Computational protocol: Long-term follow-up of females with unbalanced X;Y translocations—reproductive and nonreproductive consequences

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Protocol publication

[…] When the patient was seen at Georgia Regents University, karyotypes were performed on the proband I1 and her healthy daughter II1 using standard G banding methods from peripheral white blood cells (WBCs) []. She signed a consent to have her records reviewed and to have blood drawn for molecular studies, which were approved by the Human Assurance Committee of Georgia Regents University. A lymphoblastoid cell line was created by infecting WBCs with Epstein-Barr virus and using cyclosporine A as described previously []. FISH was performed on metaphase chromosomes using standard methods with five fluorescently labeled probes including: X centromere (DXZ1), Y centromere (DYZ3), Yq12 (DYZ1), STS, and KAL1 genes. A Genome-Wide Human SNP Array 6.0, which features 1.8 million genetic markers, including more than 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 probes, was used to exclude copy number variants (CNV) and narrow the breakpoint. The Affymetrix GeneChip Command Console (AGCC) was used to scan the chips and the Genotype Console 2.0 was used to analyze the data to generate microarray results.Long–range polymerase chain reaction (PCR) was performed to amplify the junction fragment of the derivative X chromosome. Primers were designed to amplify only fragments that contained both X and Y sequences (Table ). Primers were Y-2040for and X-4177rev (to yield a 9 kb fragment), Y-6440for and X-2150rev (to yield a 4 kb fragment), and Y-4140for and X-5891rev (to yield a 10 kb fragment). PCR conditions included a 93°C denaturation step for 3 minutes, followed by 35 cycles of 93°C for 15 seconds, 62°C for 30 seconds, and 68°C for 10 minutes. PCR products were then electrophoresed on 1.2% agarose gels in the presence of a molecular weight marker, stained with ethidium bromide, and photographed. Nested PCR using internal primers was then used to amplify the junction fragment of the breakpoint region. The resulting PCR products were then ethanol precipitated and sequenced using the dideoxy method with the Big Dye Terminator Kit and run on the ABI 310 Automated DNA Sequencer []. Multiple bioinformatic databases were utilized including Human Genome Browser (, Repeat Masker Web Server (, ClustalW2 ( and WWW READSEQ Sequence Conversion ( determine which Yq11 sequences were contained in the der(X) chromosome, 19 sequence tagged sites (STS) on various regions were amplified according to the instructions of the Y Chromosome Deletion Detection System Version 2.0 (Promega; Madison, WI) and electrophoresed on 1.2% agarose gels. […]

Pipeline specifications

Software tools RepeatMasker, Clustal W, ReadSeq
Application Genome data visualization
Organisms Homo sapiens
Diseases Congenital Abnormalities, Cardiovascular Diseases, Eye Abnormalities, Musculoskeletal Abnormalities, Kallmann Syndrome, Gonadoblastoma