Mathilde Di Filippo
email: mathilde.di-filippo@chu-lyon.fr

Sophie Collardeau-Frachon
email: sophie.collardeau-frachon@chu-lyon.fr

Alexandre Janin
email: alexandre.janin01@chu-lyon.fr

Sujith Rajan
email: rajan.sujith@yahoo.co.uk

Oriane Marmontel
email: oriane.marmontel@chu-lyon.fr

Charlotte Decourt
email: charlotte.dcrt@outlook.com

Amandine Rubio
email: arubio@chu-grenoble.fr

Séverine Nony
email: severine.nony@chu-lyon.fr

Sabrina Dumont
email: sabrina.dumont@chu-lyon.fr

Charlotte Cuerq
email: charlotte.cuerq@chu-lyon.fr

Sophie Collardeau Frachon

Sybil Charrière
email: sybil.charriere@chu-lyon.fr

Philippe Moulin
email: philippe.moulin@chu-lyon.fr

Alain Lachaux
email: alain.lachaux@chu-lyon.fr

M Mahmood Hussain

Dominique Bozon
email: bozon.dominique@orange.fr

Noël Peretti
email: noel.peretti@chu-lyon.fr

Normal serum ApoB48 and red cells vitamin E concentrations after supplementation in a novel compound heterozygous case of abetalipoproteinemia

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background and aims: Abetalipoproteinemia (ABL) is a rare recessive monogenic disease due to MTTP (micro-somal triglyceride transfer protein) mutations leading to the absence of plasma apoB-containing lipoproteins. Here we characterize a new ABL case with usual clinical phenotype, hypocholesterolemia, hypotriglyceridemia but normal serum apolipoprotein B48 (apoB48) and red blood cell vitamin E concentrations. Methods: Histology and MTP activity measurements were performed on intestinal biopsies. Mutations in MTTP were identified by Sanger sequencing, quantitative digital droplet and long-range PCR. Functional consequences of the variants were studied in vitro using a minigene splicing assay, measurement of MTP activity and apoB48

Introduction

Abetalipoproteinemia (ABL; OMIM 200100) is a rare autosomal recessive disorder characterized by extremely low levels of plasma total cholesterol (TC) and triglycerides (TG) due to an almost complete absence of apolipoprotein B (apoB)-containing lipoprotein (apoB-LP) secretion [START_REF] Burnett | Abetalipoproteinemia[END_REF]. This defect affects both apoB48-containing chylomicrons (CM) and apoB100-containing very-low density lipoproteins (VLDL) leading to an absence of low density lipoproteins (LDL) [START_REF] Shoulders | Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein[END_REF][START_REF] Sharp | Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia[END_REF]. ABL is caused by a deficiency of the microsomal triglyceride transfer protein (MTP) large subunit encoded by MTTP, the microsomal triglyceride transfer protein gene [START_REF] Shoulders | Abetalipoproteinemia is caused by defects of the gene encoding the 97 kDa subunit of a microsomal triglyceride transfer protein[END_REF][START_REF] Sharp | Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia[END_REF][START_REF] Wetterau | Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia[END_REF]. The underlying mechanism involves the absence of MTP-protein disulfide isomerase (PDI) heterodimer that acts as a chaperone for the production of apoB-LP. This complex is required for the transfer of neutral lipids to nascent primordial apoB-LP and may facilitate their fusion with luminal lipid droplets [START_REF] Hussain | Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly[END_REF][START_REF] Sirwi | Lipid transfer proteins in the assembly of apoB-containing lipoproteins[END_REF]. In case of MTP deficiency, apoB cannot be lipidated properly and is degraded by the proteasome [START_REF] Davis | Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools[END_REF][START_REF] Zhou | Regulated Co-translational ubiquitination of apolipoprotein B100. A new paradigm for proteasomal degradation of a secretory protein[END_REF].

ABL subjects are unable to secrete CM and VLDL from the intestine and liver, respectively. The absence of CM leads to lipid malabsorption, symptoms related to lipid-soluble vitamin and essential fatty acid deficiencies [START_REF] Delpre | Endoscopic assessment in abetalipoproteinemia (Bassen-Kornzweig-syndrome)[END_REF][START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF][START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF]. The absence of VLDL secretion induces liver steatosis leading to fibrosis and cirrhosis in many cases [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF]. To date, at least 59 deleterious MTTP variants have been reported in ABL patients [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Miller | Novel missense MTTP gene mutations causing abetalipoproteinemia[END_REF][START_REF] Walsh | Novel abetalipoproteinemia missense mutation highlights the importance of the N-terminal beta-barrel in microsomal triglyceride transfer protein function[END_REF][START_REF] Gunduz | Microsomal triglyceride transfer protein gene mutations in Turkish children: a novel mutation and clinical follow up[END_REF][START_REF] Paquette | A tale of 2 cousins: an atypical and a typical case of abetalipoproteinemia[END_REF][START_REF] Liu | Lack of MTTP activity in pluripotent stem cellderived hepatocytes and cardiomyocytes abolishes apoB secretion and increases cell stress[END_REF][START_REF] Di Filippo | Post-prandial lipid absorption in seven heterozygous carriers of deleterious variants of MTTP in two abetalipoproteinemic families[END_REF]. The molecular diagnosis is considered as the "gold-standard" diagnostic test, allowing the diagnosis of atypical ABL patients [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Paquette | A tale of 2 cousins: an atypical and a typical case of abetalipoproteinemia[END_REF][START_REF] Di Filippo | Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia[END_REF] and establishing the differential diagnosis versus co-dominant familial compound heterozygous or homozygous hypobetalipoproteinemia (Ho-FHBL; OMIM 107730) due to deleterious variants on the APOB gene, encoding apoB 11, 19 . Parents of ABL cases usually exhibit normal lipid profile whereas parents of Ho-FHBL cases exhibit mild hypobetalipoproteinemia [START_REF] Lee | Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management[END_REF][START_REF] Zamel | Abetalipoproteinemia: two case reports and literature review[END_REF].

We report herein a new case of ABL occurring in childhood with typical clinical and lipid profile. However, the diagnosis was delayed by her normal plasma apoB48 concentration, mild hypolipoproteinemia in both parents, and the requirement of additional methods to Sanger sequencing allowing the subsequent identification of heterozygous copy number variations (CNV).

Materials and methods

Patient

The patient is the first child of a non-consanguineous French family. She was delivered full-term after a normal pregnancy. Her birth measurements were normal (weight: 3.5 kg, height: 50 cm).

Lipid profile, fat load, apoB electrophoresis, and vitamin E determination

Lipid profile, CM, apoB48 and vitamin E concentration and apoB48 characterization can be found in Supplementary Material.

The proband received an oral fat load as described in Supplementary Material to assess the plasma TG concentration response.

MTP activity on intestinal biopsy

Intestinal biopsies of the proband and 11 control subjects without ABL were processed to obtain protein extracts and determine MTP activity as previously described [START_REF] Di Filippo | Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia[END_REF][START_REF] Athar | A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein[END_REF][START_REF] Rava | Transfer of cholesteryl esters and phospholipids as well as net deposition by microsomal triglyceride transfer protein[END_REF] and detailed in Supplementary Material. The results are expressed as UI per gram of total protein per hour.

Variant and in silico analysis

Following the extraction of genomic DNA (gDNA) from the blood, each of the coding exons, as well as the flanking intronic junctions of the MTTP, APOB, proprotein convertase subtilisin/kexin type 9 (PCSK9), and angiopoietin-like 3 (ANGPTL3) genes, was amplified by PCR and amplicons were sequenced as previously described [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF]. Analysis of the mutations was performed using Alamut v2.11 as described in Supplementary Material.

Protein activity and subcellular localization of the missense variant

Three assays were performed as previously described [START_REF] Walsh | Novel abetalipoproteinemia missense mutation highlights the importance of the N-terminal beta-barrel in microsomal triglyceride transfer protein function[END_REF][START_REF] Di Filippo | Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia[END_REF][START_REF] Walsh | Structure-function analyses of microsomal triglyceride transfer protein missense mutations in abetalipoproteinemia and hypobetalipoproteinemia subjects[END_REF] to evaluate the c.1868G > T p.(Arg623Leu) expression in COS cells: measurement of MTP activity, apoB48 secretion after cotransfection with the MTTP and P4HB (encoding PDI) cDNA, and subcellular localization after transfection with wild type (WT) or mutated MTTP cDNA C-terminally tagged with GFP (Supplementary Material).

Minigene splicing reporter assay and analysis of spliced products in HeLa cells after minigene transfection

As NM_000253.3(MTTP):c.1868G > T variant affects the first base of exon 15 and may affect splicing, minigene splicing reporter assay was performed as previously described [START_REF] Di Filippo | Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia[END_REF] and as detailed in Supplementary Material.

RNA extraction from intestinal biopsy sample, PCR amplification and sequencing

Total RNA was isolated, reverse transcribed into first strand cDNA; cDNA was then amplified and the PCR product was cloned into a TOPO vector and sequenced (Supplementary Material).

Quantitative PCR and large rearrangement characterization

Digital droplet quantitative PCR was performed from gDNA of the proband and her parents to detect CNV as detailed in Supplementary Material. Sequencing of the genomic deletion breakpoints was performed after long range PCR as previously described [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF].

Results

Patient (diagnosis and follow-up)

Diarrhea appeared after the first week of life. She was referred to the hospital at 1 month of age for an acute pyelonephritis and at 4 months of age for growth retardation (weight: 3.8 kg, < -2SD, height: 58 cm, -2SD, cranial perimeter: 37 cm, -2SD), with abdominal distension, diarrhea, and vomiting. Hepatomegaly was observed, blood transaminases were moderately elevated (Table 1) and hepatic steatosis was suspected because of the hyper-refringent liver observed at ultrasound examination. Her prothrombin ratio was decreased (18%) due to vitamin K deficiency (normal factor V). A low-fat infant formula with a high medium-chain TG content (Monogen ® , Nutricia, Dublin, Ireland) was started. Liposoluble vitamin replacement was initiated with vitamin E (Vedrop ® , Orphan Europe Laboratories, Puteaux -France, 600 U/j), vitamin K (10 mg/week), vitamin A-313 (A313 ® , Laboratoire Pharma Développement, Corbigny, France, 50 000 U/week), vitamin D (Sterogyl ® , Desma Pharma, Chiasso, Switzerland, 1600 U/day) in addition to omega 3 supplementation (Omacor ® , Laboratoire Pierre Fabre Médicament, Ussel, France, 1 capsule containing EPA 460 mg and DHA 380 mg, capsules were open and drank twice a week). The treatment stopped the diarrhea and improved growth.

At 14 months of age, her body weight was 9.1 kg (-1SD) and her height was 72 cm (-1SD). She had no hepatomegaly, no diarrhea and no steatorrhea over a 3-day period strict low-fat diet (2-3 g/24 h of lipids). The liver elastometry was normal (4.4 kPa at 1 year of age) but the levels of blood transaminases remained moderately increased (Table 1). At 18 months of age, upper-endoscopy and duodenal biopsies showed vacuolated enterocytes (Fig. 1).

The patient is now 7 years of age and is in good health, without diarrhea under a strict low-fat diet. She presents no neurological, ophthalmological or cardiac abnormalities; a slight hepatic steatosis persisted upon ultrasound examination with normal liver elastometry (3.3 kPa).

Lipid profile, fat load, and apoB electrophoresis

At diagnosis, the concentrations of TC, TG, apoB, and vitamin E were found to be dramatically low in the patient (Table 1) as were the concentrations of vitamin A 0.25 μmol/L (normal range, NR: 0.5-2.40), vitamin K 44 ng/L (NR: 100-1000) and beta-carotene < 0.10 μmol/L (NR: 0.30-0.89). After supplementation, serum vitamin E concentrations increased (Table 1) but remained under the NR whereas the red cells concentrations were found to be normal (5.4 μmol/L; NR: 2.0-7.8 μmol/L) [START_REF] Cuerq | Establishment of reference values of alpha-tocopherol in plasma, red blood cells and adipose tissue in healthy children to improve the management of chylomicron retention disease, a rare genetic hypocholesterolemia[END_REF] as were the plasma and serum concentrations of vitamin A (0.77 μmol/L) and K (143 ng/L and 424 ng/L at 3 years of age). When the patient was diagnosed, the parents had serum LDL-C below normal concentration, which was more compatible with Ho-FHBL (dominant) than ABL (recessive); the serum LDL-C concentration of the parents was later found within normal range (Table 1).

When she was 1.5 years old, after an oral fat load (Table 1), the level of serum TG slightly increased from 0.05 to 0.10 mmol/L. However, CM remained undetectable. Plasma ApoB electrophoresis revealed the presence of both apoB100 and apoB48 in the fasting state, and an increase of apoB48 and apoB100 after oral fat load (Fig. 2A andB), suggesting some intestinal lipid absorption and low amounts of intestinal and hepatic lipoprotein secretion.

ApoB48 concentration in fasting plasma at 3 different ages (2, 3.5, and 4 years) was found to range from 8.40 to 11.06 mg/L in the normal IR (mean ± 2SD for controls: 5.81 ± 6.18 mg/L, n = 11). These values were higher than the limit of quantification (0.25 mg/L), and higher than apoB48 levels found in other ABL, in chylomicron retention disease (CMRD), and in Ho-FHBL patients (Fig. 2C).

MTP activity on intestinal biopsy

TG transfer activity in the intestinal biopsy at 18 months from the patient was not detectable (< 5% of the mean of controls subjects: mean ± SD: 569 ± 101 UI/mg/h, range: 393 to 761 UI/mg/h, n = 11) whereas alkaline phosphatase activity (124 UI/g of protein) was normal (mean ± SD for controls: 114 ± 41 UI/g, range: 56 to 173 UI/g of protein, n = 7). These findings, showing a major MTP deficiency, were in agreement with ABL and not Ho-FHBL.

Identification of MTTP variants

Two heterozygous, linked, nucleotide substitutions of the MTTP gene, both inherited from her father, were identified in the patient using Sanger sequencing. The first, NM_000253.3(MTTP): c.1868G > T leads to p.(Arg623Leu) missense variant and affects the first base on exon 15. This variant is not described in the gnomAD database or in ClinVar. The second, NM_000253.3(MTTP): c.1981G > A, is a well described p.(Gly661Ser) missense variant. The frequency of this allele (rs113337987) is 2.6% in the gnomAD database. The patient had no deleterious variants in the other genes involved in familial hypocholesterolemia (APOB, PCSK9, and ANGTL3). Her sister was not carrier of these single nucleotide variants on MTTP.

Analysis of functional impacts of c.1868G > T, p.(Arg623Leu)

Impact on MTP activity and ApoB48 secretion

The p.(Arg623Leu) variant affects a likely conserved residue. The substitution changes a basic hydrophilic to a hydrophobic residue. This missense variant is predicted by Polyphen to be "probably damaging" and by SIFT to be "tolerated".

COS showed least apoB48 concentration in media, whereas COS-7 cells expressing MTTP c.1868G > T (p.(Arg623Leu)) (the patient's variant) showed 35-40% reduced apoB concentration in media compared to wild type (Fig. 3B). There was no significant difference in the level of protein expression of different MTTP mutants (Supplementary Fig. 2). These studies suggest that MTTP c.582C > A (p.(Cys194*)) variant does not transfer triglyceride and does not support apoB48 secretion. In contrast, MTTP c.1868G > T (p.(Arg623Leu)) (the patient's variant) is able to support triglyceride transfer and apoB48 secretion, albeit to a lower extent compared to WT MTTP.

Impact of the c.1868G > T on splicing 3.5.2.1. Splicing predictions.

As this nucleotide change affects the first base of exon 15, all the tested algorithms predicted an effect on splicing with a decrease in the score for the acceptor site of intron 15 ranging from -7.4% for SSF-like to a complete abolition for GeneSplicer, and NNSPLICE.

Minigene splicing reporter assay.

To study the effect of this variant on splicing, RT-PCR products were analyzed using two pairs of primers (Fig. 3C).

A 466 bp expected RT-PCR fragment was produced after transfection of the WT minigene in HeLa cells (Fig. 3D, PCR product 1 and Supplementary Fig. 3, lanes 1, 2, and 3). In contrast, transfection of the mutant c.1868G > T minigene mainly produced 2 different RT-PCR fragments: a 560 bp product retaining complete intron 14, and a shorter fragment (438bp) including an abnormal exon 15 lacking the 28 first bases (Fig. 3D, PCR products 2 and 3 respectively, Supplementary Fig. 3, lanes 4, 5, 6, and Supplementary Fig. 4). Both transcripts would result in an aberrant protein with a premature stop codon. Minigene experiments indicated that the c.1868G > T leads mainly either to an activation of an exonic cryptic acceptor splice site at c.1896 in exon 15 or to a complete retention of intron 14. A very faint 466 bp fragment, almost undetectable by gel electrophoresis, was also present in products from the mutant minigene.

To better characterize normally spliced products from the mutant minigene, cDNA was amplified with primer pair 2 (Supplementary Fig. 5). A PCR product of the expected size (240 bp) was obtained from RT PCR of both minigenes, WT and mutant, and Sanger sequencing confirmed the presence of the correct junction of exons 14 and 15 for both minigenes (with the presence of G and T at c.1868; Fig. 3D, PCR products 4 and 5, Supplementary Fig. 5). This result confirmed that a very small amount of a normally spliced transcript was also produced from the mutant minigene (Supplementary Fig. 6).

Amplification and sequence analysis of cDNA from intestinal biopsies.

To study the in vivo effect of the c.1868G > T variant, RT-PCR of RNA prepared from the patient's biopsies was carried out spanning nucleotides 1285 to 2051 of MTTP cDNA. One PCR product exhibiting the expected size was found (Fig. 3E); this confirmed that the two misspliced mRNA transcripts from the variant containing the premature termination codons were targeted for nonsense-mediated Localization of the two pairs of primers, P1, complementary to the vector, and P2, a forward primer complementary to the exon 14-exon 15 junction (MTP_E14-15chimNL MTP_E14-15chimML) and a reverse primer complementary to the vector from the RT product. (D) Ex vivo analysis performed in HeLa cells transfected either with the WT (c.1868G) or mutant (c.1868T) minigene using two pairs of primers. Gel electrophoresis of RT-PCR products obtained with either P1: primer pair 1; P2: primer pair 2; PCR products 1 to 3 were obtained using P1 primer pair: 1: wild type (466bp); 2: 560 bp product retaining complete intron 14; 3: shorter fragment (438bp) including an abnormal exon 15 lacking the 28 first bases; PCR products 4 and 5 were obtained using P2 primer pair. 4: wild type (240bp) with G at position c.1868; 5: 240bp fragment with T at position c.1868. The sequence of these 5 PCR products were shown on Supplementary Figs. 3 and4. (E) and (F) Sequence analysis of MTTP transcription product from intestinal biopsy. After total RNA extraction and reverse transcription from a control biopsy and patient intestinal biopsy, PCR was performed to sequence cDNA from nucleotides 1285 to 2051. PCR products from two different RT for the patient (lanes 1 and 2), and one for the control biopsy (Ctrl) were analyzed by migration on agarose gel (E) and sequenced (F). Neg: negative control; MW: molecular weight marker. decay and degraded. As expected, the sequence analysis of this transcript showed the presence of a normal sequence with a G/T mosaicism at positions c.1868 (Fig. 3F) and a G/A at c.1981 (from the paternal p.Gly661Ser variant).

Detection and characterization of a copy number variation (CNV)

As the patient exhibited a phenotype in agreement with ABL and only one heterozygous variant was found by Sanger sequencing, CNV were studied using digital droplet PCR. No quantitative difference was observed for exons 3, 5, 6, 11, 13 17, 18, and 19 compared to the reference, while exon 10 of the proband and her mother exhibited approximately 50% of amplification products compared to the reference suggesting a heterozygous deletion (Fig. 4A).

After long-range PCR (Fig. 4B) and sequencing, the deletion breakpoints were identified (Fig. 4C andD): the patient, her mother and her sister presented a 2198bp deletion, which begins in the intron 9 and ends in exon 10, the NM_000253.3(MTTP):c.1067+1217_1141del variant, which was previously described as c.1068-2127_1138del. Deletion of the first 71 bases of exon 10 would induce a premature stop codon (p.(Pro357Phefs*4).

Discussion

We report the result of in-depth MTTP gene analysis in a proband with very low serum TG and TC but normal apoB48 concentration. The phenotype let to suggest either an ABL or Ho-FHBL diagnosis.

The phenotypic differential diagnosis between ABL and Ho-FHBL is often difficult as they can share similar clinical and biological manifestations [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF]. The consideration of the lipid profile of the proband's parents is valuable: MTTP obligate heterozygotes usually have normal lipid profile while APOB heterozygotes are expected to have LDL-C and apoB levels below the 5th percentile according to age and sex [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Lee | Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management[END_REF][START_REF] Hooper | Contemporary aspects of the biology and therapeutic regulation of the microsomal triglyceride transfer protein[END_REF]. Accordingly, mild hypocholesterolemia in both proband's parents was suggestive of Ho-FHBL in the proband. However, the parents follow-up showed a fluctuant hypobetaliproteinemic lipid profile, and in the literature, 14% of the heterozygous carriers of deleterious MTTP variants were previously found to have low plasma LDL-C and apoB [START_REF] Di Filippo | Post-prandial lipid absorption in seven heterozygous carriers of deleterious variants of MTTP in two abetalipoproteinemic families[END_REF]. In these cases, genetic analysis is mandatory to distinguish ABL from Ho-FHBL, and to consistently establish the diagnosis.

In the patient presented herein, intestinal biopsies were available, and the absence of detectable MTP activity suggested the diagnosis to ABL. However, Sanger sequencing detected only a new heterozygous variant of unknown significance, p.(Arg623Leu), on the MTTP paternal allele. The p.(Arg623Leu) is predicted to be surface exposed (Supplemental Fig. 7) in C-terminal ß-sheets domain of MTP mediating the lipid-binding and transfer catalytic activity of MTP [START_REF] Zamel | Abetalipoproteinemia: two case reports and literature review[END_REF]. Other missense variants in this domain and have been implicated in defective lipid transfer activity [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Hegele | Arrest of neuropathy and myopathy in abetalipoproteinemia with high-dose vitamin E therapy[END_REF][START_REF] Wang | The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins[END_REF][START_REF] Khatun | Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia[END_REF]. Since the impact of this missense variant was unclear and furthermore fasting concentration of apoB48 was normal and increasing after fat load, cellular functional tests were undertaken to decipher the functionality of this new variant. Its residual activity, correct subcellular localization and the remaining apoB48 secretion in the medium found in vitro strongly suggest that, unlike arginine at position 540 supposed to form a salt bridge with PDI [START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF], this residue at position 623 is less critical for MTP function. Additionally, in vitro, the c.1868T variant produced two major abnormally spliced transcripts and a very low level of a normally spliced transcript carrying the missense variant p.623Leu. Interestingly, ex vivo, in intestinal biopsy, only the normally spliced transcript carrying the missense variant was found, but at a very low level. This variant was consequently classified as pathogenic according to the American College of Medical Genetics and Association of Medical Pathologists (ACMG) guidelines [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of medical genetics and genomics and the association for molecular pathology[END_REF].

Subsequent quantitative PCR allowed the identification of the maternal deleterious variant. This case underlines the necessity to routinely detect CNV in MTTP. Until now, CNV were reported in only 5 ABLpatients, for which the molecular mechanism (uniparental disomy, contiguous large gene deletion, or a homozygous deletion of an exon [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Yang | Abetalipoproteinemia caused by maternal isodisomy of chromosome 4q containing an intron 9 splice acceptor mutation in the microsomal triglyceride transfer protein gene[END_REF][START_REF] Benayoun | Abetalipoproteinemia in Israel: evidence for a founder mutation in the Ashkenazi Jewish population and a contiguous gene deletion in an Arab patient[END_REF][START_REF] Kotzot | Complex and segmental uniparental disomy updated[END_REF][START_REF] Aminoff | Novel mutations in microsomal triglyceride transfer protein including maternal uniparental disomy in two patients with abetalipoproteinemia[END_REF]) allowed detection by Sanger sequencing, whereas heterozygous CNV are undetectable by Sanger sequencing. Consequently, it is of utmost importance to develop methods that allow the detection of CNV such as quantitative PCR or next-generation sequencing (NGS) for routine diagnosis [START_REF] Ceulemans | Targeted screening and validation of copy number variations[END_REF][START_REF] Slatko | Overview of next-generation sequencing technologies[END_REF][START_REF] Quan | dPCR: a technology review[END_REF], and therefore to allow the simultaneous detection of both single nucleotide variations and CNV, as recently demonstrated in other dyslipidemias [START_REF] Iacocca | Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia[END_REF][START_REF] Marmontel | Single, short in-del, and copy number variations detection in monogenic dyslipidemia using an NGS strategy[END_REF].

The same partial deletion of exon 10 has previously been described in another apparently unrelated homozygous patient [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF]. The translation product of this variant is predicted to create a premature termination codon eliminating the lipid-binding cavity and the central region involved in the interaction with PDI [START_REF] Hussain | Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly[END_REF][START_REF] Mann | The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins[END_REF][START_REF] Read | A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein[END_REF]. The exon 10 deletion, caused by other molecular defects, leads to the production of a smaller MTTP protein, detectable but unable to interact with PDI, devoid of activity and shown to prevent the apoB secretion [START_REF] Wetterau | Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF][START_REF] Pons | A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP)[END_REF].

To the best of our knowledge this is the first report of normal plasma apoB48 levels associated with an increase of apoB after fat load in a clinically typical ABL case. ApoB is usually not detected in serum even after fat load in clinically typical ABL patients [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Illingworth | Abetalipoproteinemia. Report of two cases and review of therapy[END_REF][START_REF] Magnolo | Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia[END_REF]]. In the patient described herein, the normal apoB48 secretion could be explained by the residual activity of the heterozygous new missense MTTP variant. Although this residual activity was not measurable in the tested biopsy, the limited amount of tissue might have hampered the sensitivity of this ex vivo test.

The case presented is of particular interest because it has been previously established that sufficient apoB lipidation was conditional to avoid rapid apoB presecretory degradation [START_REF] Boren | The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly[END_REF]. In this patient, normal apoB48 concentration but very low apoB100 amount, undetectable using classical immunoturbidimetric measurement, was found as well as a raise in apoB plasma concentration after fat load whereas serum TG only increased to a maximum of 0.10 mmol/L and CM were not detectable. This observation raises the hypothesis of differential pathways in intestinal and hepatic cells regarding apoB proteasomal degradation and secretion of apoB. In accordance with the patient reported herein, in liver tissue-specific knockout Mttp Δ/Δ mice and in cultured murine primary hepatocytes treated with a MTP inhibitor, a partial inactivation of MTTP lowered apoB100 levels in plasma by 90 to > 95% whereas the secretion of apoB48 was only slightly decreased, indicating that secretion of apoB100 is much more sensitive to MTTP activity than apoB48 [START_REF] Raabe | Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice[END_REF][START_REF] Kulinski | Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB, as well as for ApoB lipidation[END_REF]. Despite the presence of detectable apoB48, this proband exhibited a complex profile of fat-soluble vitamin deficiency.

Vitamin E is thought to be incorporated into CM and transported by lipoproteins in serum. Despite the normal concentration of apoB48 in the patient presented herein, supporting secretion of apoB48-LP from the intestine, her vitamin E plasma concentrations were as low as those reported in other ABL patients in agreement with the very low level of its serum transporter, the LDL [START_REF] Di Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Walsh | Novel abetalipoproteinemia missense mutation highlights the importance of the N-terminal beta-barrel in microsomal triglyceride transfer protein function[END_REF][START_REF] Zamel | Abetalipoproteinemia: two case reports and literature review[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF][START_REF] Pons | A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP)[END_REF][START_REF] Narcisi | Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia[END_REF][START_REF] Ohashi | Novel mutations in the microsomal triglyceride transfer protein gene causing abetalipoproteinemia[END_REF][START_REF] Leo | Mutations in MTP gene in abetaand hypobeta-lipoproteinemia[END_REF][START_REF] Clarke | Assessment of tocopherol metabolism and oxidative stress in familial hypobetalipoproteinemia[END_REF][START_REF] Chardon | Identification of two novel mutations and long-term follow-up in abetalipoproteinemia: a report of four cases[END_REF][START_REF] Uslu | Abetalipoproteinemia in an infant with severe clinical phenotype and a novel mutation[END_REF][START_REF] Hammer | Clinical features and molecular genetics of two Tunisian families with abetalipoproteinemia[END_REF]. However, the normal concentration of vitamin E in red blood cells despite the absence of CM detected after fat load, supports additional mechanisms allowing the saving of vitamin E absorption in the context of vitamin E supplementation under a strict low-fat diet and poorly-lipidated apoB48 secretion. This might involve cooperation between regulation of vitamin E transport, HDL production and residual apoB48 secretion by enterocytes [START_REF] Reboul | Vitamin E Intestinal Absorption: Regulation of Membrane Transport across the Enterocyte[END_REF]. Additional studies will be needed to carefully characterize size and composition of the lipoproteins from the proband, when she will be adult due to the amount of plasma needed in abetalipoproteinemic patient.

In conclusion, we have shown that the main functional impairment of the c.1868G > T MTTP variant is a splicing defect that produces a small amount of normally spliced transcript enabling some residual MTP activity and apoB48 secretion. ABL patients with incomplete splicing defects may present with different clinical phenotype according to the molecular defect of the other allele. This case provides further insights into the genetic basis of the phenotypic diversity of ABL, regarding functional domains of MTTP, residual MTP activity, and their impact on apoB48 secretion and vitamin E absorption. It shows that methods allowing CNV detection are required to diagnose complex primary hypolipidemia, for which NGS may be of great interest.

Fig. 1 .

 1 Fig. 1. Histological features in intestinal biopsy. Duodenal biopsy obtained at 18 months and processed for Hematoxylin-Eosin-Saffron-staining. (A) ×100: intestinal villi mainly composed of vacuolated enterocytes and a few normal enterocytes as well as goblet cells. (B) x 200: focus on lipid droplets of variable sizes within the enterocytes. Black arrow: normal enterocytes; white arrows: vacuolated enterocytes.

Fig. 2 .

 2 Fig. 2. ApoB electrophoresis and ApoB48 concentration. (A and B) ApoB SDS-PAGE revealed by silver staining (A) or immunoblotting (B) before (T0) and after fat load for the proband (1), a normal subject (2); compared to a control (Ctrl: VLDL and chylomicrons). (C) Plasma apoB48 concentration in primary hypolipidemias and controls. Ctrl: controls; Proband n = 3 measurements (2, 3.5, and 4 years); ABL: abetalipoproteinemia; Ho-FHBL: homozygous or compound heterozygous hypobetalipoproteinemia; CMRD: chylomicron retention disease; dotted line: limit of quantification (0.25 mg/L).

Fig. 3 .

 3 Fig. 3. In vitro and in vivo characterization of the c.1868G > T, p.(Arg623Leu). (A) Activity of normal (wMTTP) and mutant MTTP in COS-7 cells 72 h after transient transfection; **p < 0.01; ***p < 0.001. (B) ApoB48 secretion in the medium of COS-7 cells 72 h after transient cotransfection in triplicate with plasmid expressing human apoB48 and different indicated MTTP expression plasmids. (C)Localization of the two pairs of primers, P1, complementary to the vector, and P2, a forward primer complementary to the exon 14-exon 15 junction (MTP_E14-15chimNL MTP_E14-15chimML) and a reverse primer complementary to the vector from the RT product. (D) Ex vivo analysis performed in HeLa cells transfected either with the WT (c.1868G) or mutant (c.1868T) minigene using two pairs of primers. Gel electrophoresis of RT-PCR products obtained with either P1: primer pair 1; P2: primer pair 2; PCR products 1 to 3 were obtained using P1 primer pair: 1: wild type (466bp); 2: 560 bp product retaining complete intron 14; 3: shorter fragment (438bp) including an abnormal exon 15 lacking the 28 first bases; PCR products 4 and 5 were obtained using P2 primer pair. 4: wild type (240bp) with G at position c.1868; 5: 240bp fragment with T at position c.1868. The sequence of these 5 PCR products were shown on Supplementary Figs.3 and 4. (E) and (F) Sequence analysis of MTTP transcription product from intestinal biopsy. After total RNA extraction and reverse transcription from a control biopsy and patient intestinal biopsy, PCR was performed to sequence cDNA from nucleotides 1285 to 2051. PCR products from two different RT for the patient (lanes 1 and 2), and one for the control biopsy (Ctrl) were analyzed by migration on agarose gel (E) and sequenced (F). Neg: negative control; MW: molecular weight marker.

Fig. 4 .

 4 Fig. 4. Characterization of the c.1068-2127_c.1138del large genomic rearrangement. (A) Digital droplet PCR: normalized percentage of positive droplets was represented for each tested exon. Deletion was suspected in case of percentage lower than 50%. White bars: father, used as control; Black bars: proband; grey bars: mother. (B) Long range amplification from gDNA expected size for control 3876 bp; MW: molecular weight; Ctrl: control; M: mother; P: proband; S: sister. (C) Sequence of the deleted PCR product; the black line shows breakpoints. (D) Schematic representation of the deletion.

Table 1

 1 Biological data for the patient and the parents.

		Age	TC	TG	HDL-C	LDL-C	ApoA1	ApoB	PL	vitamin E	ALAT	ASAT
			mmol/L	mmol/L	mmol/L	mmol/L	g/L	g/L	mmol/L	μmol/L	Multiple of ULN
	Patient	4 m	1.40	0.09	1.42	ND	1.07	< 0.26	NA	0.6	1.8	3.1
		4.5 m	1.61	0.05	1.60	ND	0.97	< 0.03	NA	8.6 a	NA	NA
		1.5 y	1.26	0.05	1.33	ND	NA	< 0.04	1.08	6.4 b	NA	NA
		1.5 y T3h e	1.31	0.10	1.27	ND	NA	< 0.04	1.09	NA	NA	NA
		1.5 y T5h e	1.22	0.07	1.28	ND	NA	< 0.04	1.09	NA	NA	NA
		3 y 3 m	NA	NA	NA	NA	NA	NA	NA	7.7 c	3.0	2.4
		3 y 9 m	1.23	< 0.04	NA	NA	NA	NA	NA	8.0 c	2.3	2.0
		4 y 3 m	1.32	< 0.04	1.35	ND	NA	< 0.04	NA	9.4 d	NA	NA
		5 y	1.22	0.05	1.21	ND	NA	< 0.04	NA	9.2 d	NA	NA
		7 y	1.24	< 0.04	NA	NA	0.94	< 0.03	NA	8.8 d	1.5	1.9
	Sister	3 m f	3.63	2.42	1.41	1.12	NA	0.63	NA	NA	NA	NA
	Father	24 y 9 m	2.30	1.33	0.65	1.04	1.35	0.50	NA	NA	NA	NA
		24 y 11 m	3.97	0.91	1.45	2.11	NA	0.58	NA	NA	NA	NA
	Mother	26 y	3.23	0.76	1.45	1.42	1.35	0.55	NA	NA	NA	NA
		27 y	3.10	0.86	1.33	1.38	1.19	0.48	NA	NA	NA	NA
		30 y	3.71	0.57	1.44	2.01	NA	NA	NA	NA	0.3	0.6

a. to d. : under supplementation: a .Vedrop ® 100UI/kg/j, b .Vedrop ® 120 UI/kg/j, c .Cambridge ® 50UI/kg, d Cambridge ® 100 mg/kg, e numbers of hours after fat load, f after meal. TC: total cholesterol; TG: triglycerides; HDL-C: High Density Lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; Apo: apolipoprotein; PL: phospholipids; ALAT: alanine aminotransferase; ASAT: aspartate aminotransferase; ULN: upper limit of normal; m: month; y: year; NA: not available; ND: not detectable. Bold type: under normal range of the laboratory. Vitamin E normal range: 16 to 30 µmol/L

Acknowledgments

We would like to thank physicians and nurses of Pediatric Gastroenterology and Nutrition unit for carefully having taken the samples. We would like to warmly thank Dr Véronique Ducros for plasma vitamin E laboratory measurement and analytical performance specifications (Service de Biochimie, Centre Hospitalier Universitaire de Grenoble-Alpes, 38043 Grenoble, France). We would also like to give special thanks to Dr Agnes Sassolas (Hospices Civils de Lyon) for her previous work on the subject and Philip Robinson (DRCI, Hospices Civils de Lyon) for help in manuscript preparation.

Conflicts of interest

Philippe Moulin has received honoraria paid to his university for participating in clinical trials and/or advisory boards and/or symposia organized by: AKCEA, AMT, Chiesi, and Uniqure. The other authors have nothing to disclose.

Author contributions

Clinical assessments: AR NP. Experiments: conception and design: MDF SCF AJ CC DB MH; performed the experiments: CD SN SD SR.

Analyzed the data: MDF SCF CD SN OM AJ DB CC MH PM. Wrote the paper, approve the version to be published, agree to be accountable for all aspects of the work: all authors.


J P Boehm

L Rabes

PhD Latifa Ferkdadji

S Abramowitz

C Dumont

MD Catherine Lenaerts

F Boileau

Joly

PharmD Mathilde Di Filippo
email: mathilde.di-filippo@chu-lyon.fr

PhD Mathilde Varret

MD Vanessa Boehm

Jean-Pierre Rab

MD Laurent Abramowitz

MD Sabrina Dumont

PharmD Catherine Boileau

PhD Francisca Joly

PhD Jacques Schmitz

PhD Marie-Elisabeth Samson-Bouma

PhD Dominique Bonnefont-Rousselot

Di Mathilde

Filippo

Postprandial lipid absorption in seven heterozygous carriers of deleterious variants of MTTP in two abetalipoproteinemic families

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Genetic hypocholesterolemia;

Abetalipoproteinemia; Microsomal triglyceride transfer protein; Fat malabsorption; Postprandial; BACKGROUND: Abetalipoproteinemia, a recessive disease resulting from deleterious variants in MTTP (microsomal triglyceride transfer protein), is characterized by undetectable concentrations of apolipoprotein B, extremely low levels of low-density lipoprotein cholesterol in the plasma, and a total inability to export apolipoprotein B-containing lipoproteins from both the intestine and the liver.

OBJECTIVE: To study lipid absorption after a fat load and liver function in 7 heterozygous relatives from 2 abetalipoproteinemic families, 1 previously unreported.

RESULTS: Both patients are compound heterozygotes for p.(Arg540His) and either c.708_709del p.(His236Glnfs*11) or c.134413_134416del on the MTTP gene. The previously undescribed patient

Introduction

Abetalipoproteinemia (ABL; OMIM 200100) is one of the 2 very rare inherited diseases characterized by very low or undetectable apolipoprotein (apo) B-containing lipoprotein (LpB) levels in blood. ABL has an autosomal recessive mode of inheritance, which distinguishes it from familial homozygous hypobetalipoproteinemia (OMIM 107730 commonly named Ho-FHBL) segregating as an autosomal dominant trait. ABL, as Ho-FHBL, is associated with a lipid malabsorption syndrome and the inability to secrete LpB. The similar phenotypes in both disorders arise, in ABL, from deleterious variants in the large polypeptide subunit of microsomal triglyceride (TG) transfer protein (MTP) and, in Ho-FHBL, from deleterious variants in the APOB gene.

MTP has been shown to be essential for LpB assembly and secretion as well as for further lipid absorption processing. MTP-mediated lipid transfer is the limiting step in the assembly and secretion of LpB: in vitro studies have demonstrated that apoB secretion is proportional to MTP activity. [START_REF] Jamil | et al Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells[END_REF] In mice, partial MTP deficiency reduces TG absorption and apoB100 levels in plasma, and it increases the accumulation of cytosolic fat in the liver. [START_REF] Raabe | Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes[END_REF][START_REF] Iqbal | An intrinsic gut leptin-melanocortin pathway modulates intestinal microsomal triglyceride transfer protein and lipid absorption[END_REF] In ABL patients, the absence of MTP activity disrupts TG-rich LpB assembly in both the intestine and the liver, which leads to a complete inability to secrete postprandial chylomicrons (CMs) and very low density lipoproteins, respectively. Endoscopy shows a white coating of the small intestine. The enterocytes are overloaded with large lipid droplets in the cytoplasm, which appear, at the ultrastructural level, to be non-membrane-bound. Affected individuals, thus, develop essentially a lipid malabsorption syndrome with fat-soluble vitamin deficiency. In most of these patients, liver function is altered and steatosis may be associated with elevated transaminases. In some cases, evolution to fibrosis and cirrhosis has been reported. [START_REF] Bouma | Description of two different patients with abetalipoproteinemia: synthesis of a normal-sized apolipoprotein B-48 in intestinal organ culture[END_REF][START_REF] Braegger | Persistence of the intestinal defect in abetalipoproteinaemia after liver transplantation[END_REF][START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF] Patients with ABL exhibit profound hypocholesterolemia, hypotriglyceridemia, the absence of LpB, and no CMs and apoB48 after a fat load. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF] Heterozygous carriers of MTTP deleterious variants have not been believed to exhibit clinical or biological abnormalities, in general, or abnormalities in the lipid profile, in particular. [START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF] Indeed, almost all of the obligate heterozygotes have been reported to have normal lipid levels. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Lee | Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management[END_REF][START_REF] Welty | Hypobetalipoproteinemia and abetalipoproteinemia[END_REF] However, to our knowledge, there have been no studies of fat loading. [START_REF] Lee | Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management[END_REF] The effects of deleterious variants in 1 allele of MTTP on intestinal absorption and the liver are unknown. Furthermore, common variants in the MTTP gene have been reported to predispose to non-alcoholic steatohepatitis in the general population including in heterozygous subjects. [START_REF] Rubin | Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene[END_REF][START_REF] Peng | MTTP polymorphisms and susceptibility to non-alcoholic fatty liver disease in a Han Chinese population[END_REF] These observations raise the question of a potential risk of fat malabsorption and hepatic complications in heterozygous carriers of deleterious MTTP variants.

In this article, we evaluated the impact, in 7 heterozygous relatives from 2 ABL families, of MTTP single deleterious allele variants on hepatic function and lipid absorption after a fat load.

Material and methods

Subjects

Members from 2 unrelated French families were evaluated (Fig. 1). The proband from family A (A.III.2) has not been reported, whereas the 1 from family B (II.1) has been partially described previously. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] Blood samples and intestinal and hepatic biopsies were collected from family A's proband and normal control subjects (for intestinal biopsies) using the procedures and the experimental methods approved by INSERM (RBM 0256) and by a bioethics committee (Comit e Consultatif de Protection des Personnes dans la Recherche Biom edicale de Paris Bichat-Claude Bernard, Paris, France, CCPPRB Bichat-C. Bernard-2003/05). Informed written consent was obtained from all participants or from their legal guardians. polymerase chain reaction. The amplicons were directly sequenced on an ABI PRISM DNA sequencer (Applied Biosystems) as previously described. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Charcosset | Anderson or chylomicron retention disease: molecular impact of five mutations in the SAR1B gene on the structure and the functionality of Sar1b protein[END_REF][START_REF] Filippo | Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia[END_REF] Analysis of intestinal and hepatic biopsies Histological and ultrastructural analyses of both intestinal and liver biopsies from the patient from family A were performed after a 12-to 15-hour fast and treated as described previously. [START_REF] Bouma | Description of two different patients with abetalipoproteinemia: synthesis of a normal-sized apolipoprotein B-48 in intestinal organ culture[END_REF] Analysis of lipids and lipoproteins after a fast or an oral fat load Venous blood samples were drawn after a 12-hour fast and after a lipid load. The lipid load for the young proband of family A (A.III.2) was performed as previously described. [START_REF] Georges | Molecular analysis and intestinal expression of SAR1 genes and proteins in Anderson's disease (Chylomicron retention disease)[END_REF] For all the relatives, a 4520-kJ meal was given at T0 immediately after drawing the fasting blood samples. The meal contained 74% lipids with 72% saturated, 25% monounsaturated, and 3% polyunsaturated fatty acids. Typically, it was composed of 200 mL of unskimmed milk with sweetener, 80 g of bread with 60 g of butter, and 100 g of cream with sweetener. The effect of the fat load on plasma TG concentration, which reflects lipid absorption ability, is expressed as the percentage of maximal increase in TG concentration, during at least 5 hours, except for III.2 (90 minute) and II.3 (190 minute). We evaluated quantitatively the absorption by calculating the total area under the curve from TG concentrations, using the trapezoidal rule. [START_REF] Matthews | Analysis of serial measurements in medical research[END_REF] However, the area under the curves (corrected or not for the basal TG value) was difficult to interpret. Indeed, oral fat loads were performed at different periods in the heterozygous subjects, with different times of follow-up, even if the fat load was always the same. To surmount these difficulties, we decided to use the maximal percentage increase in TG concentration taking into account the absolute TG concentrations (basal and during the fat load). In the same way, this process of calculation decreased the variability due to the different methods of TG determination throughout the 22-year follow-up.

Results

Abetalipoproteinemic patients

The pedigrees of the 2 families are shown in Figure 1. The clinical and biological description of the unreported proband and updated information for the previously described case [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] are provided in Tables 1 and2, respectively.

Family A

The proband (A.III.2) is a boy born on November 01, 1993 after an uneventful pregnancy. He was the second of 3 boys of unrelated healthy Caucasian parents. From normal French curves, the birth weight and height of the patient were, respectively, 4100 g (11 SD), and 52 cm (11 SD). He was breastfed during the first 6 weeks of life. The child presented with regurgitations and vomiting followed by loose stools in the first week of life. At 6 months of age, the height was below the normal French curves (63 cm, 21.5 SD) and weight average for height (6.5 kg). He was followed during 22 years. All along these years, he exhibited the usual previously described clinical and biochemical features of ABL except for an unusual hypertonicity at 7 months (Table 1). Serum TG, total cholesterol, and low-density lipoprotein cholesterol (LDL-C) levels were very low as were the fat-soluble vitamins. These vitamin deficiencies persisted throughout the 22-year follow-up. There was poor compliance to a low-fat diet and to vitamin E supplementation (Table 2). Acanthocytosis was present. Cardiac and ophthalmic investigations were normal as was psychomotor development.

Gastrointestinal endoscopies were performed in 1995, 1996, and 2006, all after a 12-hour fast. A typical white stippling-like hoar frosting covering the mucosal surface of the duodenojejunal part of the intestine was observed on each occasion. Histologically, whenever obtained, all the biopsies exhibited a mucosa with a normal thickness and villi of normal height. The enterocytes were always distended with a cytoplasm clarified due to the presence of numerous vacuoles that predominated in the upper twothirds of the villi, often on 1 side, as previously described (data not shown). [START_REF] Georges | Molecular analysis and intestinal expression of SAR1 genes and proteins in Anderson's disease (Chylomicron retention disease)[END_REF] Electron microscopy always showed non-membrane-bound lipid droplets in the cytoplasm (Fig. 2A andB). No CM-like structures were observed in the basolateral spaces (Fig. 2A). The Golgi cisternae in the enterocytes from the proband were dilated but empty (Fig. 2B) in contrast with the flat Golgi apparatus in enterocytes from normal fasted individuals (Fig. 2C). Liver biopsies were performed in 1996 and 2006 to follow up the steatosis severity. In 1996, there was hepatic macrovacuolar steatosis up to 90% of the parenchymal tissue, associated with portal and centrolobular fibrosis. No cirrhosis was observed (data not shown). Electron microscopy showed hepatocytes with marked accumulation of lipid droplets, which were not membrane bound (Fig. 2D). In 2006, the same massive centrolobular macrovacuolar steatosis and microvacuolar steatosis were present although in a decreased amount (15%) and fibrosis had disappeared (Fig. 2E).

Based on the clinical aspects, the laboratory data, the typical aspect of the intestinal and hepatic biopsies as well as the normolipidemic parents, the patient was suspected to have ABL at about 1 year of age and further confirmed by MTTP gene sequencing (see below). A low-fat diet and Intralipid infusions were thus prescribed from then up to age 13 years and orally thereafter. During this period, an oral complementation of liposoluble vitamins was prescribed (TOCO 500, 2-4 g/d). Lipid infusions were reintroduced every month in March 2016 with 1 g of vitamin E, 50,000 IU of vitamin A, 10 mg of vitamin K in 250 mL of 20% Smoflipid (containing soybean oil, medium-chain TG, olive oil, and fish oil).

At age 24 years, despite a poor compliance to vitamin E supplementation and permanently low vitamin E levels (,3.5 mmol/L), neurological examination was normal: symmetrical and unusually brisk tendon reflexes without ataxia (Tables 1 and2). Indeed, throughout the 22-year follow-up, the only striking clinical feature was the constant hypertonicity. To our knowledge, this is the first report of such a remarkable absence of neurological complications in a typical ABL patient.

Family B

The clinical features of the proband of family B (subject B.II. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] Infusion of Intralipid every 3 weeks (with 45 mg of vitamin K, 500 mg of vitamin E, and 50,000 UI of vitamin A) has been prescribed between 6 months of age and until 2011. In 2011, infusions of Intralipid (soya oil) were replaced by ClinOleic infusions (olive oil: 160 mg/mL, soy oil: 40 mg/mL). Despite a low-fat diet and liposoluble vitamin substitution, deep tendon reflexes were absent. At 28 years of age, the biological data were essentially unchanged except for a slight increase of liver cytolysis (1.4 and 1.5 times the upper limit of normal for aspartate aminotransferase and alanine aminotransferase, respectively) with diffuse moderate steatosis (Tables 1 and2).

Genetic diagnosis of abetalipoproteinemia

Family A Family A's proband (subject A.III.2, Fig. 1) is an unreported case of ABL who exhibits compound heterozygous deleterious variants in the MTTP gene. The first variant is a previously described substitution in exon 13, NM_000253.3(MTTP):c.1619G.A or p.(Arg540His). The frequency of this allele (rs199422220) is 0.0014% in the gno-mAD database (frequencies in Americans: 0.0014% and in non-Finnish Europeans: 0.0024%). This variant was inherited from the patient's father (Fig. 1). The proband's 2 healthy Typical aspect of hepatic biopsies from family A's proband (D and E). Electron microscopy of the intestinal biopsy exhibits the accumulation within the cytoplasm of numerous lipid droplets, free in the cytoplasm and which have diameters from 0.3 to 7 mm( Fig. 2A andB, labeled L, 1995). The Golgi apparatus (Fig. 2B, labeled G, 1995) is distended but without lipoprotein-like material inside. This is in contrast to the flattened Golgi in fasted enterocytes of a normal subject (Fig. 2C,labeled G). Ultrastructural (Fig. 2D andE, 1996) and histological (Fig. 2E, 2006) analysis of the liver showed that it was always engorged with numerous free cytoplasmic lipid droplets (Fig. 2D andE, labeled L), with diameters up to 8 mm. In 1996, intercellular spaces were enlarged, with fibrous tracts extending into the hepatic parenchyma, reflecting the fibrosis (Fig. 2D, dark arrows). In 2006, fibrosis disappeared (Fig. 2E,labeled L). The bars represent 1 mm. brothers, one of his uncles and his grandmother (Fig. 1), are heterozygous for this variant. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF][START_REF] Wetterau | Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia[END_REF] The second deleterious variant is a 2 bp deletion in exon 7, NM_000253.3 (MTTP):c.708_709del or p.(His236Glnfs*11), which leads to a frame shift and a premature stop codon. This variant is not described in the gnomAD database (allele frequency below 1/270,000) and was reported once in ABL in the Clin-Var database. This variant is inherited from his mother and is absent in the rest of the family (Fig. 1).

Because loss of function variants in PCSK9 have been reported to be associated with hypocholesterolemia, [START_REF] Cohen | Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9[END_REF] we sequenced the gene in this patient. No causative variants were observed in this gene nor in exon 7 of the LDLR gene, which encodes the PCSK9-interacting domain. [START_REF] Zhang | Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation[END_REF] Family B Family B's proband (subject B.II.1, Fig. 1) was previously shown, using cDNA from intestinal biopsies, to be a compound heterozygote for an in-frame deletion of exon 11 (new nomenclature) and p.(Arg540His). [START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] Direct sequencing of the genomic DNA from this family was, thus, directed to exons 11 and 13. The p.(Arg540His) mutation is inherited from his father (Fig. 1). The second mutation, a new variant, which is not described in the gnomAD database or in Clin-Var, was inherited from his mother, and is a 4 bp deletion: NM_000253.3(MTTP):c.134413_134416del downstream from the donor site (Fig. 1). An effect on splicing is predicted, in silico, for this variant with a decreased score for the exon 11's donor site ranging from 224% for Human Splicing Finder to a complete abolition for MaxEntScan and NNSplice software.

Fasting and postprandial lipid analyses

Probands

In the fasting state, at diagnosis and during the followup, both probands exhibited a marked fasting hypocholesterolemia, with a dramatic decrease in LDL-C and apoB concentrations but a high-density lipoprotein cholesterol level within the normal range (Table 2). Serum lipid electrophoresis showed the absence of CMs, undetectable beta and prebeta lipoproteins and normal alpha lipoproteins (data not shown).

In the postprandial state (30 and 90 minute after fat load), in both probands, serum TG did not increase and CMs were undetectable. Serum lipid electrophoresis was unaltered as compared to the fasting state (data not shown).

Fasting and postprandial lipid analyses of the heterozygous relatives in families A and B

Fasting lipid values were interpreted using reference values for the laboratory (see Tables).

Fasting TG values of the heterozygous members of both families were in the normal range for 5 subjects (A.II.4, A.III.1, A.III.3, B.I.1, and B.I.2), increased in 1 subject (A.II.3) and decreased in subject A.II.5. Total cholesterol was within the normal range for all subjects (see Table 3). The kinetics observed for II.3 resulted in only 66% increase after 90 min, but the initial TG concentration being high (2.5 mmol/L), they clearly reflected a lipid absorption.

After a fat load, analysis of the TG values was based on the kinetics reported for normal individuals by Nakajima et al. [START_REF] Nakajima | Postprandial lipoprotein metabolism: VLDL vs chylomicrons[END_REF] Two groups of relatives were identified (Fig. 3): in 1 group, the father (subject A.II.4), brother (subject A.III.1), and uncle (subject A.II.3) in family A and TG levels clearly rose after intake of a fat meal, and CMs clearly appeared upon serum electrophoresis. The percentages of maximal increase in TG concentrations appeared after 210 and 180 minutes for A.II.4 and A.III.1, respectively, with respective values of 173% and 263%. This increase was of the same order of magnitude as that observed by Nakajima et al. [START_REF] Nakajima | Postprandial lipoprotein metabolism: VLDL vs chylomicrons[END_REF] for control subjects, which reflects normal lipid absorption. For the father of family B (subject B.I.1), the 123% increase in TG concentration after 300 minutes closer to normal values was considered as normal. The kinetics observed for A.II.3 resulted in only a 66% increase after 90 minutes, but the initial TG concentration was high (2.5 mmol/L). The results clearly indicate lipid absorption.

In contrast, another group, the mother and the brother in family A (subjects A.II.5 and A.III.3), as well as the mother in family B (subject B.I.2), exhibited an opalescent serum with slightly increased TG levels. However, CMs were not detectable upon serum electrophoresis. Furthermore, it is important to note that abdominal pain associated with diarrhea was reported quickly after the fat load, which is consistent with lipid malabsorption syndrome. The maximal increases in TG concentrations were clearly lower for B.I.2 and A.III.3 than those observed by Nakajima et al. [START_REF] Nakajima | Postprandial lipoprotein metabolism: VLDL vs chylomicrons[END_REF] because they were only 69 and 84%, after 300 and 270 minute, respectively. For A.II.5, the apparent normal increase in value (143% after 270 minute) needs to be interpreted in light of the constant very low TG concentrations, always lower than 0.85 mmol/L.

Exploration of genes involved in chylomicron metabolism

In an attempt to understand the origin of the decreased fat loading response of 3 of the 7 heterozygous carriers of deleterious variants in the MTTP gene, we sequenced the APOB, ANGPTL3, MTTP, SAR1B, FABP1, and SAR1A genes (known to be involved in LpB processing) in these individuals. It is important to note that each of these 7 heterozygotes shared different MTTP variants.

Subjects A.II.5 and A.III.3 in family A and subjects B.I.2 and B.II.1 from family B were heterozygous for the NM_000384.2(APOB):c.6639_6641del, an in-frame deletion in the APOB gene, which leads to the absence of residue 2213, the p.(Asp2213del). This in-frame variant has been described previously as rs541497967 in gnomAD with a minor allele frequency of 0.52% and of 0.61% in the non-Finnish European population. This deletion was found to be deleterious in silico (Provean score 29.8) (Provean http://sift.jcvi.org/). Subjects B.I.2 and B.II.1 from family B were found to be homozygous and heterozygous carriers, respectively, for the NM_001443.2:c.280A.G, a common missense variant, p.(Thr94Ala) in the FABP1 gene. This variant was previously described as rs2241883 in gnomAD with an minor allele frequency of 30.5%. This variant affects a moderately conserved residue and the Grantham distance is low. It is considered as tolerated in silico using SIFT (score 0.15) and PolyPhen-2 (score 0).

In the 3 heterozygous carriers of deleterious MTTP variants (subjects A.II.5 and A.III.3 from family A, and subject B.I.2 from family B), no other point deleterious variant was found in these other genes.

Liver function in the heterozygous relatives of the 2 families

In family A, as shown in Table 3, the mother (subject A.II.5), the 2 brothers, (subjects A.III.1 and A.III.3), and the uncle (subject B.II.3) exhibited normal serum liver function tests. Furthermore, on ultrasound examination, the mother (subject A.II.5), the uncle (subject A.II.3), and a brother (subject A.III.3) showed normal liver echogenicity.

In contrast, the father (subject A.II.4), considered as healthy, neither diabetic nor obese nor an alcohol consumer, died suddenly from cardiac arrest in 2000. His liver was taken for transplantation. Pretransplant ultrasound of the liver showed a homogeneous hyperechogenic hypertrophic right lobe. Serum liver function tests were normal for alanine aminotransferase, GGT, alkaline phosphatase. Aminotransferase activity was slightly elevated (68 vs 35 IU/L for the normal value). A liver biopsy obtained during the transplantation exhibited a mixed microvesicular and macrovesicular steatosis in 70% of the hepatocytes in the centrolobular and periportal areas, without fibrosis. In addition, scattered polynuclear neutrophils were observed throughout the hepatic sinusoid. This liver steatosis was not correlated with major increased transaminase levels but was suggested by its hyperechogenicity and was confirmed by the biopsy reflecting precisely the liver fat load as in other contexts. [START_REF] Burgert | Alanine aminotransferase levels and fatty liver in childhood obesity: associations with insulin resistance, adiponectin, and visceral fat[END_REF] In family B, the father (subject B.I.1) and the mother (subject B.I.2) had normal serum liver function tests. Upon ultrasound examination, both showed normal liver echogenicity.

Discussion

We report in this article lipid absorption and liver function analyses in 7 heterozygous relatives from 2 different ABL families, 1 previously described for which we completed the molecular diagnosis [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] and another 1 previously undescribed. This patient described here has been followed for 22 years. The most striking clinical event is the constant hypertonicity, despite poor compliance to vitamin E supplementation.

Because, to our knowledge, no information is available in the literature concerning lipid absorption in heterozygous carriers of deleterious MTTP variants, we investigated this point in these 2 families, which include 5 carriers of the same MTTP variant. Indeed, in contrast to Hz-FHBL, it is believed that obligate heterozygotes for ABL, as expected from an autosomal recessive transmission, exhibit no clinical or laboratory abnormalities and, particularly, no plasma lipid abnormalities. [START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF] We, thus, evaluated the impact of the MTTP mutation on 1 allele by studying fat loading and hepatic function in the 7 heterozygous relatives of these ABL patients.

Genetic analysis of the MTTP gene in these families showed that ABL is caused by 3 different deleterious variants. The first missense variant is the previously described p.(Ar-g540His) variant [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF][START_REF] Wetterau | Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia[END_REF] , which leads, in vitro, to an absence of dimerization with protein disulphide isomerase and to the absence of MTP activity and, in vivo, to an absence of LpB secretion. [START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF][START_REF] Khatun | Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia[END_REF] Moreover, a significant reduction of MTP activity was observed for other missense variants occurring on the same residue, which lead to the loss of positive charge and change in hydrophobicity. [START_REF] Miller | Novel missense MTTP gene mutations causing abetalipoproteinemia[END_REF] This information supports the hypothesis that this residue, which is supposed to form a salt bridge with protein disulphide isomerase, constitutes a critical domain for lipid-transfer activity. [START_REF] Khatun | Loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia[END_REF][START_REF] Miller | Novel missense MTTP gene mutations causing abetalipoproteinemia[END_REF][START_REF] Mann | The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins[END_REF] This finding of 2 apparently unrelated families carrying the same missense variant, in addition to a family from Ontario of Northern European ancestry, [START_REF] Wang | The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins[END_REF] raises the question of a founder variation, as shown for p.(G865X) in the Ashkenazi Jewish population. [START_REF] Benayoun | Abetalipoproteinemia in Israel: evidence for a founder mutation in the Ashkenazi Jewish population and a contiguous gene deletion in an Arab patient[END_REF] The second variant is a 2 bp deletion leading to a frame shift and a premature termination of the protein (p.His236Glnfs*11). Therefore, this variant is likely to be functionally defective. The third variant is a 4 bp deletion: c.134413_134416d e l downstream from the donor site. In silico algorithms predicted an effect on splicing. The perfect in-frame deletion of exon 11 described by Rehberg et al [START_REF] Rehberg | A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase[END_REF] on cDNA extracted from intestinal biopsies of this patient confirmed the effect of this variant on splicing.

In heterozygous relatives in these families, as previously reported in the literature, fasted lipid levels were found to be normal except for a slight decrease of TG in 1 subject (family A, subject A.II.5) and an increase in subject A.II.3 from family A. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF][START_REF] Filippo | Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia[END_REF][START_REF] Leo | Mutations in MTP gene in abeta-and hypobeta-lipoproteinemia[END_REF][START_REF] Najah | Identification of patients with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia in Tunisia[END_REF][START_REF] Uslu | Abetalipoproteinemia in an infant with severe clinical phenotype and a novel mutation[END_REF][START_REF] Pons | A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP)[END_REF][START_REF] Sani | Identification of a novel mutation of MTP gene in a patient with abetalipoproteinemia[END_REF][START_REF] Mohamed | Molecular characterization of Tunisian families with abetalipoproteinemia and identification of a novel mutation in MTTP gene[END_REF][START_REF] Magnolo | Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia[END_REF][START_REF] Walsh | Novel abetalipoproteinemia missense mutation highlights the importance of the N-terminal betabarrel in microsomal triglyceride transfer protein function[END_REF][START_REF] Gunduz | Microsomal triglyceride transfer protein gene mutations in Turkish children: a novel mutation and clinical follow up[END_REF] However, in some articles, about 17% and 14% (see supplemental material) of heterozygotes have been reported to have, respectively, elevated [START_REF] Leo | Mutations in MTP gene in abeta-and hypobeta-lipoproteinemia[END_REF][START_REF] Narcisi | Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia[END_REF] or low plasma triglycerides, total or LDL-C levels, [START_REF] Najah | Identification of patients with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia in Tunisia[END_REF][START_REF] Sani | Identification of a novel mutation of MTP gene in a patient with abetalipoproteinemia[END_REF][START_REF] Mohamed | Molecular characterization of Tunisian families with abetalipoproteinemia and identification of a novel mutation in MTTP gene[END_REF][START_REF] Magnolo | Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia[END_REF] but there is no information about the coexistence of other genetic or environmental factors, which could influence lipid levels.

Because, to our knowledge, no evaluation of fat loading in heterozygous carriers of deleterious MTTP variants have been reported to date, oral fat loads were performed in all the 7 ABL heterozygotes. We show here that 4 heterozygous carriers of the MTTP p.(Arg540His) mutation in family A (subjects A.II.3 and A.II.4, and subject A.III.1, Fig. 1), and subject B.I.1 in family B, exhibit a normal response to a fat load.

Taken together, these data suggest that there is an adaptation in the heterozygous state with an increase in the synthesis of MTP by the normal allele or that MTP activity is present in excess in the normal state, with a single allele being able to provide a sufficient level of activity. [START_REF] Berriot-Varoqueaux | The role of the microsomal triglygeride transfer protein in abetalipoproteinemia[END_REF][START_REF] Lee | Abetalipoproteinemia and homozygous hypobetalipoproteinemia: a framework for diagnosis and management[END_REF][START_REF] Hooper | Contemporary aspects of the biology and therapeutic regulation of the microsomal triglyceride transfer protein[END_REF] In contrast, 3 other heterozygous carriers of deleterious MTTP variants (subjects A.II.5 and A.III.3 in family A and subject B.I.2 in family B) exhibited decreased postprandial TG responses. To explain these different responses, we additionally sequenced 6 other major genes known to be involved in the different steps of LpB assembly and secretion. We thus identified an additional in-frame deletion, the c.6639_6641del p.(Asp2213del) variant, in APOB. This variant, previously found in patients exhibiting hypocholesterolemia, mixed hyperlipidemia, hypercholesterolemia as well as in control subjects, was consequently considered as nondeleterious by Alves et al. [START_REF] Alves | Novel functional APOB mutations outside LDL-binding region causing familial hypercholesterolaemia[END_REF][START_REF] Johansen | LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias[END_REF] However, these nucleotides 6639 to 6641 are located near the « 6666 » cytidine mRNA editing site known to be crucial for the production of the normally occurring apoB48 in the intestine (nucleotide number 6538 in the new nomenclature). [START_REF] Blanc | APOBEC-1-mediated RNA editing[END_REF] Thus, perhaps this variant might, therefore, disturb mRNA editing, blocking intestinal LpB secretion. Unfortunately, the apoB48 levels could not be measured after the fat load in this study. Further studies, such as evaluation of the response to a fat load in other carriers of only this APOB variant as well as in vitro experiments, such as pulse chase studies of apoB secretion in cells stably expressing wild-type or mutant forms of human apoB48, would be useful to evaluate the precise role of this APOB variant in human apoB48 secretion.

A liver biopsy, the gold standard for the evaluation of liver function and a biopsy was obtained in the context of transplanted liver for one of the 7 heterozygous adult carriers of 1 deleterious MTTP variant, the proband's father (subject A.II.4). To date, the causal link between this MTTP variant and liver steatosis has not been firmly established despite literature data reporting that common SNPs in the MTTP gene predispose to non-alcoholic steatohepatitis in the general population by impairing TG-rich lipoprotein export due to a lower transcriptional activity of MTP. [START_REF] Rubin | Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene[END_REF][START_REF] Peng | MTTP polymorphisms and susceptibility to non-alcoholic fatty liver disease in a Han Chinese population[END_REF][START_REF] Bernard | Association between microsomal triglyceride transfer protein gene polymorphism and the biological features of liver steatosis in patients with type II diabetes[END_REF] Similarly, a cross-sectional study reported an association of the p.(Gln297His) in MTTP with a reduced serum cholesterol but an increased risk of nonalcoholic fatty liver disease. [START_REF] Hsiao | MTTP-297H polymorphism reduced serum cholesterol but increased risk of non-alcoholic fatty liver disease-a cross-sectional study[END_REF] The observation of a clinically and biologically silent steatosis in subject A.II.2 suggests that liver steatosis and fibrosis should be monitored in heterozygous carriers of deleterious MTTP variants. Liver biopsy being proscribed for ethical reasons, the noninvasive approach (FibroTest and Fibro-Scan), could be recommended.

In the present study, we, thus, have investigated 7 heterozygous carriers of MTTP deleterious gene variants. In the literature, a great deal of information about the functions of MTP and its role in LpB assembly and secretion has derived from the study of mouse models. However, only a few of them have investigated the consequences of a partial decrease in activity of MTP. [START_REF] Raabe | Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes[END_REF][START_REF] Raabe | Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice[END_REF][START_REF] Chang | Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse[END_REF][START_REF] Xie | Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency[END_REF] Furthermore, extrapolation to humans has been found to be difficult because MTP expression and, consequently, hepatic TG secretion depends on the animal model. [START_REF] Gao | Mechanisms underlying different responses of plasma triglyceride to high-fat diets in hamsters and mice: roles of hepatic MTP and triglyceride secretion[END_REF] Due to the key role of MTP in LpB secretion, MTP inhibitors have been developed to block the assembly of metabolic precursors of LDL particles. Thus, studies of Lomitapide have shown that efficient titration of this inhibitor decreases LDL-C 50% to 90%. [START_REF] Cuchel | Efficacy and safety of a microsomal triglyceride transfer protein inhibitor in patients with homozygous familial hypercholesterolaemia: a single-arm, open-label, phase 3 study[END_REF][START_REF] Raper | Long-term clinical results of microsomal triglyceride transfer protein inhibitor use in a patient with homozygous familial hypercholesterolemia[END_REF] However, side effects have been noted such as diarrhea, decreased vitamin E and fatty acid levels, and increased fat liver content as assessed by nuclear magnetic resonance spectroscopy and liver biopsy. [START_REF] Cuchel | Efficacy and safety of a microsomal triglyceride transfer protein inhibitor in patients with homozygous familial hypercholesterolaemia: a single-arm, open-label, phase 3 study[END_REF][START_REF] Raper | Long-term clinical results of microsomal triglyceride transfer protein inhibitor use in a patient with homozygous familial hypercholesterolemia[END_REF][START_REF] Cuchel | Lipid-lowering treatment for homozygous familial hypercholesterolaemia-authors' reply[END_REF] A case of progression of steatohepatitis to fibrosis also has been reported in a patient with pancreatitis who exhibited fatty liver before treatment. [START_REF] Sacks | Severe hypertriglyceridemia with pancreatitis: thirteen years' treatment with lomitapide[END_REF] Unfortunately, due to ethical considerations, MTP mRNA, protein or activity levels in liver and intestine have not been measured. Thus, these data are not available in our study.

The limited number of patients and relatives is an evident limitation for our conclusion. This is due to the extreme rarity of the disorder. The incidence of the ABL disease is reported as less than 1 in 1,000,000. [START_REF] Burnett | Clinical utility gene card for: abetalipoproteinaemia[END_REF] To our knowledge, fewer than 65 families have been described worldwide. [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF] However, the study of these 2 families has provided additional knowledge, which contributes to a better understanding of these rare diseases. Indeed, we report here 1 previously undescribed patient who exhibited a negative fat load test, as described in 4 typical ABL cases with known MTTP mutations [START_REF] Filippo | Homozygous MTTP and APOB mutations may lead to hepatic steatosis and fibrosis despite metabolic differences in congenital hypocholesterolemia[END_REF][START_REF] Narcisi | Mutations of the microsomal triglyceride-transfer-protein gene in abetalipoproteinemia[END_REF][START_REF] Kawashiri | Extreme contrast of postprandial remnant-like particles formed in abetalipoproteinemia and homozygous familial hypobetalipoproteinemia[END_REF] and the first description of fat load tests in heterozygous carriers of MTTP deleterious variants.

Despite this limitation, we report first that 4/7 heterozygous carriers of deleterious MTTP variants have normal lipid absorption without known additional risks. Second, and perhaps more important, some heterozygotes, despite normal fasting lipid levels and without clinical abnormalities, do not absorb normally. In these heterozygous carriers of MTTP variants, other deleterious variants of genes involved in the LpB secretory mechanism, such as apoB reported here, or environmental factors should be evaluated. Furthermore, 1 subject was found with a clinically and biologically silent steatosis. The results of this study suggest that in ABL families, there should be (1) an obligatory cascade screening to identify all heterozygous carriers, (2) an evaluation of fasting lipid status with additional fat load lipid absorption studies of heterozygotes, and (3) liver monitoring using noninvasive tests in all heterozygotes. Additional subjects need to be investigated to confirm our results, ascertain the level of risk, and definitively establish appropriate treatment and screening.

Figure 1

 1 Figure 1 Pedigrees of the families. Symbols divided in halves indicate heterozygous carriers of variants: light gray symbols indicate the MTTP c.1619G.A p.(Arg540His), black symbols the MTTP c.708_709del or p.(His236Glnfs*11), dark gray symbols the MTTP c.134413_134416del, and hatched symbols represent the APOB c.6639_6641del. For each subject, after the unique identification number and the age (years) are indicated, 1 below the other, lipid absorption and comorbidities at the time of fat load. NA, not available.

Table 2

 2 Biochemical Expressed as a multiple of the upper limit of normal A.III.Interval range 0-3 y 1.35-4.55 0.82-3.62 0.78-1.70 0.35-1.20 0.70-1.35 0.45-1.10 0.45-2.36 10.6-24.0 3-18 y 3.10-5.20 1.80-3.60 0.78-1.70 0.35-2.10 0.70-1.95 0.45-1.10 0.45-3.26 10.6-24.0 .18 y 4.05-6.05 1.80-3.60 1.05-1.75 0.55-1.55 0.70-1.95 0.45-1.10

Figure 2

 2 Figure 2 Typical aspect of intestinal biopsies from family A's proband (subject A.III.2) (A and B) and from a normal subject (C).Typical aspect of hepatic biopsies from family A's proband (D and E). Electron microscopy of the intestinal biopsy exhibits the accumulation within the cytoplasm of numerous lipid droplets, free in the cytoplasm and which have diameters from 0.3 to 7 mm( Fig.2A and B, labeled L, 1995). The Golgi apparatus (Fig.2B, labeled G, 1995) is distended but without lipoprotein-like material inside. This is in contrast to the flattened Golgi in fasted enterocytes of a normal subject (Fig.2C, labeled G). Ultrastructural (Fig.2D and E, 1996) and histological (Fig.2E, 2006) analysis of the liver showed that it was always engorged with numerous free cytoplasmic lipid droplets (Fig.2D and E, labeled L), with diameters up to 8 mm. In 1996, intercellular spaces were enlarged, with fibrous tracts extending into the hepatic parenchyma, reflecting the fibrosis (Fig.2D, dark arrows). In 2006, fibrosis disappeared (Fig.2E, labeled L). The bars represent 1 mm.

Figure 3

 3 Figure 3 Changes in the serum concentration of triglycerides (TG) after the oral fat load. The TG kinetics from the proband (A.III.2, gray diamond), father (A.II.4, diagonal hatched square), mother (A.II.5, black round), uncle, (A.II.3, white square), and brothers (A.III.1, gray square and A.III.3, black square) of family A's proband, and the mother (B.I.2, black triangle) and father (B.I.1, gray square with black cross) of the family B's proband are shown. Squares represent heterozygous carriers of the c.1619G.A p.(Arg540His) variant, the round symbol is a carrier of the c.708_709del or p.(His236Glnfs*11) variant, and the triangle is the carrier of the c.134413_134416del variant on MTTP. Black symbols represent carriers of the APOB c.6639_6641del. The TG kinetics reported by Nakajima et al. 20 after an oral fat load in normal individuals appear in gray area (mean 6 SEM).

Table 1

 1 Clinical features of patients

	Ophthalmological	imp Others	No PR Rickets: decreased	bone density.	Normal cardiac	examination		N Catch-up growth	was achieved at	around 3 y of	age	N; ERG: N; no PR Delayed puberty	(16 y).	Osteoporosis	(rachis Z score	21.5).	Osteopenia	(Z score 20.7).	Muscular pain:	only after	playing soccer	Decreased night	vision ERG N	ND
	Neurological	imp	N; EMG: N					N				N; EMG: N										EMG: normal, MRI:	no vermis	atrophy	ND
	Deep-tendons	reflexes	N 111					N 111				N 111										Present,	symmetrical	without	cerebellar ataxia	No
		HMG Liver	Yes Enlarged,	Steatosis11	(Echo)			Yes Steatosis 11				No ND										No Steatosis (Echo)	Liver MRI: 14%	No Steatosis, diffused	(Echo)
		Steatorrhea	(72 h) 0.8-2 g/	24h				ND				ND										Absorption	coefficient: 76%	(.95%)	No
	Digestive	symptoms	Diarrhea,	vomiting,	abdominal	distension, fat	malabsorption	Yes, when	interruption of	low-fat diet		Rare diarrhea,	vomiting, fat	malabsorption								No abdominal pain	Diarrhea when	interruption of	low-fat diet	No
	Failure to	Proband Age thrive (BMI)	A.III.2 7 m Yes, H 64 cm,	W 7.6 kg	(Y,18.4)			13 y No, H: 149 cm,	W:	43.5 kg (N,19.4)		18 y No, H 170.5 cm;	W: 60.3 kg; (N,	20.8)								22 y No, H 174 cm,	W 63 kg (N, 21)	B.II.1 24 y No, H 184 cm,	W 65 kg (N, 19)

Echo, Echography; EMG, electromyography; ERG, electroretinography; H, height; HMG, hepatomegaly; Imp, impairment; m, months; MRI, magnetic resonance imaging; N, normal; ND, not done; PR, pigmentary retinopathy; W, weight; y, years; Y, decreased.

  1, boy born on November 22, 1987) were previously described by Wetterau et al. and Rheberg et al. and have been summarized and updated (to 26 years of age) by Di Filippo et al.

  1.35-2.88 11.4-35.1 ALT alanine aminotransferase; Apo, apolipoprotein; AST aspartate aminotransferase; GGT, gamma glutamyl transpeptidase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein

cholesterol; m months; ND not determined; PP, postprandial; TC total cholesterol; UC, uncalculable; Vit, Vitamin; y, years. B.II.1: no TG increase was reported after a 1-h fat load (data unavailable).

*After about 1 y of low-fat diet, serum transaminase levels returned to normal.

Table 3

 3 Biochemical analyses of the heterozygous relatives of the 2 ABL families Units

		Vit A Vit E	mmol/L mmol/L		2.13 31.2	NA NA	NA NA	1.43 25.3	0.98 20.7		NA NA	NA NA
		ApoB ALT GGT Apo A1 AST	Expressed as a multiple of the upper limit of normal. g/L g/L		!1 !0.9 !0.7 1.06 1.28	!1.1 a !0.5 !1.9 a 1.19 1.61	!0.3 !0.2 !0.4 0.93 1.91	!0.4 !0.2 !0.5 0.75 1.62	!0.4 !0.2 !0.6 0.83 1.49		!0.4 !0.2 !0.3 0.71 1.62	!0.6 !0.7 !0.4 0.58 1.35	0.45-1.10 0.70-1.95	0.45-1.10 0.70-1.95
	TG increase (time,	Age TC LDL-C HDL-C TG min)	y mmol/L mmol/L mmol/L mmol/L %	Family A	66 b (90) A.II.3 43 5.82 3.41 1.3 2.46	173 (210) A.II.4 35/37 a 5.74 3.72 1.48 1.18	143 (270) A.II.5 32 5.46 3.23 2.07 0.34	263 (180) A.III.1 22 4.76 NA NA 0.82	84 (270) A.III.3 12 5.33 NA NA 0.97	Family B	123 (300) B.I.1 48 4.80 2.66 1.76 0.78	69 (300) B.I.2 45 3.85 2.09 1.43 0.65	Interval range 3-18 3.10-5.20 1.80-3.60 0.78-1.70 0.35-2.10	.18 4.05-6.05 1.80-3.60 1.05-1.75 0.55-1.55

ABL, Abetalipoproteinemia; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Apo, apolipoprotein; GGT, gamma glutamyl transpeptidase; m, months; NA, not available; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride; Vit, Vitamin; y, years.

In bold type: abnormal values. a Deceased.

b

Acknowledgments

The authors would like to express first, special thanks to Dr Lawrence Aggerbeck for his helpful discussion and reviewing of the article (Present address: INSERM UMR 1124, Toxicologie, pharmacologie et signalisation cellulaire Center Universitaire des Saints-P eres, 75006 Paris, France). The authors would like to thank Pr E Bruckert and his physicians and nurses for postprandial studies (Endocrinology Department) and Dr Yann Nadjar (Unit eN e u r o g en etique et m etabolisme, D epartement de Neurologie) for neurological investigation, both from Piti e-Salpêtri ere hospital, AP-HP, 75013 Paris, Pr F Gottrand (Service de p ediatrie -Unit ed e Gastroent erologie, CHRU de Lille -Hôpital Jeanne de Flandre, 59037 Lille Cedex France), Pr Nicole Brousse (Service d'Anatomie et de Cytologie Pathologiques, Hôpital Necker-Enfants Malades, AP-HP, 75015 Paris, France), and Dr E Leteurtre (Service d'Anatomie et de Cytologie Pathologiques, Center Hospitalier et Universitaire de 59045 Lille, France) for medical pediatric follow-up. The authors would like to thank Dr Isabel Beucler and Marie-Christine Federspiel for lipid laboratory investigation and technical assistance, respectively (Laboratoire des lipides, Service de Biochimie, Piti e-Salpêtri ere hospital, AP-HP, 75013 Paris, France), Dr Nathalie Varroqueaux and Nicole Verthier (INSERM U 327, 75018 Paris, France) for her contribution and participation in the patient's studies and the histological and ultrastructural studies, respectively.

Financial disclosure

The authors have no conflicts of interest to disclose.