Ausavarat et al. BMC Medical Genetics 2011, 12:46 http://www.biomedcentral.com/1471-2350/12/46 RESEARCHARTICLE Open Access PTPRF is disrupted in a patient with syndromic amastia Surasawadee Ausavarat1,2,3, Siraprapa Tongkobpetch2,3, Verayuth Praphanphoj4, Charan Mahatumarat5, Nond Rojvachiranonda5, Thiti Snabboon6, Thomas C Markello7, William A Gahl7, Kanya Suphapeetiporn2,3* and Vorasuk Shotelersuk2,3 Abstract Background: The presence of mammary glands distinguishes mammals from other organisms. Despite significant advances in defining the signaling pathways responsible for mammary gland development in mice, our understanding of human mammary gland development remains rudimentary. Here, we identified a woman with bilateral amastia, ectodermal dysplasia and unilateral renal agenesis. She was found to have a chromosomal balanced translocation, 46,XX,t(1;20)(p34.1;q13.13). In addition to characterization of her clinical and cytogenetic features, we successfully identified the interrupted gene and studied its consequences. Methods: Characterization of the breakpoints was performed by molecular cytogenetic techniques. The interrupted gene was further analyzed using quantitative real-time PCR and western blotting. Mutation analysis and high- density SNP array were carried out in order to find a pathogenic mutation. Allele segregations were obtained by haplotype analysis. Results: We enabled to identify its breakpoint on chromosome 1 interrupting the protein tyrosine receptor type F gene (PTPRF). While the patient’s mother and sisters also harbored the translocated chromosome, their non- translocated chromosomes 1 were different from that of the patient. Although a definite pathogenic mutation on the paternal allele could not be identified, PTPRF’s RNA and protein of the patient were significantly less than those of her unaffected family members. Conclusions: Although ptprf has been shown to involve in murine mammary gland development, no evidence has incorporated PTPRF in human organ development. We, for the first time, demonstrated the possible association of PTPRF with syndromic amastia, making it a prime candidate to investigate for its spatial and temporal roles in human breast development. Keywords: amastia athelia, development of breasts and nipples, ectodermal dysplasia, renal agenesis, balanced chromosome translocation, PTPRF, LAR Background (Additional file 1, Table S1). Amastia has likely been Excellent progress has been made in defining the signal- selected against in human evolution, but its occurrence ing pathways responsible for mammary gland develop- provides an invaluable means to identify genes involved ment in mice [1], current knowledge about human in human breast development. mammary gland development is however very restricted Amastia is the complete absence of breast which is the and requires further elucidation. This may be related to result of complete failure of mammary ridge to develop the extreme rarity of absence of breast or amastia, with at about 6 weeks in utero [2]. In addition, there is a lack only about 62 patients reported in the literature of breast development during puberty whereas other secondary sexual characters and fertility are normal [2]. * Correspondence: [email protected] Amastia can be isolated or syndromic. Syndromes asso- 2Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty ciated with the absence of breasts and nipples include of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand ectodermal dysplasia of the tricho-odonto-onychial type Full list of author information is available at the end of the article © 2011 Ausavarat et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ausavarat et al. BMC Medical Genetics 2011, 12:46 Page 2 of 7 http://www.biomedcentral.com/1471-2350/12/46 (MIM# 129510), acral-renal-ectodermal-dysplasia-lipoa- trophic-diabetes (AREDYLD syndrome) (MIM# 207780), and the scalp-ear-nipple syndrome (SEN or Finlay- Marks syndrome) (MIM# 181270). The latter is the most common amastia-associated syndrome. Addition- ally, renal involvement has been reported in some cases of scalp-ear-nipple syndrome [3,4]. In familial cases of amastia, both autosomal dominant and autosomal reces- sive inheritances have been reported (Additional file 1, Table S1). Here we reported an 18-year-old female with syndro- mic amastia who had a reciprocal balanced transloca- tion, 46, XX, t(1;20)(p34.1;q13.13). In addition to characterization of her clinical and cytogenetic features, we successfully identified the interrupted gene and stu- died its consequences. Methods Clinical descriptions We identified an 18-year-old Thai woman who pre- sented to plastic surgeons for total breast reconstruc- tion. 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 nip- ples, normal pectoralis muscles, brittle nails and normal external genitalia. Ultrasonography and computed tomo- graphy 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 func- tion with no demonstrable left kidney. The patient was the third child with an elder brother, an elder sister and Figure 1 Clinical features of the proband.(A)Faceshows a younger sister. Her father was deceased. No other absence of all four upper incisors (small, brown and easily decayed) family member was affected. after extraction at age 15 years, epicanthal folds, and small cup- shaped pinnae. (B) Computerized tomography of kidneys shows absence of the left kidney and its renal artery. Karyotype Analysis Peripheral blood samples from the patient and her family members were obtained after written informed consent. mapview/ and obtained from the BACPAC Resources Metaphase chromosomes were obtained from phytohe- Center (BPRC, Oakland, CA) (Additional file 1, Table magglutinin (PHA)-stimulated peripheral blood lympho- S2). BACs, PACs, or long-range PCR (10 kb each) pro- cytes. G-banding was performed using standard methods. ducts were labeled by nick translation with Spectrum The karyotype was at a resolution of 550 bands. Green or Spectrum Orange according to manufacturer’s protocols (Abbott Molecular/Vysis, Des Plains, IL). Fluorescence in situ hybridization (FISH) Labeled probes were denatured and hybridized to meta- Cell suspensions from the phytohemagglutinin (PHA)- phase spreads on the microscope slide. stimulated peripheral blood lymphocytes were used in all FISH experiments. Probes mapping to the region of Generation of FISH probes using long-range PCR the cytogenetically determined breakpoints were selected Three primer pairs were chosen from the genomic from the Mapviewer NCBI http://www.ncbi.nlm.nih.gov/ sequence of breakpoint-spanning clone on chromosome Ausavarat et al. BMC Medical Genetics 2011, 12:46 Page 3 of 7 http://www.biomedcentral.com/1471-2350/12/46 1 (RP5-1029K14) (Additional file 1, Table S3). Each using forward primer; 5’-GTGAAGGTCGGAGT- probe was overlapped and used for subsequent FISH CAACGG-3’, reverse primer; 5’-TCAATGAAGGGGT- analysis. We used 200 ng of plasmid DNA, 1X Ampli- CATTGATGG-3’ and probe; HEX-CGCCTGGTCA buffer C (Vivantis, Oceanside, CA), 160 mM (NH4)2SO4, CCAGGGCTGC-BHQ1. 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 Western Blot Analysis OptiTaq DNA polymerase (Vivantis, Oceanside, CA) in Lymphoblastoid protein was extracted using ice-cold a total volume of 50 μl. The PCR amplification was per- RIPA lysis buffer with Halt protease inhibitor cocktail formed as follows: initial denaturation at 95°C for 15 (Pierce, Roxford, IL). Protein concentration was deter- minutes, followed by 40 cycles of denaturation at 95°C mined using the BCA protein assay reagent (Pierce, for 30 seconds. The annealing step was performed at Roxford, IL). Western blot was performed in triplicate. optimal annealing temperature for each specific primer Protein extract of 100 μg was electrophoresed, trans- for 30 seconds. The extension was at 72°C for 11 min- ferred to PVDF membrane and incubated with 1:100 utes and the final extension was at 72°C for 10 minutes. anti-PTPRF (BD Transduction Laboratory, San Jose, CA) and 1:1000 goat anti-mouse IgG-HRP: sc-2005 Analysis of repetitive elements within the breakpoint (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The region membrane was stripped and reprobed with 1:1000 anti- We screened DNA sequences for interspersed repeats GAPDH (Trevigen, Gaitherburgs, MD) and 1:1000 goat and low complexity DNA sequences in the breakpoint anti-rabbit IgG-HRP: sc-2030 (Santa Cruz Biotechnol- region using RepeatMasker available at http://www. ogy, Inc., Santa Cruz, CA). PTPRF expression was nor- repeatmasker.org/. malizedwithGAPDHandcomparedwiththatofa control. Establishment of Ebstein-Barr virus immortalization of human B-lymphocytes Mutation analysis We isolated Peripheral