Cryptic 17q22 deletion in a boy with a t(10;17)(p15.3;q22) translocation, multiple synostosis syndrome 1, and hypogonadotropic hypogonadism.

We report on a boy who had multiple synostosis syndrome 1, an autosomal dominant disorder characterized by progressive symphalangism, multiple joint fusions, conductive deafness, and mild facial dysmorphism. In addition the boy developed delay of puberty, bone age, and closure of the epiphyseal lines of long bones with tall stature. These findings and decreased plasma LH and FSH levels at age 19 years were compatible with hypogonadotropic hypogonadism. G-banded chromosomes showed a balanced translocation t(10;17)(p15.3;q22). Chromosomal FISH analysis, using a series of BAC clones surrounding the translocation breakpoints, detected a 2.2-3.9 Mb deletion at 17q22. The deletion encompassed NOG, a gene responsible for multiple synostosis syndrome 1. It was assumed that a gene for pituitary secretion of gonoadotropic hormones was deleted at the 17q22 segment.

Deletions and epimutations affecting the human 14q32.2 imprinted region in individuals with paternal and maternal upd(14)-like phenotypes.

Human chromosome 14q32.2 carries a cluster of imprinted genes including paternally expressed genes (PEGs) such as DLK1 and RTL1 and maternally expressed genes (MEGs) such as MEG3 (also known as GTL2), RTL1as (RTL1 antisense) and MEG8 (refs. 1,2), together with the intergenic differentially methylated region (IG-DMR) and the MEG3-DMR. Consistent with this, paternal and maternal uniparental disomy for chromosome 14 (upd(14)pat and upd(14)mat) cause distinct phenotypes. We studied eight individuals (cases 1-8) with a upd(14)pat-like phenotype and three individuals (cases 9-11) with a upd(14)mat-like phenotype in the absence of upd(14) and identified various deletions and epimutations affecting the imprinted region. The results, together with recent mouse data, imply that the IG-DMR has an important cis-acting regulatory function on the maternally inherited chromosome and that excessive RTL1 expression and decreased DLK1 and RTL1 expression are relevant to upd(14)pat-like and upd(14)mat-like phenotypes, respectively.

A case of acute myeloblastic leukemia with a novel variant of t(8;21)(q22;q22).

We encountered a case of acute myeloblastic leukemia (AML), with extramedullary leukemia (EML) and a masked type of the variant translocation t(8;21)(q22;q22). Morphologically, the AML M2 subtype according to the French-American-British (FAB) classification was present. Phenotypically, leukemic cells were negative for CD19 and positive for CD56. Clinically, the case showed chemo-refractoriness and a poor outcome. The initial karyotypic interpretation was t(8;9)(q22;q34) on G-banding. Multiplex-fluorescence in situ hybridization (multiplex-FISH) analysis revealed a three-way translocation involving chromosomes 8, 9, and 21, and identified a masked type of variant t(8;21)q22;q22) translocation. The karyotype was finally determined as 45,X,-Y,der(8)t(8;21)(q22;q22), der(9)(8;9)(q22;q34), and der(21)t(9;21)(q34;q22). Results of FISH using the AML1/ETO probe and detection of the AML1/ETO fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) support the karyotype as well as the sequence of the PCR product. Additionally, C-KIT mutation was detected.

Detection of the CBFB/MYH11 fusion gene in de novo acute myeloid leukemia (AML): a single-institution study of 224 Japanese AML patients.

The cytogenetic findings in acute myeloid leukemia (AML) are a powerful prognostic indicator. Among these abnormalities, the World Health Organization has classified inv(16)(p13q22), which is closely associated with the M4E classification in the French-American-British system, as indicating a good-risk AML. However, this chromosomal abnormality can often be difficult to detect. In this study, we used RT-PCR and FISH analysis to examine 224 Japanese adult de novo AML patients for the presence of the CBFB/MYH11 fusion transcript at the time of diagnosis. The CBFB/MYH11 fusion gene was detected in 17 patients (7.6%): eight patients had the inv(16) chromosome and in all of them it was M4E; nine patients did not have abnormalities in chromosome 16. AML with the CBFB/MYH11 fusion gene but without inv(16) was found in M2, M4, and M5, but not in M4E patients. There were no statistically significant differences in the clinical features of patients with the inv(16) and those with the cryptic inv(16) chromosome. These results indicate that even if eosinophilia is not found, molecular screening for CBFB/MYH11 fusion gene should be performed in all AML patients at the time of diagnosis to help guide disease management.

Mild craniosynostosis with 1p36.3 trisomy and 1p36.3 deletion syndrome caused by familial translocation t(Y;1).

We report on a 20-year-old man and a 16-year-old woman with a chromosomal imbalance derived from a balanced translocation, t(Y;1)(q12;p36.3) of the father. The man had a partial trisomy for 1p36.3-pter [46,X,der(Y)t(Y:1)(q12;p36.3)] and mild craniosynostosis of metopic and sagittal sutures as well as a borderline mental impairment, while the woman with a deletion for 1p36.3-pter [46,XX,der(1)t(Y;1)(q12;p36.3)] showed dysmorphic face with large anterior fontanel and severe developmental delay. Fluorescence in situ hybridization (FISH) showed that his trisomy spanned the 5.3-Mb region from 1p telomere harboring the critical region for craniosynostosis. To our knowledge, the man is the first case of a pure type of simple 1p36.3 trisomy as the effect of heterochromatic Yq12-qter deletion likely does not affect phenotype.

Detection of aberrations of ubiquitin-conjugating enzyme E2C gene (UBE2C) in advanced colon cancer with liver metastases by DNA microarray and two-color FISH.

Using DNA microarrays, the expression profiles of 1,700 genes in the primary tumor, liver metastases and paired normal tissue obtained from nine patients with advanced colorectal cancer was studied. Twenty genes were upregulated and only one gene was downregulated in the primary tumors. In the liver metastases, 39 genes were upregulated and only three genes were downregulated. There was no significant difference in gene expression between the primary tumors and the liver metastases. The most highly overexpressed gene in both the primary tumors and the liver metastases was the ubiquitin-conjugating enzyme E2C gene (UBE2C), located at 20q13.1. Additionally, two-color FISH analysis using probes for the region 20q13.1 and the chromosome 20 centromere revealed that amplification at 20q13.1 had occurred in 5 of 10 (50%) colon cancers. Comparison between the levels of gene expression and FISH results revealed that UBE2C expression is significantly changed by amplification at 20q13.1, suggesting genomic amplification as one mechanism of increased UBE2C expression. Our results showing aberrations in levels of gene expression and locus copy number of UBE2C suggest that this gene may play an important role in tumor progression leading to advanced colon cancer with liver metastasis.

Molecular and phenotypic analysis of Philadelphia chromosome-positive bilineage leukemia: possibility of a lineage switch from T-lymphoid leukemic progenitor to myeloid cells.

The occurrence of acute bilineage leukemia is thought to be the malignant transformation of a myeloid or lymphoid leukemic progenitor with the potential to differentiate into the other lineages; however, the mechanisms of this lineage switch are not well understood. Here, we report on the extremely rare case of adult Philadelphia chromosome-positive acute bilineage leukemia, which is characterized by T-cell acute lymphoblastic leukemia and acute myelomonocytic leukemia. Chromosome analysis showed 46,XY,del(7)(p11.2),t(9;22)(q34;q11.2) in all metaphases and a minor BCR/ABL chimeric gene was detected in these leukemic cells by PT-PCR. When the CD5+ and CD5- cells were sorted, a fusion gene of BCR/ABL and the same clonally rearranged band of a T-cell receptor (TCR) gene were detected in both populations. Nucleotide sequencing of the TCR-gamma gene revealed the clonal rearrangement of the V8-JGT2 complex in both populations. Overexpression of PU.1, which plays a fundamental role in myelomonocyte development, was found in the sorted CD34+CD7+ and CD5-, but not CD5+ cells. These results suggest that leukemic progenitor cells in the T-lineage with the del(7) and t(9;22) have the potential to differentiate into myeloid lineage, and that enforced PU.1 expression may contribute in part of this phenomenon.

Two cases of partial trisomy 21 (pter-q22.1) without the major features of Down syndrome.

We report two cases of partial trisomy 21 with clinical features distinct from Down syndrome (DS). These patients presented with moderate mental retardation and short stature, but the typical facial appearance of DS was not observed. Each patient had a similarly sized extra chromosome 21. We performed FISH analysis to examine whether deletions of reported approximately 5 Mb DS critical region (DSCR) might be associated with unusual clinical features in these cases. The results showed that each of their extra chromosomes 21 contained a distal part of chromosome 3p or 14q at the telomeric region of chromosome 21q. The translocation breakpoint of 21q for each patient was located on the centromeric side of DSCR (DSCR was deleted) and the sizes of partial trisomy 21 in respective patients are approximately 34.5 (21pter-q22.12) and approximately 33.0 Mb (21pter-q22.11). In one patient, the additional region of the short arm of chromosome 3 was 3pter-p26.1 from maternal origin, measuring approximately 9 Mb in size. The second patient had an extra 14q32.1-qter of maternal origin, measuring approximately 14 Mb in size. These are one of the shortest partial distal trisomy among reported cases. Taken together, two patients with partial trisomy 21 lack all of DSCR on 21q22, and their distinct clinical features are likely caused by the genes located at 21pter-q22.1 and the distal part of chromosome 3p or 14q.

Molecular genetic alterations and gene expression profile of a malignant rhabdoid tumor of the kidney.

Malignant rhabdoid tumor of the kidney (MRTK) is a rare but highly aggressive tumor in children, and knowledge about the molecular signature of this tumor is limited. We report the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl. Fluorescence in situ hybridization and Southern blot analyses revealed a homozygous deletion of an approximately 0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. This deleted region encodes SMARCB1, a candidate tumor suppressor gene for MRTK. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with a noncancerous kidney (NK) sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case may have contributed to the biological characteristics of the MRTK tumor that arose in our patient.

Microdeletion in the SHOX 3' region associated with skeletal phenotypes of Langer mesomelic dysplasia in a 45,X/46,X,r(X) infant and Leri-Weill dyschondrosteosis in her 46,XX mother: implication for the SHOX enhancer.

It is known that SHOX nullizygosity results in Langer mesomelic dysplasia (LMD) and SHOX haploinsufficiency leads to Leri-Weill dyschondrosteosis (LWDC). Here, we report on a microdeletion in the SHOX 3' region identified in a Japanese infant with LMD-compatible skeletal features and a 45,X[191]/46,X,r(X)(p22.3q24)[9] karyotype and in her mother with LWDC-compatible skeletal features and a normal 46,XX karyotype. Physical and auxological examinations revealed mesomelic appearance, ulnarly deviated hands, and borderline micrognathia in the infant, and relatively short forearms and lower legs in the mother. Radiological studies indicated mesomelia, markedly curved radii, hypoplastic ulnas and fibulas, and metaphyseal splaying in the infant, and borderline to mild curvature of the radii, decreased carpal angles, and high-normal triangularization index in the mother. Cytogenetic and molecular studies showed that the ring X chromosome of the infant was missing SHOX and of paternal origin, whereas the cytogenetically normal X chromosomes of the infant and one of the two X chromosomes of the mother, though they retained SHOX with normal coding sequences, had a microdeletion in the SHOX 3' region. The microdeletion started from a position approximately 200 kb from SHOX coding sequences, and spanned 240-350 kb in physical length involving DXYS233. The results, in conjunction with those reported by Flanagan et al. [2002], suggest that a cis-acting enhancer exists in the SHOX 3' region around DXYS233.

Non-multiple lymphomatous polyposis form of mantle cell lymphoma in the gastrointestinal tract.

Mantle cell lymphoma (MCL) comprises 2.5%-7% of all non-Hodgkin's lymphomas, and the gastrointestinal tract is involved in about 20% of cases. Multiple lymphomatous polyposis (MLP) is an uncommon disease that is regarded as the intestinal form of MCL. We present a rare case of gastrointestinal MCL without MLP, and demonstrate that rituximab was effective for the treatment of this patient. A 61-year-old man presented with continuous diarrhea and hematochezia for a period of 5 months. Superficial lymph nodes were not palpable, but both tonsilla were enlarged. The level of soluble interleukin (IL)2-receptor was 3480 U/ml (normal <500 U/ml). Colonoscopy showed diffuse redness with erosion, without observation of any venous capillary, with these findings continuing from the rectum to the ileum. Upper gastrointestinal endoscopy showed a slightly rough gastric mucosal surface, and chicken-skin like mucosa was observed in the second portion of the duodenum. Small-to-medium size lymphoma cells were seen histologically from the tonsilla to the rectum. The lymphoma cells were immunohistochemically positive for CD5, CD20, CD79a, and cyclin D1. Polymerase chain reaction analysis revealed a chromosomal translocation t(11;14)(q13;q32) in the bcl-1 gene. We diagnosed this as a case of MCL from these findings. For treatment, the patient received a total of ten courses of combination chemotherapy consisting of cyclophosphamide (1000 mg), doxorubicin (70 mg), vincristine (2 mg) and prednisolone (50 mg) (CHOP), which led to a partial remission. However, 2.5 years later, massive infiltrations of the lymphoma cells were found in the colon and stomach. As the infiltrating lymphoma cells expressed CD20 molecules on their surfaces, the patient was treated with a chimeric anti-CD20 monoclohal antibody, rituximab, which showed significant efficacy, and a second partial remission was achieved.

Fluorescent in situ hybridization analysis of Philadelphia chromosome-negative chronic myeloid leukemia with the bcr/abl fusion gene.

This report describes a patient with Philadelphia chromosome-negative (Ph-) but bcr/abl fusion gene-positive chronic myeloid leukemia (CML) and a molecular analysis of the mechanisms behind the Ph status. Spectral karyotyping-fluorescent in situ hybridization (SKY-FISH) analysis showed no abnormal translocation; however, a bcr/abl fusion gene was detected by reverse transcriptase-polymerase chain reaction analysis. FISH analysis showed that signals from the 9q and 22q subtelomere probes were detected on the der(9) and der(22) chromosomes, respectively. On the other hand, FISH analysis of the abl and bcr genes with dual fusion probes, which can detect the bcr/abl fusion gene on both the der(9) and der(22) chromosomes, showed the signal for bcr/abl fusion on the der(22) chromosome but not on the der(9) chromosome. These results indicate that insertion of the abl gene into the bcr region on the der(22) chromosome or retranslocation between the der(9) chromosome and the der(22) chromosome may have caused the Ph CML in this case.

9q34.3 deletion syndrome in three unrelated children.

We described three unrelated children with cryptic 9q34.3 rearrangements and similar clinical manifestations: two with 9q34.3 terminal deletions and the other with an unbalanced translocation involving 9q34.3-qter monosomy and 6p25-pter trisomy. Common features among the three we studied and the other six patients with 9q34.3 deletions in the literature include microcephaly, mental retardation (MR), hypotonic, and epileptic seizures. Their facial characteristics included flat face, arched eyebrows, synophrys, hypertelorism, short nose, anteverted nostrils, carp mouth, protruding tongue, micrognathia, and pointed chin. Other frequent abnormalities were cardiac abnormalities, cryptorchidism or hypospadias, and abnormal toes. These findings are characteristic enough to be a clinically recognizable syndrome.

MLL/AF-1p fusion in therapy-related early pre-B acute lymphoblastic leukemia with t(1;11)(p32;q23) translocation developing in the relapse phase of acute promyelocytic leukemia.

We report the development of therapy-related early pre-B acute lymphoblastic leukemia in a patient administered a topoisomerase II inhibitor, etoposide, a consolidation therapy agent for acute promyelocytic leukemia. Our case is of interest because of simultaneous relapse of the original leukemia and onset of therapy-related leukemia and relatively rare t(1;11)(p32;q23) translocation with confirmed MLL/AF-1p fusion. This case suggests that careful monitoring for MLL gene rearrangements is necessary after administration of topoisomerase II inhibitors.

Prediction by FISH analysis of the occurrence of Wilms tumor in aniridia patients.

Aniridia is an autosomal dominant eye anomaly caused by haploinsufficiency of the PAX6 gene, of which abnormalities include base alterations, position effects and deletions. When deletion involves its adjacent genes, i.e., those in the PAX6-WT1 critical region (WTCR), patients are predisposed to Wilms tumor. We studied 18 patients with aniridia, five of whom had chromosome deletion involving 11p13, two a translocation t(10;11)(p13;p13) or a der(14;21)(q10;q10)mat, and 11 had a normal karyotype. Fluorescence in situ hybridization (FISH) using four P1-derived artificial chromosome (PAC) clones located at WTCR was carried out in the 18 patients to identify a deletion extent. Of the 18 patients, eight had a deletion of WTCR: four had microscopic deletion and four a deletion of WTCR. Deleted region in one patient with a microscopic deletion was distal to the critical region. Four of the eight patients with a deletion encompassing WTCR developed Wilms tumor, and the other four did not (two were too young to be evaluated for the tumor development). The data in the present study, together with four similar previous works, indicate that of a total of 102 aniridia patients, 29 had a deletion spanning WTCR. Wilms tumor developed in 13 (45%) of the 29 patients, whereas patients without deletion in this region did not develop the tumor. In other words, aniridia patients with WT1 deletion run a high risk of developing Wilms tumor, and those without the deletion do not.

[External quality assessment of the genetic testings for hematopoietic tumor, CML].

Genetic testings are commonly employed in various fields of clinical medicine and the test items performed at clinical laboratories are increasing rapidly in number. They are utilized to make early and/or definite diagnoses of infectious diseases, leukemia, cancers and molecular inherited diseases and also to monitor the progress of the diseases. However, these genetic testings except for infectious diseases have been developed independently at each clinical laboratory and the test results obtained at each laboratory are not always compatible each other. Under these situations it is widely expected to construct advanced genetic testing systems that can supply standardized data at any of domestic and international clinical laboratories. For the period from April, 1999 to March, 2002 three major clinical laboratories, SRL, Inc., BML, Inc. and MBC, Inc., were consigned by JBA (Japan Bioindustry Association) to collaborate in standardizing the evaluation methods for genetic testing systems among the clinical laboratories. The aim of the study is to develop the standardized genetic testing systems and to propose them as international standard operational procedures to the ISO/TC212 working group. Although one of the most important issues for standardization is the external quality assessment, they have not been carried out in reality. In this study we evaluated the difference of the genetic testing results obtained during the year of 2000 and 2001 among the clinical laboratories. The genetic testings for hematopoietic tumor, CML were selected to be evaluated since they are widely accepted as clinically useful tests.