Brief Communication Hereditary protein S deficiency from a novel large deletion mutation of the PROS1 gene detected by multiplex ligation-dependent probe amplification (MLPA) Jong-Ha Yoo a,d , Hee-Jin Kim b, ⁎, Ho-Young Maeng c , Young-Ah Kim a,d , Young-Kyu Sun a,d , Jae-Woo Song d , Jong-Rak Choi d , Sun-Hee Kim b , Kyung-A Lee d, ⁎ a Department of Laboratory Medicine, National Health Insurance Corporation Ilsan Hospital, Goyang, Korea b Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea c Department of Internal Medicine, National Health Insurance Corporation Ilsan Hospital, Goyang, Korea d Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea article info Article history: Received 4 August 2008 Received in revised form 25 November 2008 Accepted 5 December 2008 Available online 24 January 2009 Keywords: Protein S deficiency PROS1 multiplex ligation-dependent probe amplification MLPA Korea Introduction Hereditary protein S deficiency (MIM 176880) is an autosomal dominant trait associated with a risk of recurrent venous thrombotic disease [1]. The genetic background is a mutation in the gene encoding protein S (PS), PROS1 , which contains 15 exons over approximately 80 kb on the chromosome band 3q11.2 [2,3]. The mutation spectrum of PROS1 is heterogeneous, being mostly comprised of small point muta- tions [4,5], while large rearrangements including deletions or duplications have been described in a limited number of reports thus far [6,7]. Given the previous observation that point mutations in PROS1 were found only in approximately half of patients with suspected PS deficiency, it was speculated that large deletion or duplication muta- tions might explain those point mutation-negative cases [8,9]. How- ever the technical drawbacks in the traditional molecular diagnostic Thrombosis Research 123 (2009) 793–795 ⁎ Corresponding authors. Kim is to be contacted at Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710, Korea. Tel.: +82 2 3410 2702; fax: +82 2 3410 2719. Lee, Department of Laboratory Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea. Tel.: +82 2 2019 3531; fax: +82 2 2019 4822. E-mail addresses: heejinkim@skku.edu (H.-J. Kim), kal1119@yuhs.ac (K.-A. Lee). approaches, such as Southern blot analysis, have limited the detection of large gene rearrangements. Meanwhile, a relatively new molecular technique that can assess so-called ‘a gene (exon) dosage change’ has been developed [10]; the technique, multiplex ligation-dependent probe amplification (MLPA), can easily detect dosage changes from deletions or duplications involving one or more exons to the whole gene. Briefly, multiple pairs of oligonucleotides containing universal primer sequences are hybridized on genomic target sequences, and then the adjacent probes hybridized are ligated with a thermostable ligase. The resulting fragments are amplified by using the universal primer, and the amplicons can be separated and quantified on an automatic sequencer. The amount of amplicons is proportional to the amount of the target DNA, and thus an abnormal copy number of exons or entire gene can be detected by relative quantification of target amplicons as compared with in control DNA. Indeed, a couple of recent papers described successful detection of large deletion and duplication mutations in PROS1 in patients with point mutation-negative PS deficiency [11,12]. The authors herein describe our experience of detection of a large deletion mutation of PROS1 by the MLPA technique in a Korean patient with thrombophilia. We believe our experience additionally proves the clinical utility of dosage testing in point mutation-negative PS deficiency and the novel mutation involving exon 1 further extends the genetic heterogeneity of PROS1 mutations. Materials and Methods Patient The patient was a 44-year-old Korean woman who had been on warfarin medication since 40 years of age. She first experience painful swelling of her right leg at age 35. At 40, she developed dyspnea and visited a local tertiary hospital where she was diagnosed as having pulmonary embolism (PE). On her visit to our hospital as an outpatient, coagulation tests without anticoagulation revealed a decreased free PS antigen level at 7.1% (LIATEST Free protein S, Diagnostica Stago, Asnieres, France; reference range, 50-150%) and a decreased PS activity at 16% (STACLOT Protein S, Diagnostica Stago; 55-123%). The total PS antigen level on a separate blood sample was 1.56 mg/dl (Human Protein S NL Nanorid, Binding Site, Birmingham, UK; 0.9-2.1 mg/dl). Other coagulation tests including protein C activity and antithrombin 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.12.003 Contents lists available at ScienceDirect Thrombosis Research journal homepage: www.elsevier.com/locate/thromres