Widening of the genetic and clinical spectrum of Lamb-Shaffer syndrome, a neurodevelopmental disorder due to SOX5 haploinsufficiency.

PURPOSE: Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved. METHODS: Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types ofSOX5 alterations. Functional consequences of selected substitutions were investigated. RESULTS: Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated. CONCLUSIONS: This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

Correction: IQSEC2-related encephalopathy in males and females: a comparative study including 37 novel patients.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly.

IQSEC2-related encephalopathy in males and females: a comparative study including 37 novel patients.

PURPOSE: Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences. METHODS: We collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms. RESULTS: IQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments. CONCLUSION: This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

HCN1 mutation spectrum: from neonatal epileptic encephalopathy to benign generalized epilepsy and beyond.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.

Mutations in SLC34A3/NPT2c are associated with kidney stones and nephrocalcinosis.

Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na(+))-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P<0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.

regSNPs: a strategy for prioritizing regulatory single nucleotide substitutions.

MOTIVATION: One of the fundamental questions in genetics study is to identify functional DNA variants that are responsible to a disease or phenotype of interest. Results from large-scale genetics studies, such as genome-wide association studies (GWAS), and the availability of high-throughput sequencing technologies provide opportunities in identifying causal variants. Despite the technical advances, informatics methodologies need to be developed to prioritize thousands of variants for potential causative effects. RESULTS: We present regSNPs, an informatics strategy that integrates several established bioinformatics tools, for prioritizing regulatory SNPs, i.e. the SNPs in the promoter regions that potentially affect phenotype through changing transcription of downstream genes. Comparing to existing tools, regSNPs has two distinct features. It considers degenerative features of binding motifs by calculating the differences on the binding affinity caused by the candidate variants and integrates potential phenotypic effects of various transcription factors. When tested by using the disease-causing variants documented in the Human Gene Mutation Database, regSNPs showed mixed performance on various diseases. regSNPs predicted three SNPs that can potentially affect bone density in a region detected in an earlier linkage study. Potential effects of one of the variants were validated using luciferase reporter assay.

Dosage effect of a Phex mutation in a murine model of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is caused by mutations in the PHEX gene, which increase circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Because XLH is a dominant disease, one mutant allele is sufficient for manifestation of the disease. However, the dosage effect of a PHEX mutation in XLH is not completely understood. To examine the effect of Phex genotypes, we compared serum biochemistries and skeletal measures between all five possible genotypes of a new murine model of XLH (Phex (K496X) or Phex (Jrt) ). Compared to sex-matched littermate controls, all Phex mutant mice had hypophosphatemia, mild hypocalcemia, and increased parathyroid hormone and alkaline phosphatase levels. Furthermore, mutant mice had markedly elevated serum Fgf23 levels due to increased Fgf23 expression and reduced cleavage of Fgf23. Although females with a homozygous Phex mutation were slightly more hypocalcemic and hypophosphatemic than heterozygous females, the two groups had comparable intact Fgf23 levels. Similarly, there was no difference in intact Fgf23 or phosphorus concentrations between hemizygous males and heterozygous females. Compared to heterozygous females, homozygous counterparts were significantly smaller and had shorter femurs with reduced bone mineral density, suggesting the existence of dosage effect in the skeletal phenotype of XLH. However, overall phenotypic trends in regards to mineral ion homeostasis were mostly unaffected by the presence of one or two mutant Phex allele(s). The lack of a gene dosage effect on circulating Fgf23 (and thus phosphorus) levels suggests that a Phex mutation may create the lower set point for extracellular phosphate concentrations.

A novel Phex mutation in a new mouse model of hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies.

A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis.

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans.

Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects.

BACKGROUND: Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation. OBJECTIVES: The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. RESULTS: We successively developed an ELISA to measure serum salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/mL) than those in adults (mean+/-SD; 562+/-146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls. CONCLUSION: We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism.

Clinical variability of familial tumoral calcinosis caused by novel GALNT3 mutations.

The GALNT3 gene encodes GalNAc-T3, which prevents degradation of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Biallelic mutations in either GALNT3 or FGF23 result in hyperphosphatemic familial tumoral calcinosis or its variant, hyperostosis-hyperphosphatemia syndrome. Tumoral calcinosis is characterized by the presence of ectopic calcifications around major joints, whereas hyperostosis-hyperphosphatemia syndrome is characterized by recurrent long bone lesions with hyperostosis. Here we investigated four patients with hyperphosphatemia and clinical manifestations including tumoral calcinosis and/or hyperostosis-hyperphosphatemia syndrome to determine underlying genetic cause and delineate phenotypic heterogeneity of these disorders. Mutational analysis of FGF23 and GALNT3 in these patients revealed novel homozygous mutations in GALNT3. Although the presence of massive calcifications, cortical hyperostosis, or dental anomalies was not shared by all patients, all had persistent hyperphosphatemia. Three of the patients also had inappropriately normal 1,25-dihyroxyvitamin D [1,25(OH)(2)D] and confirmed low circulating intact FGF23 concentrations. The four novel GALNT3 mutations invariably resulted in hyperphosphatemia as a result of low intact FGF23, but other clinical manifestations were variable. Therefore, tumoral calcinosis and hyperostosis-hyperphosphatemia syndrome represent a continuous spectrum of the same disease caused by increased phosphate levels, rather than two distinct disorders.

Reticular dysgenesis caused by an intronic pathogenic variant in AK2.

Reticular dysgenesis is a form of severe combined immunodeficiency (SCID) caused by biallelic pathogenic variants in AK2 Here we present the case of a boy diagnosed with SCID following a positive newborn screen (NBS). Genetic testing revealed a homozygous variant: AK2 c.330 + 5G > A. In silico analyses predicted weakened native donor splice site. However, this variant was initially classified as a variant of uncertain significance (VUS) given lack of direct evidence. To determine the impact on splicing, we analyzed RNA from the proband and his parents, using massively parallel RNA-seq of cloned RT-PCR products. Analysis showed that c.330 + 5G > A results in exon 3 skipping, which encodes a critical region of the AK2 protein. With these results, the variant was upgraded to pathogenic, and the patient was given a diagnosis of reticular dysgenesis. Interpretation of VUS at noncanonical splice site nucleotides presents a challenge. RNA sequencing provides an ideal platform to perform qualitative and quantitative assessment of intronic VUS, which can lead to reclassification if a significant impact on mRNA is observed. Genetic disorders of hematopoiesis and immunity represent fruitful areas to apply RNA-based analysis for variant interpretation given the high expression of RNA in blood.

Association of adenylate cyclase 10 (ADCY10) polymorphisms and bone mineral density in healthy adults.

Phenotypic variation in bone mineral density (BMD) among healthy adults is influenced by both genetic and environmental factors. Sequence variations in the adenylate cyclase 10 (ADCY10) gene, which is also called soluble adenylate cyclase, have previously been associated with low spinal BMD in hypercalciuric patients. Since ADCY10 is located in the region linked to spinal BMD in our previous linkage analysis, we tested whether polymorphisms in this gene are also associated with normal BMD variation in healthy adults. Sixteen single-nucleotide polymorphisms (SNPs) distributed throughout ADCY10 were genotyped in two healthy groups of American whites: 1692 premenopausal women and 715 men. Statistical analyses were performed in the two groups to test for association between these SNPs and the femoral neck and lumbar spine areal BMD. We observed significant evidence of association (p < 0.01), with one SNP each in men and women. Genotypes at these SNPs accounted for <1% of hip BMD variation in men but 1.5% of spinal BMD in women. However, adjacent SNPs did not corroborate the association in either men or women. In conclusion, we found a modest association between an ADCY10 polymorphism and the spinal areal BMD in premenopausal white women.

Discordance for X-linked hypophosphataemic rickets in identical twin girls.

BACKGROUND: We report monozygotic twin girls with a family history consistent with X-linked hypophosphataemic rickets (XLH). One twin had a skeletal and biochemical phenotype consistent with XLH, whilst the second twin appeared normal. Complete non-penetrance in XLH has not been previously reported and our aim was to explore potential reasons for this. METHODS: Serum and urine biochemistry were analysed at regular intervals. Microsatellite analysis was performed to confirm monozygosity and bi-parental inheritance of the X chromosome. The X chromosome inactivation pattern was studied in peripheral blood. Exons of the paternal PHEX and FGF23 genes were sequenced. RESULTS: Biochemistry was persistently abnormal in the slow-growing twin 1 and normal in twin 2 who has grown normally. Maximal tubular phosphate reabsorption was 0.68 mmol/l in twin 1 and 1.64 mmol/l in twin 2 at 10.8 years of age (normal 1.15-2.58 mmol/l). Microsatellite analysis confirmed monozygosity and the X chromosome inactivation pattern was random. These studies also excluded uniparental isodisomy. The exon sequence of paternal PHEX and FGF23 genes was normal. CONCLUSIONS: Discordant X inactivation is a well-recognised phenomenon in identical twins, and we suspect that non-random expression of the normal PHEX gene in critical tissues is the most likely explanation for non-penetrance.

An Unusual Combination of Neurological Manifestations and Sudden Vision Loss in a Child with Familial Hyperphosphatemic Tumoral Calcinosis.

Hyperphosphatemia in the absence of renal failure is an unusual occurrence, particularly in children, but is a common primary feature of familial hyperphosphatemic tumor calcinosis. We report a child with hyperphosphatemia who presented with multiple episodes of neurologic dysfunction involving lower motor neuron facial nerve palsy along with sequential visual loss. He also had an episode of stroke. There was an extensive metastatic calcification of soft tissue and vasculature. Hyperphosphatemia with normal serum alkaline phosphatase, calcium, parathyroid hormone, and renal function was noted. He was managed with hemodialysis and sevelamer (3 months) without much success in reducing serum phosphate level, requiring continuous ambulatory peritoneal dialysis (3 years). Intact fibroblast growth factor 23 (FGF23) was undetectable, with C-terminal FGF23 fragments significantly elevated (2575 RU/ml, normal <180 RU/ml). Sequencing demonstrated homozygous c.486C >A (p.N162K) mutation in FGF23 exon 3, confirming the diagnoses of primary FGF23 deficiency, the first case to be reported from India.

Mutational survey of the PHEX gene in patients with X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disorder characterized by renal phosphate wasting, aberrant vitamin D metabolism, and abnormal bone mineralization. XLH is caused by inactivating mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). In this study, we sequenced the PHEX gene in subjects from 26 kindreds who were clinically diagnosed with XLH. Sequencing revealed 18 different mutations, of which thirteen have not been reported previously. In addition to deletions, splice site mutations, and missense and nonsense mutations, a rare point mutation in the 3'-untranslated region (3'-UTR) was identified as a novel cause of XLH. In summary, we identified a wide spectrum of mutations in the PHEX gene. Our data, in accord with those of others, indicate that there is no single predominant PHEX mutation responsible for XLH.

Autoimmune hyperphosphatemic tumoral calcinosis in a patient with FGF23 autoantibodies.

Hyperphosphatemic familial tumoral calcinosis (HFTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is an autosomal recessive disorder of ectopic calcification due to deficiency of or resistance to intact fibroblast growth factor 23 (iFGF23). Inactivating mutations in FGF23, N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO (KL) have been reported as causing HFTC/HHS. We present what we believe is the first identified case of autoimmune hyperphosphatemic tumoral calcinosis in an 8-year-old boy. In addition to the classical clinical and biochemical features of hyperphosphatemic tumoral calcinosis, the patient exhibited markedly elevated intact and C-terminal FGF23 levels, suggestive of FGF23 resistance. However, no mutations in FGF23, KL, or FGF receptor 1 (FGFR1) were identified. He subsequently developed type 1 diabetes mellitus, which raised the possibility of an autoimmune cause for hyperphosphatemic tumoral calcinosis. Luciferase immunoprecipitation systems revealed markedly elevated FGF23 autoantibodies without detectable FGFR1 or Klotho autoantibodies. Using an in vitro FGF23 functional assay, we found that the FGF23 autoantibodies in the patient's plasma blocked downstream signaling via the MAPK/ERK signaling pathway in a dose-dependent manner. Thus, this report describes the first case, to our knowledge, of autoimmune hyperphosphatemic tumoral calcinosis with pathogenic autoantibodies targeting FGF23. Identification of this pathophysiology extends the etiologic spectrum of hyperphosphatemic tumoral calcinosis and suggests that immunomodulatory therapy may be an effective treatment.

Novel GALNT3 mutations causing hyperostosis-hyperphosphatemia syndrome result in low intact fibroblast growth factor 23 concentrations.

CONTEXT: Hyperostosis-hyperphosphatemia syndrome (HHS) is a rare metabolic disorder characterized by hyperphosphatemia and localized hyperostosis. HHS is caused by mutations in GALNT3, which encodes UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 3. Familial tumoral calcinosis (TC), characterized by ectopic calcifications and hyperphosphatemia, is caused by mutations in the GALNT3 or fibroblast growth factor 23 (FGF23) genes. OBJECTIVE: Our objective was to identify mutations in FGF23 or GALNT3 and determine serum FGF23 levels in an HHS patient. DESIGN: Mutation detection in FGF23 and GALNT3 was performed by DNA sequencing, and serum FGF23 concentrations were measured by ELISA. PATIENTS OR OTHER PARTICIPANTS: A 5-year-old French boy with HHS and his family members participated. RESULTS: The patient presented with painful cortical lesions in his leg. Radiographs of the affected bone showed diaphyseal hyperostosis. The lesional tissue comprised trabeculae of immature, woven bone surrounded by fibrous tissue. Biochemistry revealed elevated phosphate, tubular maximum rate for phosphate reabsorption per deciliter of glomerular filtrate, and 1,25-dihydroxyvitamin D levels. The patient was a compound heterozygote for two novel GALNT3 mutations. His parents and brother were heterozygous for one of the mutations and had no biochemical abnormalities. Intact FGF23 level in the patient was low normal, whereas C-terminal FGF23 was elevated, a pattern similar to TC. CONCLUSION: The presence of GALNT3 mutations and elevated C-terminal, but low intact serum FGF23, levels in HHS resemble those seen in TC, suggesting that HHS and TC are different manifestations of the same disorder. The absence of biochemical abnormalities in the heterozygous individuals suggests that one normal allele is sufficient for secretion of intact FGF23.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice.

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression.

Human ALOX12, but not ALOX15, is associated with BMD in white men and women.

UNLABELLED: The Alox15 gene was recently identified as a negative regulator of peak BMD in mice. Polymorphisms in human ALOX12, but not ALOX15, were significantly associated with spine BMD in white men and women, suggesting that ALOX12 may contribute to normal variation in BMD. INTRODUCTION: Osteoporosis is a complex disease with both genetic and environmental risk factors. A major determinant of osteoporosis is peak BMD, which is a highly heritable trait. Recently, the arachidonate 15-lipoxygenase (Alox15) gene was identified as a negative regulator of peak BMD in mice. MATERIALS AND METHODS: To assess the contribution of lipoxygenase genes to normal BMD variation in healthy white men and women, we performed population- and family-based association studies of two arachidonate lipoxygenase genes: ALOX15, which is the human homolog of mouse Alox15, and ALOX12, which is functionally similar to Alox15. Single nucleotide polymorphisms (SNPs) distributed across the two genes were genotyped. BMD was measured at the femoral neck and lumbar spine in 411 men 18-61 years of age and 1291 premenopausal women 20-50 years of age. RESULTS: Moderate evidence of association was found between spine BMD and six SNPs in the ALOX12 gene in both men and women (p = 0.0052-0.050). Furthermore, the most common SNP haplotype in ALOX12 showed evidence of significant association with high spine BMD in men (p = 0.0083), whereas the second most common haplotype was associated with high spine BMD in women (p = 0.0081). CONCLUSIONS: Polymorphisms in the ALOX12 gene may contribute to normal variation in spine BMD.