A sequence variation in the promoter of the placental alkaline phosphatase gene (ALPP) is associated with allele-specific expression in human term placenta.

Placental alkaline phosphatase (PLAP), encoded by the ALPP gene, is produced by the fetal side of the placenta. This enzyme displays strong genetic variability. Some of the variants were reported to be associated with pathology of pregnancy. We show here that the two most common ALPP allelic variants, Pl(1) and Pl(2), differ in mRNA expression level. This differential expression was independent of the parental origin and probably results from linkage disequilibrium with the sequence variation rs2014683G>A in the ALPP gene promoter that was shown to have allele-specific binding patterns to placental nuclear proteins. The possible role of this allelic-specific expression in placenta-related pathology is discussed.

A case of lethal hypophosphatasia providing new insights into the perinatal benign form of hypophosphatasia and expression of the ALPL gene.

Hypophosphatasia is a rare inherited bone disease caused by mutations in the alkaline phosphatase liver-type gene (ALPL) gene, with extensive allelic heterogeneity leading to a range of clinical phenotypes. We report here a patient who died from severe lethal hypophosphatasia, who was compound heterozygous for the mutation c.1133A>T (D361V) and the newly detected missense mutation c791A>G, and whose parents were both healthy. Because the c.1133A>T (D361V) mutation was previously reported to have a dominant-negative effect and to be responsible for the uncommon perinatal benign form of the disease, we studied the expression of the ALPL gene in this family. Analysis at the messenger RNA (mRNA) level, both quantitative and qualitative, showed that the paternal c.1133A>T (D361V) mutation was associated with over-expression of the ALPL gene and that the maternal c.791A>G mutation lead to complete skipping of exon 7. The results provide an explanation of the lethal phenotype in the patient where the two ALPL alleles are non-functional and in the asymptomatic father where over-expression of the normal allele could counteract the effect of the c.1133A>T (D361V) mutation by providing an increased level of normal mRNA. This may also explain the variable expression of hypophosphatasia observed in parents of patients with the perinatal benign form.

Clinical and experimental evaluation of a new chromogenic medium (OCCA, Oxoid) for direct identification of Candida albicans, C. tropicalis and C. krusei.

Oxoid Chromogenic Candida Agar (OCCA) is a new commercial ready-to-use medium that contains chromogenic substrates for rapid detection and specific identification of Candida albicans, C. tropicalis and C. krusei. We evaluated the performance of this medium with 364 clinical specimens and 31 subcultures, and examined its ability to support the growth of small inocula of six reference strains. CHROMagar Candida was used as the reference medium. OCCA permitted the growth of most important yeasts, and readily discriminated among Candida spp. in clinical specimens, including mixed cultures.

Evolution of the snow area index of the subarctic snowpack in central Alaska over a whole season. consequences for the air to snow transfer of pollutants.

The detailed physical characteristics of the subarctic snowpack must be known to quantify the exchange of adsorbed pollutants between the atmosphere and the snow cover. For the first time, the combined evolutions of specific surface area (SSA), snow stratigraphy, temperature, and density were monitored throughout winter in central Alaska. We define the snow area index (SAI) as the vertically integrated surface area of snow crystals, and this variable is used to quantify pollutants' adsorption. Intense metamorphism generated by strong temperature gradients formed a thick depth hoar layer with low SSA (90 cm(2) g-1) and density (200 kg m(-3)), resulting in a low SAI. After snowpack buildup in autumn, the winter SAI remained around 1000 m(2)/m(2) of ground, much lower than the SAI of the Arctic snowpack, 2500 m(2) m-(2). With the example of PCBs 28 and 180, we calculate that the subarctic snowpack is a smaller reservoir of adsorbed pollutants than the Arctic snowpack and less efficiently transfers adsorbed pollutants from the atmosphere to ecosystems. The difference is greater for the more volatile PCB 28. With climate change, snowpack structure will be modified, and the snowpack's ability to transfer adsorbed pollutants from the atmosphere to ecosystems may be reduced, especially for the more volatile pollutants.

Childhood hypophosphatasia due to a de novo missense mutation in the tissue-nonspecific alkaline phosphatase gene.

Hypophosphatasia is an inherited disorder due to mutations in the bone alkaline phosphatase (ALPL) gene. We report here a patient with childhood hypophosphatasia diagnosed at 1.4 yr because of pectus excavatum, large anterior fontanel, rachitic skeletal changes, and low serum alkaline phosphatase. Sequencing of the ALPL gene produced evidence of two distinct missense mutations, E174K (c.571G>A), of maternal origin, and a de novo mutation, M45I (c.186G>C). The study of various microsatellite polymorphisms ruled out false paternity and therefore confirmed that M45I occurred de novo in the paternal germline or in the early development of the patient. Site-directed mutagenesis showed that M45I results in the absence of in vitro alkaline phosphatase activity, suggesting that the mutation is a severe allele. In conclusion, childhood hypophosphatasia in this patient is the result of compound heterozygosity for the moderate mutation E174K and a novel severe de novo mutation M45I.

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.

A molecular approach to dominance in hypophosphatasia.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. The disease is highly variable in its clinical expression, because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was found, despite exhaustive sequencing of the gene, suggesting that missing mutations are harbored in intron or regulatory sequences or that the disease is dominantly transmitted. The distinction between these two situations is of importance, especially in terms of genetic counseling, but dominance is sometimes difficult to conclusively determine by using familial analysis since expression of the disease may be highly variable, with parents of even severely affected children showing no or extremely mild symptoms of the disease. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, I473F) found in patients with no other detectable mutation. Three of these mutations, G46 V, S164L, and I473F, have not previously been described. Pedigree and/or serum alkaline phosphatase data suggested possible dominant transmission in families with A99T, R167 W, and G232 V. By means of site-directed mutagenesis, transfections in COS-1 cells, and three-dimensional (3D) modeling, we evaluated the possible dominant effect of these eight mutations. The results showed that four of these mutations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effect by inhibiting the enzymatic activity of the heterodimer, whereas the four others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of the disease. Analysis of the 3D model of the enzyme showed that mutations exhibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular biological function such as dimerization, tetramerization, or membrane anchoring.

Twelve novel mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL) in patients with various forms of hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report here the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 11 families affected by various forms of hypophosphatasia. Nineteen distinct mutations were found, 7 of which were previously reported. Eleven of the 12 new mutations were missense mutations (Y11C, A34V, R54H, R135H, N194D, G203V, E218G, D277Y, F310G, A382S, V406A), the last one (998-1G>T) was a mutation affecting acceptor splice site.

Specific surface area of snow samples determined by CH4 adsorption at 77 K and estimated by optical microscopy and scanning electron microscopy.

Snow is a divided medium that can adsorb atmospheric trace gases. Evaluating the impact of the snow cover on atmospheric chemistry therefore requires the knowledge of the specific surface area (SSA) of snow. This paper compares the results of three methods used to measure or estimate the SSA of four snow samples: CH4 adsorption at 77 K, optical microscopy (OM), and scanning electron microscopy (SEM, used only on two samples). Within error bars, CH4 adsorption and OM yield similar results on three of the four snow samples. Values for the 4th sample are within a factor of 2. For both samples where CH4 adsorption, OM, and SEM are used, all three methods yield similar results, but CH4 adsorption always has a better accuracy and a much better precision. Thus, despite its ease of use, estimates from OM images are often not accurate enough to monitor the evolution of snow SSA. The main sources of error in the OM method are the difficulty to determine snow crystal thicknesses and to take into account the topography of the snow crystal surface. The combination of CH4 adsorption and OM or SEM can provide useful information on the evolution of both the SSA and the shape of snow crystals. This will be useful to evaluate the respective contributions of adsorption/desorption and sublimation/condensation processes to the impact of the snow cover on atmospheric chemistry.

Parental origin of the extra chromosome in prenatally diagnosed fetal trisomy 21.

Trisomy 21 (Down syndrome) is one of the most common chromosomal abnormalities. Of cases of free trisomy 21 causing Down syndrome, about 95% result from nondisjunction during meiosis, and about 5% are due to mitotic errors in somatic cells. Previous studies using DNA polymorphisms of chromosome 21 showed that paternal origin of trisomy 21 occurred in only 6.7% of cases. However, these studies were conducted in liveborn trisomy 21-affected infants, and the possible impact of fetal death was not taken into account. Using nine distinct DNA polymorphisms, we tested 110 families with a prenatally diagnosed trisomy 21 fetus. Of the 102 informative cases, parental origin was maternal in 91 cases (89.2%) and paternal in 11 (10.8%). This percentage differs significantly from the 7.0% observed in previous studies (P<0.001). In order to test the influence of genomic parental imprinting, we determined the origin of the extra chromosome 21 in relation to different factors: advanced maternal age, maternal serum human chorionic gonadotropin (hormone of placental origin), severity of the disease, gestational age at diagnosis and fetal gender. We found that the increased frequency of paternal origin of nondisjunction in trisomy 21-affected fetuses cannot obviously be explained by factors leading to selective loss of paternal origin fetuses.

Fifteen new mutations (-195C>T, L-12X, 298-2A>G, T117N, A159T, R229S, 997+2T>A, E274X, A331T, H364R, D389G, 1256delC, R433H, N461I, C472S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and liver/bone/kidney-type alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 12 families affected by severe or mild hypophosphatasia. Twenty distinct mutations were found, 5 of which were previously reported. Nine of the 15 new mutations were missense mutations (T117N, A159T, R229S, A331T, H364R, D389G, R433H, N461I, and C472S). The others were 2 nonsense mutations (L-12X and E274X), one single nucleotide deletion (1256delC), 2 mutations affecting splicing (298-2A>G, 997+2T>A), and a mutation in the major transcription start site (-195C>T). Hum Mutat 15:293, 2000.

Correlation of alkaline phosphatase (ALP) determination and analysis of the tissue non-specific ALP gene in prenatal diagnosis of severe hypophosphatasia.

Prenatal diagnosis of severe hypophosphatasia by mutation analysis of the tissue non-specific alkaline phosphatase (TNSALP) gene is reliable and mostly informative. However, alkaline phosphatase (ALP) assay of CVS may be a useful complementary and independent method, especially when a mutation is unidentified and DNA from the index case is unavailable, rendering impossible the use of DNA polymorphisms as genetic markers of the disease. We report here mutation analysis of the TNSALP gene and ALP assay in nine cases of prenatal diagnosis of severe hypophosphatasia. The results showed a good correlation between ALP assay and DNA analysis in all but one case, which suggested that in at least some cases low values of ALP may correspond to affected fetuses as well as to heterozygotes.

Correlations of genotype and phenotype in hypophosphatasia.

Hypophosphatasia, a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical expression. The disease is due to various mutations in the tissue-non-specific alkaline phosphatase ( TNSALP ) gene. We report here the use of clinical data, site-directed mutagenesis and computer-assisted modelling to propose a classification of 32 TNSALP gene mutations found in 23 European patients, 17 affected with lethal hypophosphatasia and six with non-lethal hypophosphatasia. Transfection studies of the missense mutations found in non-lethal hypophosphatasia showed that six of them allowed significant residual in vitro enzymatic activity, suggesting that these mutations corresponded to moderate alleles. Each of the six patients with non-lethal hypophosphatasia carried at least one of these alleles. The three-dimensional model study showed that moderate mutations were not found in the active site, and that most of the severe missense mutations were localized in crucial domains such as the active site, the vicinity of the active site and homodimer interface. Some mutations appeared to be organized in clusters on the surface of the molecule that may represent possible candidates for regions interacting with the C-terminal end involved in glycosylphosphatidylinositol (GPI) attachment or with other dimers to form tetramers. Finally, our results show a good correlation between clinical forms of the disease, mutagenesis experiments and the three-dimensional structure study, and allowed us to clearly distinguish moderate alleles from severe alleles. They also confirm that the extremely high phenotypic heterogeneity observed in patients with hypophosphatasia was due mainly to variable residual enzymatic activities allowed by missense mutations found in the human TNSALP gene.

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).

Identification of fifteen novel mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in European patients with severe hypophosphatasia.

Hypophosphatasia is an inherited disorder characterised by defective bone mineralisation and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterisation of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 13 European families affected by perinatal, infantile or childhood hypophosphatasia. Eighteen distinct mutations were found, only three of which had been reported previously in North American and Japanese populations. Most of the 15 new mutations were missense mutations, but we also found two mutations affecting donor splice sites and a nonsense mutation. A missense mutation in the last codon of the putative signal peptide probably affects the final maturation of the protein. Despite extensive sequencing of the gene and its promotor region, only one mutation was identified in two cases, one of which was compatible with a possible dominant effect of certain mutations and the putative role of polymorphisms of the TNSALP gene. In 12 of the 13 tested families, genetic diagnosis was possible by characterisation of the mutations or by use of polymorphisms as genetic markers. Hypophosphatasia diagnosis was assigned in two families where clinical, laboratory and radiographic data were unclear and prenatal diagnosis was performed in one case. The results also show that severe hypophosphatasia is due to a very large spectrum of mutations in European populations with no prevalent mutation and that genetic diagnosis of the disease must be performed by extensive analysis of the gene.

Analysis of germline variation at the FMR1 CGG repeat shows variation in the normal-premutated borderline range.

In order to characterize the dynamics of CGG repeat instability at the fragile X syndrome locus (FMR1 gene), we have used small pool PCR to estimate the mutation rate within germline (sperm) and somatic tissue (leukocytes) of two normal males, one carrying the most common 29 CGG repeats allele, the other carrying a borderline normal-premutated allele of 55 repeats. Large contractions and moderate expansions of the repeat were found in sperm and blood for the 55 repeat allele while almost no variation was found in sperm or blood with the 29 repeat allele. Somatic blood DNA exhibited fewer expansions and contractions than sperm. Contractions were more frequent than expansions, and all the expansions were found in the +4 to +10 repeats range, while most of the contractions were found in the -10 to -30 range, suggesting that a subset of contractions results from a distinct mechanism. These results also suggest that the dynamics of the CGG repeat could be partly due to germline instability within the high normal or premutated ranges.

Recurrent and unexpected segregation of the FMR1 CGG repeat in a family with fragile X syndrome.

Fragile X syndrome, the most common cause of hereditary mental retardation, results from amplification of a CGG trinucleotide repeat in the FMR1 gene. The transmission of the CGG repeat from premutated individuals to their premutated descendants is usually unstable, showing an increase in the size of the repeat. We report here a family which exhibits recurrent and unexpected transmission of the maternal premutation to three daughters. The first daughter exhibited mosaicism with two premutated alleles, one contracted and the other expanded. The second daughter showed a reversion from the maternal premutation to the normal range, and the third carried an expanded premutated allele associated with an expanded paternal allele within the normal range. These variations in the size of the CGG repeat may result from many different mechanisms such as DNA polymerase slippage on the leading or lagging strand during replication, large contractions of repeats on the parental strand during replication, or recombination through unequal crossover between sister chromatids. Our results suggest that the variation of the CGG premutated alleles in this family may be the result of intrinsic instability associated with a trans-acting factor such as a mismatch repair gene product.

Transition from normal to premutated alleles in fragile X syndrome results from a multistep process.

In fragile X syndrome, the most common cause of inherited mental retardation, phenotypic expression has been linked to a region containing a repetitive sequence, (CGG)n, that appears to lengthen dramatically in fragile X patients and to show length variation in normal individuals. In order to investigate possible mechanisms responsible for further expansion of CGG in the normal population, we selected 31 normal unrelated X chromosomes carrying either the high-risk DX204-AC155 or DX196-AC151 haplotypes, as defined by the flanking microsatellites, DXS548 and FRAXAC2. Nearly 100% of CGGs with more than 35 repeats were found on DX204-AC155 haplotypes, with a mean length significantly higher and much more variable than in normal individuals carrying other haplotypes including the high-risk haplotype DX196-AC151. These findings suggest that the transition from the normal to the abnormal range occurs by a multistep process, a primary event, such as unequal crossing-over, leading to increased size and moderate instability of the repeat, and from which DNA polymerase slippage could lead to recurrent premutations. Our results also suggest that the upper limit of the normal range is roughly 35 repeats in the fragile X gene. The 36-54 repeats range would define an intermediate allele only observed, up to now, in DX204-AC155 fragile X chromosomes.