Mariko Suchi

An Autoinflammatory Disease Due to Homozygous Deletion of the IL1RN Locus

We describe a patient with an autoinflammatory disease in which the main clinical features are pustular rash, marked osteopenia, lytic bone lesions, respiratory insufficiency, and thrombosis. Genetic studies revealed a 175-kb homozygous deletion at chromosome 2q13, which encompasses several interleukin-1 family members, including the gene encoding the interleukin-1-receptor antagonist (IL1RN). Mononuclear cells, obtained from the patient and cultured, produced large amounts of inflammatory cytokines, with increasing amounts secreted after stimulation with lipopolysaccharide. A similar increase was not observed in peripheral-blood mononuclear cells from a patient with neonatal-onset multisystem inflammatory disorder (NOMID). Treatment with anakinra completely resolved the symptoms and lesions.

Isolation of cDNA clones encoding human acid sphingomyelinase: occurrence of alternatively processed transcripts.

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine and 12 tryptic peptides were microsequenced (128 residues). Based on regions of minimal codon redundancy, four oligonucleotide mixtures were synthesized and oligonucleotide mixture 1 (20mer; 256 mix) was used to screen 3 X 10(6) independent recombinants from a human fibroblast cDNA library. Putative positive clones (92) were purified and analyzed by Southern hybridization with oligonucleotide mixtures 2‐4. These studies revealed two groups of clones; group 1 (80 clones; inserts ranging from approximately 1.2 to 1.6 kb) hybridized with oligonucleotides mixtures 1‐4, while group II (12 clones; inserts ranging from approximately 1.2 to 1.4 kb) hybridized with oligonucleotide mixtures 1‐3. Several group II clones had larger inserts than those in group I, but did not hybridize with oligonucleotide mixture 4. Screening of a human placental cDNA library with a 450 bp group I fragment, also resulted in the isolation of group I and II clones. Representative clones from group I (pASM‐1) and group II (pASM‐2) were sequenced. pASM‐1 contained a 1879 bp insert which was colinear with 96 microsequenced amino acids, while the pASM‐2 1382 bp insert was colinear with 78 microsequenced residues. Notably, pASM‐2 did not have an internal 172 bp sequence encoding 57 amino acid residues, but had instead an in‐frame 40 bp sequence encoding 13 amino acids which was not present in pASM‐1. These findings demonstrate the presence of two distinct acid sphingomyelinase transcripts in human fibroblasts and placenta and suggest the occurrence of alternative processing of the mRNA encoding this lysosomal hydrolase.

Extremes of Clinical and Enzymatic Phenotypes in Children With Hyperinsulinism Caused by Glucokinase Activating Mutations

OBJECTIVE Heterozygous activating mutations of glucokinase have been reported to cause hypoglycemia attributable to hyperinsulinism in a limited number of families. We report three children with de novo glucokinase hyperinsulinism mutations who displayed a spectrum of clinical phenotypes corresponding to marked differences in enzyme kinetics. RESEARCH DESIGN AND METHODS Mutations were directly sequenced, and mutants were expressed as glutathionyl S-transferase–glucokinase fusion proteins. Kinetic analysis of the enzymes included determinations of stability, activity index, the response to glucokinase activator drug, and the effect of glucokinase regulatory protein. RESULTS Child 1 had an ins454A mutation, child 2 a W99L mutation, and child 3 an M197I mutation. Diazoxide treatment was effective in child 3 but ineffective in child 1 and only partially effective in child 2. Expression of the mutant glucokinase ins454A, W99L, and M197I enzymes revealed a continuum of high relative activity indexes in the three children (26, 8.9, and 3.1, respectively; wild type = 1.0). Allosteric responses to inhibition by glucokinase regulatory protein and activation by the drug RO0281675 were impaired by the ins454A but unaffected by the M197I mutation. Estimated thresholds for glucose-stimulated insulin release were more severely reduced by the ins454A than the M197I mutation and intermediate in the W99L mutation (1.1, 3.5, and 2.2 mmol/l, respectively; wild type = 5.0 mmol/l). CONCLUSIONS These results confirm the potency of glucokinase as the pancreatic β-cell glucose sensor, and they demonstrate that responsiveness to diazoxide varies with genotype in glucokinase hyperinsulinism resulting in hypoglycemia, which can be more difficult to control than previously believed.

Microsatellite instability in sporadic human breast cancers

Human breast‐cancer specimens from 100 patients were analyzed for microsatellite instability (referred to as replication error; RER) at 12 genomic loci on 7 chromosomes, and results were correlated with clinicopathologic characteristics. In 42 of 100 breast‐cancer patients, we investigated whether RER was associated with the amplification of oncogenes and/or suppression of tumor‐suppressor genes. Of the 100 patients, 8 (8%) were RER‐positive at one or more chromosomal loci. The majority of RER‐positive patients had early‐stage disease with ER‐positive tumors, suggesting that RER occurs early in breast tumorigenesis. However, no significant correlation was observed between RER and oncogenes or tumor‐suppressor genes. Thus, the mechanism of RER in sporadic human breast cancer may be independent of the multi‐step carcinogenesis caused by the alterations of oncogenes and tumor‐suppressor genes.

Histopathology of Congenital Hyperinsulinism: Retrospective Study with Genotype Correlations

The majority of the most severe cases of congenital hyperinsulinism (HI) are caused by defects in the β-cell adenosine triphosphate (ATP)-sensitive potassium channel and usually require pancreatectomy to control blood sugar levels. In contrast to the recent advances in understanding the pathophysiology and genetic bases of HI, the histologic classification of this condition remains controversial. A recent proposal to classify the HI pancreata into diffuse and focal forms has drawn much interest because of its relative simplicity and its good correlation with the genetic abnormalities. We undertook a retrospective study to determine whether this classification scheme could be applied to 38 pancreata resected for HI at our institution. We also obtained leukocyte genomic DNA from 29 cases and screened the exons of ABCC8 and KCNJ11 genes for the presence of mutations. Nineteen cases (50.0%) were histologically classified as diffuse HI and 14 cases (36.8%) were categorized as focal form. The mutational analysis revealed that 14 of the 16 diffuse cases analyzed had either homozygous or compound heterozygous mutations of ABCC8 or KCNJ11 and 7 of 10 focal cases had only the paternally inherited mutations, consistent with the previous observations. Two patients (5.3%) had normal pancreatic histology but had persistent hypoglycemia postoperatively, leaving the possibility of residual focal lesion. Three of 38 cases (7.9%) did not fit well into either diffuse or focal category. Two cases differed from the described pattern for the diffuse form in that the nuclear enlargement was confined to a single area of the pancreas. The other case had a focal lesion but β-cell nuclear enlargement was present in nonadjacent areas. Mutations for typical diffuse or focal HI were not identified in two of these three equivocal cases. We conclude from this study that nearly 90% of HI cases can be classified into either a diffuse or a focal form. However, a small percentage of cases represented a diagnostic challenge.

Molecular and immunohistochemical analyses of the focal form of congenital hyperinsulinism.

Congenital hyperinsulinism is a rare pancreatic endocrine cell disorder that has been categorized histologically into diffuse and focal forms. In focal hyperinsulinism, the pancreas contains a focus of endocrine cell adenomatous hyperplasia, and the patients have been reported to possess paternally inherited mutations of the ABCC8 and KCNJ11 genes, which encode subunits of an ATP-sensitive potassium channel (KATP). In addition, the hyperplastic endocrine cells show loss of maternal 11p15, where imprinted genes such as p57kip2 reside. In order to evaluate whether all cases of focal hyperinsulinism are caused by this mechanism, 56 pancreatectomy specimens with focal hyperinsulinism were tested for the loss of maternal allele by two methods: immunohistochemistry for p57kip2 (n=56) and microsatellite marker analysis (n=27). Additionally, 49 patients were analyzed for KATP mutations. Out of 56 focal lesions, 48 demonstrated clear loss of p57kip2 expression by immunohistochemistry. The other eight lesions similarly showed no nuclear labeling, but the available tissue was not ideal for definitive interpretation. Five of these eight patients had paternal KATP mutations, of which four demonstrated loss of maternal 11p15 within the lesion by microsatellite marker analysis. All of the other three without a paternal KATP mutation showed loss of maternal 11p15. KATP mutation analysis identified 32/49 cases with paternal mutations. There were seven patients with nonmaternal mutations whose paternal DNA material was not available, and one patient with a mutation that was not present in either parents DNA. These eight patients showed either loss of p57kip2 expression or loss of maternal 11p15 region by microsatellite marker analysis, as did the remaining nine patients with no identifiable KATP coding region mutations. The combined results from the immunohistochemical and molecular methods indicate that maternal 11p15 loss together with paternal KATP mutation is the predominant causative mechanism of focal hyperinsulinism.

Congenital hyperinsulinism: intraoperative biopsy interpretation can direct the extent of pancreatectomy.

Most cases of congenital hyperinsulinism (HI) manifest as either a diffuse or focal form. Diffuse HI is characterized by the presence of enlarged islet cell nuclei, defined as those occupying an area 3 times larger than the surrounding nuclei, throughout the pancreas, and usually requires near total pancreatectomy. Focal HI contains, within an otherwise normal pancreas with islet cell nuclei of normal size, a focus of adenomatous hyperplasia characterized by endocrine cell overgrowth occupying more than 40% of a given area. This form of HI is amenable to partial pancreatectomy. The current study assesses whether intraoperative frozen section evaluation can distinguish the 2 forms and guide the extent of pancreatectomy. By frozen section analysis, diffuse HI is diagnosed when enlarged islet cell nuclei are present in random intraoperative biopsies from the head, body, and tail of the pancreas. Focal HI is suggested when random biopsies contain no large islet cell nuclei, prompting a further search for a focal lesion. Fifty-two HI patients who underwent pancreatectomy from October 1, 1998 to September 30, 2002 were reviewed. On permanent sections, 18 were classified as diffuse HI, 30 had focal HI, and 4 could not be categorized as either. Among 18 diffuse HI patients, 17 were correctly diagnosed by frozen section; all underwent near total pancreatectomy. One case was interpreted as not belonging to typical diffuse or focal HI; however, the permanent sections showed diffuse HI. Twenty-six of 30 focal HI cases were correctly diagnosed by frozen section. The remaining 4 focal HI cases posed diagnostic difficulties on frozen sections because of one the following reasons: 1) presence of equivocally large islet cell nuclei or rare truly large islet cell nuclei in areas nonadjacent to the focal lesion, and 2) large and/or ill defined focus of adenomatous hyperplasia. Twenty-one of 30 focal HI patients eventually had 10% to 93% (mean, 41.8%) of their pancreas resected. In addition to cases typical for diffuse and focal HI, there were 4 other cases whose pancreata did not fit well with either category. These pancreata showed islet cell nuclear enlargement, as characteristically seen in diffuse HI, but only in confined areas of the pancreas. Examination of routinely processed tissue confirmed frozen section findings in all 4 cases. Intraoperative frozen section evaluation, therefore, can assume an essential role in identifying patients with focal HI to limit the extent of pancreatectomy. However, a small number of cases with unusual histology warrant caution when performing frozen section evaluation.

Molecular cloning of a cDNA encoding human histidase

We isolated overlapping cDNA clones encoding human histidase (histidine ammonia-lyase) from a human lambda gt10 library. The cDNA predicted a 657 amino acid protein of 72,651 Da. The human histadase amino acid sequence was 93% conserved with both rat and mouse histidase sequences, including four N-glycosylation consensus sites.

Fetal Lung Interstitial Tumor (FLIT): A Proposed Newly Recognized Lung Tumor of Infancy to Be Differentiated From Cystic Pleuropulmonary Blastoma and Other Developmental Pulmonary Lesions

The differential diagnosis of congenital lung lesions includes a variety of pulmonary malformations, and uncommon or rare neoplasms such as the pleuropulmonary blastoma (PPB) and congenital peribronchial myofibroblastic tumor (CPMT). Although most of the congenital lesions have a predominantly cystic appearance, the exceptions of a more solid process are the type 3 congenital cystic adenomatoid or pulmonary airway malformation (CCAM-CPAM) and the CPMT. The clinical and pathologic features of a unique solid or mixed solid/cystic lung mass composed of immature interstitial mesenchyme in association with irregular airspace-like structures mimicking abnormal incompletely developed lung are presented in this report of 10 infants (7 males, 3 females) whose tumor-like lesions were detected in the prenatal period to 3 months of age (median, 1-day old). A lobectomy was done in all 10 infants and 1 infant received adjuvant chemotherapy. One of the surgical resections occurred as an ex utero, antenatal procedure because of fetal ascites. There have been no reported recurrences in those patients with greater than 12 months of follow-up ranging from 15 to 182 months (9 cases). Because of the morphologic resemblance of this mass-like lesion to fetal lung at 20 to 24 weeks gestation (as though any further pulmonary development was arrested in these localized lesions), we are proposing the designation of fetal lung interstitial tumor (FLIT) whose pathogenetic relationship, if any, to type 1 (cystic) pleuropulmonary blastoma remains uncertain to date.

Microsatellite instability in in situ and invasive sporadic breast cancers of Japanese women.

We studied the timing of microsatellite instability (referred to as replication error; RER) presentation during human breast carcinogenesis using tissue microdissected from both in situ and invasive breast cancers of Japanese women. We analyzed 100 breast cancer specimens for RER at nine genomic loci on seven chromosomes. Eight of the 100 cases (8%) were RER-positive at one or more chromosomal loci. Additionally; we obtained genomic DNA from two of four RER-positive patients with an intraductal component, both of which showed microsatellite instability in in situ foci. This finding indicates that microsatellite instability may be an early event during human breast carcinogenesis.