for a particular mutation by using Guthrie cards, the possibility arises of using these samples collected by national newborn screening centers for prospective and retrospective detection of other mutations in the human genome.