UBE3A/E6-AP mutations cause Angelman syndrome.

Angelman syndrome (AS), characterized by mental retardation, seizures, frequent smiling and laughter, and abnormal gait, is one of the best examples of human disease in which genetic imprinting plays a role. In about 70% of cases, AS is caused by de novo maternal deletions at 15q11-q13 (ref. 2). Approximately 2% of AS cases are caused by paternal uniparental disomy (UPD) of chromosome 15 (ref. 3) and 2-3% are caused by "imprinting mutations'. In the remaining 25% of AS cases, no deletion, uniparental disomy (UPD), or methylation abnormality is detectable, and these cases, unlike deletions or UPD, can be familial. These cases are likely to result from mutations in a gene that is expressed either exclusively or preferentially from the maternal chromosome 15. We have found that a 15q inversion inherited by an AS child from her normal mother disrupts the 5' end of the UBE3A (E6-AP) gene, the product of which functions in protein ubiquitination. We have looked for novel UBE3A mutations in nondeletion/non-UPD/non-imprinting mutation (NDUI) AS patients and have found one patient who is heterozygous for a 5-bp de novo tandem duplication. We have also found in two brothers a heterozygous mutation, an A to G transition that creates a new 3' splice junction 7 bp upstream from the normal splice junction. Both mutations are predicted to cause a frameshift and premature termination of translation. Our results demonstrate that UBE3A mutations are one cause of AS and indicate a possible abnormality in ubiquitin-mediated protein degradation during brain development in this disease.

Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA.

Holocarboxylase synthetase (HCS) plays an essential role in biotin utilization in eukaryotic cells and its deficiency causes biotin-responsive multiple carboxylase deficiency in humans. We have cloned the human HCS cDNA and show that antiserum against the recombinant protein immunoprecipitates human HCS. A one base deletion resulting in a premature termination and a missense mutation (Leu to Pro) were found in cells from siblings with HCS deficiency. Human HCS shows homology to BirA, which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. The human HCS gene maps to chromosome 21q22.1.

Loss of p53 induces leukemic transformation in a murine model of Jak2 V617F-driven polycythemia vera.

As leukemic transformation of myeloproliferative neoplasms (MPNs) worsens the clinical outcome, reducing the inherent risk of the critical event in MPN cases could be beneficial. Among genetic alterations concerning the transformation, the frequent one is TP53 mutation. Here we show that retroviral overexpression of Jak2 V617F mutant into wild-type p53 murine bone marrow cells induced polycythemia vera (PV) in the recipient mice, whereas Jak2 V617F-transduced p53-null mice developed lethal leukemia after the preceding PV phase. The leukemic mice had severe anemia, hepatosplenomegaly, pulmonary hemorrhage and expansion of dysplastic erythroid progenitors. Primitive leukemia cells (c-kit+Sca1+Lin- (KSL) and CD34-CD16/32-c-kit+Sca1-Lin- (megakaryocyte-erythroid progenitor; MEP)) and erythroid progenitors (CD71+) from Jak2 V617F-transduced p53-null leukemic mice had leukemia-initiating capacity, however, myeloid differentiated populations (Mac-1+) could not recapitulate the disease. Interestingly, recipients transplanted with CD71+ cells rapidly developed erythroid leukemia, which was in sharp contrast to leukemic KSL cells to cause lethal leukemia after the polycythemic state. The leukemic CD71+ cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2 V617F-transduced p53-null erythroleukemia. Taken together, our results show that p53 loss is sufficient for inducing leukemic transformation in Jak2 V617F-positive MPN.

Imprinting in neurons.

Although most imprinted genes display parent-origin-specific gene expression in tissues where they are transcribed, some genes are imprinted in a tissue-specific manner. Genes that show brain-specific imprinting or brain-specific lack of imprinting present a unique opportunity to study the process of imprinting during tissue differentiation. In this review, I introduce the systematic study of brain-cell-lineage-specific imprinting using a primary brain cell culture system, where neurons or glial cells are cultured separately. Two reports using the primary brain cell culture revealed brain-cell-lineage-specific imprinting in Ube3a and Igf2r, which had previously been described to show brain-specific imprinting and brain-specific lack of imprinting, respectively. Such brain-cell-lineage-specific imprinting was associated with cell-specific epigenetic modifications, especially with their reciprocally imprinted antisense non-coding RNAs, Ube3a-ATS and Air. These results emphasize the necessity of imprinting analysis at the cell level rather than in whole brain tissue during brain differentiation. The brain cell culture system provides us with a new powerful tool to understand the molecular mechanism of brain-specific imprinting.

Comparative study of bone mineral density estimated by various methods of single- and dual-energy quantitative computed tomography: the capability of the four-equation four-unknown method.

A dual-energy (DE) quantitative computed tomography (QCT) method, the four-equation four-unknown method (DEQCT 4E-4U), was assessed and compared to single-energy (SE) QCT and standard DEQCT (two-line method). The results of this study indicate that bone mineral density (BMD) was more accurately estimated by the present method than by the SEQCT or standard DEQCT techniques on the basis of a phantom study when a large fat content was present. The results of both the phantom study and a human study also showed that the present method corrected for fat in estimating BMD in the presence of high-fat content. These findings suggest that use of this method for estimating BMD can provide useful information in studies assessing the metabolic state of bone. We propose that CT numbers estimated from excised vertebral bone marrow can serve as a soft-tissue correction for the present method.

A new syndrome of dwarfism, brachydactyly, nail dysplasia, and mental retardation in sibs.

We describe a new multiple congenital anomaly/mental retardation (MCA/MR) syndrome in chromosomally normal sibs. Both had microcephaly, a "coarse" face with synophrys, ear anomalies, type B brachydactyly, nail dysplasia, skeletal anomalies, dwarfism, and mental retardation. Their mother had nail dysplasia, mild mental retardation, and short stature. An uncle, a younger brother of the mother, died at 17 years of age and also had a "coarse" face, digital anomalies, dwarfism, and severe mental retardation. The malformation complex in this family apparently has not been described previously, and the manifestations of the patients do not correspond to those of any known malformation syndrome. The disorder may be attributable to the pleiotropic effect of an autosomal dominant or an X-linked semidominant gene.

Two Thai families with Norrie disease (ND): association of two novel missense mutations with severe ND phenotype, seizures, and a manifesting carrier.

We describe two Thai families with Norrie disease (ND) in three generations, including 10 affected males and one manifesting female. All affected males in each family had severely defective eye development with complete loss of vision. In addition, three male patients (one from family 1 and two from family 2) suffered from epilepsy, and one female carrier from one family manifested blindness with phthisis bulbi in her right eye. Mutation analysis of the ND gene (NDP) revealed two different novel missense mutations (L16P and S75P) that co-segregated with ND in each family, suggesting that the newly appearing proline at codon 16 or codon 75 alters the conformation of the ND protein and contributes to the severe phenotype of ND in each family. Other studies suggest that epileptic seizures or growth retardation that is associated with ND is the consequence of loss of contiguous genes, because most such patients had deletions extending beyond the Norrie locus. Our finding that the three affected males in the two families with the missense mutations had epilepsy does not support a contiguous gene effect, but favors the pleiotropism of NDP, at least as far as the epileptic manifestation is concerned. The unilateral blindness in the female carrier may have been due to non-random X-inactivation.

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.

Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines.

Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies. We established cultured cell lines from four untreated tumors. The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities. The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors. The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein. The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant. One cell line produced G-CSF. The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents. The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs. This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism. The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

Changes in liver fatty acid unsaturation after partial hepatectomy in the rat.

The purpose of this study was to assess changes in the degree of fatty acid unsaturation in rat liver after partial hepatectomy. This is the first study in which liver fatty acid unsaturation has been analyzed over a long period of regeneration until day 28 after operation. The relationship between changes in unsaturation and fatty acid composition in the regenerating liver were also investigated in this study. Proton nuclear magnetic resonance spectroscopy revealed significantly elevated levels of unsaturation with a maximum on day 5 after partial hepatectomy, compared with untreated controls (11.72+/-0.55 vs. 11.05+/-0.26%, P < 0.05). No significant changes in unsaturation were found in day 1 regenerating liver, which is rich in absolute amounts of fatty acids. Based on gas-liquid chromatography, the relative amounts of oleic acid (18:1n-9) and linoleic acid (LA; 18:2n-6) were increased, while polyunsaturated fatty acids such as arachidonic acid (20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3) were decreased on day 1. On the other hand, on day 5 of regeneration, while most fatty acids were returning to their preoperative control levels, only DHA was higher than the control value (7.69+/-0.58 vs. 5.57+/-0.37%, P < 0.001). The high levels of unsaturation on day 5 were found to be partly due to the increase in DHA. The findings suggest that some significant signals are transmitted during the regeneration process owing to alterations in the membrane structure by the high levels of fatty acid unsaturation and the increase in DHA levels on day 5 after partial hepatectomy.

A neonatal case of hydranencephaly caused by atheromatous plaque obstruction of aortic arch: possible association with a congenital cytomegalovirus infection?

A neonate was seen with hydranencephaly and aortic arch obstruction possibly caused by congenital cytomegalovirus infection. At autopsy the aortic arch was occluded by atheromatous plaque between the right brachiocephalic artery and left common carotid artery. Congenital cytomegalovirus infection may have caused cardiovascular disease and hydranencephaly in this case.

Submucosal tumor (SMT)-like esophageal squamous cell carcinoma with gastric metastasis.

We present here a case of submucosal tumor-like esophageal cancer with metastasis to the stomach. A 60-year-old man, whose ability to swallow was impaired, was admitted to Kyorin University School of Medicine Hospital. Gastrointestinal endoscopy demonstrated a small bulging mass in the lower esophagus and a large submucosal mass in the gastric cardia. The gastric lesion was growing rapidly and becoming easily hemorrhagic. It appeared rich in blood flow by angiography. Surgical treatment was adopted after a diagnosis of esophageal cancer and a gastric submucosal tumor was made. However, in the end, the gastric mass was identified as a metastasis from the esophageal squamous cell carcinoma. Moreover, the primary esophageal lesion displayed quite a special type of histology, a so-called submucosal tumor-like pattern, which was covered by normal epithelium and grew mainly in the submucosal layer of the esophagus. Gastric metastasis from esophageal cancer is relatively rare, and it is quite rare that an esophageal squamous cell carcinoma grows as a submucosal tumor. Finally, the patient died of pneumonia and metastasis to the liver on the 110th day of hospitalization. Intramural metastasis to the stomach from esophageal cancer should be treated in its advanced stage, and poor prognosis can be expected from aggressive treatment. It is necessary to recognize this complication so that appropriate therapy can be carried out on patients with esophageal cancer, and the situation needs to be carefully evaluated, including the stomach, both before and after treatment.

Pre-elastofibroma-like colonic polyp: another cause of colonic polyp.

We present a case of pre-elastofibroma-like lesion, a kind of elastic-producing fibrous tumor. The small colonic polyp, which was found in a 49-year-old asymptomatic man in association with a large colonic adenoma, showed submucosal nodular deposits of fine granular or fibrillar eosinophilic materials with interspersed fibroblastic cells. Elastic stain revealed these deposits to consist mainly of dark gray granular or partially fibrillar dense elastinophilic materials, most of which were digested with elastase. This stromal lesion somewhat resembled a pre-elastofibroma. Therefore, pre-elastofibroma-like lesions should be kept in mind as a possible origin of colonic polyp.

[A case of polyarteritis nodosa presenting as a mass in scrotum].

A 16-year-old boy with a painful tumor in the left scrotum was referred to our department. CT scans showed a low density area in the left testis, so we diagnosed a left testicular tumor and performed left inguinal orchiectomy. Histological examination revealed polyarteritis nodosa (PN) of the testis and epididymis. Systemic examination revealed no other evidence of PN. Although induration developed in the right epididymis after the operation, it resolved with steroid therapy. The patient is currently asymptomatic and is being followed at our clinic. The pathogenesis and management of this rare condition are discussed.

[Long-term outcome of monotherapy by Cohen ureterocystoneostomy for vesicoureteral reflux in spina bifida patients].

BACKGROUND: The conventional surgical treatment for vesioureteral reflux (VUR) is spina bifida patients is ureterocystoneostomy. Various newer therapies, including augmentation enterocystoplasty and minimally invasive subureteral collagen injection, have been introduced. However, all of these procedures have specific advantages and disadvantages, and no guidelines for deciding on the surgical treatment of VUR in spina bifida patients have yet been established. In this study, the long-term outcome of the Cohen procedure, a method of ureterocystoneostomy, was examined. PATIENTS AND METHODS: Among spina bifida patients in whom VUR was treated by the Cohen procedure alone from 1984 to 1989, 27 patients who could be followed up for 5 years or longer were enrolled in the study (11 males and 16 females, with a mean age of 13.4 years at surgery). In principle, they were followed up using annual cystography, excretory urography, and blood and urine tests. At the final assessment, they were examined for the presence at VUR and for morphological abnormalities of the urinary tract. Their renal function was also assessed. They were followed for 6 to 13 years (mean: 8.9 years), and the mean age at final assessment was 22.2 years. RESULTS: Among 42 ureters in the 27 patients examined, 38 ureters (90.5%) in 22 patients (81.5%) did not have VUR postoperatively. Four ureters in 4 patients had the recurrence of VUR, and in another patient new occurrence was detected postoperatively. Augmentation ileocystoplasty was performed to treat the postoperative decrease of bladder compliance in 4 patients. Among 22 patients who had hydronephrosis preoperatively, 9 (40.9%) showed improvement and none suffered from aggravation of this condition. None of the patients showed a decline of renal function, except for 1 who had a serum creatinine of 2.5 mg/dl preoperatively and developed end-stage renal failure at 7 years postoperatively. CONCLUSIONS: The Cohen procedure has an excellent anti-reflux effect. It is one of the therapeutic options for VUR in patients with good bladder compliance.

[Transient abnormal myelopoiesis followed by acute leukemia in children with Down syndrome].

This report describes three cases with Down's syndrome. These cases initially had transient abnormal myelopoiesis (TAM), from which they recovered spontaneously. They finally developed into overt acute leukemia characterized by an increase of blasts, hepatosplenomegaly, and elevated lactic dehydrogenase. Of these three cases, one was thought to have ANLL, which broke out 5 months after spontaneous remission. The other two had ALL, each occurring 8 and 9 years later. Chromosomal abnormality, in addition to trisomy 21, was detected in blast cells from one of the patients with acute leukemia. All three patients with acute leukemia experienced complete remission. However, two of the three patients relapsed and died. It is noted in the literature that remission is permanent in most cases of TAM, and is rarely terminated by leukemic relapse. In view of our observations, the importance of following up on such patients who evidence apparent remission of their leukemia-like disorder is emphasized.

[Septicemia in children with malignant disease].

Severe infection is a major cause of morbidity and mortality in patients with malignancy. In this study, 34 episodes of septicemia occurred in 1,468 childhood patients with malignancy who admitted and were treated at National Sapporo Hospital between 1979 and 1988. The occurrence of septicemia and its mortality rate were higher in malignant hematologic disease than in malignant solid tumor. Most cases of septicemia occurred in relapse. The most frequent organism causing septicemia were Klebsiella pneumoniae (16.3%) and Staphylococcus epidermidis (16.3%). Septicemia due to Gram-negative organism was more frequent than that of Gram-positive organism or fungus. Polymicrobic septicemia occurred 3 times and multiple episodes 6 times. They had a high mortality rate. Neutropenia was strongly associated with episode of septicemia. In our series, absolute neutrophil count under 500 per microliter developed septicemia. Especially, children with less than 100 granulocytes per microliter had a major risk factor for the development of infection and death. No children with granulocyte count greater than 1000 per microliter died in connection with septicemia.

[Evaluation of the role of surgery in 25 patients with metastatic neuroblastoma].

The role of surgery was evaluated using a recently proposed TNM staging system in metastatic neuroblastomas. Of twenty-five patients, twenty-four were over 1 year, 1 case was 3 months old, nine were boys, sixteen were girls, and all were stage IV using Evans-D'Angio staging system (excluding IV-s). They were retrospectively assigned a TNM clinical stage (CS) preoperatively and a pathologic stage (PS) postoperatively. All twenty-five patients were CS 4 using this TNM staging system. The role of surgery was evaluated by analyzing survival according to the postoperative PS. PS 1-2-3A were regarded as satisfactory resections, since all macroscopic tumor was removed, while PS 3B-3C-4-5 were regarded as unsatisfactory resections. With Kaplan-Meier analysis, there was a slight survival advantage when satisfactory resection of the primary tumor was achieved in the cases with any evidence of metastasis at the time of operation. However, in the cases with no evidence of metastasis at operation, there was a survival advantage when satisfactory resection of the primary tumor was done (p = 0.05). If metastatic disease is controlled prior to operation, total resection improves prognosis of metastatic neuroblastoma.

Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.