Ring chromosome 17: phenotype variation by deletion size.

Ring chromosome 17 is a rare cytogenetic abnormality, with 12 previous reports in the literature. Some have a relatively mild phenotype characterized by seizures, mental retardation, skin changes and short stature. Other patients have Miller-Dieker syndrome (MDS), which includes lissencephaly, multiple dysmorphic features, severe mental retardation and shortened life expectancy. We describe two new cases of ring chromosome 17 and review the literature. Our cases and the other reports of patients without a deletion encompassing the Miller-Dieker region, delineate a fairly distinctive subgroup of individuals with ring 17, whose phenotype consists of growth and mental retardation, seizures, minor dysmorphic features, café-au-lait spots and retinal flecks. This classification of ring 17 into two distinct groups based on the size of the deletion and the phenotypic manifestations should facilitate clinical suspicion of this rare chromosomal abnormality.

Fatal EBV-related post-transplant lymphoproliferative disorder (LPD) after matched related donor nonmyeloablative peripheral blood progenitor cell transplant.

A 39-year-old male underwent a nonmyeloablative stem cell transplant (NMAPBPCT) from his HLA-matched sister for recurrent anaplastic large cell lymphoma in CR-2, receiving fludarabine, cyclophosphamide, and rabbit antithymocyte globulin for the preparative therapy. The patient was readmitted on day+33 for persistent culture-negative fevers. He rapidly developed marked elevations of alkaline phosphatase and bilirubin. Liver biopsy showed a periportal infiltrate of large immunoblastic appearing cells. The tumor cells did not stain for CD3/CD20/CD30 and alk protein, but did stain for CD79a/LCA and CD43. In situ hybridization for Epstein-Barr virus (EBV) RNA (EBER 1) was strongly positive in the periportal infiltrating lymphocytes. Fluorescence in situ hybridization (FISH) studies revealed female (XX) cells in the tumor cells and male (XY) in the surrounding hepatic parenchymal cells. The patient developed severe lactic acidosis, oliguric renal failure and expired on day+44. Both donor and patient had positive IgG serologies for EBV VCA and EBNA pretransplant. The donor also had a positive IgM titer for EBV VCA in the pretransplant specimen. The LPD may have been related to the intense immunosuppression of the preparative therapy and the presence of recent EBV infection in the donor.

Biochemical and mutational analyses of the cathepsin c gene (CTSC) in three North American families with Papillon Lefèvre syndrome.

Papillon Lefèvre syndrome (PLS) is an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and severe periodontitis. The disease is caused by mutations in the cathepsin C gene (CTSC) that maps to chromosome 11q14. CTSC gene mutations associated with PLS have been correlated with significantly decreased enzyme activity. Mutational analysis of the CTSC gene in three North American families segregating PLS identified four mutations, including a novel mutation p.G139R. All mutations were associated with dramatically reduced CTSC protease enzyme activity. A homozygous c.96T>G transversion resulting in a p.Y32X change was present in a Mexican PLS proband, while one Caucasian PLS proband was a compound heterozygote for the p.Y32X and p.R272P (c.815G>C) mutations. The other Caucasian PLS proband was a compound heterozygote for c.415G>A transition and c.1141delC mutations that resulted in a p.G139R and a frameshift and premature termination (p.L381fsX393), respectively. The c.415G>A was not present in more than 300 controls, suggesting it is not a CTSC polymorphism. Biochemical analysis demonstrated almost no detectable CTSC activity in leukocytes of all three probands. These mutations altered restriction enzyme sites in the highly conserved CTSC gene. Sequence analysis of CTSC exon 3 confirmed the previously reported p.T153I polymorphism in 4 of the 5 ethnically diverse populations studied.

Primary malignant lymphoma of uterine corpus: case report and review of the literature.

We describe a patient presenting with postmenopausal vaginal bleeding and a uterine mass subjected to endometrial biopsy that showed a high-grade non-Hodgkin's lymphoma, consistent with a diffuse large B-cell lymphoma. Staging computed tomography (CT) scans of the chest, abdomen, and pelvis revealed three lung nodules in addition to the uterine mass. Fine needle aspirate of one lung lesion showed lymphomatous involvement. She was treated with intensive chemotherapy alone and has remained in complete remission 21 months after diagnosis. The literature on primary lymphoma of the uterine corpus is reviewed.

The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein.

Human minor histocompatibility Ags (mHag) present significant barriers to successful bone marrow transplantation. However, the structure of human mHag and the basis for antigenic disparities are still largely unknown. Here we report the identification of the gene encoding the human mHag HA-2 as a previously unknown member of the class I myosin family, which we have designated MYO1G. The gene is located on the short arm of chromosome 7. Expression of this gene is limited to cells of hemopoietic origin, in keeping with the previously defined tissue expression of the HA-2 Ag. RT-PCR amplification of MYO1G from different individuals led to the identification of two genetic variants, designated MYO1G(V) and MYO1G(M). The former encodes the peptide sequence previously shown to be the HA-2 epitope (YIGEVLVSV), whereas the latter shows a single amino acid change in this peptide (YIGEVLVSM). This change has only a modest effect on peptide binding to the class I MHC-restricted element HLA-A*0201, and a minimal impact on recognition by T cells when added exogenously to target cells. Nonetheless, as detected using either T cells or mass spectrometry, this amino acid change results in a failure of the latter peptide to be presented at the surface of cells that express MYO1G(M) endogenously. These studies have thus identified a new mHag-encoding gene, and thereby provide additional information about both the genetic origins of human mHag as well as the underlying basis of an Ag-positive vs Ag-negative state.

Prenatal diagnosis of complete sole trisomy 1q.

The prenatal diagnosis of a complete trisomy of the long arm of chromosome 1 is reported. Major ultrasound findings included: nuchal thickening, bi-temporal narrowing, a single choroid plexus cyst, and mild ventriculomegaly. There was a mass in the chest and abdomen, pleural effusion, ascites and a hyperechoic bowel. Skin edema was present. The fetus died at 26 weeks' gestation. A literature review is presented of 17 de novo and two inherited cases with only trisomy 1q. Of note is the fact that 3/5 prenatally detected 1q trisomies have teratomas. A review of the literature reveals a dismal outcome for trisomy 1q cases if the duplication involves bands 1q25-->q32.

Maternal homozygosity for the common MTHFR mutation as a potential risk factor for offspring with limb defects.

A common mutation, C677T, in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene leads to altered homocysteine metabolism, and has been associated with the occurrence of neural tube defects (NTD). Administration of folic acid decreases this risk. There is also evidence that periconceptional supplementation of mothers with folic acid can decrease the risk of limb defects in the offspring. Here we describe a child with a transverse terminal defect of one hand, whose mother is homozygous for the C677T MTHFR mutation. We suggest that homozygosity for the MTHFR mutation may be a risk factor for transverse terminal limb defect/s by an effect mediated through altered folate and homocysteine metabolism. Further studies of mothers of infants with limb reduction defects for the MTHFR mutation may be of help in establishing this association. A simple intervention in the form of folic acid supplementation would be protective, should an association be established.

Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv(16) in a prospective series of adults with de novo acute myeloid leukemia: a Cancer and Leukemia Group B Study.

PURPOSE: To prospectively compare cytogenetics and reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFbeta/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFbeta/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P =.83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFbeta/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR-positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFbeta/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFbeta/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.

Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature.

Since 1993, the position of the American College of Medical Genetics (ACMG) has been that prenatal interphase fluorescence in situ hybridization (FISH) is investigational. In 1997, the FDA cleared the AneuVysion assay (Vysis, Inc.) to enumerate chromosomes 13, 18, 21, X and Y for prenatal diagnosis. Data is presented from the clinical trial that led to regulatory clearance (1379 pregnancies) and from retrospective case review on 5197 new pregnancies. These studies demonstrated an extremely high concordance rate between FISH and standard cytogenetics (99.8%) for specific abnormalities that the AneuVysion assay is designed to detect. In 29 039 informative testing events (6576 new and 22 463 cases in the literature) only one false positive (false positive rate = 0.003%) and seven false negative results (false negative rate = 0.024%) occurred. A historical review of all known accounts of specimens tested is presented (29 039 using AneuVysion and 18 275 specimens tested with other probes). These performance characteristics support a prenatal management strategy that includes utilization of FISH for prenatal testing when a diagnosis of aneuploidy of chromosome 13, 18, 21, X or Y is highly suspected by virtue of maternal age, positive maternal serum biochemical screening or abnormal ultrasound findings.

Down syndrome congenital heart disease: a narrowed region and a candidate gene.

PURPOSE: Down syndrome (DS) is a major cause of congenital heart disease (CHD) and the most frequent known cause of atrioventricular septal defects (AVSDs). Molecular studies of rare individuals with CHD and partial duplications of chromosome 21 established a candidate region that included D21S55 through the telomere. We now report human molecular and cardiac data that narrow the DS-CHD region, excluding two candidate regions, and propose DSCAM (Down syndrome cell adhesion molecule) as a candidate gene. METHODS: A panel of 19 individuals with partial trisomy 21 was evaluated using quantitative Southern blot dosage analysis and fluorescence in situ hybridization (FISH) with subsets of 32 BACs spanning the region defined by D21S16 (21q11.2) through the telomere. These BACs span the molecular markers D21S55, ERG, ETS2, MX1/2, collagen XVIII and collagen VI A1/A2. Fourteen individuals are duplicated for the candidate region, of whom eight (57%) have the characteristic spectrum of DS-CHD. RESULTS: Combining the results from these eight individuals suggests the candidate region for DS-CHD is demarcated by D21S3 (defined by ventricular septal defect), through PFKL (defined by tetralogy of Fallot). CONCLUSIONS: These data suggest that the presence of three copies of gene(s) from the region is sufficient for the production of subsets of DS-CHD. This region does not include genes located near D21S55, previously proposed as a "DS critical region," or the genes encoding collagens VI and XVIII. Of the potential gene candidates in the narrowed DS-CHD region, DSCAM is notable in that it encodes a cell adhesion molecule, spans more than 840 kb of the candidate region, and is expressed in the heart during cardiac development. Given these properties, we propose DSCAM as a candidate for DS-CHD.

Bone marrow cytogenetic abnormalities of aplastic anemia.

Cytogenetic abnormalities in association with aplastic anemia have been reported fairly infrequently. Clonal cytogenetic abnormalities at initial diagnosis are uncommon. A retrospective study was performed of the cytogenetic findings in patients with typical morphological and clinical features of severe aplastic anemia from a single institution for the years 1988 through 1998. A total of 30 cases of aplastic anemia, 16 men and 14 women, were identified. The median age was 60 with females being significantly older (67.5 years) in comparison to males (44 years). Bone marrow specimens failed to yield metaphases in 16 cases and normal karyotypes were detected in 11 cases. Cytogenetic abnormalities were detected in 3 cases. Clonal abnormalities, as defined, occurred in only 2 cases (6.7%). A review of the literature identified a total of 24 cases of aplastic anemia with abnormal cytogenetic findings. Overall, the most common chromosome abnormalities are trisomies of 6 and 8 and loss of chromosome 7. Trisomy 6 is more common at diagnosis while loss of chromosome 7 is more common after therapy.

Localization, genomic organization, and alternative transcription of a novel human SAM-dependent methyltransferase gene on chromosome 2p22-->p21.

As part of our studies to identify the gene responsible for hereditary gingival fibromatosis, GINGF (OMIM 135300), we have identified and cloned a novel human gene that contains the highly conserved methyltransferase domain characteristic of S-adenosylmethionine-dependent methyltransferases. We localized this gene (C2orf8 encoding 288L6 SAM-methyltransferase) to chromosome 2p22-->p21 by FISH, and sublocalized it to BAC RP11 288L6 flanked by D2S2238 and D2S2331. Computational analysis of aligned ESTs identified ten exons in the hypothetical C2orf8 gene. Results of RACE analyses in placenta identified multiple transcripts of this gene with heterogeneity at the 5'-UTR. Alternative transcription and tissue specific expression of C2orf8 were detected by RT-PCR and Northern blot analyses. C2orf8 is expressed in a variety of tissues including brain, colon, gingiva, heart, kidney, liver, lung, placenta, small intestine, spleen, and thymus. Open reading frame analysis of the alternative transcripts identified a shared coding region spanning exons 6-10. This ORF consists of 732 nucleotides encoding a putative 244 amino acid protein. Bioinformational searches of both C2orf8 and the putative protein product identified three methyltransferase motifs conserved across many prokaryotic and eukaryotic species. Sequence analyses of C2orf8 excluded coding region mutations as causative of GINGF.

Identification of female cells in postcoital penile swabs using fluorescence in situ hybridization.

Traditionally, the finding of semen, that is, spermatozoa and acid phosphatase, in cervicovaginal specimens has been considered the laboratory evidence needed to prove recent sexual contact. Recent research with fluorescence in situ hybridization (FISH) has shown that in the absence of semen, male epithelial and inflammatory cells can be found within the female genital tract. A striking paucity of literature exists pertaining to the examination of the penis of an alleged assailant for potential evidence indicative of sexual assault. The current study uses FISH to analyzepostcoital swabs of the penis for such laboratory evidence. A male and female volunteer couple consented to participate in this study. Following coitus, the male partner presented to one of the investigators for penile swabbing. Swabs were taken at varying postcoital intervals (1-24 hours) subsequent to 10 coital episodes. The male participant was instructed not to shower following coitus, but to otherwise go about daily activities until specimen collection. To obtain each sample, 4 sterile cotton-tipped applicators were slightly moistened in sterile saline and swabbed along the length of the penile shaft and around the base of the penis. From the swabs, 3 air-dried slides were prepared, coded, and blinded. As controls, swabs were taken from the buccal surfaces of both volunteers. Multicolor FISH was performed using dual X- and Y-chromosome probes, and slides were counterstained with 4'-6-diamidino-2-phenylindole (DAPI). Cells were easily visualized under a fluorescent microscope, but only cells with 2 nonoverlapping fluorescent signals were counted. Fluorescence in situ hybridization is highly sensitive and specific, and the dual probes easily distinguished between male and female cells. Female cells were identified on smears from every penile swab over the entire 1- to 24-hour postcoital interval. The FISH technique, previously successful in identifying male cells within the female genital tract, may also be employed on penile swabs. Once the presence of female cells is confirmed by FISH, the identity of the female can be confirmed by DNA analysis. Potentially, with such current molecular analyses, both the assailant and the victim can be positively identified.

Isolation and identification of male and female DNA on a postcoital condom.

BACKGROUND: Identification of male perpetrators of sexual assault may be made from cells and fluids recovered from postcoital condoms. To date, the focus has been on identifying the person who had worn the condom. OBJECTIVE: To describe a method for scientifically identifying both the male and female participants in a sex act by employing polymerase chain reaction-based technology on swabs taken from the internal and external surfaces of a condom. Fluorescence in situ hybridization may be used to screen for the presence of female cells on a condom. METHODS: Swabs were taken from the internal and external surfaces of a condom 8 hours postcoitus. DNA was isolated from each swab through standard organic extraction. Extracted DNA was amplified for 8 different genetic loci using the Promega PowerPlex kit and the sex identification amelogenin marker. Amplified samples were electrophoresed on precast sequencing gels and analyzed fluorescently using a Hitachi FMBIO 2 fluorescent scanner and software. Each DNA sample obtained from the condom was compared with male and female buccal controls. At the time of collection, air-dried slides were prepared from the swabs for subsequent multicolor fluorescence in situ hybridization using dual X- and Y-chromosome probes with 4'-6-diamidino-2-phenylindole (DAPI) counterstaining. RESULTS: A pure sample of female DNA was isolated from the external surface of the condom as determined by exclusive amplification of the X-chromosome-specific 212-base pair amelogenin marker. Swabs taken from the internal surface yielded DNA originating from the male participant. Identification was conclusive at 8 of 8 genetic loci. Fluorescence in situ hybridization identified pure populations of male epithelial cells from the internal surface of the condom and female cells from the external surface. CONCLUSIONS: Cells shed from a female during sexual intercourse can be retrieved from the external surface of a condom following sexual intercourse. Fluorescence in situ hybridization can be used to screen for the presence of female cells, and positive identification of the female sexual partner can then be made using polymerase chain reaction-based methods. We suggest that swabs taken from both surfaces of a condom used during sexual assault may be used to provide information that will definitively link the victim to the suspect.

Isolation and identification of female DNA on postcoital penile swabs.

After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.

Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9.

The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans.

Physical and cDNA mapping in the DBH region of human chromosome 9q34.

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.