Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell.

We identified multiple paternity in 52.9% of the clutches of Hermann's tortoise Testudo hermanni boettgeri using polymorphic microsatellite markers. In addition we demonstrated sperm storage across seasons. DNA was extracted from the amniotic fluid adhering to the eggshell's inner surface, a procedure suitable for easy, non-invasive DNA sampling in conservation and breeding programs. To improve the informative value of monomorphic single tandem repeat (STR) markers we additionally analyzed single nucleotide polymorphism (SNP) variability.

Diagnostic difficulties in glucokinase hyperinsulinism.

Glucokinase hyperinsulinism is a rare variant of congenital hyperinsulinism caused by activating mutations in the glucokinase gene and has been reported so far to be a result of overactivity of glucokinase within the pancreatic beta-cell. Here we report on a new patient with difficulties to diagnose persistent hyperinsulinism and discuss diagnostic procedures of this as well as the other reported individuals. After neonatal hypoglycemia, the patient was reevaluated at the age of 3 years for developmental delay. Morning glucose after overnight fast was 2.5-3.6 mmol/l. Fasting tests revealed supressed insulin secretion at the end of fasting (1.4-14.5 pmol/l). In addition, diagnostic data of the patients reported so far were reviewed. A novel heterozygous missense mutation in exon 10 c.1354G>C (p.Val452Leu) was found and functional studies confirmed the activating mutation. There was no single consistent diagnostic criterion found for our patient and glucokinase hyperinsulinism individuals in general. Often at the time of hypoglycemia low insulin levels were found. Therefore insulin concentrations at hypoglycemia, or during fasting test as well as reactive hypoglycemia after an oral glucose tolerance test were not conclusive for all patients. A glucose lowering effect in extra-pancreatic tissues independent from hyperinsulinism that results in diagnostic difficulties may contribute to underestimation of glucokinase hyperinsulinism. Mutational analysis of the GCK-gene should be performed in all individuals with unclear episodes of hypoglycemia even without documented hyperinsulinism during hypoglycemia. Delay of diagnosis might result in mental handicap of the affected individuals.

Isolation and characterization of microsatellite marker loci in the greylag goose (Anser anser).

Ten novel polymorphic microsatellite loci were isolated and characterized from the greylag goose, Anser anser, a long-term monogamous and biparental bird. Additionally, five new primers pairs were designed based on previously published microsatellite locus sequences from closely related species. Multiplex polymerase chain reactions conditions were optimized for all 15 primer pairs. The number of alleles ranged from two to 12 per locus with an observed heterozygosity ranging from 0.07 to 0.85. This marker set will be used to determine rates and origins of extra-pair and parasitic young in a population of individually banded greylag geese with known life histories.

Chromosomal anchoring of linkage groups and identification of wing size QTL using markers and FISH probes derived from microdissected chromosomes in Nasonia (Pteromalidae: Hymenoptera).

Nasonia vitripennis is a small parasitic hymenopteran with a 50-year history of genetic work including linkage mapping with mutant and molecular markers. For the first time we are now able to anchor linkage groups to specific chromosomes. Two linkage maps based on a hybrid cross (N. vitripennis x N. longicornis) were constructed using STS, RAPD and microsatellite markers, where 17 of the linked STS markers were developed from single microdissected banded chromosomes. Based on these microdissections we anchored all linkage groups to the five chromosomes of N. vitripennis. We also verified the chromosomal specificity of the microdissection through in situ hybridization and linkage analyses. This information and technique will allow us in the future to locate genes or QTL detected in different mapping populations efficiently and fast on homologous chromosomes or even chromosomal regions. To test this approach we asked whether QTL responsible for the wing size in two different hybrid crosses (N. vitripennis x N. longicornis and N. vitripennis x N.giraulti) map to the same location. One QTL with a major effect was found to map to the centromere region of chromosome 3 in both crosses. This could indicate that indeed the same gene/s is involved in the reduction of wing in N. vitripennis and N. longicornis.

Carnitine palmitoyltransferase II deficiency: molecular and biochemical analysis of 32 patients.

The authors investigated 32 patients with the muscle form of CPT II deficiency. Total carnitine palmitoyltransferase enzyme system (CPT) activity was normal but abnormally inhibited by malonyl-CoA, palmitoyl-CoA, and the detergents Triton X and Tween 20. Mutation analysis identified three described mutations (S113L, P50H, and F448L) and two novel mutations (M214T and Y479F). Using modeling techniques, a structure could be identified anchoring the protein in the membrane. Only one of the five mutations (Y479F) is located within this region.

Molecular pathology of haemophilia A in Turkish patients: identification of 36 independent mutations.

Haemophilia A is an X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene. In an attempt to reveal the molecular pathology of Turkish haemophilia A patients, the coding sequence of the gene, excluding a large portion of exon 14, was amplified from genomic DNA and subjected to denaturing gradient gel electrophoresis prior to DNA sequencing. Fifty-nine haemophilia A patients were included in the study with severe, moderate and mild phenotypes observed in 24, 15 and 16 patients, respectively. Factor VIII activity and clinical phenotypes were not available for four patients. A total of 36 independent mutations were found, with a mutation detection efficacy of 61%. The mutations that were reported for the first time include 20 point mutations, one 8-bp insertion (TCAAGATA) in exon 4 and one large deletion greater than 2.8 kb involving exon 14. The novel point mutations were composed of three nonsense (Ser681Ter, Cys2021Ter and Gln2113Ter), one splicing error (IVS-1G-->A), 15 missense mutations (Lys48Asn; Leu-98Phe; Thr118Ala; Cys248Tyr; Glu456Lys; Asp560Ala; Tyr664Cys; Phe679Leu; Gly691Trp; Asp1769His; Val1857Leu; Gly2026Gln; Arg2163Pro; Asp2288Ala; and Arg2304Leu) and a T deletion in exon 25 that caused a frameshift followed by a stop codon. All missense mutations except Val1857Leu, which maintained a conserved nonpolar R group, occurred at amino acids conserved among four species and were most probably pathogenic. In addition, two sequence changes (IVS3-9C-->T) and (Leu2230Leu) were also detected in patients carrying Val1857Leu and Phe679Leu missense mutations, respectively. Identification of mutation origins in eight sporadic cases revealed an equal sex ratio of mutations.

Somatic mosaicism in hemophilia A: a fairly common event.

Mutations in the large gene of clotting factor VIII (FVIII) are the most common events leading to severe human bleeding disorder. The high proportion of de novo mutations observed in this gene raises the possibility that a significant proportion of such mutations does not derive from a single germ cell but instead should be attributed to a germline or somatic mosaic originating from a mutation during early embryogenesis. The present study explores this hypothesis by using allele-specific PCR to analyze 61 families that included members who had sporadic severe hemophilia A and known FVIII gene defects. The presence of somatic mosaicisms of varying degrees (0.2%-25%) could be shown in 8 (13%) of the 61 families and has been confirmed by a mutation-enrichment procedure. All mosaics were found in families with point mutations (8 [25%] of 32 families). In the subgroup of 8 families with CpG transitions, the percentage with mosaicism increased to 50% (4 of 8 families). In contrast, no mosaics were observed in 13 families with small deletions/insertions or in 16 families with intron 22 inversions. Our data suggest that mosaicism may represent a fairly common event in hemophilia A. As a consequence, risk assessment in genetic counseling should include consideration of the possibility of somatic mosaicism in families with apparently de novo mutations, especially families with the subtype of point mutations.

De novo factor VIII gene intron 22 inversion in a female carrier presents as a somatic mosaicism.

The intron 22 inversion represents the most prevalent factor VIII gene defect in severe hemophilia A, accounting for about 40% of all mutations. It is hypothesized that the inversion mutations occur almost exclusively in germ cells during meiotic cell division by intrachromosomal recombination between 1 of 2 telomeric copies of the Int22h region and its intragenic homologue. The majority of inversion mutations originate in male germ cells, where the lack of bivalent formation may facilitate flipping of the telomeric end of the single X chromosome. This is the first intron 22 inversion that presents as a somatic mosaicism in a female, affecting only about 50% of lymphocyte and fibroblast cells of the proposita. Supposing a post-zygotic de novo mutation as the usual cause of somatic mosaicism, the finding would imply that the intron 22 inversion mutation is not restricted to meiotic cell divisions but can also occur during mitotic cell divisions, either in germ cell precursors or in somatic cells. (Blood. 2000;96:2905-2906)

Methylation levels at selected CpG sites in the factor VIII and FGFR3 genes, in mature female and male germ cells: implications for male-driven evolution.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.

Comparison between intratracheal and intravenous administration of liposome-DNA complexes for cystic fibrosis lung gene therapy.

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

[Maternal transmission or bovine spongiform encephalopathy in the case of "Cindy" disproved]. / Maternale Transmission der bovinen spongiformen Encephalopathie im Fall "Cindy" widerlegt.

On December 27th, 1996 a Galloway cattle named "Cindy" died of bovine spongiform encephalopathy (BSE) in Höxter. So far all cases of BSE reported in Germany have been imported from the UK. However, the identity and origin of "Cindy" was not clear. DNA sequence analysis of the mitochondrial D-loop region and DNA typing of micro-satellite sites finally revealed that "Cindy" was imported from the UK as well.

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type-dependent sex ratio of mutation frequencies.

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients--all exclusively from sporadic families--were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9 percent) of 76 patients, mothers, comprising 3 of 16 de novo mutations in the patients mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patients mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold-higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types.

Haemophilia A: mutation type determines risk of inhibitor formation.

The formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations. large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.

Characterization of mutations within the factor VIII gene of 73 unrelated mild and moderate haemophiliacs.

To screen for mutations within the factor VIII gene of 101 patients (85 unrelated), we used denaturing gradient gel electrophoresis (DGGE) after DNA amplification of target regions, including all coding regions except for the middle part (amino acid 757 to amino acid 1649) of the B domain. With this method, missense mutations were identified in 86% of unrelated patients. 41 different mutations were identified: 25 of them have not been described previously. Five of the genotypes are associated with CRM+ and 26 with CRMred status. Patients who are definitely related to each other showed no differences in DNA sequence. One patient showed two different base pair alterations, the first at amino acid 469 [ala(GCA-->gly(GGA)] and the second at position 473 [tyr(TAT)-->cys(TGT)]. One patient with an amino acid change at position 1689 [arg(CGC)-->his(CAC)] has developed an inhibitor against factor VIII.

[A new point mutation in the mitochondrial gene ND1, detected in a patient with type II diabetes]. / Novaia tochechnaia mutatsiia v mitokhondrial'nom gene ND1, obnaruzhennaia u bol'nogo diabetom tipa II.

A novel mutation in a mitochondrial gene was identified in a patient with type II diabetes mellitus. G-to-A transition was localized at the nt3316 position of gene ND1 and resulted in alanine threonine replacement at position 4 of mitochondrial NAD-H-dehydrogenase.

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PI*Q0 alleles and one deficient PI*M allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.