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A 3-day-old neonate with severe hypertriglyceridemia from novel mutations of the GPIHBP1 gene
Journal of Clinical Lipidology (2015) 9, 265–270
A 3-day-old neonate with severe hypertriglyceridemia from novel mutations of the GPIHBP1 gene Paola Sabrina Buonuomo, MD, Andrea Bartuli, MD, Claudio Rabacchi, PhD, Stefano Bertolini, MD, Sebastiano Calandra, MD* Rare Diseases and Medical Genetics, Bambino Ges u Children Hospital, Rome, Italy (Drs Buonuomo and Bartuli); Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy (Dr Rabacchi); Department of Internal Medicine, University of Genova, Genova, Italy (Bertolini); and Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy (Dr Calandra) KEYWORDS: Hypertriglyceridemia; Lipoprotein lipase; (GPIHBP1) glycosylphosphatidylinositolanchored high-density lipoprotein-binding protein 1; Intravascular lipolysis
BACKGROUND: Familial chylomicronemia is a genetic defect of the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. Intravascular lipolysis involves the TG-hydrolase lipoprotein lipase (LPL) as well as other factors such as apolipoprotein CII and apolipoprotein AV (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface), and LMF1 (a factor required for intracellular formation of active LPL). METHODS: We sequenced the familial chylomicronemia candidate genes in a neonate with chylomicronemia. RESULTS: A 3-day-old newborn was found to have chylomicronemia (plasma TG 18.8 mmol/L, 1.667 mg/dL). The discontinuation of breastfeeding for 24 hours reduced plasma TG to 2.3 mmol/L (201 mg/dL), whereas its resumption induced a sharp TG increase (7.9 mmol/L, 690 mg/dL). The child was switched to a low-fat diet, which was effective in maintaining TG level below 3.5 mmol/L (294 mg/dL) during the first months of life. The child was found to be a compound heterozygous for 2 novel mutations in GPIHBP1 gene. The first mutation was a 9-bp deletion and 4-bp insertion in exon 2, causing a frameshift that abolished the canonical termination codon TGA. The predicted translation product of the mutant messenger RNA is a peptide that contains 51 amino acids of the N-terminal end of the wild-type protein followed by 252 novel amino acids. The second mutation was a nucleotide change (c.319T.C), causing an amino acid substitution p.(Ser107Pro) predicted in silico to be damaging. CONCLUSIONS: GPIHBP1 mutations should be considered in neonates with chylomicronemia negative for mutations in LPL gene. Ó 2015 National Lipid Association. All rights reserved.
* Corresponding author. Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Campi 287, I-41125 Modena, Italy.
E-mail address: [email protected] Submitted July 29, 2014. Accepted for publication October 6, 2014.
1933-2874/$ - see front matter Ó 2015 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2014.10.003
266 Hypertriglyceridemia (HTG) is conventionally defined as severe when the level of fasting plasma triglycerides (TG) is .10 mmol/L. This condition is often referred to as chylomicronemia because of plasma accumulation of chylomicrons in the fasting state.1,2 Familial chylomicronemia (OMIM238600), also known as hyperlipoproteinemia type 1, is a rare recessive disorder characterized by the accumulation of chylomicrons in the circulation, which often appears during early infancy and childhood.1,2 In this condition, fasting serum TG level is very high and sometimes can exceed 100 mmol/L (8857 mg/dL). The clinical features include failure to thrive, eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, recurrent abdominal pain, and episodes of acute pancreatitis.1-5 In newborns and very young children (,1 year of age), chylomicronemia is often an incidental finding of milky plasma during routine blood tests or laboratory investigations carried out in the presence of clinical manifestations not related to chylomicronemia.3,7,8 Familial chylomicronemia is due to a defect in the lipolytic cascade of TG-rich lipoproteins that may result from mutations in at least 5 different genes: LPL (encoding the enzyme lipoprotein lipase, LPL; OMIM #238600); APOC2 (encoding the apolipoprotein CII, the activator of LPL; OMIM #207750); APOA5 (encoding apolipoprotein AV, also an activator of LPL; OMIM #144650); GPIHBP1 (encoding glycosylphosphatidylinositol-anchored highdensity lipoprotein-binding protein 1), the molecular platform that, on the endothelial surface of capillaries, allows the interactions of LPL with TG-rich lipoproteins, apolipoprotein CII, and apolipoprotein AV; OMIM #612757); and LMF1 (encoding the lipase maturation factor 1, a tissue factor that allows the secretion of functional LPL and hepatic lipase; OMIM #611761).2,5,9,10 Inactivating mutations of LPL are the most common cause of familial chylomicronemia (95% of cases).2 Here we describe the clinical features and genetic analysis of a 3-day-old child found to have elevated plasma TG (.10 mmol/L, .885 mg/dL) during a routine blood test for high bilirubin levels. The child was found to be a compound heterozygous for 2 novel mutations in the GPIHBP1 gene.
Methods Clinical features of the patient The patient was the first female neonate of unrelated parents delivered after a full-term uneventful pregnancy. Her birth weight was 3100 g, length was 50 cm, and the head circumference was 35 cm; the physical examination was negative. Breastfeeding was started on day 1. On day 3, she presented mild jaundice (bilirubin level 15.9 mg/dL), but capillary blood was remarkably pink-creamy colored. Laboratory tests revealed elevated plasma level of TG (18 mmol/L, 1667 mg/dL). Ophthalmoscopic examination
Journal of Clinical Lipidology, Vol 9, No 2, April 2015 and abdominal and cerebral ultrasound examinations were negative. Glucose, liver transaminases, alkaline phosphatase, lipase, amylase, and serum electrolytes were within normal ranges. Blood cell count was normal. Family history was unremarkable. The parents’ plasma lipid levels were in the normal range. In view of the severe HTG, it was assumed that the baby had a genetic defect of the intravascular lipolysis of TG-rich plasma lipoproteins (familial chylomicronemia).
Laboratory investigations A blood sample was collected from the patient and her parents to analyze the candidate genes of familial chylomicronemia. A written informed consent was obtained from the parents before DNA analysis. All procedures were in accordance with the ethical standards of the responsible institutional committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2013. Genomic DNA was extracted from peripheral blood leukocytes using a standard method. The candidate genes for familial chylomicronemia (LPL, APOC2, APOA5, GPIHBP1, and LMF1) were resequenced sequentially as previously reported.11,12 The mutations were designated according to the Human Genome Variation Society, 2012 version (http://www.hgvs.org/mutnomen/recs-DNA.html). GPIHBP1 protein sequence variants were designated according to http://www.hgvs.org/mutnomen/recs-prot.html. The screening of the 2 GPIHBP1 mutations found in the proband was performed in 80 individuals with primary severe HTG and 200 normolipidemic controls. The in silico prediction of the effect of the missense mutation of GPIHBP1 protein was performed using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT Human Protein and SIFT BLink (http://sift.jcvi.org/), SNPs3D (http://snps3d.org), PMut (http://mmb2.pcb.ub.es/ pmut), and SNAP (https://www.rostlab.org/services/snap/).
Results Clinical findings and follow-up Because the high risk of pancreatitis related to the high levels of plasma TG13,14 enteral feeding was interrupted at day 3 and infusion of isotonic intravenous solution was started. After 24 hours, plasma TG level was found to be greatly reduced (3.87 mmol/L, 342 mg/dL); the switching to breastfeeding for 1 day was followed by a substantial rise of plasma TG up to 7.9 mmol/L (700 mg/dL). In view of this response to breastfeeding, the baby was switched to a low-lipid formula diet (Basic F. Milupa – 49 Kcal/100 g). This dietary treatment induced a prompt and persistent reduction of plasma TG level (2.86 mmol/ L, 235 mg/dL) at discharge from the hospital. At 3 months of age the baby was found to be in good health with a normal growth (weight, height, and head circumference
Buonuomo et al
GPIHBP1 mutations in chylomicronemia
within the 50th percentile) and a normal development. Her plasma lipid profile showed a mild elevation of plasma TG and a low level of high-density lipoprotein cholesterol (HDL-C) (Table 1). (At 5 months of age, the lipid values were as follows: TG 3.12 mmol/L [276 mg/dL] and total cholesterol 2.8 mmol/L (108 mg/dL). The plasma lipid levels of proband’s parents were within the normal range (Table 1).
Gene analysis The sequence of LPL and APOC2 revealed that the proband was heterozygous for 3 common variants of the LPL gene [c.405 C.A, p.(Val108Val); c.435 G.A, p.(Glu118Glu), and c.1164 C.A, p.(Thr361Thr)] and homozygous for 2 common variants of APOC2 gene [c.1 –80G.T and c.215138_40 or 41_43delACC] located in introns 1 and 3, respectively. No variants were detected in APOA5 and LMF1 genes. The sequence of GPIHBP1 gene revealed that the proband was heterozygous for 2 mutations: 1) a 9-bp deletion and 4-bp insertion (c.154_162AA CAGGCTCdelTCTTins) in exon 2 (Figs. 1 and 2) and 2) a single nucleotide substitution in exon 4 (c.319T.C) (Fig. 1). The del/ins mutation in exon 2 causes a frameshift that abolishes the canonical termination codon TGA at position c.553-555. The translation product of the mutant messenger RNA (mRNA) is predicted to be a peptide that contains 51 amino acids of the N-terminal end of the wild-type protein followed by 252 novel amino acids (Fig. 2). The point mutation in exon 4 leads to the conversion of the serine residue at position 107 into a proline residue [p.(Ser107Pro)]. The in silico analyses (Polyphen 2, SIFT, SNPs3D, PMut, and SNAP) all were consistent in predicting a pathogenic effect of this mutation. The mutation screening of the parents revealed that del/ins mutation was inherited from the mother and the missense mutation from the father. These novel mutations were not detected in 80 adult patients with severe primary HTG and in 200 normolipidemic individuals. Because the proband’s parents had a normal plasma triglycerides, we thought that they might carry some variants of TG-linked genes that reduced plasma TG levels.15–17 For this reason, we sequenced the LPL and APOC3 genes. Both parents were found to be heterozygous for the common truncation variant p.(Ser474*) (Ser447X) of the LPL gene. No rare loss-of-function variants were found in the APOC3 gene (Supplementary Tables S2 and S3).
Table 1
267
Discussion Clinical aspects In this study, we describe a 3-day-old newborn who incidentally was found to have severe HTG, suggesting familial chylomicronemia. Because severe HTG is considered at high risk for pancreatitis,1,13,14 enteral feeding was suspended and replaced by infusion of an isotonic solution. This therapeutic option was sufficient to rapidly reduce plasma TG to a safety level in terms of possible risk of pancreatitis. The replacement of breastfeeding with a low-lipid formula diet was successful in maintaining the TG level below 4 mmol/L (354 mg/dL) and ensuring a normal growth over time. Our patient is the third case of a biallelic carrier of GPIHBP1 mutations with severe HTG diagnosed in the first few months of life. Rios et al18 described a breastfed infant 2 months of age with failure to thrive and lethargy whose plasma TG level was .280 mmol/L (.24,800 mg/dL) on hospital admission. He was found to be homozygous for a complete deletion of GPIHBP1 gene. Upon admission, he was placed on low-fat diet and given fluids intravenously. After 24 hours, plasma TG level was modestly reduced. He was then switched to a low-fat diet milk protein–based dietary formula containing 40% fat with 87% medium-chain triglycerides. After 12 days on this diet, plasma TG level was 18 mmol/L (1594 mg/dL). Gonzaga-Jauregui et al19 reported a 3-month-old breastfed infant admitted to the hospital for intestinal bleeding who was found to have severe HTG (TG level 136 mmol/L) (12,046 mg/dL). He was compound heterozygous for 2 mutations in the GHPIBP1 gene [c.331A.C, p.(Thr111Pro) and c.413_429del, p.(P140Sfs*161)]. The third infant, recently reported by Ahmad et al,20 was admitted to the hospital at 6 months of age for acute pancreatitis associated with hypertriglyceridemia (TG.22.5 mmol/L, .2000 mg/dL). She was found to be compound heterozygote for a 4 base deletion in exon 2 (c.85_88GAGGdel, p.(E29Tfs*50) and a base substitution in exon 3, resulting in a nonsense mutation (c.267 C.A, p.(C89*). Both mutations resulted in truncated protein devoid of function. The much lower plasma TG level found in our patient with respect to the infants described in the studies mentioned previously18–20 may be due to the fact that in our patient hypertriglyceridemia was detected very early (on day 3 of life) and breastfeeding was promptly
Plasma lipids in the proband and her parents
Subject
Total cholesterol mmol/L (mg/dL)
Triglyceride mmol/L (mg/dL)
LDL-C mmol/L (mg/dL)
HDL-C mmol/L (mg/dL)
Proband Mother Father
3.36 (130) 4.31 (166) 4.82 (186)
3.37 (298) 0.90 (79) 0.61 (64)
1.31 (51) 2.51 (97) 2.82 (108)
0.77 (30) 1.40 (54) 1.62 (63)
HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.
268
Journal of Clinical Lipidology, Vol 9, No 2, April 2015
Figure 2 Partial nucleotide sequence of GPIHBP1 gene showing the nucleotide deletion/insertion in exon 2 and its effect on messenger RNA (mRNA) sequence. The mutation abolishes the canonical termination codon TGA (term 185) in exon 4 and creates a new termination codon (term 304). The predicted translation product of the mutant (Mut) mRNA is an extremely long protein (303 amino acids vs 184 amino acids of the wild-type [WT] counterpart) [p.(Asn52Serfs*253)]. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
Figure 1 Partial nucleotide sequence of GPIHBP1 gene in the proband. The proband was a compound heterozygous for a minute deletion/insertion in exon 2 (A) and a single nucleotide substitution in exon 4 (B). For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
discontinued and replaced by an alternative low-fat formula diet. It is reasonable to assume that the few months of breastfeeding of the patients described by Rios et,18 GonzagaJauregui et al,19 and Ahmad et al20 could have induced a progressive accumulation of plasma TG, which reached an extremely high level. This hypothesis seems to be confirmed by other reports of infants with LPL deficiency resulting from mutations in the LPL gene. Avis et al6 reported a 3-month-old baby with vomiting, loose stools, and abdominal pain with severe HTG (350 mmol/L) (31,000 mg/dL). This child had been breastfed for 2 months and then switched to standard formula bottle feeding. The administration of a nearly fatfree diet led to a dramatic reduction of plasma TG within a week. Stefanutti et at al7 described a 3 month-old breastfed baby with extremely severe HTG (TG level .300 mmol/L) (.26,500 mg/dL). This patient was treated with plasma exchange, which induced a dramatic decrease in plasma TG. After 24 and 72 hours, the plasma TG level dropped to 30.4
and 11.6 mmol/L (2657 and 1027 mg/dL), respectively. The switch to a low-fat formula diet was associated with persistent plasma TG reduction with a level ranging from 5 to 10 mmol (442 to 884 mg/dL). Similar considerations apply to the patient reported by Pugni et al,8 a 23-day-old breastfed patient with plasma TG level of 93 mmol/L (8237 mg/dL). This child underwent blood exchange transfusion, which reduced plasma TG level to 3 mmol/L (265 mg/dL) within 24 hours. The switch to a low-fat diet maintained plasma TG level between 3 and 5 mmol/L (265 and 442 mg/dL). In the light of the plasma TG level and the satisfactory response to dietary changes, invasive therapeutic options (plasma exchange or exchange blood transfusion)7,8 were not considered as a first line of treatment in our patient. The plasma lipid profile of the proband’s parents was unremarkable as it is often observed in young and lean heterozygous carriers of LPL, APOA5, and GPIHBP1 mutations.11,12,20,21 One factor that might contribute to normo-triglyceridemia in a proband’s parents is the presence of the common variant Ser447X of the LPL gene that is known to be associated with higher LPL activity and reduced increase of postprandial TG after an oral fat load.17
Molecular aspects Our patient was found to be compound heterozygous for 2 novel mutations of GPIHBP1, 1 of the candidate genes in familial chylomicronemia. The del/ins mutation in exon 2 causes a frameshift that results in the ‘‘abolition’’ of the canonical termination codon (TGA at position c.553-555) and the formation of a novel termination codon 357 nucleotides
Buonuomo et al Table 2
GPIHBP1 mutations in chylomicronemia
269
Patients with familial hyperchylomicronemia from mutations in the GPIHBP1 gene
Ethnic origin (no. of families, no. of patients, genetic status, and clinical findings) Columbian (1 fam, 1 HO: M 33 y, HSM, LR, TG 38) Arabian (1 fam, 1HO: M 3 y: PC, LR, TG 45) Swedish (1 fam, 3 CHE: F 18 y, HSM, TG 22-57; M 13 y, SM, TG 19.5; F 2 y, PC, TG 48.5. French (1 fam, 1 CHE: M 6 mo, PC, TG 19.6) Algerian (1 fam, 1 HO: M 26 y, RPC, TG 26) Spanish (1 fam, 1 HO: F 30 y, PC, TG 15.8) Asian Indian (1 fam, 2 HO: M 2 mo, LR, TG .282; F 44 y, PC, TG 11) Salvadoran (1 fam, 1 HO: F 36 y, PC, LR, ERX, TG 73.2) Caucasian (1 fam, 1 HO: M 1 y, PC) Caucasian (1 fam, 1 HE: F 45y, PC, TG 35.8) Japanese (1 fam, 1 HO: F 54 y, PC, CHD, TG 29.8) Pakistani (1 fam, 4 HO: M 37 y, RPC, TG 100; F 22 y, RPC, TG 60; M 40 y, TG .40; F 37 y, RPC, TG 27) Hispanic (1 fam, 1 CHE: F 5 wk, TG 135, ERX, PC at 2 y) Thai (1 fam, 3 HO: F 46 y, TG 14.0-72.8; M 64 y, TG 9.51; M 43y, TG 7.60) Caucasian (1 fam, 1 CHE: F6 mo, TG 30.09, PC, ERX)
Supplementary references
Allele 1
Allele 2
c.344A.C, p.(Q115P) c.194G.A, p.(C65Y) c.194G.C, p.(C65S)
c.344A.C, p.(Q115P) c.194G.A, p.(C65Y) c.202T.G, p.(C68G)
1 2 3
c.41G.C, p.(C14F) 1 c.266G.T, p.(C89F) c.523G.C, p.(G175R) c.203G.A, p.(C68Y) 17,499bp del including GPIHBP1 gene, p.0 c.203G.A, p.(C68Y) c.323C.G, p.(T108R) c.431C.T, p.(S144F) c.41G.C, p.(C14F) 1 c.202T.C, p.(C68R) c.182- ?_5551 ?del (Ex3_4del), p.0 c.331A.C, p.(T111P)
c.1-?_22821?del (gene del), p.0 c.523G.C, p.(G175R) c.203G.A, p.(C68Y) 17,499bp del including GPIHBP1 gene, p.0 c.203G.A, p.(C68Y) c.323C.G, p.(T108R) – c.41G.C, p.(C14F) 1 c.202T.C, p.(C68R) c.182- ?_5551 ?del (Ex3_4del), p.0 c.413_429del, p.(P140Sfs*161) c.320C.G, p.(S107C)
4
c.320C.G, p.(S107C) c.85_88GAGGdel, p.(E29Tfs*50)
c.267C.A, p.(C89*)
4 5 6 6 7 7 8 9 10 11 12
CHD, coronary heart disease; CHE, compound heterozygote; ERX, eruptive xanthomas; F, female; fam, family; HE, heterozygote; HO, homozygote; HSM, hepatosplenomegaly; LR, lipemia retinalis; M, male; PC, pancreatitis; RPC, recurrent pancreatitis; SM, splenomegaly; TG, triglycerides, mmol/L.
downstream (TAA, c.910-912) (Fig. 2). The predicted translation product of the mutant mRNA would be an extremely long protein (303 amino acids vs 184 amino acids of the wild-type counterpart: p.(Asn52Serfs*253) (Fig. 2) that, if synthesized and secreted, is expected to have no function, because it will be devoid of key functional domains (part of Ly6 linker and GPI anchor) of the normal GPIHBP1.22,23 It also likely that this grossly abnormal protein undergoes a rapid intracellular degradation. The p.(Ser107Pro) substitution affects a serine residue located in the second finger of the Ly6 domain of GPIHBP1. All in silico algorithms were consistent in predicting that this amino acid substitution was not tolerated or damaging. This prediction is supported by previous mutagenesis studies that demonstrated that the substitution of serine at position 107 with alanine reduced the binding of LPL by .90%.23 The Ser107 residue belongs to finger 2 of the Ly6 domain of GPIHBP1, a region that appears to be crucial for LPL binding, as the substitution of several amino acid residues located in the region spanning from Cys89 to Cys110 with alanine, resulted in a variable but consistent reduction in LPL binding.23 Only another amino acid substitution (Thr108Arg) located in finger 2 of Ly6 domain has been reported so far in a patient with chylomicronemia.24 The number of GPIHBP1 mutations reported so far in chylomicronemia patients has steadily increased over the
past few years (Table 2, Supplementary Table S1, and related references), suggesting that defects of GPIHB1 function should be taken into consideration as a possible cause of severe hypertriglyceridemia in patients negative for LPL mutations.
Acknowledgment The authors would like to thank the patient and her family for their participation in the study and Dr. Enrico Zecca of the Division of Neonatology, ‘‘A. Gemelli’’ University Hospital, Roma, Italy, for referring the patient to ‘‘Bambino Gesu’’ Hospital. Funding Support: Genetic analysis was supported in part by a grant from Emilia-Romagna Region: RARER–Area1 (E35E09000880002) project to Sebastiano Calandra.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jacl.2014.10.003.
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A 3-day-old neonate with severe hypertriglyceridemia from novel mutations of the GPIHBP1 gene Paola Sabrina Buonuomo, MD, Andrea Bartuli, MD, Claudio Rabacchi, PhD, Stefano Bertolini, MD, Sebastiano Calandra, MD* Rare Diseases and Medical Genetics, Bambino Ges u Children Hospital, Rome, Italy (Drs Buonuomo and Bartuli); Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy (Dr Rabacchi); Department of Internal Medicine, University of Genova, Genova, Italy (Bertolini); and Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy (Dr Calandra) KEYWORDS: Hypertriglyceridemia; Lipoprotein lipase; (GPIHBP1) glycosylphosphatidylinositolanchored high-density lipoprotein-binding protein 1; Intravascular lipolysis
BACKGROUND: Familial chylomicronemia is a genetic defect of the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. Intravascular lipolysis involves the TG-hydrolase lipoprotein lipase (LPL) as well as other factors such as apolipoprotein CII and apolipoprotein AV (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface), and LMF1 (a factor required for intracellular formation of active LPL). METHODS: We sequenced the familial chylomicronemia candidate genes in a neonate with chylomicronemia. RESULTS: A 3-day-old newborn was found to have chylomicronemia (plasma TG 18.8 mmol/L, 1.667 mg/dL). The discontinuation of breastfeeding for 24 hours reduced plasma TG to 2.3 mmol/L (201 mg/dL), whereas its resumption induced a sharp TG increase (7.9 mmol/L, 690 mg/dL). The child was switched to a low-fat diet, which was effective in maintaining TG level below 3.5 mmol/L (294 mg/dL) during the first months of life. The child was found to be a compound heterozygous for 2 novel mutations in GPIHBP1 gene. The first mutation was a 9-bp deletion and 4-bp insertion in exon 2, causing a frameshift that abolished the canonical termination codon TGA. The predicted translation product of the mutant messenger RNA is a peptide that contains 51 amino acids of the N-terminal end of the wild-type protein followed by 252 novel amino acids. The second mutation was a nucleotide change (c.319T.C), causing an amino acid substitution p.(Ser107Pro) predicted in silico to be damaging. CONCLUSIONS: GPIHBP1 mutations should be considered in neonates with chylomicronemia negative for mutations in LPL gene. Ó 2015 National Lipid Association. All rights reserved.
* Corresponding author. Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Via Campi 287, I-41125 Modena, Italy.
E-mail address: [email protected] Submitted July 29, 2014. Accepted for publication October 6, 2014.
1933-2874/$ - see front matter Ó 2015 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/j.jacl.2014.10.003
266 Hypertriglyceridemia (HTG) is conventionally defined as severe when the level of fasting plasma triglycerides (TG) is .10 mmol/L. This condition is often referred to as chylomicronemia because of plasma accumulation of chylomicrons in the fasting state.1,2 Familial chylomicronemia (OMIM238600), also known as hyperlipoproteinemia type 1, is a rare recessive disorder characterized by the accumulation of chylomicrons in the circulation, which often appears during early infancy and childhood.1,2 In this condition, fasting serum TG level is very high and sometimes can exceed 100 mmol/L (8857 mg/dL). The clinical features include failure to thrive, eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, recurrent abdominal pain, and episodes of acute pancreatitis.1-5 In newborns and very young children (,1 year of age), chylomicronemia is often an incidental finding of milky plasma during routine blood tests or laboratory investigations carried out in the presence of clinical manifestations not related to chylomicronemia.3,7,8 Familial chylomicronemia is due to a defect in the lipolytic cascade of TG-rich lipoproteins that may result from mutations in at least 5 different genes: LPL (encoding the enzyme lipoprotein lipase, LPL; OMIM #238600); APOC2 (encoding the apolipoprotein CII, the activator of LPL; OMIM #207750); APOA5 (encoding apolipoprotein AV, also an activator of LPL; OMIM #144650); GPIHBP1 (encoding glycosylphosphatidylinositol-anchored highdensity lipoprotein-binding protein 1), the molecular platform that, on the endothelial surface of capillaries, allows the interactions of LPL with TG-rich lipoproteins, apolipoprotein CII, and apolipoprotein AV; OMIM #612757); and LMF1 (encoding the lipase maturation factor 1, a tissue factor that allows the secretion of functional LPL and hepatic lipase; OMIM #611761).2,5,9,10 Inactivating mutations of LPL are the most common cause of familial chylomicronemia (95% of cases).2 Here we describe the clinical features and genetic analysis of a 3-day-old child found to have elevated plasma TG (.10 mmol/L, .885 mg/dL) during a routine blood test for high bilirubin levels. The child was found to be a compound heterozygous for 2 novel mutations in the GPIHBP1 gene.
Methods Clinical features of the patient The patient was the first female neonate of unrelated parents delivered after a full-term uneventful pregnancy. Her birth weight was 3100 g, length was 50 cm, and the head circumference was 35 cm; the physical examination was negative. Breastfeeding was started on day 1. On day 3, she presented mild jaundice (bilirubin level 15.9 mg/dL), but capillary blood was remarkably pink-creamy colored. Laboratory tests revealed elevated plasma level of TG (18 mmol/L, 1667 mg/dL). Ophthalmoscopic examination
Journal of Clinical Lipidology, Vol 9, No 2, April 2015 and abdominal and cerebral ultrasound examinations were negative. Glucose, liver transaminases, alkaline phosphatase, lipase, amylase, and serum electrolytes were within normal ranges. Blood cell count was normal. Family history was unremarkable. The parents’ plasma lipid levels were in the normal range. In view of the severe HTG, it was assumed that the baby had a genetic defect of the intravascular lipolysis of TG-rich plasma lipoproteins (familial chylomicronemia).
Laboratory investigations A blood sample was collected from the patient and her parents to analyze the candidate genes of familial chylomicronemia. A written informed consent was obtained from the parents before DNA analysis. All procedures were in accordance with the ethical standards of the responsible institutional committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2013. Genomic DNA was extracted from peripheral blood leukocytes using a standard method. The candidate genes for familial chylomicronemia (LPL, APOC2, APOA5, GPIHBP1, and LMF1) were resequenced sequentially as previously reported.11,12 The mutations were designated according to the Human Genome Variation Society, 2012 version (http://www.hgvs.org/mutnomen/recs-DNA.html). GPIHBP1 protein sequence variants were designated according to http://www.hgvs.org/mutnomen/recs-prot.html. The screening of the 2 GPIHBP1 mutations found in the proband was performed in 80 individuals with primary severe HTG and 200 normolipidemic controls. The in silico prediction of the effect of the missense mutation of GPIHBP1 protein was performed using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT Human Protein and SIFT BLink (http://sift.jcvi.org/), SNPs3D (http://snps3d.org), PMut (http://mmb2.pcb.ub.es/ pmut), and SNAP (https://www.rostlab.org/services/snap/).
Results Clinical findings and follow-up Because the high risk of pancreatitis related to the high levels of plasma TG13,14 enteral feeding was interrupted at day 3 and infusion of isotonic intravenous solution was started. After 24 hours, plasma TG level was found to be greatly reduced (3.87 mmol/L, 342 mg/dL); the switching to breastfeeding for 1 day was followed by a substantial rise of plasma TG up to 7.9 mmol/L (700 mg/dL). In view of this response to breastfeeding, the baby was switched to a low-lipid formula diet (Basic F. Milupa – 49 Kcal/100 g). This dietary treatment induced a prompt and persistent reduction of plasma TG level (2.86 mmol/ L, 235 mg/dL) at discharge from the hospital. At 3 months of age the baby was found to be in good health with a normal growth (weight, height, and head circumference
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GPIHBP1 mutations in chylomicronemia
within the 50th percentile) and a normal development. Her plasma lipid profile showed a mild elevation of plasma TG and a low level of high-density lipoprotein cholesterol (HDL-C) (Table 1). (At 5 months of age, the lipid values were as follows: TG 3.12 mmol/L [276 mg/dL] and total cholesterol 2.8 mmol/L (108 mg/dL). The plasma lipid levels of proband’s parents were within the normal range (Table 1).
Gene analysis The sequence of LPL and APOC2 revealed that the proband was heterozygous for 3 common variants of the LPL gene [c.405 C.A, p.(Val108Val); c.435 G.A, p.(Glu118Glu), and c.1164 C.A, p.(Thr361Thr)] and homozygous for 2 common variants of APOC2 gene [c.1 –80G.T and c.215138_40 or 41_43delACC] located in introns 1 and 3, respectively. No variants were detected in APOA5 and LMF1 genes. The sequence of GPIHBP1 gene revealed that the proband was heterozygous for 2 mutations: 1) a 9-bp deletion and 4-bp insertion (c.154_162AA CAGGCTCdelTCTTins) in exon 2 (Figs. 1 and 2) and 2) a single nucleotide substitution in exon 4 (c.319T.C) (Fig. 1). The del/ins mutation in exon 2 causes a frameshift that abolishes the canonical termination codon TGA at position c.553-555. The translation product of the mutant messenger RNA (mRNA) is predicted to be a peptide that contains 51 amino acids of the N-terminal end of the wild-type protein followed by 252 novel amino acids (Fig. 2). The point mutation in exon 4 leads to the conversion of the serine residue at position 107 into a proline residue [p.(Ser107Pro)]. The in silico analyses (Polyphen 2, SIFT, SNPs3D, PMut, and SNAP) all were consistent in predicting a pathogenic effect of this mutation. The mutation screening of the parents revealed that del/ins mutation was inherited from the mother and the missense mutation from the father. These novel mutations were not detected in 80 adult patients with severe primary HTG and in 200 normolipidemic individuals. Because the proband’s parents had a normal plasma triglycerides, we thought that they might carry some variants of TG-linked genes that reduced plasma TG levels.15–17 For this reason, we sequenced the LPL and APOC3 genes. Both parents were found to be heterozygous for the common truncation variant p.(Ser474*) (Ser447X) of the LPL gene. No rare loss-of-function variants were found in the APOC3 gene (Supplementary Tables S2 and S3).
Table 1
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Discussion Clinical aspects In this study, we describe a 3-day-old newborn who incidentally was found to have severe HTG, suggesting familial chylomicronemia. Because severe HTG is considered at high risk for pancreatitis,1,13,14 enteral feeding was suspended and replaced by infusion of an isotonic solution. This therapeutic option was sufficient to rapidly reduce plasma TG to a safety level in terms of possible risk of pancreatitis. The replacement of breastfeeding with a low-lipid formula diet was successful in maintaining the TG level below 4 mmol/L (354 mg/dL) and ensuring a normal growth over time. Our patient is the third case of a biallelic carrier of GPIHBP1 mutations with severe HTG diagnosed in the first few months of life. Rios et al18 described a breastfed infant 2 months of age with failure to thrive and lethargy whose plasma TG level was .280 mmol/L (.24,800 mg/dL) on hospital admission. He was found to be homozygous for a complete deletion of GPIHBP1 gene. Upon admission, he was placed on low-fat diet and given fluids intravenously. After 24 hours, plasma TG level was modestly reduced. He was then switched to a low-fat diet milk protein–based dietary formula containing 40% fat with 87% medium-chain triglycerides. After 12 days on this diet, plasma TG level was 18 mmol/L (1594 mg/dL). Gonzaga-Jauregui et al19 reported a 3-month-old breastfed infant admitted to the hospital for intestinal bleeding who was found to have severe HTG (TG level 136 mmol/L) (12,046 mg/dL). He was compound heterozygous for 2 mutations in the GHPIBP1 gene [c.331A.C, p.(Thr111Pro) and c.413_429del, p.(P140Sfs*161)]. The third infant, recently reported by Ahmad et al,20 was admitted to the hospital at 6 months of age for acute pancreatitis associated with hypertriglyceridemia (TG.22.5 mmol/L, .2000 mg/dL). She was found to be compound heterozygote for a 4 base deletion in exon 2 (c.85_88GAGGdel, p.(E29Tfs*50) and a base substitution in exon 3, resulting in a nonsense mutation (c.267 C.A, p.(C89*). Both mutations resulted in truncated protein devoid of function. The much lower plasma TG level found in our patient with respect to the infants described in the studies mentioned previously18–20 may be due to the fact that in our patient hypertriglyceridemia was detected very early (on day 3 of life) and breastfeeding was promptly
Plasma lipids in the proband and her parents
Subject
Total cholesterol mmol/L (mg/dL)
Triglyceride mmol/L (mg/dL)
LDL-C mmol/L (mg/dL)
HDL-C mmol/L (mg/dL)
Proband Mother Father
3.36 (130) 4.31 (166) 4.82 (186)
3.37 (298) 0.90 (79) 0.61 (64)
1.31 (51) 2.51 (97) 2.82 (108)
0.77 (30) 1.40 (54) 1.62 (63)
HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.
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Figure 2 Partial nucleotide sequence of GPIHBP1 gene showing the nucleotide deletion/insertion in exon 2 and its effect on messenger RNA (mRNA) sequence. The mutation abolishes the canonical termination codon TGA (term 185) in exon 4 and creates a new termination codon (term 304). The predicted translation product of the mutant (Mut) mRNA is an extremely long protein (303 amino acids vs 184 amino acids of the wild-type [WT] counterpart) [p.(Asn52Serfs*253)]. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
Figure 1 Partial nucleotide sequence of GPIHBP1 gene in the proband. The proband was a compound heterozygous for a minute deletion/insertion in exon 2 (A) and a single nucleotide substitution in exon 4 (B). For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
discontinued and replaced by an alternative low-fat formula diet. It is reasonable to assume that the few months of breastfeeding of the patients described by Rios et,18 GonzagaJauregui et al,19 and Ahmad et al20 could have induced a progressive accumulation of plasma TG, which reached an extremely high level. This hypothesis seems to be confirmed by other reports of infants with LPL deficiency resulting from mutations in the LPL gene. Avis et al6 reported a 3-month-old baby with vomiting, loose stools, and abdominal pain with severe HTG (350 mmol/L) (31,000 mg/dL). This child had been breastfed for 2 months and then switched to standard formula bottle feeding. The administration of a nearly fatfree diet led to a dramatic reduction of plasma TG within a week. Stefanutti et at al7 described a 3 month-old breastfed baby with extremely severe HTG (TG level .300 mmol/L) (.26,500 mg/dL). This patient was treated with plasma exchange, which induced a dramatic decrease in plasma TG. After 24 and 72 hours, the plasma TG level dropped to 30.4
and 11.6 mmol/L (2657 and 1027 mg/dL), respectively. The switch to a low-fat formula diet was associated with persistent plasma TG reduction with a level ranging from 5 to 10 mmol (442 to 884 mg/dL). Similar considerations apply to the patient reported by Pugni et al,8 a 23-day-old breastfed patient with plasma TG level of 93 mmol/L (8237 mg/dL). This child underwent blood exchange transfusion, which reduced plasma TG level to 3 mmol/L (265 mg/dL) within 24 hours. The switch to a low-fat diet maintained plasma TG level between 3 and 5 mmol/L (265 and 442 mg/dL). In the light of the plasma TG level and the satisfactory response to dietary changes, invasive therapeutic options (plasma exchange or exchange blood transfusion)7,8 were not considered as a first line of treatment in our patient. The plasma lipid profile of the proband’s parents was unremarkable as it is often observed in young and lean heterozygous carriers of LPL, APOA5, and GPIHBP1 mutations.11,12,20,21 One factor that might contribute to normo-triglyceridemia in a proband’s parents is the presence of the common variant Ser447X of the LPL gene that is known to be associated with higher LPL activity and reduced increase of postprandial TG after an oral fat load.17
Molecular aspects Our patient was found to be compound heterozygous for 2 novel mutations of GPIHBP1, 1 of the candidate genes in familial chylomicronemia. The del/ins mutation in exon 2 causes a frameshift that results in the ‘‘abolition’’ of the canonical termination codon (TGA at position c.553-555) and the formation of a novel termination codon 357 nucleotides
Buonuomo et al Table 2
GPIHBP1 mutations in chylomicronemia
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Patients with familial hyperchylomicronemia from mutations in the GPIHBP1 gene
Ethnic origin (no. of families, no. of patients, genetic status, and clinical findings) Columbian (1 fam, 1 HO: M 33 y, HSM, LR, TG 38) Arabian (1 fam, 1HO: M 3 y: PC, LR, TG 45) Swedish (1 fam, 3 CHE: F 18 y, HSM, TG 22-57; M 13 y, SM, TG 19.5; F 2 y, PC, TG 48.5. French (1 fam, 1 CHE: M 6 mo, PC, TG 19.6) Algerian (1 fam, 1 HO: M 26 y, RPC, TG 26) Spanish (1 fam, 1 HO: F 30 y, PC, TG 15.8) Asian Indian (1 fam, 2 HO: M 2 mo, LR, TG .282; F 44 y, PC, TG 11) Salvadoran (1 fam, 1 HO: F 36 y, PC, LR, ERX, TG 73.2) Caucasian (1 fam, 1 HO: M 1 y, PC) Caucasian (1 fam, 1 HE: F 45y, PC, TG 35.8) Japanese (1 fam, 1 HO: F 54 y, PC, CHD, TG 29.8) Pakistani (1 fam, 4 HO: M 37 y, RPC, TG 100; F 22 y, RPC, TG 60; M 40 y, TG .40; F 37 y, RPC, TG 27) Hispanic (1 fam, 1 CHE: F 5 wk, TG 135, ERX, PC at 2 y) Thai (1 fam, 3 HO: F 46 y, TG 14.0-72.8; M 64 y, TG 9.51; M 43y, TG 7.60) Caucasian (1 fam, 1 CHE: F6 mo, TG 30.09, PC, ERX)
Supplementary references
Allele 1
Allele 2
c.344A.C, p.(Q115P) c.194G.A, p.(C65Y) c.194G.C, p.(C65S)
c.344A.C, p.(Q115P) c.194G.A, p.(C65Y) c.202T.G, p.(C68G)
1 2 3
c.41G.C, p.(C14F) 1 c.266G.T, p.(C89F) c.523G.C, p.(G175R) c.203G.A, p.(C68Y) 17,499bp del including GPIHBP1 gene, p.0 c.203G.A, p.(C68Y) c.323C.G, p.(T108R) c.431C.T, p.(S144F) c.41G.C, p.(C14F) 1 c.202T.C, p.(C68R) c.182- ?_5551 ?del (Ex3_4del), p.0 c.331A.C, p.(T111P)
c.1-?_22821?del (gene del), p.0 c.523G.C, p.(G175R) c.203G.A, p.(C68Y) 17,499bp del including GPIHBP1 gene, p.0 c.203G.A, p.(C68Y) c.323C.G, p.(T108R) – c.41G.C, p.(C14F) 1 c.202T.C, p.(C68R) c.182- ?_5551 ?del (Ex3_4del), p.0 c.413_429del, p.(P140Sfs*161) c.320C.G, p.(S107C)
4
c.320C.G, p.(S107C) c.85_88GAGGdel, p.(E29Tfs*50)
c.267C.A, p.(C89*)
4 5 6 6 7 7 8 9 10 11 12
CHD, coronary heart disease; CHE, compound heterozygote; ERX, eruptive xanthomas; F, female; fam, family; HE, heterozygote; HO, homozygote; HSM, hepatosplenomegaly; LR, lipemia retinalis; M, male; PC, pancreatitis; RPC, recurrent pancreatitis; SM, splenomegaly; TG, triglycerides, mmol/L.
downstream (TAA, c.910-912) (Fig. 2). The predicted translation product of the mutant mRNA would be an extremely long protein (303 amino acids vs 184 amino acids of the wild-type counterpart: p.(Asn52Serfs*253) (Fig. 2) that, if synthesized and secreted, is expected to have no function, because it will be devoid of key functional domains (part of Ly6 linker and GPI anchor) of the normal GPIHBP1.22,23 It also likely that this grossly abnormal protein undergoes a rapid intracellular degradation. The p.(Ser107Pro) substitution affects a serine residue located in the second finger of the Ly6 domain of GPIHBP1. All in silico algorithms were consistent in predicting that this amino acid substitution was not tolerated or damaging. This prediction is supported by previous mutagenesis studies that demonstrated that the substitution of serine at position 107 with alanine reduced the binding of LPL by .90%.23 The Ser107 residue belongs to finger 2 of the Ly6 domain of GPIHBP1, a region that appears to be crucial for LPL binding, as the substitution of several amino acid residues located in the region spanning from Cys89 to Cys110 with alanine, resulted in a variable but consistent reduction in LPL binding.23 Only another amino acid substitution (Thr108Arg) located in finger 2 of Ly6 domain has been reported so far in a patient with chylomicronemia.24 The number of GPIHBP1 mutations reported so far in chylomicronemia patients has steadily increased over the
past few years (Table 2, Supplementary Table S1, and related references), suggesting that defects of GPIHB1 function should be taken into consideration as a possible cause of severe hypertriglyceridemia in patients negative for LPL mutations.
Acknowledgment The authors would like to thank the patient and her family for their participation in the study and Dr. Enrico Zecca of the Division of Neonatology, ‘‘A. Gemelli’’ University Hospital, Roma, Italy, for referring the patient to ‘‘Bambino Gesu’’ Hospital. Funding Support: Genetic analysis was supported in part by a grant from Emilia-Romagna Region: RARER–Area1 (E35E09000880002) project to Sebastiano Calandra.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jacl.2014.10.003.
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