Three Novel Mutations in the PHEX Gene in Chinese Subjects with Hypophosphatemic Rickets Extends Genotypic Variability

Mutations in the phosphate-regulating endopeptidase homolog, X-linked, gene (PHEX), which encodes a zinc-dependent endopeptidase that is involved in bone mineralization and renal phosphate reabsorption, cause the most common form of hypophosphatemic rickets, X-linked hypophosphatemic rickets (XLH). The distribution of PHEX mutations is extensive, but few mutations have been identified in Chinese with XLH. We extracted genomic DNA and total RNA from leukocytes obtained from nine unrelated Chinese subjects (three males and six females, age range 11–36 years) who were living in Taiwan. The PHEX gene was amplified from DNA by PCR, and the amplicons were directly sequenced. Expression studies were performed by reverse-transcription PCR of leukocyte RNA. Serum levels of FGF23 were significantly greater in the patients than in normal subjects (mean 69.4 ± 18.8 vs. 27.2 ± 8.4 pg/mL, P < 0.005), and eight of the nine patients had elevated levels of FGF23. Germline mutations in the PHEX gene were identified in five of 9 patients, including novel c.1843 delA, donor splice site mutations c.663+2delT and c.1899+2T>A, and two previously reported missense mutations, p.C733Y and p.G579R. These data extend the spectrum of mutations in the PHEX gene in Han Chinese and confirm variability for XLH in Taiwan

A Novel Nonsense Mutation in the DMP1 Gene Identified by a Genome-Wide Association Study Is Responsible for Inherited Rickets in Corriedale Sheep

Inherited rickets of Corriedale sheep is characterized by decreased growth rate, thoracic lordosis and angular limb deformities. Previous outcross and backcross studies implicate inheritance as a simple autosomal recessive disorder. A genome wide association study was conducted using the Illumina OvineSNP50 BeadChip on 20 related sheep comprising 17 affected and 3 carriers. A homozygous region of 125 consecutive single-nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 6 Mb on ovine chromosome 6. Among 35 candidate genes in this region, the dentin matrix protein 1 gene (DMP1) was sequenced to reveal a nonsense mutation 250C/T on exon 6. This mutation introduced a stop codon (R145X) and could truncate C-terminal amino acids. Genotyping by PCR-RFLP for this mutation showed all 17 affected sheep were “T T” genotypes; the 3 carriers were “C T”; 24 phenotypically normal related sheep were either “C T” or “C C”; and 46 unrelated normal control sheep from other breeds were all “C C”. The other SNPs in DMP1 were not concordant with the disease and can all be ruled out as candidates. Previous research has shown that mutations in the DMP1 gene are responsible for autosomal recessive hypophosphatemic rickets in humans. Dmp1_knockout mice exhibit rickets phenotypes. We believe the R145X mutation to be responsible for the inherited rickets found in Corriedale sheep. A simple diagnostic test can be designed to identify carriers with the defective “T” allele. Affected sheep could be used as animal models for this form of human rickets, and for further investigation of the role of DMP1 in phosphate homeostasis

Functional Analysis: Evaluation of Response Intensities - Tailoring ANOVA for Lists of Expression Subsets

Background: Microarray data is frequently used to characterize the expression profile of a whole genome and to compare the characteristics of that genome under several conditions. Geneset analysis methods have been described previously to analyze the expression values of several genes related by known biological criteria (metabolic pathway, pathology signature, co-regulation by a common factor, etc.) at the same time and the cost of these methods allows for the use of more values to help discover the underlying biological mechanisms. Results: As several methods assume different null hypotheses, we propose to reformulate the main question that biologists seek to answer. To determine which genesets are associated with expression values that differ between two experiments, we focused on three ad hoc criteria: expression levels, the direction of individual gene expression changes (up or down regulation), and correlations between genes. We introduce the FAERI methodology, tailored from a two-way ANOVA to examine these criteria. The significance of the results was evaluated according to the self-contained null hypothesis, using label sampling or by inferring the null distribution from normally distributed random data. Evaluations performed on simulated data revealed that FAERI outperforms currently available methods for each type of set tested. We then applied the FAERI method to analyze three real-world datasets on hypoxia response. FAERI was able to detect more genesets than other methodologies, and the genesets selected were coherent with current knowledge of cellular response to hypoxia. Moreover, the genesets selected by FAERI were confirmed when the analysis was repeated on two additional related datasets. Conclusions: The expression values of genesets are associated with several biological effects. The underlying mathematical structure of the genesets allows for analysis of data from several genes at the same time. Focusing on expression levels, the direction of the expression changes, and correlations, we showed that two-step data reduction allowed us to significantly improve the performance of geneset analysis using a modified two-way ANOVA procedure, and to detect genesets that current methods fail to detect

Genetic Evidence of Serum Phosphate-Independent Functions of FGF-23 on Bone

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23−/−) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23−/− mice and to examine serum phosphate–independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23−/− mice on phosphate homeostasis and skeletal mineralization. Fgf-23−/−/NaPi2a−/− double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23−/− animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23−/− mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23−/−/NaPi2a−/−, their skeletal phenotype still resembles the one of Fgf23−/− animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23−/− mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis

FGF-23 in bone biology

Recent studies have demonstrated that levels of fibroblast growth factor 23 (FGF-23), a key regulator of phosphorus and vitamin D metabolism, rise dramatically as renal function declines and may play a key initiating role in disordered mineral and bone metabolism in patients with chronic kidney disease (CKD). The physiologic importance of FGF-23 in mineral metabolism was first identified in human genetic and acquired rachitic diseases and further characterized in animal models. FGF-23 and its regulators, including phosphate regulating endopeptidase homolog, dentin matrix 1 (DMP1), and matrix extracellular phosphoglycoprotein, are made primarily in bone, specifically in osteocytes. Dysregulation of these proteins results in osteomalacia, implicating the osteocyte in the regulation of skeletal mineralization. Studies in pediatric patients with CKD, the majority of whom have altered skeletal mineralization in early stages of CKD, have demonstrated that skeletal expression of both FGF-23 and its regulator, DMP1, are increased in early stages of CKD and that expression of these proteins is associated with alterations in skeletal mineralization. Thus, dysregulation of osteocytic proteins occur very early in the course of CKD and appear to be central to altered bone and mineral metabolism in this patient population

Detecting Low Frequent Loss-of-Function Alleles in Genome Wide Association Studies with Red Hair Color as Example

Multiple loss-of-function (LOF) alleles at the same gene may influence a phenotype not only in the homozygote state when alleles are considered individually, but also in the compound heterozygote (CH) state. Such LOF alleles typically have low frequencies and moderate to large effects. Detecting such variants is of interest to the genetics community, and relevant statistical methods for detecting and quantifying their effects are sorely needed. We present a collapsed double heterozygosity (CDH) test to detect the presence of multiple LOF alleles at a gene. When causal SNPs are available, which may be the case in next generation genome sequencing studies, this CDH test has overwhelmingly higher power than single SNP analysis. When causal SNPs are not directly available such as in current GWA settings, we show the CDH test has higher power than standard single SNP analysis if tagging SNPs are in linkage disequilibrium with the underlying causal SNPs to at least a moderate degree (r2>0.1). The test is implemented for genome-wide analysis in the publically available software package GenABEL which is based on a sliding window approach. We provide the proof of principle by conducting a genome-wide CDH analysis of red hair color, a trait known to be influenced by multiple loss-of-function alleles, in a total of 7,732 Dutch individuals with hair color ascertained. The association signals at the MC1R gene locus from CDH were uniformly more significant than traditional GWA analyses (the most significant P for CDH = 3.11×10−142 vs. P for rs258322 = 1.33×10−66). The CDH test will contribute towards finding rare LOF variants in GWAS and sequencing studies

Hormone-like (endocrine) Fgfs: their evolutionary history and roles in development, metabolism, and disease

Fibroblast growth factors (Fgfs) are proteins with diverse functions in development, repair, and metabolism. The human Fgf gene family with 22 members can be classified into three groups, canonical, intracellular, and hormone-like Fgf genes. In contrast to canonical and intracellular Fgfs identified in invertebrates and vertebrates, hormone-like Fgfs, Fgf15/19, Fgf21, and Fgf23, are vertebrate-specific. The ancestral gene of hormone-like Fgfs was generated from the ancestral gene of canonical Fgfs by gene duplication early in vertebrate evolution. Later, Fgf15/19, Fgf21, and Fgf23 were generated from the ancestral gene by genome duplication events. Canonical Fgfs act as autocrine/paracrine factors in an Fgf receptor (Fgfr)-dependent manner. In contrast, hormone-like Fgfs act as endocrine factors in an Fgfr-dependent manner. Canonical Fgfs have a heparin-binding site necessary for the stable binding of Fgfrs and local signaling. In contrast, hormone-like Fgfs acquired endocrine functions by reducing their heparin-binding affinity during their evolution. Fgf15/19 and Fgf23 require βKlotho and αKlotho as cofactors, respectively. However, Fgf21 might physiologically require neither. Hormone-like Fgfs play roles in metabolism at postnatal stages, although they also play roles in development at embryonic stages. Fgf15/19 regulates bile acid metabolism in the liver. Fgf21 regulates lipid metabolism in the white adipose tissue. Fgf23 regulates serum phosphate and active vitamin D levels. Fgf23 signaling disorders caused by hereditary diseases or tumors result in metabolic disorders. In addition, serum Fgf19 or Fgf21 levels are significantly increased by metabolic disorders. Hormone-like Fgfs are newly emerging and quite unique in their evolution and function

Genetic diagnosis of X-linked dominant hypophosphatemic rickets in a cohort study: Tubular reabsorption of phosphate and 1,25(OH)2D serum levels are associated with PHEX mutation type

<p>Abstract</p> <p>Background</p> <p>Genetic Hypophosphatemic Rickets (HR) is a group of diseases characterized by renal phosphate wasting with inappropriately low or normal 1,25-dihydroxyvitamin D₃(1,25(OH)₂D) serum levels. The most common form of HR is X-linked dominant HR (XLHR) which is caused by inactivating mutations in the <it>PHEX </it>gene. The purpose of this study was to perform genetic diagnosis in a cohort of patients with clinical diagnosis of HR, to perform genotype-phenotype correlations of those patients and to compare our data with other HR cohort studies.</p> <p>Methods</p> <p>Forty three affected individuals from 36 non related families were analyzed. For the genetic analysis, the <it>PHEX </it>gene was sequenced in all of the patients and in 13 cases the study was complemented by mRNA sequencing and Multiple Ligation Probe Assay. For the genotype-phenotype correlation study, the clinical and biochemical phenotype of the patients was compared with the type of mutation, which was grouped into clearly deleterious or likely causative, using the Mann-Whitney and Fisher's exact test.</p> <p>Results</p> <p>Mutations in the <it>PHEX </it>gene were identified in all the patients thus confirming an XLHR. Thirty four different mutations were found distributed throughout the gene with higher density at the 3' end. The majority of the mutations were novel (69.4%), most of them resulted in a truncated PHEX protein (83.3%) and were family specific (88.9%). Tubular reabsorption of phosphate (TRP) and 1,25(OH)₂D serum levels were significantly lower in patients carrying clearly deleterious mutations than in patients carrying likely causative ones (61.39 ± 19.76 vs. 80.14 ± 8.80%, p = 0.028 and 40.93 ± 30.73 vs. 78.46 ± 36.27 pg/ml, p = 0.013).</p> <p>Conclusions</p> <p><it>PHEX </it>gene mutations were found in all the HR cases analyzed, which was in contrast with other cohort studies. Patients with clearly deleterious <it>PHEX </it>mutations had lower TRP and 1,25(OH)₂D levels suggesting that the <it>PHEX </it>type of mutation might predict the XLHR phenotype severity.</p

A Low Concentration of Ethanol Impairs Learning but Not Motor and Sensory Behavior in Drosophila Larvae

Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (∼7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects