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A complement C5 gene mutation, c.754G>A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases E. Patricia Owen a , Reinhard Würzner b , Felicity Leisegang a , Pierre Rizkallah c , Andrew Whitelaw a , John Simpson a , Andrew D. Thomas c , Claire L. Harris c , Joanna L. Giles c , Bernt C. Hellerud d , Tom E. Mollnes d , B. Paul Morgan c , Paul C. Potter e , Ann Orren a,c,e,∗ a
Department of Clinical Sciences, University of Cape Town, South Africa Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria Institute of Infection and Immunity, Cardiff University, Cardiff, UK d Department of Immunology, Oslo University Hospital, National Hospital, University of Oslo, 0424 Oslo, Norway e Allergy Diagnostic and Clinical Research Unit, Department of Medicine, Lung Institute, University of Cape Town, South Africa b c
a r t i c l e
i n f o
Article history: Received 6 November 2013 Received in revised form 10 November 2014 Accepted 11 November 2014 Available online xxx Keywords: Complement C5 deficiency Meningococcal disease Black African
a b s t r a c t Patients with genetically determined deficiency of complement component 5 are usually diagnosed because of recurrent invasive Neisseria meningitidis infections. Approximately 40 individual cases have been diagnosed worldwide. Nevertheless, reports of the responsible genetic defects have been sporadic, and we know of no previous reports of C5 deficiency being associated with a number of independent meningococcal disease cases in particular communities. Here we describe C5 deficiency in seven unrelated Western Cape, South African families. Three different C5 mutations c.55C>T:p.Q19X, c.754G>A:p.A252T and c.4426C>T:p.R1476X were diagnosed in index cases from two families who had both presented with recurrent meningococcal disease. p.Q19X and p.R1476X have already been described in North American Black families and more recently p.Q19X in a Saudi family. However, p.A252T was only reported in SNP databases and was not associated with disease until the present study was undertaken in the Western Cape, South Africa. We tested for p.A252T in 140 patients presenting with meningococcal disease in the Cape Town area, and found seven individuals in five families who were homozygous for the mutation p.A252T. Very low serum C5 protein levels (0.1–4%) and correspondingly low in vitro functional activity were found in all homozygous individuals. Allele frequencies of p.A252T in the Black African and Cape Coloured communities were 3% and 0.66% and estimated homozygosities are 1/1100 and 1/22,500 respectively. In 2012 we reported association between p.A252T and meningococcal disease. Molecular modelling of p.A252T has indicated an area of molecular stress in the C5 molecule which may provide a mechanism for the very low level in the circulation. This report includes seven affected families indicating that C5D is not rare in South Africa. © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction
Abbreviations: C5D, complement C5 deficiency; MD, meningococcal disease; TCC, terminal complement complex; MAC, membrane attack complex. ∗ Corresponding author at: Department of Clinical Laboratory Sciences, University of Cape Town, PO Box 34560, Groote Schuur 7937, South Africa. Tel.: +44 2920750832. E-mail address:
[email protected] (A. Orren).
The terminal complement pathway comprises the final five components of the complement cascade, C5 through to C9. Complement activation results in the splitting of C5 into the small (74 amino acids) C5a molecule, and the larger C5b molecule which is the initial component of the membrane attack complex (MAC). C5a has an immuno-stimulatory role which also participates in the pathological process including the response to sepsis (Lappegård
http://dx.doi.org/10.1016/j.molimm.2014.11.010 0161-5890/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Owen, E.P., et al., A complement C5 gene mutation, c.754G>A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases. Mol. Immunol. (2014), http://dx.doi.org/10.1016/j.molimm.2014.11.010
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et al., 2009). Terminal pathway complement proteins C5b through to C9 combine sequentially to form the MAC (Hadders et al., 2012). The MAC has a major role in defending the host from infections. There is now also evidence of an anti-inflammatory role of at least one MAC component and C7 is reported as expressed on membranes of endothelial cells as a trap, and thus may have anti-inflammatory properties (Bossi et al., 2009). There is also experimental evidence that late terminal component activation has other more subtle effects which can lead to vascular inflammation in experimental diabetes (Fischetti et al., 2011). Complete MAC formation is not possible if any of the terminal components, such as C5b, are absent. Patients who are deficient in any of the components of the MAC are usually identified clinically after they have presented with recurrent invasive Neisseria meningitidis infections (Petersen et al., 1979; Orren et al., 1987). In South Africa meningococcal disease (MD) is endemic with 1668 cases having been reported over a 5-year period (2008–2012) (URL 1). Recurrent MD infections in individuals with genetic deficiency of one of the terminal complement components can lead to very serious long term sequelae, and affected patients need protection from further MD episodes. Thus, diagnosis of terminal complement defects during the first episode of MD is very important (Orren et al., 2012). C5 deficiency (C5D) has been diagnosed worldwide. Wang et al. (1995) described two genetic defects p.Q19X (rs121909587) and p.R1476X (rs12109588) responsible for C5D in Black American patients who presented with MD. Other C5 defects were described ˜ et al. (2005), and Lopez-Lera et al. (2009), Pfarr by Delgado-Cervino and co-workers commented that exonic sequences not only encode for protein information, but also contain sequences essential for correct mRNA splicing (Pfarr et al., 2005). C5D was also found due to the absence of exon 30 (Aguilar-Ramirez et al., 2009). Recently two patients homozygous for the mutation C5 p.Q19X were found in a Saudi Arabian family (Arnaout et al., 2013), and two new C5 mutations have been reported in Norway and Denmark (Schejbel et al., 2013). We have recently reported three C5D cases among 109 MD cases tested for C5 and C6 gene defects recognised in South Africa (Owen et al., 2012). We have continued to test new MD patients for the C5 mutations p.Q19X, p.A252T (rs112959008) and p.R1476X. Altogether 140 MD patients have now been tested and five index cases were found to be homozygous for C5 p.A252T. We now report the gene frequencies of these three C5 mutations in two Cape South African communities. 2. Patients and methods Molecular genetic analyses of the initial two families (P1 and P2) had been previously undertaken to determine defects in the C5 gene. The three C5 gene mutations found in these families were then screened for in all available patients presenting with MD in the Western Cape. 2.1. Patients 2.1.1. Family 1 An adult Black African woman (P1) was referred to Groote Schuur Hospital, Cape Town following a second episode of MD. No close family members had suffered meningococcal infections. She was initially diagnosed as C5D by haemolytic functional assay. We also investigated her mother, half-sister, half-brother and two children but unfortunately her father was untraceable. 2.1.2. Family 2 The index case (P2) was a Cape Coloured man who was referred to Groote Schuur Hospital because of a history of recurrent MD
episodes which were recorded at 17, 23 and 26 years of age and diagnosed as C5D by haemolytic functional assay. He died at the age of 38 of a subsequent episode. No other family member suffered from meningococcal infections. Samples for genetic testing were only available from the maternal family, but not from the index case himself nor his paternal family. 2.1.3. MD patients (families 3–7) These comprised 140 individuals who had presented with culture confirmed disease either currently or within the previous five years. Patients included were either Black African (Xhosa speaking) or Cape Coloured from the Cape region, South Africa. The Cape Coloured population is a heterogeneous South African ethnic group, with diverse ancestral links. Ancestry may include European settlers, indigenous KhoiSan, Bantu-Nguni (Xhosa speaking), as well as slaves from the East Indies (Malay region). From these MD patients five additional unrelated cases (P3–P7) were diagnosed as C5D. Unfortunately, P5 died of MD during hospital admission. All C5D patients with their immediate family members were asked to attend the Immunology clinic. Family members were interviewed and tested for the three C5 mutations detailed above. All C5D patients identified were prescribed antibiotic prophylaxis. 2.2. Population gene frequency During 2002/3, 750 Black African and 750 Cape Coloured umbilical blood samples were obtained at Medical Obstetric Units near Cape Town. Blood was spotted on Whatman 3 mm filter paper, dried, sealed and stored at −20 ◦ C. Race was determined by the name of the mother. DNA samples from 103 adult KhoiSan and 200 adult South African Caucasians were donated to the study. 2.2.1. Ethics The study was approved by the University of Cape Town Ethics Committee REC REF 093/2002, 282/2003 and 259/2007. All C5D parents and family members gave signed informed consent to participate in the study. 2.3. Molecular studies to determine C5 defects 2.3.1. C5 DNA analysis DNA was isolated from heparinised blood using Trizol phase separation reagent (Life Technologies, Madison, WI, USA) in accordance with the manufacturer’s instructions. DNA analysis of the C5 gene was by PCR amplification of the exons (41) including the putative promoter (Carney et al., 1991). Following amplification, the PCR products were subjected to automated fluorescent sequencing using BigDye Terminator V3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) in accordance with manufacturer’s instructions. sequencing reactions were performed in both the sense and antisense orientations and at least 50 bp of intronic sequence were included on either side of each exon. 2.3.2. C5 mRNA studies Lymphocytes were isolated from heparinised blood and immortalised with Epstein Barr Virus. The mRNA was then isolated using Trizol (Life Technologies, Madison, WI, USA) and reversed transcribed to cDNA using Superscript III (Invitrogen, Madison, WI, USA) and Oligo d(T)20 in accordance with the manufacturer’s instructions. The primers used to amplify six overlapping fragments covering the coding region of C5 cDNA were a kind gift from Margarita ˜ et al. (2005). López-Trascasa and are described by Delgado-Cervino Nested primers were used to amplify the first fragment which
Please cite this article in press as: Owen, E.P., et al., A complement C5 gene mutation, c.754G>A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases. Mol. Immunol. (2014), http://dx.doi.org/10.1016/j.molimm.2014.11.010
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Family 5
*01/07/1970 C5 65µg/ml
*14/10/1994 C5 63µg/ml
*10/01/1997 C5 250µg/ml
Family 6
*21/01/74 C5 71µg/ml
*27/01/1999 C5 62µg/ml
= Heterozygous for C5 p.A252T
3
*16/10/2004 C5 0.22µg/ml
= Heterozygous for C5 p.A252T
Not available
*12/11/2007 No serum
= Homozygous for C5 p.A252T
*12/10/1985 C5 3.2µg/ml
*04/03/2009 C5 2.2µg/ml
= Homozygous for C5 p.A252T
Fig. 1. The family trees for families 5 and 6. The index cases P5 and P6 are indicted with an arrow. No serum was available from P5 who died on hospital admission. Normal levels for C5 protein given in Section 2.
encompassed exons 1–10. All six fragments were sequenced in both the sense and antisense orientations using BigDye Terminator V3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) in accordance with manufacturer’s instructions. The sequences were compared to the published cDNA sequence build number NM001735.2. All mutations seen in the cDNA were confirmed in the genomic DNA by amplification of the relevant exon followed by sequencing. 2.3.3. Detection of C5 mutations DNA was extracted from a 3 mm punch obtained from a blood spot or from buffy coat using QiaAmp DNA blood Mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. The DNA samples were tested for three C5 mutations p.Q19X, p.A252T and p.R1476X by amplification of the relevant region, digestion with restriction enzymes Faq1, Mwo1 or Rsa1 respectively and visualisation on a 3.5% agarose gel using ethidium bromide. All positive samples were confirmed by sequencing. 2.4. Complement protein levels and analysis Serum and/or plasma C5 levels in patients, relatives and controls were measured by sandwich ELISAs using two different monoclonal anti-C5 antibodies in two different laboratories. 2.4.1. ELISA assay 1 The ELISA plates were coated with mouse monoclonal antihuman C5 (N 20-9; 10 g/ml) (Würzner et al., 1991), blocked, and then incubated with test sample. C5 was detected with rabbit polyclonal anti-C5 (15 g/ml) followed by alkaline phosphatase conjugated goat anti-rabbit IgG (1/2000; Sigma, Poole, UK). Values were interpolated from the standard curve; Caucasian normal range 38–122 g/ml (mean 80 ± 42, 2SD; intra-assay C.V.
