Genetic diversity in widespread species is not congruent with species richness in alpine plant communities.

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

A novel third type of recurrent NF1 microdeletion mediated by nonallelic homologous recombination between LRRC37B-containing low-copy repeats in 17q11.2.

Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1-REPs B and C. The breakpoints of these approximately 1.0-Mb ("type-3") NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109-bp, respectively, were identified within the LRRC37B-P paralogues of NF1-REPs B and C, and were found to contain sequences capable of non-B DNA formation. Both LCRs contain LRRC37-core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37-containing LCRs at 17q21.31 is known to have mediated the 970-kb polymorphic inversions of the MAPT-locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37-containing LCRs in the human genome.

Mechanisms of loss of heterozygosity in neurofibromatosis type 1-associated plexiform neurofibromas.

Plexiform neurofibromas constitute a serious burden for patients with neurofibromatosis type 1 (NF1), a common autosomal dominant disorder characterized by pigmentary changes and tumorous skin lesions (neurofibromas). Despite the prominence of these benign tumors in NF1 patients, the mechanisms underlying the tumor-associated loss of heterozygosity (LOH) in plexiform neurofibromas have not been extensively studied. We performed LOH analysis on 43 plexiform neurofibromas from 31 NF1 patients, the largest study of its kind to date. A total of 13 (30%) plexiform neurofibromas exhibited LOH involving 17q markers. In three tumors, LOH was found to be confined to the NF1 gene region. However, in none of the tumors was a somatic NF1 microdeletion, mediated by non-allelic homologous recombination between either NF1-REPs or SUZ12 genes, detected. Thus, NF1 microdeletions do not appear to be frequent somatic events in plexiform neurofibromas. Determination of NF1 gene copy number by multiplex ligation-dependent probe amplification indicated that although tumors with smaller regions of LOH were characterized by 17q deletions, no NF1 gene copy number changes were detected in six plexiform neurofibromas with more extensive LOH. To our knowledge, mitotic recombination has not previously been reported to be a frequent cause of LOH in plexiform neurofibromas.

A gene conversion hotspot in the human growth hormone (GH1) gene promoter.

To assess the evolutionary importance of nonallelic (or interlocus) gene conversion for the highly polymorphic human growth hormone (GH1) gene promoter, sequence variation in this region was studied in four different ethnic groups. For 14 SNPs in the proximal GH1 promoter (535 bp), 60 different haplotypes were observed in 577 individuals (156 Britons, 116 Spaniards, 163 West-Africans, 142 Asians). Using a novel coalescence-based statistical test, significant evidence was found in the British, Spanish, and African groups for GH1 having acted as an acceptor of gene conversion, with at least one of the four paralogous GH gene promoters serving as the donor (and specifically GH2 in the Britons and Spaniards). The average gene conversion tract length was estimated to be 84 bp. A gene conversion hotspot was identified, spanning the GH1 transcriptional initiation site (positions -6 to +25). Although these findings serve to highlight the importance of gene conversion for the recent evolution of the human GH1 promoter, its relative frequency does not appear to be related simply to the presence of specific DNA sequence motifs or secondary structures, the degree of homology between GH paralogs, the distance between them, or their transcriptional orientation. The GH1 promoter was also found to be highly polymorphic in chimpanzee but not in macaque. This may reflect the lower degree of pair-wise similarity between the GH1 promoter and its paralogs in macaque (mean, 92.0%) as compared to chimpanzee (93.5%) and human (94.0%), and hence provides further support for the idea of a threshold (perhaps around 92%) below which gene conversion is reduced or abolished.

Copy number variations in the NF1 gene region are infrequent and do not predispose to recurrent type-1 deletions.

Gross deletions of the NF1 gene at 17q11.2 belong to the group of 'genomic disorders' characterized by local sequence architecture that predisposes to genomic rearrangements. Segmental duplications within regions associated with genomic disorders are prone to non-allelic homologous recombination (NAHR), which mediates gross rearrangements. Copy number variants (CNVs) without obvious phenotypic consequences also occur frequently in regions of genomic disorders. In the NF1 gene region, putative CNVs have been reportedly detected by array comparative genomic hybridization (array CGH). These variants include duplications and deletions within the NF1 gene itself (CNV1) and a duplication that encompasses the SUZ12 gene, the distal NF1-REPc repeat and the RHOT1 gene (CNV2). To explore the possibility that these CNVs could have played a role in promoting deletion mutagenesis in type-1 deletions (the most common type of gross NF1 deletion), non-affected transmitting parents of patients with type-1 NF1 deletions were investigated by multiplex ligation-dependent probe amplification (MLPA). However, neither CNV1 nor CNV2 were detected. This would appear to exclude these variants as frequent mediators of NAHR giving rise to type-1 deletions. Using MLPA, we were also unable to confirm CNV1 in healthy controls as previously reported. We conclude that locus-specific techniques should be used to independently confirm putative CNVs, originally detected by array CGH, to avoid false-positive results. In one patient with an atypical deletion, a duplication in the region of CNV2 was noted. This duplication could have occurred concomitantly with the deletion as part of a complex rearrangement or may alternatively have preceded the deletion.

Type 2 NF1 deletions are highly unusual by virtue of the absence of nonallelic homologous recombination hotspots and an apparent preference for female mitotic recombination.

Approximately 5% of patients with neurofibromatosis type 1 (NF1) exhibit gross deletions that encompass the NF1 gene and its flanking regions. The breakpoints of the common 1.4-Mb (type 1) deletions are located within low-copy repeats (NF1-REPs) and cluster within a 3.4-kb hotspot of nonallelic homologous recombination (NAHR). Here, we present the first comprehensive breakpoint analysis of type 2 deletions, which are a second type of recurring NF1 gene deletion. Type 2 deletions span 1.2 Mb and are characterized by breakpoints located within the SUZ12 gene and its pseudogene, which closely flank the NF1-REPs. Breakpoint analysis of 13 independent type 2 deletions did not reveal any obvious hotspots of NAHR. However, an overrepresentation of polypyrimidine/polypurine tracts and triplex-forming sequences was noted in the breakpoint regions that could have facilitated NAHR. Intriguingly, all 13 type 2 deletions identified so far are characterized by somatic mosaicism, which indicates a positional preference for mitotic NAHR within the NF1 gene region. Indeed, whereas interchromosomal meiotic NAHR occurs between the NF1-REPs giving rise to type 1 deletions, NAHR during mitosis appears to occur intrachromosomally between the SUZ12 gene and its pseudogene, thereby generating type 2 deletions. Such a clear distinction between the preferred sites of mitotic versus meiotic NAHR is unprecedented in any other genomic disorder induced by the local genomic architecture. Additionally, 12 of the 13 mosaic type 2 deletions were found in females. The marked female preponderance among mosaic type 2 deletions contrasts with the equal sex distribution noted for type 1 and/or atypical NF1 deletions. Although an influence of chromatin structure was strongly suspected, no sex-specific differences in the methylation pattern exhibited by the SUZ12 gene were apparent that could explain the higher rate of mitotic recombination in females.

Detection of heterozygous SALL1 deletions by quantitative real time PCR proves the contribution of a SALL1 dosage effect in the pathogenesis of Townes-Brocks syndrome.

Townes-Brocks syndrome (TBS) is an autosomal dominantly inherited disorder characterized by ear, anal, limb, and renal malformations, and results from mutations in the gene SALL1. All SALL1 mutations previously found in TBS patients create preterminal termination codons. In accordance with the findings of pericentric inversions or balanced translocations, TBS was initially assumed to be caused by SALL1 haploinsufficiency. This assumption was strongly contradicted by a Sall1 mouse knock-out, because neither hetero- nor homozygous knock-out mutants displayed a TBS-like phenotype. A different mouse mutant mimicking the human SALL1 mutations, however, showed a TBS-like phenotype in the heterozygous situation, suggesting a dominant-negative action of the mutations causing TBS. We applied quantitative real time PCR to detect and map SALL1 deletions in 240 patients with the clinical diagnosis of TBS, who were negative for SALL1 mutations. Deletions were found in three families. In the first family, a 75 kb deletion including all SALL1 exons had been inherited by two siblings from their father. A second, sporadic patient carried a de novo 1.9-2.6 Mb deletion including the whole SALL1 gene, and yet another sporadic case was found to carry an intragenic deletion of 3384 bp. In all affected persons, the TBS phenotype is rather mild as compared to the phenotype resulting from point mutations. These results confirm that SALL1 haploinsufficiency is sufficient to cause a mild TBS phenotype but suggest that it is not sufficient to cause the severe, classical form. It therefore seems that there is a different contribution of SALL1 gene function to mouse and human embryonic development.

Two immunochemical assays to measure advanced glycation end-products in serum from dialysis patients.

Advanced glycation end-products are uremic toxins that accumulate in the serum and tissues of patients with chronic renal failure. Here, we established two enzyme-linked immunosorbent assays (ELISAs) for N(epsilon)-carboxymethyllysine and imidazolone to analyze advanced glycation end-products in human serum. Both ELISAs detected advanced glycation end-products bound to human serum albumin in a dose-dependent way. Whereas the formation of imida-zolone was independent of the presence of oxygen, concentrations of N(epsilon)-carboxymethyllysine epitopes increased 20-fold under oxidative conditions. The N(epsilon)-carboxymethyllysine ELISA showed a similar response to free, peptide-bound and protein-bound N(epsilon)-carboxymethyllysine, whereas the imidazolone antibody showed slightly higher affinity toward peptide-bound compared to protein-bound imidazolone. In human serum, linear dilution ranges from 1:10 to 1:40 (N(epsilon)-carboxymethyllysine ELISA) and from 1:2 to 1:8 (imidazolone ELISA) were found. The recovery of N(epsilon)-carboxymethyllysine from serum was 101 +/- 10% and 94 +/- 12%, respectively, and 93 +/- 15% and 97 +/- 12% for imidazolone. The coefficients of variation for intra-assay variability were 0.26-2.7% (N(epsilon)-carboxymethyllysine) and 0.1-2.4% (imidazolone), and 8.3-13.4% (N(epsilon)-carboxymethyllysine) and 7.8-12.5% (imidazolone) for inter-assay variability. In serum samples from hemodialysis patients (n = 20) and controls (n =20), an approximately two-fold increase was detected in the patient group (p < 0.001). The combination of the N(epsilon)-carboxymethyllysine and imidazolone ELISAs is a valuable tool to measure serum concentrations of advanced glycation end-products for clinical studies.

Carbon fluxes to the soil in a mature temperate forest assessed by 13C isotope tracing.

Photosynthetic carbon uptake and respiratory C release from soil are major components of the global carbon balance. The use of 13C depleted CO2)(delta13C = -30 per thousand) in a free air CO2 enrichment experiment in a mature deciduous forest permitted us to trace the carbon transfer from tree crowns to the rhizosphere of 100-120 years old trees. During the first season of CO2 enrichment the CO2 released from soil originated substantially from concurrent assimilation. The small contribution of recent carbon in fine roots suggests a much slower fine root turnover than is often assumed. 13C abundance in soil air correlated best with temperature data taken from 4 to 10 days before air sampling time and is thus rapidly available for root and rhizosphere respiration. The spatial variability of delta13C in soil air showed relationships to above ground tree types such as conifers versus broad-leaved trees. Considering the complexity and strong overlap of roots from different individuals in a forest, this finding opens an exciting new possibility of associating respiration with different species. What might be seen as signal noise does in fact contain valuable information on the spatial heterogeneity of tree-soil interaction.