Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings.

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive progeroid syndrome, characterized by mandibular hypoplasia, acroosteolysis affecting distal phalanges and clavicles, delayed closure of the cranial sutures, atrophic skin, and lipodystrophy. Recently, mutations in lamin A/C (LMNA) and zinc metalloprotease (ZMPSTE24), involved in post-translational processing of prelamin A to mature lamin A, have been identified in MAD kindreds. We now report novel compound heterozygous mutations in exon 1 (c.121C>T; p.Q41X) and exon 6 (c.743C>T; p.P248L) in ZMPSTE24 in two Japanese sisters, 7- and 3-year old, with severe MAD and characteristic facies and atrophic skin. The older sister had lipodystrophy affecting the chest and thighs but sparing abdomen. Their parents and a brother, who were healthy, had heterozygous mutations. The missense mutation, P248L, was not found in 100 normal subjects of Japanese origin. The mutant Q41X was inactive in a yeast halo assay; however, the mutant P248L retained near normal ZMPSTE24 activity. Immunoblots demonstrated accumulation of prelamin A in the patients' cell lysates from lymphoblasts. The lymphoblasts from the patients also revealed less intense staining for lamin A/C on immunofluorescence. We conclude that ZMPSTE24 deficiency results in accumulation of farnesylated prelamin A, which may be responsible for cellular toxicity and the MAD phenotype.

Hypercalcemia associated with infantile fibrosarcoma producing parathyroid hormone-related protein.

We describe a 7-month-old boy who manifested severe hypercalcemia associated with mesenchymal neoplasm. A huge hypervascular tumor on the neck had been detected in prenatal ultrasonography. Surgical removal of the entire tumor at birth was not indicated, because the tumor was diagnosed as hemangioma. Chemotherapy and radiotherapy were attempted, but there was no effect on tumor growth. When the infant was 6 months old, the serum calcium level increased rapidly, associated with the expansion of the tumor. Hypophosphatemia due to phosphaturia was also observed. Serum PTH was undetectable, whereas the serum concentration of carboxyl-terminal (C-terminal) fragments of PTH-related protein (PTH-rP) was markedly elevated. Northern blot analysis and immunostaining demonstrated the expression of PTH-rP in the tumor. The tumor was transplantable to nude mice and caused elevation of circulating PTH-rP in the animals. Histological examination of the patient's bone revealed an increased number of osteoclasts. These findings were consistent with humoral hypercalcemia of malignancy caused by the excess production of PTH-rP. The tumor was identified histologically as infantile fibrosarcoma, which has not been reported as a cause of humoral hypercalcemia of malignancy to date. The expression of PTH/PTH-rP receptor messenger ribonucleic acid was detected in the tumor by the RT-PCR, suggesting that PTH-rP may have exerted its effect in the tumor in an autocrine/paracrine manner. In addition to the systemic effect of PTH-rP manifested as hypercalcemia, the PTH-rP secreted from the neoplasm could have been a local factor involved in the growth of the tumor.

Analysis of localization of mutated tissue-nonspecific alkaline phosphatase proteins associated with neonatal hypophosphatasia using green fluorescent protein chimeras.

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458-4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

Genes for members of the GATA-binding protein family (GATA-GT1 and GATA-GT2) together with H+/K(+)-ATPase are specifically transcribed in gastric parietal cells.

mRNAs for novel DNA-binding proteins (GATA-GT1 and GATA-GT2) recognizing the (G/C)PuPu(G/C)NGAT(A/T)PuPy sequence and H+/K(+)-ATPase (proton pump) alpha subunit were detected in parietal cells of the rat gastric body mucosa by in situ hybridization. These results suggest that GATA-GT1 and GATA-GT2 together with H+/K(+)-ATPase are transcribed specifically in gastric parietal cells and that the two DNA-binding proteins may have important roles in cell specific gene regulation. Furthermore, we could detect parietal cells in different states of gene expression.

The rat intrinsic factor gene: its 5'-upstream region and chief cell-specific transcription.

A DNA segment containing the 5'-upstream region and amino terminal reading frame of the gastric intrinsic factor gene was cloned from rat and its nucleotide sequence was determined. S1 mapping demonstrated that the transcription initiation site is located downstream of the second TATA-box sequence. Similar sequence motifs to those in the pepsinogen genes transcribed in gastric chief cells were found in the deduced sequence, suggesting that the rat intrinsic factor gene is transcribed in these cells. The genes for the intrinsic factor and its homologous protein transcobalamin I were apparently derived from a common ancestoral gene, since the positions of their intron insertions as well as the amino acid residues are conserved. Northern blot hybridization showed that the gene for the intrinsic factor is transcribed in the stomach but not detectably in the intestine, kidney, testis, brain, heart, liver, lung, or spleen. In situ hybridization using radioactive complementary RNA clearly indicated that the major transcription site in gastric glands is chief cells. Different locations of expression of intrinsic factor proteins in various mammals were observed previously using antibodies: in rat parietal cells and chief cells, in mouse chief cells, and in human parietal cells. The present results clearly demonstrated the intrinsic factor mRNA mainly in chief cells of adult rats, as in mice, suggesting that transcriptional regulation of the intrinsic factor gene is essentially the same in rodents.

Rhabdomyoma of the orbit in a child.

PURPOSE: To study a case of rhabdomyoma of the orbit in a 16-month-old boy. METHOD: The child had progressive right proptosis for 1 month. He underwent a computed tomographic scan, which showed an irregular right retrobulbar mass and partial resection. RESULTS: Histologic examination disclosed well-differentiated striated muscle cells with a mixture of collagen fibers and immature striated muscle cells with centrally placed nuclei. The specimen lacked nuclear atypia, indicating benign rhabdomyoma of the orbit. During the 1 1/2-year follow-up, the patient did not receive additional treatment, and no regrowth occurred. CONCLUSION: Rhabdomyoma can occur in the orbit of a child. Because of differences in treatment, rhabdomyoma must be distinguished from rhabdomyosarcoma.

Hypomelanosis of Ito associated with hemimegalencephaly: a clinicopathological study.

A girl with hypomelanosis of Ito was studied both clinically and at postmortem examination. She manifested severe epilepsy early after birth. Magnetic resonance imaging demonstrated left-sided hemimegalencephaly. The seizures were secondarily generalized or unilateral initially, followed by infantile spasms with asymmetrical hypsarrhythmia at 1.5 months of age. Frequent complex partial seizures, refractory to anti-epileptic drug treatments appeared at 4 months of age. She died of pneumonia at the age of 14 months. Postmortem examination revealed marked asymmetry of the cerebrum and gyral abnormalities in the left cerebral hemisphere. Histopathologically, severe disorganization of the neuronal cytoarchitecture was evident. Absence of delineation between cortical gray and white matter was evident, as was increase and hypertrophy of the neurons and glial cells. We believe that the association of skin and brain lesions was not one of chance; that is, they may share a common pathogenetic mechanism.

Two-dimensional demonstration of myenteric nerve plexus: application for pseudo-Hirschsprung's disease.

The use of two-dimensional demonstration of myenteric nerve plexus for the morphological estimation of pseudo-Hirschsprung's disease is introduced. By fluorescent immunohistochemistry for S-100 protein on flat-mounted frozen sections, a decrease in the amount of ganglionic plexuses in the small intestinal segments in cases of pseudo-Hirschsprung's disease was able to be distinguished from normoganglionic plexuses in control cases. Moreover, a morphological difference between the two types of cases was clearly demonstrated. This method may be an indispensable tool in the reliable diagnosis of pseudo-Hirschsprung's disease.

Measurement of serum hyaluronic acid as a sensitive marker of liver fibrosis in biliary atresia.

PURPOSE: The aim of this study was to clarify whether serum hyaluronic acid level (SHA) can reflect the degree of liver fibrosis in biliary atresia (BA). METHODS: SHA was measured in 44 postoperative BA patients at 7 months to 22 years of age, with sandwich enzyme method (Hy-A 100 kit). SHA was compared with T.Bil (group 1, T Bil < 2; group 2, 2 < or = T Bil < 5; group 3, T Bil > or = 5 mg/dL), fibrosis score (0-6, the number of abnormal values among Alb, PT, ChE, T Chol, Fischer's ratio, prealbumin), and histologic grading (0-IV). RESULTS: SHA was 499.8 +/- 332.5 in group 3, significantly higher than in the control, group 1, or group 2. As fibrosis score rose, SHA became higher, and SHA in Score 6 (430.1 +/- 366.1 ng/mL) and score-5 (172.9 +/- 141.8 ng/mL) was significantly higher than in the control and other scores, respectively. As the histologic grade rose, SHA became higher, and SHA in grade IV (444.8 +/- 323.5 ng/mL) and grade III (166.0 +/- 70.3 ng/mL) was significantly higher than in the control or other Grades. Serial change of SHA since before HPE was parallel to the clinical course in 8 patients. CONCLUSION: SHA may be a useful serum marker reflecting the degree of liver fibrosis in BA.

Development of intrapulmonary arteriovenous shunting in postoperative biliary atresia: evaluation by contrast-enhanced echocardiography.

PURPOSE: This report presents biliary atresia (BA) patients with intrapulmonary arteriovenous shunting (IPS), which was evaluated suitably by contrast-enhanced echocardiography (CEC). METHODS: Of 88 BA patients seen in the last 20 years, 8 (9.1%) had IPS at 8 months to 16 years of age. Two were associated with polysplenia syndrome, 1 had persistent jaundice after hepatic portoenterostomy, and 2 underwent splenorenal shunt. According to the comparison between microbubbles in left atrium (LA) and in right atrium (RA) detected by CEC, IPS was classified as grade I, mild (LA << RA); grade II, moderate (LA < RA); grade II, severe (LA = RA). RESULTS: Grade I consisted of 4 patients whereas grade II and III held 2 patients each. Clinical symptoms such as cyanosis, exertional dyspnea, or clubbing were present in 50% of grade I and all of grade II and IlI. Mean PaO2 in grade I, II, and III was 70.5, 50.4, and 35.3 mm Hg, respectively. In 1 patient with grade I, IPS spontaneously disappeared, but pulmonary hypertension developed later. One patient in grade II died of pulmonary complications, and the other is considered a candidate for livertransplantation (LTx). One patient in grade III died of liver failure, whereas the other is free of IPS after LTx. CONCLUSION: IPS can lead to a life-threatening complication in postoperative BA patients, and CEC may be a convenient and useful method to evaluate the degree of IPS and determine therapeutic strategy.

Clinicopathologic relationship of hypoganglionosis.

BACKGROUND/PURPOSE: Congenital motor dysfunction of the intestine associated with a morphologically abnormal myenteric nervous plexus (MP) is known as Hirschsprung's disease allied disorder (HAD). However, the clinicopathologic features of HAD are not well understood, partially because a standardized method of histologic evaluation of MP has not been established. To elucidate the clinicopathologic relationship of HAD the authors reviewed 6 cases of HAD using a newly devised histologic evaluation method. METHODS: Flat-mounted frozen sections of the ileum were stained for S-100 protein by fluorescent immunohistochemistry. Quantitative evaluation of MP was performed by measuring the fluorescence-positive area (MP ratio), and the results were compared with those of age-matched normal controls. RESULTS: All of 6 patients required laparotomy within 1 month after birth and enterostomy between 23 days and 10 months. Three died of intractable enteritis by the age of 2.2 years and were totally dependent on parenteral nutrition (PN) throughout their lives. The other 3 have survived for 6 to 10 years but have required PN occasionally. MP ratio in controls was more than 0.34 at all ages, whereas that in HAD was significantly lower than that in controls according to the clinical severity. CONCLUSION: MP size measured on 2-dimensional demonstration is suggested to be an indicator of clinical severity of HAD. J Pediatr Surg 36:898-900.

Pulmonary hypertension associated with postoperative biliary atresia: report of two cases.

The authors report on 2 patients with biliary atresia in whom pulmonary hypertension (PH) developed in the long-term follow-up after hepatoportoenterostomy. Both had portal hypertension and had undergone distal splenorenal shunt. Dyspnea developed around 14 to 15 years of age. Cardiac catheterization showed pulmonary artery pressure (PAP) of 99/37 (58) and 67/32 (48) mm Hg, respectively, which did not respond to vasodilators. One patient suffered from respiratory tract infection followed by right heart failure and subsequent death at 20 years of age. Postmortum histological findings exhibited severe thickening of the pulmonary artery wall. PH may grow insidiously even after successful hepatoportoenterostomy. Careful monitoring of PAP and hemodynamic response of PAP to vasodilators is essential for evaluating the reversibility of PH and making treatment decisions.

Functional ileus in neonates: Hirschsprung's disease-allied disorders versus meconium-related ileus.

Sixty-eight neonates with functional ileus were reviewed. Twelve required laparotomy; in seven, histological studies revealed decreased ganglia and ganglion cells of the myenteric plexus (MP) (Group A), and in five, MP was normal (Group B). In the remaining 56 cases, obstructive symptoms were relieved following conservative therapy (Group C). All Group A cases except one had normal birth weight, while Group B and C cases showed significantly lower birth weights. A marked caliber change of the small intestine and/or small-caliber distal intestine with meconium stagnation in the proximal intestine was commonly demonstrated at operation in Group A and B, or on contrast enema in Group C. Four Group A cases died of enteritis, and three survivors suffered from prolonged obstructive symptoms. The grade of histological abnormality of MP correlated with the clinical outcome. In Group B, three died of sepsis shortly after surgery, but two survivors have been free from symptoms. Group A can be categorized as Hirschsprung's disease-allied disorders (HAD). Group B and C can be categorized as meconium-related ileus (MRI). The similarity of the macroscopic findings of HAD and MRI, and the occurrence of MRI exclusively in low birth weight neonates, strongly suggest that functional immaturity of MP plays a role in the etiology of MRI.

[Hypomelanosis of Ito associated with hemimegalencephaly].

A girl aged 1 year and 2 months with hypomelanosis of Ito was reported. She suffered from intractable epileptic seizures since the second day after birth. Characteristic bizarre hypopigmented skin lesions were seen on her left shoulder and extended in a linear fashion to the flexion side of the left upper extremity. Brain MRI revealed diffuse enlargement of the left hemisphere, dilatation of the left lateral ventricle, and neuronal migration anomalies, indicating hemimegalencephaly. The types of her seizures were secondarily generalized or unilateral initially, followed by infantile spasms at 1.5 month. Around 4 months of age, frequent partial seizures appeared. Her seizures were intractable despite vigorous anticonvulsant therapy. As patients with hypomelanosis of Ito are frequently associated with neurological abnormalities, brain MRI studies were thought to be essential in detecting central nervous system anomalies, particularly in the presence of early onset intractable seizures.

Inhibition of thalamic ventrobasal complex neurons by glutamate infusion into the thalamic reticular nucleus in rats.

In urethane-anesthetized rats a 0.36-mm metallic cannula for infusion was positioned in the somatosensory component of the thalamic reticular nucleus (sTR), where movement of the vibrissae evoked neuronal discharge. Infusion there of 0.125-0.5 microliter of a 50 mM solution of glutamate over a 1-min period suppressed both spontaneous and evoked discharge of neurons in the ventrobasal complex (VB), but only for those which also responded to vibrissal stimulation. VB neurons activated by somatosensory stimuli at other locations were unaffected. Thus, excitation of neurons in sTR inhibits those in VB, but the effect appears to be highly coordinated somatotopically.

Diagnosis of congenital cytomegalovirus infection by examination of placenta: application of polymerase chain reaction and in situ hybridization.

We examined the placentas of 12 patients in whom congenital cytomegalovirus (CMV) infection was suspected from serological and/or pathological evaluation. Seven patients died (including four intrauterine deaths) and five survived. On histological examination, the characteristic inclusion bodies were detected in only three placentas, and villitis with plasma cell infiltration was seen in eight placentas. Immunohistochemistry using a specific antibody against CMV improved the sensitivity of CMV deletion (10 cases were positive). With the polymerase chain reaction (PCR) following the extraction of DNA from formaldehyde-fixed placenta samples, CMV DNA was detected in seven cases. All 12 subjects were diagnosed with CMV infection by additional Southern blot analysis after the PCR. CMV DNA was also detected by an in situ hybridization method in all cases. With current molecular biological techniques the placenta can be reliably used for the diagnosis of congenital CMV infection.

The effect of spermine on the disaccharidase activities in suckling rats of different age.

The influence of the age of the rat on the maturation of disaccharidase activities induced by spermine was studied. Three-day- and 9-day-old rats were used in the experiment. Spermine was administered orally or directly into the stomach using thin tubing daily for 3 days, and disaccharidase activities in the jejunum were measured. While spermine caused maturation of not only lactase activity but also maltase and sucrase activities in 9-day-old rats, it only caused maturation of lactase activity in 3-day-old rats. Histological studies showed no significant changes in the jejunum of 3-day-old rats treated with spermine.

Loss of EDB+ fibronectin isoform is associated with differentiation of alveolar epithelial cells in human fetal lung.

Cell-matrix interactions have been shown to regulate the development of the lung, particularly airway branching and alveolarization. Fibronectin is the major constituent of pulmonary extracellular matrix and exists in multiple isoforms arising from alternative RNA splicing. EDA and EDB are the two major alternatively spliced segments, the expression of which is regulated in a spatiotemporal and oncodevelopmental manner. In this study, we investigated immunohistochemically the distribution of the EDA- and EDB-containing fibronectin isoforms (referred to as EDA+ fibronectin and EDB+ fibronectin, respectively) in normal and hypoplastic human lungs at different gestational ages to explore the role of these fibronectin isoforms in alveolarization. EDA+ fibronectin was expressed around the distal airspaces throughout the development of both normal and hypoplastic lungs. In contrast, the expression of EDB+ fibronectin was restricted to the lung with morphologically immature acinar complex, typically observed in normally developing lungs of < 30 gestational weeks or in hypoplastic lungs. To further confirm the restricted expression of EDB+ fibronectin in immature acinar complex, we examined the correlation of EDB+ fibronectin expression with that of the surfactant protein SP-A, a biochemical marker for the differentiated type II pneumocytes. A clear inverse relationship between the immunoreactivities for EDB+ fibronectin and SP-A was observed in both control and hypoplastic lungs. Given the proposed importance of fibronectins in the differentiation of alveolar epithelial cells, our results suggest that the EDB segment plays a regulatory role in the differentiation of immature acinar epithelial cells into type II pneumocytes. The EDB segment may also serve as a new histochemical marker for the functional maturity of fetal lung tissues.

Indication for redo hepatic portoenterostomy for insufficient bile drainage in biliary atresia: re-evaluation in the era of liver transplantation.

To determine the role of redo hepatic portoenterostomy (HPE) in biliary atresia (BA) patients with insufficient bile excretion after the initial HPE, 25 patients (type I, correctable: 2; type III, uncorrectable: 23) undergoing the initial HPE at 25 to 119 days of age were studied. Four patients achieved disappearance of jaundice (total bilirubin [T.Bil] < 2 mg/dl) postoperatively. A redo HPE was performed at 2 to 8 months of age with sufficient and extensive removal of granulation and scar tissue at the hepatic hilum. Five patients became free of jaundice in 3 to 6 months (group 1), while the remaining 20 did not (group 2). Disappearance of jaundice after the initial HPE had been achieved in 2 of 5 patients (40%) in group 1 and 2 of 20 (10%) in group 2 ( P < 0.05). Age, serum T.Bil, aspartate aminotransferase albumin, prothrombin time, cholinesterase, total cholesterol, and Fischer's ratio at redo HPE showed no significant differences between the two groups. On liver histology obtained at redo HPE, cirrhosis and hepatocyte degeneration were seen in 1 of 5 cases (20%) in group 1 and 12 of 20 (60%) in group 2 ( P < 0.05). Redo HPE may thus be effective in BA patients with insufficient bile drainage who achieved disappearance of jaundice after the initial HPE and have not developed cirrhosis.

Three cases with TT virus infection and idiopathic neonatal hepatitis.

We present three cases of infants with idiopathic neonatal hepatitis showing diffuse intrahepatic fatty degeneration. Prolonged cholestasis has improved immediately upon intravenous administration of a high-dose gammaglobulin treatment in all three patients. The TT virus (TTV) genome was detectable in the serum of two patients, in the duodenal fluid of one and in the liver of all three. By analyzing sequence homology, we observed that the respective TTV isolated from serum, duodenal fluid and liver tissue were completely identical in cases 2 and 3. These findings suggest that TTV infection was one of the contributing factors for neonatal cholestasis in these patients. TTV was isolated from the serum of two out of the three mothers. The viruses were either completely or almost identical in sequence to those isolated from their respective infants, suggesting that they had been transmitted from mother to infant in these 2 cases. The patients presented here, whose livers were infected with the TTV and showed a favorable response to gammaglobulin therapy, may represent a subset of idiopathic neonatal hepatitis patients.