X-linked hypophosphataemia in South Africa.

OBJECTIVES: To investigate the pattern of clinical presentation in a series of South African subjects with X-linked hypophosphataemia (XLH) with particular reference to ethnic differences in presentation and inheritance, and to determine the perceptions and psychosocial problems associated with the disease. DESIGN AND SETTING: The clinical details of 50 subjects were collected from their records as well as from examining those currently attending the clinics held at Chris Hani Baragwanath Hospital and the National Health Laboratory Services in Johannesburg. There were 17 males and 33 females in the study. The psychosocial part of the study involved interviews with 20 parents and 7 subjects (aged 16 years or more). RESULTS: Thirty-one of the subjects were black, 17 white and 2 Indian. The mean age of clinical onset was 2.02 years (range 0.25 - 10 years). Fifty-four per cent of the cases were apparently sporadic. The prevalence of sporadic mutations was 64% among the black subjects and 41% among the white subjects. No differences were found in either clinical or biochemical presentation between genders or ethnic groups, despite an apparently higher sporadic presentation in the black children. The study also showed that this disorder had not only affected family life but also the lives of the subjects and their interpersonal relationships. The hereditary nature of the condition was not clear to most parents even after having attended the clinic for many years. CONCLUSIONS: South African subjects with XLH have similar features to those reported in other studies but there is a higher prevalence of sporadic mutations in the black subjects. Better counselling services are needed to improve the understanding of this condition among parents of affected children.     

Hypertension in connexin40-null mice: a renin disorder

Studies described in this issue indicate that the gap junction protein connexin40 (Cx40) appears to play an unexpected role in blood pressure regulation. In mice lacking this gap junction protein, renin secretion is high and not regulated by arteriolar pressure.     

Parabiosis suggests a humoral factor is involved in X-linked hypophosphatemia in mice

Reduced renal tubular reabsorption of phosphate of unknown etiology is characteristic of X-linked hypophosphatemia in both humans and mice. To test whether a humoral abnormality is involved in the renal effect, parabiosis was performed between Hyp and normal mice at 4 weeks of age. The normal mice joined to Hyp mice showed a progressive diminution of plasma phosphate over the next 3 weeks to approach the level of the Hyp mice. These normal mice had a greater renal phosphate excretion index (urine P/plasma P/urine creatinine) than normal-normal pairs, thus suggesting a reduced renal tubular reabsorption of phosphate. At the same time the expected rises in plasma calcium and plasma 1,25-dihydroxyvitamin D did not occur. There was a significant reduction in their femoral mineral content but not in their femoral length or body growth relative to normal-normal pairs. This change in renal handling of phosphate was specific since the urinary losses of potassium and magnesium were not significantly changed. Separation of normal-Hyp pairs 3 or 6 weeks after parabiosis caused the normal mice to achieve normal plasma phosphate levels within 24 h. At 48 h and 7 days after separation these normal mice had plasma phosphate levels higher than normal mice separated from normal-normal pairs. In summary, the data suggest the presence of a phosphaturic factor in the Hyp mice that can cross a parabiotic union into normal mice and induce many of the symptoms of X-linked hypophosphatemia.     

The effects of bone marrow transplantation on X-linked hypophosphatemic mice.

The genes responsible for X-linked hypophosphatemic (XLH) vitamin D-resistant rickets and the murine homolog, hypophosphatemic mice (Hyp), were identified as PHEX and Phex (phosphate-regulating gene with homology to endopeptidases on the X chromosome), respectively. However, the mechanism by which inactivating mutations of PHEX cause XLH remains unknown. We investigated the mechanisms by syngeneic bone marrow transplantation (BMT) from wild mice to Hyp mice. The expression of the Phex gene was detected in mouse BM cells. BMT introduced a chimerism in recipient Hyp mice and a significant increase in the serum phosphorus level. The renal sodium phosphate cotransporter gene expression was significantly increased. The effect of BMT on the serum phosphorus level depended on engraftment efficiencies, which represent the dosage of normal gene. Similarly, the serum alkaline phosphatase (ALP) activity was decreased and bone mineral density was increased. Furthermore, the renal expression of 25-hydroxyvitamin D3 24-hydroxylase, which is a key enzyme in the catabolic pathway and is increased in XLH/Hyp, was improved. From these results, we conclude that transplantation of normal BM cells improved abnormal bone mineral metabolism and deranged vitamin D metabolism in Hyp by replacing defective gene product(s) with normal gene product(s). This result may provide strong evidence for clinical application of BMT in metabolic bone disorders.     

Metabolites of vitamin D in normal and X-linked hypophosphatemic mice

Summary Hyp mice are a model for human X-linked hypophosphatemia (vitamin D-resistant rickets.) To determine whether an abnormality of vitamin D metabolism exists in this disease, the profiles of the metabolites of vitamin D were determined in normal andHyp mouse plasma.Hyp and normal mice were fed a vitamin D-deficient diet and received 1,2³H-vitamin D₃ at 16 Ci/mmol by stomach tube at 5 ng/g body weight (0.21 μCi/g b.w.) on alternate days for 14 days. The dose of vitamin D given maintained near normal plasma 25-OH-vitamin D. Thus the mice were in a vitamin D-replete state with all metabolite pools labeled with³H. Plasma was collected from 4 normal and 4Hyp mice. The plasma was extracted, and the extracts were chromatographed separately for each mouse on an LH-20 column. Each major peak of radioactivity was rechromatographed using high performance liquid chromatography on a Zorbax-Sil column using solvent systems known to resolve several vitamin D metabolites. Twenty-one radioactive peaks were identified. The disintegrations per minute of³H in each peak were quantified and converted to plasma concentration using the known specific activity of the administered vitamin D. The 25-OH-vitamin D accounted for 55% of the circulating radioactivity, and 24,25-(OH)₂-vitamin D accounted for 22%. The plasma levels of 24,25-(OH)₂-vitamin D were similar to levels previously reported by us using protein binding assays. No peaks of radioactivity were missing in the plasma extracts of theHyp mice. Also there was no evidence that plasma 24,25-(OH)₂-vitamin D was elevated in theHyp mice.     

Phosphate transport in osteoblasts from normal and X-linked hypophosphatemic mice

Human hypophosphatemic vitamin D-resistant rickets (X-linked hypophosphatemia-XLH) is characterized by hypophosphatemia, a decreased tubular reabsorption of phosphate (P_i) and defective skeleton mineralization. Utilizing a mouse model (Hyp) of XLH, which demonstrates biological abnormalities and skeletal defects of XLH, we analyzed sodium-dependent phosphate transport in isolated osteoblasts derived from the calvaria of normophosphatemic and hypophosphatemic mice. Initial rates of phosphate uptake by normal and Hyp osteoblasts showed similar slopes. Osteoblasts from both normal and Hyp mice exhibited saturable, sodium-dependent phosphate transport with apparent V_max and K_m values not significantly different (normal mice, V_max=24.30±3.45 nmol/mg prot. 10 min, K_m=349.49±95.20 mol/liter; Hyp mice, V_max=23.03±3.41 nmol/mg prot. 10 min, K_m=453.64±106.93 mol/liter, n=24). No differences were found in the ability of normal and Hyp osteoblasts to respond to P_i transport after 5 hours of P_i deprivation. Both cell types exhibited a similar increase in cAMP in response to PTH. The accumulated results demonstrate that P_i uptake and transport in normal and Hyp mouse osteoblasts is a sodium-dependent saturable process. As osteoblast P_i uptake and transport is apparently normal in the Hyp mouse model of XLH, the osteoblastic failure described for the Hyp mouse should be attributed to other mechanism(s).     

X-linked spondyloepiphyseal dysplasia tarda: molecular cause of a heritable disorder associated with early degenerative joint disease

Spondyloepiphyseal dysplasia tarda (SEDT) is a genetically heterogeneous disorder often associated with the early onset of osteoarthrosis. The X-linked recessive form (SEDL) affects men and is characterized by reduced height, arm span exceeding total height, and barrel chest deformity. The radiographic phenotype comprises a hump-shaped deformity of vertebral bodies and mild epiphyseal dysplasia of the femoral head associated with early signs of hip arthrosis. The disorder is caused by mutations in the SEDL (or sedlin) gene on Xp22.12-p22.31. In 4 male patients from a German family, we identified a new nonsense mutation in SEDL exon 4 (C74A). The carrier status for the mutation could be confirmed in 2 women of the family, both of whom show no obvious signs of bone and joint diseases. SEDT should be kept in mind as a differential diagnosis in men with early "primary" bilateral osteoarthrosis.     

Altered osteoblast gluconeogenesis in X-linked hypophosphatemic mice is associated with a depressed intracellular pH

We have studied gluconeogenesis and intracellular pH levels in normal (+/Y) and X-linked hypophosphatemic (Hyp/Y) mice. Compared with +/Y littermates, Hyp/Y mouse osteoblasts showed a higher rate of glucose production from fructose (10-fold), glutamine, and malate, but no significant difference when -ketoglutarate was used as substrate. The activities of the pentose cycle enzymes, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, were not different in the two osteoblast preparations. Examination of intracellular pH (pH_i) using the double excitation of the pH-sensitive dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) revealed a significantly lower pHi in Hyp/Y mouse osteoblasts compared with +/Y mouse osteoblasts (7.01±0.03 n=10 versus 7.15±0.04 n=8, respectively; P<0.05). These results show for the first time that osteoblasts are capable of glucose production and that glucose production is altered in the Hyp/Y mouse osteoblast. As altered gluconeogenesis has been associated with reduced intracellular pH in other systems, a similar mechanism may be operative in the Hyp/Y mouse osteoblast. The observed defects may be intrinsic to the Hyp phenotype as the alterations in intracellular pH and gluconeogenesis persisted in vitro, or they may represent impressed memory from the in vivo state and the presumed circulating factor that influences phosphate transport.     

X-linked spondyloepiphyseal dysplasia tardaMolecular cause of a heritable disorder associated with early degenerative joint disease

Spondyloepiphyseal dysplasia tarda (SEDT) is a genetically heterogeneous disorder often associated with the early onset of osteoarthrosis. The X-linked recessive form (SEDL) affects men and is characterized by reduced height, arm span exceeding total height, and barrel chest deformity. The radiographic phenotype comprises a hump-shaped deformity of vertebral bodies and mild epiphyseal dysplasia of the femoral head associated with early signs of hip arthrosis. The disorder is caused by mutations in the SEDL (or sedlin) gene on Xp22.12-p22.31. In 4 male patients from a German family, we identified a new nonsense mutation in SEDL exon 4 (C74A). The carrier status for the mutation could be confirmed in 2 women of the family, both of whom show no obvious signs of bone and joint diseases. SEDT should be kept in mind as a differential diagnosis in men with early "primary" bilateral osteoarthrosis     

Developmental expression of ACRV1 in humans and mice

To identify the developmental expression of the ACRV1 gene in humans and mice, testes cDNA samples were collected at different post-natal days (days 4, 9, 18, 35, 54, and 6 months) from Balb/c mice and were hybridised to the mouse whole genome 430 2.0 Array (Affymetrix Inc.) chip. The characteristics of ACRV1 were analysed using various cellular and molecular biotechnologies. The results showed that the expression of mouse ACRV1 was not detected in mouse testes on days 4, 9, and 18 but was present on days 35, 54, and 6 months. Using RT-PCR analysis of mouse ACRV1, we determined that mouse ACRV1 was expressed specifically in the mouse testis, and its expression began at days 35. Western blot analysis demonstrated that human ACRV1 was primarily expressed in human testes, and immunofluorescent and immunohistochemistry staining showed that human ACRV1 protein was predominantly located in round and elongated spermatids in human testes, indicating that ACRV1 may play an important role in mammalian spermatogenesis and may be a target of a contraceptive vaccine.     

Normal milk composition in lactating X-linked hypophosphatemic mice despite continued hypophosphatemia

Patients with X-linked hypophosphatemia and mice bearing the Hyp gene have reduced renal tubular reabsorption of phosphate and an osteomalacic bone disease. To test if altered phosphate transport also exists in the mammary glands, milk was analyzed from normal (n = 9) and heterozygous Hyp (n = 8) mice 14 days after giving birth. Inorganic phosphate, total phosphate, calcium, magnesium, sodium, and potassium were measured; percent cream, fat, water, and nonfat organic solids were measured; and protein was measured. No significant differences (NSD) were found except for greater sodium in Hyp milk. There was also NSD in litter weight. The lactating Hyp had a lower body weight and remained hypophosphatemic relative to lactating normals, but both groups had higher plasma phosphate than nonlactating controls of the same genotype. The data suggest that Hyp mice can accumulate a normal amount of phosphate in their milk despite the plasma phosphate being two-thirds of normal. These data, with other recent reports of different organ systems, suggest that the altered phosphate transport activity may be restricted to the kidney.     

Muscular high-energy phosphates and red-cell 2,3-DPG in post-traumatic hypophosphataemia. An experimental study in pigs

The relationship between post-traumatic hypophosphataemia, muscular high-energy phosphates (HEP) and red-cell 2,3-diphosphoglycerate (2,3-DPG) were studied in an experimental trauma model. Twenty-three anaesthetized pigs were submitted to trauma and observed for 72 hours. In group I (n = 10) and group II (n = 6) major trauma was inflicted with a high-energy missile. No phosphate supplement was given in group I, but group II pigs received 20 mmol phosphate/24 hours parenterally. Group III (n = 7) sustained only minor trauma, consisting of tracheotomy and catheterization of artery, vein and bladder, and received no phosphate in the post-trauma observation period. Reduction of serum phosphate was found in all groups after trauma. In skeletal muscle, decreased levels of HEP were observed after major trauma, whether or not phosphate supplement was given. Following minor trauma there was no alteration of muscle HEP. In red-cells, 2,3-DPG decreased slightly only after major trauma in pigs without phosphate supplement. Conclusions from the study were that hypophosphataemia was associated with cellular metabolic disturbance only after major trauma. In red-cells this disturbance seemed to be prevented by administration of phosphate. In skeletal muscle the phosphate supplement could not prevent fall in HEP levels, possibly because of a post-traumatic insulin resistance in muscle tissue, which may prevent uptake of phosphate into muscle cells.     

Normal milk composition in lactating X-linked hypophosphatemic mice despite continued hypophosphatemia

Summary Patients with X-linked hypophosphatemia and mice bearing theHyp gene have reduced renal tubular reabsorption of phosphate and an osteomalacic bone disease. To test if altered phosphate transport also exists in the mammary glands, milk was analyzed from normal (n=9) and heterozygousHyp (n=8) mice 14 days after giving birth. Inorganic phosphate, total phosphate, calcium, magnesium, sodium, and potassium were measured; percent cream, fat, water, and nonfat organic solids were measured; and protein was measured. No significant differences (NSD) were found except for greater sodium inHyp milk. There was also NSD in litter weight. The lactatingHyp had a lower body weight and remained hypophosphatemic relative to lactating normals, but both groups had higher plasma phosphate than nonlactating controls of the same genotype. The data suggest thatHyp mice can accumulate a normal amount of phosphate in their milk despite the plasma phosphate being two-thirds of normal. These data, with other recent reports of different organ systems, suggest that the altered phosphate transport activity may be restricted to the kidney.     

The renal phosphate transport defect in normal mice parabiosed to X-linked hypophosphatemic mice persists after parathyroidectomy

The X-linked hypophosphatemic (Hyp) mouse is a model for human X-linked hypophosphatemia. Surgical joining of normal to Hyp mice by parabiosis results in the normal mice developing low renal retention of phosphate and hypophosphatemia. These results suggest a humoral component to the renal defect. To test whether this component could be parathyroid hormone, surgical parathyroidectomy (PTX) or sham surgery was performed in mice 3 weeks after parabiotic union (n greater than 20 per group). After an overnight fast, PTX mice were hypocalcemic and hyperphosphatemic relative to sham-operated control mice. PTX normal mice joined to PTX Hyp mice were significantly lower in plasma phosphate and higher in fractional excretion of phosphate [U/P phosphate/(U/P creatinine)] when compared with PTX normal mice joined to other PTX normals. To test for more specific evidence of altered renal transport function, renal brush-border membrane vesicles (BBMV) were prepared from these mice, and phosphate and glucose uptakes were measured. The phosphate/glucose transport ratio was lower in BBMV from Hyp mice, joined to either normal mice or to Hyp mice, when compared with that from normal-normal pairs. Moreover, BBMV from normal mice joined to Hyp mice had a significantly lower phosphate/glucose uptake ratio than BBMV from normal mice joined to other normal mice, and their activity approached that of BBMV derived from Hyp mice. Glucose uptake in BBMV was unaffected by parabiosis or genotype. In summary, parabiosis of normal mice to Hyp mice resulted in the development of phosphaturia and decreased BBMV phosphate transport in the normal mice. The persistence of the phosphate transport defect in parathyroidectomized mice suggests that parathyroid hormone is not the humoral factor contributing to these results.     

Compartment syndrome in a patient with X-linked agammaglobulinaemia and ecthyma gangrenosum. Case report.

Compartment syndrome is a surgical emergency that requires immediate decompression. We know of no documented cases that describe ecthyma gangrenosum as a primary cause of compartment syndrome. We present a case of a baby with x-linked agammaglobulinaemia who developed compartment syndrome associated with systemic Pseudomonas aeruginosa infection and ecthyma gangrenosum of the leg. He was treated by debridement and fasciotomies followed by primary closure and skin grafting and made an uneventful recovery.