Clinical descriptions
We identified an 18-year-old Thai woman who presented to plastic surgeons for total breast reconstruction. Menarche occurred at age 14 years and menstruation was regular. She had been generally healthy with normal intelligence. Height was normal (157 cm, 50th centile). Blood pressure was 140/90 mmHg. The patient had epicanthal folds, small and cup-shaped pinnae, absence of all four upper incisors (small, brown and easily decayed) after extraction at age 15 years (Figure 1A), bilateral absence of breasts and nipples, normal pectoralis muscles, brittle nails and normal external genitalia. Ultrasonography and computed tomography of the kidneys revealed absence of the left kidney and left renal artery, yet normal right kidney and uterus (Figure 1B). A renal function study by a post captopril Tc-99 mMAG3 test showed normal right kidney function with no demonstrable left kidney. The patient was the third child with an elder brother, an elder sister and a younger sister. Her father was deceased. No other family member was affected.
Karyotype Analysis
Peripheral blood samples from the patient and her family members were obtained after written informed consent. Metaphase chromosomes were obtained from phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes. G-banding was performed using standard methods. The karyotype was at a resolution of 550 bands.
Fluorescence in situ hybridization (FISH)
Cell suspensions from the phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes were used in all FISH experiments. Probes mapping to the region of the cytogenetically determined breakpoints were selected from the Mapviewer NCBI http://www.ncbi.nlm.nih.gov/mapview/ and obtained from the BACPAC Resources Center (BPRC, Oakland, CA) (Additional file 1, Table S2). BACs, PACs, or long-range PCR (10 kb each) products were labeled by nick translation with Spectrum Green or Spectrum Orange according to manufacturer's protocols (Abbott Molecular/Vysis, Des Plains, IL). Labeled probes were denatured and hybridized to metaphase spreads on the microscope slide.
Generation of FISH probes using long-range PCR
Three primer pairs were chosen from the genomic sequence of breakpoint-spanning clone on chromosome 1 (RP5-1029K14) (Additional file 1, Table S3). Each probe was overlapped and used for subsequent FISH analysis. We used 200 ng of plasmid DNA, 1X Amplibuffer C (Vivantis, Oceanside, CA), 160 mM (NH4)2SO4, 500 mM Tris-HCl,17.5 mM MgCl2 and stabilizers, 0.4 mM dNTPs, 0.4 μM of each primer and 2.5 U Perpetual OptiTaq DNA polymerase (Vivantis, Oceanside, CA) in a total volume of 50 μl. The PCR amplification was performed as follows: initial denaturation at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds. The annealing step was performed at optimal annealing temperature for each specific primer for 30 seconds. The extension was at 72°C for 11 minutes and the final extension was at 72°C for 10 minutes.
Analysis of repetitive elements within the breakpoint region
We screened DNA sequences for interspersed repeats and low complexity DNA sequences in the breakpoint region using RepeatMasker available at http://www.repeatmasker.org/.
Establishment of Ebstein-Barr virus immortalization of human B-lymphocytes
We isolated Peripheral Blood Mononuclear Cells (PBMCs) using Ficoll-Parque gradient. Peripheral blood lymphocytes (B-lymphocytes) were immortalized by infecting them with active Epstein Barr Virus supernatant [5]. Cyclosporin A was added to suppress the growth of the T cells present in the preparation. Half of the supernatant was replaced by fresh medium once a week. Transformation occurred within 2 to 3 weeks after starting the culture. The human immortalized cells were seeded with the primary culture medium (RPMI1640 with 20% fetal bovine serum, 100 units/ml of penicillin-streptomycin) and were cultured in 37°C with 5% CO2.
Quantitative Real-Time PCR
PTPRF RNA level was quantified by StepOnePlus™ Real-Time PCR Systems (Applied Biosystems, Foster City, CA). Total RNA extracted from EBV transformed lymphoblastoid cell lines was converted to cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). PTPRF RNA levels were studied by quantitative real-time analysis (qRT), using two different TaqMan probes. The first one, Hs00160858_m1 (Applied Biosystems, Foster city, CA), spanning exons 5 and 6, is 5' to the chromosomal breakpoint, while the other, Hs00892984_m1 (Applied Biosystems), spanning exons 32 and 33, is 3' to the breakpoint. The probes were run in triplicate in separate tubes. Relative expression analysis was calculated in terms of ΔΔCt normalized to GAPDH transcript levels, using forward primer; 5'-GTGAAGGTCGGAGTCAACGG-3', reverse primer; 5'-TCAATGAAGGGGTCATTGATGG-3' and probe; HEX-CGCCTGGTCACCAGGGCTGC-BHQ1.
Western Blot Analysis
Lymphoblastoid protein was extracted using ice-cold RIPA lysis buffer with Halt protease inhibitor cocktail (Pierce, Roxford, IL). Protein concentration was determined using the BCA protein assay reagent (Pierce, Roxford, IL). Western blot was performed in triplicate. Protein extract of 100 μg was electrophoresed, transferred to PVDF membrane and incubated with 1:100 anti-PTPRF (BD Transduction Laboratory, San Jose, CA) and 1:1000 goat anti-mouse IgG-HRP: sc-2005 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was stripped and reprobed with 1:1000 anti-GAPDH (Trevigen, Gaitherburgs, MD) and 1:1000 goat anti-rabbit IgG-HRP: sc-2030 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). PTPRF expression was normalized with GAPDH and compared with that of a control.
Mutation analysis
Total RNA and genomic DNA were extracted from peripheral blood using Qiagen RNA and DNA extraction kits according to manufacturer's instructions, respectively (Qiagen, Valencia, CA). Reverse transcription was performed using ImProm-II™ reverse transcriptase (Promega, Madison, WI), according to the manufacturer's instructions. PCR amplification of the entire coding sequence of the PTPRF gene, its promoter [6] (-654 to + 294) and cDNA amplification was performed using set of primers and parameters (Additional file 1, Table S4). In brief, we used 50 ng of either genomic DNA or 100 ng of cDNA, 1×PCR buffer (Promega, Madison, WI), 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of each primer and 0.5 U Taq DNA polymerase (Promega) in a volume of 20 μl. Amplification of the promotor sequences of the PTPRF gene was performed in a reaction mixture of 1× Qiagen PCR buffer (containing 1.5 mM MgCl2), 1× Q-Solution, 0.1 unit HotStarTaq (Qiagen), 200 μM dNTPs, 0.2 μM of each primer and 50 ng of genomic DNA. PCR products were treated with ExoSAP-IT (USP Corporation, Cleveland, OH) according to the manufacturer's recommendations, and sent for direct sequencing (Macrogen Inc., Seoul, Korea). Sequence data were analyzed using Sequencher (version 4.2; Gene Codes Corporation, Ann Arbor, MI).
High-density SNP genotyping array
We performed SNP array for genome wide genotyping using Illumina Human 1 M BeadArray. SNP genotyping was performed according to manufacturer's protocols. The results include within-sample normalized fluorescence ('x' and 'y'), between-sample normalized fluorescence ('Log R ratio' and 'B-allele frequency') and SNP genotype calling. We analyzed data using Bead Studio software and PennCNV. The PennCNV package is available at http://www.openbioinformatics.org/penncnv.
Haplotype analysis
Panels 1 and 2 of the ABI Prism Linkage Mapping Set version 2.5 (Applied Biosystems, Foster City, CA) were amplified. These two panels consist of 31 tandem repeat markers on chromosome 1 that define a 10 cM resolution human index map. Each panel consists of a group of markers that were fluorescently labeled with FAM, HEX, or NED. Amplifications were carried out as single reactions in 96 well plates, according to the manufacturer's protocols. An additional four microsatellite markers selected from Mapviewer, NCBI (D1S3721, D1S2645, D1S2713, D1S3728) were used. These markers were amplified in a 20 μl reaction mixture containing 50 ng DNA, 0.2 μM of each primer, 0.2 mM dNTPs, 1×PCR buffer (Fermentas Inc., Maryland), 1.5 mM MgCl2 and 0.5 U Taq DNA polymerase (Fermentas Inc., Maryland). PCR condition was performed as follows: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 45 seconds, annealing at 60°C for 45 seconds, extension at 72°C for 45 minutes and a final extension at 72°C for 10 minutes. After PCR-amplification of genomic DNA samples, amplified fragments were resolved on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) together with ROX-500 size standard (Applied Biosystems, Foster City, CA). The resulting genotype data were analyzed using GeneScan Analysis (Applied Biosystems, Foster City, CA). Allele numbers were assigned for each marker based on the size of the amplified fragment.