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Home , Abetalipoproteinemia, Hypertriglyceridemia, Hypocholesterolemia

A severe form of caused by new splicing mutations of microsomal transfer protein Véronique Pons, Corinne Rolland, Michel Nauze, Marie Danjoux, Gérald Gaibelet, Anne H. Durandy, Agnes Sassolas, Emile Lévy, François Tercé, Xavier Collet, et al.

To cite this version:

Véronique Pons, Corinne Rolland, Michel Nauze, Marie Danjoux, Gérald Gaibelet, et al.. A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein. Human Mutation, Wiley, 2011, 32 (7), pp.751. ￿10.1002/humu.21494￿. ￿hal-00651635￿

HAL Id: hal-00651635 https://hal.archives-ouvertes.fr/hal-00651635 Submitted on 14 Dec 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Human Mutation

A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein For Peer Review

Journal: Human Mutation

Manuscript ID: humu-2010-0522.R1

Wiley - Manuscript type: Research Article

Date Submitted by the 07-Feb-2011 Author:

Complete List of Authors: Pons, Véronique; INSERM, U1048; Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires Rolland, Corinne; INSERM, U1043; Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP) Nauze, Michel; INSERM, U1048; Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires Danjoux, Marie; CHU Toulouse, Hôpital Purpan, Département d’Anatomopathologie Gaibelet, Gérald; INSERM, U1048; Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires Durandy, Anne; INSERM U768, Université Paris V-René Descartes, Hôpital Necker-Enfants Malades Sassolas, Agnes; UF Dyslipidémies, CBPE, Hospices Civils de Lyon and Lyon University, INSERM U1060, CarMeN Laboratory, Univ Lyon-1 Lévy, Emile; Départements de Nutrition et de Biochimie, Hôpital Sainte-Justine et Université de Montréal Tercé, François; INSERM, U1048; Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires Collet, Xavier; INSERM, U1048; Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires MAS, Emmanuel; INSERM, U1043; Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP); CHU Toulouse, Hôpital des Enfants, Unité de Gastroentérologie, Hépatologie et Nutrition, Département de Pédiatrie

abetalipoproteinemia, MTTP, triglyceride transfer, endoplasmic Key Words: reticulum, intestinal HDL

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 3 of 78

New splicing mutations of MT TP 1 Formatted: Font: Italic 1 2 A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal 3 4 triglyceride transfer protein 5 1, 6 1,2 3,4 1,2 5 Deleted : Véronique Pons , Corinne Rolland , Michel Nauze , Marie Danjoux , Gérald Gaibelet 2…3 Deleted : ... [1] 7 1,2 6 7 8 1,2 1,2 8 , Anne Durandy , Agnès Sassolas , Emile Lévy , François Tercé , Xavier Collet * Deleted : 4 9 and Emmanuel Mas 3,4,9* Deleted : 4 5 10 Deleted : 11 Deleted : 5 1 12 INSERM, U1048 , Toulouse, F-31 000 , France; Deleted : 6 13 6 2Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires, Toulouse, Deleted : 14 7…1, Deleted : ... [2] 15 F-31300 France; 2…7 Deleted : ... [3] 16 3 INSERM, U1043, Toulouse, F-31300 France; Deleted : 8 17 8 18 4Université de Toulouse , UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP) , Deleted : 19 Formatted: Font: (Default) Times Toulouse, F-31300 , France; New Roman, Complex Script Font: 20 For Peer Review Times New Roman, French France 5CHU Toulouse, Hôpital Purpan, Département d’Anatomopathologie, Toulouse, F-31300 21 Deleted : U563… ... [4] 22 France; Deleted : 3 23 Formatted 6INSERM U768, Université Paris V-René Descartes, Hôpital Necker-Enfants Malades, 75015 ... [5] 24 Deleted : 25 Paris (France); Formatted 26 ... [6] 7 27 UF Dyslipidémies, CBPE, Hospices Civils de Lyon and Lyon University, INSERM U1060, Deleted : France 28 Formatted ... [7] CarMeN Laboratory, Univ Lyon-1 F-69621, Villeurbanne (France); 3 29 Deleted : 8 30 Départements de Nutrition et de Biochimie, Hôpital Sainte-Justine et Université de Montréal, Formatted: Superscript 31 Montréal, Québec (Canada); Deleted : III Paul-Sabatier, Deleted : 4 32 9 33 CHU Toulouse, Hôpital des Enfants, Unité de Gastroentérologie, Hépatologie et Nutrition, Deleted : 3 34 Département de Pédiatrie, Toulouse, F-31300 , France. Deleted : Hôpital Purpan, 35 4 * These authors equally contributed to the work Deleted : 36 5… Deleted : , ... [8] 37 Deleted : Université Paris Descartes, 38 Faculté de Médecine Paris V-René 39 Corresponding author: Descartes, 40 Emmanuel Mas, MD Deleted : 5 41 Deleted : 6 Hôpital des Enfants - Unité de Gastroentérologie, Hépatologie et Nutrition – 330 avenue de 42 Formatted ... [9] 43 Grande-Bretagne – TSA 70034 – 31059 Toulouse cedex 9 (France) Deleted : Laboratoire des 44 Dyslipidémies, Centre de Biologie Est, Tel. (33) 5 34 55 84 45 Lyon 45 Deleted : 6 46 Fax. (33) 5 34 55 85 67 Deleted : 7 47 E-mail: [email protected] 7 48 Deleted : 8… 49 Deleted : Hôpital des Enfants, ... [10] 50 Deleted : 9¶ 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 2 Formatted: Font: Italic 1 2 ABSTRACT 3 4 Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels 5 and by the absence of apoB-containing . It is the consequence of microsomal 6 7 triglyceride transfer protein (MT TP) deficiency. Deleted : ed 8 We report 2 patients with new MTTP mutations. We studied their functional consequences on 9 Deleted : MTP MTTP 10 the triglyceride transfer function using duodenal . We transfected mutants in Formatted: Font: Italic 11 HepG2 and HeLa cells to investigate their association with protein disulfide isomerase ( PDI ) Deleted : MTP 12 Formatted: Font: Italic and their localization at the endoplasmic reticulum. 13 Formatted: Font: Italic 14 These children have a severe abetalipoproteinemia. Both of them had also a mild 15 hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237- 16 Deleted : mttp 17 28A>G mutations within MTTP gene. mRNA analysis revealed abnormal splicing with 18 Formatted: Font: Italic deletion of exon 6 and 10, respectively. Deletion of exon 6 (6-MTTP ) introduced a frame 19 Formatted: Font: Italic 20 shift in the reading frame andFor a premature Peerstop codon at position Review 234. Despite 6-MTTP and Deleted : MTP 21 Formatted: Font: Italic 22 10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally Deleted : MTP 23 localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP Formatted: Font: Italic 24 Deleted : MTP triglyceride transfer activity. 25 Deleted : MTP 26 These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI Deleted : MTP 27 and responsible for the loss of function of MTTP , thereby explaining the severe Deleted : MTP 28 Deleted : MTP 29 abetalipoproteinemia phenotype of these children. 30

31 Deleted : MTP 32 Keywords: abetalipoproteinemia, MTTP , triglyceride transfer, intestinal HDL, endoplasmic 33 Formatted: Font: Italic 34 reticulum 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 3 Formatted: Font: Italic 1 2 INTRODUCTION 3 4 Abetalipoproteinemia (ABL, OMIM 200100) is a rare autosomal recessive disease 5 Deleted : MTP 6 caused by the deficiency of the microsomal triglyceride transfer protein (MTTP, OMIM 7 8 157147 ). ABL is characterized by the absence of (apoB)-containing 9 Deleted : MTP 10 lipoproteins from plasma. MTTP is a 97 kDa protein, containing 894 amino acids, that forms 11 Deleted : MTP 12 a heterodimer with the 55 kDa protein disulfide isomerase (PDI). MTTP is mainly found in 13 14 the endoplasmic reticulum (ER) of hepatocytes and enterocytes (Wetterau, et al., 1992). 15 Deleted : MTP 16 MTTP is required for the transfer of neutral lipids to nascent apoB-lipoproteins, resulting in 17 18 the secretion of very low-density lipoproteins (VLDL) in the or (CM) in 19 Deleted : MTP 20 the intestine. When MTTP is Fordeficient, nascent Peer apoB is degraded Review by the proteasome (Benoist Formatted: Font: Italic 21 Deleted : (Zhou, et al., 1998) 22 and Grand-Perret, 1997) . It differs from familial , displaying 23 24 dysfunctional apoB and leading to the absence of apoB-lipoproteins in the plasma. Familial 25 26 hypobetalipoproteinemia is a co-dominant disorder and even if heterozygotes can be 27 28 asymptomatic, they had low levels of total (TC), low density (LDL)- 29 30 cholesterol and apoB (OMIM 107730). 31 Deleted : MTP 32 ABL was characterized as the consequence of MTTP absence or dysfunction in 1992 33 Deleted : mttp 34 by Wettereau et al. (Wetterau, et al., 1992). The genetic defects in MTTP gene, located on Formatted: Font: Italic 35 36 chromosome 4q22-24, were confirmed one year later (Sharp, et al., 1993; Shoulders, et al., 37 38 1993). ABL is a very uncommon disease, which highlights the crucial role of this protein. 39 Deleted : MTP Indeed, the mttp -deficient mice died at embryonic day 10.5 with abnormal neurological 40 41 development (exencephalia) (Raabe, et al., 1998). When a specific inducible intestinal 42 Deleted : MTP 43 deletion of mttp is generated in mice, they presented a phenotype similar to ABL, with 44 Formatted: Font: Italic 45 , growth arrest and decreased cholesterol absorption (Xie, et al., 2006). 46 Deleted : MTP 47 During evolution, MTTP has progressively acquired TG transfer activity in vertebrates 48 Deleted : MTP 49 whereas invertebrate MTTP is known to only transfer phospholipids (Rava and Hussain, 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 4 Formatted: Font: Italic 1 Deleted : MTP 2 2007). Another way to study MTTP functions is to characterize human mutations. This 3 4 approach could help us to determine its active domains that transfer phospholipids, 5 6 cholesterol esters or TG, as well as its binding domains to PDI or to apoB. To our knowledge, 7 Deleted : lementary 8 so far, 40 mutations have been described in 52 patients (Supp . Table S1) (Najah, et al., 2009; Deleted : table 9 10 Zamel, et al., 2008). Here, we report 2 children with a severe form of ABL that are compound 11 Deleted : MTP 12 heterozygotes for MTTP . The new mutations induce deletion of exon 6 and 10 and translation Formatted: Font: Italic 13 14 of truncated proteins. 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 5 Formatted: Font: Italic 1 2 MATERIALS AND METHODS 3 4 Patients: 5 Deleted : ed 6 We report 2 new cases of ABL, whose diagnosis was performed when AM was a 13 month- 7 8 old boy and his sister PM was 6 years-old. They were born from Caucasian non- 9 10 consanguineous parents. We received the agreement of both parents for genetic 11 12 investigations. 13 14 Endoscopy and duodenal biopsies: 15 16 Upper gastrointestinal endoscopy was realized under general anesthesia with a Pentax EG- 17 18 1870K. Duodenal biopsies were fixed in Bouin for pathology or immediately frozen in liquid 19 Deleted : MTP 20 nitrogen for further investigationsFor (Western Peer blot and MTTP Review activity assay). For routine 21 22 microscopy, biopsies were embedded in paraformaldehyde and sections were 23 24 hematoxylin/eosin-stained. 25 Deleted : MTP 26 Intestinal MTTP analyses: 27 Deleted : MTP 28 Proteins were extracted from duodenal biopsies to perform a western blot of MTTP . MTTP Deleted : MTP 29 30 activity was measured by radiolabelled TG transfer from donor to acceptor vesicles as 31 32 previously described (Levy, et al., 2002). 33 34 Plasma sample analyses: 35 36 samples were collected for lipids, lipoproteins, A and E investigations, and 37 38 ALT. TG and TC were measured by an enzymatic method (Roche). HDL and VLDL- 39 cholesterol were measured by a dextran/PEG method (Roche). LDL-cholesterol was 40 41 calculated by Friedwald method. ApoA-I and apoB were determined by immuno-turbidimetry 42 43 (Roche). 44 45 Lipid chromatography: 46 Deleted : s 47 The FPLC system consisted of a Hitachi L7250 auto-sampler, a L7100 pump system and two 48 Deleted : 49 detectors: L7400 (Hitachi) and UVD170U (Dionex).  20µL of plasma were injected and 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 6 Formatted: Font: Italic 1 2 lipoproteins were separated on SuperoseTM6 10/300GL column (GE Healthcare) with 0.15M 3 4 NaCl solution at a flow rate of 0.4mL/min. The column effluent was split equally into two 5 6 lines by a microsplitter 50:50, mixing with cholesterol or triglyceride reagents (Biolabo), thus 7 8 achieving a simultaneous profile from single injection. The two enzymatic reagents were each 9 10 pumped at a rate of 0.2mL/min. Both enzymatic reactions proceeded at 37°C in a Teflon 11 12 reactor coil (15mLx0.4mm id). Proteins were measured directly at 280nm. 13 Deleted : MTP 14 Genomic DNA sequencing of MTTP : Formatted: Font: Italic 15 Deleted : mttp 16 DNA from patients was extracted from blood samples and then exons and introns of MTTP Formatted: Font: Italic 17 Deleted : 18 gene (NCBI Reference Sequence: NG_011469.1) were sequenced with Applied ABI Prism. 19 Deleted : MTP MTTP 20 mRNA sequencing of For: Peer Review Formatted: Font: Italic 21 22 Total RNA was extracted using RNeasy Mini Kit (Qiagen) from either EBV-immortalized B 23 24 lymphocytes for both children or primary lymphocytes for both children and their parents. 25 Deleted : lementary Primary lymphocytes were isolated from blood samples using a ficoll technique. A reverse 26 Deleted : ¶ 27 Formatted: Font: Italic 28 transcription was performed and nested PCR was used to amplify exon 6 and exon 10 with 5’ Deleted : MTP 29 30 and 3’ flanking regions (see primers in Supp . Table S2). PCR products were separated on Formatted: Font: Italic 31 Formatted: Font: Italic 32 agarose gel, then extracted with a QIAquick Gel Extraction Kit (Qiagen) and directly inserted Deleted : MTP 33 Formatted: Font: Italic 34 into pCR2.1 vector (Invitrogen) before sequencing analyses. Deleted : MTP 35 Formatted: Font: Italic Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 36 Formatted: Font: Italic 37 Deleted : MTP translation initiation codon in the reference sequence (www.hgvs.org/mutnomen). The 38 Formatted: Font: Italic 39 initiation codon is codon 1. Formatted: Font: Italic 40 Deleted : MTP 41 Constructs: Formatted: Font: Italic 42 43 Formatted: Font: Italic WT-MTTP cDNA was a generous gift of Dr M.Hussain (New-York). We produced by PCR 44 Deleted : MTP 45 Formatted: Font: Italic WT-MTTP and MTTP mutants with deletions of patients: 6-MTTP and 10-MTTP . PCR 46 Formatted: Font: Italic 47 Deleted : MTP fragments (WT-MTTP , 6-MTTP and 10-MTTP ) were digested using BamHI and HindIII 48 Deleted : MTP 49 Formatted: Font: Italic restriction , purified with a QIAquick Gel Extraction Kit (Qiagen) and then inserted 50 Formatted: Font: Italic 51 Formatted: Font: Italic 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 7 Formatted: Font: Italic 1 2 in pEGFP-N1 or in p2xMyc-N1, generated by replacing the EGFP from pEGFP-N1 with a 3 4 2xMyc epitope. pDsRed2-ER vector (Clontech) was used to encode an ER marker. 5 6 Cell culture and transfection: 7 8 HepG2 (human hepatocellular carcinoma), HeLa (human cervix carcinoma) or Caco-2 9 10 (human epithelial colorectal adenocarcinoma) cells were grown in Dulbecco’s modified 11 12 Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 0.1 13 14 mM non essential amino acids (Invitrogen), and 100 µg/ml penicillin/streptomycin 15 16 (Invitrogen) in a humidified atmosphere containing 5% CO 2. HepG2 and HeLa cells were 17 18 transfected using GenJuice (Novagen, Merck) or Fugene (Roche) respectively. 19 20 Immunoprecipitation: For Peer Review 21 22 The cells were lysed for 30 min in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% 23 24 Nonidet P-40, 10% glycerol) containing protease and phosphatase inhibitors. Soluble material 25 26 was then incubated overnight at 4°C with 5 µg of monoclonal anti-Myc antibody (clone 27 28 9E10) or polyclonal anti-PDI antibody (DL-11) . The antigen-antibody complex was incubated 29 30 with a mix of protein-G and protein-A Sepharose for 1h and then washed in lysis buffer 31 32 containing 0.1% Nonidet P-40. The bound proteins were eluted by boiling the beads in 33 34 Laemmli buffer and analyzed by immunoblotting. 35 36 Immunoblotting: 37 38 The proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing 39 conditions, transferred onto nitrocellulose membrane, and analyzed by immunoblotting 40 Deleted : MTP 41 according to standard protocols using indicated antibodies. Anti-MTTP antibody was a 42 43 generous gift of Drs. J. R. Wetterau and H. Jamil. We used a mouse monoclonal antibody 44 45 against Myc (clone 9E10) from BD Biosciences, a rabbit polyclonal antibody against PDI 46 47 (DL-11) from Sigma and a mouse monoclonal antibody against  GFP from Roche. HRP- 48 49 labelled secondary antibodies were from Santa-Cruz or Roche Diagnostics. 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 8 Formatted: Font: Italic 1 2 Immunofluorescence experiments: 3 4 HepG2 and HeLa cells were grown on collagen I coated or glass coverslips respectively. Cells 5 6 were fixed in 4% paraformaldehyde, permeabilized with 0.05% saponin, saturated with PBS 7 Deleted : first 8 containing 10% fetal bovine serum and incubated with the anti-PDI rabbit polyclonal 9 Deleted : (Molecular Probes) 10 antibody (Sigma) or the anti-Golgin 97 mouse monoclonal antibody (Molecular Probes) and 11 12 then with Cy3-labelled secondary antibody (Jackson Immunoresearch Laboratories). Pictures 13 14 were captured using a Zeiss LSM 510 META confocal microscope equipped with a 63x Plan- 15 16 Apochromat objective. 17 18 Immunological investigation : 19 20 Serum immunoglobulin levelsFor were determined Peer by neph elemetry.Review B lymphocyte phenotype 21 22 and function were analyzed as previously described (Peron, et al., 2008). 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 9 Formatted: Font: Italic 1 2 RESULTS 3 4 Severe form of abetalipoproteinemia: 5 6 When AM was 13-months-old, he presented with a . An intestinal 7 Deleted : o 8 was suspected on the basis of abdominal distension, abnormal stools (diarrhea 9 10 or constipation) and hydro-aeric levels on abdominal X-ray. An upper gastrointestinal 11 12 endoscopy was performed and revealed a white aspect of duodenal villi characteristic of 13 Deleted : . 14 intestinal malabsorption and abnormal lipid storage (Fig ure 1A), as confirmed by 15 Deleted : . 16 histology (large fat inclusions located within enterocytes, Fig ure 1B). Blood analyses revealed 17 18 a severe and hypotriglyceridemia without LDL particles and apoB 19 Deleted : As the 20 (Table 1). Since parents haveFor normal lipid Peer values as well asReview apoB and LDL levels, we ruled Deleted : panels 21 Deleted : , the 22 out a hypo-betalipoproteinemia which is a dominant inheritance disease. Thus ABL was Deleted : of their parents were normal 23 Deleted : homozygote form of suspected . They also had an increased level of alanine amino-transferase (ALT) and a 24 Deleted : was ruled out 25 Deleted : and an 26 hyperechogeneic aspect of the liver on ultra-sound scan. Furthermore, both children presented Deleted : diagnosed 27 28 a severe deficiency secondary to intestinal fat malabsorption. His sister (PM), 6- 29 30 years-old at the time of diagnosis, had no rotula reflex (the very early diagnosis of AM 31 32 prevented the development of peripheral neuropathy). However, both of them have no sign of 33 34 . 35 36 Abnormal lipoprotein profiles: 37 38 We assessed the blood lipoprotein profiles by FPLC analysis. Both children presented 39 a complete absence of LDL and an almost complete absence VLDL in TC and TG moieties 40 Deleted : . 41 (Fig ure 1C and 1D). Although HDL-cholesterol levels are only 20% of control, HDL particles Deleted : 2 42 43 remain the only lipid class, with a normal ratio of cholesterol ester to TC (Table 1) reflecting 44 45 a functional lecithin cholesterol acyl-transferase (LCAT) activity. The HDL pattern differs 46 47 between patient and control. Patient had 2 peaks of HDL particles. The early peak is specific 48 49 of the patient while the later one appears at the same time than control, as it is suggested by 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 10 Formatted: Font: Italic 1 2 the concordance of protein chromatogram. HDL particles of the early peak seem to be 3 4 cholesterol-enriched and to have less protein than HDL of the second peak. 5 6 Immunoglobulin synthesis abnormalities: 7 8 Both of these children had also a congenital hypogammaglobulinemia requiring 9 10 regular immunoglobulin substitution. They were diagnosed as immunodeficient on serum 11 12 IgG, IgM and IgA levels that were performed because of a failure to thrive in the first months 13 14 of age (Table 2). There was no protein loosing enteropathy. Mild hypogammaglobulinemia 15 16 was discovered, affecting IgM and IgG in both children. IgA was also slightly decreased in 17 18 the brother AM during the first years of age. AM and PM had normal T and B cell 19 20 populations (data not shown).For B cell phenotype Peer was found Review normal in PM but absence of 21 22 memory switched B cells was observed in the younger brother AM. In contrast, in vitro B cell 23 24 immunoglobulin production was found normal (data not shown). 25 Deleted : MTP 26 MTTP mutations: Formatted: Font: Italic 27 Deleted : mttp 28 Genomic DNA sequencing of MTTP gene showed 2 different mutations: c.619G>T Formatted: Font: Italic 29 Deleted : lementary and c.1237-28A>G ( Supp . Table S3). The first one c.619G>T was located at the first 30 Deleted : 3 31 32 nucleotide of exon 6 and the second one c.1237-28A>G was located within intron 9. In order 33 34 to understand the consequences of such mutations, we extracted mRNA from lymphocytes 35 Deleted : MTP 36 and analyzed MTTP transcripts. MTTP is mainly expressed in the liver and in the intestine, Formatted: Font: Italic 37 Deleted : MTP 38 but can also be detected from EBV-immortalized B lymphocytes. After reverse transcription 39 and PCR amplification, products were analyzed by gel migration, thereby revealing a 40 Deleted : cDNA 41 decrease in size for both mutations in patient compared to the control (Fig ure 2A and 2C ). As Deleted : . 42 43 Deleted : 3 expected, we obtained in control a band at 228 pb and 888 pb for exon s 6 and 10 respectively, 44 Deleted : 3B 45 whereas lower fragments were revealed in patient. Those fragments were detected at the size 46 47 of ∼90pb and ∼780pb, suggesting the loss of exon s 6 and 10 respectively. Sequencing 48 49 confirmed that these two mutations are responsible for abnormal splicing of exons 6 and 10 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 11 Formatted: Font: Italic 1 Deleted : . 2 respectively (Fig ure 2B and 2D). The first genomic DNA mutation c.619G>T induce s a frank Deleted : 3 3 Deleted : C 4 deletion of exon 6 and a shift in the open reading frame leading to a premature stop codon at Deleted : 3 5 Deleted : d 6 position 234 and resulting in an abnormal truncated protein consisting of 233 amino acids 7 Deleted : d 8 (∼25kDa). The second DNA mutation c.1237-28A>G induces a frank deletion of exon 10 (36 9 10 amino acids), with no shift in the reading frame, leading to a protein of 858 amino acids 11 12 (∼94kDa) (Figure 2E) . 13 14 These mutations were also confirmed with sequencing analyses from primary 15 Deleted : MTP 16 lymphocytes from parents and children, meaning that abnormal splicing of MTTP was a Formatted: Font: Italic 17 18 consequence of genomic mutations and not of EBV immortalization. 19 20 As children are compoundFor heterozygotes, Peer these resul Reviewts confirm the genetic diagnosis 21 22 of ABL. 23 Deleted : MTP 24 Loss of function of MTTP protein: 25 Deleted : MTP 26 Duodenal biopsies were performed and samples were analyzed for MTTP expression 27 Deleted : MTP 28 and activity. In patient, we detected a band with a slight decrease of the apparent MTTP size Deleted : . 29 Deleted : 4 30 compared to control or Caco-2 cells (Fig ure 3A), which probably corresponds to 10-MTTP 31 Deleted : MTP 32 with the loss of 36 amino acids (3kDa) due to exon 10 deletion . We observed no band at a Deleted : deletion 33 Deleted : of the 34 size of 25kDa, corresponding to the expected size of 6-MTTP (data not shown), suggesting Deleted : MTP 35 Deleted : MTP 36 that 6-MTTP might be degraded by the proteasome. MTTP activity was also measured on Deleted : MTP 37 Deleted : . 38 these samples and showed a complete loss of TG transfer activity (5-8%/mg protein in control Deleted : 4 39 Deleted : MTP 40 versus 0.08-0.8%/mg protein in AM, Fig ure 3B). Deleted : MTP 41 Deleted : MTP 42 We further investigated the ability of both 6-MTTP and 10-MTTP mutants to Deleted : MTP 43 Deleted : MTP 44 interact with PDI and their subcellular localization by first generating constructs encoding 45 Deleted : MTP Deleted : MTP 46 GFP- or 2xMyc-tagged WT-MTTP , 6-MTTP or 10-MTTP . Tagged MTTP mutants as well 47 Deleted : are 48 as WT-MTTP were expressed in HeLa cells and analyzed by immunoblotting. We observed Deleted : MTP 49 Deleted : MTP 50 that 6-MTTP and 10-MTTP were detected, as well as WT form (Fig ure 4A and 4B ). This Deleted : . 51 Deleted : 5 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 12 Formatted: Font: Italic 1 Deleted : MTP…MTP…MTP…MTP… MTP…MTP…MTP 2 is in contrast to the mutant MTTP analysis in duodenal since 6-MTTP could not be ... [11] 3 4 detected. The overexpression level of tagged proteins might overcome the degradation 5 6 machinery, thereby allowing the detection of a part of 6-MTTP and 10-MTTP . However, 7 8 10-MTTP seemed to be lower expressed compared to WT-MTTP , either expressed as GFP- 9 Deleted : .… 5 10 ... [12] or 2xMyc-10-MTTP (quantification in Fig ure 4C), probably because a part of abnormal 11 12 proteins was degraded by proteasome. 13 Deleted : MTP…was …MTP…MTP 14 ... [13] Since MTTP is localized in the ER through its binding with PDI, we tested the 15 16 association of MTTP mutants with PDI. 2xMyc-tagged MTTP mutant and WT proteins were 17 Deleted : immunoprecipitated 18 expressed in both HeLa and HepG2 cells and immunoprecipitation was performed using anti- 19 20 myc antibody (Figure 4D) or Foranti-PDI antibody Peer (Figure 4E) Review. As expected, we found that WT- 21 Deleted : MTP 22 MTTP interacted with PDI (Fig ure 4D and 4E, upper panel ). By contrast, neither 6-MTTP Deleted : .…6A 23 ... [14] Deleted : MTP…MTP 24 ... [15] nor 10-MTTP was able to associate with PDI (Fig ure 4D and 4E, middle and lower panels ). Deleted : bring down…after 25 immunoprecipitation ….…6B…6C ... [16] 26 We then investigated the cellular localization of WT-MTTP as well as 6-MTTP and Deleted : MTP…MTP…MTP…MTP… 27 MTP…MTP…lementary ... [17] 28 29 10-MTTP . After expression in both HeLa and HepG2 cells, WT-MTTP , 6-MTTP and 10- 30 31 MTTP displayed an identical pattern, showing a reticular localization throughout the cytosol, 32 Deleted : Fig. 6D and 6E 33 reminiscent of ER staining ( Supp . Figure S1). However, in HepG2 cells but not in HeLa cells, Deleted : S…MTP…lementary ... [18] 34 35 6-MTTP also displays a non specific localization both in the nucleus and throughout the 36 Deleted : .…6E ... [19] 37 cytosol ( Supp . Fig ure S1B , see inset in 6-MTTP -GFP), much like GFP alone. This suggests Deleted : S…MTP…MTP…MTP…MT P…MTP 38 ... [20] 39 that in HepG2 cells, 6-MTTP -GFP protein can be degraded, as we observed by 40 41 immunoblotting in duodenal biopsies. Surprisingly, WT-MTTP as well as 6-MTTP and 42 Deleted : of ER ….…7 ... [21] 43 10-MTTP co-localized with ER markers such as ER-DsRed, a red marker consisting of a 44 45 fusion of the DsRed and the ER retention sequence, KDEL (Fig ure 5A ) and PDI (Figure 5B) . 46 Deleted : lementary 47 By contrast, no co-localization was observed with cis-Golgi marker such as Golgin 97 ( Supp . 48 Deleted : .…8 ... [22] 49 Fig ure S2) or trans-Golgi marker such as TGN46 (data not shown). Deleted : S 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 13 Formatted: Font: Italic 1 Deleted : MTP 2 Altogether, these data showed that MTTP mutants lost TG transfer activity. They are 3 4 still localized in the ER despite their inability to interact with PDI. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 14 Formatted: Font: Italic 1 2 DISCUSSION 3 Deleted : MTP 4 MTTP is mainly expressed in enterocytes and hepatocytes where it participates in the 5 6 synthesis of chylomicrons and VLDL respectively. These patients presented fat storage in the 7 Deleted : . 8 small intestine (Fig ure 1 A and 1B ) and in the liver (as suggested by increased ALT levels and 9 Deleted : MTP 10 hyperechogeneic aspect), as a consequence of MTTP dysfunction. 11 12 Lipids are almost exclusively transported in blood into HDL lipoproteins in ABL 13 Deleted : . 14 patients (Fig ure s 1C and 1D and Table 1). These particles are mainly synthesized in Deleted : 2 15 16 peripheral tissues to ensure the reverse cholesterol transport to the liver. But, HDL are also 17 18 produced at the basolateral membrane of enterocytes, via ATP Binding Cassette-A1 (ABCA1) 19 20 efflux of cholesterol to apoA-IFor (Levy, et al.,Peer 2007). Cholesterol Review is transported by enterocytes 21 Deleted : 2 22 in two different pathways: the apoB-dependent and the apoB-independent pathways resulting 23 24 respectively in the production of CM or HDL lipoproteins (Iqbal, et al., 2003). Some 25 Deleted : 2 26 differences exist between these two pathways. Cholesterol ester is only secreted within CM 27 28 while free cholesterol is secreted by both pathways. The apoB-dependent pathway is also 29 Deleted : MTP 30 regulated by MTTP and contributes to cholesterol absorption during post-prandial states. 31 Deleted : MTP 32 MTTP is not required for cholesterol secretion by the apoB-independent pathway that may be 33 34 more important during fasting states. In the human epithelial Caco-2 cell line, Liver X 35 Formatted: Font: Italic 36 Receptor/Retinoid X Receptor activation increases ABCA1 gene expression and basolateral 37 38 efflux of cholesterol in intestinal HDL (Murthy, et al., 2002). In ABL patients, neurological 39 impairment ( and peripheral neuropathy) and retinopathy are the consequences of 40 41 . Like cholesterol, these two routes also exist for vitamin E absorption by 42 Deleted : MTP 43 enterocytes (Anwar, et al., 2007). In MTTP -deficient enterocytes, the HDL pathway is used to Formatted: Font: Italic 44 45 Deleted : MTP deliver vitamin E. Vitamin E secretion within HDL lipoproteins is not increased by MTTP 46 47 inhibition (Anwar, et al., 2007). This pathway is not as important as synthesis 48 49 but, in ABL patients, intestinal HDL represent the only route for lipids or vitamin E from 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 15 Formatted: Font: Italic 1 2 intestine to the liver (Anwar, et al., 2007; Anwar, et al., 2006). These ABL patients have a 3 4 specific class of HDL particles that have a lower density and a lower protein content. 5 Deleted : MTP 6 MTTP is also expressed in natural killer T (NKT) cells. Inhibition of MTTP in fetal Deleted : MTP 7 8 thymocyte organ culture results in a complete loss of NKT cells (Dougan, et al., 2007). In 9 Deleted : MTP 10 these antigen presenting cells, MTTP loads lipids onto nascent CD1d and regulates 11 Deleted : MTP 12 presentation of glycolipid antigens (Dougan, et al., 2005). MTTP deficiency could impair the 13 14 recycling of CD1d from lysosome to the plasma membrane (Sagiv, et al., 2007). Interestingly, 15 16 both patients present with a hypogammaglobulinemia that was mild but however required 17 18 immunoglobulin substitution. We only found an absence of memory switched B cells in one 19 20 patient while both of them hadFor a normal inPeer vitro immunoglobulin Review production (Table 2). Since 21 22 such an association between mild B lymphocyte immunodeficiency and abetalipoproteinemia 23 Deleted : MTP 24 has not been previously reported in ABL patients, it is unlikely that MTTP defect is directly 25 26 involved in hypogammaglobulinemia. However, this point should be further investigated, 27 28 looking for a subtle defect in immunoglobulin levels in abetalipoproteinemia patients. 29 Formatted: Font: Italic 30 Recently, Zeissig et al . characterized the loss of CD1 function in ABL patients (Zeissig, et al., 31 Deleted : r 32 2010). They found a defect of all antigen-presenting CD1 family members in dend ritic cells 33 Deleted : A 34 from ABL patients. Similarly to apoB, MTTP deficiency in the ER leads to the degradation of 35 36 group 1 CD1 by the proteasome pathway, which altered activation of NKT cells. 37 Deleted : MTP 38 In these new patients with ABL, we characterized mutations of MTTP by analyzing Formatted: Font: Italic 39 genomic DNA and mRNA products. Sequencing revealed abnormal splicing leading to 40 Deleted : 2 41 deletion of exon 6 or 10, as a result of two genomic mutations, c.619G>T and c.1237-28A>G 42 43 respectively. c.619G>T is not only a missense mutation (207 Val>Phe), but induces as well an 44 45 abnormal splicing (Mitchell, et al., 1986). The deletion of 140 bp of exon 6 causes a shift in 46 47 the reading frame and a truncated protein because of a premature stop codon in position 234. 48 49 Recently, Najah et al. (Najah, et al., 2009) found similar results with c.619-3T>G mutation 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 16 Formatted: Font: Italic 1 Deleted : MTP 2 located in intron 5 of MTTP . We showed that this truncated protein is not detected in Formatted: Font: Italic 3 Deleted : MTP 4 duodenal biopsies (data not shown), suggesting that 6-MTTP is degraded by the proteasome 5 6 pathway (Pan, et al., 2007). c.1237-28A>G located in intron 9 leads to the frank deletion of 7 Deleted : MTP 8 108 bp of exon 10, with no shift in the reading frame, resulting in the loss of 36 amino acids. Deleted : 2 9 Deleted : MTP…MTP ... [23] N 10 Human MTTP structure contains an N-terminal β-barrel ( β ) (residues 22-297), a Deleted : 11 Deleted : MTP… (Zamel, et al., 12 central α-helical domain ( α) (residues 298-603), and two C-terminal β-sheets ( βC and βA) 2008)…MTP ... [24] 13 Formatted: Font: Italic N 14 (residues 604-894). β is conserved in apoB, lipovitellin, and apolipophorin and may be one Deleted : MTP…MTP ... [25] 15 Deleted : .…7….…6A…-C ... [26] 16 C A of the two phospholipid binding sites of MTTP . Helices 4-6, β and β domains of MTTP are Deleted : MTP…MTP…MTP ... [27] 17 Comment [A1]: Reviewer 1 : moved 18 conserved in vertebrates, but not in invertebrates, suggesting that they are involved in TG or removed Reviewer 2 : supprimer jusqu’à la ligne 4 19 page 16 20 transfer activity (Rava and Hussain,For 2007). Peer βN mediates the interactionReview with the N -terminus of Deleted : A better understanding of 21 Deleted : MTP 22 apoB; the middle α-helical domain mediates the interaction with both PDI (residues 520-598) Deleted : MTTP regulation could enable 23 us to improve treatment of ABL patients. 24 and apoB (residues 517-603); and the C-terminal mediates the lipid-binding and transfer Moreover, inhibition of 25 Deleted : MTP 26 catalytic activity of MTTP (Mann, et al., 1999; Read, et al., 2000) . Expression of MTTP Deleted : MTTP could be used to lower 27 cholesterol levels in patients with (Cuchel, et al.,... [28] 28 constructs in HepG2 and HeLa cells showed that 6-MTTP and 10-MTTP proteins are Deleted : MTP 29 Formatted: Font: Italic 30 located at ER (Fig ure 5) but are not associated with PDI (Fig ure 4D and 4E ). MTTP mutants 31 Deleted : MTTP transcription is ... [29] 32 might be retained in the ER as defective or misfolded proteins and a part might be Deleted : MTP 33 Deleted : MTTP mRNA when mice... [30]are fed with high -cholesterol and high -fat 34 retrotranslocated to the cytosol for proteasomal degradation. The truncated 6-MTTP protein Deleted : MTP 35 Formatted: Font: Italic 36 only conserved one apoB-binding domain. By contrast, 10-MTTP protein is characterized Deleted : MTTP promoter variant... - [31] 579T/T is associated with peripheral 37 Deleted : ed 38 by the loss of residues 413-448, that are not directly involved in PDI binding but their loss Deleted : 2 39 Deleted : MTP 40 could alter the tertiary structure of the protein. Finally, these residues, corresponding to Formatted: Font: Italic 41 Deleted : MTTP promoter serves... as [32]a 42 helices 7-9 of the central α-helical domain, belong to a critical domain for TG transfer activity novel negative insulin -responsive element Deleted : 43 MTP Deleted : MTTP production, that... may [33] 44 as described by Rava and Hussain (Rava and Hussain, 2007). be a causative factor for VLDL 45 Deleted : MTP 46 In conclusion, we report here two new mutations of MTTP , c.619G>T and c.1237- Formatted: Font: Italic 47 Deleted : MTTP expression and CM... [34] production is controlled by inositol - 48 28A>G, resulting respectively in 6-MTTP protein, a truncated protein of 233 amino acids Deleted : MTP 49 Formatted: Font: Italic 50 and 10-MTTP protein, deleted of exon 10. Despite these mutations d o not change ER Formatted: Font: Italic 51 Deleted : MTP…MTP…id ... [35] 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 17 Formatted: Font: Italic 1 Deleted : MTP 2 localization of mutant MTTP proteins , they abolish their binding with PDI and totally Deleted : ed 3 4 impaired their TG transfer activity. 5 6 7 8 ACKNOWLEDGEMENTS 9 10 The authors thank these children and their parents, and also pediatricians taking care of them 11 12 (Dr. E. Fournié-Gardini and Dr. A.I. Bertozzi). We also thank J. Bertrand-Michel and V. 13 14 Roques (Lipidomic Plateau of IFR-BMT/IFR150) for lipidomic analysis, advice and technical 15 16 assistance. We are grateful to Pr. T. Levade (Toulouse, France) for immortalization of B 17 18 lymphocytes. 19 20 This work was supported byFor grants from Peer INSERM, ANR Review(PNRA-2006 ABSINTE) and by 21 22 grants from Canadian Institutes of Health Research, MOP 10584 (E.L). V.P and E.M were the 23 24 recipients of a fellowship from the Fondation pour la Recherche Médicale. V.P is a young 25 26 researcher with temporary contract from INSERM. X.C was a recipient of a “Contrat 27 28 d’Interface” from CHU of Toulouse. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 18 Formatted: Font: Italic 1 Deleted : Immunoglobulin levels of 2 both patients were reported at the diagnosis of hypogammaglobulinemia, 3 just after the diagnosis of 4 abetalipoproteinemia and at the last follow-up. 5 Deleted : TABLES¶ 6 ¶ 7 Table 1. Biochemical diagnosis of abetalipoproteinemia 8 9 Deleted : ¶ ... [36] Deleted : Table 2. Immunoglobulin 10 levels¶ 11

12 ... [37] 13 Formatted: English U.K. 14 Formatted Table 15 Deleted : ¶ 16 Deleted : ¶ 17 Page Break 18 Comment [A2]: Reviewer 2 : 19 supplementary data 20 For Peer Review Deleted : ¶ 21 Table 3. Genomic mutations of 22 Deleted : MTP 23 Formatted: Font: Italic 24 Deleted : MTTP 25 Deleted : Titles and legends to figures 26 Deleted : ¶ 27 ¶ ... [38] 28 Formatted: English U.K. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 19 Formatted: Font: Italic 1 2 FIGURES LEGENDS 3 4 5 6 Figure 1. Diagnosis of abetalipoproteinemia 7 8 (A) Upper gastrointestinal endoscopy performed in patient AM revealed a white aspect of 9 10 duodenal villi, characteristic of lipid storage. (B) Biopsy was performed in and 11 12 processed for hematoxylin/eosin-staining. The arrow points at the presence of lipid droplets 13 Deleted : ¶ ¶ 14 within the enterocytes. (C and D) Serum from control or patient (AM) was analyzed using Figure 2. Blood lipid analysis ¶ 15 Deleted : A 16 FPLC system. Lipoproteins were separated and total cholesterol ( C) and ( D) Deleted : B 17 18 were measured. Profiles are shown for control (Ctrl) and patient (AM). Proteins were only Deleted : were 19 Deleted : A 20 showed for patient in C. NoteFor the difference Peer of y-axis (lipid Review content) between control and Deleted : B 21 Deleted : A 22 patient. 23 24 25 Deleted : 3 26 Figure 2. Sequence analysis of mutant MTTP transcription products Deleted : MTP 27 Formatted: Font: Italic 28 (A and C) After mRNA extraction and reverse transcription from control and patient (AM) Deleted : B 29 Deleted : B 30 EBV-immortalized lymphocytes, PCR was performed to amplify exon 6 (A) and exon 10 ( C) 31 32 with 5’ and 3’ flanking regions. PCR products were analyzed by migration on agarose gel. 33 Deleted : C 34 Arrowhead points at the lower band found in patient compared to the control. ( B and D) 35 36 Upper bands in the control and lower bands in patients were purified and then cloned into 37 Deleted : C 38 pCR2.1 vector before sequencing. In (B), electrophoregrams showed the exon 6 and 5’ and 3’ 39 flanking regions with exon 5 and 7 in the control. In the patient, the exon 6 is deleted, leading 40 41 to a frameshift and a premature stop codon. In (D), similarly, electrophoregrams showed the 42 43 exon 10 and 5’ and 3’ flanking regions with exon 9 and 11 in the control. In the patient, the 44 45 exon 10 is deleted. (E) The amino acid sequences corresponding to exons 6 and 10 are shown 46 47 underlined and in blue in the normal MTTP sequence. // determined the deletion of exon s 6 or 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 20 Formatted: Font: Italic 1 Formatted: Font: Not Italic 2 10 in each mutant. For 6-MTTP , the frame-shift mutation results in an abnormal sequence 3 4 described in red and in italic. 5 Deleted : 6 Deleted : ¶ 7 Deleted : 4 8 Figure 3. MTTP expression and activity in duodenal biopsy Deleted : MTP 9 10 (A) Duodenal biopsy from control (Ctrl) or patient (AM) and Caco-2 cell lysate were 11 Deleted : MTP 12 analyzed by SDS gel electrophoresis and western blotting with antibodies against MTTP . (B) 13 Deleted : MTP 14 The triglyceride transfer activity of MTTP was measured on duodenal biopsy from control or Deleted : (Ctrl) 15 16 patient (AM). Values are expressed as the mean of three independent experiments; standard 17 18 errors are indicated. 19 20 For Peer Review Deleted : 5 21 Deleted : MTP 22 Figure 4. Interaction of WT and mutant MTTP with PDI Deleted : MTP 23 Deleted : MTP 24 (A and B) HeLa cells were transfected or not with WT-MTTP , 6-MTTP or 10-MTTP Formatted: Font: Not Bold 25 Formatted: Font: Not Bold 26 tagged with EGFP (A) or 2xMyc (B). Cell lysates (100 µg) were analyzed by SDS gel Deleted : WT and mutant 27 Deleted : MTP 28 electrophoresis and western blotting with antibodies against GFP (A), Myc (B) and Rab5 as Formatted: Font: Not Bold, Italic 29 Deleted : MTTP expression in HeLa 30 loading control. Arrowheads point at WT-MTTP , 6-MTTP or 10-MTTP and arrows point cells¶ 31 Deleted : MTP 32 at non specific band (ns). Blots in (B) were scanned and the quantification is shown in (C). Deleted : MTP 33 Deleted : MTP 34 Each experiment was repeated at least 3 times, and (C) shows a representative example. (D Deleted : ¶ 35 ¶ 36 Figure 6. Interaction of WT and and E) HeLa cells were transfected with 2xMyc-tagged WT-MTTP , 6-MTTP or 10-MTTP . mutant 37 Deleted : MTP 38 Cell lysates (Lys) were subjected to immunoprecipitation with (+) or without (-) the anti-myc 39 Formatted: Font: Italic Deleted : MTTP with PDI¶ 40 antibody (D) or the anti-PDI antibody (E) . Analysis was performed by SDS gel (A-C) 41 Deleted : MTP 42 electrophoresis and western blotting with antibodies against Myc or PDI. Arrowheads point at Deleted : (A) 43 Deleted : MTP 44 immunoprecipitated WT-MTTP , 6-MTTP or 10-MTTP (D) or PDI (E) and arrows point at 45 Deleted : (B) 46 non specific band (ns). IgG HC represents heavy chain of anti-PDI antibody used for Deleted : MTP 47 Deleted : (C) 48 immunoprecipitation. Deleted : : 5% of total 49 Deleted : MTP 50 Deleted : MTP 51 Deleted : MTP 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 21 Formatted: Font: Italic 1 Deleted : (D and E) HeLa cells (D) or 2 hepG2 cells (E) were transfected with Figure 5. Localization of WT and mutant MTTP and ER marker GFP-tagged WT- 3 Deleted : MTP 4 (A) HeLa cells were co-transfected with GFP-tagged WT-MTTP , 6-MTTP or 10-MTT P 5 Formatted: Font: Italic Deleted : MTTP , 6- 6 and a vector encoding DsRed-ER marker. Cells were then processed for fluorescence 7 Deleted : MTP 8 analysis. (B) HeLa cells were transfected with GFP-tagged WT-MTTP, 6-MTTP or 10- Deleted : MTTP or 10- 9 Deleted : MTP 10 MTTP and were then processed for immunofluorescence using anti-PDI antibody. Bar: 10µm. Formatted: Font: Italic 11 Deleted : MTTP . Cells were then 12 processed for fluorescence analysis. Bar: 10µm.¶ 13 ¶ 14 Deleted : 7 15 Deleted : MTP 16 Formatted: Font: Italic 17 Deleted : MTP 18 19 Deleted : MTP 20 For Peer Review Deleted : MTP 21 Formatted: Font: Italic 22 Formatted: Font: Not Italic 23 Formatted: Font: Not Italic 24 Formatted: Font: Not Italic 25 Deleted : ¶ 26 Figure 8. Localization of WT and mutant 27 Deleted : MTP 28 29 Formatted: Font: Italic Deleted : MTTP . Cells were then 30 processed for immunofluorescence using 31 anti-golgin 97 antibody and analyzed. Bar: 10µm.¶ 32 ¶ 33 ¶ 34 ¶ 35 Formatted: Font: Italic 36 Deleted : MTTP and Golgi marker¶ HeLa cells were transfected with GFP- 37 tagged WT- 38 Deleted : MTP 39 Formatted: Font: Italic 40 Deleted : MTTP , 6- 41 Deleted : MTP 42 43 Formatted: Font: Italic 44 Deleted : MTTP or 10- 45 Deleted : MTP 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 22 Formatted: Font: Italic 1 Deleted : SUPPLEMENTARY 2 INFORMATION¶ REFERENCES ¶ 3 Supplementary Table 1. Description of 4 MTP mutations reported in the Al-Shali K, Wang J, Rosen F, Hegele RA. 2003. Ileal adenocarcinoma in a mild phenotype of literature¶ 5 6 abetalipoproteinemia. Clin Genet 63(2):135-8. Deleted : ¶ Deleted : ¶ 7 Anwar K, Iqbal J, Hussain MM. 2007. Mechanisms involved in vitamin E transport by Supplementary Table 2. PCR primers¶ 8 ¶ primary enterocytes and in vivo absorption. J Lipid Res 48(9):2028-38. Supplementary Table 3. Genomic 9 mutations of MTTP ¶ 10 Anwar K, Kayden HJ, Hussain MM. 2006. Transport of vitamin E by differentiated Caco-2 ¶ 11 Formatted: Font: Italic cells. J Lipid Res 47(6):1261-73. 12 Deleted : Supplementary Figure 1. 13 MTP translation products¶ Au WS, Lu L, Yeung CM, Liu CC, Wong OG, Lai L, Kung HF, Lin MC. 2008. Hepatocyte The amino acids corresponding to exon 6 14 and 10 are underlined in the normal MTP nuclear factor 1 binding element within the promoter of microsomal triglyceride 15 sequence. // determined the deletion of exon 6 or 10. For 6-MTP , the frame- 16 transfer protein (MTTP) gene is crucial for MTTP basal expression and insulin shift mutation results in an abnormal 17 sequence described in red and in italic. ¶ responsiveness. J Mol Endocrinol 41(4):229-38. 18 Formatted: Font: Italic 19 Benayoun L, Granot E, Rizel L, Allon-Shalev S, Behar DM, Ben-Yosef T. 2007. Formatted: Font: Italic 20 For Peer Review Deleted : Supplementary Figure S1: Abetalipoproteinemia in Israel: evidence for a founder mutation in the Ashkenazi Localization of WT and mutant MTTP¶ 21 HeLa cells (A) or HepG2 cells (B) were 22 Jewish population and a contiguous gene deletion in an Arab patient. Mol Genet transfected with GFP-tagged WT-MTTP, 6-MTTP or 10-MTTP and were then 23 Metab 90(4):453-7. processed for immunofluorescence. Bar: 24 10µm.¶ Benoist F, Grand-Perret T. 1997. Co-translational degradation of apolipoprotein B100 by the ¶ 25 Supplementary Figure S2: Localization 26 of WT and mutant MTTP and Golgi proteasome is prevented by microsomal triglyceride transfer protein. Synchronized marker¶ 27 ¶ 28 translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride HeLa cells were transfected with GFP- tagged WT-MTTP, 6-MTTP or 10- 29 transfer protein. J Biol Chem 272(33):20435-42. MTTP and were then processed for 30 immunofluorescence using anti-Golgin Berthier MT, Couture P, Houde A, Paradis AM, Sammak A, Verner A, Depres JP, Gagne C, 97 antibody. Bar: 10µm 31 Page Break 32 Gaudet D, Vohl MC. 2004. The c.419-420insA in the MTP gene is associated with Formatted: Font: Italic 33 Formatted: German Germany 34 abetalipoproteinemia among French-Canadians. Mol Genet Metab 81(2):140-3. Formatted: Font: Not Bold 35 Chardon L, Sassolas A, Dingeon B, Michel-Calemard L, Bovier-Lapierre M, Moulin P, Formatted: Justified, Line spacing: 36 1.5 lines 37 Lachaux A. 2008. Identification of two novel mutations and long-term follow-up in 38 abetalipoproteinemia: a report of four cases. Eur J Pediatr. 39 40 Cuchel M, Bloedon LT, Szapary PO, Kolansky DM, Wolfe ML, Sarkis A, Millar JS, Ikewaki 41 K, Siegelman ES, Gregg RE and others. 2007. Inhibition of microsomal triglyceride 42 transfer protein in familial hypercholesterolemia. N Engl J Med 356(2):148-56. 43 44 Di Leo E, Lancellotti S, Penacchioni JY, Cefalu AB, Averna M, Pisciotta L, Bertolini S, 45 Calandra S, Gabelli C, Tarugi P. 2005. Mutations in MTP gene in abeta- and 46 47 hypobeta-lipoproteinemia. 180(2):311-8. 48 Dische MR, Porro RS. 1970. The cardiac lesions in Bassen-Kornzweig syndrome. Report of a 49 50 case, with autopsy findings. Am J Med 49(4):568-71. 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 23 Formatted: Font: Italic 1 2 Dougan SK, Rava P, Hussain MM, Blumberg RS. 2007. MTP regulated by an alternate 3 4 promoter is essential for NKT cell development. J Exp Med 204(3):533-45. 5 Dougan SK, Salas A, Rava P, Agyemang A, Kaser A, Morrison J, Khurana A, Kronenberg M, 6 7 Johnson C, Exley M and others. 2005. Microsomal triglyceride transfer protein 8 lipidation and control of CD1d on antigen-presenting cells. J Exp Med 202(4):529-39. 9 10 Heath KE, Luong LA, Leonard JV, Chester A, Shoulders CC, Scott J, Middleton-Price HR, 11 Humphries SE, Talmud PJ. 1997. The use of a highly informative CA repeat 12 polymorphism within the abetalipoproteinaemia locus (4q22-24). Prenat Diagn 13 14 17(12):1181-6. 15 Iqbal J, Anwar K, Hussain MM. 2003. Multiple, independently regulated pathways of 16 17 cholesterol transport across the intestinal epithelial cells. J Biol Chem 278(34):31610- 18 20. 19 20 Iqbal J, Dai K, Seimon T, JungreisFor R, Oyadomari Peer M, Kuriakose Review G, Ron D, Tabas I, Hussain 21 MM. 2008. IRE1beta inhibits chylomicron production by selectively degrading MTP 22 23 mRNA. Cell Metab 7(5):445-55. 24 Kamagate A, Qu S, Perdomo G, Su D, Kim DH, Slusher S, Meseck M, Dong HH. 2008. 25 26 FoxO1 mediates insulin-dependent regulation of hepatic VLDL production in mice. J 27 Clin Invest 118(6):2347-64. 28 29 Levy E, Spahis S, Sinnett D, Peretti N, Maupas-Schwalm F, Delvin E, Lambert M, Lavoie 30 MA. 2007. Intestinal cholesterol transport proteins: an update and beyond. Curr Opin 31 32 Lipidol 18(3):310-8. 33 Levy E, Stan S, Delvin E, Menard D, Shoulders C, Garofalo C, Slight I, Seidman E, Mayer G, 34 35 Bendayan M. 2002. Localization of microsomal triglyceride transfer protein in the 36 Golgi: possible role in the assembly of chylomicrons. J Biol Chem 277(19):16470-7. 37 38 Mitchell PJ, Urlaub G, Chasin L. 1986. Spontaneous splicing mutations at the dihydrofolate 39 reductase locus in Chinese hamster ovary cells. Mol Cell Biol 6(6):1926-35. 40 Murthy S, Born E, Mathur SN, Field FJ. 2002. LXR/RXR activation enhances basolateral 41 42 efflux of cholesterol in CaCo-2 cells. J Lipid Res 43(7):1054-64. 43 Najah M, Di Leo E, Awatef J, Magnolo L, Imene J, Pinotti E, Bahri M, Barsaoui S, Brini I, 44 45 Fekih M and others. 2009. Identification of patients with abetalipoproteinemia and 46 homozygous familial hypobetalipoproteinemia in Tunisia. Clin Chim Acta 401(1- 47 48 2):51-6. 49 Narcisi TM, Shoulders CC, Chester SA, Read J, Brett DJ, Harrison GB, Grantham TT, Fox 50 51 MF, Povey S, de Bruin TW and others. 1995. Mutations of the microsomal 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 24 Formatted: Font: Italic 1 2 triglyceride-transfer-protein gene in abetalipoproteinemia. Am J Hum Genet 3 4 57(6):1298-310. 5 Ohashi K, Ishibashi S, Osuga J, Tozawa R, Harada K, Yahagi N, Shionoiri F, Iizuka Y, 6 7 Tamura Y, Nagai R and others. 2000. Novel mutations in the microsomal triglyceride 8 transfer protein gene causing abetalipoproteinemia. J Lipid Res 41(8):1199-204. 9 10 Pan X, Hussain FN, Iqbal J, Feuerman MH, Hussain MM. 2007. Inhibiting proteasomal 11 degradation of microsomal triglyceride transfer protein prevents CCl4-induced 12 steatosis. J Biol Chem 282(23):17078-89. 13 14 Peron S, Metin A, Gardes P, Alyanakian MA, Sheridan E, Kratz CP, Fischer A, Durandy A. 15 2008. Human PMS2 deficiency is associated with impaired immunoglobulin class 16 17 switch recombination. J Exp Med 205(11):2465-72. 18 Raabe M, Flynn LM, Zlot CH, Wong JS, Veniant MM, Hamilton RL, Young SG. 1998. 19 20 Knockout of the abetalipoproteinemiaFor Peer gene in mice: Review reduced lipoprotein secretion in 21 heterozygotes and embryonic lethality in homozygotes. Proc Natl Acad Sci U S A 22 23 95(15):8686-91. 24 Rava P, Hussain MM. 2007. Acquisition of triacylglycerol transfer activity by microsomal 25 26 triglyceride transfer protein during evolution. Biochemistry 46(43):12263-74. Formatted: German Germany 27 Rehberg EF, Samson-Bouma ME, Kienzle B, Blinderman L, Jamil H, Wetterau JR, 28 29 Aggerbeck LP, Gordon DA. 1996. A novel abetalipoproteinemia genotype. 30 Identification of a missense mutation in the 97-kDa subunit of the microsomal 31 32 triglyceride transfer protein that prevents complex formation with protein disulfide 33 isomerase. J Biol Chem 271(47):29945-52. 34 35 Ricci B, Sharp D, O'Rourke E, Kienzle B, Blinderman L, Gordon D, Smith-Monroy C, 36 Robinson G, Gregg RE, Rader DJ and others. 1995. A 30-amino acid truncation of the 37 38 microsomal triglyceride transfer protein large subunit disrupts its interaction with 39 protein disulfide-isomerase and causes abetalipoproteinemia. J Biol Chem 40 270(24):14281-5. 41 42 Sagiv Y, Bai L, Wei DG, Agami R, Savage PB, Teyton L, Bendelac A. 2007. A distal effect 43 of microsomal triglyceride transfer protein deficiency on the lysosomal recycling of 44 45 CD1d. J Exp Med 204(4):921-8. 46 Sakamoto O, Ohura T, Matsubara Y, Takayanagi M, Tsuchiya S. 2007. Mutation and 47 48 haplotype analyses of the MUT gene in Japanese patients with methylmalonic 49 acidemia. J Hum Genet 52(1):48-55. 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 25 Formatted: Font: Italic 1 2 Schgoer W, Eller P, Mueller T, Tancevski I, Wehinger A, Ulmer H, Sandhofer A, Ritsch A, 3 4 Haltmayer M, Patsch JR. 2008. The MTP -493TT genotype is associated with 5 peripheral arterial disease: results from the Linz Peripheral Arterial Disease (LIPAD) 6 7 Study. Clin Biochem 41(9):712-6. 8 Sharp D, Blinderman L, Combs KA, Kienzle B, Ricci B, Wager-Smith K, Gil CM, Turck 9 10 CW, Bouma ME, Rader DJ and others. 1993. Cloning and gene defects in microsomal 11 triglyceride transfer protein associated with abetalipoproteinaemia. Nature 12 365(6441):65-9. 13 14 Sheena V, Hertz R, Nousbeck J, Berman I, Magenheim J, Bar-Tana J. 2005. Transcriptional 15 regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear 16 17 factor-4alpha. J Lipid Res 46(2):328-41. 18 Shoulders CC, Brett DJ, Bayliss JD, Narcisi TM, Jarmuz A, Grantham TT, Leoni PR, 19 20 Bhattacharya S, PeaseFor RJ, Cullen Peer PM and others. Review 1993. Abetalipoproteinemia is 21 caused by defects of the gene encoding the 97 kDa subunit of a microsomal 22 23 triglyceride transfer protein. Hum Mol Genet 2(12):2109-16. 24 Vongsuvanh R, Hooper AJ, Coakley JC, Macdessi JS, O'Loughlin EV, Burnett JR, Gaskin 25 26 KJ. 2007. Novel mutations in abetalipoproteinaemia and homozygous familial 27 hypobetalipoproteinaemia. J Inherit Metab Dis 30(6):990. 28 29 Wang J, Hegele RA. 2000. Microsomal triglyceride transfer protein (MTP) gene mutations in 30 Canadian subjects with abetalipoproteinemia. Hum Mutat 15(3):294-5. 31 32 Wetterau JR, Aggerbeck LP, Bouma ME, Eisenberg C, Munck A, Hermier M, Schmitz J, Gay 33 G, Rader DJ, Gregg RE. 1992. Absence of microsomal triglyceride transfer protein in 34 35 individuals with abetalipoproteinemia. Science 258(5084):999-1001. 36 Xie Y, Newberry EP, Young SG, Robine S, Hamilton RL, Wong JS, Luo J, Kennedy S, 37 38 Davidson NO. 2006. Compensatory increase in hepatic lipogenesis in mice with 39 conditional intestine-specific Mttp deficiency. J Biol Chem 281(7):4075-86. 40 Yang XP, Inazu A, Yagi K, Kajinami K, Koizumi J, Mabuchi H. 1999. Abetalipoproteinemia 41 42 caused by maternal isodisomy of chromosome 4q containing an intron 9 splice 43 acceptor mutation in the microsomal triglyceride transfer protein gene. Arterioscler 44 45 Thromb Vasc Biol 19(8):1950-5. 46 Zamel R, Khan R, Pollex RL, Hegele RA. 2008. Abetalipoproteinemia: two case reports and 47 48 literature review. Orphanet J Rare Dis 3:19. 49 Zampino R, Ingrosso D, Durante-Mangoni E, Capasso R, Tripodi MF, Restivo L, Zappia V, 50 51 Ruggiero G, Adinolfi LE. 2008. Microsomal triglyceride transfer protein (MTP) - 52 53 54 55 56 57 58 59 60

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New splicing mutations of MT TP 26 Formatted: Font: Italic 1 2 493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 3 4 infected patients. J Viral Hepat 15(10):740-6. 5 Zhou M, Fisher EA, Ginsberg HN. 1998. Regulated Co-translational ubiquitination of 6 7 apolipoprotein B100. A new paradigm for proteasomal degradation of a secretory 8 protein. J Biol Chem 273(38):24649-53. 9 Formatted: Font: Not Bold Zeissig S, Dougan SK, Barral DC, Junker Y, Chen Z, Kaser A, Ho M, Mandel H, McIntyre A, 10 Formatted: Justified, Line spacing: 11 Kennedy SM and others. 2010. Primary deficiency of microsomal triglyceride transfer 1.5 lines 12 Formatted: Font: Not Bold protein in human abetalipoproteinemia is associated with loss of CD1 function. J Clin 13 14 Invest 120(8):2889-99. 15

16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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26 27 Page 1: [4] Deleted Administrateur 2/7/2011 8:31:00 AM 28 29 30 31 Page 1: [5] Formatted Administrateur 2/7/2011 8:41:00 AM 32 Font: (Default) Times New Roman, Complex Script Font: Times New Roman, French 33 France, Not Highlight 34 Page 1: [5] Formatted Administrateur 2/7/2011 8:41:00 AM 35 36 Font: (Default) Times New Roman, Complex Script Font: Times New Roman, French 37 France

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9 Page 1: [8] Deleted Administrateur 1/31/2011 11:17:00 AM 10 11 , 12 13 Page 1: [9] Formatted Administrateur 2/7/2011 8:40:00 AM 14 Font: (Default) Times New Roman, 12 pt, Font color: Auto, Complex Script Font: Times 15 New Roman, 12 pt 16 17 Page 1: [9] Formatted Administrateur 2/7/2011 8:40:00 AM 18 Font: (Default) TimesFor New Roman, Peer 12 pt, Font Review color: Auto, Complex Script Font: Times 19 New Roman, 12 pt 20 Page 1: [10] Deleted Administrateur 2/7/2011 8:38:00 AM 21 8 22 23 24 Page 1: [10] Deleted Administrateur 2/7/2011 8:33:00 AM 25 Hôpital des Enfants,

26 27 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 28 MTP 29

30 31 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 32 33 MTP 34 35 36 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 37 MTP 38 39 40 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 41 MTP 42 43 44 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 45 MTP 46

47 48 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 49 MTP 50

51 52 Page 12: [11] Deleted Administrateur 1/3/2011 12:26:00 PM 53 54 MTP 55

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1 2 3 . 4

5 6 Page 12: [12] Deleted PONS 1/28/2011 5:08:00 PM 7 8 5 9

10 11 Page 12: [13] Deleted Administrateur 1/3/2011 12:26:00 PM 12 MTP 13 14 15 Page 12: [13] Deleted Administrateur 2/7/2011 9:12:00 AM 16 was 17 18 For Peer Review 19 Page 12: [13] Deleted Administrateur 1/3/2011 12:26:00 PM 20 MTP 21

22 23 Page 12: [13] Deleted Administrateur 1/3/2011 12:26:00 PM 24 MTP 25

26 27 Page 12: [14] Deleted PONS 1/28/2011 6:13:00 PM 28 29 . 30

31 32 Page 12: [14] Deleted PONS 1/28/2011 5:09:00 PM 33 6A 34 35 36 Page 12: [15] Deleted Administrateur 1/3/2011 12:26:00 PM 37 MTP 38 39 40 Page 12: [15] Deleted Administrateur 1/3/2011 12:26:00 PM 41 MTP 42

43 44 Page 12: [16] Deleted PONS 1/28/2011 5:09:00 PM 45 bring down 46

47 48 Page 12: [16] Deleted PONS 1/28/2011 5:10:00 PM 49 50 after immunoprecipitation 51

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1 2 3 6B 4

5 6 Page 12: [16] Deleted PONS 1/28/2011 5:10:00 PM 7 8 6C 9

10 11 Page 12: [17] Deleted Administrateur 1/3/2011 12:26:00 PM 12 MTP 13 14 15 Page 12: [17] Deleted Administrateur 1/3/2011 12:26:00 PM 16 MTP 17 18 For Peer Review 19 Page 12: [17] Deleted Administrateur 1/3/2011 12:26:00 PM 20 MTP 21

22 23 Page 12: [17] Deleted Administrateur 1/3/2011 12:26:00 PM 24 MTP 25

26 27 Page 12: [17] Deleted Administrateur 1/3/2011 12:26:00 PM 28 29 MTP 30

31 32 Page 12: [17] Deleted Administrateur 1/3/2011 12:26:00 PM 33 MTP 34 35 36 Page 12: [17] Deleted Administrateur 2/7/2011 10:28:00 AM 37 lementary 38 39 40 Page 12: [18] Deleted Administrateur 2/7/2011 9:13:00 AM 41 S 42

43 44 Page 12: [18] Deleted Administrateur 1/3/2011 12:26:00 PM 45 MTP 46

47 48 Page 12: [18] Deleted Administrateur 2/7/2011 10:28:00 AM 49 50 lementary 51

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1 2 3 6E 4

5 6 Page 12: [20] Deleted Administrateur 2/7/2011 9:13:00 AM 7 8 S 9

10 11 Page 12: [20] Deleted Administrateur 1/3/2011 12:26:00 PM 12 MTP 13 14 15 Page 12: [20] Deleted Administrateur 1/3/2011 12:26:00 PM 16 MTP 17 18 For Peer Review 19 Page 12: [20] Deleted Administrateur 1/3/2011 12:26:00 PM 20 MTP 21

22 23 Page 12: [20] Deleted Administrateur 1/3/2011 12:26:00 PM 24 MTP 25

26 27 Page 12: [20] Deleted Administrateur 1/3/2011 12:26:00 PM 28 29 MTP 30

31 32 Page 12: [21] Deleted PONS 1/28/2011 5:15:00 PM 33 of ER 34 35 36 Page 12: [21] Deleted PONS 1/28/2011 6:15:00 PM 37 . 38 39 40 Page 12: [21] Deleted PONS 1/28/2011 5:15:00 PM 41 7 42

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47 48 Page 12: [22] Deleted PONS 1/28/2011 5:16:00 PM 49 50 8 51

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1 2 3 MTP 4

5 6 Page 16: [24] Deleted Administrateur 1/3/2011 12:26:00 PM 7 8 MTP 9

10 11 Page 16: [24] Deleted Administrateur 1/10/2011 1:07:00 PM 12 (Zamel, et al., 2008) 13 14 15 Page 16: [24] Deleted Administrateur 1/3/2011 12:26:00 PM 16 MTP 17 18 For Peer Review 19 Page 16: [25] Deleted Administrateur 1/3/2011 12:26:00 PM 20 MTP 21

22 23 Page 16: [25] Deleted Administrateur 1/3/2011 12:26:00 PM 24 MTP 25

26 27 Page 16: [26] Deleted PONS 1/28/2011 6:17:00 PM 28 29 . 30

31 32 Page 16: [26] Deleted PONS 1/28/2011 6:17:00 PM 33 7 34 35 36 Page 16: [26] Deleted PONS 1/28/2011 6:18:00 PM 37 . 38 39 40 Page 16: [26] Deleted PONS 1/28/2011 6:17:00 PM 41 6A 42

43 44 Page 16: [26] Deleted PONS 1/28/2011 6:18:00 PM 45 -C 46

47 48 Page 16: [27] Deleted Administrateur 1/3/2011 12:26:00 PM 49 50 MTP 51

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1 2 3 MTP 4

5 6 Page 16: [28] Deleted PONS 1/31/2011 5:23:00 PM 7 8 MTTP could be used to lower cholesterol levels in patients with 9 10 hypercholesterolemia (Cuchel, et al., 2007). However, adverse events like liver steatosis 11 12 or vitamin E deficiency should be prevented. 13

14 15 Page 16: [29] Deleted PONS 1/31/2011 5:23:00 PM 16 α α 17 MTTP transcription is controlled by hepatocyte nuclear factor-4 (HNF-4 ) 18 For Peer Review 19 (Sheena, et al., 2005). Hepatocyte nuclear factor binding element (HNF1A) within the 20 21 22 Page 16: [30] Deleted PONS 1/31/2011 5:23:00 PM 23 MTTP mRNA when mice are fed with high-cholesterol and high-fat diets (Iqbal, 24 25 26 et al., 2008). The 27

28 29 Page 16: [31] Deleted PONS 1/31/2011 5:23:00 PM 30 MTTP promoter variant -579T/T is associated with peripheral arterial disease 31 32 (Schgoer, et al., 2008). The -579G/T polymorphism contributes to fat liver accumulation 33 34 35 in 3 infected patients with HCV genotype (Zampino, et al., 2008), but could also have a 36 37 role in the steatosis of these children. [A1] 38 39 40 Page 16: [32] Deleted PONS 1/31/2011 5:23:00 PM 41 MTTP promoter serves as a novel negative insulin-responsive element (Au, et al., 42 43 2008). Forkhead box O1 (FoxO1) mediates insulin-regulated 44 45 46 Page 16: [33] Deleted PONS 1/31/2011 5:23:00 PM 47 MTTP production, that may be a causative factor for VLDL overproduction and 48 49 50 in (Kamagate, et al., 2008). Regulation of 51 52 53 Page 16: [34] Deleted PONS 1/31/2011 5:23:00 PM 54 MTTP expression and CM production is controlled by inositol-requiring 55 56 1beta (IRE1beta), which degrades 57 58 59 60 John Wiley & Sons, Inc. Page 36 of 78 Human Mutation

1 2 3 4 Page 16: [35] Deleted Administrateur 1/3/2011 12:26:00 PM 5 MTP 6

7 8 Page 16: [35] Deleted Administrateur 1/3/2011 12:26:00 PM 9 MTP 10

11 12 Page 16: [35] Deleted Administrateur 2/7/2011 9:39:00 AM 13 14 id 15 16 17 Page 18: [36] Deleted Administrateur 2/7/2011 10:18:00 AM 18 For Peer Review 19 20 Children 21 22 23 Normal Adults 24 25 AM PM Range Father Mother Normal Range 26 27 Triglycerides 6 1 < 75 mg/dL 77 87 50-150 mg/dL 28 29 30 Total cholesterol 30 30 < 170 mg/dL 166 235 105-240 mg/dL 31 32 HDL-cholesterol 29 30 > 40 mg/dL 46 69 40-80 mg/dL 33 34 35 VLDL-cholesterol 10 0 ND 16 17 10-30 mg/dL 36 37 LDL-cholesterol 0 0 < 110 mg/dL 105 148 108-162 mg/dL 38 39 40 ApoA-I 0.43 0.48 NA 1.25 1.71 1.1-2.1 g/L 41 42 Apo-B 0.02 0.01 < 90 mg/dL 0.68 0.92 0.5-1.35 g/L 43 44 45 /RBP 0.68 1.06 0.95-1.06 ND ND 46 47 0.41 0.16 6-15 mg/L ND ND 48 49 50 51 52 NA: no available; ND: not done. 53 54 55 Page 18: [37] Deleted Administrateur 2/7/2011 10:18:00 AM 56 AM, born the 21/01/2004 PM, born the 25/07/1999 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 37 of 78

1 2 3 Normal 4 5 6 29/04/2004 11/10/2005 27/07/2010 30/09/1999 29/11/2005 27/07/2010 range 7 8 IgG 0.95 7.86 6.48 0.33 8.63 5.47 5.6 - 10.4 g/L 9 10 11 IgA 0.04 0.11 1.11 0.02 1.73 1.67 0.4 - 1.4 g/L 12 13 IgM 0.11 0.67 0.73 0.04 0.44 0.36 0.6 - 1.6 g/L 14 15 16 17

18 Page 18: [38] DeletedFor Peer PONS Review 1/31/2011 5:29:00 PM 19 20 21 22 23 24 exon 6 deletion exon 10 deletion 25 26 27 AM c.619G>T c.1237-28A>G 28 29 PM c.619G>T c.1237-28A>G 30 31 32 Father c.619G>T normal 33 34 Mother normal c.1237-28A>G 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 38 of 78 Human Mutation

New splicing mutations of MTTP 1 1 2 3 A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal 4 5 triglyceride transfer protein 6 7 8 Véronique Pons 1,2 , Corinne Rolland 3,4 , Michel Nauze 1,2 , Marie Danjoux 5, Gérald Gaibelet 9 1,2 6 7 8 1,2 1,2 10 , Anne Durandy , Agnès Sassolas , Emile Lévy , François Tercé , Xavier Collet * 11 3,4,9 12 and Emmanuel Mas * 13 14 15 1INSERM, U1048, Toulouse, F-31000, France; 16 2 17 Université Toulouse III, Institut de Maladies Métaboliques et Cardiovasculaires, Toulouse, 18 19 F-31300 France; 20 3INSERM, U1043, Toulouse,For F-31300 Peer France; Review 21 22 4Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), 23 24 Toulouse, F-31300, France; 25 5 26 CHU Toulouse, Hôpital Purpan, Département d’Anatomopathologie, Toulouse, F-31300 27 28 France; 29 6INSERM U768, Université Paris V-René Descartes, Hôpital Necker-Enfants Malades, 75015 30 31 Paris (France); 32 7 33 UF Dyslipidémies, CBPE, Hospices Civils de Lyon and Lyon University, INSERM U1060, 34 35 CarMeN Laboratory, Univ Lyon-1 F-69621, Villeurbanne (France); 36 8Départements de Nutrition et de Biochimie, Hôpital Sainte-Justine et Université de Montréal, 37 38 Montréal, Québec (Canada); 39 40 9CHU Toulouse, Hôpital des Enfants, Unité de Gastroentérologie, Hépatologie et Nutrition, 41 42 Département de Pédiatrie, Toulouse, F-31300, France. 43 44 * These authors equally contributed to the work 45 46 47 Corresponding author: 48 49 Emmanuel Mas, MD 50 51 Hôpital des Enfants - Unité de Gastroentérologie, Hépatologie et Nutrition – 330 avenue de 52 Grande-Bretagne – TSA 70034 – 31059 Toulouse cedex 9 (France) 53 54 Tel. (33) 5 34 55 84 45 55 56 Fax. (33) 5 34 55 85 67 57 58 E-mail: [email protected] 59 60

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New splicing mutations of MTTP 2 1 2 3 ABSTRACT 4 5 Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels 6 7 and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal 8 9 triglyceride transfer protein (MTTP) deficiency. 10 We report 2 patients with new MTTP mutations. We studied their functional consequences on 11 12 the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in 13 14 HepG2 and HeLa cells to investigate their association with protein disulfide isomerase ( PDI ) 15 16 and their localization at the endoplasmic reticulum. 17 18 These children have a severe abetalipoproteinemia. Both of them had also a mild 19 hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237- 20 For Peer Review 21 28A>G mutations within MTTP gene. mRNA analysis revealed abnormal splicing with 22 23 deletion of exon 6 and 10, respectively. Deletion of exon 6 (6-MTTP ) introduced a frame 24 25 shift in the reading frame and a premature stop codon at position 234. Despite 6-MTTP and 26 27 10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally 28 29 localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP 30 triglyceride transfer activity. 31 32 These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI 33 34 and responsible for the loss of function of MTTP, thereby explaining the severe 35 36 abetalipoproteinemia phenotype of these children. 37 38 39 40 Keywords: abetalipoproteinemia, MTTP , triglyceride transfer, intestinal HDL, endoplasmic 41 42 reticulum 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

John Wiley & Sons, Inc. Page 40 of 78 Human Mutation

New splicing mutations of MTTP 3 1 2 3 INTRODUCTION 4 5 6 Abetalipoproteinemia (ABL, OMIM 200100) is a rare autosomal recessive disease 7 8 caused by the deficiency of the microsomal triglyceride transfer protein (MTTP, OMIM 9 10 157147). ABL is characterized by the absence of apolipoprotein B (apoB)-containing 11 12 13 lipoproteins from plasma. MTTP is a 97 kDa protein, containing 894 amino acids, that forms 14 15 a heterodimer with the 55 kDa protein disulfide isomerase (PDI). MTTP is mainly found in 16 17 18 the endoplasmic reticulum (ER) of hepatocytes and enterocytes (Wetterau, et al., 1992). 19 20 MTTP is required for Forthe transfer Peer of neutral lipids Review to nascent apoB-lipoproteins, resulting in 21 22 the secretion of very low-density lipoproteins (VLDL) in the liver or chylomicrons (CM) in 23 24 25 the intestine. When MTTP is deficient, nascent apoB is degraded by the proteasome (Benoist 26 27 and Grand-Perret, 1997). It differs from familial hypobetalipoproteinemia, displaying 28 29 dysfunctional apoB and leading to the absence of apoB-lipoproteins in the plasma. Familial 30 31 32 hypobetalipoproteinemia is a co-dominant disorder and even if heterozygotes can be 33 34 asymptomatic, they had low levels of total cholesterol (TC), low density lipoprotein (LDL)- 35 36 cholesterol and apoB (OMIM 107730). 37 38 39 ABL was characterized as the consequence of MTTP absence or dysfunction in 1992 40 41 by Wettereau et al. (Wetterau, et al., 1992). The genetic defects in MTTP gene, located on 42 43 44 chromosome 4q22-24, were confirmed one year later (Sharp, et al., 1993; Shoulders, et al., 45 46 1993). ABL is a very uncommon disease, which highlights the crucial role of this protein. 47 48 Indeed, the mttp-deficient mice died at embryonic day 10.5 with abnormal neurological 49 50 51 development (exencephalia) (Raabe, et al., 1998). When a specific inducible intestinal 52 53 deletion of mttp is generated in mice, they presented a phenotype similar to ABL, with 54 55 steatorrhea, growth arrest and decreased cholesterol absorption (Xie, et al., 2006). 56 57 58 During evolution, MTTP has progressively acquired TG transfer activity in vertebrates 59 60 whereas invertebrate MTTP is known to only transfer phospholipids (Rava and Hussain,

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New splicing mutations of MTTP 4 1 2 3 2007). Another way to study MTTP functions is to characterize human mutations. This 4 5 6 approach could help us to determine its active domains that transfer phospholipids, 7 8 cholesterol esters or TG, as well as its binding domains to PDI or to apoB. To our knowledge, 9 10 so far, 40 mutations have been described in 52 patients (Supp. Table S1) (Najah, et al., 2009; 11 12 13 Zamel, et al., 2008). Here, we report 2 children with a severe form of ABL that are compound 14 15 heterozygotes for MTTP . The new mutations induce deletion of exon 6 and 10 and translation 16 17 18 of truncated proteins. 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MTTP 5 1 2 3 MATERIALS AND METHODS 4 5 6 Patients: 7 8 We report 2 new cases of ABL, whose diagnosis was performed when AM was a 13 month- 9 10 old boy and his sister PM was 6 years-old. They were born from Caucasian non- 11 12 13 consanguineous parents. We received the agreement of both parents for genetic 14 15 investigations. 16 17 18 Endoscopy and duodenal biopsies: 19 20 Upper gastrointestinalFor endoscopy Peer was realized Review under general anesthesia with a Pentax EG- 21 22 1870K. Duodenal biopsies were fixed in Bouin for pathology or immediately frozen in liquid 23 24 25 nitrogen for further investigations (Western blot and MTTP activity assay). For routine 26 27 microscopy, biopsies were embedded in paraformaldehyde and sections were 28 29 hematoxylin/eosin-stained. 30 31 32 Intestinal MTTP analyses: 33 34 Proteins were extracted from duodenal biopsies to perform a western blot of MTTP. MTTP 35 36 activity was measured by radiolabelled TG transfer from donor to acceptor vesicles as 37 38 39 previously described (Levy, et al., 2002). 40 41 Plasma sample analyses: 42 43 44 Blood samples were collected for lipids, lipoproteins, vitamin A and E investigations, and 45 46 ALT. TG and TC were measured by an enzymatic method (Roche). HDL and VLDL- 47 48 cholesterol were measured by a dextran/PEG method (Roche). LDL-cholesterol was 49 50 51 calculated by Friedwald method. ApoA-I and apoB were determined by immuno-turbidimetry 52 53 (Roche). 54 55 Lipid chromatography: 56 57 58 The FPLC system consisted of a Hitachi L7250 auto-sampler, a L7100 pump system and two 59 60 detectors: L7400 (Hitachi) and UVD170U (Dionex).  20µL of plasma were injected and

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New splicing mutations of MTTP 6 1 2 3 lipoproteins were separated on SuperoseTM6 10/300GL column (GE Healthcare) with 0.15M 4 5 6 NaCl solution at a flow rate of 0.4mL/min. The column effluent was split equally into two 7 8 lines by a microsplitter 50:50, mixing with cholesterol or triglyceride reagents (Biolabo), thus 9 10 achieving a simultaneous profile from single injection. The two enzymatic reagents were each 11 12 13 pumped at a rate of 0.2mL/min. Both enzymatic reactions proceeded at 37°C in a Teflon 14 15 reactor coil (15mLx0.4mm id). Proteins were measured directly at 280nm. 16 17 MTTP 18 Genomic DNA sequencing of : 19 20 DNA from patients wasFor extracted Peer from blood Reviewsamples and then exons and introns of MTTP 21 22 gene (NCBI Reference Sequence: NG_011469.1) were sequenced with Applied ABI Prism. 23 24 MTTP 25 mRNA sequencing of : 26 27 Total RNA was extracted using RNeasy Mini Kit (Qiagen) from either EBV-immortalized B 28 29 lymphocytes for both children or primary lymphocytes for both children and their parents. 30 31 32 Primary lymphocytes were isolated from blood samples using a ficoll technique. A reverse 33 34 transcription was performed and nested PCR was used to amplify exon 6 and exon 10 with 5’ 35 36 and 3’ flanking regions (see primers in Supp. Table S2). PCR products were separated on 37 38 39 agarose gel, then extracted with a QIAquick Gel Extraction Kit (Qiagen) and directly inserted 40 41 into pCR2.1 vector (Invitrogen) before sequencing analyses. 42 43 44 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG 45 46 translation initiation codon in the reference sequence (www.hgvs.org/mutnomen). The 47 48 initiation codon is codon 1. 49 50 51 Constructs: 52 53 WT-MTTP cDNA was a generous gift of Dr M.Hussain (New-York). We produced by PCR 54 55 WT-MTTP and MTTP mutants with deletions of patients: 6-MTTP and 10-MTTP . PCR 56 57 58 fragments (WT-MTTP , 6-MTTP and 10-MTTP ) were digested using BamHI and HindIII 59 60 restriction enzymes, purified with a QIAquick Gel Extraction Kit (Qiagen) and then inserted

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New splicing mutations of MTTP 7 1 2 3 in pEGFP-N1 or in p2xMyc-N1, generated by replacing the EGFP from pEGFP-N1 with a 4 5 6 2xMyc epitope. pDsRed2-ER vector (Clontech) was used to encode an ER marker. 7 8 Cell culture and transfection: 9 10 HepG2 (human hepatocellular carcinoma), HeLa (human cervix carcinoma) or Caco-2 11 12 13 (human epithelial colorectal adenocarcinoma) cells were grown in Dulbecco’s modified 14 15 Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 0.1 16 17 18 mM non essential amino acids (Invitrogen), and 100 µg/ml penicillin/streptomycin 19 20 (Invitrogen) in a humidifiedFor atmosphere Peer containing Review 5% CO 2. HepG2 and HeLa cells were 21 22 transfected using GenJuice (Novagen, Merck) or Fugene (Roche) respectively. 23 24 25 Immunoprecipitation: 26 27 The cells were lysed for 30 min in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% 28 29 Nonidet P-40, 10% glycerol) containing protease and phosphatase inhibitors. Soluble material 30 31 32 was then incubated overnight at 4°C with 5 µg of monoclonal anti-Myc antibody (clone 33 34 9E10) or polyclonal anti-PDI antibody (DL-11). The antigen-antibody complex was incubated 35 36 with a mix of protein-G and protein-A Sepharose for 1h and then washed in lysis buffer 37 38 39 containing 0.1% Nonidet P-40. The bound proteins were eluted by boiling the beads in 40 41 Laemmli buffer and analyzed by immunoblotting. 42 43 44 Immunoblotting: 45 46 The proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing 47 48 conditions, transferred onto nitrocellulose membrane, and analyzed by immunoblotting 49 50 51 according to standard protocols using indicated antibodies. Anti-MTTP antibody was a 52 53 generous gift of Drs. J. R. Wetterau and H. Jamil. We used a mouse monoclonal antibody 54 55 against Myc (clone 9E10) from BD Biosciences, a rabbit polyclonal antibody against PDI 56 57 58 (DL-11) from Sigma and a mouse monoclonal antibody against  GFP from Roche. HRP- 59 60 labelled secondary antibodies were from Santa-Cruz or Roche Diagnostics.

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New splicing mutations of MTTP 8 1 2 3 Immunofluorescence experiments: 4 5 6 HepG2 and HeLa cells were grown on collagen I coated or glass coverslips respectively. Cells 7 8 were fixed in 4% paraformaldehyde, permeabilized with 0.05% saponin, saturated with PBS 9 10 containing 10% fetal bovine serum and incubated with the anti-PDI rabbit polyclonal 11 12 13 antibody (Sigma) or the anti-Golgin 97 mouse monoclonal antibody (Molecular Probes) and 14 15 then with Cy3-labelled secondary antibody (Jackson Immunoresearch Laboratories). Pictures 16 17 18 were captured using a Zeiss LSM 510 META confocal microscope equipped with a 63x Plan- 19 20 Apochromat objective.For Peer Review 21 22 Immunological investigation: 23 24 25 Serum immunoglobulin levels were determined by nephelemetry. B lymphocyte phenotype 26 27 and function were analyzed as previously described (Peron, et al., 2008). 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MTTP 9 1 2 3 RESULTS 4 5 6 Severe form of abetalipoproteinemia: 7 8 When AM was 13-months-old, he presented with a failure to thrive. An intestinal 9 10 malabsorption was suspected on the basis of abdominal distension, abnormal stools (diarrhea 11 12 13 or constipation) and hydro-aeric levels on abdominal X-ray. An upper gastrointestinal 14 15 endoscopy was performed and revealed a white aspect of duodenal villi characteristic of 16 17 18 intestinal fat malabsorption and abnormal lipid storage (Figure 1A), as confirmed by 19 20 histology (large fat inclusionsFor located Peer within enterocytes, Review Figure 1B). Blood analyses revealed 21 22 a severe hypocholesterolemia and hypotriglyceridemia without LDL particles and apoB 23 24 25 (Table 1). Since parents have normal lipid values as well as apoB and LDL levels, we ruled 26 27 out a hypo-betalipoproteinemia which is a dominant inheritance disease. Thus ABL was 28 29 suspected. They also had an increased level of alanine amino-transferase (ALT) and a 30 31 32 hyperechogeneic aspect of the liver on ultra-sound scan. Furthermore, both children presented 33 34 a severe vitamin E deficiency secondary to intestinal fat malabsorption. His sister (PM), 6- 35 36 years-old at the time of diagnosis, had no rotula reflex (the very early diagnosis of AM 37 38 39 prevented the development of peripheral neuropathy). However, both of them have no sign of 40 41 retinopathy. 42 43 44 Abnormal lipoprotein profiles: 45 46 We assessed the blood lipoprotein profiles by FPLC analysis. Both children presented 47 48 a complete absence of LDL and an almost complete absence VLDL in TC and TG moieties 49 50 51 (Figure 1C and 1D). Although HDL-cholesterol levels are only 20% of control, HDL particles 52 53 remain the only lipid class, with a normal ratio of cholesterol ester to TC (Table 1) reflecting 54 55 a functional lecithin cholesterol acyl-transferase (LCAT) activity. The HDL pattern differs 56 57 58 between patient and control. Patient had 2 peaks of HDL particles. The early peak is specific 59 60 of the patient while the later one appears at the same time than control, as it is suggested by

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New splicing mutations of MTTP 10 1 2 3 the concordance of protein chromatogram. HDL particles of the early peak seem to be 4 5 6 cholesterol-enriched and to have less protein than HDL of the second peak. 7 8 Immunoglobulin synthesis abnormalities: 9 10 Both of these children had also a congenital hypogammaglobulinemia requiring 11 12 13 regular immunoglobulin substitution. They were diagnosed as immunodeficient on serum 14 15 IgG, IgM and IgA levels that were performed because of a failure to thrive in the first months 16 17 18 of age (Table 2). There was no protein loosing enteropathy. Mild hypogammaglobulinemia 19 20 was discovered, affectingFor IgM andPeer IgG in both Review children. IgA was also slightly decreased in 21 22 the brother AM during the first years of age. AM and PM had normal T and B cell 23 24 25 populations (data not shown). B cell phenotype was found normal in PM but absence of 26 27 memory switched B cells was observed in the younger brother AM. In contrast, in vitro B cell 28 29 immunoglobulin production was found normal (data not shown). 30 31 32 MTTP mutations: 33 34 Genomic DNA sequencing of MTTP gene showed 2 different mutations: c.619G>T 35 36 and c.1237-28A>G (Supp. Table S3). The first one c.619G>T was located at the first 37 38 39 nucleotide of exon 6 and the second one c.1237-28A>G was located within intron 9. In order 40 41 to understand the consequences of such mutations, we extracted mRNA from lymphocytes 42 43 MTTP 44 and analyzed transcripts. MTTP is mainly expressed in the liver and in the intestine, 45 46 but can also be detected from EBV-immortalized B lymphocytes. After reverse transcription 47 48 and PCR amplification, products were analyzed by gel migration, thereby revealing a 49 50 51 decrease in size for both mutations in patient compared to the control (Figure 2A and 2C). As 52 53 expected, we obtained in control a band at 228 pb and 888 pb for exons 6 and 10 respectively, 54 55 whereas lower fragments were revealed in patient. Those fragments were detected at the size 56 57 58 of ∼90pb and ∼780pb, suggesting the loss of exons 6 and 10 respectively. Sequencing 59 60 confirmed that these two mutations are responsible for abnormal splicing of exons 6 and 10

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New splicing mutations of MTTP 11 1 2 3 respectively (Figure 2B and 2D). The first genomic DNA mutation c.619G>T induces a frank 4 5 6 deletion of exon 6 and a shift in the open reading frame leading to a premature stop codon at 7 8 position 234 and resulting in an abnormal truncated protein consisting of 233 amino acids 9 10 11 (∼25kDa). The second DNA mutation c.1237-28A>G induces a frank deletion of exon 10 (36 12 13 amino acids), with no shift in the reading frame, leading to a protein of 858 amino acids 14 15 (∼94kDa) (Figure 2E). 16 17 18 These mutations were also confirmed with sequencing analyses from primary 19 20 lymphocytes from parentsFor and children,Peer meaning Review that abnormal splicing of MTTP was a 21 22 23 consequence of genomic mutations and not of EBV immortalization. 24 25 As children are compound heterozygotes, these results confirm the genetic diagnosis 26 27 of ABL. 28 29 30 Loss of function of MTTP protein: 31 32 Duodenal biopsies were performed and samples were analyzed for MTTP expression 33 34 and activity. In patient, we detected a band with a slight decrease of the apparent MTTP size 35 36 37 compared to control or Caco-2 cells (Figure 3A), which probably corresponds to 10-MTTP 38 39 with the loss of 36 amino acids (3kDa) due to exon 10 deletion. We observed no band at a 40 41 42 size of 25kDa, corresponding to the expected size of 6-MTTP (data not shown), suggesting 43 44 that 6-MTTP might be degraded by the proteasome. MTTP activity was also measured on 45 46 47 these samples and showed a complete loss of TG transfer activity (5-8%/mg protein in control 48 49 versus 0.08-0.8%/mg protein in AM, Figure 3B). 50 51 52 We further investigated the ability of both 6-MTTP and 10-MTTP mutants to 53 54 interact with PDI and their subcellular localization by first generating constructs encoding 55 56 GFP- or 2xMyc-tagged WT-MTTP, 6-MTTP or 10-MTTP. Tagged MTTP mutants as well 57 58 59 as WT-MTTP were expressed in HeLa cells and analyzed by immunoblotting. We observed 60 that 6-MTTP and 10-MTTP were detected, as well as WT form (Figure 4A and 4B). This

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New splicing mutations of MTTP 12 1 2 3 is in contrast to the mutant MTTP analysis in duodenal biopsy since 6-MTTP could not be 4 5 6 detected. The overexpression level of tagged proteins might overcome the degradation 7 8 machinery, thereby allowing the detection of a part of 6-MTTP and 10-MTTP. However, 9 10 11 10-MTTP seemed to be lower expressed compared to WT-MTTP, either expressed as GFP- 12 13 or 2xMyc-10-MTTP (quantification in Figure 4C), probably because a part of abnormal 14 15 16 proteins was degraded by proteasome. 17 18 Since MTTP is localized in the ER through its binding with PDI, we tested the 19 20 For Peer Review 21 association of MTTP mutants with PDI. 2xMyc-tagged MTTP mutant and WT proteins were 22 23 expressed in both HeLa and HepG2 cells and immunoprecipitation was performed using anti- 24 25 myc antibody (Figure 4D) or anti-PDI antibody (Figure 4E). As expected, we found that WT- 26 27 28 MTTP interacted with PDI (Figure 4D and 4E, upper panel). By contrast, neither 6-MTTP 29 30 nor 10-MTTP was able to associate with PDI (Figure 4D and 4E, middle and lower panels). 31 32 33 We then investigated the cellular localization of WT-MTTP as well as 6-MTTP and 34 35 10-MTTP. After expression in both HeLa and HepG2 cells, WT-MTTP, 6-MTTP and 10- 36 37 38 MTTP displayed an identical pattern, showing a reticular localization throughout the cytosol, 39 40 reminiscent of ER staining (Supp. Figure S1). However, in HepG2 cells but not in HeLa cells, 41 42 43 6-MTTP also displays a non specific localization both in the nucleus and throughout the 44 45 cytosol (Supp. Figure S1B, see inset in 6-MTTP-GFP), much like GFP alone. This suggests 46 47 48 that in HepG2 cells, 6-MTTP-GFP protein can be degraded, as we observed by 49 50 immunoblotting in duodenal biopsies. Surprisingly, WT-MTTP as well as 6-MTTP and 51 52 53 10-MTTP co-localized with ER markers such as ER-DsRed, a red marker consisting of a 54 55 fusion of the DsRed and the ER retention sequence, KDEL (Figure 5A) and PDI (Figure 5B). 56 57 By contrast, no co-localization was observed with cis-Golgi marker such as Golgin 97 (Supp. 58 59 60 Figure S2) or trans-Golgi marker such as TGN46 (data not shown).

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New splicing mutations of MTTP 13 1 2 3 Altogether, these data showed that MTTP mutants lost TG transfer activity. They are 4 5 6 still localized in the ER despite their inability to interact with PDI. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MTTP 14 1 2 3 DISCUSSION 4 5 6 MTTP is mainly expressed in enterocytes and hepatocytes where it participates in the 7 8 synthesis of chylomicrons and VLDL respectively. These patients presented fat storage in the 9 10 small intestine (Figure 1A and 1B) and in the liver (as suggested by increased ALT levels and 11 12 13 hyperechogeneic aspect), as a consequence of MTTP dysfunction. 14 15 Lipids are almost exclusively transported in blood into HDL lipoproteins in ABL 16 17 18 patients (Figures 1C and 1D and Table 1). These particles are mainly synthesized in 19 20 peripheral tissues to ensureFor the reversePeer cholesterol Review transport to the liver. But, HDL are also 21 22 produced at the basolateral membrane of enterocytes, via ATP Binding Cassette-A1 (ABCA1) 23 24 25 efflux of cholesterol to apoA-I (Levy, et al., 2007). Cholesterol is transported by enterocytes 26 27 in two different pathways: the apoB-dependent and the apoB-independent pathways resulting 28 29 respectively in the production of CM or HDL lipoproteins (Iqbal, et al., 2003). Some 30 31 32 differences exist between these two pathways. Cholesterol ester is only secreted within CM 33 34 while free cholesterol is secreted by both pathways. The apoB-dependent pathway is also 35 36 regulated by MTTP and contributes to cholesterol absorption during post-prandial states. 37 38 39 MTTP is not required for cholesterol secretion by the apoB-independent pathway that may be 40 41 more important during fasting states. In the human epithelial Caco-2 cell line, Liver X 42 43 ABCA1 44 Receptor/Retinoid X Receptor activation increases gene expression and basolateral 45 46 efflux of cholesterol in intestinal HDL (Murthy, et al., 2002). In ABL patients, neurological 47 48 impairment (ataxia and peripheral neuropathy) and retinopathy are the consequences of 49 50 51 vitamin E deficiency. Like cholesterol, these two routes also exist for vitamin E absorption by 52 53 enterocytes (Anwar, et al., 2007). In MTTP -deficient enterocytes, the HDL pathway is used to 54 55 deliver vitamin E. Vitamin E secretion within HDL lipoproteins is not increased by MTTP 56 57 58 inhibition (Anwar, et al., 2007). This pathway is not as important as chylomicron synthesis 59 60 but, in ABL patients, intestinal HDL represent the only route for lipids or vitamin E from

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New splicing mutations of MTTP 15 1 2 3 intestine to the liver (Anwar, et al., 2007; Anwar, et al., 2006). These ABL patients have a 4 5 6 specific class of HDL particles that have a lower density and a lower protein content. 7 8 MTTP is also expressed in natural killer T (NKT) cells. Inhibition of MTTP in fetal 9 10 thymocyte organ culture results in a complete loss of NKT cells (Dougan, et al., 2007). In 11 12 13 these antigen presenting cells, MTTP loads lipids onto nascent CD1d and regulates 14 15 presentation of glycolipid antigens (Dougan, et al., 2005). MTTP deficiency could impair the 16 17 18 recycling of CD1d from lysosome to the plasma membrane (Sagiv, et al., 2007). Interestingly, 19 20 both patients present For with a hypogammaglobulinemia Peer Review that was mild but however required 21 22 immunoglobulin substitution. We only found an absence of memory switched B cells in one 23 24 25 patient while both of them had a normal in vitro immunoglobulin production (Table 2). Since 26 27 such an association between mild B lymphocyte immunodeficiency and abetalipoproteinemia 28 29 has not been previously reported in ABL patients, it is unlikely that MTTP defect is directly 30 31 32 involved in hypogammaglobulinemia. However, this point should be further investigated, 33 34 looking for a subtle defect in immunoglobulin levels in abetalipoproteinemia patients. 35 36 Recently, Zeissig et al . characterized the loss of CD1 function in ABL patients (Zeissig, et al., 37 38 39 2010). They found a defect of all antigen-presenting CD1 family members in dendritic cells 40 41 from ABL patients. Similarly to apoB, MTTP deficiency in the ER leads to the degradation of 42 43 44 group 1 CD1 by the proteasome pathway, which altered activation of NKT cells. 45 46 In these new patients with ABL, we characterized mutations of MTTP by analyzing 47 48 genomic DNA and mRNA products. Sequencing revealed abnormal splicing leading to 49 50 51 deletion of exon 6 or 10, as a result of two genomic mutations, c.619G>T and c.1237-28A>G 52 53 respectively. c.619G>T is not only a missense mutation (207 Val>Phe), but induces as well an 54 55 abnormal splicing (Mitchell, et al., 1986). The deletion of 140 bp of exon 6 causes a shift in 56 57 58 the reading frame and a truncated protein because of a premature stop codon in position 234. 59 60 Recently, Najah et al. (Najah, et al., 2009) found similar results with c.619-3T>G mutation

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New splicing mutations of MTTP 16 1 2 3 located in intron 5 of MTTP . We showed that this truncated protein is not detected in 4 5 6 duodenal biopsies (data not shown), suggesting that 6-MTTP is degraded by the proteasome 7 8 pathway (Pan, et al., 2007). c.1237-28A>G located in intron 9 leads to the frank deletion of 9 10 11 108 bp of exon 10, with no shift in the reading frame, resulting in the loss of 36 amino acids. 12 N 13 Human MTTP structure contains an N-terminal β-barrel ( β ) (residues 22-297), a 14 15 central α-helical domain ( α) (residues 298-603), and two C-terminal β-sheets ( βC and βA) 16 17 N 18 (residues 604-894). β is conserved in apoB, lipovitellin, and apolipophorin and may be one 19 20 of the two phospholipidFor binding sitesPeer of MTTP. Review Helices 4-6, βC and βA domains of MTTP are 21 22 conserved in vertebrates, but not in invertebrates, suggesting that they are involved in TG 23 24 N 25 transfer activity (Rava and Hussain, 2007). β mediates the interaction with the N-terminus of 26 27 apoB; the middle α-helical domain mediates the interaction with both PDI (residues 520-598) 28 29 30 and apoB (residues 517-603); and the C-terminal mediates the lipid-binding and transfer 31 32 catalytic activity of MTTP (Mann, et al., 1999; Read, et al., 2000). Expression of MTTP 33 34 constructs in HepG2 and HeLa cells showed that 6-MTTP and 10-MTTP proteins are 35 36 37 located at ER (Figure 5) but are not associated with PDI (Figure 4D and 4E). MTTP mutants 38 39 might be retained in the ER as defective or misfolded proteins and a part might be 40 41 42 retrotranslocated to the cytosol for proteasomal degradation. The truncated 6-MTTP protein 43 44 only conserved one apoB-binding domain. By contrast, 10-MTTP protein is characterized 45 46 47 by the loss of residues 413-448, that are not directly involved in PDI binding but their loss 48 49 could alter the tertiary structure of the protein. Finally, these residues, corresponding to 50 51 helices 7-9 of the central α-helical domain, belong to a critical domain for TG transfer activity 52 53 54 as described by Rava and Hussain (Rava and Hussain, 2007). 55 56 In conclusion, we report here two new mutations of MTTP , c.619G>T and c.1237- 57 58 28A>G, resulting respectively in 6-MTTP protein, a truncated protein of 233 amino acids 59 60 and 10-MTTP protein, deleted of exon 10. Despite these mutations do not change ER

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New splicing mutations of MTTP 17 1 2 3 localization of mutant MTTP proteins, they abolish their binding with PDI and totally 4 5 6 impaired their TG transfer activity. 7 8 9 10 ACKNOWLEDGEMENTS 11 12 13 The authors thank these children and their parents, and also pediatricians taking care of them 14 15 (Dr. E. Fournié-Gardini and Dr. A.I. Bertozzi). We also thank J. Bertrand-Michel and V. 16 17 18 Roques (Lipidomic Plateau of IFR-BMT/IFR150) for lipidomic analysis, advice and technical 19 20 assistance. We are gratefulFor to Pr.Peer T. Levade Review (Toulouse, France) for immortalization of B 21 22 lymphocytes. 23 24 25 This work was supported by grants from INSERM, ANR (PNRA-2006 ABSINTE) and by 26 27 grants from Canadian Institutes of Health Research, MOP 10584 (E.L). V.P and E.M were the 28 29 recipients of a fellowship from the Fondation pour la Recherche Médicale. V.P is a young 30 31 32 researcher with temporary contract from INSERM. X.C was a recipient of a “Contrat 33 34 d’Interface” from CHU of Toulouse. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MTTP 18 1 2 3 4 5 6 FIGURES LEGENDS 7 8 9 10 Figure 1. Diagnosis of abetalipoproteinemia 11 12 13 (A) Upper gastrointestinal endoscopy performed in patient AM revealed a white aspect of 14 15 duodenal villi, characteristic of lipid storage. (B) Biopsy was performed in duodenum and 16 17 18 processed for hematoxylin/eosin-staining. The arrow points at the presence of lipid droplets 19 20 within the enterocytes.For (C and D)Peer Serum from Review control or patient (AM) was analyzed using 21 22 FPLC system. Lipoproteins were separated and total cholesterol (C) and triglycerides (D) 23 24 25 were measured. Profiles are shown for control (Ctrl) and patient (AM). Proteins were only 26 27 showed for patient in C. Note the difference of y-axis (lipid content) between control and 28 29 patient. 30 31 32 33 34 Figure 2. Sequence analysis of mutant MTTP transcription products 35 36 (A and C) After mRNA extraction and reverse transcription from control and patient (AM) 37 38 39 EBV-immortalized lymphocytes, PCR was performed to amplify exon 6 (A) and exon 10 (C) 40 41 with 5’ and 3’ flanking regions. PCR products were analyzed by migration on agarose gel. 42 43 44 Arrowhead points at the lower band found in patient compared to the control. (B and D) 45 46 Upper bands in the control and lower bands in patients were purified and then cloned into 47 48 pCR2.1 vector before sequencing. In (B), electrophoregrams showed the exon 6 and 5’ and 3’ 49 50 51 flanking regions with exon 5 and 7 in the control. In the patient, the exon 6 is deleted, leading 52 53 to a frameshift and a premature stop codon. In (D), similarly, electrophoregrams showed the 54 55 exon 10 and 5’ and 3’ flanking regions with exon 9 and 11 in the control. In the patient, the 56 57 58 exon 10 is deleted. (E) The amino acid sequences corresponding to exons 6 and 10 are shown 59 60 underlined and in blue in the normal MTTP sequence. // determined the deletion of exons 6 or

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New splicing mutations of MTTP 19 1 2 3 10 in each mutant. For 6-MTTP , the frame-shift mutation results in an abnormal sequence 4 5 6 described in red and in italic. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MTTP 20 1 2 3 Figure 3. MTTP expression and activity in duodenal biopsy 4 5 6 (A) Duodenal biopsy from control (Ctrl) or patient (AM) and Caco-2 cell lysate were 7 8 analyzed by SDS gel electrophoresis and western blotting with antibodies against MTTP. (B) 9 10 The triglyceride transfer activity of MTTP was measured on duodenal biopsy from control or 11 12 13 patient (AM). Values are expressed as the mean of three independent experiments; standard 14 15 errors are indicated. 16 17 18 19 20 Figure 4. InteractionFor of WT and Peer mutant MTTP Review with PDI 21 22 (A and B) HeLa cells were transfected or not with WT-MTTP, 6-MTTP or 10-MTTP 23 24 25 tagged with EGFP (A) or 2xMyc (B). Cell lysates (100 µg) were analyzed by SDS gel 26 27 electrophoresis and western blotting with antibodies against GFP (A), Myc (B) and Rab5 as 28 29 30 loading control. Arrowheads point at WT-MTTP, 6-MTTP or 10-MTTP and arrows point 31 32 at non specific band (ns). Blots in (B) were scanned and the quantification is shown in (C). 33 34 Each experiment was repeated at least 3 times, and (C) shows a representative example. (D 35 36 37 and E) HeLa cells were transfected with 2xMyc-tagged WT-MTTP, 6-MTTP or 10-MTTP. 38 39 Cell lysates (Lys) were subjected to immunoprecipitation with (+) or without (-) the anti-myc 40 41 42 antibody (D) or the anti-PDI antibody (E). Analysis was performed by SDS gel 43 44 electrophoresis and western blotting with antibodies against Myc or PDI. Arrowheads point at 45 46 immunoprecipitated WT-MTTP, 6-MTTP or 10-MTTP (D) or PDI (E) and arrows point at 47 48 49 non specific band (ns). IgG HC represents heavy chain of anti-PDI antibody used for 50 51 immunoprecipitation. 52 53 54 55 56 Figure 5. Localization of WT and mutant MTTP and ER marker 57 58 (A) HeLa cells were co-transfected with GFP-tagged WT-MTTP, 6-MTTP or 10-MTT P 59 60 and a vector encoding DsRed-ER marker. Cells were then processed for fluorescence

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New splicing mutations of MTTP 21 1 2 3 analysis. (B) HeLa cells were transfected with GFP-tagged WT-MTTP, 6-MTTP or 10- 4 5 6 MTTP and were then processed for immunofluorescence using anti-PDI antibody. Bar: 10µm. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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New splicing mutations of MTTP 22 1 2 3 REFERENCES 4 5 6 Al-Shali K, Wang J, Rosen F, Hegele RA. 2003. Ileal adenocarcinoma in a mild phenotype of 7 abetalipoproteinemia. Clin Genet 63(2):135-8. 8 9 Anwar K, Iqbal J, Hussain MM. 2007. Mechanisms involved in vitamin E transport by 10 11 primary enterocytes and in vivo absorption. J Lipid Res 48(9):2028-38. 12 13 Anwar K, Kayden HJ, Hussain MM. 2006. Transport of vitamin E by differentiated Caco-2 14 15 cells. J Lipid Res 47(6):1261-73. 16 Au WS, Lu L, Yeung CM, Liu CC, Wong OG, Lai L, Kung HF, Lin MC. 2008. Hepatocyte 17 18 nuclear factor 1 binding element within the promoter of microsomal triglyceride 19 20 transfer proteinFor (MTTP) Peer gene is crucial Review for MTTP basal expression and insulin 21 22 responsiveness. J Mol Endocrinol 41(4):229-38. 23 Benayoun L, Granot E, Rizel L, Allon-Shalev S, Behar DM, Ben-Yosef T. 2007. 24 25 Abetalipoproteinemia in Israel: evidence for a founder mutation in the Ashkenazi 26 27 Jewish population and a contiguous gene deletion in an Arab patient. Mol Genet 28 29 Metab 90(4):453-7. 30 31 Benoist F, Grand-Perret T. 1997. Co-translational degradation of apolipoprotein B100 by the 32 proteasome is prevented by microsomal triglyceride transfer protein. Synchronized 33 34 translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride 35 36 transfer protein. J Biol Chem 272(33):20435-42. 37 38 Berthier MT, Couture P, Houde A, Paradis AM, Sammak A, Verner A, Depres JP, Gagne C, 39 Gaudet D, Vohl MC. 2004. The c.419-420insA in the MTP gene is associated with 40 41 abetalipoproteinemia among French-Canadians. Mol Genet Metab 81(2):140-3. 42 43 Chardon L, Sassolas A, Dingeon B, Michel-Calemard L, Bovier-Lapierre M, Moulin P, 44 45 Lachaux A. 2008. Identification of two novel mutations and long-term follow-up in 46 47 abetalipoproteinemia: a report of four cases. Eur J Pediatr. 48 Cuchel M, Bloedon LT, Szapary PO, Kolansky DM, Wolfe ML, Sarkis A, Millar JS, Ikewaki 49 50 K, Siegelman ES, Gregg RE and others. 2007. Inhibition of microsomal triglyceride 51 52 transfer protein in familial hypercholesterolemia. N Engl J Med 356(2):148-56. 53 54 Di Leo E, Lancellotti S, Penacchioni JY, Cefalu AB, Averna M, Pisciotta L, Bertolini S, 55 Calandra S, Gabelli C, Tarugi P. 2005. Mutations in MTP gene in abeta- and 56 57 hypobeta-lipoproteinemia. Atherosclerosis 180(2):311-8. 58 59 Dische MR, Porro RS. 1970. The cardiac lesions in Bassen-Kornzweig syndrome. Report of a 60 case, with autopsy findings. Am J Med 49(4):568-71.

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New splicing mutations of MTTP 23 1 2 3 Dougan SK, Rava P, Hussain MM, Blumberg RS. 2007. MTP regulated by an alternate 4 5 promoter is essential for NKT cell development. J Exp Med 204(3):533-45. 6 7 Dougan SK, Salas A, Rava P, Agyemang A, Kaser A, Morrison J, Khurana A, Kronenberg M, 8 9 Johnson C, Exley M and others. 2005. Microsomal triglyceride transfer protein 10 lipidation and control of CD1d on antigen-presenting cells. J Exp Med 202(4):529-39. 11 12 Heath KE, Luong LA, Leonard JV, Chester A, Shoulders CC, Scott J, Middleton-Price HR, 13 14 Humphries SE, Talmud PJ. 1997. The use of a highly informative CA repeat 15 16 polymorphism within the abetalipoproteinaemia locus (4q22-24). Prenat Diagn 17 18 17(12):1181-6. 19 Iqbal J, Anwar K, Hussain MM. 2003. Multiple, independently regulated pathways of 20 For Peer Review 21 cholesterol transport across the intestinal epithelial cells. J Biol Chem 278(34):31610- 22 23 20. 24 25 Iqbal J, Dai K, Seimon T, Jungreis R, Oyadomari M, Kuriakose G, Ron D, Tabas I, Hussain 26 MM. 2008. IRE1beta inhibits chylomicron production by selectively degrading MTP 27 28 mRNA. Cell Metab 7(5):445-55. 29 30 Kamagate A, Qu S, Perdomo G, Su D, Kim DH, Slusher S, Meseck M, Dong HH. 2008. 31 32 FoxO1 mediates insulin-dependent regulation of hepatic VLDL production in mice. J 33 34 Clin Invest 118(6):2347-64. 35 Levy E, Spahis S, Sinnett D, Peretti N, Maupas-Schwalm F, Delvin E, Lambert M, Lavoie 36 37 MA. 2007. Intestinal cholesterol transport proteins: an update and beyond. Curr Opin 38 39 Lipidol 18(3):310-8. 40 41 Levy E, Stan S, Delvin E, Menard D, Shoulders C, Garofalo C, Slight I, Seidman E, Mayer G, 42 Bendayan M. 2002. Localization of microsomal triglyceride transfer protein in the 43 44 Golgi: possible role in the assembly of chylomicrons. J Biol Chem 277(19):16470-7. 45 46 Mitchell PJ, Urlaub G, Chasin L. 1986. Spontaneous splicing mutations at the dihydrofolate 47 48 reductase locus in Chinese hamster ovary cells. Mol Cell Biol 6(6):1926-35. 49 Murthy S, Born E, Mathur SN, Field FJ. 2002. LXR/RXR activation enhances basolateral 50 51 efflux of cholesterol in CaCo-2 cells. J Lipid Res 43(7):1054-64. 52 53 Najah M, Di Leo E, Awatef J, Magnolo L, Imene J, Pinotti E, Bahri M, Barsaoui S, Brini I, 54 55 Fekih M and others. 2009. Identification of patients with abetalipoproteinemia and 56 57 homozygous familial hypobetalipoproteinemia in Tunisia. Clin Chim Acta 401(1- 58 2):51-6. 59 60 Narcisi TM, Shoulders CC, Chester SA, Read J, Brett DJ, Harrison GB, Grantham TT, Fox MF, Povey S, de Bruin TW and others. 1995. Mutations of the microsomal

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New splicing mutations of MTTP 24 1 2 3 triglyceride-transfer-protein gene in abetalipoproteinemia. Am J Hum Genet 4 5 57(6):1298-310. 6 7 Ohashi K, Ishibashi S, Osuga J, Tozawa R, Harada K, Yahagi N, Shionoiri F, Iizuka Y, 8 9 Tamura Y, Nagai R and others. 2000. Novel mutations in the microsomal triglyceride 10 transfer protein gene causing abetalipoproteinemia. J Lipid Res 41(8):1199-204. 11 12 Pan X, Hussain FN, Iqbal J, Feuerman MH, Hussain MM. 2007. Inhibiting proteasomal 13 14 degradation of microsomal triglyceride transfer protein prevents CCl4-induced 15 16 steatosis. J Biol Chem 282(23):17078-89. 17 18 Peron S, Metin A, Gardes P, Alyanakian MA, Sheridan E, Kratz CP, Fischer A, Durandy A. 19 2008. Human PMS2 deficiency is associated with impaired immunoglobulin class 20 For Peer Review 21 switch recombination. J Exp Med 205(11):2465-72. 22 23 Raabe M, Flynn LM, Zlot CH, Wong JS, Veniant MM, Hamilton RL, Young SG. 1998. 24 25 Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in 26 heterozygotes and embryonic lethality in homozygotes. Proc Natl Acad Sci U S A 27 28 95(15):8686-91. 29 30 Rava P, Hussain MM. 2007. Acquisition of triacylglycerol transfer activity by microsomal 31 32 triglyceride transfer protein during evolution. Biochemistry 46(43):12263-74. 33 34 Rehberg EF, Samson-Bouma ME, Kienzle B, Blinderman L, Jamil H, Wetterau JR, 35 Aggerbeck LP, Gordon DA. 1996. A novel abetalipoproteinemia genotype. 36 37 Identification of a missense mutation in the 97-kDa subunit of the microsomal 38 39 triglyceride transfer protein that prevents complex formation with protein disulfide 40 41 isomerase. J Biol Chem 271(47):29945-52. 42 Ricci B, Sharp D, O'Rourke E, Kienzle B, Blinderman L, Gordon D, Smith-Monroy C, 43 44 Robinson G, Gregg RE, Rader DJ and others. 1995. A 30-amino acid truncation of the 45 46 microsomal triglyceride transfer protein large subunit disrupts its interaction with 47 48 protein disulfide-isomerase and causes abetalipoproteinemia. J Biol Chem 49 270(24):14281-5. 50 51 Sagiv Y, Bai L, Wei DG, Agami R, Savage PB, Teyton L, Bendelac A. 2007. A distal effect 52 53 of microsomal triglyceride transfer protein deficiency on the lysosomal recycling of 54 55 CD1d. J Exp Med 204(4):921-8. 56 57 Sakamoto O, Ohura T, Matsubara Y, Takayanagi M, Tsuchiya S. 2007. Mutation and 58 haplotype analyses of the MUT gene in Japanese patients with methylmalonic 59 60 acidemia. J Hum Genet 52(1):48-55.

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New splicing mutations of MTTP 25 1 2 3 Schgoer W, Eller P, Mueller T, Tancevski I, Wehinger A, Ulmer H, Sandhofer A, Ritsch A, 4 5 Haltmayer M, Patsch JR. 2008. The MTP -493TT genotype is associated with 6 7 peripheral arterial disease: results from the Linz Peripheral Arterial Disease (LIPAD) 8 9 Study. Clin Biochem 41(9):712-6. 10 Sharp D, Blinderman L, Combs KA, Kienzle B, Ricci B, Wager-Smith K, Gil CM, Turck 11 12 CW, Bouma ME, Rader DJ and others. 1993. Cloning and gene defects in microsomal 13 14 triglyceride transfer protein associated with abetalipoproteinaemia. Nature 15 16 365(6441):65-9. 17 18 Sheena V, Hertz R, Nousbeck J, Berman I, Magenheim J, Bar-Tana J. 2005. Transcriptional 19 regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear 20 For Peer Review 21 factor-4alpha. J Lipid Res 46(2):328-41. 22 23 Shoulders CC, Brett DJ, Bayliss JD, Narcisi TM, Jarmuz A, Grantham TT, Leoni PR, 24 25 Bhattacharya S, Pease RJ, Cullen PM and others. 1993. Abetalipoproteinemia is 26 caused by defects of the gene encoding the 97 kDa subunit of a microsomal 27 28 triglyceride transfer protein. Hum Mol Genet 2(12):2109-16. 29 30 Vongsuvanh R, Hooper AJ, Coakley JC, Macdessi JS, O'Loughlin EV, Burnett JR, Gaskin 31 32 KJ. 2007. Novel mutations in abetalipoproteinaemia and homozygous familial 33 34 hypobetalipoproteinaemia. J Inherit Metab Dis 30(6):990. 35 Wang J, Hegele RA. 2000. Microsomal triglyceride transfer protein (MTP) gene mutations in 36 37 Canadian subjects with abetalipoproteinemia. Hum Mutat 15(3):294-5. 38 39 Wetterau JR, Aggerbeck LP, Bouma ME, Eisenberg C, Munck A, Hermier M, Schmitz J, Gay 40 41 G, Rader DJ, Gregg RE. 1992. Absence of microsomal triglyceride transfer protein in 42 individuals with abetalipoproteinemia. Science 258(5084):999-1001. 43 44 Xie Y, Newberry EP, Young SG, Robine S, Hamilton RL, Wong JS, Luo J, Kennedy S, 45 46 Davidson NO. 2006. Compensatory increase in hepatic lipogenesis in mice with 47 48 conditional intestine-specific Mttp deficiency. J Biol Chem 281(7):4075-86. 49 Yang XP, Inazu A, Yagi K, Kajinami K, Koizumi J, Mabuchi H. 1999. Abetalipoproteinemia 50 51 caused by maternal isodisomy of chromosome 4q containing an intron 9 splice 52 53 acceptor mutation in the microsomal triglyceride transfer protein gene. Arterioscler 54 55 Thromb Vasc Biol 19(8):1950-5. 56 57 Zamel R, Khan R, Pollex RL, Hegele RA. 2008. Abetalipoproteinemia: two case reports and 58 literature review. Orphanet J Rare Dis 3:19. 59 60 Zampino R, Ingrosso D, Durante-Mangoni E, Capasso R, Tripodi MF, Restivo L, Zappia V, Ruggiero G, Adinolfi LE. 2008. Microsomal triglyceride transfer protein (MTP) -

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New splicing mutations of MTTP 26 1 2 3 493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 4 5 infected patients. J Viral Hepat 15(10):740-6. 6 7 Zhou M, Fisher EA, Ginsberg HN. 1998. Regulated Co-translational ubiquitination of 8 9 apolipoprotein B100. A new paradigm for proteasomal degradation of a secretory 10 protein. J Biol Chem 273(38):24649-53. 11 12 Zeissig S, Dougan SK, Barral DC, Junker Y, Chen Z, Kaser A, Ho M, Mandel H, McIntyre A, 13 14 Kennedy SM and others. 2010. Primary deficiency of microsomal triglyceride transfer 15 16 protein in human abetalipoproteinemia is associated with loss of CD1 function. J Clin 17 18 Invest 120(8):2889-99. 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 TABLES 4 5 6 7 8 Table 1. Clinical and biological findings in abetalipoproteinemia patients and their 9 10 parents 11 12 13 Children 14 15 Normal Adults 16 17 18 AM PM Range Father Mother Normal Range 19 20 Age (year) For1.3 5.9Peer Review 40 37 21 22 Weight (kg) 8.2 19 NA NA 23 24 25 Height (cm) 75 111 NA NA 26 27 Triglycerides 6 1 < 75 mg/dL 77 87 50-150 mg/dL 28 29 30 Free cholesterol 7,9 6,6 NA ND ND 31 32 Esterified cholesterol 22,1 23,4 NA ND ND 33 34 35 Total cholesterol 30 30 < 170 mg/dL 166 235 105-240 mg/dL 36 37 HDL-cholesterol 29 30 > 40 mg/dL 46 69 40-80 mg/dL 38 39 40 VLDL-cholesterol 10 0 ND 16 17 10-30 mg/dL 41 42 LDL-cholesterol 0 0 < 110 mg/dL 105 148 108-162 mg/dL 43 44 45 ApoA-I 0.43 0.48 NA 1.25 1.71 1.1-2.1 g/L 46 47 Apo-B 0.02 0.01 < 90 mg/dL 0.68 0.92 0.5-1.35 g/L 48 49 Retinol/RBP 0.68 1.06 0.95-1.06 ND ND 50 51 52 Tocopherol 0.41 0.16 6-15 mg/L ND ND 53 54 ALT 60 60 < 36 IU/L ND ND 55 56 57 AST 76 76 < 50 IU/L ND ND 58 59 60 NA: not available; ND: not done.

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1 2 3 4 5 6 Table 2. Immunoglobulin levels 7 8 AM, born the 21/01/2004 PM, born the 25/07/1999 9 10 11 Normal 12 13 29/04/2004 11/10/2005 27/07/2010 30/09/1999 29/11/2005 27/07/2010 range 14 15 IgG 0.95 7.86 6.48 0.33 8.63 5.47 5.6 - 10.4 g/L 16 17 18 IgA 0.04 0.11 1.11 0.02 1.73 1.67 0.4 - 1.4 g/L 19 20 IgM 0.11 0.67For Peer 0.73 Review 0.04 0.44 0.36 0.6 - 1.6 g/L 21 22 23 24 Immunoglobulin levels of both patients were reported at the diagnosis of 25 26 hypogammaglobulinemia, just after the diagnosis of abetalipoproteinemia and at the last 27 28 29 follow-up. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 67 of 78

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 68 of 78 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 69 of 78

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 70 of 78 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 71 of 78

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 72 of 78 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 254x190mm (96 x 96 DPI) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 73 of 78

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 254x190mm (96 x 96 DPI) 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 74 of 78 Human Mutation

1 2 3 Supplementary Table S1. Description of MTTP mutations reported in the literature 4 5 6 number 7 of 8 exon/intron mutations exon/intron mutations consequency patients references 9 intron 9, c.12371G>A, intron 9, c.12371G>A, abnormal splicing site, 10 maternal unidisomy maternal unidisomy exon 10 deletion 1 Yang, et al., 1999 11 exon 13, c.1820delG exon 13, c.1820delG abnormal reading frame 1 Wang, et al., 1999 12 exon 16, c.2237G>A exon 18, c.2524A>T p.G746E / p.K842X 1 Wang, et al., 1999 13 exon 12, c.1619G>A exon 12, c.1619G>A p.R540H 1 Wang, et al., 1999 14 15 Benayoun, et al., 2007 16 Ricci, et al., 1995 17 exon 18, c.2593G>T exon 18, c.2593G>T p.G865X 7 Wang, et al., 1999 18 p.K448X / abnormal 19 exon 10, c.1342A>T intron 12, 1769+1G>A splicing site 1 Wang, et al., 1999 20 For Peer Review Al Shali, et al., 2003 21 exon 12, c.1769G>T exon 12, c.1769G>T p.S590I 2 Wang, et al., 1999 22 exon 11, c.1389delA exon 11, c.1389delA abnormal reading frame 1 Ohashi, et al., 2000 23 Al Shali, et al., 2003 24 Berthier, et al., 2004 25 26 Narcisi, et al., 1995 27 exon 4, c.419insA exon 4, c.419insA abnormal reading frame 3 Ohashi, et al., 2000 28 p.R595X, abnormal 29 exon 13, c.1783C>T intron 15, c.22182A>G splicing site 1 Ohashi, et al., 2000 30 exon 16, c.2338A>T exon 16, c.2338A>T p.D780Y 1 Ohashi, et al., 2000 31 exon 9, exon 9, 32 c.1228delCCCinsT c.1228delCCCinsT abnormal reading frame 1 Di Leo, et al., 2005 33 abnormal splicing site, 34 intron 9, c.12371G>A NA exon 10 deletion 1 Di Leo, et al., 2005 35 36 exon 9, c.1151 A>C exon14, c.1982 G>C p.D384A / p.G661A 1 Di Leo, et al., 2005 37 Benayoun, et al., 2007 38 exon 15, c.2212delT exon 15, c.2212delT abnormal reading frame 3 Narcisi, et al., 1995 39 intron 1, c.1482A>G intron 1, c.1482A>G abnormal splicing site 2 Benayoun, et al., 2007 40 exon 3, c.307A>T exon 3, c.307A>T p.K103X 1 Benayoun, et al., 2007 41 loss of MTP and 8 other 42 large deletion of 481 kb large deletion of 481 kb genes 1 Benayoun, et al., 2007 43 exon 15, c.2076 exon 15, c.2076 Vongsuvanh, et 44 39_2303+52del319 39_2303+52del319 exon 15 deletion 1 al., 2007 45 46 exon 2, c.215delC exon 2, c.215delC abnormal reading frame 1 Sharp, et al., 1993 47 Chardon, et al., 2009 48 exon 13, c.1783C>T exon 13, c.1783C>T p.R595X 2 Sharp, et al., 1993 49 exon 17, exon 17, 50 c.2346insACTG c.2346insACTG abnormal reading frame 1 Heath, et al., 1997 51 exon1, c.59del17 exon1, c.59del17 abnormal reading frame 1 Chardon, et al., 2009 52 exon 5, c.582C>A exon 5, c.582C>A p.Cys194X 2 Chardon, et al., 2009 53 abnormal splicing site, 54 55 intron 5, c.6193T>G intron 5, c.6193T>G exon 6 deletion 1 Najah, et al., 2009 56 exon 8, c.923G>A exon 8, c.923G>A p.W308X 1 Najah, et al., 2009 57 exon 10, c.1237 exon 10 deletion / 58 1344del107 exon 12, c.1619G>A p.R540H 1 Rehberg JBC 1996 59 exon 9, c.1147delA exon 9, c.1147delA abnormal reading frame 1 Narcisi, et al., 1995 60 intron 10, c.1344+5del7 intron 10, c.1344+5del7 abnormal splicing site 1 Narcisi, et al., 1995 exon 4, c.419insA exon 11, 1401insA abnormal reading frame 1 Narcisi, et al., 1995

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1 2 3 abnormal splicing site / 4 intron 13, c.1867+1G>A intron 14, c.19901G>A abnormal splicing site 1 Narcisi, et al., 1995 5 abnormal reading frame 6 7 exon 4, c.419insA intron 13, c.1867+5G>A / abnormal splicing site 1 Narcisi, et al., 1995 8 Narcisi, et al., 1995 9 intron 13, c.1867+5G>A intron 13, c.1867+5G>A abnormal splicing site 2 Shoulders, et al., 1993 10 abnormal splicing site / 11 intron 1, 147+2T>C exon 4, c.419insA abnormal reading frame 1 Dische, et al., 1970 12 abnormal splicing site / 13 intron 1, 147+1G>C exon 12, c.1692T>C p.I564T 1 Sakamoto, et al., 2006 14 abnormal splicing site / 15 exon 6, c.619G>T intron 9, c.123728A>G abnormal splicing site 2 this report 16 17 18 19 20 For Peer Review 21 Supplementary Table S2. PCR primers 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Supplementary Table S3. Genomic mutations of MTTP 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Supplementary Figure S1: Localization of WT and mutant MTTP 4 5 6 HeLa cells (A) or HepG2 cells (B) were transfected with GFPtagged WTMTTP, ∆6MTTP 7 8 or ∆10MTTP and were then processed for immunofluorescence. Bar: 10m. 9 10 11 12 13 Supplementary Figure S2: Localization of WT and mutant MTTP and Golgi marker 14 15 16 17 18 HeLa cells were transfected with GFPtagged WTMTTP, ∆6MTTP or ∆10MTTP and were 19 20 then processed for immunofluorescenceFor Peer using antiGolginReview 97 antibody. Bar: 10m 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 78 of 78 Human Mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 190x254mm (96 x 96 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc.