A case of familial tumoral calcinosis/hyperostosis-hyperphosphatemia syndrome due to a compound heterozygous mutation in GALNT3 demonstrating new phenotypic features.

SUMMARY: A new case of familial tumoral calcinosis (FTC)/hyperostosis-hyperphosphatemia syndrome (HHS) due to a novel compound heterozygous mutation in N-acetylgalactosaminyltransferase 3 (GALNT3) and with new phenotypic findings is presented. The response in serum phosphate and fibroblast growth factor 23 (FGF23) to medical treatment is detailed. This case expands the genotype and phenotype of FTC/HHS and gives insight into its treatment and pathophysiology. INTRODUCTION: FTC and HHS are caused by mutations in FGF23, GALNT3, or KLOTHO. They are characterized by hyperphosphatemia, increased phosphate reabsorption, and elevated or inappropriately normal serum 1,25-dihydroxyvitamin D(3) (1,25-D(3)); FTC is associated with calcific masses, and HHS with diaphyseal hyperostosis. METHODS: A 36-year-old woman presented with abnormal dental X-rays at age 12 and was hyperphosphatemic at 22. She underwent radiographic, biochemical and genetic testing, and medical treatment. RESULTS: Serum phosphorus was 7.3 mg/dL (2.5-4.8), TmP/GFR 6.99 mg/100 mL (2.97-4.45), 1,25-D(3) 35 pg/mL (22-67). Radiographs revealed tooth anomalies, thyroid cartilage calcification, calcific masses in vertebral spaces, calcification of the interstitial septa of the soft tissue in the lower extremities, and cortical thickening of the long bones. Her total hip Z score was 1.9. C-terminus serum FGF23 was 1,210 RU/mL (20-108), but intact FGF23 was 7.4 pg/mL (10-50). DNA sequencing determined she was a compound heterozygote for mutations in GALNT3. Treatment with niacinamide and acetazolamide decreased TmP/GFR and serum phosphate, which was paralleled by a decrease in serum C-terminus FGF23. CONCLUSIONS: This case broadens the spectrum of phenotypic and genotypic features of FTC/HHS and suggests treatments to decrease renal phosphate reabsorption in the setting of a low intact FGF23.

Functional, structural and molecular aspects of diastolic heart failure in the diabetic (mRen-2)27 rat.

OBJECTIVE: Diabetic cardiomyopathy is an increasingly recognized cause of cardiac failure despite preserved left ventricular systolic function. Given the over-expression of angiotensin II in human diabetic cardiomyopathy, we hypothesized that combining hyperglycaemia with an enhanced tissue renin-angiotensin system would lead to the development of diastolic dysfunction with adverse remodeling in a rodent model. METHODS: Homozygous (mRen-2)27 rats and non-transgenic Sprague Dawley (SD) rats were randomized to receive streptozotocin (diabetic) or vehicle (non-diabetic) and followed for 6 weeks. Prior to tissue collection, animals underwent pressure-volume loop acquisition. RESULTS: Diabetic Ren-2 rats developed impairment of both active and passive phases of diastole, accompanied by reductions in SERCA-2a ATPase and phospholamban along with activation of the fetal gene program. Structural features of diabetic cardiomyopathy in the Ren-2 rat included interstitial fibrosis, cardiac myocyte hypertrophy and apoptosis in conjunction with increased activity of transforming growth factor-beta (p<0.01 compared with non-diabetic Ren-2 rats for all parameters). No significant functional or structural derangements were observed in non-transgenic, SD diabetic rats. CONCLUSION: These findings indicate that the combination of enhanced tissue renin-angiotensin system and hyperglycaemia lead to the development of diabetic cardiomyopathy. Fibrosis, and myocyte hypertrophy, a prominent feature of this model, may be a consequence of activation of the pro-sclerotic cytokine, transforming growth factor-beta, by the diabetic state.

Molecular dissection of Ca2+ efflux in immortalized proximal tubule cells.

Plasma membrane Ca(2+)-ATPase (PMCA) and the Na+/Ca2+ exchanger participate in regulating cell function by maintaining proper intracellular Ca2+ concentrations ([Ca2+]i). In renal epithelial cells these proteins have been additionally implicated in cellular calcium absorption. The purpose of the present studies was to determine the Ca2+ extrusion mechanisms in cells derived from the proximal tubule. Homology-based RT-PCR was used to amplify PMCA transcripts from RNA isolated from mouse cell lines originating from the S1, S2, and S3 proximal tubule segments. S1, S2, and S3 cells exhibited only PMCA1 and PMCA4 products. PCR product identity was confirmed by sequence analysis. Northern analysis of proximal tubule cell RNAs revealed appropriate transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4, but were negative for PMCA2 and PMCA3. Western analysis with a monoclonal antibody to PMCA showed that all proximal cell lines expressed a reacting plasma membrane protein of 140 kD, the reported PMCA molecular mas. Na+/Ca2+ exchanger (NCX1) mRNA expression, analyzed by RT-PCR, protein expression by Western analysis, and functional exchange activity were uniformly absent from all proximal tubule cell lines. These observations support the idea that immortalized cells derived from the proximal tubule express PMCA1 and PMCA4, which may serve as the primary mechanism of cellular Ca2+ efflux.

Na+/Ca2+ exchange in rat osteoblast-like UMR 106 cells.

Ca2+ efflux from osteoblasts is thought to be mediated by Na+/Ca2+ exchange and by a plasma membrane Ca(2+)-ATPase. The presence of plasma membrane Na+/Ca2+ exchange was determined in rat UMR 106 osteosarcoma cells by functional and molecular studies. Na+/Ca2+ exchange activity was tested by measuring changes of [Ca2+]i in single cells. After Na+ loading the cells and removing extracellular Na+, the direction of exchange was reversed and [Ca2+]i increased by 100%. Multiple isoforms of the NCX1 gene product, encoding plasma membrane Na+/Ca2+ exchangers, were cloned from UMR 106 cells and a sample of primary human osteoblasts using homology-based RT-PCR. Isoforms NACA3, NACA7, and NACA10 were found in UMR 106 cells, whereas human osteoblasts expressed NACA3 and NACA7. Transcripts for NCX2 and the Na+/Ca2+, K+ exchanger were not detected. Northern analysis of UMR 106 cells with a probe to the NCX1 gene product revealed the presence of a transcript of 7 kb, the size of the exchanger message. Western analysis of UMR 106 cell membrane preparations with a polyclonal antibody specific for the NCX1 exchanger showed the presence of reacting proteins consistent with the reported masses of the exchanger at 125 and 85 kD. These results demonstrate Na(+)-dependent Ca2+ efflux from UMR 106 cells and the presence of several NACA isoforms in UMR 106 and primary human osteoblasts.

Linkage of structure at the proximal femur to chromosomes 3, 7, 8, and 19.

Risk for osteoporotic fracture is determined in part by femoral structure, which is under genetic control. We conducted a genome scan in 638 sister-pairs for structure phenotypes. Significant evidence of linkage was detected with several chromosomal regions, including confirmation of our prior linkage findings. Bone strength and resistance to fracture at the proximal femur is determined in part by structural variables. We previously reported that several structural variables, including pelvic axis length, femur axis length, femur head width, and femur midshaft width, had significant or suggestive linkage to regions of chromosomes 3, 4, 5, 7, 9, 17, and 19 in a sample of 309 white premenopausal sister pairs. We now report the results of a genome-wide linkage analysis of femoral structure variables in 437 white and 201 black healthy premenopausal sister pairs, of which 191 white pairs overlapped with our previously published sample. Multipoint quantitative linkage analysis was performed using microsatellite markers genotyped throughout the genome. In the current sample, linkage of femoral structure to chromosomes 3, 7, and 19 was confirmed in the white sister pairs, and a new linkage to chromosome 8 was identified. There was linkage at chromosome 3 to femoral head width (logarithm of the odds [LOD] = 5.0) and femur shaft width (LOD = 3.6). On chromosome 19, there was linkage to femoral neck axis length (LOD = 3.2); on chromosome 7, to femoral head width (LOD = 5.0); and on chromosome 8, to femoral head width (LOD = 6.0). The current findings emphasize the importance of increasing sample size to replicate linkage findings and identify new regions of linkage.

Locus heterogeneity of autosomal dominant osteopetrosis (ADO).

Autosomal dominant osteopetrosis (ADO), is a heritable disorder that results from a failure of osteoclast-mediated bone resorption. The etiology of the disorder is unknown. A previous linkage study of one Danish family mapped an ADO locus to chromosome 1p21. We have studied two families from Indiana with ADO. The present study sought to determine if the ADO gene in these families was also linked to chromosome 1p21. We used six microsatellite repeat markers, which demonstrated linkage to the 1p21 ADO locus in the Danish study, to perform linkage analysis in the new kindreds. Multipoint analysis excluded linkage of ADO to chromosome 1p21 (logarithm of the odds score < -7.00) in both families. In addition, no haplotype segregated with the disorder in either family. In summary, the present investigation ruled out linkage of ADO to chromosome 1p21 in two families from Indiana. Our results demonstrate that there is locus heterogeneity of this disorder; therefore, mutations in at least two different genes can give rise to the ADO phenotype.

The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting.

The gene mutated in autosomal dominant hypophosphatemic rickets (ADHR), a phosphate wasting disorder, has been identified as FGF-23, a protein that shares sequence homology with fibroblast growth factors (FGFs). Patients with ADHR display many of the clinical and laboratory characteristics that are observed in patients with oncogenic hypophosphatemic osteomalacia (OHO), a disorder thought to arise by the secretion of a phosphate wasting factor from different mesenchymal tumors. In the present studies, we therefore investigated whether FGF-23 is a secreted factor and whether it is abundantly expressed in OHO tumors. After transient transfection of OK-E, COS-7, and HEK293 cells with the plasmid encoding full-length FGF-23, all three cell lines efficiently secreted two protein species into the medium that were approximately 32 and 12 kDa upon SDS-PAGE and subsequent Western blot analysis using an affinity-purified polyclonal antibody to FGF-23. Furthermore, Northern blot analysis using total RNA from five different OHO tumors revealed extremely high levels of FGF-23 mRNA, and Western blot analysis of extracts from a sixth tumor detected the 32 kDa FGF-23 protein species. In summary, FGF-23, the gene mutated in ADHR, is a secreted protein and its mRNA is abundantly expressed by several different OHO tumors. Our findings indicate that FGF-23 may be a candidate phosphate wasting factor, previously designated "phosphatonin".

Molecular cloning of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene.

The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family of enzymes initiates mucin-like O-glycosylation of specific proteins. Using exon-prediction analysis on genomic sequence from human chromosome 12p13.3, we identified novel exons that shared significant homology with the ppGaNTases. cDNA library screening and RT-PCR produced the complete coding sequence of a novel human ppGaNTase family member, designated GalNAc-T8. The open reading frame (ORF) of GalNAc-T8 codes for a 637 amino acid, type-II membrane protein that is 45-60% identical to the other mammalian ppGaNTases. GalNAc-T8 shares high homology within the functional regions of the known ppGaNTases; however, the enzyme possesses a novel residue substitution within a characteristic motif of the catalytic domain. Northern analysis of multiple human tissue mRNAs demonstrated that the 5.0 and 2.1kb GalNAc-T8 transcripts are widely expressed. The metabolic disorder autosomal dominant hypophosphatemic rickets (ADHR) was previously mapped to the region of chromosome 12p13.3 in which GalNAc-T8 resides. Using a positional-candidate strategy for identifying the ADHR gene, GalNAc-T8 was subjected to mutational analysis in DNA from ADHR individuals. We detected multiple polymorphisms in the human GalNAc-T8 ORF, but did not find ADHR mutations. In summary, these studies identified the human GalNAc-T8 gene, as well as multiple genomic polymorphisms that will be useful for further understanding the structure-function relations of the ppGaNTases.

Resolution of severe, adolescent-onset hypophosphatemic rickets following resection of an FGF-23-producing tumour of the distal ulna.

Oncogenic hypophosphatemic osteomalacia (OHO) is an uncommon hypophosphatemic syndrome characterized by bone pain, proximal muscle weakness and rickets. It has been postulated that OHO results from overproduction of a humoral phosphaturic factor by an occult tumour. Recently, some OHO tumours have been shown to elaborate fibroblast growth factor-23 (FGF-23), which causes renal phosphate wasting when administered to mice. The purpose of this study was to undertake detailed investigations to confirm the diagnosis of OHO in a pediatric patient and to document the biochemical, radiographic and bone histological phenotype before and after tumour removal. We describe an 11-year-old, previously healthy girl with significant pain and functional disability associated with hypophosphatemic rickets. Circulating 1,25-(OH)(2) vitamin D was very low (14 pM; N: 40-140) while the FGF-23 serum level was markedly elevated [359.5 reference units (RU)/ml, N: 33-105]. An iliac bone biopsy revealed severe osteomalacia, but periosteocytic lesions, as are typical for X-linked hypophosphatemic rickets, were not seen. Sequence analyses of the PHEX and FGF23 genes were normal. A radiographic skeletal survey revealed a small exostosis of the left, distal ulnar metaphysis. A tumour was subsequently removed from this site and the pathology was consistent with benign, fibro-osseous tissue. Serum FGF-23 was normal when measured at 7 h post-operatively, while serum phosphate reached the low-normal range at 16 days following surgery. An iliac bone biopsy taken 5 months after the operation showed improvement, but not yet resolution, of the osteomalacia. Biochemical parameters of bone and mineral metabolism suggested that complete resolution of the osteomalacia was not achieved until 12 months following surgery. One year after tumour removal, the patient was pain-free and had resumed a normal level of activity. The rapid normalization of FGF-23 levels following removal of a benign tumour and the subsequent improvement in the biochemical and histological parameters of bone and mineral metabolism suggest that FGF-23 played a key role in this girl's disease.

A case-control study to assess possible triggers and cofactors in chronic fatigue syndrome.

PURPOSE: To assess possible triggers and cofactors for chronic fatigue syndrome (CFS) and to compare levels of selected cytokines between cases and an appropriately matched control group. PATIENTS AND METHODS: We conducted a case-control study of 47 cases of CFS obtained through a regional CFS research program maintained at a tertiary care medical center. One age-, gender-, and neighborhood-matched control was identified for each case through systematic community telephone sampling. Standardized questionnaires were administered to cases and controls. Sera were assayed for transforming growth factor-beta (TGF-beta), interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and antibody to Borrelia burgdorferi and Babesia microti. RESULTS: Cases were more likely to have exercised regularly before illness onset than controls (67% versus 40%; matched odds ratio (MOR) = 3.4; 95% CI = 1.2 to 11.8; P = 0.02). Female cases were more likely to be nulliparous prior to onset of CFS than controls (51% versus 31%; MOR = 8.0; 95% CI = 1.03 to 170; P = 0.05). History of other major factors, including silicone-gel breast implants (one female case and one female control), pre-morbid history of depression (15% of cases, 11% of controls) and history of allergies (66% of cases, 51% of controls) were similar for cases and controls. However, cases were more likely to have a diagnosis of depression subsequent to their diagnosis of CFS compared to a similar time frame for controls (MOR = undefined; 95% CI lower bound = 2.5; P < 0.001). Positive antibody titers to B burgdorferi (one case and one control) and B microti (zero cases and two controls) were also similar. CONCLUSIONS: Further investigation into the role of prior routine exercise as a cofactor for CFS is warranted. This study supports the concurrence of CFS and depression, although pre-morbid history of depression was similar for both groups.

Disorders of phosphate metabolism.

Correct identification of the disorders of hypophosphatemia and hyperphosphatemia is important for determining therapy. Further research will provide insights into normal phosphate homeostasis, a complex and fascinating process.

Prospective surveillance of Haemophilus influenzae type b disease in Dallas County, Texas, and in Minnesota.

Among children less than 12 years of age residing in Dallas County, Texas, and in the state of Minnesota we conducted prospective, active surveillance of invasive Haemophilus influenzae disease. During 18 months, 616 cases were identified, of which 600 were caused by type b organisms. The annual incidence of disease was significantly greater in Dallas than in Minnesota (109 v 68/100,000 children younger than 5 years of age, P less than .001) and was greater in Dallas, even when rates for white children in the two regions were compared (P less than .001). Other regional differences were observed. In Dallas, a larger proportion of cases were in children attending day-care centers (27% compared with 12% in Minnesota, P less than .001) and more patients attended day care for greater than 40 h/wk (56% compared with 30% in Minnesota, P less than .001). Outer membrane protein subtyping of isolates revealed that in Dallas 6U isolates were associated significantly with cases in black children who attended day care. In Minnesota, but not in Dallas, isolates with subtype 1H were associated significantly with cases in children in day care. These data indicate that there are regional differences in the epidemiology of type b Haemophilus disease that may relate to differences in strains, day-care practices, or other unknown cultural or environmental factors. Finally, because only 15% of systemic Haemophilus disease in these regions occurred in children in the age groups recommended for vaccination (24 to 59 months), the new Haemophilus type b polysaccharide vaccine is expected to have a limited impact on the overall incidence of disease.

Physician attitudes and practices regarding universal infant vaccination against hepatitis B infection in Minnesota: implications for public health policy.

Physician attitudes and practices regarding universal infant vaccination against hepatitis B virus infection in Minnesota were assessed approximately 1 year after publication of the Immunization Practices Advisory Committee recommendations. Four-hundred eighteen Minnesota family physicians and pediatricians were sent self-administered questionnaires, with follow-up by telephone. Among physicians who provide care to infants, 67 (29%) of 234 family physicians and 29 (50%) of 58 pediatricians routinely offered hepatitis B vaccine to all infants (overall 33%) (P = 0.002). The recommendations of the Immunization Practices Advisory Committee, the American Academy of Pediatrics and the American Academy of Family Physicians had the greatest positive influence on physicians' opinions regarding routine hepatitis B vaccination. The factors with the greatest negative influence on their opinions were the low prevalence of hepatitis B virus infection in Minnesota and the addition of three injections to the current childhood immunization schedule. Universal infant hepatitis B vaccination remains controversial among Minnesota family physicians and pediatricians. We believe, given the variability in hepatitis B virus incidence and prevalence in the United States and the relatively low risk of most infants, that a single national policy based solely on universal infant immunization may be difficult to implement.

Outbreak of erythromycin-resistant staphylococcal conjunctivitis in a newborn nursery.

We investigated an outbreak of erythromycin-resistant Staphylococcus aureus conjunctivitis in a hospital newborn nursery that used erythromycin eye ointment to prevent ophthalmia neonatorum. Cases occurred in 2 clusters; 20 (14%) of 146 infants in the nursery developed conjunctivitis from July through October, 1987; and 5 (7%) of 69 infants in the nursery developed conjunctivitis during April and May, 1988. A case-control study of the first cluster demonstrated that culture-confirmed cases were more likely than controls to have received prophylactic erythromycin eye ointment or their initial bath from one nurse (odds ratio, 9.0; P = 0.01) or to have been delivered by one physician (odds ratio, 12.7; P = 0.03). The nurse was the only staff person to have a nasopharyngeal culture which yielded erythromycin-resistant S. aureus. Control measures, instituted in October, 1987, included using silver nitrate drops instead of erythromycin eye ointment for prophylaxis; however, in January, 1988, the hospital resumed use of erythromycin eye ointment. No additional cases were identified until mid-April, 1988, when the second cluster of cases occurred. At that time the hospital reinstituted the use of silver nitrate and no additional cases were identified. This investigation illustrates the potential for conjunctival infection with an antimicrobial-resistant pathogen when antimicrobials are used to prevent ophthalmia neonatorum.

Estimating immunization coverage from school-based childhood immunization records.

To determine the accuracy of school-based childhood immunization records and to describe the effects of their use on estimates of community-wide immunization coverage, we verified the immunizations to 72 months of age for children born in 1986 to residents in Dallas County, TX, and in Minnesota. Verified immunizations were compared with those documented in the school record. Major transcription errors accounted for fewer than 1% of discrepancies between school and provider records. For 99 subjects with 987 verified immunizations in Minnesota, age-appropriate immunization coverage estimated from the school records was within two percent of actual coverage. For 86 subjects with 981 verified immunizations in Dallas County, age-appropriate immunization coverage from the school records underestimated actual coverage by as much as 21%. The primary factor explaining the underestimate in Dallas was incomplete school immunization records for 33 (38%) subjects and 126 (13%) immunizations. Selective recording of immunizations related to the minimum state requirements in Texas contributed to incomplete school records in Dallas County. Verification of the completeness of records selected to estimate immunization coverage is essential if the estimates are used to monitor trends or to make public policy decisions.

A statewide survey of immunization rates in Minnesota school age children: implications for targeted assessment and prevention strategies.

BACKGROUND: A retrospective statewide immunization survey of the 69115 Minnesota children who entered kindergarten in 1992 was conducted. METHODS: Information was collected from school immunization records on date of birth, dates of vaccination for each dose of vaccine, address of residence and race/ethnicity (when available). Immunization rates were assessed retrospectively for each month of a child's life from 2 to 48 months of age. Age-appropriate immunization was defined as receipt of all scheduled vaccines within 30 days of the recommended age. RESULTS: Immunization levels varied by vaccine, age of the child and race/ethnicity. For example at 19 months of age, 73% of students had received measles, mumps, rubella vaccine; however, only 39% had received their fourth dose of diphtheria, tetanus and pertussis vaccine. White, non-Hispanic students consistently had higher vaccination rates than children of other racial/ ethnic groups. For example 45% of white, non-Hispanic students were age-appropriately vaccinated at 16 months of age compared with 25% of Blacks, 30% of American Indians, 30% of white Hispanics and 28% of Asian-Pacific Islanders (Mantel-Haenzel chi square, P < 0.001 for each comparison). Furthermore coverage rates frequently varied significantly by neighborhood, thereby identifying pockets of underimmunization within communities. CONCLUSION: Our data demonstrate that vaccination rates can vary substantially by age, race/ ethnicity and neighborhood. Detailed immunization assessment is necessary so that effective targeted interventions can be developed.

Identification and antisense inhibition of Na-Ca exchange in renal epithelial cells.

In summary, DCT cells express multiple isoforms of the Na-Ca exchanger and exhibit functional exchange, and antisense oligonucleotides to a downstream region of the exchanger transcript inhibit activity. These experiments provide direct evidence for Na-Ca exchange in DCT cells mediated by NACA2, NACA3, or NACA6.