Acute pancreatitis induced thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare syndrome of unknown cause with an estimated incidence of one case per million. The disease is characterized by a pentad of symptoms: Thrombocytopenia, microangiopathic hemolytic anemia, neurologic changes, renal dysfunction, and fever. It causes thrombosis in the microvasculature of several organs, producing diverse manifestations. Acute pancreatitis (AP) is a well-described consequence of TTP. Acute pancreatitis triggering TTP is uncommon.

Reflex fluorescent in situ hybridization testing for unsuccessful product of conception cultures: a retrospective analysis of 5555 samples attempted by conventional cytogenetics and fluorescent in situ hybridization.

INTRODUCTION: The use of chromosome analysis on products of conception from spontaneous abortions is recommended to identify a genetic etiology. However, 20% of products of conception cultures are unsuccessful due to microbial contamination or lack of viable dividing cells. Our laboratory implemented a reflex fluorescent in situ hybridization (FISH) assay to detect numeric chromosome abnormalities for unsuccessful cultures. MATERIALS AND METHODS: All products of conception samples were simultaneously processed for both chromosome analysis and FISH analysis. If the chromosome analysis was unsuccessful, interphase FISH was performed for chromosomes 13, 16, 18, 21, 22, X, and Y. To assess the performance of the FISH assay, a 3-year retrospective comparative analysis of the FISH results versus chromosome results was performed. RESULTS: Of 5555 total specimens, 4189 (75%) represented chorionic villi/fetal tissue and 1366 (25%) represented tissue of unidentified origin. Of the 1189 tissues of unidentified origin with chromosome or FISH results, 1096 (92%) were XX, indicating that the majority of these tissues are likely maternal in origin. Of the 3361 successful chromosome studies on the chorionic villi/fetal tissue specimens, 1734 (52%) samples had a chromosome abnormality. Of the 762 successful FISH studies on chorionic villi/fetal tissue specimens that were unsuccessful by chromosome studies, 181 (25%) had an abnormal result with the targeted FISH panel. Overall, the FISH panel detected approximately 70% of the chromosome abnormalities in products of conception detectable by karyotype. When the FISH panel results were combined with chromosome analysis for the 4189 chorionic villi/fetal tissue specimens, the overall abnormality rate is 47%. CONCLUSIONS: Our reflex FISH assay proved useful for the detection of common chromosome aneuploidies in products of conception samples that failed conventional chromosome analysis. Because of its limited view of the genome, cautious interpretation of FISH results is required for all samples, in particular, trisomy of an acrocentric chromosome, which may represent a Robertsonian translocation. An algorithmic approach to the genetic evaluation of products of conception specimens, with the potential for initial evaluation by a FISH panel, may be warranted.

Ready reference to common segmental aneusomies by syndromic names, major features and chromosomal locations.

Abnormal gene dosage usually results in recognizable phenotypic abnormalities, especially if it involves a series of contiguous genes. Schmickel (1986) defined contiguous gene syndromes as diseases resulting from loss or gain of a series of adjacent genes. The terms microdeletion and microduplication as well as segmental aneusomy have also been used to describe such losses or gains that may not be readily detectable by Gbanded analysis. The loss (haploinsufficiency) or gain of a series of adjoining genes may result in a direct phenotypic effect and/or cause a genetic regulatory disturbance. Such syndromic gains or losses are often detectable when in situ hybridization of fluorescent labeled DNA probes or array comparative genomic hybridization technique are used (Gersen and Keagle 2005; Stumm et al. 1999; Barch, Knutsen and Spurbeck 1997). Segmental aneusomies generally occur due to homologous pairing between non-allelic low copy repeats (LCR) followed by crossing over. The LCRs, as part of the repetitive DNA sequences range from 1-500 Kb repeats, share >97% base sequence identity and constitute up to five percent of the genomic DNA. They are distributed throughout the genome, but are more concentrated near the centromeres and telomeres. A segment of 300 bp completely identical sequence within the LCRs is adequate for mediating non-allelic homologous or paralogous pairing. This process results in generating both deletion and duplication of a defined segment.

Delineation of the cryptic 1qter deletion phenotype.

The 1qter microdeletion is often reported in the literature as a part of a complex chromosome rearrangement. We describe a patient with a normal initial cytogenetic analysis later found by subtelomeric FISH to have a de novo isolated 1qter microdeletion. Further characterization was completed through microarray comparative genomic hybridization (CGH) and specific bacterial artificial chromosomes (BACs) to a region of 5.2-5.3 Mbp. Six additional cases were reviewed from a literature search. While no particular feature is specifically unique, the most frequently associated features include short stature, developmental delay and mental retardation, microcephaly, seizures, abnormal corpus callosum, and abnormal ear shape. This further delineates the phenotype and further narrows the chromosomal region responsible for a 1qter microdeletion phenotype.

Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma.

Deciphering the molecular basis of cancer is critical for developing novel diagnostic and therapeutic strategies. To better understand the early molecular events involving osteogenic sarcoma (OGS), we have initiated a program to identify potential tumor suppressor genes. Expression profiling of total RNA from ten normal bone cell lines and eleven OGS-derived cell lines by microarray showed 135-fold lower expression of FRZB/sFRP3 mRNA in OGS cells compared to bone cells; this down-regulation of Frzb/sFRP3 mRNA expression was found to be serum-independent. Subsequently, fourteen OGS biopsy specimens showed nine-fold down-regulation of Frzb/sFRP3 mRNA expression compared to expression in eight normal bone specimens as determined by microarray. FRZB /sFRP3 protein level was also found to be at a very low level in 4/4 OGS cell lines examined. Quantitation by RT-PCR indicated approximately 70% and approximately 90% loss of Frzb/sFRP3 mRNA expression in OGS biopsy specimens and OGS-derived cell lines respectively, compared to expression in bone (p<0.0001). Hybridization experiments of a cDNA microarray containing paired normal and tumor specimens from nineteen different organs did not show any significant difference in the level of Frzb/sFRP3 mRNA expression between the normal and the corresponding tumor tissues. Exogenous expression of FRZB/sFRP3 mRNA in two OGS-derived cell lines lacking endogenous expression of the mRNA produced abundant mRNA from the exogenous gene, eliminating degradation as a possibility for very low level of FRZB/sFRP3 mRNA in OGS specimens. Results from PCR-based experiments suggest that the FRZB/sFRP3 gene is not deleted in OGS cell lines, however, karyotyping shows gross abnormalities involving chromosome 2 (location of the FRZB gene) in five of twelve OGS-derived cell lines. Together, these data suggest a tumor-suppressive potential for FRZB/sFRP3 in OGS.

Mosaic ring 20 with no detectable deletion by FISH analysis: Characteristic seizure disorder and literature review.

Ring chromosome 20 is a rare chromosome disorder characterized by a typical seizure phenotype consisting of complex partial seizures, frequent progression to generalized tonic or tonic-clonic seizures, and nocturnal frontal lobe seizures with frequent episodes of non-convulsive status epilepticus. Development may be normal or mildly delayed, followed by cognitive and behavioral decline after seizure onset. Here, we describe a patient with a typical severe seizure phenotype and a mosaic ring chromosome 20 without loss of p or q subtelomere regions or telomeric sequences. The ring had a longer telomere length than either of the telomere ends of its homologous chromosome 20 by quantitative fluorescence in situ hybridization analysis, suggesting that it might be derived from telomere-telomere fusion. The phenotypic comparison of this patient and other chromosome 20 cases that had terminal deletions of 20qter (n = 1) and 20pter (n = 7), shows that the epilepsy phenotype and electroencephalographic abnormalities are characteristic in patients with ring chromosome 20. Several hypotheses have been proposed to address the elusive mechanisms underlying the seizure disorder in ring chromosome 20. These possibilities include haploinsufficiency of two epilepsy genes CHRNA4 and KCNQ2 located at 20qter, silencing of these genes by a telomere position effect, or microdeletions or rearrangements of genetic material during the ring formation.

Familial adenomatous polyposis and mental retardation caused by a de novo chromosomal deletion at 5q15-q22: report of a case.

Familial adenomatous polyposis, caused by mutations in the adenomatous polyposis coli gene located at chromosome 5q21, is an autosomal dominant syndrome characterized by polyposis of the colon and rectum and nearly 100 percent progression to colorectal cancer. We report a case of familial adenomatous polyposis and mental retardation caused by a chromosomal deletion at 5q15-q22. Chromosomal analysis is considered part of the evaluation of children with mental retardation and developmental delay. The resulting karyotypes from high-resolution chromosomal analysis can help characterize large deletions, some of which involve known tumor suppressor genes. Because familial adenomatous polyposis may arise from de novo chromosomal deletions involving the adenomatous polyposis coli gene locus, individuals with chromosomal deletions involving 5q21 should be considered at-risk for familial adenomatous polyposis and offered standard screening with flexible sigmoidoscopy by 10 to 12 years of age.

A validated FISH trisomy index demonstrates the hyperdiploid and nonhyperdiploid dichotomy in MGUS.

Two major genetic categories of multiple myeloma (MM) exist. Hyperdiploid MM (48 to 74 chromosomes, median 53 chromosomes) is associated with trisomies especially of chromosomes 3, 7, 9, 11, 15, and 19, whereas the nonhyperdiploid (< 48 chromosomes or more than 74 chromosomes) MM is associated with primary translocations such as t(11;14), t(4;14), and t(14;16). Whether this dichotomy exists in monoclonal gammopathy of undetermined significance (MGUS) is uncertain due to limitations of current methods in the study of ploidy. This is especially true in MGUS where the number of clonal plasma cells is small. In this study, we derived a fluorescent in situ hybridization (FISH)-based trisomy index from pooled cytogenetic data (karyotype analysis) from 2 large cohorts of patients with MM with abnormal karyotype, and then validated it in 2 independent cohorts of patients who had known ploidy status either by karyotyping or DNA content measurement using flow cytometry. Using the criteria of 2 or more trisomies from a 3-chromosome combination, hyperdiploid myeloma can be detected with high specificity. Applying this index on 28 patients with smoldering multiple myeloma (SMM) or MGUS (11 SMM, 17 MGUS) who had normal karyotype, 11 cases of hyperdiploid SMM/MGUS were detected. This percentage (40%) is remarkably similar to the percentage of hyperdiploid MM reported in the literature, suggesting that hyperdiploid MM may originate early during disease evolution.

Subtelomere deletions and translocations are frequently familial.

In recent years, strategies have been developed to investigate the possible role of chromosomal subtelomere regions in genetic disorders. The present study was to determine the incidence of familial subtelomeric abnormalities among individuals with developmental delay, idiopathic mental retardation, or non-specific congenital abnormalities. A review was conducted for patients and their relatives on whom subtelomeric DNA fluorescence in situ hybridization (telo-FISH) studies were performed. Patients were identified through a search of the Mayo Genetics System (MGS) database. Of 2,170 consecutive telo-FISH index case studies completed in our laboratory between January 2002 and December 2003, 121 or 5.6% had abnormalities of the subtelomere region. The present report includes 18 other abnormal index cases seen prior to 2002 to yield a total of 139 abnormal index cases. This represents 71 index patients with deletions, 53 index patients with derivative chromosomes, and 15 index patients with balanced rearrangements. A familial abnormality was identified in 29 (51.8%) of 56 families in whom parents and/or sibs were available for testing. Among 28 patients with deletions, 9 (32%) had an inherited deletion, whereas 19 (68%) were de novo. Family members of 20 index patients with derivative chromosomes were tested. Of these, 13 (65%) patients inherited the abnormality from a parent (12 from a parent who had a balanced translocation and 1 from a parent with the same abnormality), while 7 (35%) apparently arose de novo. Seven (88%) of 8 with balanced translocations inherited the translocation from one parent. The most common familial abnormalities involved 8pter deletion or rearrangement. The incidence of familial subtelomeric abnormalities is significantly high making parental telo-FISH studies an essential part of the investigation of patients with subtelomeric chromosome abnormalities.

Prevalence and clinical manifestations of 22q11.2 microdeletion in adults with selected conotruncal anomalies.

OBJECTIVES: This study was designed to determine the prevalence and clinical manifestations of 22q11.2 microdeletion in adults with selected conotruncal anomalies and to assess the clinician's ability to predict the presence or absence of 22q11.2 microdeletion on the basis of clinical features. BACKGROUND: It is known that 22q11.2 microdeletion is a chromosomal anomaly with cardiac and extracardiac manifestations. The prevalence and manifestations in adults have not been well characterized. METHODS: A total of 103 consecutive adults with either tetralogy of Fallot (TOF), pulmonary atresia/ventricular septal defect (PA/VSD), or truncus arteriosus (TA) were prospectively screened for 22q11.2 microdeletion using a fluorescence in situ hybridization (FISH) assay. Clinicians were asked to predict 22q11.2 microdeletion status on the basis of clinical features. A geneticist blinded to FISH assay results reviewed photographs of the patients for typical dysmorphic features of 22q11.2 microdeletion. RESULTS: Six patients (prevalence 5.8%, 95% confidence interval 1.3 to 10.3) had 22q11.2 microdeletion (3 with TOF, 2 with PA/VSD, 1 with TA). In two of these patients, the clinician incorrectly predicted absence of the deletion. In three, typical dysmorphic features of 22q11.2 microdeletion were absent. CONCLUSIONS: Our work showed that 22q11.2 microdeletion is under-recognized in adults with congenital heart disease. The absence of typical phenotypic features makes it difficult to correctly predict if the deletion is present. Screening for 22q11.2 microdeletion should be considered in adults with high-risk cardiac lesions, as it has important implications in reproductive counseling and surveillance for associated extracardiac manifestations.

Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia.

Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.

Molecular diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor in formalin-fixed paraffin-embedded tissues by RT-PCR and fluorescence in situ hybridization.

Recent studies have suggested that formalin-fixed paraffin-embedded (FFPE) tissues can be used for molecular analyses by fluorescence in situ hybridization (FISH) and RT-PCR. We analyzed 18 cases of ES/PNET for the t(11;22)(q24;q12) and t(21;22)(q22;q12) fusion transcripts by RT-PCR and analyzed for EWS translocation by interphase FISH with a dual color fusion probe to compare these two approaches directly. RT-PCR detected 13 (72%) EWS-FLI-1 fusions (type I=10, type II=3) and 2 (11%) EWS-ERG fusions. Three cases could not be evaluated because the housekeeping gene phosphoglycerate kinase (internal mRNA control) was not amplified. FISH was diagnostic in 15 of 18 cases (83%). There were three discordant cases between RT-PCR and FISH (concordance of 83%). Using a combination of RT-PCR and FISH, the results were complementary. One advantage of RT-PCR analysis was that subtypes of EWS translocation could be determined specifically (type I, type II and ERG). These findings indicate that because of the difficulties and limitations associated with the molecular analysis of FFPE tissues, a combination of RT-PCR and FISH may be a better approach to enhance the sensitivity and accuracy of detecting ES/PNET translocations in FFPE tissues with suboptimally preserved nucleic acids.

Trisomy 20 mosaicism caused by a maternal meiosis II error is associated with normal intellect but multiple congenital anomalies.

Mosaicism for trisomy 20 is generally ascertained following prenatal sampling and rarely is associated with significant phenotypic abnormalities. Uniparental disomy for chromosome 20 in the euploid lines has been found in several cases, which showed relatively mild clinical features, primarily growth delay. Here we report on a case of mosaic trisomy 20 in a child with normal neurologic development who was ascertained because of multiple physical anomalies including spinal segmentation anomalies and altered skin pigmentation. Trisomic cells were found in buccal epithelial cells and in cultured skin fibroblasts but not in peripheral blood. Molecular analysis of blood cells, fibroblasts, and parental cells gave evidence of a maternal meiosis II error as the cause of the trisomy. Disomic cells presumably arose through trisomy rescue, and no evidence was found for uniparental disomy in these cells.

Familial 22q11.2 deletions in DiGeorge/velocardiofacial syndrome are predominantly smaller than the commonly observed 3Mb.

PURPOSE: DiGeorge/velocardiofacial syndrome (DG/VCFS) is the most common cytogenetically characterized microdeletion of 22q11.2 region. In approximately 90% of patients, the deletion size is 3 Mb, whereas the remaining range from 1.5 to 2.5 Mb. The purpose of this study was to test the hypothesis that small deletions may be more easily tolerated in a familial fashion than larger deletions, especially for this syndrome. METHOD: Sixteen FISH probes designed from bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) mapped to 22q11.2 were used to determine the deletion sizes in 22 individuals from ten families with familial 22q11.2 deletion detected by standard FISH tests. RESULT: Seven families had deletions of < 3 Mb ( approximately 1.5 Mb) in size and 3 families had the common 3-Mb deletion. The 70% frequency of smaller sized deletions among this group of patients with familial del(22)(q11.2) is significantly higher than that reported among unselected group of patients with del(22)(q11.2) (P < 0.0001, Fisher exact test). CONCLUSION: Familial del(22)(q11.2) are predominantly smaller than the common deletion size of 3 Mb, indicating that there may be some underlying mechanisms that favor parent-to-child transmission of smaller deletions in individuals with del(22)(q11.2), therefore, underscoring the need to exclude a familial basis in cases of del(22)(q11.2) smaller than 3 Mb.

Human SULT1A3 pharmacogenetics: gene duplication and functional genomic studies.

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines. Inheritance is an important factor responsible for individual variation in SULT1A3 activity, and gene resequencing studies have shown the presence of one functionally significant SULT1A3 nonsynonymous cSNP. However, following completion of the Human Genome Project, it appeared that SULT1A3 might be duplicated. We used specific PCR-based assays and fluorescence in situ hybridization to verify that 2 SULT1A3 genes-SULT1A3 and SULT1A4-were present on chromosome 16 in all human DNA samples studied. Furthermore, reanalysis of previous gene resequencing data confirmed the presence of the SULT1A3 SNPs identified previously, but also revealed 11 novel polymorphisms, including 3 nonsynonymous cSNPs. Functional genomic studies showed that two of those cSNPs, C302T, and C302A, resulted in decreased enzyme activity without striking changes in substrate kinetics but with parallel changes in levels of immunoreactive protein. In addition, RT-PCR revealed that both SULT1A3 and SULT1A4 can be transcriptionally active. The duplication of SULT1A3 will have to be taken into account in future efforts to understand individual variation in SULT1A3 activity or properties.

Nonmosaic smallest duplication of 12q24.31-qter: the first reported case.

Duplication of the terminal region of the long arm of chromosome 12 is not common. In 13 previous cases, duplication of this region was generally associated with deletions of the derivative chromosomes, larger sized duplications or mosaicism. We have studied a young man with a nonmosaic duplication of 12q24.31-qter translocated to chromosome 5pter. This is the first reported case of pure subtle duplication involving less than two terminal subbands of 12q24.31 to qter. The origin of this genetic material was confirmed by whole chromosome paints and subtelomere specific FISH probes. As both the subtelomere signals for 5p and 12q were present in the der(5) chromosome, it is unlikely that there was any loss of unique DNA sequences from the terminal region of chromosome 5p. This case is compared with 13 other reported cases with a duplication of the 12q terminal segment.

Analysis of parathyroid neoplasms by interphase fluorescence in situ hybridization.

Recent studies have indicated that numerical chromosomal abnormalities, including changes in cyclin D1 and p53, may be involved in parathyroid tumorigenesis. We analyzed a series of parathyroid neoplasms with DNA fluorescent probes to evaluate the diagnostic and prognostic utility of numerical abnormalities of chromosomes 1, 6, 9, 11, 13, 15, 17, and 22 and cyclin D1 and p53 gene loci. Interphase fluorescence in situ hybridization (FISH) analysis was performed on paraffin-embedded tissue sections from 15 parathyroid adenomas and 18 parathyroid carcinomas. Directly labeled fluorescent DNA probes for the centromere region of chromosomes 1, 6, 9, 11, 15, and 17, and locus-specific probes for chromosome 22 and chromosome 13 and for cyclin D1 and p53 gene loci were used for dual-probe hybridization. Sixty-seven percent (10 of 15) parathyroid adenomas and 78% (14 of 18) of parathyroid carcinomas showed chromosome gains. Seventy-three percent (11 of 15) of parathyroid adenomas and 33% (6 of 18) of parathyroid carcinomas showed chromosome losses. Normal parathyroid tissues used as controls showed no chromosomal abnormalities. Parathyroid hyperplasias averaged 1.8 gains and 0.2 losses per case. Parathyroid adenomas averaged 2.8 gains and 0.8 losses per case, and parathyroid carcinomas averaged 3.6 gains and 0.6 losses per case. In summary, chromosome abnormalities, both gains and losses, are common in parathyroid adenomas and carcinomas. Parathyroid carcinomas tend to show gains of more chromosome than adenomas. Chromosome 11 was the most frequent chromosome loss identified in parathyroid adenomas and a frequent chromosomal gain in parathyroid carcinomas. These results indicate that gain of chromosome 11 is associated with more aggressive biologic behavior in parathyroid neoplasms.

Unbalanced cryptic 5p deletion/17p duplication identified by subtelomeric FISH in a family with a boy with chimerism and a balanced t(4;5).

The use of subtelomeric FISH probes has greatly supplemented conventional chromosome analysis in detecting cryptic anomalies in patients with mental retardation (MR), dysmorphic features, and congenital malformations. We report a 3-month-old boy who was diagnosed with ambiguous genitalia, dysmorphic features, and developmental delay. Standard chromosome studies on blood revealed a chimeric karyotype of 46,XY,t(4;5)(q31.1;q14)[46]/46,XX[4]. The boy had intra-abdominal gonads that were testicular in origin by biopsy. Multiple dysmorphic features, marked hypotonia, developmental delay, poor growth, and relative macrocephaly were noted on physical exam. His 2.5-year-old sister also presented with hypotonia, developmental delay, relative macrocephaly, and similar dysmorphic stigmata. In addition, she was diagnosed with several internal malformations. Her karyotype was 46,XX. Due to the striking phenotypic similarity, subtelomeric FISH studies were initiated in the siblings. In addition to the known balanced karyotypic abnormalities, the boy was found to have a derivative chromosome 5 with a 5pter deletion and a 17pter duplication. This cryptic abnormality was also detected in his sister. Chromosome analysis of the father revealed a subtle balanced t(5;17)(p15.31;p13.1) which was confirmed by subtelomeric FISH, whereas the mother's chromosome complement was normal. This familial constellation illustrates the usefulness of subtelomeric FISH in the diagnosis of cryptic chromosome abnormalities in patients for whom conventional karyotype does not disclose findings sufficient to explain the observed phenotypic anomalies.