Maxime Kwapich

Dominique Lacroix

Stéphanie Espiard

Sandro Ninni

François Brigadeau

Claude Kouakam

Pascal Degroote

Jean-Marc Laurent

Vincent Tiffreau

Laurent Jm

Ben Hamou

Ben Yaou

Vigouroux C 10

Bonne G 11

Vantyghem Mc
email: mc-vantyghem@chru-lille.fr

Diamenord-Aednl Working

Group Averous

Balavoine As

Boury S, Bouzaib S, Caiazzo R, Cortet C, Cuisset JM, Defrance F, Derveaux A, Devemy F, Douillard C, Bourdelle Mf

Prof. M C Vantyghem

Cardiometabolic assessment of lamin A/C gene mutation carriers: A phenotype-genotype correlation

Keywords: Coronary artery disease, Laminopathy, Lipodystrophy, LMNA gene, Rhythm disorders

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

INTRODUCTION

Lamin A/C gene mutations are associated with heterogeneous phenotypes ranging from cardiomyopathies with or without muscular dystrophies to lipodystrophies-collectively referred to as 'laminopathies' [START_REF] Worman | Laminopathies: a wide spectrum of human diseases[END_REF][START_REF] Florwick | LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes[END_REF]. The LMNA gene encodes A-type lamins (lamins A/C), members of the intermediate filament protein family that are required for nuclear lamina formation. Recent studies have suggested that LMNA mutations affect epigenetic regulation of developmental pathways [START_REF] Perovanovic | Laminopathies disrupt epigenomic developmental programs and cell fate[END_REF][START_REF] Paulsen | Chrom3D: three-dimensional genome modeling from Hi-C and nuclear lamin-genome contacts[END_REF][START_REF] Oldenburg | A lipodystrophy-causing lamin A mutant alters conformation and epigenetic regulation of the anti-adipogenic MIR335 locus[END_REF].

Lamin A/C gene mutations have been described over the entire gene. Nevertheless, the rarity of the disease makes phenotype-genotype correlation difficult. Familial partial lipodystrophy type 2 (FPLD2) is characterized by progressive fat loss in the extremities at puberty, associated with severe insulin resistance, diabetes and hypertriglyceridaemia with muscle hypertrophy [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF][START_REF] Vantyghem | Patients with familial partial lipodystrophy of the Dunnigan type due to a LMNA R482W mutation show muscular and cardiac abnormalities[END_REF]. FPLD2 is related to heterozygous LMNA gene mutations, especially those at the 'hotspot' codon Arg482 (R482) in exon 8.

The severity of LMNA-related cardiomyopathies has been demonstrated in several studies showing frequent atrioventricular blocks and ventricular arrhythmias often requiring cardiac implantable electronic devices (CIEDs), as well as severe heart failure, especially in the context of dilated cardiomyopathy [START_REF] Jakobs | Novel lamin A/C mutations in two families with dilated cardiomyopathy and conduction system disease[END_REF][START_REF] Van Rijsingen | Risk factors for malignant ventricular arrhythmias in lamin a/c mutation carriers a European cohort study[END_REF][START_REF] Kitaguchi | A missense mutation in the exon 8 of lamin A/C gene in a Japanese case of autosomal dominant limbgirdle muscular dystrophy and cardiac conduction block[END_REF][START_REF] Brodt | Temporal relationship of conduction system disease and ventricular dysfunction in LMNA cardiomyopathy[END_REF][START_REF] Van Berlo | Meta-analysis of clinical characteristics of 299 carriers of LMNA gene mutations: do lamin A/C mutations portend a high risk of sudden death?[END_REF][START_REF] Subramanyam | Overlapping syndrome with familial partial lipodystrophy, Dunnigan variety and cardiomyopathy due to amino-terminal heterozygous missense lamin A/C mutations[END_REF][START_REF] Carboni | Evolution of the phenotype in a family with an LMNA gene mutation presenting with isolated cardiac involvement[END_REF][START_REF] Pankuweit | A/C mutations in patients with dilated cardiomyopathy[END_REF][START_REF] Macleod | Lamin A/C truncation in dilated cardiomyopathy with conduction disease[END_REF][START_REF] Francisco | Complex phenotype linked to a mutation in exon 11 of the lamin A/C gene: Hypertrophic cardiomyopathy, atrioventricular block, severe dyslipidemia and diabetes[END_REF][START_REF] Hoorntje | Lamin A/C-Related Cardiac Disease: Late Onset with a Variable and Mild Phenotype in a Large Cohort of Patients With the Lamin A/C p.(Arg331Gln) Founder Mutation[END_REF][START_REF] Madej-Pilarczyk | Limbgirdle muscular dystrophy with severe heart failure overlapping with lipodystrophy in a patient with LMNA mutation p.Ser334del[END_REF][START_REF] Ambrosi | A novel overlapping phenotype characterized by lipodystrophy, mandibular dysplasia, and dilated cardiomyopathy associated with a new mutation in the LMNA gene[END_REF][START_REF] Villa | A G613A missense in the Hutchinson's progeria lamin A/C gene causes a lone, autosomal dominant atrioventricular block[END_REF][START_REF] Türk | Multisystem disorder and limb girdle muscular dystrophy caused by LMNA p.R28W mutation[END_REF][START_REF] Kumar | Long-Term Arrhythmic and Nonarrhythmic Outcomes of Lamin A/C Mutation Carriers[END_REF]. Recommendations have already been given concerning the need for implanting defibrillators rather than pacemakers in laminopathies with cardiac involvement [START_REF] Meune | Primary prevention of sudden death in patients with lamin A/C gene mutations[END_REF][START_REF] Feingold | Management of Cardiac Involvement Associated with Neuromuscular Diseases: A Scientific Statement From the American Heart Association[END_REF].

However, some of these reports could have been biased by exclusive cardiac recruitment [START_REF] Kumar | Long-Term Arrhythmic and Nonarrhythmic Outcomes of Lamin A/C Mutation Carriers[END_REF]. Indeed, patients with the R482 genotype, whose metabolic phenotype is well described, have rarely been reported to have primary cardiomyopathies, although ischaemic heart disease might be more prevalent [START_REF] Hegele | Premature atherosclerosis associated with monogenic insulin resistance[END_REF][START_REF] Afonso | LMNA mutations resulting in lipodystrophy and HIV protease inhibitors trigger vascular smooth muscle cell senescence and calcification: Role of ZMPSTE24 downregulation[END_REF]. Nevertheless, unlike the cardiovascular phenotype, the metabolic phenotype of cardiac laminopathies remains largely unknown.

Thus, the aim of the present study was to compare cardiometabolic complications in patients with LMNA mutations at R482, the hotspot for FPLD2, with those in carriers of other LMNA mutations.

PATIENTS and METHODS

Study design

This retrospective longitudinal study was conducted at one university hospital over a 15-year period (from 2000 to 2015). All patients referred to the institution's endocrinology and metabolism department with a diagnosis of laminopathy were included. Patients were classified into two groups according to genotype: (i) those with the heterozygous LMNA R482 mutation; or (ii) those with other lamin A/C mutations. The cardiometabolic phenotypes in these two groups were then compared, while the metabolic phenotypes in these two groups were further compared with a control group.

Patients

A total of 157 patients were referred because of suspected laminopathy by either endocrinologists who suspected lipodystrophic syndromes, or by cardiologists or geneticists because of unexplained cardiac abnormalities or familial screening. After subjecting these patients to careful clinical and biological examinations, and obtaining their written informed consent, their LMNA genes were studied by direct sequencing. Clinical and biological data were collected from patients' medical files. In addition to gender, their age at the time of their first and last cardiometabolic evaluations were recorded for calculation of the average follow-up duration. In addition, 19 healthy subjects matched for age and gender, and recruited from the PHRC-Clin.gov2009-AO-1169-48 trial, served as the control group for metabolic assessment.

Cardiac outcomes

The following cardiac parameters were collected from the patients' medical files: (i) family history of CIED or sudden death, considered positive for any patient with at least one first-degree relative with an implanted device or who had died suddenly; (ii) abnormal electrocardiography (ECG), defined as the presence of at least one of the following during follow-up: atrial fibrillation, atrial flutter, highgrade atrioventricular (AV) block, ventricular ectopy, or complete left bundle branch block; (iii) abnormal ECG on Holter monitoring, defined as the presence of non-sustained (NSVT) or sustained ventricular tachycardia (VT), high-grade AV block, or atrial flutter or atrial fibrillation; (iv) use of a CIED, defined as a pacemaker or automatic implantable cardiac defibrillator (ICD) acquired during follow-up (before or after diagnosis of the LMNA mutation); (v) CIED interrogation, when ventricular rhythm disorders correspond to the presence of at least one NSVT or VT episode retrieved from stored electrograms during scheduled follow-up visits; (vi) abnormalities on echocardiography, defined by the presence of at least one of the following as per international guidelines for echocardiography measurements: left ventricular ejection fraction (LVEF) < 50%; left atrial enlargement; and/or LV hypertrophy or LV enlargement [START_REF] Lang | Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular[END_REF]; patients with non-ischaemic heart disease (cardiomyopathies), defined by LV dilatation and/or altered LVEF, were also noted, and LV diastolic function was assessed by E/A ratio if available; (vii) smoking status, recorded as either currently active or over the past 3 years; (viii) screening for ischaemic heart disease, which included myocardial ischaemia screening with an exercise stress test in five patients with R482 and six patients with non-R482 mutations, radionuclide angiography (six R482, one non-R482) or dobutamine stress echocardiography (two R482); and coronary angiography in four patients because of positive noninvasive screening or acute coronary syndrome (two patients); and (ix) screening for atheromatosis by Doppler ultrasound of the carotid and lower-limb arteries.

Metabolic outcomes

The following metabolic parameters were also collected from patients' medical records of the last endocrinological evaluation or, for the control group, from the PHRC-Clin.gov2009-AO-1169-48 database: (i) body mass index (BMI), assessment of fasting blood glucose (FBG) and triglycerides, as measured by routine techniques, and fasting C-peptide and leptin using radioimmunoassay [RIA-coat C-peptide (Mallinckrodt France SARL, Paris, France), detection limit: 0.2 ng/mL] and Human Leptin RIA kits (EMD Millipore Corporation, Burlington, MA, USA; normal range in normal-weight subjects: women 7.4 ± 3.7 ng/mL, men 3.8 ± 1.8 ng/mL), respectively; (ii) diabetes and glucose intolerance by subjecting participants not already being treated for diabetes at inclusion to a 75-g oral glucose tolerance test (OGTT), which was interpreted according to American Diabetes Association criteria; (iii) hypertension, defined as blood pressure > 140/90 mmHg or use of an antihypertensive drug; (iv) use of lipid-lowering agents (such as statins, fibrates, ezetimib); (v) use of antidiabetic treatments (such as metformin or any other antidiabetic drugs, including glucagon-like peptide-1 receptor agonists and insulin); (vi) body fat-mass percentage, as measured by dual-energy X-ray absorptiometry (DXA; Lunar DPX-IQ, GE Healthcare, Chicago, IL, USA); and (vii) ratio of intraabdominal/total abdominal fat mass, as calculated by measurement of subcutaneous and visceral fat surface areas from 1-cm reconstructed slices of abdominal L4 magnetic resonance imaging (MRI), which is contraindicated in cases of CIED and was therefore only performed in 22 R482 and 12 non-R482 patients.

Statistical analysis

Qualitative variables, expressed in absolute numbers and percentages, were compared by chi-squared or Fisher's exact test for values < 5. For quantitative variables, the Shapiro-Wilk test for normality was performed, with results expressed as medians with interquartile ranges (Q25-Q75), and compared using the Mann-Whitney test. Analyses were carried out with GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). Any differences with P values < 0.05 were considered significant.

RESULTS

Description of the two groups

In all, 60 patients carried LMNA mutations; however, as two were excluded because their medical files were incomplete, 58 patients were ultimately included in this study: 29 patients from eight families carried the heterozygous R482W mutation, and 29 patients from 16 families carried another heterozygous LMNA mutation.

The gender ratio between the LMNA R482 and non-R482 groups did not differ, although the number of women tended to be higher in the R482 group (22 vs 16; P = 0.09). There were also no differences between the two groups in terms of age at first evaluation [R482: 43 (24-50.5) years vs non-R482: 39 (28.2-47.5) years; P = 0.9] and duration of follow-up [R482: 5 (2-11) years vs non-R482: 4 (1-9) years; P = 0.42]. These patients' genotypes and their main phenotypic features are presented in Table I, and their diagnostic circumstances are depicted in Fig. 1. The proportion of diabetes patients in the R482 group increased during the follow-up period from 11 to 24 out of 29 patients (from 37.9% to 82.7%), and from 6 to 12 out of 29 patients (from 20.7% to 41.4%) in the non-R482 group, as determined at the last evaluation.

Cardiovascular phenotype (Table II, Fig. 2 A)

The frequency of a family history of sudden death (P < 0.01) or CIED use (P < 0.001) was significantly lower in the R482 vs non-R482 mutation groups, as was the frequency of abnormal ECG (P < 0.001). The frequency of abnormal Holter monitoring also differed significantly between the two groups, with a greater frequency of abnormalities, especially NSVT, in the non-R482 mutation group compared with the R482 group (P < 0.01).

In addition, the frequency of implantable CIEDs was significantly lower in R482 vs non-R482 mutation patients (P < 0.001). More specifically, the only patient in the R482 group (R482Q mutation) with a CIED, a pacemaker implanted at age 46, died at age 60 after several years of dialysis. In the non-R482 group, there were 16 CIEDs, including eight pacemakers, seven of which were later upgraded to defibrillators when their indication for cardiac laminopathies was standardized [START_REF] Kumar | Long-Term Arrhythmic and Nonarrhythmic Outcomes of Lamin A/C Mutation Carriers[END_REF], and eight defibrillators as first-line devices. The only remaining non-R482 patient with a pacemaker, implanted at age 33, developed end-stage renal disease (ESRD) and died suddenly during dialysis at age 50, just before genetic results were obtained. Seven VT episodes were recorded by CIEDs in seven different non-R482 patients.

There was no difference in frequency of cardiac ultrasound abnormalities between the two patient groups (P = 0.18). Nevertheless, the frequency of left atrial enlargement was significantly lower in patients with R482 vs non-R482 mutations (P < 0.05), and median LVEF was significantly higher in patients with R482 vs non-R482 mutations (P < 0.01), but with no difference in LV hypertrophy (P = 1.00) or LV diastolic diameter (P = 0.76). Five patients, all in the non-R482 group, had a cardiac phenotype of non-ischaemic heart disease (Table I). Of the remaining population, 16 (seven R482 and nine non-R482 carriers) had normal systolic function and E/A measurements. In the R482 group, 4/7 had impaired relaxation (E/A < 1) vs none in the non-R482 group whereas, in the non-R482 group, 3/9 had restrictive filling patterns (E/A > 2) vs none in the R482 group (Table II). The rate of deaths during the follow-up period was similar in both groups (P = 1.00), but was more often related to cardiac causes in the non-R482 group. ESRD related to long-term severely insulin-resistant diabetes worsened the prognosis in two cases.

There was no difference in the number of smokers (P = 0.33) between the two groups. Also, no patient in the non-R482 group tested positive for cardiac ischaemia or significant coronary stenosis, which contrasted with positive diagnoses in 38% and 14% of patients, respectively, with R482 mutations. However, screening tests for cardiac ischaemia tended to be performed more often (P = 0.09) in patients with R482 mutations, who more often had diabetes (P < 0.01) than the non-R482 patients.

Comparison of peripheral atherosclerotic features showed no differences between the two groups (lower limb: P = 0.78; carotid atheromatosis: P = 0.42). III, Fig. 2 B,C) BMI (P < 0.05), blood leptin (P < 0.01) and high-density lipoprotein (HDL) cholesterol levels (P < 0.05) and fat-mass percentages (P < 0.001) were all significantly lower in patients with R482 vs non-R482 mutations. In contrast, median fasting C-peptide (P < 0.05) and glucose (< 0.001) levels, intraabdominal/total abdominal fat-mass ratio (P < 0.001), and frequencies of diabetes or glucose intolerance (P < 0.01), metformin treatment (P < 0.01) and hypertriglyceridaemia (P < 0.05) were all significantly higher in patients with R482 vs non-R482 mutations. There were no differences between the two groups in rates of hypertension (P = 0.11) or in median triglyceride (P = 0.15), total cholesterol (P = 0.73) and low-density lipoprotein (LDL) cholesterol (P = 0.82) levels, or use of lipidlowering (P = 0.10) or antidiabetic treatments other than metformin (P = 0.24), including insulin (P = 0.14).

Metabolic phenotype (Table

Comparison of the two LMNA mutation groups to an age-and gender-matched healthy control group (Table III) revealed that BMI (P < 0.02), intra-/total abdominal fat-mass ratio (P < 0.05; Fig. S1; see supplementary materials associated with this article online), leptin levels (P < 0.01), frequency of hypertriglyceridaemia (P < 0.05) and diabetes or glucose intolerance (P < 0.001) were significantly higher, whereas HDL levels (P < 0.02) were significantly lower, in the non-R482 group compared with the controls. Similarly, intra-/total abdominal fat-mass ratio (P < 0.001; Fig. S1) and frequency of hypertriglyceridemia (P < 0.05) and diabetes or glucose intolerance (P < 0.001) were significantly higher, but HDL level (P < 0.02) and fat-mass percentage significantly lower, in the R482 vs the control group.

DISCUSSION

This large-scale, single-centre, LMNA mutation cohort comparing cardiometabolic complications in patients with R482 mutations with carriers of other LMNA mutations showed more arrhythmias in the non-R482 than R482 group, which tended to have a greater frequency of coronary artery disease. On the other hand, the rate of diabetes in the non-R482 group was 40%, but twofold higher than that in the R482 group. Each group also showed higher intra-/total abdominal fat-mass ratios, a hallmark of lipodystrophy, than the control group.

Recognized only since 1999, laminopathies are rare diseases with multiple phenotypes, some of which overlap [START_REF] Worman | Laminopathies: a wide spectrum of human diseases[END_REF]. Yet, despite their rarity, nearly 60 patients from 24 families were identified at our centre alone, with a greater number of families with non-R482 than R482 mutations (16 vs 8 patients, respectively). As the northern region of France, where our patients were from, has four million inhabitants, the prevalence of patients with LMNA mutations and LMNA-related FPLD2 in this area is estimated to be 14.5 and 7.25 cases/million people, respectively, which is twice the prevalence reported in a recent study [START_REF] Chiquette | Estimating the prevalence of generalized and partial lipodystrophy: findings and challenges[END_REF]. Moreover, our results do not reflect any founder-effect bias, as 16 different mutations were identified in the non-R482 group, two of the eight R482 families were of Portuguese descent, and two of the remaining six families had two different types of mutations (R482W and R482Q). Indeed, laminopathies are most likely underrecognized, given their variable phenotype [START_REF] Worman | Laminopathies: a wide spectrum of human diseases[END_REF][START_REF] Florwick | LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes[END_REF][START_REF] Vantyghem | Patients with familial partial lipodystrophy of the Dunnigan type due to a LMNA R482W mutation show muscular and cardiac abnormalities[END_REF], although the phenotype may perhaps be better recognized in this region, known for its high levels of obesity and diabetes, due to an anticipation phenomenon [START_REF] Jeru | LMNA-associated partial lipodystrophy: anticipation of metabolic complications[END_REF].

One limitation of our study is that it was retrospective. All investigations were not performed in every patient, but were adjusted according to clinical situation and guidelines for follow-up of type 2 diabetes. For this reason, the number of patients who underwent each investigation has been systematically mentioned. On the other hand, the size of our cohort was relatively large for such a rare disease, and all evaluations were performed at the same centre using the same methods, thereby strengthening all data comparisons. Concerning the cardiac phenotype, there was a marked difference between the R482 and non-R482 groups in familial history of CIED use or sudden cardiac death, arguing in favour of a better cardiac prognosis for R482 than for other LMNA mutations. This point, which has never been emphasized before, was easy to explore during clinical assessment of the patients' medical histories and was confirmed by cardiac investigations. Indeed, the frequencies of abnormal ECG and left atrial dilatation were significantly lower, and LVEF significantly higher, in patients with R482 vs non-R482 mutations. The latter patients were also more likely to have CIEDs, and five cases of non-ischaemic heart disease were found in this group. In contrast, patients with R482 mutations tended to have more frequent positive non-invasive tests for ischaemia or documented severe coronary artery disease. However, diastolic function was altered in some patients in both groups, but with different patterns: there was restrictive filling in non-R482 carriers, but impaired relaxation in R482 carriers, a possible consequence of the high incidence of diabetes in the latter. Also, the large number of carriers in the R482 families suggests that the prognosis for the R482W group in terms of mortality may be better.

Finally, although gender did not significantly differ, there tended to be more men in the non-R482 group, which might reflect a bias, as male gender has been reported to be a risk factor for sudden cardiac death in laminopathies [START_REF] Van Rijsingen | Gender-specific differences in major cardiac events and mortality in lamin A/C mutation carriers[END_REF]. However, this link was possibly related to the fact that male gender is more often associated with non-R482 mutations, including non-missense mutations, which are also known to be associated with higher risk of severe cardiac disorders [START_REF] Van Rijsingen | Gender-specific differences in major cardiac events and mortality in lamin A/C mutation carriers[END_REF]. Nevertheless, no definitive conclusions concerning coronary artery disease can be drawn, as there was also a trend for more frequent screening in the R482 group, most likely because those patients more often also had diabetes.

From a metabolic perspective, the prevalence of diabetes and hypertriglyceridaemia was higher in R482 than in non-R482 carriers. The R482 carriers also had lower BMIs, and lower levels of leptin and fat mass, but higher MRI-assessed visceral fat levels, than non-R482 carriers, which is in accordance with the lipodystrophic phenotype of R482 carriers. Interestingly, the intra-/total abdominal fat-mass ratio, a hallmark of lipodystrophy [START_REF] Sa | Quantitative and qualitative differences in subcutaneous adipose tissue stores across lipodystrophy types shown by magnetic resonance imaging[END_REF], was significantly higher in the non-R482 vs control group, thereby arguing for a specific lipodystrophy fat distribution even in patients with non-R482 mutations. It is also noteworthy that the frequency of diabetes reached 40% in the non-R482 group, but often required OGTT to make the diagnosis. Diabetes is known to promote atherosclerosis and findings in vitro favour a direct proatherogenic effect of the LMNA R482W mutation in endothelial cells, which is consistent with the trend towards a greater frequency of coronary heart disease in the R482 group [START_REF] Hegele | Premature atherosclerosis associated with monogenic insulin resistance[END_REF][START_REF] Afonso | LMNA mutations resulting in lipodystrophy and HIV protease inhibitors trigger vascular smooth muscle cell senescence and calcification: Role of ZMPSTE24 downregulation[END_REF]. However, the diagnosis of lipodystrophy is easier to make, as the metabolic syndrome (MetS) is more severe in women than in men [START_REF] Nishiuchi | Gene-based risk stratification for cardiac disorders in lmna mutation carriers[END_REF][START_REF] Araújo-Vilar | Phenotypic gender differences in subjects with familial partial lipodystrophy (Dunnigan variety) due to a nuclear lamin A/C R482W mutation[END_REF][START_REF] Donadille | Etiological investigations in apparent type 2 diabetes: when to search for lamin A/C mutations?[END_REF]. This gender difference in disease phenotype might be modulating the expression of coronary artery disease, thereby worsening the cardiovascular prognosis for women which, before menopause, is usually considered better than that for men. Also, our results support more systematic screening for MetS, especially by OGTT, in the non-R482 group, and for silent myocardial ischaemia in both groups of mutations, especially in cases with diabetes.

Finally, comparisons between R482 and non-R482 carriers have revealed different cardiometabolic phenotypes with different types of risk, a point that has never been emphasized before. Patients with R482 mutations have an FPLD2 phenotype, which may sometimes be associated with other features, such as myopathy [START_REF] Vantyghem | Patients with familial partial lipodystrophy of the Dunnigan type due to a LMNA R482W mutation show muscular and cardiac abnormalities[END_REF]. In this group, diabetes is present in 80% of cases, yet the cardiac phenotype seems less severe than in non-R482 cases, and is mostly the result of insulin resistance and atherosclerosis. These findings are concordant with the low frequency of arrhythmia events reported in patients with R482 mutations in the literature in contrast to the non-negligible frequency of coronary artery disease [START_REF] Hegele | Premature atherosclerosis associated with monogenic insulin resistance[END_REF][START_REF] Afonso | LMNA mutations resulting in lipodystrophy and HIV protease inhibitors trigger vascular smooth muscle cell senescence and calcification: Role of ZMPSTE24 downregulation[END_REF]. Indeed, patients with non-R482 mutations often have a severe cardiac phenotype, such as an initial conduction or rhythm disorder with a risk of sudden cardiac death, and diabetes is present in 40% of these patients. Thus, such patients should be screened for ventricular arrhythmias and dilated cardiomyopathy by Holter monitoring and echocardiography, with consideration of electrophysiological studies, CIED use or cardiac transplantation where appropriate.

In any case, the presence of ventricular arrhythmias, especially when associated with diabetes or a familial history of CIED use, should prompt LMNA genetic testing and subsequent ICD implantation.

The reason for the different expressions of the disease in our two patient groups remains unexplained.

Limited peripheral adipose tissue storage capacity has recently been emphasized in the pathogenesis of human insulin resistance [START_REF] Lotta | Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance[END_REF]. Frequent and/or early atherosclerotic complications could be mediated through oxidative stress and mitochondrial dysfunction. Recent studies suggest that LMNA mutations affect epigenetic regulation of developmental pathways, and might alter myogenesis and adipogenesis processes in a genotype-specific manner [START_REF] Perovanovic | Laminopathies disrupt epigenomic developmental programs and cell fate[END_REF][START_REF] Paulsen | Chrom3D: three-dimensional genome modeling from Hi-C and nuclear lamin-genome contacts[END_REF][START_REF] Oldenburg | A lipodystrophy-causing lamin A mutant alters conformation and epigenetic regulation of the anti-adipogenic MIR335 locus[END_REF]. These mechanisms, however, have yet to be explored.

In conclusion, these results suggest that cardiac follow-up of laminopathies might be adjusted according to genotype, with more aggressive screening for arrhythmias in non-R482 patients. Also, it is important to consider laminopathies as a differential diagnosis when faced with an unexplained rhythm disorder, especially if there is a familial history of CIED use, sudden cardiac death and/or a personal history of diabetes. In addition, coronary artery disease should not be overlooked, especially when diabetes is present.

FIGURE LEGENDS

Fig. 1 .

 1 Fig. 1. Flow chart of patient recruitment for the study.

Fig. 2 .

 2 Fig. 2. Main differences between R482 and non-R482 groups in cardiac and metabolic outcomes.

Fig. S1 .

 S1 Fig. S1. Comparison of intra-abdominal/total abdominal fat-mass ratios as measured by magnetic

Figure 1 FlowchartFig 1 Figure 2

 112 Figure 1

ACKNOWLEDGEMENTS

Our team is part of the European Consortium of Lipodystrophies (ECLIP) of the FIRENDO Reference Centre for Rare Diseases PRISIS (Rare Pathologies of Insulin Resistance and Insulin Sensitivity), and participates in the OPALE registry (ClinicalTrials.gov identifier: NCT03058185). We are indebted to the Clinical Research Department of Lille University Hospital and to Clinical Research nurses Mrs Paranthoen and Mrs Lavergne.

FUNDING

This study was supported by the French Ministry of Health (PHRC 2009; Clinical Trial Registration Number: Clin.gov2009-AO-1169-48).

Appendix supplementary material

Supplementary material (Fig. S1) associated with this article can be found at http://www.scincedirect.com at doi . . .


MD, MSc Thiébaud Picart
email: thiebaud.picart@chu-lyon.fr

MD Marc Barritault
email: marc.barritault@chu-lyon.fr

MD Emile Simon
email: emile.simon@chu-lyon.fr

PhD Philip Robinson
email: philip.robinson@chu-lyon.fr

Cédric Barrey
email: cedric.barrey@chu-lyon.fr

MD David Meyronet
email: david.meyronet@chu-lyon.fr

MD Patrick Mertens
email: patrick.mertens@chu-lyon.fr

Cécric Barrey

Claude Bernard Lyon

MD Cécric Barrey

Anatomical and Histological Analysis of a Complex Structure Too Long Considered a Simple Ligament: The Filum Terminale

Keywords: Anatomy, Ependymoma, Filum Terminale, Histology, Spine, Tethered Cord Syndrom

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

INTRODUCTION

The development of the Filum Terminale (FT) results from the dedifferentiation of the caudal part of the embryonic and fetal Spinal Cord (SC). [START_REF] Streeter | Factors involved in the formation of the filum terminale[END_REF][START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Jang | The Filum Terminale Revisited: A Histological Study in Human Fetuses[END_REF][START_REF] Pearson | Observations on the caudal end of the spinal cord[END_REF] After birth, the FT connects the Conus Medullaris (CM) and the coccygeal periosteum. [START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF] More precisely, the intradural FT (iFT) is the cranial portion of the FT that lies between the CM and the DS, while the extradural FT (eFT = Ligamentum Coccygeale), constitutes the caudal portion of the FT, thus linking the Dural Sac (DS) and the coccyx. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF][START_REF] Reimann | Vertebral level of termination of the spinal cord with report of a case of sacral cord[END_REF] The anatomical studies dedicated to the FT are relatively scarce and most are not recent. [START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Reimann | Vertebral level of termination of the spinal cord with report of a case of sacral cord[END_REF][START_REF] Fontes | Ultrastructural study of the filum terminale and its elastic fibers[END_REF][START_REF] Harmeier | The normal histology of the intradural filum terminale[END_REF][START_REF] Mccotter | Regarding the length and extent of the human medulla spinalis[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Trotter | Variations of the sacral canal; their significance in the administration of caudal analgesia[END_REF][START_REF] Needles | The caudal level of termination of the spinal cord in american whites and american negroes[END_REF][START_REF] Thomson | Fifth Annual Report of the Committee of Collective Investigation of the Anatomical Society of Great Britain and Ireland for the Year 1893-94[END_REF] Histologically, the adult FT has long been considered as a fibrous structure and the identification of cellular elements in its vicinity is recent. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF] Obviously, malformations or tumors of the FT are likely to generate neurological disorders because of its close relationship with the SC and the Cauda Equina (CE). [START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF] The aim of the present study was therefore to update the anatomical and histological description of the FT, and to integrate these data in a physiopathological context.

MATERIAL AND METHODS

Ten human cadavers from adults who donated their bodies to science for anatomical purposes were included. For each specimen, demographic characteristics and morphometric parameters were collected. As summarized in Table 1, 50% of specimens were women and 50% were men. With the exception of degenerative scoliosis in one specimen, no obvious neural or orthopedic abnormality was noted.

Dissection protocol

After prone positioning, a midline dorsal longitudinal skin and aponeurotic incision was performed from the ninth thoracic vertebra to the coccyx. After dissection of the subcutaneous and muscular plans, spinous processes and laminae from the tenth thoracic vertebra to the fifth sacral vertebra and the coccyx were exposed. A laminectomy was performed from the tenth thoracic vertebra cranially to the sacral hiatus caudally in order to expose the DS, the emerging nervous roots, and the eFT. After careful longitudinal opening of the DS from the tenth thoracic vertebra to its termination, the CM, the CM-iFT junction, the iFT and the CE were exposed. By convention, the end of the CM was defined as the level of emergence of the last coccygeal roots [START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF] .

Anatomical study

The shape of the FT, its relationships with the arachnoid and the CE were thoroughly examined. Then, the FT was carefully removed "en bloc" with a collar of dura-matter to preserve the FT-DS junction. Caudally, a fragment of coccygeal periosteum was detached to guarantee the integrity of the eFT. The levels of the CM-iFT junction, FT-DS junction, and the insertion of the FT on the coccygeal periosteum were noted thanks to benchmarks precisely set before the removal of the FT. As previously proposed [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Reimann | Vertebral level of termination of the spinal cord with report of a case of sacral cord[END_REF][START_REF] Mccotter | Regarding the length and extent of the human medulla spinalis[END_REF][START_REF] Needles | The caudal level of termination of the spinal cord in american whites and american negroes[END_REF][START_REF] Thomson | Fifth Annual Report of the Committee of Collective Investigation of the Anatomical Society of Great Britain and Ireland for the Year 1893-94[END_REF] , each vertebra was divided into thirds to increase the precision of the description. The lengths of the iFT and eFT, the diameters of the iFT at different points (origin, first quarter, half, third quarter, and termination), and the diameters of the eFT (origin, half, and termination) were determined with a digital Vernier caliper.

Histological study

The specimens were fixed in zinc formalin. After paraffin embedding, longitudinal 4µm-thick sections were made. Sections were stained with hemalum-phloxine-saffron to highlight cellular and fibrous elements. Complementary staining using luxol-fast-blue, neurofilament, NeuN, and actin were performed to highlight myelinated structures, neural structures, Ganglion Cells (GC) and Smooth Muscular Cells (SMC), respectively.

Statistical analysis

A statistical analysis was performed using R software (R foundation for Statistical Computing, Vienna, Austria, version3.5.1) to investigate correlations using the Pearson correlation coefficient and the Spearman correlation coefficient for quantitative and ordinal variables, respectively. The level of significance was set at 5% (p<.05).

RESULTS

Microsurgical anatomy

The iFT was easily differentiated from the CE roots (CEr) thanks to its characteristic pearly white color. Conversely, the eFT did not always have the same hue and could also appear greyish or translucent.

iFT

As the very proximal portion of the iFT was anterior to the lumbar and sacrococcygeal roots, the CM-iFT transition was not directly seen after opening the dura mater (Figure 1A). The exposure of the CM-iFT transition revealed the progressive appearance of fibrous tissue organized in linear spans within the CM. In parallel, the nervous tissue became less abundant and disappeared completely about 1 millimeter below the emergence of the last coccygeal roots. At this level, the fibrous spans converged towards a common origin which should be considered as the starting point of the FT (Figure 1B).

The iFT had a close relationship with the CEr and its relative position compared to the roots changed gradually during its intradural course. Its central and distal parts were clearly posterior to the roots. Adhesion of nervous rootlets to the iFT were observed. They were located in the proximal portion of the iFT in three cases (30%) and in the most distal portion of the iFT in five cases (50%). In two cases (20%), a rootlet was intertwined with the FTi.

Only the posterior surface of the iFT was in contact with the arachnoid membrane. The contact was direct in three cases (30%) while a film of cerebrospinal fluid was interposed between these two structures in the remaining cases (70%). The iFT was in the same subarachnoidal compartment as the CEr except in one specimen in which there was a longitudinal septum isolating the iFT and some CEr from the remaining roots. The iFT was either hourglass shaped (70%) or cone-shaped (30%).

FT-DS junction

There was a continuum between the FT and the DS (Figure 1C). This fusion area was most frequently centered on the median line (80%) and sometimes lateralized on the right (10%) or left (10%)

eFT

The eFT lay against the posterior vertebral wall, anteriorly to the CE, in an adipose environment until it came out of the sacral channel through the sacral hiatus. Its path continued either in contact with the coccygeal periosteum or with the posterior sacrococcygeal ligament. The eFT was free of any attachment, except in its last centimeter where osteo-periosteal attachments were inconstantly found.

The periosteal insertion of the eFT was located on the posterior surface of the coccyx and was polymorphic. In its most common form, it was Y-shaped and located on the midline (50%) or lateralized on the right (10%). In the remaining cases, there were either a single bundle inserted in a punctual zone (20%, Figure 1D) or several bundles (at least three) that were fan-shaped (20%, Figure 1E). The shape of the eFT was more constant since 9 eFTs (90%) were cone-shaped and one (10%) hourglass-shaped.

Vascular elements

Proximally, the arterial vasculature of the FT was provided by a small artery that was connected to the anterior spinal artery. A bifurcation was sometimes noticed at the proximal part of the iFT but, in this case, one of the two branches was dominant. Distally, the artery of the iFT was connected with several vessels distributed in the iFT.

A vein of variable caliber, often thin (80%) but appearing dilated in two specimens (20%), was found posteriorly to the iFT artery. Insofar as this vein had no collateral branches, it seemed improbable that it could provide the venous drainage of the iFT.

Morphometric parameters

As summarized in Table 2, the mean lengths of the iFT and eFT were 167.13 millimeters and 87.59 millimeters, respectively. The mean diameter of the iFT ranged from 1.84 millimeter at its origin to 0.74 millimeter at its termination with an intermediate shrinkage. In 2 specimens, the initial diameter of the iFT excessed 2 millimeters and in one of them, the CM was in a low position (L2L3 disc). The mean diameter of the eFT decreased progressively from 1.05 millimeter to 0.59 millimeter, consistently with the predominant cone-shape.

The distribution of the CM-iFT junction was Gaussian, centered on the L1L2 in female specimens and more variable in male specimens. The levels of the iFT-DS-eFT and eFT-coccyx junctions were more constant and centered on the lower third of S2 (60% of cases) and the second coccygeal vertebra (80% of cases), respectively (Figure 2).

There was no significant correlation between the height of the specimen and the iFT length (ρ=0.43, p=0.21) or the eFT length (ρ=0.37, p=0.29). The iFT and eFT lengths were also not correlated (ρ=0. 34, p=0.32). The comparison of the different diameters of iFT and eFT with each other in all possible combinations found only one statistically significant correlation: the proximal diameter of the eFT was correlated with the distal diameter of the eFT (ρ=.94, p=0.02).

The level of CM-iFT junction was significantly correlated with the iFT length (ρ=-0.67, p=0.03) but neither with the specimen height (ρ=-0.12, p=0.73) nor the length of the eFT (ρ=0.23, p=0.51). The level of iFT-DS-eFT junction was neither correlated with the specimen height (ρ=-0.22, p=0.52), the length of the iFT (ρ=-0.22, p=0.53), the length of the eFT (ρ=-0.56, p=0.09), nor the level of CM-iFT junction (ρ=-0.06, p=0.85).

Mobilization of the FT

Although cranial and caudal tractions of the iFT resulted in concomitant mobilization of the CM and DS, respectively, the movements were neither transmitted to the eFT nor to the CEr. Reciprocally, cranial and caudal tractions of the eFT were transmitted to all the CEr in their extradural portion. Whereas very few movements were transmitted to the DS, the iFT and the CM remained static.

Histological structure

As macroscopically observed, the CM-iFT transition is progressive since there is a gradual depletion of glial and neuronal cell elements with a strong enrichment in connective tissue. Peripherally, the pia mater was thickened. At this level, included axons, myelinated or not, were found in all specimens (Figure 3A). The iFT-CM junction was also enriched in ependymal cells organized in tubes or rosettes (Figure 3B). The persistence of ependymal cells, randomly arranged in islets, beyond the first third of the iFT, was noted in five specimens (50%). NeuN-immunopositive GC were randomly distributed within the CM-iFT junction (Figure 3C and insert) and in the first half of the iFT (Figure 3D) in five specimens (50%). In nine specimens (90%), amyloid bodies were present within the cellular areas of the proximal part of the iFT (Figure 3E). Nervous elements were abundant at the close periphery of the iFT (Figure 3F) and were sometimes myelinated, as highlighted by luxol-fast-blue staining (Figure 3G), but also within the iFT (Figure 3H,3I).

In five specimens (50%), the iFT-DS junction was particularly enriched in GC which were anarchically disposed (Figure 3J).

In the eFT of one specimen, a structure composed of a circular cluster of neuronal cells organized in sheets was found (Figure 3K) and probably corresponded to a mechanoreceptor. The proximal eFT contained included axons (Figure 3L) and calcospherites within connective tissue areas (Figure 3M). SMC were constantly present in the whole eFT (Figure 3L and3N) as highlighted by the immunostaining for actin (Figure 3N insert).

Adipocytes were found around the eFT and within the most distal portion of the eFT (Figure 3O), as was fibrous tissue (Figure 3P).

DISCUSSION

Anatomical considerations

The definition of the iFT origin remains elusive. [START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF] In fine, the CM-iFT junction is not punctual but instead corresponds to a transition zone where the nervous tissue of the CM is progressively replaced by fibrous tissue spans converging at a point that seems to relevantly and objectively define the origin of the iFT.

The iFT-DS-eFT junction has been very rarely studied. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF] It has either been described as a "perforation" of the DS by the iFT [START_REF] Moore | The clinical use of sodium fluorescein and radioactive diiodofluorescein in the localization of tumors of the central nervous system[END_REF] or as a "fusion", given the continuum between the FT fibrous stroma and the dura-mater. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF] The description herein is in accordance with this second term and remains consistent with the embryological data. Indeed, the FT originates from the dedifferentiated distal portion of the embryonic SC which fuses with the dura-mater that previously covered it. [START_REF] Streeter | Factors involved in the formation of the filum terminale[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF] In series reported herein, the iFT-DS-eFT junction was located outside the sagittal medial line in 20% of cases while it was so in 11% of cases in the study reported by Hansasuta et al. [START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF] This anatomical data must be kept in mind when looking for the iFT in its distal portion through a restricted surgical approach.

The termination of the eFT has been considered as a blend of the eFT fibers and the coccygeal periosteum. [START_REF] Tubbs | The filum terminale externum[END_REF] The polymorphism of this termination with one or several bundles described in the present study is original.

Analysis of morphological and biomechanical parameters

The mean lengths of the iFT and eFT herein are approximately 10 mm longer than the values previously published 2,7,9,10,12,17 (Table 3) probably because the specimens were not fixed with formaldehyde that is responsible for retraction and restriction of the measured lengths. [START_REF] Benet | Novel embalming solution for neurosurgical simulation in cadavers[END_REF] We confirmed the absence of correlation between the specimen height and the iFT length. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Nasr | Morphometric parameters and histological Study of the Filum Terminale of Adult human cadavers and MR images[END_REF] Conversely, the correlation between the specimen height and the eFT length previously described by De Vloo et al. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF] , Pinto et al. [START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF] and Nasr et al. [START_REF] Nasr | Morphometric parameters and histological Study of the Filum Terminale of Adult human cadavers and MR images[END_REF] was not found. The diameters of the iFT and the eFT are comparable to those obtained in other studies [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Fontes | Ultrastructural study of the filum terminale and its elastic fibers[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Nasr | Morphometric parameters and histological Study of the Filum Terminale of Adult human cadavers and MR images[END_REF] (Table 3). However, the correlation between the 4 most caudal diameters of the iFT and between the 3 diameters of the eFT, identified by De Vloo et al. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF] were not validated in this series.

The median level of the CM-iFT junction is concordant with those of other cadaveric [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Reimann | Vertebral level of termination of the spinal cord with report of a case of sacral cord[END_REF][START_REF] Mccotter | Regarding the length and extent of the human medulla spinalis[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Trotter | Variations of the sacral canal; their significance in the administration of caudal analgesia[END_REF][START_REF] Needles | The caudal level of termination of the spinal cord in american whites and american negroes[END_REF][START_REF] Thomson | Fifth Annual Report of the Committee of Collective Investigation of the Anatomical Society of Great Britain and Ireland for the Year 1893-94[END_REF] and remnologic series [START_REF] Macdonald | Level of termination of the spinal cord and the dural sac: a magnetic resonance study[END_REF] (Table 4). While this junction is usually lower in men than in women [START_REF] Thomson | Fifth Annual Report of the Committee of Collective Investigation of the Anatomical Society of Great Britain and Ireland for the Year 1893-94[END_REF][START_REF] Macdonald | Level of termination of the spinal cord and the dural sac: a magnetic resonance study[END_REF] , we observed the converse (L1L2 disc in women, T12L1 disc in men).

The median level of the iFT-DS junction corresponded to the lower third of S2, which is onethird vertebra lower than in the other studies [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Trotter | Variations of the sacral canal; their significance in the administration of caudal analgesia[END_REF][START_REF] Macdonald | Level of termination of the spinal cord and the dural sac: a magnetic resonance study[END_REF] (Table 4) and did not vary according to the sex, contrary to what has been observed by Mac Donald et al. [START_REF] Macdonald | Level of termination of the spinal cord and the dural sac: a magnetic resonance study[END_REF] These conflicting data could suggest that the level of the CM-iFT and iFT-DS-eFT junctions are gender independent.

The eFT ended at the second coccygeal vertebra in 80% of cases, which confirms that its termination level is more stable than the levels of the CM-iFT and iFT-DS-eFT junctions. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF] The level of the CM-iFT junction was correlated with the length of the iFT, which is consistent with the relatively low variability of the iFT-DS junction level. This correlation had already been demonstrated by Pinto et al [START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF] but is not consensual. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF] Besides, we do not confirm the significant correlation between the level of the iFT-DS junction and the length of the eFT 2 , nor the correlation between the levels of the CM-iFT and the iFT-DS-eFT junctions. [START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF] These discordant results could be related to statistical power and a larger sample may be required to conclude. Ethnical differences may also have contributed as not all previously published data were obtained in European specimens. Nevertheless, no correlation has been constantly found in the other studies. Subsequently, the clinical application of these results requires caution because of several biases including measurements after removal, in old specimens and overestimation of nervous structures in cadavers. [START_REF] Choi | Spinal cord diameters in cadaveric specimens and magnetic resonance scans, to assess embalming artefacts[END_REF] The tractions performed during the dissections highlighted the damping role played by the FT, since its mobilization in one of its compartments (iFT or eFT) was not transmitted to the elements of the other compartment. Accordingly, Tubbs et al. noticed that the traction of the eFT induced no movement of the iFT [START_REF] Tubbs | The filum terminale externum[END_REF] . The application of increasing loads demonstrated that the FT loses its elasticity more rapidly than the SC in animals and in humans, which confirms that the FT protects the SC against mechanical stress. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF][START_REF] Tani | Changes in lumbosacral canal length during flexion and extension--dynamic effect on the elongated spinal cord in the tethered spinal cord[END_REF][START_REF] Tani | Extensibility of the lumbar and sacral cord. Pathophysiology of the tethered spinal cord in cats[END_REF] The identification of a multicellular structure, that may correspond to a mechanoreceptor, close to SMC in one specimen, could suggest that the eFT is able to self-regulate its tension. For some authors, the loss of FT elastic properties is linked to histological changes [START_REF] Selçuki | Is a filum terminale with a normal appearance really normal? Childs Nerv Syst ChNS[END_REF][START_REF] Tehli | A comparative study of histopathological analysis of filum terminale in patients with tethered cord syndrome and in normal human fetuses[END_REF][START_REF] Liu | SEM study on filum terminale with tethered cord syndrome[END_REF][START_REF] George | The immunohistochemical profile of the tethered filum terminale[END_REF] , results in a lowering of the CM-iFT junction (below L2 body [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF] ), and could lead to myelopathy development described as thethered cord syndrome. [START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF][START_REF] Fontes | Ultrastructural study of the filum terminale and its elastic fibers[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Filippidis | Spinal cord traction, vascular compromise, hypoxia, and metabolic derangements in the pathophysiology of tethered cord syndrome[END_REF][START_REF] Yamada | Pathophysiology of tethered cord syndrome and similar complex disorders[END_REF] From embryology to histology and histopathology During secondary neurulation, the FT originates from the SC located downstream of the 31 st metameric segment. [START_REF] Streeter | Factors involved in the formation of the filum terminale[END_REF] Vacuolization and apoptotic phenomena [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Selçuki | Is a filum terminale with a normal appearance really normal? Childs Nerv Syst ChNS[END_REF][START_REF] Hertzler | Tethered cord syndrome: a review of the literature from embryology to adult presentation[END_REF] are responsible for the dedifferentiation of this structure that is redifferentiated into fibrous tissue. [START_REF] Streeter | Factors involved in the formation of the filum terminale[END_REF][START_REF] Pearson | Observations on the caudal end of the spinal cord[END_REF][START_REF] Saker | The Human Central Canal of the Spinal Cord: A Comprehensive Review of its Anatomy[END_REF] Microscopic examination of human fetal SC reveals GC, ependymal and neuroblastic or neuronal cell bodies that are or not subjected to degenerative processes. [START_REF] Streeter | Factors involved in the formation of the filum terminale[END_REF][START_REF] Tehli | A comparative study of histopathological analysis of filum terminale in patients with tethered cord syndrome and in normal human fetuses[END_REF][START_REF] Gamble | Electron microscope observations upon the conus medullaris and filum terminale of human fetuses[END_REF] Subsequently, the adult FT is likely to contain the same tissue elements as the SC. [START_REF] Pearson | Observations on the caudal end of the spinal cord[END_REF][START_REF] Harmeier | The normal histology of the intradural filum terminale[END_REF] Furthermore, the recent identification of neuronal and glial stem cells in rat and human FT [START_REF] Chrenek | Characterization of the Filum terminale as a neural progenitor cell niche in both rats and humans[END_REF] confirms that this structure is far more complex than a simple ligament.

The performance of longitudinal sections is original. Neuronal bodies, axons that were sometimes myelinated and ependymocytes were identified within the iFT, as previously described [START_REF] Jang | The Filum Terminale Revisited: A Histological Study in Human Fetuses[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF][START_REF] Gamble | Electron microscope observations upon the conus medullaris and filum terminale of human fetuses[END_REF][START_REF] Choudhary | Extradural myxopapillary ependymoma with sacral osteolysis--a case report[END_REF] This could explain the development of ependymomas within the iFT [START_REF] Tubbs | The filum terminale externum[END_REF] .

Nevertheless, the decreasing frequency of these elements following a cranio-caudal gradient usually described [START_REF] Jang | The Filum Terminale Revisited: A Histological Study in Human Fetuses[END_REF][START_REF] Tubbs | The filum terminale externum[END_REF][START_REF] Choudhary | Extradural myxopapillary ependymoma with sacral osteolysis--a case report[END_REF] , needs to be nuanced; even though the cell density is higher proximally, islands of ependymocytes are identifiable all along the iFT and are rather randomly distributed.

Consistent with the only description of the iFT-DS junction 2 , we also observed that the outer fibrous layer of the DS blends almost imperceptibly into the fibrous stroma of the FT.

Strikingly, we noted that the density of GC was particularly high at this level, that would explain the occurrence of gangliocytomas, paragangliomas [START_REF] Pytel | Ganglioneuromatous paraganglioma of the cauda equina--a pathological case study[END_REF][START_REF] Shankar | Composite ganglioneuroma-paraganglioma of the filum terminale[END_REF] and ganglioneuroblastomas. [START_REF] Okudera | Ganglioneuroblastoma of filum terminale: case report[END_REF] Examination of the eFT found axonal structures, SMC, and adipose tissue. The absence of ependymal cells, as previously described [START_REF] Tubbs | The filum terminale externum[END_REF] , indicates that coccygeal ependymomas would more probably arise from subcutaneous coccygeal ependymal residues rather than from the eFT. [START_REF] Tubbs | The filum terminale externum[END_REF] Contrary to Tubbs et al., we did not find residues of meningothelial tissue or GC within the eFT. [START_REF] Tubbs | The filum terminale externum[END_REF] The presence of adipose and meningothelial tissue within FT is sometimes considered as pathological. [START_REF] Tehli | A comparative study of histopathological analysis of filum terminale in patients with tethered cord syndrome and in normal human fetuses[END_REF][START_REF] Pang | Limited dorsal myeloschisis: a notso-rare form of primary neurulation defect[END_REF] This remark should consequently only apply to the iFT.

CONCLUSIONS

In accordance with their common embryological origin, there is a true anatomical and histological continuum between the CM and the FT. The biomechanical role played by the FT is particularly important and the identification of a mechanoreceptor, thanks to longitudinal sections, suggests potential self-regulatory mechanisms. Subsequently, this study definitively confirms that the FT should no longer be considered as a simple ligament but rather as a complex fibro-cellular structure.

The CM-iFT junction contains small rootlets included in the iFT stroma (A), ependymal cells organized as a rosette (B) and ganglion cells randomly distributed at the CM-iFT junction (C) which display positive immunostaining for NeuN (insert). The First half of the iFT contains isolated ganglion cells (D) and amyloid bodies which are frequently found in cellular areas (E). Nervous rootlets are directly contiguous to the iFT (F).

The second half of the iFT is also contiguous to myelinated rootlet, as highlighted by luxol-

fast-blue coloration (G). Groups of axons are included in the iFT (H), as attested by the immunopositivity for neurofilament (I).

In the FT-DS junction, the density of ganglion cells is particularly high (J).

In the first half of the eFT, a structure composed of a circular cluster of neuronal cells organized in sheets was found and probably corresponded to a mechanoreceptor (K). Group of axons are surrounded by linear groups of smooth muscle cells (L). Calcospherites (one of which is partially broken) are found in connective tissue (M).

The Second half of the eFT contains smooth muscle cells (N), as attested by the positive immunostaining for actin (insert)

  Word count: 2993 wordsFinancial support: No funding was received for this research.

FIGURE CAPTIONS Fig. 1 Fig. 2

 CAPTIONS12 FIGURE CAPTIONS

Fig. 3

 3 Fig. 3 Schematic representation of the Filum Terminale with pictures corresponding to the different levels (x400).

Table 1 :

 1 Demographic characteristics of the specimens

	. A huge amount of adipose cells is found around the distal
	eFT. Adipose cells are also present within the eFT (O). Winding-up of fibrous tissue are
	observed (P).
	CM = Conus Medullaris, DS = Dural Sac, eFT = extradural Filum Terminale, iFT =
	intradural Filum Terminale

Table 2 :

 2 Morphometric parameters of the different segments of the intradural Filum Terminale and extradural Filum Terminale in the present series (expressed in millimeters) eFT = extradural Filum Terminale, iFT = intradural Filum Terminale, SD = Standard

	Deviation

Table 3 :

 3 Comparison of the length and diameter of the intradural Filum Terminale andextradural Filum Terminale (expressed in millimetres), according to other published series2, 7,

9, 10, 12, 17

eFT = extradural Filum Terminale, iFT = intradural Filum Terminale

Conflict of interest:

The authors declare that they have no conflict of interest.

Ethical approval:

No ethics committee approval was required as the human cadavers were donated to the laboratory for anatomical and/or histological purposes. [START_REF] Vloo | The Filum Terminale: A Cadaver Study of Anatomy, Histology, and Elastic Properties[END_REF][START_REF] Hansasuta | Filum terminale fusion and dural sac termination: study in 27 cadavers[END_REF][START_REF] Reimann | Vertebral level of termination of the spinal cord with report of a case of sacral cord[END_REF][START_REF] Mccotter | Regarding the length and extent of the human medulla spinalis[END_REF][START_REF] Pinto | Anatomic study of the filum terminale and its correlations with the tethered cord syndrome[END_REF][START_REF] Trotter | Variations of the sacral canal; their significance in the administration of caudal analgesia[END_REF][START_REF] Needles | The caudal level of termination of the spinal cord in american whites and american negroes[END_REF][START_REF] Thomson | Fifth Annual Report of the Committee of Collective Investigation of the Anatomical Society of Great Britain and Ireland for the Year 1893-94[END_REF][START_REF] Macdonald | Level of termination of the spinal cord and the dural sac: a magnetic resonance study[END_REF] CM = Conus Medullaris, iFT = intradural Filum Terminale, DS = Dural Sac