A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.     

Novel mutations in the ABCC6 gene of German patients with pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue affecting the skin, eyes, and cardiovascular system. Recently, the PXE candidate gene ABCC6 was identified and a limited number of ABCC6 mutations were observed in different PXE cohorts. To identify novel PXE-causing ABCC6 mutations in German patients with PXE, we investigated a cohort of 54 German PXE patients and 23 family members from 49 apparently nonconsanguineous families. From the mutational analysis we found 27 different ABCC6 sequence variations. Among these, 11 were polymorphisms or neutral alterations and 16 were PXE-causing mutations. The most common mutation in our PXE cohort was the nonsense mutation p.R1141X, which occurred with an allele frequency of 25.9%. Furthermore, we found nine missense, one additional nonsense, and two putative splice site mutations as well as three single-nucleotide deletions. Most of these mutations were unique and occurred in cytoplasmic regions of the MRP6 protein; these mutations are proposed to be critical for the physiological function of the MRP6 protein. In these regions we also found the three novel PXE-causing mutations p.R1114C, p.Y1239H, and p.G1311E, which were identified in three alleles from patients with PXE and were absent in 200 healthy control subjects. In addition, the first genotype-phenotype correlation was observed. By obtaining these genetic mutation data, we are contributing to an overview of all ABCC6 mutations leading to PXE and the pathogenetics of this disease.     

Zebrafish enpp1 mutants exhibit pathological mineralization,mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression.KEY WORDS: Zebrafish, Ectopic mineralization, Generalized arterial calcification of infancy, GACI, Pseudoxanthoma elasticum, PXE, Pyrophosphate     

Identification of ABCC6 pseudogenes on human chromosome 16p: implications for mutation detection in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a heritable disorder affecting the skin, eyes, and the cardiovascular system, has recently been linked to mutations in the ABCC6 gene on chromosome 16p13.1. The original mutation detection strategy employed by us consisted of the amplification of each exon of the ABCC6 gene with primer pairs placed on the flanking introns, followed by heteroduplex scanning and direct nucleotide sequencing. However, this approach suggested the presence of multiple copies of the 5'-region of the gene when total genomic DNA was used as a template. In this study, we have identified two pseudogenes containing sequences highly homologous to the 5'-end of ABCC6. First, by the use of allele-specific polymerase chain reaction (PCR), two bacterial artificial chromosome (BAC) clones containing a putative pseudogene of ABCC6, designated as ABCC6-psi 1, were isolated from the human BAC library. Sequence analysis of ABCC6-psi 1 revealed it to be a truncated copy of ABCC6, which contains the upstream region and exon 1 through intron 9 of the gene. Secondly, a homology search of a high-throughput sequence database revealed the presence of another truncated copy of ABCC6, which was designated as ABCC6-psi 2, and which was shown to harbor upstream sequences and a segment spanning exon 1 through intron 4 of ABCC6. In addition to several nucleotide differences in the flanking introns and the upstream region, both pseudogenes contain several nucleotide changes in the exonic sequences, including stop codon mutations, which complicate mutation analysis in patients with PXE. Nucleotide differences in flanking introns between these two pseudogenes and ABCC6 allowed us to design allele-specific primers that eliminated the amplification of both pseudogene sequences by PCR and provided reliable amplification of ABCC6-specific sequences only. The use of allele-specific PCR has revealed, thus far, two novel 5'-end PXE mutations, 179del9 and T364R in exons 2 and 9, respectively, and several polymorphisms within the upstream region and exons 1-9 of ABCC6. These strategies facilitate comprehensive analysis of ABCC6 for mutations in PXE.     

Compound heterozygosity for a recurrent 16.5-kb Alu-mediated deletion mutation and single-base-pair substitutions in the ABCC6 gene results in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder affecting the elastic structures in the skin, eyes, and cardiovascular system, with considerable morbidity and mortality. Recently, mutations in the ABCC6 gene (also referred to as "MRP6" or "eMOAT") encoding multidrug-resistance protein 6 (MRP6), a putative transmembrane ABC transporter protein of unknown function, have been disclosed. Most of the genetic lesions delineated thus far consist of single-base-pair substitutions resulting in nonsense, missense, or splice-site mutations. In this study, we examined four multiplex families with PXE inherited in an autosomal recessive pattern. In each family, the proband was a compound heterozygote for a single-base-pair-substitution mutation and a novel, approximately 16.5-kb deletion mutation spanning the site of the single-base-pair substitution in trans. The deletion mutation was shown to extend from intron 22 to intron 29, resulting in out-of-frame deletion of 1,213 nucleotides from the corresponding mRNA and causing elimination of 505 amino acids from the MRP6 polypeptide. The deletion breakpoints were precisely the same in all four families, which were of different ethnic backgrounds, and haplotype analysis by 13 microsatellite markers suggested that the deletion had occurred independently. Deletion breakpoints within introns 22 and 29 were embedded within AluSx repeat sequences, specifically in a 16-bp segment of DNA, suggesting Alu-mediated homologous recombination as a mechanism.     

Hennekam syndrome can be caused by FAT4 mutations and be allelic to Van Maldergem syndrome

The Hennekam lymphangiectasia–lymphedema syndrome is a genetically heterogeneous disorder. It can be caused by mutations in CCBE1 which are found in approximately 25 % of cases. We used homozygosity mapping and whole-exome sequencing in the original HS family with multiple affected individuals in whom no CCBE1 mutation had been detected, and identified a homozygous mutation in the FAT4 gene. Subsequent targeted mutation analysis of FAT4 in a cohort of 24 CCBE1 mutation-negative Hennekam syndrome patients identified homozygous or compound heterozygous mutations in four additional families. Mutations in FAT4 have been previously associated with Van Maldergem syndrome. Detailed clinical comparison between van Maldergem syndrome and Hennekam syndrome patients shows that there is a substantial overlap in phenotype, especially in facial appearance. We conclude that Hennekam syndrome can be caused by mutations in FAT4 and be allelic to Van Maldergem syndrome.     

The effects of bisphosphonates on ectopic soft tissue mineralization caused by mutations in the ABCC6 gene

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by ABCC6 mutations by feeding Abcc6^−/− mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the ABCC6 gene, when combined with careful monitoring of efficacy and potential side-effects.     

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.     

Structural variability of Tvv1 grapevine retrotransposons can be caused by illegitimate recombination

Structural variability of Tvv1, a grapevine retrotransposon Ty1 copia-like family, was investigated within the grape genome and the canonical sequence of Tvv1 determined. Then, two remarkable elements, Tvv1-Δ3001 and Tvv1-Δ3640, which had suffered large deletions 3,001 bp and 3,460 bp in length of their coding sequences were compared to the canonical copy. In both deleted elements, the deletion breakpoint was characterized by a stretch 13 bp-long in Tvv1-Δ3001 and 11 bp-long in Tvv1-Δ3640 found duplicated in the canonical copy at each bound of the deleted regions. Tvv1-Δ3001 and Tvv1-Δ3460 were both shown to be unique copies fixed at a single locus in the grapevine genome. Their presence was very variable in a set of 58 varieties and wild vines. These elements have most likely been dispersed through natural intermixing after their initial insertion whose chronology was estimated. The model that we propose to explain the structure of Tvv1-Δ3001 and Tvv1-Δ3640, implies illegitimate recombination involving template switching between two RNA molecules co-packaged in the VLP prior to the integration of the deleted daughter copy into the host genome.     

Na + /H + exchanger isoform 1 activity in AQP2-expressing cells can be either proliferative or anti-proliferative depending on extracellular pH

We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.     

Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2.

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.     

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.     

Type-1c glycogen storage disease is not caused by mutations in the glucose-6-phosphate transporter gene

Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive disorders caused by deficiencies in glucose-6-phosphatase (G6Pase) and the associated substrate/product transporters. Molecular genetic studies have demonstrated that GSD-1a and GSD-1b are caused by mutations in the G6Pase enzyme and a glucose-6-phosphate transporter (G6PT), respectively. While kinetic studies of G6Pase catalysis predict that the index GSD-1c patient is deficient in a pyrophosphate/phosphate transporter, the existence of a separate locus for GSD-1c remains unclear. We have previously shown that the G6Pase gene of the index GSD-1c patient is intact; we now show that the G6PT gene of this patient is normal, strongly suggesting the existence of a distinct GSD-1c locus.     

Cell surface colony-stimulating factor 1 can be cleaved by TNF-alpha converting enzyme or endocytosed in a clathrin-dependent manner

CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-alpha converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.