Analphoid supernumerary marker chromosome characterized by aCGH and FISH as inv dup(3)(q25.33qter) de novo in a child with dysmorphic features and streaky pigmentation: case report.

BACKGROUND: Small supernumerary marker chromosomes (sSMC) occur in 0.075% of unselected prenatal and in 0.044% of consecutively studied postnatal cases. Individuals with sSMC present with varying phenotype, ranging from normal to extremely mild or severe depending on the chromosomal region involved, the euchromatic content present and degree of mosaicism. Except for chromosomes 15 and 22, the number of reported cases of sSMC is extremely small to provide us with a good genotype-phenotype correlation. Analphoid sSMC are even rarer. To our knowledge only eight cases of analphoid inversion-duplication 3q sSMC are reported so far. RESULTS: We describe here a one month old female child with several dysmorphic features and with a de novo analphoid supernumerary marker chromosome only in cultured skin fibroblast cells and not in lymphocytes. The marker was characterized as analphoid inversion-duplication 3q25.33-qter by oligo array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH) studies. The final skin fibroblast karyotype was interpreted as 47,XX,+der(3).ish inv dup(3)(qter-q25.33::q25.33-qter)(subtel 3q+,subtel 3q+) de novo. CONCLUSION: In addition to the eight reported cases of analphoid inversion-duplication 3q supernumerary marker in the literature, this is yet another case of 3q sSMC with a new breakpoint at 3q25.33 and with varying phenotype as described in the case report. Identification of more and more similar cases of analphoid inversion-duplication 3q marker will help in establishing a better genotype-phenotype correlation. The study further demonstrates that aCGH in conjunction with routine cytogenetics and FISH is very useful in precisely identifying and characterizing a marker chromosome, and more importantly help in providing with an accurate genetic diagnosis and better counseling to the family.

Incidence of Down syndrome in Dubai, UAE.

OBJECTIVE: To describe incidence of Down syndrome in Dubai, United Arab Emirates (UAE). SUBJECTS AND METHODS: A total of 63,398 newborn babies in Dubai (24,250 UAE nationals and 39,148 non-UAE) during a 5-year period of 1999-2003 were routinely examined by experienced nurses, neonatologists, pediatricians and/or general practitioners for symptoms of Down syndrome. Those suspected with Down syndrome were referred to the cytogenetic laboratory for karyotyping. RESULTS: A total of 141 cases were confirmed cytogenetically as Down syndrome. Of these, 139 were trisomy 21 and of the remaining 2, 1 was a translocation and the other a mosaic. Theoverall incidence of Down syndrome in Dubai was 1/449 live births (2.2 per 1,000); 1/319 live births (3.13 per 1,000) among UAE nationals and 1/602 live births (1.66 per 1,000) among non-UAE nationals. The mean maternal age of UAE national mothers was 33.48 +/- 8.08, with 41.66% of the mothers being in the advanced maternal age group (>35 years). The higher incidence of Down syndrome among UAE nationals is comparable to incidences reported for other Arab populations in the Middle Eastern region. Advanced maternal age, with mothers bearing children until their 50s and higher parity, appear to be the major contributing factors for the increased incidence. CONCLUSION: The study indicates the need to provide efficient genetic counseling and to introduce an effective antenatal screening program and prenatal diagnostic services to reduce the psychological and genetic burden on the families and community.

Ectrodactyly with aplasia of long bones (OMIM; 119100) in a large inbred Arab family with an apparent autosomal dominant inheritance and reduced penetrance: clinical and genetic analysis.

Ectrodactyly with aplasia of long bones syndrome is one of the most recognizable defects involving the extremities. We have studied a very large eight-generation consanguineous Arab family from the United Arab Emirates (UAE) with multiple severe limb anomalies resembling this condition (OMIM; 119100), for which the affected gene is unknown. The pedigree consists of 145 individuals including 23 affected (14 males/9 females) with limb anomalies. Of these, 18 had tibial aplasia (TA) usually on the right side. The expression of the phenotype was variable and ranged from bilateral to unilateral TA with ectrodactyly and other defects of the extremities. The mode of inheritance appears to be autosomal dominant with reduced penetrance. There were 10 consanguineous marriages observed in this pedigree. This could suggest possible pseudodominance due to high frequency of the mutant allele. Candidate loci for the described syndrome include GLI3 (OMIM: 165240) on 7p13, sonic hedgehog; (OMIM: 600725) on 7q36, Langer-Giedion syndrome (OMIM: 150230) on 8q24.1 and split-hand/foot malformation 3 (OMIM: 600095) on 10q24. In addition, bilateral tibial hemimelia and unilateral absence of the ulna was previously observed to co-segregate with deletion of 8q24.1. Two-point linkage and haplotype analyses did not show the involvement of the above regions in this family.

New approaches to fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a nonisotopic labeling and detection method that provides a direct way to determine the relative location or copy number of specific DNA sequences in nuclei or chromosomes. With recent advancements, this technique has found increased application in a number of research areas, including cytogenetics, prenatal diagnosis, cancer research and diagnosis, nuclear organization, gene loss and/or amplification, and gene mapping. The availability of different types of probe and the increasing number of FISH techniques has made it a widespread and diversely applied technology. Multicolor karyotyping by multicolor FISH and spectral karyotyping interphase FISH and comparative genomic hybridization allow genetic analysis of previously intractable targets. We present a brief overview of FISH technology and describe in detail methods of probe labeling and detection for different types of tissue sample, including microdissected nuclei from formalin-fixed paraffin-embedded tissue sections.

Copy number analysis of c-erb-B2 (HER-2/neu) and topoisomerase IIalpha genes in breast carcinoma by quantitative real-time polymerase chain reaction using hybridization probes and fluorescence in situ hybridization.

CONTEXT: The Topoisomerase IIalpha (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents. TOP2A is frequently coamplified with c-erb-B2 and consequently might be a prognostic and/or predictive factor for breast cancer patients when anthracycline-based chemotherapy is a consideration. A total of 20% to 35% of breast carcinomas show amplification of the erb-B2 gene, some of which also have coamplification of the TOP2A gene. Investigation of the prognostic or predictive significance of these gene amplifications requires a reliable and sensitive method for the measurement of gene copy number in clinical tumor samples. OBJECTIVE: To assess 2 different assay methods that might allow accurate, reproducible, quantitative, and high-throughput estimation of gene copy number in fresh, frozen, or paraffin-embedded breast cancer specimens. DESIGN: We developed an assay and analyzed the gene copy numbers of the erb-B2 and TOP2A genes in 8 breast cancer cell lines, 6 fresh frozen samples, and 38 paraffin-embedded breast tumor specimens by a novel real-time polymerase chain reaction (PCR) assay using hybridization probes. The results were compared with standard fluorescence in situ hybridization. RESULTS: We discovered a 100% concordance between assessment of gene copy number of erb-B2 and TOP2A between quantitative PCR and fluorescence in situ hybridization (FISH). Quantitative PCR also had the additional feature of uncovering an erb-B2 gene polymorphism. Finally, we observed that TOP2A amplification only occurred in conjunction with erb-B2 amplification in our paraffin-embedded cases of invasive breast carcinoma and that this event was present in 5 (42%) of 12 erb-B2 amplified cases. CONCLUSIONS: We conclude that the potentially automatic, real-time PCR analysis using hybridization probes is an efficient method to perform copy number analysis, with results that appear identical to the FISH technique and with the benefit of identifying HER-2 polymorphisms.

Loss of heterozygosity associated with uniparental disomy in breast carcinoma.

Loss of heterozygosity is commonly assumed to be due to deletion of the appropriate genomic region in one chromosome within a neoplastic cell but may be due to other mechanisms such as mitotic non-disjunction or somatic recombination leading to uniparental heterodisomy. We chose to study the genomic regions surrounding the p53 and RB1 tumor suppressor genes in breast carcinoma to evaluate the different mechanisms that could mediate loss of heterozygosity. A microsatellite analysis of polymorphic markers in 50 breast cancer samples showed loss of heterozygosity for at least 1 of the 10 markers analyzed in 50% of the tumors studied, and an overall 8.47% of the informative loci showed loss of heterozygosity. All of the cases with loss of heterozygosity were further analyzed for gene copy number of the tumor suppressor genes RB1 and p53 by fluorescence in situ hybridization of either tumor touch preparations or microdissected tumor nuclei with specific genomic probes. Surprisingly, all samples showed the presence of both copies of tumor suppressor genes, including 4/50 cases showing loss of heterozygosity of tumor suppressor gene-spanning markers. One of the 4 cases showed loss of heterozygosity of markers spanning a distance of 6 cM over the RB1 gene, with normal copy numbers of the gene. Three other cases showed loss of heterozygosity of markers within the tumor suppressor gene (RBI or p53) and at least one other spanning marker. No cases showed a simultaneous reduction to homozygosity of markers both near the tumor suppressor gene and distal loci. We suggest that the presence of both copies of the tumor suppressor gene in the cases with loss of heterozygosity of spanning markers and internal markers for that tumor suppressor gene could be explained by somatic recombination resulting in uniparental disomy, but not mitotic nondisjunction or deletion. As the mechanism for physical deletion of a chromosome may be different from those mediating somatic recombination, study of this phenomenon may identify different pathways of genomic instability that may be of diagnostic or treatment significance in breast or other cancers, particularly in those treatments based upon DNA-altering agents.

The retinal rod and cone Na+/Ca2+-K+ exchangers.

The past few years has seen significant progress in our understanding of the retinal rod and cone Na+/Ca2+-K+ exchanger (NCKX) genes. The human rod and cone NCKX genes were localized to chromosomes 15q22 and 9p22, respectively. In situ hybridization localized the rod and cone NCKX transcripts in both human and chicken retinas: rod NCKX transcripts were found only in the inner segments of rods, whereas cone NCKX transcripts were found in a subset of retinal ganglion cells as well as in the inner segments of cones. We identified two sets of putative transmembrane spanning segments (TM's) as the only sequence elements strongly conserved between the rod and cone NCKX cDNAs, as well as between mammalian NCKX cDNAs and NCKX cDNAs cloned from lower organisms (C. elegans and Drosophila). The two sets of TM's make up less than onethird of the rod NCKX sequence and less than half of the cone NCKX sequence. Basic cation binding properties as inferred from an analysis of 45Ca transport rates and NCKX currents were very similar for all our NCKX clones, implying that conserved residues within the two sets of TM's contain all the residues involved in cation binding and cation transport.