A molecular approach to dominance in hypophosphatasia.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. The disease is highly variable in its clinical expression, because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was found, despite exhaustive sequencing of the gene, suggesting that missing mutations are harbored in intron or regulatory sequences or that the disease is dominantly transmitted. The distinction between these two situations is of importance, especially in terms of genetic counseling, but dominance is sometimes difficult to conclusively determine by using familial analysis since expression of the disease may be highly variable, with parents of even severely affected children showing no or extremely mild symptoms of the disease. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, I473F) found in patients with no other detectable mutation. Three of these mutations, G46 V, S164L, and I473F, have not previously been described. Pedigree and/or serum alkaline phosphatase data suggested possible dominant transmission in families with A99T, R167 W, and G232 V. By means of site-directed mutagenesis, transfections in COS-1 cells, and three-dimensional (3D) modeling, we evaluated the possible dominant effect of these eight mutations. The results showed that four of these mutations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effect by inhibiting the enzymatic activity of the heterodimer, whereas the four others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of the disease. Analysis of the 3D model of the enzyme showed that mutations exhibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular biological function such as dimerization, tetramerization, or membrane anchoring.

Severe congenital hyperthyroidism caused by a germ-line neo mutation in the extracellular portion of the thyrotropin receptor.

Gain of function mutations in the TSH receptor (TSHR) have been identified as the molecular basis for congenital and acquired forms of autonomous thyroid function. Herein, we report the molecular characterization of a case of severe congenital hyperthyroidism with a history of hyperthyroidism in the paternal aunt and the paternal grandmother, who were both found to be heterozygous for a mutation (R528H) located in exon 10 of the TSHR gene. Functional expression of the mutant TSHR-R528H in COS-7 cells, however, did not result in constitutive activity of the TSHR. Subsequent analysis of exons 1-9 led to the detection of an additional heterozygous mutation (S281N) in the patient, but not in other family members. Interestingly, the latter mutation is located in the extracellular domain of the TSHR, and functional studies revealed a marked increase in basal cAMP levels when the mutant receptor was expressed in COS-7 cells. To address the question of whether both mutations were present on the same allele, a double mutant TSHR (S281N/R528H) was generated and characterized. These functional studies in conjunction with RT-PCR analysis of thyroid tissue obtained from subtotal thyroidectomy performed at the age of 6 yr revealed that the patient bears two distinct mutations on different alleles: the familial paternal R528H mutation to be regarded as a polymorphism and a de novo mutation (S281N) on the maternal allele accounting for the clinical picture. Thus, the main conclusions to be drawn from this case are 1) a search for mutations in cases of congenital nonautoimmune hyperthyroidism should not remain restricted to exon 10 of the TSHR gene, because germ-line gain of function mutations of the TSH receptor can be located outside of the transmembrane core of the receptor; and 2) this case illustrates the necessity for careful functional characterization of any novel mutation before a causal relationship to hyperthyroidism can be established.

Congenital hyperthyroidism.

Congenital hyperthyroidism is a very rare disease. But, for each affected child it has to be considered as a serious condition because of the negative impact of hyperthyroidism on fetal and postnatal development. If the manifestation occurs during fetal life tachycardia, cardiac arrhythmia, growth retardation and, most significant, prematurity are the consequences. Postnatal signs of hyperthyroidism are irritability, tachycardia, hypertension, poor weight gain and thyroid enlargement. Even cardiac failure may occur if hyperthyroidism is severe and treatment not adequate which explains the high early mortality rate of 16%. The main complication of persistent hyperthyroidism in the neonatal period and during infancy is craniosynostosis. Severe developmental delay or even mental retardation can be the consequence of inadequate high T4-levels during fetal and neonatal life. Congenital hyperthyroidism was first recognized in infants born to mothers with Graves' disease. The description of transplacental passage of the maternal thyroid stimulating antibodies elucidated the molecular mechanism in this major group of patients with "autoimmune congenital hyperthyroidism". In contrast to this transient, self-limited character of "autoimmune congenital hyperthyroidism", due to the clearance of maternal antibodies from the infant's circulation, some cases of persistent congenital hyperthyroidism without signs of thyroid autoimmunity have been recognized. Activating mutations in the thyroid-stimulating hormone receptor were described recently as the underlying molecular pathogenesis in this group of "non-immune congenital hyperthyroidism". Therefore the possibility of a molecular differential diagnosis of both groups of congenital hyperthyroidism now exists and opens the opportunity of optimal treatment for each patient.

X linked hypophosphataemia: treatment, height gain, and nephrocalcinosis.

The clinical data of 18 patients with X linked hypophosphataemia were analysed retrospectively. The height data were expressed as SD scores. There was no difference in the final height of patients treated with vitamin D (or 1,25-dihydroxyvitamin D) and phosphate for at least two years (n = 12) and that of 16 hypophosphataemic family members who had never been treated. The mean final SD score (-2.07) of treated patients, however, was significantly higher than the value before treatment (-2.79), which indicated an average absolute height gain of 4-4.5 cm compared with the expected height values. Six of the treated patients developed ultrasonographically detectable nephrocalcinosis with normal renal function. The daily phosphate intake and excretion of patients with nephrocalcinosis was significantly higher than that of patients with normal renal morphology. There was no difference in the doses of vitamin D between the two groups. The average urinary calcium:creatinine ratio of the two groups was similar to and below the hypercalciuric 0.6 mmol:mmol limit. The group with nephrocalcinosis, however, had a higher incidence of hypercalciuric episodes than the group without nephrocalcinosis (12 in 130 observations compared with six in 334 observations, respectively). The benefits and risks of treatment of patients with X linked hypophosphataemia must be further evaluated. The high dose of phosphate seems to be an important factor in the development of nephrocalcinosis in this group of patients.

[Multicystic kidney dysplasia]. / Die multizystische Nierendysplasie.

The clinical course of 48 children (27 boys and 21 girls) with multicystic kidney dysplasia was analysed retrospectively. The patients were seen and treated at the Children's Hospital of Medical School Hannover between 1976 to 1989. There was no familial occurrence of the disease, yet in eight families various other renal diseases could be observed. In 20 patients the diagnosis of multicystic renal dysplasia was presumed by prenatal sonographic examination, in the other patients the diagnosis was established at the age between 1 day to 12 years. The first manifestations were palpable abdominal mass (n = 16), urinary tract infection (n = 4), casually because of a sonographic evaluation for other reasons (n = 4) and vomiting (n = 2). Associated malformations were found in 18 patients: cardiac malformations (n = 6), dysplasia of the other kidney (n = 5), ureter obstruction of the other kidney (n = 3), horseshoe kidney (n = 1) and others (n = 3). There was no hypertension and serum creatinine levels were normal in those children, who did not suffer from associated malformations of the other kidney. The multicystic kidney was removed operatively in 42 patients at the age of 3 days to 9.5 years (median 4 weeks). The prognosis of multicystic kidney dysplasia depends on associated renal and other malformations.

[Cystic kidneys in children]. / Zystennieren im Kindesalter.

From 1976-1987 a total of 26 infants and children with polycystic kidney disease were treated at the Children's Hospital of the Medical School Hannover. 13 of them suffered from infantile recessive polycystic kidney disease (IRPKD), and 13 from adult dominant polycystic kidney disease (ADPKD). IRPKD was diagnosed at a median age of 0.33 years (range 1 day-13 years), ADPKD at 6.0 years (3 days-14 years). Of those with IRPKD two infants died from bacterial infection and two others developed terminal renal insufficiency at the age of 8 years, while the others are living and 1-20 years old. All those suffer from severe arterial hypertension and have reduced renal function, but only 5 developed signs of liver fibrosis. Of those with ADPKD one infant died from sepsis and renal insufficiency, while the others are well and now 2-17 years old. Only one child needs an antihypertensive treatment. The most important criteria to differentiate IRKPD and ADKPD in children are the genetic transmission, age of first manifestation, hypertension and renal function. The prognosis is much more severe in IRPKD than in ADPKD, but is not as infaust in IRPKD as often assumed.

Neonatal pyruvate dehydrogenase deficiency with lipoate responsive lactic acidaemia and hyperammonaemia.

A 2-day-old girl developed a severe lactic acidosis with a normal lactate/pyruvate ratio and hyperammonaemia. Plasma arginine and citrulline levels were below the limit of detection. In muscle total pyruvate dehydrogenase complex (PDHC) and pyruvate decarboxylase (E1) activities were reduced to a fraction of lower control values. The acute neonatal period was bridged with peritoneal dialysis, dichloroacetate therapy, supplements of arginine and branched chain amino acids, a complete vitamin B complex and lipoic acid. Lactate homeostasis responded to pharmacological supplements of lipoic acid. At age 1 year the child was hypotonic, showed severe developmental retardation, optic atrophy and cranial dysmorphism. She died aged 1 year 8 months with signs of respiratory paralysis but with normal lactate levels under assisted breathing. Pathological findings at autopsy were suggestive of Leigh syndrome, interstitial pneumonia and extensive fatty infiltration of hepatocytes. Regression analysis of data from 187 plasma amino acid determinations from the patient over a period of 1 year 8 months revealed a persistent-imbalance involving alanine, glutamic acid, glutamine, proline, citrulline and branched chain amino acids. Aspects of acute and long-term therapy in this patient and some implications of the imbalances in plasma amino acids are discussed.

Effect of cyclosporine on the renal tubular amino acid handling after kidney transplantation.

The renal tubular handling of free amino acids was studied 5-6 weeks after successful renal transplantation (tx) in 20 children treated with CsA and in 10 children treated with azathioprine (Aza). The results were compared with those of 34 control children. The amino-acid clearance studies were performed in combination with short-term inulin clearance. The CsA group revealed a mean inulin clearance of 49 +/- 16.8 ml/min/1.73 m2, the Aza group of 76.9 +/- 18.2, and the controls of 114 +/- 15.6. The plasma amino-acid concentrations were not different between CsA- and Aza-treated groups; however, most of the essential amino acids were lower in transplanted children than in controls. The decrease was correlated with the GFR. The amino-acid-clearance rates were statistically not different between both transplanted groups, but lower values than in controls were found for alanine, glycine, histidine, lysine, and phenylalanine, and significantly higher values for methionine. The fractional clearance rates of most amino acids were significantly elevated in transplanted children compared to controls. In CsA-treated patients, the fractional clearance rates of arginine, glycine, and serine were higher than in Aza-treated patients. No influence of CsA blood levels or rejection episodes on the amino-acid handling were detectable. We conclude that CsA has no specific influence on the renal handling of amino acids. Most disturbances observed depend on the graft function or may be caused by injuries to the graft following the tx procedure.

The DiGeorge syndrome. I. Clinical evaluation and course of partial and complete forms of the syndrome.

This study describes clinical signs and symptoms in 16 patients with the DiGeorge syndrome (DGS). Diagnosed on the basis of typical facial stigmata, a broad spectrum of severity is seen with respect to congenital heart disease, hypoparathyroidism and immunologic parameters. A simple index of severity is introduced that clearly differentiates complete forms of the syndrome (cDGS) with poor prognosis from partial forms of the syndrome (pDGS). Of 13 pDGS patients, 12 are still living; 8 underwent corrective heart surgery without infectious complications. Moderate to severe mental retardation is seen in all pDGS patients. Due to the lack of thymus function, immunodeficiency is a result of cDGS, whereas immunoregulatory disturbances (hypergammaglobulinaemia, high titres of specific antibody production) prevail in pDGS patients.

End stage renal failure: 14 years' experience of dialysis and renal transplantation.

One hundred and thirteen children (59 boys and 54 girls aged from 2 to 16 years) with end stage renal failure entered the renal dialysis and transplantation programme between 1972 and 1983. They were followed up until December 1985. Ninety eight children were initially treated by haemodialysis in hospital and 15 by renal transplantation. The average wait on dialysis was seven months (range 0.1-43 months). One hundred and six children were given 129 renal transplants, 32 of which were from living related donors. At the end of 1985 94 of the 113 patients (83%) were alive, 81 (72%) with functioning grafts, 11 (10%) were receiving haemodialysis in hospital, two (1%) were being treated by continuous ambulatory peritoneal dialysis, and three had been lost to follow up. The 14 years actuarial survival was 81%. Four patients receiving dialysis and 12 who had received transplants died, a mortality of 14%. The main complications of treatment were retardation of growth in 49 (43%), hypertension in 75 (66%), and osteopathy in 36 (32%). Retardation of growth could not be reversed by successful renal transplantation. Seventy two patients (88%) assessed their health as good to excellent, and 9 (12%) as poor. Patients with a functioning graft did much better than those receiving dialysis. Treatment of end stage renal failure led to full rehabilitation in most patients, and renal transplantation was more effective than dialysis.

Renal handling of uric acid under cyclosporin A treatment.

The renal handling of uric acid during cyclosporin A (CyA) treatment was investigated by clearance studies using 24-h urine collections in 28 paediatric renal transplant recipients (CyA group), and the results were compared with those of 19 renal transplanted children treated with azathioprine and prednisolone (AZA group), 35 children with chronic renal failure (CRF) and 10 children with normal renal function (N group). Serum uric acid levels were significantly higher in the CyA group (567 +/- 156 mumol/l) compared with the AZA group (378 +/- 98), the CRF group (415 +/- 119) and the N group (290 +/- 68). Mean uric acid clearances in each group measured 3.9 +/- 2.8 ml/min per 1.73 m2 (CyA), 5.6 +/- 3.4 (AZA), 4.0 +/- 2.2 (CRF) and 8.4 +/- 3.7 (N). Calculation of the net tubular uric acid reabsorption per millilitre glomerular filtration rate revealed a significantly increased value of 0.53 +/- 0.15 mumol/ml in the CyA group (P less than 0.01) compared with 0.34 +/- 0.08, 0.29 +/- 0.15 and 0.27 +/- 0.07 mumol/l for the AZA, CRF and N groups respectively. We therefore conclude that CyA treatment is associated with an increased net tubular reabsorption of uric acid, which may lead to hyperuricaemia.

Acute rejection episodes after renal transplantation in children under cyclosporin A treatment.

Thirty-two pediatric renal transplant patients receiving cyclosporin and 34 receiving azathioprine treatment (historical controls) were investigated for the occurrence of rejection episodes; their clinical symptoms and findings, time of onset, influence of donorship, relation to cyclosporin blood levels and graft function outcome were also studied. In the cyclosporin group, four grafts were lost in the 2nd year, while in the azathioprine group five grafts were lost within the first 5 weeks after transplantation due to acute irreversible rejection. Clinical signs of rejection episodes under cyclosporin were mild and usually presented a silent increase of serum creatinine. First rejection episodes occurred later in patients treated with cyclosporin than in azathioprine-treated patients (50% probability after 7 weeks as opposed to 2 weeks). The percentage of patients receiving cyclosporin who had experienced no rejection episodes was 18.8% as opposed to 11.8% of patients receiving azathioprine. The lowest incidence of rejection episodes was observed in patients with living related grafts receiving cyclosporin treatment, 75% of whom were free of rejection episodes after 2 years. Cyclosporin blood levels below 400 ng/ml were observed in 74% of rejection episodes. Biopsies were often used to differentiate between cyclosporin nephrotoxicity and rejection when the cyclosporin levels were above 400 ng/ml. Both treatment groups exhibited a parallel decline in graft function, which correlated with the number of rejection episodes.

Somatic growth after kidney transplantation. Beneficial effect of cyclosporine in comparison with conventional immunosuppression.

Growth performance was evaluated in 69 children, aged 3 to 16 years, who had undergone kidney transplantation between 1974 and 1984. Forty children (21 boys, 19 girls) who received transplants before September 1982 were treated conventionally with azathioprine and high-dose prednisolone; 29 children (13 boys, 16 girls) who received transplants after September 1982 were treated with cyclosporine and low-dose prednisolone. The mean (+/- SD) survival times of grafts in the azathioprine and cyclosporine groups were 6.0 +/- 2.1 years and 1.4 +/- 0.5 years, respectively. Height and bone age were evaluated at the time of transplantation, one year after transplantation, and at reevaluation in December 1985. Growth rates expressed by standard deviation scores (SDS) declined in the azathioprine group for boys (mean, -2.2 at transplantation, -2.5 after one year, and -2.8 in December 1985) and girls (-2.1, -2.4, and -2.7) and improved in the cyclosporine group for boys (-2.5, -2.2, and -2.1) and girls (-2.2, -1.9, and -1.8). The difference between both groups one year after transplantation was significant. This trend continued beyond the first year after transplantation. Graft function was better in the azathioprine group than in the cyclosporine group. Bone age in December 1985 was less retarded in the cyclosporine group, but this could have been related to the shorter observation time. We conclude that somatic growth after kidney transplantation is below expected rates under azathioprine-prednisolone immunosuppression but is significantly better under cyclosporine-prednisolone immunosuppression.

Renal function after kidney transplantation in children. A comparison of conventional immunosuppression with cyclosporine.

Clearance studies were performed in 32 transplanted children treated with CsA in combination with low-dose prednisolone (CsA group), and the results were compared with those of 29 children transplanted earlier and treated with azathioprine and prednisolone (CIS group). Serum creatinine and urea levels 6 weeks and 1 year after transplantation (Tx) were significantly higher in the CsA than in the CIS group. Clearance studies 6 weeks after Tx exhibited significantly lower rates in the CsA group: Cin = 47 +/- 16.5 versus 83 +/- 25 ml/min/1.73 sqm, CPAH = 271 +/- 110 versus 503 +/- 181 ml/min/1.73 sqm (P less than 0.001). The filtration fractions were not different (19.1 versus 17.1%). The tubular phosphate reabsorption per ml GFR (Tp/Cin) was only slightly lower in the CsA group (0.76 +/- 0.23 mumol/ml versus 0.93 +/- 0.29; P = 0.09). The endogenous glucose clearance rates were equally elevated in both groups and returned to normal after 1 year. The creatinine clearance (Ccr) had dropped in both groups by a mean for 13 ml/min/1.73 sqm between 6 weeks and 1 year after Tx. No correlation was found between the Ccr and the CsA blood levels, but Ccr was inversely correlated with the number of rejection episodes (r = -0.72, P = 0.001). In conclusion, renal allografts in CsA-treated children exhibited a significantly lower function than in CIS-treated children. The effect was related to the global kidney function without any signs of additional tubular toxicity and was apparent within the first weeks after Tx. Thereafter, the decline in graft function was comparable in both groups and could not be related to CsA treatment.

Thromboembolic complications in children with nephrotic syndrome. Risk and incidence.

Coagulation studies were performed in 16 children with steroid responsive minimal change nephrotic syndrome in order to elucidate the incidence of thromboembolic complications. Fibrinogen and alpha 2-macroglobulin concentrations were inversely correlated with serum albumin concentrations, antithrombin III correlated positively (p less than 0.001). Factor VIII:R:AG concentration was elevated. Coagulation disturbances in children are not less severe than in adults with nephrotic syndrome. Combined scintigraphic pulmonary ventilation and perfusion studies were employed in 26 children to detect noninvasively events of pulmonary embolism, respectively their residual changes. The lung scintigraphic investigation demonstrated a pattern consistent with pulmonary embolism in 7 patients (27.9%), residual changes in 10 (38.5%) and normal findings in 9 (34.9%). The incidence of thromboembolic complications in children with severe nephrotic syndrome is as high as reported for adults. Pulmonary symptoms may well be due to pulmonary embolism.

[Mid C regional parathyroid hormone in the clinical workup: diagnostic value in extrarenal (primary) and renal (secondary) hyperparathyroidism]. / Mitt-C-regionales Parathormon in der klinischen Routine: Diagnostische Wertigkeit beim extrarenalen (primären) und renalen (sekundären) Hyperparathyreodismus.

The selective determination of mid-C-regional parathyroid hormone (mid-C-PTH) in combination with other laboratory parameters is a reliable tool for diagnosis and treatment of extra-renal (primary) and renal (secondary) hyperparathyroidism. Early stages, which show either high-to-normal serum calcium and elevated mid-C-PTH or increased serum calcium but normal mid-C-PTH, can be distinguished from overt hyperparathyroidism. Alkaline phosphatase (AP) activity and mid-C-regional PTH provide biochemical confirmation of histologically classified renal osteodystrophy. Since the index AP X PTH signifies osseous changes in dialysis patients at an early stage, therapeutic regimens may be altered without additional invasive procedures. After renal transplantation mid-C-PTH normalizes and serum creatinine decreases. Increased mid-C-PTH in patients with normal renal graft function reflects autonomous PTH secretion, which requires careful monitoring to prevent PTH-induced hypercalciuria.

Multiple pre- and postreceptor defects in pseudohypoparathyroidism (a multicenter study with twenty four patients).

Three different pathophysiological mechanisms are probably responsible for hereditary pseudohypoparathyroidism: 1) a defect at the prereceptor-level, 2) a defective membrane N-protein accounting for diminished second messenger production, and 3) a defect in the cytosolic response to the hormone. In a cooperative, study 24 patients (mean age, 13 yr; range, 3-23 yr, 8 girls, 16 boys) receiving vitamin D metabolites (5,000-80,000 U/day) were examined and compared to a control group of 36 normal children. Immunoreactive N-terminal PTH (N-PTH), mid-C-regional PTH (mid-C-PTH), intact PTH and bio-PTH, vitamin D metabolites, and serum calcium and phosphate, alkaline phosphatase activity, and the N-protein activity of erythrocyte membranes were measured in each subject. By clinical and biochemical criteria three groups were differentiated. Eight patients had the completely expressed features of Albright's Hereditary Osteodystrophy (AHO+), including brachydactyly and/or sc calcifications, and increased N-PTH, mid-C-PTH, and alkaline phosphatase activity. Bio-PTH, intact PTH, and N-protein were normal. Nine additional patients with complete (AHO+) had elevated levels of bio-PTH, N-PTH, and mid-C PTH, normal hydroxylation of vitamin D, but decreased N-protein activity. Seven patients with pseudohypoparathyroidism had no features of AHO (AHO-), no increase of urinary cAMP excretion after exogenous PTH, normal PTH peptide levels and N-protein activity, but elevated 25-hydroxyvitamin D and decreased 1,25-dihydroxyvitamin D concentrations. In conclusion, we identified three subpopulations of PsHP: group a had a dissociation of N-PTH and bio-PTH suggesting a defective N-PTH causing renal resistance, whereas their bones respond to PTH. Group b had defective N-protein causing generalized PTH resistance. Group c was characterized by high 25-hydroxyvitamin D and relatively low 1,25-dihydroxyvitamin D levels, thus providing evidence for a defect in the cytosolic interaction of the two different second messengers for PTH, cAMP, and calcium.

Scintigraphic imaging of renal angioma.

Renal angiomyolipomas are frequent findings in patients with tuberous sclerosis. They must be localized before a biopsy is done. Radionuclide blood pool scintigraphy is a simple noninvasive procedure for the localization of hemangiomatous tumours. A patient is presented with four renal angiomas shown by this technique.