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     

Association of genetic variation in ENPP1 with obesity-related phenotypes

Ectonucleotide pyrophosphatase phosphodiesterase (ENPP1) is a positional candidate gene at chromosome 6q23 where we previously detected strong linkage with fasting-specific plasma insulin and obesity in Mexican Americans from the San Antonio Family Diabetes Study (SAFDS). We genotyped 106 single-nucleotide polymorphisms (SNPs) within ENPP1 in all 439 subjects from the linkage study, and measured association with obesity and metabolic syndrome (MS)-related traits. Of 72 polymorphic SNPs, 24 were associated, using an additive model, with at least one of eight key metabolic traits. Three traits were associated with at least four SNPs. They were high-density lipoprotein cholesterol (HDL-C), leptin, and fasting plasma glucose (FPG). HDL-C was associated with seven SNPs, of which the two most significant P values were 0.0068 and 0.0096. All SNPs and SNP combinations were analyzed for functional contribution to the traits using the Bayesian quantitative-trait nucleotide (BQTN) approach. With this SNP-prioritization analysis, HDL-C was the most strongly associated trait in a four-SNP model (P=0.00008). After accounting for multiple testing, we conclude that ENPP1 is not a major contributor to our previous linkage peak with MS-related traits in Mexican Americans. However, these results indicate that ENPP1 is a genetic determinant of these traits in this population, and are consistent with multiple positive association findings in independent studies in diverse human populations.     

Auxin-induced heritable variants in yeast with special reference to physiological and genetic characters

A high concentration (400 mg/l) of an auxin, -naphthaleneaceticacid produced heritable variants differing in cell form andsize and in sporulation ability in a diploid strain of Saccharomycescerevisiae. These variants showed altered absorption spectrain the region of cytochromes a + a₃, b and/or c, but could growin a glycerol medium. Cultures of dissected spores from thesevariants involved many non-maters. These variants showed irregularsegregation of nutritional markers and/or a high DNA contentper cell. Since most of the variants grow more slowly on anauxin medium than does the original strain, they seem to beproduced by the induction by auxin rather than by selectiveenrichment. Genetic control of the ability to respond to auxinand the biological significance of auxin-induced variation inyeast is discussed. (Received January 12, 1968; )     

Nature of the heterogeneity within genetic variants of bovine serum transferrin

A comparison is made of the four main components of an homozygous variant (A or D₂, D₂) of bovine serum transferrin. These are designated I-IV in order of increasing mobility in electrophoresis at pH 7.5. Components I, II, HI and IV have 2,2,3 and 3 residues of sialic acid per transferrin molecule and appear to correspond to components 2a, 2b, 3a and 3b respectively of Stratil & Spooner (1971). The difference between components I and II and between III and IV does not reside in sialic acid differences. On the basis of peptide maps of reduced carboxami-domethylated components, urea-starch gel electrophoresis and quantitative sequence studies, it is concluded that components II and IV have a scission in the peptide chain. By homology with the sequence of MacGillivray et al. (1977) for human serum transferrin it is suggested that the scission occurs between residues 55 and 54 from the C-terminus and this portion of the chain has a 'molecular' weight of ca. 6000. The implications are briefly discussed.     

Gender differences in the relationship of ENPP1/PC-1 variants to obesity in a Turkish population

The ectoenzyme ENPP1 (also termed membrane glycoprotein PC-1 or ENPP1/PC-1) is an inhibitor of insulin-induced activation of the insulin receptor. There is evidence from previous studies that coding variants of ENPP1/PC-1 (K121Q) are associated with type 2 diabetes (T2D) and obesity. Studies in the general Turkish population have demonstrated: unique plasma lipid characteristics, a high prevalence of cardiovascular risk factors, and an increased prevalence of obesity and T2D. We investigated, therefore, the association of ENPP1/PC-1 variants with obesity and T2D in Turkish individuals. The TaqMan allelic discrimination assay was used for genotyping the relationship of ENPP1/PC-1 variants to obesity and T2D in a genetic association study of 1,553 genotyped, randomly selected subjects from the Turkish Heart Study. The K121Q (rs1044498) variant and other previously reported variants (rs997509, rs1799774, rs1044548, rs11964389, rs7754561) were analyzed. In this cohort, the minor allele frequency (MAF) of the K121Q variant was associated with obesity in male, but not in female subjects (male, odds ratio 1.64, 95% confidence interval 1.004-2.698, P = 0.048; female, odds ratio 1.003, 95% confidence interval 0.684-1.471, P = ns). In addition, the previously reported ENPP1/PC-1 "risk haplotype" (Q (rs1044498), delT (rs1799774), and G (rs7754561) alleles) was found to be associated with obesity in male, but not in female, subjects (P = 0.035). In contrast, there was no association of either the K121Q variant or the ENPP1/PC-1 haplotype with T2D. We find evidence that variants of ENPP1/PC-1 are associated with obesity in the male Turkish population; thus, these variants may contribute to the development of the obesity in these individuals.     

Single nucleotide polymorphisms in clinical genetic testing: the characterization of the clinical significance of genetic variants and their application in clinical research for BRCA1

Clinical genetic testing is increasingly employed in the medical management of cancer patients. These tests support a variety of clinical decisions by providing results that indicate risk for future disease, confirmation of diagnoses, and more recently, therapeutic selection and prognosis. Most genetic variation detected during clinical testing involves single nucleotide polymorphisms (SNPs). Continued advances in the technologies of genetic analyses make these tests increasingly sensitive, cost-effective and timely, which contribute to their increased utilization. Conversely, it has proven difficult to characterize the clinical significance of genetic variants that do not obviously truncate the open reading frames of genes. These genetic variants of uncertain clinical significance diminish the value of genetic test results. This article highlights a variety of approaches that have emerged from research in diverse disciplines to solve the problem, including the application of information about common SNPs in multiple methods to better characterize clinically uncertain variants. Hereditary breast/ovarian cancer, and in particular BRCA1, provides a framework for this discussion. BRCA1 is particularly interesting in this respect since clinical genetic testing by direct DNA sequencing for over 50,000 patients in North America has revealed approximately 1500 genetic variants to date. This large data set combined with the clinical significance of BRCA1 have resulted in research groups selecting BRCA1 as a preferred gene to evaluate novel methods in this field. Finally, the lessons learned through work with BRCA1 are highly applicable to many other genes associated with cancer risk.     

Discovery of allelic variants of HOXA1 and HOXB1: genetic susceptibility to autism spectrum disorders

BACKGROUND: Family studies have demonstrated that the autism spectrum disorders (ASDs) have a major genetic etiologic component, but expression and penetrance of the phenotype are variable. Mice with null mutations of Hoxa1 or Hoxb1, two genes critical to hindbrain development, have phenotypic features frequently observed in autism, but no naturally occurring variants of either gene have been identified in mammals. METHODS: By sequencing regions of genomic DNA of patients with autism spectrum disorders, we detected a substitution variant at HOXA1 and an insertion variant at HOXB1, both in coding regions of the genes. Fifty-seven individuals ascertained for a diagnosis of an ASD, along with 166 of their relatives, were typed for these variants. Two non-ASD populations were typed, and the frequency of the newly identified alleles was determined in all groups. The genotypes of the ASD families were tested for conformation to Hardy-Weinberg proportions and Mendelian expectations for gene transmission. RESULTS: The frequency of the variants was 10-25% in persons of European or African origin. In the ASD families, there was a significant deviation from the HOXA1 genotype ratios expected from Hardy-Weinberg proportions (P = 0.005). Among affected offspring, a significant deviation from Mendelian expectation in gene transmission (P = 0.011) was observed. No statistically significant effects were detected when the same analyses were applied to the HOXB1 locus, but there was evidence of an interaction between HOXA1, HOXB1, and gender in susceptibility to ASDs. CONCLUSIONS: The results support a role for HOXA1 in susceptibility to autism, and add to the existing body of evidence implicating early brain stem injury in the etiology of ASDs.     

Identification of rare variants causing urea cycle disorders: A clinical,genetic, and biophysical study

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle.     

The presence of γ-carboxyglutamic acid in the proteins associated with ectopic calcification

γ-Carboxyglutamic acid (Gla) is identified in the proteins associated with several types of ectopic calcifications in which hydroxyapatite is the major mineral component. These included the calcified skin and subcutaneous plaques from a patient with dermatomyositis, the calcium containing material extruded from the skin of a patient with scleroderma, and the calcified, atheromatous plaques from aorta. Alkaline hydrolysis of biopsy material from these and from normal tissue revealed the presence of Gla only in the plaque specimens. Since a γ-carboxyglutamic acid-containing protein is normally present in bone and absent in unmineralized tissues, the presence of Gla in soft tissue calcifications is a potentially significant finding, especially in view of its known calcium and phospholipid binding properties.     

Generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6

Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. However, the genetic basis in subsets of both disease phenotypes remains elusive. We hypothesized that GACI and PXE are in a closely related spectrum of disease. We used a standardized questionnaire to retrospectively evaluate the phenotype of 92 probands with a clinical history of GACI. We obtained the ENPP1 genotype by conventional sequencing. In those patients with less than two disease-causing ENPP1 mutations, we sequenced ABCC6. We observed that three GACI patients who carried biallelic ENPP1 mutations developed typical signs of PXE between 5 and 8 years of age; these signs included angioid streaks and pseudoxanthomatous skin lesions. In 28 patients, no disease-causing ENPP1 mutation was found. In 14 of these patients, we detected pathogenic ABCC6 mutations (biallelic mutations in eight patients, monoallelic mutations in six patients). Thus, ABCC6 mutations account for a significant subset of GACI patients, and ENPP1 mutations can also be associated with PXE lesions in school-aged children. Based on the considerable overlap of genotype and phenotype of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification and other organ pathologies, rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways in tissues beyond the artery.     

Mouse genetic model for clinical and immunological heterogeneity of leishmaniasis

Systematic assessment of the role of host genes in clinico-pathological and immunological manifestations of Leishmania major-induced disease in mice was performed using 20 recombinant congenic (RC) strains. As the RC strains are homozygous and each carries a different, random set of 12.5% genes from the resistant strain, STS/A, and 87.5% genes from the susceptible strain, BALB/cHeA, they allowed us to study the pathological and immunological characteristics of infected hosts in 20 fixed different random combinations of BALB/c and STS genes. The 20 RC strains differ widely in expression of different symptoms of disease and in immunological characteristics. Disease or healing in different strains occurred in association with different components of immune response -- with the exception of a frequently occurring correlation between the disease and IgE levels. Moreover, some parameters of the immune response were highly correlated in some strains but not at all in others. This shows that several patterns of the immune response may be associated with the same clinical outcome, depending on the host genotype. Our data also suggest that despite the complexity of regulation, when a sufficient number of controlling loci is known, the prediction of a phenotype is possible. Combining functional and clinical information with multilocus genotyping may improve our ability to predict the progression of the disease and to optimize the treatment.     

Polycystic ovary syndrome is associated with genetic polymorphism in the insulin signaling gene IRS-1 but not ENPP1 in a Japanese population

Recent studies indicate that insulin resistance resulting from altered post-receptor signaling is associated with polycystic ovary syndrome (PCOS). We hypothesized that insulin receptor substrate-1 (IRS-1) Gly972Arg polymorphism and/or ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) Lys121Gln polymorphism predisposes women to PCOS and that these polymorphisms also affect anthropometric variables, glucose metabolism and androgen synthesis. To test those ideas, we studied the genotypes, indexes of insulin resistance, and hormone profiles in 123 Japanese women with PCOS and 380 healthy Japanese controls. We found that there were significantly more IRS-1 972Arg carriers among the PCOS patients than among the healthy controls (10.6% vs. 4.8%, p=0.029), which is consistent with our finding that women carrying the IRS-1 972Arg allele had a significantly increased risk of developing PCOS (odds ratio: 3.31, 95% confidence interval: 1.49-7.35). By contrast, the ENPP1 Lys121Arg polymorphism was distributed equally among PCOS patients and controls. In addition, neither of these polymorphisms studied affected the anthropometric variables, metabolic parameters or androgen levels of women with PCOS. We conclude that the IRS-1 Gly972Arg polymorphism is associated with PCOS in the Japanese population.     

Allele polymorphism of the serotonin transporter gene and clinical heterogeneity of depressive disorders

Depression disorders are a clinically heterogeneous disease group. Their development is to a substantial extent underlain by dysfunction of the serotonin system, in particular, disturbed serotonin transport. The heterogeneity of depressions is associated, among other factors, with the age at disease onset. Allele polymorphism of the serotonin transporter (5-HTT) gene was tested for association with age at disease onset, clinical signs, and anxiety-related traits of depression patients. A sample included 77 patients (mean age 61.2 +/- 8.8 years) with late-onset depression (LOD, mean age at onset 56.58 +/- 9.7 years) and 74 patients (mean age 31.0 +/- 11.8 years) with early-onset depression (EOD, mean age at onset 23.9 +/- 7.4 years). In genotype frequency distribution of two 5-HTT gene polymorphism, the LOD and EOD groups did not differ from each other (chi 2 = 0.33, P = 0.85 for VNTR-17; chi 2 = 3.33, P = 0.19 for HTTLPR) and from a control group (chi 2 = 0.34, P = 0.84 for VNTR-17; chi 2 = 2.1, P = 0.35 for HTTLPR). In either group, patients differing in VNTR-17 and HTTLPR genotypes did not differ in psychological traits and, in particular, in anxiety-related traits. In the case of the HTTLPR polymorphism, LOD patients with genotype ss tended to display less severe neuroticism (t = 2.03, P = 0.0507) and scored significantly less on the Hamilton depression scale (t = 2.19, P = 0.039). Thus, the 5-HTT gene polymorphisms do not affect the risk of depression but is possibly associated with specific clinical signs of the disease, at least in elderly patients.     

ENPP1/PC-1 K121Q polymorphism and genetic susceptibility to type 2 diabetes in North Indians

Genetic susceptibility may be responsible for high prevalence of type 2 diabetes worldwide. A common missense single nucleotide polymorphism, K121Q in the ectoenzyme nucleotide pyrophosphate phosphodiesterase (ENPP1) gene, has recently been associated with type 2 diabetes in Italian, South Indian, and American populations. The objective of this study was to investigate the possible role of K121Q polymorphism in ENPP1 gene with type 2 diabetes in North Indians. The genotype of the ENPP1/PC-1 K121Q polymorphism was determined using polymerase chain reaction-restriction fragment length polymorphism analysis for 328 T2DM patients and 326 non-diabetic participants. Anthropometric and clinical characteristics (Body mass index (BMI), glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL), Creatinine, HbA1c, and insulin levels) were measured using standard protocols. Their Chi-square analyses were used to test the significance differences in genotypic and allelic frequencies. Association studies were undertaken using the t test and logistic regression analyses. Our results revealed there was no significant difference in the genotypic distribution between T2DM patients and control subjects. The KK and KQ genotype frequencies were similar in T2DM cases and controls (60.7 and 39.3% in T2DM and 59.8 and 40.2% in controls). No subjects with the QQ genotype were found. Binary logistic regression analysis of data did not show any association of K121Q polymorphism with type 2 diabetes (OR; 0.97, 95% CI; 0.7–1.32, P = 0.82). No significant correlation among the BMI, WHR, BP, TG, TC, HDL-C, LDL-C, Glucose, HbA1c, Creatinine, and insulin indices (HOMA-IR) was observed in the individuals carrying KK and KQ genotypes. In conclusion, our results showed that ENPP1/PC-1 K121Q polymorphism is not associated with type 2 diabetes and related quantitative metabolic traits in North Indian Punjabi population.     

Molecular genetic and clinical aspects of mitochondrial disorders in childhood

Mitochondrial OXPHOS disorders are caused by mutations in mitochondrial or nuclear genes, which directly or indirectly affect mitochondrial oxidative phosphorylation (OXPHOS). Primary mtDNA abnormalities in children are due to rearrangements (deletions or duplications) and point mutations or insertions. Mutations in the nuclear-encoded polypeptide subunits of OXPHOS result in complex I and II deficiency, whereas mutations in the nuclear proteins involved in the assembly of OXPHOS subunits cause defects in complexes I, III, IV, and V. Here, we review recent progress in the identification of mitochondrial and nuclear gene defects and the associated clinical manifestations of these disorders in childhood.     

The genetic architecture of autism spectrum disorders(ASDs) and the potential importance of common regulatory genetic variants

Currently, there is great interest in identifying genetic variants that contribute to the risk of developing autism spectrum disorders(ASDs), due in part to recent increases in the frequency of diagnosis of these disorders worldwide. While there is nearly universal agreement that ASDs are complex diseases, with multiple genetic and environmental contributing factors, there is less agreement concerning the relative importance of common vs rare genetic variants in ASD liability. Recent observations that rare mutations and copy number variants(CNVs) are frequently associated with ASDs, combined with reduced fecundity of individuals with these disorders, has led to the hypothesis that ASDs are caused primarily by de novo or rare genetic mutations. Based on this model, large-scale whole-genome DNA sequencing has been proposed as the most appropriate method for discovering ASD liability genes. While this approach will undoubtedly identify many novel candidate genes and produce important new insights concerning the genetic causes of these disorders, a full accounting of the genetics of ASDs will be incomplete absent an understanding of the contributions of common regulatory variants, which are likely to influence ASD liability by modifying the effects of rare variants or, by assuming unfavorable combinations, directly produce these disorders. Because it is not yet possible to identify regulatory genetic variants by examination of DNA sequences alone, their identification will require experimentation. In this essay, I discuss these issues and describe the advantages of measurements of allelic expression imbalance(AEI) of m RNA expression for identifying cis-acting regulatory variants that contribute to ASDs.