Clinica Chimica Acta 438 (2015) 148–153
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Apolipoprotein C-II Tuzla: A novel large deletion in APOC2 caused by Alu-Alu homologous recombination in an infant with apolipoprotein C-II deficiency Minoru Okubo a,b,⁎, Alma Toromanovic c, Tetsu Ebara a, Toshio Murase a a b c
Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan Department of Endocrinology and Metabolism, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan Department of Pediatrics, University Clinical Center Tuzla, 75000 Tuzla, Bosnia and Herzegovina
a r t i c l e
i n f o
Article history: Received 3 August 2014 Accepted 19 August 2014 Available online 27 August 2014 Keywords: Alu repetitive element Apolipoprotein C-II Chylomicronemia Hypertriglyceridemia Large deletion Homologous recombination
a b s t r a c t Backgrounds: Familial apolipoprotein (apo) C-II deficiency is a very rare inherited disorder characterized by chylomicronemia. Since the discovery in 1978, reports on apo C-II deficient patients have been limited and only 13 different mutations in APOC2, a gene encoding apo C-II protein, were identified. Objectives: The objective is to investigate the biochemical and genetic features of a 3-month-old Bosniak girl with chylomicronemia whose apo C-II protein was undetectable in her plasma. Methods: APOC2, LPL, APOA5, and GPIHBP1 were sequenced. Isoelectrofocusing and immunoblotting of chylomicrons and VLDL fraction from the patient were performed. Results: Sequence analysis demonstrated a large deletion of 2978 base pairs in APOC2, which encompassed exons 2, 3, and 4. The patient was homozygous for the deletion. The 5′ part of the breakpoint was located in an Alu Sx repetitive element in intron 1 of APOC2, whereas the 3′ part of the breakpoint was in another Alu Sx between APOC2 and CLPTM1, a gene flanking APOC2. We speculate that the deletion was caused by a homologous recombination between two Alu Sx elements. No mutations were detected in LPL, APOA5, and GPIHBP1. Isoelectrofocusing and immunoblotting confirmed the absence of apo C-II protein. Conclusions: We diagnosed the patient as having apo C-II deficiency and designated the novel large deletion as apo C-II Tuzla. This is the first description of apo C-II deficiency caused by Alu-Alu recombination in APOC2. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Apolipoprotein (apo) C-II is a 79-amino-acid polypeptide and functions as an activator for lipoprotein lipase (LPL), an enzyme catalyzing the hydrolysis of triglyceride (TG) on chylomicrons and very low-density lipoproteins (VLDL) [1]. Deficiency of apo C-II results in the malfunction of the LPL-apo C-II system and causes an accumulation of chylomicrons in the plasma, i.e., chylomicronemia [2]. Clinical manifestations of chylomicronemia are fasting hypertriglyceridemia and recurrent attacks of pancreatitis. Familial apo C-II deficiency (MIM #207750) is a very rare inherited disorder characterized by chylomicronemia. The frequency of LPL Abbreviations: apo, apolipoprotein; bp, base pair; CLPTM1, cleft lip- and palateassociated transmembrane protein-1; GPIHBP1, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1; HDL-C, high-density lipoprotein cholesterol; IEF, isoelectrofocusing; LPL, lipoprotein lipase; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein. ⁎ Corresponding author at: Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111; fax: + 81 3 3582 7068. E-mail address: [email protected] (M. Okubo).
http://dx.doi.org/10.1016/j.cca.2014.08.022 0009-8981/© 2014 Elsevier B.V. All rights reserved.
deficiency is estimated one case in one million persons; however, the frequency of apo C-II deficiency is much lower than that of LPL deficiency. Since the discovery of this autosomal recessive genetic disorder in 1978 [3], reports on apo C-II deficient patients have been limited. Only 13 different mutations in APOC2, a gene encoding apo C-II protein, were revealed in apo C-II deficient patients [4]. Most mutations are specific to each affected family and point mutations at individual sites. Here, we present with the first case of apo C-II deficiency in Bosnia and Herzegovina, and demonstrate a novel large deletion encompassing exons 2, 3, and 4 in the patient's APOC2. We speculate that the deletion is caused by a homologous recombination between two Alu Sx repetitive elements, leading to apo C-II deficiency in the patient. 2. Patient and methods 2.1. Patient A 3-month-old girl presented with vomiting and had chylomicronemia. She was admitted to a hospital in Tuzla. She exhibited neither eruptive xanthomas nor hepatosplenomegaly. No anomalies including
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cleft lip and palate (signs reported to be associated with disrupted cleft lip- and palate-associated transmembrane protein-1, CLPTM1) were present. Her plasma TG concentration was 52.6 mmol/L and total cholesterol (TC) was 13.7 mmol/L. A week after switching breast milk to artificial milk rich in medium chain triglycerides (brand name: Monogen, Nutricia Co., http://nutricia.com/), her TG level was reduced to 5.8 mmol/L. Her TC and high-density lipoprotein cholesterol (HDL-C) were 9.2 and 0.35 mmol/L, respectively. At 5 months of age, her TG was 4.65 mmol/L, while TC and HDL-C were 4.07 and 0.39 mmol/L, respectively. Later, at the age of 9 months on a strict low-fat diet (daily 3 g of fat), her TG, TC, and HDL-C levels were 3.78 mmol/L, 2.90 mmol/L, and 0.27 mmol/L, respectively. Thus far, she has never suffered from acute pancreatitis. There is consanguinity in the family: a common ancestor was traced back to six generations, i.e., parents were 5th grade kinship. The ethnicity of the family is Bosniak. The patient was the only child for her parents. Her mother's plasma TG, TC, and HDL-C were 0.64, 3.9, and 1.22 mmol/L, respectively. Her father's TG, TC, and HDL-C were 2.75, 6.5, and 1.39 mmol/L, respectively. Serum apo C-II concentration was undetectable in the patient, while her mother and father's apo C-II were 1.9 and 4.1 mg/dl, respectively (normal range; 1.6–4.2 mg/dl, determined by an immunoturbidimetric method [5]). These findings suggested that the patient should be apo CII deficiency and prompted us to further investigation.
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Hubley and Green P (http://repeatmasker.org). To rule out structural variations (copy number variations, insertion–deletion, and inversions) in human genomes, we searched the database of genomic variants [11].
2.6. Isoelectrofocusing of apo C-II and immunoblotting Chylomicrons and VLDL fraction (d b 1.006) were isolated from the patient's plasma by using a Beckman TL-100 ultracentrifuge and delipidated. Proteins were dissolved in 8 M urea and isoelectrofocusing (IEF) was performed by using a PhastGel IEF pH 4–6.5 precast-gel on a PhastSystem (GE Healthcare UK Ltd, Buckinghamshire, England). Gels were stained with a silver staining kit. Apolipoprotein C-II protein from human plasma was purchased from Sigma-Aldrich (St. Louis, USA) and used as a positive control. The fraction (d b 1.006) was isolated from healthy controls as well. After IEF, proteins were transferred onto a polyvinyl difluoride membrane. Immunoblotting was performed with rabbit anti-human apolipoprotein C-II antibody (ab76452, Abcam, Cambridge, England) as a primary antibody, and with anti-rabbit IgG antibody (GE Healthcare UK Ltd, Buckinghamshire, England).
3. Results 2.2. Sequencing analysis of APOC2, LPL, APOA5, and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) The protocol of molecular analyses for chylomicronemia was approved by our institutional ethics committee. Genomic DNA was isolated from peripheral blood samples of the patient and parents after informed consent was obtained. PCR for each exon in APOC2 and sequencing analysis of PCR products were performed, as described previously [6]. Long-range PCR was performed with a LA-PCR kit (Takara, Otsu, Japan) by using a sense primer A (5′-gtagaagaggtgacacctgatgg3′) in the 5′-promoter region of APOC2 and an antisense primer B (5′acctcgcatccgtgggctaggcttcacg-3′) in exon 1 of CLPTM1, a gene flanking APOC2. LA-PCR products were electrophoresed on a 0.9% agarose gel. The band of interest was excised and sequenced using internal sequencing primers with a BigDye terminator v1.1 cycle sequencing kit on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Calif., USA). Direct sequencing analyses of LPL and APOA5 were performed, as described elsewhere [7,8]. GPIHBP1 was sequenced by the method of Wang and Hegele [9]. 2.3. Screening for apo C-II Tuzla In order to investigate apo C-II Tuzla carrier, a pair of Primer C (5′gcaacaaagcaagtctcccatctc-3′) and Primer D (5′-atggagcctctgcttatttgc acc-3′) was designed. Carriers of apo C-II Tuzla produce a PCR product of 543 base pairs (bp), whereas the wild type sequence does not produce PCR products due to a long distance between two primers. 2.4. Apo E genotyping Apo E genotypes were determined according to the method of Hixon and Vernier [10]. 2.5. Bioinformatical analysis The nucleotide sequences from APOC2 to CLPTM1 were retrieved from the Homo sapiens chromosome 19 genomic contig NT_011109.16. Positions and subfamilies of Alu elements in APOC2-CLPTM1 sequence were analyzed using RepeatMasker, a program that screens DNA sequences for interspersed repeats and low complexity DNA sequences, by Smit,
3.1. Identification of a novel mutation in the patient's APOC2 Exon 1 of APOC2 was PCR-amplified from the patient's DNA and no mutations were detected. PCR products corresponding to exons 2, 3 and 4, however, could not be obtained from the patients' DNA, while those were successfully PCR-amplified from parents' samples. We postulated that the patient had a gross DNA rearrangement involving exons 2-4 in APOC2. We then performed LA-PCR between the 5′-promoter region of APOC2 and exon 1 of CLPTM1. The PCR fragment from the patient was approximately 3-kb shorter than 9.5-kb long fragments from controls (Fig. 1A). The patient had only the shorter fragment, while parents had both normal and shorter fragments. Sequence analysis of the shorter fragment indicated a novel deletion of 2978 bp (17,719,326–17,722,303 in the H. sapiens chromosome 19 genomic contig NT_011109.16) [g.17,719,326– 17,722,303del2978] (Fig. 1B). APOC4-APOC2 was located between 17,713,713 and 17,721,040 in NT_011109.16, and the deletion encompassed exons 2, 3, and 4 of APOC2. Bioinformatical analysis demonstrated that the 5′ part of the breakpoint was located in an Alu Sx repetitive element in intron 1 of APOC2, whereas the 3′ part of the breakpoint was in another Alu Sx between APOC2 and CLPTM1 (Fig. 1C). Sequence alignment of two Alu Sx indicated high homology and the breakpoint was located in the left flank of Alu repetitive element (Fig. 2). Sequences flanking 5′- and 3′-breakpoint had overlapping 5 nucleotides (5′-GATCA-3′). We designate this large deletion as apo C-II Tuzla after the name of the town where the patient was diagnosed. The patient was a homozygote for the deletion and parents were heterozygotes. Furthermore, the database of genomic variants did not contain this deletion among more than 6500 genomes. In order to screen apo C-II Tuzla mutation, we designed a pair of primers C and D. PCR products containing the breakpoint were visualized on agarose electrophoresis (Fig. 1D). Unfortunately, we could not collect normal controls of Bosniaks; we then decided to screen Japanese controls. Screening of C-II Tuzla in 50 controls (100 chromosomes) showed that none had the large deletion. Apo E genotyping showed that the patient and parents were all homozygous for apo E3. No mutations were detected in the patient's LPL, APOA5, and GPIHBP1.
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Fig. 1. A. Electrophoretic analysis of LA-PCR products from the family with apo C-II Tuzla and control subjects. Primers A and B are used to amplify the region encompassing the deletion in APOC2. M, DNA size marker; Lane 1, patient; lane 2, father; lane 3, mother; lanes 4 and 5, control subjects. The patient shows a 6.5 kb-fragment alone, suggesting that she is homozygous for the deletion. Parents have both 6.5 kb- and normal 9.5-kb fragments, which means that parents are heterozygotes. B. Electropherogram of a novel large deletion in APOC2 from the patient. The deletion of 2978 base pairs is indicated by a vertical line and triangle. Overlapping 5 nucleotides (5′-GATCA-3′) between APOC2 intron 2 sequences and sequences between APOC2 and CLPTM1 are boxed. C. Schematic representation of 2978 bp-deletion in the patient's APOC2. The upper panel shows the normal structure of APOC2, and the lower illustrates the deletion of exons 2, 3, and 4 in the patient. D. Electrophoretic analysis of PCR products from the family with apo C-II Tuzla and control subjects. Primers C and D are used to amplify PCR fragments containing the breakpoints. The patient and parents produce 543-bp fragments, while controls do not.
3.2. Confirmation of the absence of apo C-II proteins in the patient
4. Discussion
Apo C-II proteins have two major isoforms: proapo C-II (apo C-II0) and glycosylated apo C-II (apo C-II 1 ). IEF of chylomicrons and VLDL stained by silver indicated that the patient lacked apo C-II bands (Fig. 3 upper panel). Immunoblotting using anti-apo C-II antibody verified that the patient had no apo C-II proteins (Fig. 3 lower panel).
We investigated a molecular basis of a 3-month-old girl with chylomicronemia and identified a novel deletion of 2978 bp in APOC2. This mutation deleted exons 2, 3, and 4 of APOC2, thereby resulting in deficiency of apo C-II protein (C-II Tuzla ). The patient was homozygous for the deletion and inherited it from heterozygous parents who were distant relatives, 5th degree of kinship.
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Fig. 2. The comparison of Alu sequences. The alignment demonstrates the Alu SX consensus sequence, an Alu SX in intron 1 of APOC2, and another Alu Sx between APOC2 and CLPTM1. Identical nucleotides are indicated by dashes; deletions are denoted by dots. Nucleotide sequences detected in the patient are boxed and the breakpoint is shown by an arrow.
Among the mechanisms causing large deletions in humans, the involvement of Alu repetitive elements has been reported [12]. Alu repetitive elements are characterized by approximately 300 bp-long sequences with a poly (A) tract of variable length and flanking direct repeats. Alu repetitive elements are considered to be retrotransposable and to play significant roles in gene rearrangements during genomic evolution. In our case with apo C-II deficiency, the 5′- and the 3′-parts of the breakpoint were located in Alu Sx repetitive elements. Presumably, a
Fig. 3. Isoelectrofocusing (IEF) of chylomicrons and VLDL fraction (d b 1.006) and immunoblotting. The cathode is at the top and the anode is at the bottom. Upper panel, IEF stained with silver. CII, human apo C-II protein; C, control; P, patient. 1, apo C-II0; 2, apo C-II1; 3, apo C-III0; 4, apo C-III1. Bands corresponding to apo C-II0 and apo C-II1 are not detected in a sample from the patient. Lower panel, immunoblotting using anti-human apo CII antibody. The patient lacks apo C-II proteins.
homologous recombination occurred between two Alu Sx repetitive elements in the past (Fig. 4). The event resulted in the elimination of three exons of APOC2 in the common ancestor in this family more than six generations ago. To date, 13 different mutations in APOC2 were found in patients with apo C-II deficiency (Table 1); our case indicated by boldface is the 14th mutation identified in apo C-II deficiency. All patients were homozygotes for each mutation. Among them, a large deletion was reported in Nijmegen CIV-CII whose deletion encompasses the APOC4 gene [13]; however, the precise breakpoint was not sequenced in that case. Point mutations cause the premature termination of the synthesis of apo C-II protein, leading to apo C-II deficiency in Paris 2 [14], Barcelona [15], Japan [16,17], Venezuela [17], Nijmegen [18], Padova [19], and Bari [20]. A single nucleotide substitution in the promoter [21] or the initiation codon (Paris 1 [22]) results in the inability of apo C-II mRNA synthesis. In Hamburg [23] and Tokyo [6], a G-to-C transversion at the donor splice site of intron 2 causes skipping of exon 2 that contains the initiation codon. Missense mutations have been reported in Wakayama [24] and in a Pakistani patient [25]. Mutant apo C-II proteins were produced in Toronto [26] and St. Michael [27,28]; however, they were non-functional to activate LPL. The majority of mutations is specific for an individual family with apo C-II deficiency, whereas several mutations were found in different ethnic groups, such as the splicing
Fig. 4. A proposed model of an Alu-Alu homologous recombination in the patient. Four exons in APOC2 and exon 1 in CLPTM1 are illustrated by rectangles and doted lines indicated the deleted region. Exons 2, 3, and 4 of APOC2 are deleted in the patient.
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Table 1 Summary of 14 mutations in apo C-II deficiency. Codona
Nucleotideb change
Mutation type Promoter Large deletion
−22 −19
c.-86A N G Breakpoint not sequenced g.17,719,326-17, 722,303del2978c c.1A N G c.10C N T
Number
Mutation location
1 2
Promoter Exon 1
3
Exon 2, 3, and 4
4 5
Exon 2 Exon 2
6
Intron 2
7
Exon 3
2
8 9
Exon 3 Exon 3
10
Large deletion No initiation codon Nonsense mutation R-19X
c.55 + 1G N C
Donor splice site mutation
c.70delC
One base deletion
18 26
c.118delG c.141T N C
Exon 3
37
c.177C N A
11
Exon 3
37
c.177C N G
12 13 14
Exon 4 Exon 4 Exon 4
68 70 72
c.270delT c.274insC c.281T N C
One base deletion Missense mutation p.W26R Nonsense mutation p.Y37X Nonsense mutation p.Y37X One base deletion One base insertion Missense mutation p.L72P
a b c
Name
Ethnic group
Consanguinity
Age (year)
Gender
Greek Caucasian
+ +
42 32
F F
18.9 42.5
(21) (13)
Bosniak
+
F
52.6
This study
Paris 1 Paris 2
Senegalese French
– +
27 12
F F
10.7 20.3
(22) (14)
Barcelona Hamburg
Spain? Turkish
? +
6 30
F F
11.3 20.9
(15) (23)
Tokyo Japan Venezuela Nijmegen Wakayama
Japanese Japanese Venezuelan Dutch Japanese
– + + – +
5 13 0.3 33 36
M F M M M
35.4 12.4 112.9 11.8 10.3
(6) (17) (17) (18) (24)
Padova
Italian
–
36
F
18.4
(19)
Bari
Italian
+
8
F
30.7
(20)
Toronto St. Michael
British Anglo-Saxons Pakistani
+ + +
62 58 2.5
M F F
107.0 15.0 83.2
(26) (28) (25)
Nijmegen CIV-CII Tuzla
0.3
Triglyceride (mmol/L)
Reference
Codons −22 to −1 are in the signal peptide and codons 1 to 79 are in the mature apo C-II protein. M: male; F: female. Nucleotide 1 is the A of the ATG-translation initiation codon in Homo sapiens APOC2 mRNA reference sequence NM_000483.4. Genomic reference sequence is Homo sapiens chromosome 19 genomic contig NT_011109.16.
mutation c.55 + 1G N C in Turkish patients [23,29] and a Japanese patient [6]. In terms of clinical phenotypes, apo C-II deficient patient generally had been detected at a later age, compared with LPL deficient patients [2], but the diagnosis of apo C-II deficiency at earlier age can be made as our report: the patient presented here is one of the youngest among those with apo C-II deficiency (Table 1). Plasma TG levels usually exceeded above 10.0 mmol/L and apo C-II concentrations were undetectable or virtually absent in most patients with apo C-II deficiency. Mutant apo C-II Toronto was not detected by several assay methods as well [30]. In contrast, mutant apo C-II St. Michael was detectable by Western blot but had different molecular weight [27]. In our case, apo C-II protein was undetectable. In conclusion, we identified a novel large deletion in APOC2 (apo C-II Tuzla). The deletion was mediated by a homologous recombination between two Alu Sx elements. This is the first description of apo C-II deficiency caused by Alu-Alu recombination in APOC2. Contributions of authors M.O. performed biochemical and genetic analyses, and wrote the manuscript. A.T. managed the patient and wrote the manuscript. T.E. and T.M. contributed to interpretation of data. Conflict of interest The authors declare no conflict of interest. References [1] Jong MC, Hofker MH, Havekes LM. Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb Vasc Biol 1999;19:472–84. [2] Brunzell JD, Deeb SS. Familial lipoprotein lipase deficiency, Apo C-II deficiency, and hepatic lipase deficiency. In: Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Ballabio A, editors. Scriver's online metabolic and molecular bases of inherited disease. McGraw-Hill; January 2006. http://dx.doi.org/10.1036/ ommbid.145 [Published].
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M. Okubo et al. / Clinica Chimica Acta 438 (2015) 148–153 [19] Fojo SS, Lohse P, Parrott C, et al. A nonsense mutation in the apolipoprotein C-II Padova gene in a patient with apolipoprotein C-II deficiency. J Clin Invest 1989;84: 1215–9. [20] Crecchio C, Capurso A, Pepe G. Identification of the mutation responsible for a case of plasmatic apolipoprotein CII deficiency (Apo CII-Bari). Biochem Biophys Res Commun 1990;168:1118–27. [21] Streicher R, Geisel J, Weisshaar C, et al. A single nucleotide substitution in the promoter region of the apolipoprotein C-II gene identified in individuals with chylomicronemia. J Lipid Res 1996;37:2599–607. [22] Fojo SS, de Gennes JL, Chapman J, et al. An initiation codon mutation in the apoC-II gene (apoC-II Paris) of a patient with a deficiency of apolipoprotein C-II. J Biol Chem 1989;264:20839–42. [23] Fojo SS, Beisiegel U, Beil U, et al. Donor splice site mutation in the apolipoprotein (Apo) C-II gene (Apo C-II Hamburg) of a patient with Apo C-II deficiency. J Clin Invest 1988;82:1489–94. [24] Inadera H, Hibino A, Kobayashi J, et al. A missense mutation (Trp 26– N Arg) in exon 3 of the apolipoprotein CII gene in a patient with apolipoprotein CII deficiency (apo CII-Wakayama). Biochem Biophys Res Commun 1993;193:1174–83.
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Apolipoprotein C-II Tuzla: A novel large deletion in APOC2 caused by Alu-Alu homologous recombination in an infant with apolipoprotein C-II deficiency Minoru Okubo a,b,⁎, Alma Toromanovic c, Tetsu Ebara a, Toshio Murase a a b c
Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan Department of Endocrinology and Metabolism, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan Department of Pediatrics, University Clinical Center Tuzla, 75000 Tuzla, Bosnia and Herzegovina
a r t i c l e
i n f o
Article history: Received 3 August 2014 Accepted 19 August 2014 Available online 27 August 2014 Keywords: Alu repetitive element Apolipoprotein C-II Chylomicronemia Hypertriglyceridemia Large deletion Homologous recombination
a b s t r a c t Backgrounds: Familial apolipoprotein (apo) C-II deficiency is a very rare inherited disorder characterized by chylomicronemia. Since the discovery in 1978, reports on apo C-II deficient patients have been limited and only 13 different mutations in APOC2, a gene encoding apo C-II protein, were identified. Objectives: The objective is to investigate the biochemical and genetic features of a 3-month-old Bosniak girl with chylomicronemia whose apo C-II protein was undetectable in her plasma. Methods: APOC2, LPL, APOA5, and GPIHBP1 were sequenced. Isoelectrofocusing and immunoblotting of chylomicrons and VLDL fraction from the patient were performed. Results: Sequence analysis demonstrated a large deletion of 2978 base pairs in APOC2, which encompassed exons 2, 3, and 4. The patient was homozygous for the deletion. The 5′ part of the breakpoint was located in an Alu Sx repetitive element in intron 1 of APOC2, whereas the 3′ part of the breakpoint was in another Alu Sx between APOC2 and CLPTM1, a gene flanking APOC2. We speculate that the deletion was caused by a homologous recombination between two Alu Sx elements. No mutations were detected in LPL, APOA5, and GPIHBP1. Isoelectrofocusing and immunoblotting confirmed the absence of apo C-II protein. Conclusions: We diagnosed the patient as having apo C-II deficiency and designated the novel large deletion as apo C-II Tuzla. This is the first description of apo C-II deficiency caused by Alu-Alu recombination in APOC2. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Apolipoprotein (apo) C-II is a 79-amino-acid polypeptide and functions as an activator for lipoprotein lipase (LPL), an enzyme catalyzing the hydrolysis of triglyceride (TG) on chylomicrons and very low-density lipoproteins (VLDL) [1]. Deficiency of apo C-II results in the malfunction of the LPL-apo C-II system and causes an accumulation of chylomicrons in the plasma, i.e., chylomicronemia [2]. Clinical manifestations of chylomicronemia are fasting hypertriglyceridemia and recurrent attacks of pancreatitis. Familial apo C-II deficiency (MIM #207750) is a very rare inherited disorder characterized by chylomicronemia. The frequency of LPL Abbreviations: apo, apolipoprotein; bp, base pair; CLPTM1, cleft lip- and palateassociated transmembrane protein-1; GPIHBP1, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1; HDL-C, high-density lipoprotein cholesterol; IEF, isoelectrofocusing; LPL, lipoprotein lipase; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein. ⁎ Corresponding author at: Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111; fax: + 81 3 3582 7068. E-mail address: [email protected] (M. Okubo).
http://dx.doi.org/10.1016/j.cca.2014.08.022 0009-8981/© 2014 Elsevier B.V. All rights reserved.
deficiency is estimated one case in one million persons; however, the frequency of apo C-II deficiency is much lower than that of LPL deficiency. Since the discovery of this autosomal recessive genetic disorder in 1978 [3], reports on apo C-II deficient patients have been limited. Only 13 different mutations in APOC2, a gene encoding apo C-II protein, were revealed in apo C-II deficient patients [4]. Most mutations are specific to each affected family and point mutations at individual sites. Here, we present with the first case of apo C-II deficiency in Bosnia and Herzegovina, and demonstrate a novel large deletion encompassing exons 2, 3, and 4 in the patient's APOC2. We speculate that the deletion is caused by a homologous recombination between two Alu Sx repetitive elements, leading to apo C-II deficiency in the patient. 2. Patient and methods 2.1. Patient A 3-month-old girl presented with vomiting and had chylomicronemia. She was admitted to a hospital in Tuzla. She exhibited neither eruptive xanthomas nor hepatosplenomegaly. No anomalies including
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cleft lip and palate (signs reported to be associated with disrupted cleft lip- and palate-associated transmembrane protein-1, CLPTM1) were present. Her plasma TG concentration was 52.6 mmol/L and total cholesterol (TC) was 13.7 mmol/L. A week after switching breast milk to artificial milk rich in medium chain triglycerides (brand name: Monogen, Nutricia Co., http://nutricia.com/), her TG level was reduced to 5.8 mmol/L. Her TC and high-density lipoprotein cholesterol (HDL-C) were 9.2 and 0.35 mmol/L, respectively. At 5 months of age, her TG was 4.65 mmol/L, while TC and HDL-C were 4.07 and 0.39 mmol/L, respectively. Later, at the age of 9 months on a strict low-fat diet (daily 3 g of fat), her TG, TC, and HDL-C levels were 3.78 mmol/L, 2.90 mmol/L, and 0.27 mmol/L, respectively. Thus far, she has never suffered from acute pancreatitis. There is consanguinity in the family: a common ancestor was traced back to six generations, i.e., parents were 5th grade kinship. The ethnicity of the family is Bosniak. The patient was the only child for her parents. Her mother's plasma TG, TC, and HDL-C were 0.64, 3.9, and 1.22 mmol/L, respectively. Her father's TG, TC, and HDL-C were 2.75, 6.5, and 1.39 mmol/L, respectively. Serum apo C-II concentration was undetectable in the patient, while her mother and father's apo C-II were 1.9 and 4.1 mg/dl, respectively (normal range; 1.6–4.2 mg/dl, determined by an immunoturbidimetric method [5]). These findings suggested that the patient should be apo CII deficiency and prompted us to further investigation.
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Hubley and Green P (http://repeatmasker.org). To rule out structural variations (copy number variations, insertion–deletion, and inversions) in human genomes, we searched the database of genomic variants [11].
2.6. Isoelectrofocusing of apo C-II and immunoblotting Chylomicrons and VLDL fraction (d b 1.006) were isolated from the patient's plasma by using a Beckman TL-100 ultracentrifuge and delipidated. Proteins were dissolved in 8 M urea and isoelectrofocusing (IEF) was performed by using a PhastGel IEF pH 4–6.5 precast-gel on a PhastSystem (GE Healthcare UK Ltd, Buckinghamshire, England). Gels were stained with a silver staining kit. Apolipoprotein C-II protein from human plasma was purchased from Sigma-Aldrich (St. Louis, USA) and used as a positive control. The fraction (d b 1.006) was isolated from healthy controls as well. After IEF, proteins were transferred onto a polyvinyl difluoride membrane. Immunoblotting was performed with rabbit anti-human apolipoprotein C-II antibody (ab76452, Abcam, Cambridge, England) as a primary antibody, and with anti-rabbit IgG antibody (GE Healthcare UK Ltd, Buckinghamshire, England).
3. Results 2.2. Sequencing analysis of APOC2, LPL, APOA5, and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) The protocol of molecular analyses for chylomicronemia was approved by our institutional ethics committee. Genomic DNA was isolated from peripheral blood samples of the patient and parents after informed consent was obtained. PCR for each exon in APOC2 and sequencing analysis of PCR products were performed, as described previously [6]. Long-range PCR was performed with a LA-PCR kit (Takara, Otsu, Japan) by using a sense primer A (5′-gtagaagaggtgacacctgatgg3′) in the 5′-promoter region of APOC2 and an antisense primer B (5′acctcgcatccgtgggctaggcttcacg-3′) in exon 1 of CLPTM1, a gene flanking APOC2. LA-PCR products were electrophoresed on a 0.9% agarose gel. The band of interest was excised and sequenced using internal sequencing primers with a BigDye terminator v1.1 cycle sequencing kit on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Calif., USA). Direct sequencing analyses of LPL and APOA5 were performed, as described elsewhere [7,8]. GPIHBP1 was sequenced by the method of Wang and Hegele [9]. 2.3. Screening for apo C-II Tuzla In order to investigate apo C-II Tuzla carrier, a pair of Primer C (5′gcaacaaagcaagtctcccatctc-3′) and Primer D (5′-atggagcctctgcttatttgc acc-3′) was designed. Carriers of apo C-II Tuzla produce a PCR product of 543 base pairs (bp), whereas the wild type sequence does not produce PCR products due to a long distance between two primers. 2.4. Apo E genotyping Apo E genotypes were determined according to the method of Hixon and Vernier [10]. 2.5. Bioinformatical analysis The nucleotide sequences from APOC2 to CLPTM1 were retrieved from the Homo sapiens chromosome 19 genomic contig NT_011109.16. Positions and subfamilies of Alu elements in APOC2-CLPTM1 sequence were analyzed using RepeatMasker, a program that screens DNA sequences for interspersed repeats and low complexity DNA sequences, by Smit,
3.1. Identification of a novel mutation in the patient's APOC2 Exon 1 of APOC2 was PCR-amplified from the patient's DNA and no mutations were detected. PCR products corresponding to exons 2, 3 and 4, however, could not be obtained from the patients' DNA, while those were successfully PCR-amplified from parents' samples. We postulated that the patient had a gross DNA rearrangement involving exons 2-4 in APOC2. We then performed LA-PCR between the 5′-promoter region of APOC2 and exon 1 of CLPTM1. The PCR fragment from the patient was approximately 3-kb shorter than 9.5-kb long fragments from controls (Fig. 1A). The patient had only the shorter fragment, while parents had both normal and shorter fragments. Sequence analysis of the shorter fragment indicated a novel deletion of 2978 bp (17,719,326–17,722,303 in the H. sapiens chromosome 19 genomic contig NT_011109.16) [g.17,719,326– 17,722,303del2978] (Fig. 1B). APOC4-APOC2 was located between 17,713,713 and 17,721,040 in NT_011109.16, and the deletion encompassed exons 2, 3, and 4 of APOC2. Bioinformatical analysis demonstrated that the 5′ part of the breakpoint was located in an Alu Sx repetitive element in intron 1 of APOC2, whereas the 3′ part of the breakpoint was in another Alu Sx between APOC2 and CLPTM1 (Fig. 1C). Sequence alignment of two Alu Sx indicated high homology and the breakpoint was located in the left flank of Alu repetitive element (Fig. 2). Sequences flanking 5′- and 3′-breakpoint had overlapping 5 nucleotides (5′-GATCA-3′). We designate this large deletion as apo C-II Tuzla after the name of the town where the patient was diagnosed. The patient was a homozygote for the deletion and parents were heterozygotes. Furthermore, the database of genomic variants did not contain this deletion among more than 6500 genomes. In order to screen apo C-II Tuzla mutation, we designed a pair of primers C and D. PCR products containing the breakpoint were visualized on agarose electrophoresis (Fig. 1D). Unfortunately, we could not collect normal controls of Bosniaks; we then decided to screen Japanese controls. Screening of C-II Tuzla in 50 controls (100 chromosomes) showed that none had the large deletion. Apo E genotyping showed that the patient and parents were all homozygous for apo E3. No mutations were detected in the patient's LPL, APOA5, and GPIHBP1.
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Fig. 1. A. Electrophoretic analysis of LA-PCR products from the family with apo C-II Tuzla and control subjects. Primers A and B are used to amplify the region encompassing the deletion in APOC2. M, DNA size marker; Lane 1, patient; lane 2, father; lane 3, mother; lanes 4 and 5, control subjects. The patient shows a 6.5 kb-fragment alone, suggesting that she is homozygous for the deletion. Parents have both 6.5 kb- and normal 9.5-kb fragments, which means that parents are heterozygotes. B. Electropherogram of a novel large deletion in APOC2 from the patient. The deletion of 2978 base pairs is indicated by a vertical line and triangle. Overlapping 5 nucleotides (5′-GATCA-3′) between APOC2 intron 2 sequences and sequences between APOC2 and CLPTM1 are boxed. C. Schematic representation of 2978 bp-deletion in the patient's APOC2. The upper panel shows the normal structure of APOC2, and the lower illustrates the deletion of exons 2, 3, and 4 in the patient. D. Electrophoretic analysis of PCR products from the family with apo C-II Tuzla and control subjects. Primers C and D are used to amplify PCR fragments containing the breakpoints. The patient and parents produce 543-bp fragments, while controls do not.
3.2. Confirmation of the absence of apo C-II proteins in the patient
4. Discussion
Apo C-II proteins have two major isoforms: proapo C-II (apo C-II0) and glycosylated apo C-II (apo C-II 1 ). IEF of chylomicrons and VLDL stained by silver indicated that the patient lacked apo C-II bands (Fig. 3 upper panel). Immunoblotting using anti-apo C-II antibody verified that the patient had no apo C-II proteins (Fig. 3 lower panel).
We investigated a molecular basis of a 3-month-old girl with chylomicronemia and identified a novel deletion of 2978 bp in APOC2. This mutation deleted exons 2, 3, and 4 of APOC2, thereby resulting in deficiency of apo C-II protein (C-II Tuzla ). The patient was homozygous for the deletion and inherited it from heterozygous parents who were distant relatives, 5th degree of kinship.
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Fig. 2. The comparison of Alu sequences. The alignment demonstrates the Alu SX consensus sequence, an Alu SX in intron 1 of APOC2, and another Alu Sx between APOC2 and CLPTM1. Identical nucleotides are indicated by dashes; deletions are denoted by dots. Nucleotide sequences detected in the patient are boxed and the breakpoint is shown by an arrow.
Among the mechanisms causing large deletions in humans, the involvement of Alu repetitive elements has been reported [12]. Alu repetitive elements are characterized by approximately 300 bp-long sequences with a poly (A) tract of variable length and flanking direct repeats. Alu repetitive elements are considered to be retrotransposable and to play significant roles in gene rearrangements during genomic evolution. In our case with apo C-II deficiency, the 5′- and the 3′-parts of the breakpoint were located in Alu Sx repetitive elements. Presumably, a
Fig. 3. Isoelectrofocusing (IEF) of chylomicrons and VLDL fraction (d b 1.006) and immunoblotting. The cathode is at the top and the anode is at the bottom. Upper panel, IEF stained with silver. CII, human apo C-II protein; C, control; P, patient. 1, apo C-II0; 2, apo C-II1; 3, apo C-III0; 4, apo C-III1. Bands corresponding to apo C-II0 and apo C-II1 are not detected in a sample from the patient. Lower panel, immunoblotting using anti-human apo CII antibody. The patient lacks apo C-II proteins.
homologous recombination occurred between two Alu Sx repetitive elements in the past (Fig. 4). The event resulted in the elimination of three exons of APOC2 in the common ancestor in this family more than six generations ago. To date, 13 different mutations in APOC2 were found in patients with apo C-II deficiency (Table 1); our case indicated by boldface is the 14th mutation identified in apo C-II deficiency. All patients were homozygotes for each mutation. Among them, a large deletion was reported in Nijmegen CIV-CII whose deletion encompasses the APOC4 gene [13]; however, the precise breakpoint was not sequenced in that case. Point mutations cause the premature termination of the synthesis of apo C-II protein, leading to apo C-II deficiency in Paris 2 [14], Barcelona [15], Japan [16,17], Venezuela [17], Nijmegen [18], Padova [19], and Bari [20]. A single nucleotide substitution in the promoter [21] or the initiation codon (Paris 1 [22]) results in the inability of apo C-II mRNA synthesis. In Hamburg [23] and Tokyo [6], a G-to-C transversion at the donor splice site of intron 2 causes skipping of exon 2 that contains the initiation codon. Missense mutations have been reported in Wakayama [24] and in a Pakistani patient [25]. Mutant apo C-II proteins were produced in Toronto [26] and St. Michael [27,28]; however, they were non-functional to activate LPL. The majority of mutations is specific for an individual family with apo C-II deficiency, whereas several mutations were found in different ethnic groups, such as the splicing
Fig. 4. A proposed model of an Alu-Alu homologous recombination in the patient. Four exons in APOC2 and exon 1 in CLPTM1 are illustrated by rectangles and doted lines indicated the deleted region. Exons 2, 3, and 4 of APOC2 are deleted in the patient.
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Table 1 Summary of 14 mutations in apo C-II deficiency. Codona
Nucleotideb change
Mutation type Promoter Large deletion
−22 −19
c.-86A N G Breakpoint not sequenced g.17,719,326-17, 722,303del2978c c.1A N G c.10C N T
Number
Mutation location
1 2
Promoter Exon 1
3
Exon 2, 3, and 4
4 5
Exon 2 Exon 2
6
Intron 2
7
Exon 3
2
8 9
Exon 3 Exon 3
10
Large deletion No initiation codon Nonsense mutation R-19X
c.55 + 1G N C
Donor splice site mutation
c.70delC
One base deletion
18 26
c.118delG c.141T N C
Exon 3
37
c.177C N A
11
Exon 3
37
c.177C N G
12 13 14
Exon 4 Exon 4 Exon 4
68 70 72
c.270delT c.274insC c.281T N C
One base deletion Missense mutation p.W26R Nonsense mutation p.Y37X Nonsense mutation p.Y37X One base deletion One base insertion Missense mutation p.L72P
a b c
Name
Ethnic group
Consanguinity
Age (year)
Gender
Greek Caucasian
+ +
42 32
F F
18.9 42.5
(21) (13)
Bosniak
+
F
52.6
This study
Paris 1 Paris 2
Senegalese French
– +
27 12
F F
10.7 20.3
(22) (14)
Barcelona Hamburg
Spain? Turkish
? +
6 30
F F
11.3 20.9
(15) (23)
Tokyo Japan Venezuela Nijmegen Wakayama
Japanese Japanese Venezuelan Dutch Japanese
– + + – +
5 13 0.3 33 36
M F M M M
35.4 12.4 112.9 11.8 10.3
(6) (17) (17) (18) (24)
Padova
Italian
–
36
F
18.4
(19)
Bari
Italian
+
8
F
30.7
(20)
Toronto St. Michael
British Anglo-Saxons Pakistani
+ + +
62 58 2.5
M F F
107.0 15.0 83.2
(26) (28) (25)
Nijmegen CIV-CII Tuzla
0.3
Triglyceride (mmol/L)
Reference
Codons −22 to −1 are in the signal peptide and codons 1 to 79 are in the mature apo C-II protein. M: male; F: female. Nucleotide 1 is the A of the ATG-translation initiation codon in Homo sapiens APOC2 mRNA reference sequence NM_000483.4. Genomic reference sequence is Homo sapiens chromosome 19 genomic contig NT_011109.16.
mutation c.55 + 1G N C in Turkish patients [23,29] and a Japanese patient [6]. In terms of clinical phenotypes, apo C-II deficient patient generally had been detected at a later age, compared with LPL deficient patients [2], but the diagnosis of apo C-II deficiency at earlier age can be made as our report: the patient presented here is one of the youngest among those with apo C-II deficiency (Table 1). Plasma TG levels usually exceeded above 10.0 mmol/L and apo C-II concentrations were undetectable or virtually absent in most patients with apo C-II deficiency. Mutant apo C-II Toronto was not detected by several assay methods as well [30]. In contrast, mutant apo C-II St. Michael was detectable by Western blot but had different molecular weight [27]. In our case, apo C-II protein was undetectable. In conclusion, we identified a novel large deletion in APOC2 (apo C-II Tuzla). The deletion was mediated by a homologous recombination between two Alu Sx elements. This is the first description of apo C-II deficiency caused by Alu-Alu recombination in APOC2. Contributions of authors M.O. performed biochemical and genetic analyses, and wrote the manuscript. A.T. managed the patient and wrote the manuscript. T.E. and T.M. contributed to interpretation of data. Conflict of interest The authors declare no conflict of interest. References [1] Jong MC, Hofker MH, Havekes LM. Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb Vasc Biol 1999;19:472–84. [2] Brunzell JD, Deeb SS. Familial lipoprotein lipase deficiency, Apo C-II deficiency, and hepatic lipase deficiency. In: Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Ballabio A, editors. Scriver's online metabolic and molecular bases of inherited disease. McGraw-Hill; January 2006. http://dx.doi.org/10.1036/ ommbid.145 [Published].
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