Siriane Lefèvre

Marie-Pierre Audrézet

Jean-Michel Halimi

Hélène Longuet

Frank Bridoux

Laure Ecotière

Jf Augusto

Agnès Duveau

Eric Renaudineau

Diagnosis and Risk Factors for Intracranial Aneurysms in Autosomal Polycystic Kidney Disease: A cross-sectional study from the Genkyst Cohort

Keywords: autosomal dominant polycystic kidney disease (ADPKD), genetics, intracranial aneurysms, PKD1, PKD2, risk factors, subarachnoid hemorrhage

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

A cross-sectional study from the Genkyst Cohort Siriane Lefèvre (1, 2); Marie-Pierre Audrézet [START_REF] Suwabe | Epidemiology of Autosomal Dominant Polycystic Kidney Disease in Olmsted County[END_REF][START_REF] Gall | Genetic Complexity of Autosomal Dominant Polycystic Kidney and Liver Diseases[END_REF]; Jean-Michel Halimi [START_REF] Gall | Autosomal dominant polycystic kidney disease[END_REF][START_REF] Gall | Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease[END_REF]; Hélène Longuet (4);

Frank Bridoux [START_REF] Porath | Mutations in GANAB, Encoding the Glucosidase IIalpha Subunit, Cause Autosomal-Dominant Polycystic Kidney and Liver Disease[END_REF]; Laure Ecotière [START_REF] Porath | Mutations in GANAB, Encoding the Glucosidase IIalpha Subunit, Cause Autosomal-Dominant Polycystic Kidney and Liver Disease[END_REF]; Jean-François Augusto [START_REF] Besse | ALG9 Mutation Carriers Develop Kidney and Liver Cysts[END_REF]; Agnès Duveau [START_REF] Besse | ALG9 Mutation Carriers Develop Kidney and Liver Cysts[END_REF]; Eric Renaudineau [START_REF] Besse | Isolated polycystic liver disease genes define effectors of polycystin-1 function[END_REF]; Cécile Vigneau [START_REF] Irazabal | Extended Follow-Up of Unruptured Intracranial Aneurysms Detected by Presymptomatic Screening in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF]; Thierry Frouget [START_REF] Vernooij | Incidental findings on brain MRI in the general population[END_REF]; Christophe Charasse [START_REF] Vlak | Prevalence of unruptured intracranial aneurysms, with emphasis on sex, age, comorbidity, country, and time period: a systematic review and metaanalysis[END_REF]; Lorraine Gueguen [START_REF] Xu | Screening for intracranial aneurysm in 355 patients with autosomal-dominant polycystic kidney disease[END_REF], Régine Perrichot [START_REF] Chauveau | Intracranial aneurysms in autosomal dominant polycystic kidney disease[END_REF]; Grégoire Couvrat [START_REF] Lozano | Familial intracranial aneurysms[END_REF]; Guillaume Seret [START_REF] Schievink | Saccular intracranial aneurysms in autosomal dominant polycystic kidney disease[END_REF]; GENKYST Study Group; Yannick Le Meur [START_REF] Shaw | Epidemiology of patients in England and Wales with autosomal dominant polycystic kidney disease and end-stage renal failure[END_REF][START_REF] Abulhasan | Mortality after Spontaneous Subarachnoid Hemorrhage: Causality and Validation of a Prediction Model[END_REF]; Emilie Cornec-Le Gall ( that age, the risk continued to increase more markedly in female patients, reaching 10.8% vs 5.4% at 70 y. The diagnosis rate of IAs was more than twofold higher in PKD1 compared to PKD2 with no influence of PKD1 mutation type or location. In multivariate analysis, female sex, hypertension <35 y, smoking and PKD1 genotype were associated with an increased risk for diagnosis of IAs.

Conclusions. This study presents epidemiological data reflecting real-life clinical practice. The increased risk for IAs in postmenopausal women suggests a possible protective role of estrogen.

O R I G I N A L U N E D I T E D M A N U S C R I P T KEY LEARNING POINTS

What is already known about this subject?

 The most frequent and severe vascular feature in ADPKD is the development of intracranial aneurysm (IA).  IA prevalence is fivefold higher in ADPKD than in the general population, with estimates ranging from 9 to 12%.  The only identified risk factor is the presence of a familial history of unruptured or ruptured IA.

What this study adds?

 This study provides information about prevalence of detected IAs in one of the largest population-based cohorts of ADPKD patients. The cumulative probabilities of a previous diagnosis of IAs in the Genkyst cohort were 3.9, 6.2 and 8.1% at 50, 60 and 70 y.  The risk of diagnosis of ruptured or unruptured IAs appear to be higher in women, especially after 50 y. This parallels observations made in the non-ADPKD population and suggests a possible protective role of estrogens.  Multivariate analysis shows that female sex, age at diagnosis of hypertension <35y, smoking exposure and PKD1 genotype are associated with an increased risk for diagnosis of IAs.

What impact this may have on practice or policy?

 This study provides a real-life picture of diagnosis of IAs in ADPKD and may increase awareness and knowledge regarding prevalence and risk factors for IAs in ADPKD patients.  Policies regarding screening for IAs in ADPKD patients differ accross countries and centers, and no clear recommandations have been made. Our study suggests that in at-risk women, screening should be probably repeated later in life than in men.

O R I G

I N A L U N E D I T E D M A N U S C R I P T

INTRODUCTION

Autosomal dominant polycystic kidney disease (ADPKD) affects ~39 to 68 individuals in 10000. [START_REF] Shaw | Epidemiology of patients in England and Wales with autosomal dominant polycystic kidney disease and end-stage renal failure[END_REF][START_REF] Suwabe | Epidemiology of Autosomal Dominant Polycystic Kidney Disease in Olmsted County[END_REF] Mutations in PKD1 and PKD2, which encode polycystin 1 (PC1) and polycystin 2 (PC2), respectively, account for ~75% and ~18% of the ADPKD pedigrees. [START_REF] Gall | Genetic Complexity of Autosomal Dominant Polycystic Kidney and Liver Diseases[END_REF] Four other genes, GANAB, DNAJB11, ALG8 and ALG9, involved in protein biogenesis, have been more recently described in atypical forms of ADPKD and/or autosomal dominant polycystic liver disease (ADPLD). [START_REF] Gall | Autosomal dominant polycystic kidney disease[END_REF][START_REF] Gall | Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease[END_REF][START_REF] Porath | Mutations in GANAB, Encoding the Glucosidase IIalpha Subunit, Cause Autosomal-Dominant Polycystic Kidney and Liver Disease[END_REF][START_REF] Besse | ALG9 Mutation Carriers Develop Kidney and Liver Cysts[END_REF][START_REF] Besse | Isolated polycystic liver disease genes define effectors of polycystin-1 function[END_REF] In addition to the relentless development of renal and hepatic cysts, ADPKD is characterized by a wide spectrum of vascular complications, the most frequent and serious of which are intracranial aneurysms (IAs). In the non-ADPKD population, unruptured IAs are detected in 1.8 to 3.2% of the individuals screened by cerebral imaging, whereas the prevalence of IAs in ADPKD patients is nearly fivefold higher, ranging from 9.3% to 12.4% when systematic screening strategies are employed. [START_REF] Irazabal | Extended Follow-Up of Unruptured Intracranial Aneurysms Detected by Presymptomatic Screening in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Vernooij | Incidental findings on brain MRI in the general population[END_REF][START_REF] Vlak | Prevalence of unruptured intracranial aneurysms, with emphasis on sex, age, comorbidity, country, and time period: a systematic review and metaanalysis[END_REF][START_REF] Xu | Screening for intracranial aneurysm in 355 patients with autosomal-dominant polycystic kidney disease[END_REF] IA rupture, leading to subarachnoid hemorrhage (SAH), is associated with significant morbidity and mortality and occurs approximately ten years earlier in the ADPKD population than in the general population, with respective median ages of 41 versus 51 y. [START_REF] Chauveau | Intracranial aneurysms in autosomal dominant polycystic kidney disease[END_REF][START_REF] Lozano | Familial intracranial aneurysms[END_REF][START_REF] Schievink | Saccular intracranial aneurysms in autosomal dominant polycystic kidney disease[END_REF] In the United States, the early mortality rate associated with SAH is estimated to be ~20-30%. [START_REF] Abulhasan | Mortality after Spontaneous Subarachnoid Hemorrhage: Causality and Validation of a Prediction Model[END_REF][START_REF] Chang | Impact of case volume on aneurysmal subarachnoid hemorrhage outcomes[END_REF][START_REF] Lindbohm | Risk Factors of Sudden Death From Subarachnoid Hemorrhage[END_REF][START_REF] Mackey | Stable incidence but declining case-fatality rates of subarachnoid hemorrhage in a population[END_REF] Long-term complications of SAH include neurocognitive dysfunction, epilepsy, and other focal neurologic deficits.

This susceptibility to IAs in ADPKD is thought to be mediated by a maladaptive response of the vascular wall to hemodynamic stress, resulting from defects in PC1 and PC2, which are abundantly expressed in vascular smooth muscle cells and the endothelium and likely act as pressure sensors. [START_REF] Hassane | Pathogenic sequence for dissecting aneurysm formation in a hypomorphic polycystic kidney disease 1 mouse model[END_REF][START_REF] Kim | Polycystin 1 is required for the structural integrity of blood vessels[END_REF][START_REF] Qian | Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells[END_REF][START_REF] Wu | Cardiac defects and renal failure in mice with targeted mutations in Pkd2[END_REF][START_REF] Nauli | Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1[END_REF] Until now, the main risk factor identified for IA occurrence in ADPKD patients has been the presence of a positive personal or family history of unruptured or ruptured IAs, suggesting a predominant role of genetic determinants. [START_REF] Irazabal | Extended Follow-Up of Unruptured Intracranial Aneurysms Detected by Presymptomatic Screening in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Xu | Screening for intracranial aneurysm in 355 patients with autosomal-dominant polycystic kidney disease[END_REF] Indeed, the prevalence of IAs is nearly four times higher in ADPKD patients with a family history of IAs (~22%) than in those without such a family history (~6%). [START_REF] Irazabal | Extended Follow-Up of Unruptured Intracranial Aneurysms Detected by Presymptomatic Screening in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Xu | Screening for intracranial aneurysm in 355 patients with autosomal-dominant polycystic kidney disease[END_REF] Previous studies in the early 2000s suggested an association of the localization of PKD1 mutations in the 5' half of the gene with an increased risk for vascular complications. [START_REF] Rossetti | Association of mutation position in polycystic kidney disease 1 (PKD1) gene and development of a vascular phenotype[END_REF] O R I G

I N A L U N E D I T E D M A N U S C R I P T
However, PKD1 gene sequencing analysis is highly complex and was often incomplete at that time. [START_REF] Audrézet | Autosomal dominant polycystic kidney disease: comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients[END_REF] Compared to the general population, where nonmodifiable (e.g., female sex) and modifiable risk factors (such as smoking, hypertension and possibly dyslipidemia) are well identified, limited information is available in the ADPKD population. [START_REF] Brown | Unruptured intracranial aneurysms: epidemiology, natural history, management options, and familial screening[END_REF][START_REF] Etminan | Unruptured intracranial aneurysms: development, rupture and preventive management[END_REF][START_REF] Sanchis | Presymptomatic Screening for Intracranial Aneurysms in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF] Screening for IA using cerebral magnetic resonance imaging is currently recommended only for patients with a family history of IAs or SAH, patients with a high-risk profession (e.g., airline pilots), and patients who demand screening despite adequate information. [START_REF] Chapman | Autosomal-dominant polycystic kidney disease (ADPKD): executive summary from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference[END_REF] These recommendations relate to the cost associated with unnecessary screening and the risk of identifying IAs at low risk of rupture that are ineligible for treatment, causing anxiety to patients and their families. However, recent cost-effectiveness studies suggest a benefit of systematic screening strategies. [START_REF] Flahault | Screening for intracranial aneurysms in autosomal dominant polycystic kidney disease is cost-effective[END_REF][START_REF] Malhotra | MR Angiography Screening and Surveillance for Intracranial Aneurysms in Autosomal Dominant Polycystic Kidney Disease: A Cost-effectiveness Analysis[END_REF] It should be noted that the annual aneurysm rupture rates employed in these models were higher than in other ADPKD cohorts. [START_REF] Sanchis | Presymptomatic Screening for Intracranial Aneurysms in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF] This study aimed to evaluate the frequency of diagnosis of ruptured and unruptured IAs in

MATERIALS AND METHODS

Patients

This is a cross-sectional study involving 26 centers from the Genkyst Consortium in western

France. [START_REF] Gall | Type of PKD1 mutation influences renal outcome in ADPKD[END_REF][START_REF] Gall | The PROPKD Score: A New Algorithm to Predict Renal Survival in Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Gall | PKD2-Related Autosomal Dominant Polycystic Kidney Disease: Prevalence, Clinical Presentation, Mutation Spectrum, and Prognosis[END_REF] ADPKD patients over 16 years of age were recruited from January 2010 to June 2019.

ADPKD diagnosis was made by abdominal imaging, by employing the Pei criteria in the presence of a family history of ADPKD, [START_REF] Pei | Unified criteria for ultrasonographic diagnosis of ADPKD[END_REF] or following the identification of a proven pathogenic PKD1 or PKD2 mutation. In the absence of familial history or mutation identification, diagnosis required the presence of at least 10 bilateral kidney cysts. The patients' clinical data obtained during medical interviews at the time of their inclusion and from medical records were entered in a standardized clinical report form (CRF). For participants in whom previous history of IAs was reported in the CRF, medical records were reviewed on study sites to confirm the previous history of IAs and to collect additional information regarding aneurysm size (in millimeters), type (saccular versus fusiform), localization (anterior or posterior circulation) and management (monitoring, neurosurgical or neuroradiointerventional treatment). The screening strategy for detecting unruptured IAs was, in most cases, a targeted screening strategy, in accordance with the current Kidney Disease Improving Global Outcomes (KDIGO) guidelines, i.e., by performing magnetic resonance imaging on patients with a positive family history of IAs, patients with at-risk occupations, and patients requesting screening after comprehensive information. All participants provided informed consent, and the local ethics committee approved the study (CCTIRS 10.385).

Molecular analysis

All consenting patients underwent molecular genetic testing by Sanger sequencing for the

O R I G

I N A L U N E D I T E D M A N U S C R I P T
The latest version of the targeted NGS panel, in addition to the above-mentioned genes, also includes ALG8, ALG9, OFD1, COL4A1, DZIP1L, LRP5, PMM2, REN, SEC61B, SEC61A1, TSC1, TSC2 and VHL and was used after September 2019 for reanalysis of genetically unresolved patients. Patients with no clear pathogenic variant detected after Sanger sequencing or targeted NGS were screened for large rearrangements using multiplex ligation-dependent probe amplification and array-based comparative genomic hybridization. Mutations were classified as truncating (frameshift, indels, nonsense mutations, canonical splicing changes and in-frame indels>=5 amino acids) or nontruncating (missense, in-frame <=4 amino acids, noncanonical splicing events).

Statistical analyses

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, N.Y., USA). The frequency of IAs detected in our cohort was estimated and reported with a 95% confidence interval (CI) constructed for the estimate. Baseline data are expressed as the median and standard deviation (SD) or range. Quantitative data were compared using the nonparametric Mann-Whitney test or Kruskal-Wallis test, depending on the number of groups.

Qualitative data were compared using a t test or the chi-squared test.

Survival without a diagnosis of IA and survival without ruptured IAs were analyzed using the Kaplan-Meier method. The differences between the survival curves were assessed using a log-rank test with a 0.05 significance level.

HNF1B (n=8), PKHD1 (n=1), SEC63 (n=2), PRKCSH (n=3), OFD1 (n=4), UMOD (n=2), ALG8 (n=6), ALG9 (n=1)
, COL4A4 (n=2), COL4A1 (n=1)).

Diagnosis of ruptured or unruptured IA

A total of 114 patients from 107 pedigrees had a previous diagnosis of ruptured or unruptured IAs (4.65%, 95% CI: 3.56-5.74) (Figure 1). The cumulative probability of diagnosis of ruptured or unruptured IAs was 1.3% at 40 y (95% CI, 0.9-1.7), 3.9% at 50 y (95% CI, 2.9-4.9), and 6.2% at 60 y (95% CI, 5-7.4) and reached 8.1% at 70 y (95% CI, 6.5-9.7) (Figure 2a). The cumulative probability of diagnosis of ruptured aneurysm was 0.9%

O R I G I N A L U N E D I T E D M A N U S C

Sex and diagnosis of IA

The diagnosis rates of ruptured and unruptured IAs were 3.71% and 5.54% in male (n=44)

and female (n=70) patients, respectively. The cumulative probability of diagnosis of ruptured or unruptured IAs was higher in female patients (p=0.012, Figure 3A). Interestingly, while cumulative probabilities for IA diagnosis appeared to be similar in individuals <50 y, after 50 y, probabilities continued to increase more markedly in female than in male patients. The cumulative probabilities of

O R I G I N A L U N E D I T E D M A N U S C R I P T
IAs in male and female patients were hence 1.2% (95% CI, 0. 3C). The cumulative probabilities of diagnosis of ruptured IAs were also significantly higher in the PKD1 group (p=0.008), reaching 3.6% (95% CI, 3-4.3) vs 0.7% (95% CI, 0.2-1.2) at age 60 (Figure 3D).

The distribution of PKD1 mutations along the PKD1 gene did not differ in patients with and without IAs, and the median amino acid position did not differ between groups (p=0.057) (Figure 4).

We did not observe recurring mutation of PKD1 or PKD2 among unrelated individuals with IAs.

Furthermore, there was no association between PKD1 mutation type (truncating vs nontruncating)

and IA occurrence (p=0.957) (Table 3).

Risk factors associated with diagnosis of IAs

To evaluate the association between the different clinical and genetic factors and the diagnosis of ruptured or unruptured IAs, univariate and multivariate cox regression analyses were

O R I G I N A L U N E D I T E D M A N U S C R I P T performed.
In the univariate analysis, female sex, hypertension before age 35 y, and PKD1 genotype were associated with an increased risk for the diagnosis of IA (Table 3). After the multivariate Cox regression that included 1737 subjects, these three variables remained independent predictors of diagnosis of ruptured or unruptured IAs. In addition, smoking status, particularly when >20 PY, was also independently associated with diagnosis of IAs (p=0.009) (Table 3).

DISCUSSION

Widespread screening of IAs in ADPKD patients is currently not recommended, and presymptomatic screening was therefore conducted here only in patients with a positive family or personal history of IAs or SAH, before major elective surgeries, or upon specific request after adequate information. [START_REF] Chapman | Autosomal-dominant polycystic kidney disease (ADPKD): executive summary from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference[END_REF] While these recommendations might change in the future, our study reflects current real-life clinical practice. We report here a prevalence of detected IAs in the Genkyst cohort, one of the largest population-based cohorts of ADPKD patients, of 4.65% (95% CI: 3.56-5.74).

As expected, this prevalence is therefore lower than that in systematic screening studies. [START_REF] Irazabal | Extended Follow-Up of Unruptured Intracranial Aneurysms Detected by Presymptomatic Screening in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Xu | Screening for intracranial aneurysm in 355 patients with autosomal-dominant polycystic kidney disease[END_REF] Two recent studies, conducted at expert referral centers-Mayo Clinic, Rochester Minnesota, US and Necker Hospital, Paris, France-and possibly reflecting more severely affected patients, report IA detection rates of ~9.2% (n=75) and ~12.7% (n=23) in 812 and 181 patients who underwent presymptomatic screening (presymptomatic screening was performed in ~27 and 35% of the Mayo and Necker cohorts). [START_REF] Sanchis | Presymptomatic Screening for Intracranial Aneurysms in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Flahault | Screening for intracranial aneurysms in autosomal dominant polycystic kidney disease is cost-effective[END_REF] We confirm here that a large majority of IAs in ADPKD patients are small and saccular and located in the anterior circulation. [START_REF] Irazabal | Extended Follow-Up of Unruptured Intracranial Aneurysms Detected by Presymptomatic Screening in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF][START_REF] Xu | Screening for intracranial aneurysm in 355 patients with autosomal-dominant polycystic kidney disease[END_REF][START_REF] Sanchis | Presymptomatic Screening for Intracranial Aneurysms in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF] Approximately 26% of IA patients had multiple aneurysms. The median age at aneurysm rupture was 41.5 y, which is ten years earlier than in the non-ADPKD population and consistent with previous studies. [START_REF] Chauveau | Intracranial aneurysms in autosomal dominant polycystic kidney disease[END_REF][START_REF] Schievink | Saccular intracranial aneurysms in autosomal dominant polycystic kidney disease[END_REF] While IAs from the anterior circulation are considered at lower risk of rupture in non-ADPKD patients, ~87% of the ruptured IAs in our cohort originated from the anterior circulation.

O R I G

I N A L U N E D I T E D M A N U S C R I P T
Cumulative probabilities of diagnosis of IA in this population-based cohort provide new and interesting information, which should, however, be interpreted with caution, as individuals with ruptured IAs, particularly at younger ages, might be underrepresented. At 70 y, the cumulative probabilities for diagnosis of IA or past history of ruptured IAs were 8.1 and 3.2%, respectively.

Strikingly, cumulative probabilities of diagnosis of IA and ruptured IAs appear to be higher in women, especially after 50 y, which was not reported in ADPKD patients until now. This parallels observations made in the non-ADPKD population and suggests a possible protective role of estrogens. [START_REF] Vlak | Prevalence of unruptured intracranial aneurysms, with emphasis on sex, age, comorbidity, country, and time period: a systematic review and metaanalysis[END_REF] Several former studies support a role for estrogen deficiency, causing a reduction in the collagen content of tissues, which might contribute to aneurysm development in postmenopausal women, similar to patients with connective tissue disorders. In the non-ADPKD population, an earlier age at menopause is associated with a higher risk for IAs, and animal and clinical studies have suggested a protective role of hormone replacement therapy (HRT) against SAH. [START_REF] Ding | Younger age of menopause in women with cerebral aneurysms[END_REF][START_REF] Jamous | Role of estrogen deficiency in the formation and progression of cerebral aneurysms. Part II: experimental study of the effects of hormone replacement therapy in rats[END_REF][START_REF] Mhurchu | Hormonal factors and risk of aneurysmal subarachnoid hemorrhage: an international population-based, case-control study[END_REF] Whether HRT should be recommended for women with ADPKD at increased risk for IAs, i.e., with a positive familial history for IAs will require further studies.

Importantly

O R I G

I N A L U N E D I T E D M A N U S C R I P T
Severe vascular phenotypes have been described in both Pkd1 and Pkd2 murine models. [START_REF] Hassane | Pathogenic sequence for dissecting aneurysm formation in a hypomorphic polycystic kidney disease 1 mouse model[END_REF][START_REF] Kim | Polycystin 1 is required for the structural integrity of blood vessels[END_REF][START_REF] Qian | Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells[END_REF][START_REF] Wu | Cardiac defects and renal failure in mice with targeted mutations in Pkd2[END_REF][START_REF] Perrone | Vascular complications in autosomal dominant polycystic kidney disease[END_REF] The reasons of this apparent higher risk for IAs in PKD1 patients remain to be understood. In contrast with a study from the early 2000s, we did not identify any correlation between the occurrence of IAs and the localization of mutations along the PKD1 gene. [START_REF] Rossetti | Association of mutation position in polycystic kidney disease 1 (PKD1) gene and development of a vascular phenotype[END_REF] While truncating pathogenic variants of PKD1 are known to be associated with poorer renal prognosis, no association between PKD1 mutation type and diagnosis of IAs was identified. [START_REF] Gall | Type of PKD1 mutation influences renal outcome in ADPKD[END_REF] We did not identify any recurring PKD1 or PKD2 mutations among individuals with IAs. Former studies reported IAs in unrelated families harboring a two base-pair deletion in PKD1 (c.5014_5015delAG), suggesting an association with this specific mutation, but more systematic genetic testing in ADPKD patients has now established that this variant is in fact the most common pathogenic variant of PKD1 (~1-2%). [START_REF] Rossetti | Association of mutation position in polycystic kidney disease 1 (PKD1) gene and development of a vascular phenotype[END_REF][START_REF] Watnick | Mutation detection of PKD1 identifies a novel mutation common to three families with aneurysms and/or very-early-onset disease[END_REF] In the small subgroup of 39 patients harboring mutations in other genes, no diagnosis of IA was reported.

However, IAs were occasionally reported in GANAB pedigrees, and various vascular phenotypes, including IAs, dilatation of the thoracic aorta and dissection of a carotid artery, were recently described in a cohort of DNAJB11 mutation carriers. [START_REF] Huynh | Clinical spectrum, prognosis and estimated prevalence of DNAJB11-kidney disease[END_REF] Studies in the non-ADPKD population have identified risk factors associated with IAs. These include nonmodifiable factors such as female sex, ethnicity and personal or family history (FH) of ruptured or unruptured aneurysm and modifiable factors such as smoking exposure and hypertension. [START_REF] Vlak | Prevalence of unruptured intracranial aneurysms, with emphasis on sex, age, comorbidity, country, and time period: a systematic review and metaanalysis[END_REF][START_REF] Brown | Unruptured intracranial aneurysms: epidemiology, natural history, management options, and familial screening[END_REF][START_REF] Hackenberg | Unruptured Intracranial Aneurysms[END_REF][START_REF] Zhou | Genetics of Intracranial Aneurysms[END_REF][START_REF] Greving | Development of the PHASES score for prediction of risk of rupture of intracranial aneurysms: a pooled analysis of six prospective cohort studies[END_REF] In a recent study conducted in the Mayo Clinic cohort, hypertension and smoking history were significantly more frequent in ADPKD patients with IAs. [START_REF] Sanchis | Presymptomatic Screening for Intracranial Aneurysms in Patients with Autosomal Dominant Polycystic Kidney Disease[END_REF] In our study, after multivariate Cox analysis, four factors remained independently associated with the diagnosis of IA: female sex, hypertension before age 35, smoking status and PKD1 genotype.

Because IA diagnosis was made by targeted screening in the context of a positive familial history in a majority of the IA subjects, this risk factor could not be entered in the multivariate analysis. However, the importance of a positive family history should be stressed. The observation of pedigrees with a strong familial clustering of IAs in our cohort and others suggests the role of genetic

O R I G I N A L U N E D I T E D M A N U S C R I P T
modifiers hitherto unidentified. Using genome-wide association studies, whole-exome sequencing and/or RNA sequencing, some candidate genes involved in the development of IAs have already been identified in the general population. [START_REF] Zhou | Genetics of Intracranial Aneurysms[END_REF][START_REF] Bourcier | Rare Coding Variants in ANGPTL6 Are Associated with Familial Forms of Intracranial Aneurysm[END_REF] Similar strategies might be successful in identifying such modifiers in ADPKD but will require the enrollment of large numbers of participants and international collaborative efforts.

This study has some limitations. Data on the number of ADPKD-affected individuals who underwent screening for IAs and in whom no IA was detected are lacking, and this information could not be collected retrospectively. Because targeted screening was employed, the number of IAs may have been underestimated. However, the data presented here reflect real-life practice and are hence likely to be of interest in clinical practice.

In conclusion, the determinants of the development of IAs in ADPKD patients are complex

O R I G

I N A L U N E D I T E D M A N U S C R I P T

O R I G

I N A L U N E D I T E D M A N U S C R I P T

©

  The Author(s) 2022. Published by Oxford University Press on behalf of the ERA. All rights reserved. For permissions, please e-mail: journals.permissions@oup.comDiagnosis and Risk Factors for Intracranial Aneurysms in Autosomal Polycystic Kidney Disease:

  the large population-based Genkyst ADPKD cohort, to describe ADPKD-associated IAs and to analyze the risk factors associated with the occurrence of IAs in ADPKD patients.

PKD1

  and PKD2 genes (patients included before March 2017) and/or targeted next-generation sequencing (NGS) of the PKD1, PKD2, GANAB, DNAJB11, HNF1B, PKHD1, SEC63, PKRKCSH and UMOD genes (patients included after March 2017 or before 2017 with no pathogenic variant detected by Sanger sequencing) in the Department of Molecular Genetics at Brest University Hospital, France.

  and multifactorial. Female sex, PKD1 genotype, early-onset hypertension, smoking exposure and positive familial history for IAs are associated with a higher risk for diagnosis of IA

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Flow diagram summarizing the diagnosis of ruptured and unruptured IAs in the Genkyst cohort. Abbreviations: ADPKD, autosomal dominant polycystic kidney disease; IAs, intracranial aneurysms.

Figure 4 .

 4 Figure 4. Distribution of mutations identified along the PKD1 gene in each of the three groups: no known intracranial aneurysm (IAs), all IAs, unruptured IAs and ruptured IAs. The different PKD1 pathogenic mutations are plotted against their respective nucleotide positions within PKD1 coding sequence (cDNA designation, from 1 to 12912, indicated on the lower part of the figure). The median nucleotide position for each group is represented by a star. PKD1 transcript is shown above. Abbreviations: IAs, intracranial aneurysms.

RESULTS Baseline clinical parameters in the Genkyst cohort and genetic analyses

  Univariate Cox proportional hazards regression analysis was used to identify variables associated with IA diagnosis. Variables were entered into the Cox multivariate analysis when they were associated with ruptured or unruptured IA diagnosis in the univariate analysis at a conservative threshold of 20%. Complete observations were entered in the Cox multivariate analysis.

	Two thousand four hundred forty-nine ADPKD patients (1185 males) from 1810 pedigrees
	were included. The median age at inclusion was 55.1 y [range 16.2-94.3]. The characteristics of the
	patients at inclusion are presented in Table 1. The results of genetic analysis were available in 2386
	patients. PKD1 pathogenic variants were identified in 67.6% of the participants (n=1612; truncating
	variants 44.1% (n=1052), nontruncating variants 23.5% (n=560)), while PKD2 pathogenic variants R I P T were identified in 19% (n=454). In 11.8% of cases (n=281), no mutation was identified. Genetic
	S C
	U
	A N
	M
	I T E D
	N E D
	U
	A L
	R I G I N
	O

analysis led to a differential diagnosis in 1.6% of the cases (n=39: GANAB (n=4), DNAJB11 (n=5),

  Although nonsignificant, there were more smokers with exposure> 20 pack-years in the IA group than in the non-IA group (15% versus 8.8%, P=0.095) (Table1). IAs were diagnosed at all chronic kidney disease (CKD) stages. Family history of IAs was positive in 47.4% of the individuals with a diagnosis of ruptured or unruptured IAs.

	at 40 y (95% CI, 0.5-1.3), 1.8% at 50 y (95% CI, 1.2-2.4), 2.6% at 60 y (95% CI, 1.8-3.4) and 3.2% at 70 y
	(95% CI, 2.2-4.2) (Figure 2b).
	Patients with IA were more frequently female patients (61.4% versus (vs) 51.1%, P=0.013), of
	PKD1 genotype (77.2% vs 63.8%, P=0.001) and those being treated for hypertension (96.5% vs 76.1%,
	P=0.007).
	R I P T

Imaging characteristics of the aneurysms

One hundred fifty-nine IAs were detected in 114 patients. Multiple aneurysms were found in 30 patients; either diagnosed simultaneously (n=16), or successively (i.e. recurrent diagnoses of different IAs, prior inclusion in the Genkyst cohort) (n=14). Amongst these 30 patients, 22 had two IAs, 3 had three IAs, 4 had three IAs, and 2 had five IAs. A large majority of the 159 aneurysms were saccular (~98%), and originated from the anterior circulation (89.3%) (Table

2

). Size was available for 104/159 IAs (size was missing for 31 IAs diagnosed at rupture, and 24 unruptured IAs, mostly diagnosed before <2000). Most of these IAs were small, measuring less than 7 mm (n=85, 81.2%), including 47 IAs measuring less than 3 mm (45.2%). Only five patients were diagnosed with giant IAs (e.g., >10 mm, 3.6%). Of the 62 ruptured IAs in 48 patients, 49 were in the anterior circulation (87.5%), and 25 were <7mm.

  6-1.8) vs 1.4% (95% CI, 0.6-2.2) at age 40, 3.6% (95% CI, 2.4-4.8) vs 4.2% (95% CI, 2.8-5.6) at age 50, 5.1% (95% CI, 3.5-6.7) vs 7.3% (95% CI, 5.3-9.3) at age 60, and 5.4% (95% CI, 3.8-7) vs 10.8% (95% CI, 8-13.6) at age 70. The cumulative

	probability of diagnosis of ruptured IAs was also higher in the female group (p=0.041): 3.2% (95% CI,
	2-4.4) vs 1.9% (95% CI, 0.9-2.9) at age 60 and 4.5% (95% CI, 2.7-6.3) vs 1.9% (95% CI, 0.9-2.9) at age
	70 (Figure 3B).
	Genotype of patients with IA
	The respective diagnosis rates of ruptured or unruptured IAs were 5.46% and 2.64% in PKD1
	8) at age
	40, 4.9% (95% CI, 3.6-4.8) vs 1.9% (95% CI, 1.2-2.8) at age 50, and 8.1% (95% CI, 7.2-9) vs 2.8% (95%
	CI, 1.1-3.8) at age 60 (Figure

(n=88/1612) and PKD2 (n=12/454) mutation carriers. No cases of IAs were reported in patients with GANAB (n=4) or DNAJB11 (n=5) mutations or patients harboring pathogenic mutations in other genes screened on the panel (n=30). The cumulative probability of diagnosis of ruptured or unruptured IAs was higher in PKD1 mutation carriers (p=0.001; Figure

3C and 3D

). The cumulative probabilities of IAs in PKD1 and PKD2 mutation carriers were indeed 1.7% (95% CI, 1.4-2) vs 0.5% (95% CI, 0.2-0.

  PKD1 genotype remained significantly associated with diagnosis of IAs after the multivariate analysis. Confounding factors may partially account for this observation: first, early IA rupture, before the diagnosis of ADPKD, might occur in some PKD2 mutation carriers. Second, presymptomatic screening might be more frequently conducted in PKD1 mutation carriers who generally have more significant family histories and more severe renal disease, resulting in earlier ADPKD diagnosis and closer medical follow-up.[35] However, the consistent observation of a higher rate of diagnosis of ruptured IAs in PKD1 mutation carriers and

, our study allows revisiting the influence of genetics on IA development in ADPKD in light of modern genomics tools in a large cohort of patients. The diagnosis rate of ruptured and unruptured IAs was more than twofold higher in carriers of mutations in PKD1 than in PKD2, with respective cumulative probabilities of 8.1 vs 2.8% at age 60. the results of the multivariate Cox analyses, showing that this association is independent from age at hypertension diagnosis, suggest that PKD1 mutation carriers might be more at-risk of developing IAs.

Table 1 . Clinical characteristics of the study participants at inclusion or at diagnosis of intracranial aneurysm

 1

	Tables
			Variable	Total cohort	b	No past history of IAs	b	All IAs	c Past history of IAs Unruptured IAs	Ruptured IAs
	Patients (number of pedigrees), n (n)	2449 (1810)		2335		114	66	48
	Male			1185 (48.4%)	1141 (48.9%)		44 (35.6%)	27 (40.9%)	17 (35.4%)
	Median age (years)	b, c		55.1 [16.2-94.3]	55.2 [16.2-94.3]		48 [40.8-54.3]	49 [45-57]	43.5 [35.5-52.6]
	CKD Stages a, b, c		1	456 (18.9%)		447 (19.4%)		20 (21.3%)	8 (13.3%)	12 (35.3%)
			2	394 (16.3%)		384 (16.7%)		21 (22.3%)	10 (16.7%)	11 (32.4%)
			3a	236 (9.8%)		230 (10%)		7 (7.4%)	4 (6.7%)	3 (8.8%)
			3b	256 (10.6%)		247 (10.7%)		15 (16%)	11 (18.3%)	4 (11.8%)
			4	211 (8.8%)		198 (8.6%)		15 (16%)	11 (18.3%)	4 (11.8%)
			5	859 (35.6%)		793 (34.5%)		16 (17%)	16 (26.7%)	0
			Requiring RRT Missing	762 (31.6%) 37 b		700 (30.4%) 36 b		9 (9.6%) 20 c	9 (15%) 6 c	0 14	c
	Hypertension		Yes	1873 (77%)		1763 (76.1%)		110 (96.5%)	64 (97%)	46 (95.8%)
			No	559 (23%)		555 (23.9%)		4 (3.5%)	2 (3%)	2 (4.2%)
			Missing	17		17		0	0	0
	Hypertension before 35 y	Yes	535 (27.8%)		493 (27.2%)		42 (37.5%)	29 (43.9%)	13 (28.3%)
			No	1390 (72.2%)	1320 (72.8%)		70 (62.5%)	37 (56.1%)	33 (71.7%)
			Missing	326		325		1	0	1
			NA	198		197		1	0	1
	Smoking status		Never smoker	1275 (56%)		1215 (56%)		60 (53.1%)	35 (53%)	25 (53.2%)
			Past or active <20PY	799 (35%)		763 (35.2%)		36 (31.9%)	24 (36.4%)	12 (25.5%)
			Past or active >20PY	207 (9%)		190 (8.8%)		17 (15%)	7 (10.6%)	10 (21.3%)
			Missing	168		167		1	0	1
	Family history of IAs	No	2150 (87.8%)	2090 (89.5%)		60 (52.6%)	31 (53%)	19 (39.6%)
			Yes	299 (12.2%)		245 (10.5%)		54 (47.4%)	35 (47%)	29 (60.4%)
	Genotype		PKD1	1612 (67.6%)	1524 (63.8%)		88 (77.2%)	50 (75.8%)	38 (82.6%)
			truncating	1052 (44.1%)	997 (41.8%)		55 (48.2%)	34 (51.5%)	21 (45.7%)
			non-truncating	560 (23.5%)		527 (22.1%)		33 (28.9%)	16 (24.2%)	17 (37%)
			PKD2	454 (19%)		442 (18.5%)		12 (10.5%)	10 (15.2%)	2 (4.3%)
			Unresolved	281 (11.8%)		269 (11.3%)		12 (10.5%)	6 (9.1%)	6 (13%)
			Other genes	39 (1.6%)		39 (1.6%)		2 (1.8%)	0	0
			Missing	63		61		0	0	2
	Note: Values for categorical variables are given as number (percentage), and for continuous variables, as median [interquartile range].

a According to estimated glomerular filtration rate (calculated using the CKD Epidemiology Collaboration creatinine equation) b Data at inclusion in the Genkyst cohort c Data at diagnosis of IA . Abbreviations:

CKD, chronic kidney disease; IA, intracranial aneurysm, NA non applicable (corresponding to individuals <35y with no hypertension).

Table 2 . Characteristics of 159 IAs in 114 patients 1

 2

	Characteristics	All aneurysms	Unruptured	Ruptured
			N=159	aneurysms	aneurysms
				N=97	N=62
	Type
	Saccular	149 (98%)	93 (96.9%)	56 (100%)
	Fusiform	3 (2%)	3 (3.1%)	0
	Missing	7	1	6
	Location
	Anterior circulation	135 (90%)	86 (91.5%)	49 (87.5%)
	Internal carotid artery	21	13	8
	Middle cerebral artery	74	51	23
	Anterior communicating artery	39	22	17
	Posterior communicating artery	1	0	1
	Posterior circulation	15 (10%)	9 (8.5%)	7 (12.5%)
	Missing	9	3	6
	Size	2
	<7mm	85 (81.2%)	60 (82.2%)	25 (80.6%)
	<3mm	47	31	16
	≥7mm	19 (18.3%)	13 (17.8%)	6 (19.4%)
	>10mm	5	3	2
	Missing	55	24	31
	Notes:
	1 Thirty patients (26.3%) had more than one intracranial aneurysm, either diagnosed
	simultaneously (n=16), or diagnosed successively (de novo formation of intracranial
	aneurysm) prior inclusion in Genkyst cohort (n=14).
	Abbreviations: IA, intracranial aneurysm.

Table 3 . Effect of Clinical and Genetic variables on the occurrence of IAs

 3 Univariate and multivariable cox regressions. Abbreviations: CI, confidence interval; SE, standard error; PY, pack-years.

				Univariate			Multivariate
	Variables	n	HR	95% CI	P-value	HR	95% CI	P-value
	Sex
	Male	1185
	Female	1264	1.6	1.1 to 2.4	0.013	1.8	1.2 to 2.7	0.005
	Hypertension before 35y
	No	1390
	Yes	535	2.5	1.7 to 3.7	<0.001	2.2	1.5 to 3.2 <0.001
	Smoking status
	Never	1275
	Past or active,<20PY	799	1.1	0.7 to 1.7	0.575	1.3	0.9 to 2.1	0.176
	Past or active,>20PY	207	1.6	0.9 to 2.7	0.095	2.1	1.2 to 3.7	0.009
	Genotype
	PKD1	1612
	PKD2	454	0.4	0.2 to 0.7	0.002	0.4	0.2 to 0.8	0.009
	Unresolved	281	0.4	0.2 to 0.8	0.005	0.7	0.4 to 1.3	0.230
	PKD1 mutation type
	Non truncating	560
	Truncating	1052	1	0.6 to 1.5	0.957
	Note:

ACKNOWLEDGEMENTS

We thank the patients and families for involvement in the study. We would like to thank Genkyst/GeneQuest investigators: Drs. Grall A, Moal MC, Hanrotel C, Segalen I, Tanquerel T, Lanfranco L (Centre Hospitalier Régional Universitaire, Brest); Morin MP, Le Pogamp P, Gie S, Rivalan J, Laruelle E, Richer C, Lorcy N, Golbin L, Terrasse M, Morice S, Brenier H, Michel A, Lavergne A, Tomkiewicz E (Centre Hospitalier Universitaire, Rennes); Gatault P, Merieau E, Barbet C, Buchler M, Golea G, Ghouti L, Gautard D, Sautenet B, François M, Fournier A, Baron C, Salmon C, Rabot N, Prat L, Valentin JF, Birmele B, Genest C, Goumard A (Centre Hospitalier Régional Universitaire, Tours); Desport E, Thierry A, Touchard G, Belmouaz M, Javaugue V, Bauwens MA, Fride-Leroy F, Bouteau I (Centre Hospitalier Universitaire, Poitiers); Subra JF, Besson V, Cousin M, Sayegh J, Onno C, Maghakian MN, Demiselle J, Garnier AS, Planchais M, Guibert F (Centre Hospitalier Universitaire, Angers); Stanescu C, Le Cacheux P, Baluta S, Leonetti F (ⴕ), Boulahrouz R, Ferrier ML, Freguin C, Simon A, Potier J, Coulibaly JM, Colombo A (Centre Hospitalier Yves le Foll, Saint Brieuc); Dolley-Hitze T (Centre Hospitalier Broussais, Saint Malo) ; Michez E, Mandart L, Menoyo V, Pincon E, Muresan C, Durand PY, Wegner I (Centre Hospitalier de Bretagne Atlantique, Vannes); Metes I, Guyon-Roger T, Wehbe B, Siohan P, Drouet C (Centre Hospitalier de Cornouaille, Quimper); Le Guillou A, Le Jeune M, Beillard G, Corlu L, Sawadogo T, Georgescu S (Centre Hospitalier de Bretagne Sud, Lorient); Jousset P, Latif R, Massad M (Centre Hospitalier du Centre Bretagne, Pontivy); Jaulin JP, Couvrat-Desvergnes G, Querard AH, Ottavioli JN, Target N, Chapal A, Le Fur A (Centre Hospitalier Départemental de Vendée, La Roche sur Yon); Besnier D, Regnier-Le Coz S, Blanpain A, Durault S, Larmet D, Skandri A (Centre Hospitalier Georges Charpak, Saint-Nazaire); Pouteau LM (Centre Hospitalier de Laval); Labatut D (Centre Hospitalier de Niort); Bachelet-Rousseau C, Delbes S, Pourreau F (Centre Hospitalier de La Rochelle) ; Mzoughi S (Centre Perharidy, Roscoff); Guillodo MP, Depraetre P, Strullu B, Chaffara E, Le Mee M (Association des Urémiques de Bretagne, Brest-Morlaix); Terki N, Goulesque K (Centre de Néphrologie et de Dialyse d'Armorique, Brest); Benarbia S, Dimulescu M, Rifaat M (Association des Urémiques de Bretagne, Quimper); Legrand D, Duneau G, Georges E (Association des Urémiques de Bretagne, Lorient); Babinet F, Lanoiselee S (ECHO, Le Mans); Savoiu C, Testa A, Oancea I, Coupel I, Parahy S, Lefrancois G, Hristea D (ECHO, Nantes) ; Briand E, Bugnon D (ECHO les Sables d'Olonne). We would like to acknowledge the study coordinators Christelle Guillerm-Regost, Christelle Ratajczak and Noée Gales (CHU Brest, France), Fabien Duthe (CHU Poitiers, France), Bernadette Pilorge et Julien Bontemps (CHU Tours), Sylvie Le Stum and David Naveiro (CHU Rennes), Béatrice Mazé and Eléonore Ourouda (CHU Angers), Véronique Bihel et Stéphane Natur (CH saint Malo), Alexandra Bréant (CH Lorient), Bénédicte Hue (CH saint Brieuc), Pauline Floch (CH Saint Nazaire), Angélique Colin (ECHO), Edwige Migne et Hélène Pelerin (CH La Roche sur Yon), Fanny Biais-Sauvetre (CH La Rochelle).

FUNDING

The study was supported by a National Plan for Clinical Research (PHRC inter-regional GeneQuest, NCT02112136, ECLG), a grant by the French Association for Information and Research on Genetic Kidney Diseases (AIRG France, ECLG), a grant from Otsuka Pharmaceuticals Industry (YLM), and a National Research Agency grant (ANR JCJC 2019 GENOVAS-PKD, ECLG).

CONFLICT OF INTEREST STATEMENT

None declared. The results presented in this paper have not been published previously in whole or part, except in abstract format (ERA EDTA 2019).


Yongqiang Tu

Alaa Hassan
email: alaa.hassan@univ-lorraine.fr

Javier A Arrieta-Escobar

Uzair Khaleeq

Uz Zaman

Ali Siadat

Gongliu Yang

Uzair Khaleeq Uz Zaman

Modeling and evaluation of freeform extruded filament based on numerical simulation method for direct ink writing

Keywords: Modeling, evaluation, numerical simulation, freeform extruded filament, direct ink

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

Direct ink writing (DIW), which was firstly proposed by [START_REF] Cesarano | ROBOCASTING PROVIDES MOLDLESS FABRICATION FROM SLURRY DEPOSITION[END_REF] [START_REF] Cesarano | ROBOCASTING PROVIDES MOLDLESS FABRICATION FROM SLURRY DEPOSITION[END_REF] and was patented in 2000 [START_REF] Cesarano | FREEFORMING OBJECTS WITH LOW BINDER SLURRY[END_REF], is an extrusion-based additive manufacturing (AM) technique by depositing slurry or paste type materials named inks onto the substrate layer by layer [START_REF] Hossain | 3D-Printed Objects for Multipurpose Applications[END_REF]. Compared with other AM techniques, DIW has advantages in materials flexibility, low cost and reliability [START_REF] Kumar | 3D Printing Technology for Biomedical Practice: A Review[END_REF]. Due to the advantages, various materials have been made into inks suitable for DIW and been successfully used in various applications: polymer inks for rubber industry [START_REF] Kim | Rubber ink formulations with high solid content for direct-ink write process[END_REF]; bio-inks with living cells for tissue engineering and regenerative medicine [START_REF] Campos | Synchronized Dual Bioprinting of Bioinks and Biomaterial Inks as a Translational Strategy for Cartilage Tissue Engineering[END_REF];metal inks for batteries and electronic circuits [START_REF] Kim | 3D Printed Disposable Wireless Ion Sensors with Biocompatible Cellulose Composites[END_REF];ceramic inks for ceramic engineering [START_REF] Ye | Study on 3D-Direct Ink Writing based on adding silica submicron-particles to improve the rheological properties of alumina ceramic ink[END_REF];cellulose inks for clothing, sensors and 4D printing [START_REF] Mulakkal | Responsive cellulose-hydrogel composite ink for 4D printing[END_REF][START_REF] Wang | 3D printing with cellulose materials[END_REF][START_REF] Thibaut | A fibrous cellulose paste formulation to manufacture structural parts using 3D printing by extrusion[END_REF];food inks for food processing engineering [START_REF] Lille | Applicability of protein and fiber-rich food materials in extrusion-based 3D printing[END_REF]; cement ink for buildings [START_REF] Vergara | Additive manufacturing of Portland cement pastes with additions of kaolin, superplastificant and calcium carbonate[END_REF]. In the foreseeable future, more materials will be prepared into inks suitable for DIW and used in more fields. Thus, DIW is a research focus in AM studies and regarded as an important technique to realize advanced and intelligent manufacturing nowadays [START_REF] Shahzad | Direct ink writing (DIW) of structural and functional ceramics: Recent achievements and future challenges[END_REF]. Freeform extruded filaments (FEF) were extruded filaments in the air between the nozzle bottom and the substrate in DIW. During the DIW, producing continuous, tubular FEF with diameters close to the actual nozzle inner diameter is a prerequisite and also a characterization for a high printing quality [START_REF] Smith | Chemical modification and printability of shearthinning hydrogel inks for direct-write 3D printing[END_REF]. For instance, Ouyang et al. [START_REF] Ouyang | Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells[END_REF] characterized ink printability through shape statuses of FEF; Paxton et al. [START_REF] Paxton | Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability[END_REF] focused on the formation properties of FEF as an initial screening to assess ink printability; Smith et al. [START_REF] Smith | Chemical modification and printability of shearthinning hydrogel inks for direct-write 3D printing[END_REF] proposed a term considering the measured diameter of the FEF and nozzle inner diameter to define printability; Dávila et al. [START_REF] Dávila | Rheological evaluation of Laponite/alginate inks for 3D extrusionbased printing[END_REF] evaluated ink printability through observation of FEF formation; He et al. [START_REF] He | Research on the printability of hydrogels in 3D bioprinting[END_REF] chose suitable ranges for process parameters setting in DIW 3D printers by observing shapes of FEF. Therefore, modeling and evaluation of FEF are necessary for quality evaluation and control for DIW. Recently, filaments in extrusion-based AM have been modeled and evaluated mainly through analytical methods [START_REF] Percoco | Analytical model to predict the extrusion force as a function of the layer height, in extrusion based 3D printing[END_REF], experimental methods [START_REF] Ashrafi | Experimental prediction of material deformation in large-scale additive manufacturing of concrete[END_REF] and numerical simulation methods [START_REF] Roussel | Numerical simulations of concrete processing: From standard formative casting to additive manufacturing[END_REF]. Analytical methods give a model based on the mathematical description and derivation of physical process, but model accuracy is restricted by theoretical simplification. Experimental method presents a regression model between input factors and experimental results, but this method is restricted by measurement method and cannot provide a dynamic prediction. In complement to analytical and experimental methods, numerical simulation methods can deliver a vast amount of information about the process and has proven to be an accurate and reliable dynamic predictive tool in the analysis of extrusion-based AM techniques [START_REF] Liravi | Additive manufacturing of 3D structures with non-Newtonian highly viscous fluids: Finite element modeling and experimental validation[END_REF][START_REF] Göhl | Simulations of 3D bioprinting: predicting bioprintability of nanofibrillar inks[END_REF][START_REF] Comminal | Modelling of 3D concrete printing based on computational fluid dynamics[END_REF]. However, there is few works on modeling and evaluation of FEF based on numerical simulation methods for DIW. Numerical simulation modeling of FEF should fully consider ink's properties and process parameters as the quality of FEF depends both on ink's material properties and process parameters setting [START_REF] Suntornnond | A Mathematical Model on the Resolution of Extrusion Bioprinting for the Development of New Bioinks[END_REF]. Evaluation of FEF should consider both the mean diameter and the stability as these two indicators have the most significant influence on the printing quality [START_REF] Schwab | Printability and Shape Fidelity of Bioinks in 3D Bioprinting[END_REF]. The aim of this study is to establish a model of FEF with numerical simulation method and then evaluate the FEF based on this model. First, ink properties including density, surface tension coefficient and rheological properties were determined by properties tests. Then, model of FEF was established using numerical simulation method and two evaluation indicators, mean diameter and stability, were proposed to evaluate the FEF. Finally, two inks, the cellulose ink and Nivea Crème, were used under three levels of piston velocity to verify the method. After experimental verification, the model and evaluation indicators were used to analyze effect of the piston velocity on FEF, characterize the ink printability and find suitable printable windows for process parameters.

Ink preparation and properties tests 2.1. Ink preparation

To verify the proposed method considering the universality for different materials, two well printable inks, the cellulose ink with low viscosity and Nivea Crème with high viscosity, were prepared. Cellulose inks, which mainly contain cellulose fibers and cellulose derivatives, are representatives of novel inks for DIW [START_REF] Demitri | Novel superabsorbent cellulose-based hydrogels crosslinked with citric acid[END_REF][START_REF] Mcoscar | Hydrogels from norbornene-functionalized carboxymethyl cellulose using a UV-initiated thiol-ene click reaction[END_REF]. The cellulose ink used was prepared by mixing 2.36% natural cellulose fibers, 0.59% hydroxyethyl cellulose (HEC), 1.77% carboxymethyl cellulose (CMC), 0.47% montmorillonite, 0.47% citric acid (CA) and 94.34% demineralized water [START_REF] Mulakkal | Responsive cellulose-hydrogel composite ink for 4D printing[END_REF]. Cotton linter packs (CLP) (Goetz & Sons, Inc., USA) were purchased as natural cellulose fibers. CLP were first left in demineralized water for 24 hours and then dried at room temperature (25 °C). Once dried, CLP were added to a beaker with demineralized water and the montmorillonite (reference: 42531) and mixed with a spatula. HEC (reference: 54290) and CMC (reference: Acros Organics 332641000) were mixed and then added to the mixture in the beaker. One quarter of the polymer mixture was added into the beaker and mixed for 1 min at 700 rpm by planetary mill with 5 grinding balls of zirconium oxide (Ø20 mm) using a Pulverisette 7 Premium Line (Fritsch, Germany). The remaining polymer mixture was then incorporated, followed by another grinding cycle under the same conditions. CA (reference: C0759) was added to the combination of all batches prepared as a crosslinking agent on the same day by means of a spatula. Nivea Crème with well printability is often used as printability reference for inks to test the process parameters in DIW [START_REF] Paxton | Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability[END_REF] as many inks in DIW are designed and produced to have the same printability of Nivea Crème [START_REF] Tu | Optimizing Process Parameters of Direct Ink Writing for Dimensional Accuracy of Printed Layers, 3D Printing and Additive Manufacturing[END_REF]. In this study, Nivea Crème (Art. No. 80104) (Beiersdorf Global AG, Germany) with high viscosity and well printability was chosen and bought.

Ink material properties tests

A 5 mL pycnometer and analytical balance XSR (both from Mettler Toledo, France) were used to assess ink density by measuring a mass of 5 mL of the ink (measuring accuracy: 0.008 mg). A tensiometer K9 (KRÜSS, Germany) was used to measure the surface tension coefficient using the Du Noüy ring method (measuring accuracy: 0.1 mN/m). For both tests, measurements were performed at 25 ℃. The Hershel-Bulkley model (HBM) has been verified and adopted as a suitable and reliable model to represent the viscosity of the shear thinning inks in DIW [START_REF] Kim | Enhanced rheological behaviors of alginate hydrogels with carrageenan for extrusion-based bioprinting[END_REF][START_REF] Koch | Generic method of printing window adjustment for extrusion-based 3D-bioprinting to maintain high viability of mesenchymal stem cells in an alginate-gelatin hydrogel[END_REF]. Using the HBM, the shear stress and the viscosity of inks can be expressed by Eq. ( 1) and Eq. ( 2), respectively.

0 n K    =+ . (1)
( )

1 00 =min , / n K      - + . ( 2
)
Where  (unit: Pa ),  (unit: s -(5 points per decade) through regression between Eq. ( 2) and experimental viscosity-shear rate data.

Modeling and evaluation indicators

Meshes and boundary conditions

The numerical simulation was implemented within the software OpenFOAM, which is a free, open-source and popular numerical simulation software for computational fluid dynamics (CFD) simulations [START_REF] Busse | Numerical modeling of an inductively coupled plasma torch using OpenFOAM[END_REF]. In the modeling, OpenFOAM was used to build geometrical model, generate meshes, determine boundary conditions and solve governing equations. ParaView, an open-source, multi-platform data analysis and visualization application, was used to build visualizations and analyze data obtained by OpenFOAM. As shown in Fig. 1a, FEF was produced by extruding ink in the syringe through the nozzle into the air with the piston movement. Dimensions of the geometrical model of FEF in DIW include: the inner diameter of syringe OpenFOAM is designed as a code for 3D space and defines all meshes as 3D meshes, but wedge type meshes are used for 2D axisymmetric problems specifically [START_REF] Ge | Two-dimensional axisymmetric formulation of high order spherical harmonics methods for radiative heat transfer[END_REF]. Thus, wedge type meshes were used for the simplified 2D axisymmetric model as shown in Fig. 2a, where lengths and widths of meshes in the same area were set nearly same and meshes in the junction of different areas were refined to improve calculation accuracy. Total number of the meshes was 18996. Boundary conditions were grouped into 5 categories and were defined as shown in Fig. 2b and Table 1.

Volume of fluid method

The volume of fluid (VOF) method [START_REF] Scapin | A volume-of-fluid method for interface-resolved simulations of phase-changing two-fluid flows[END_REF] was chosen to capture the interface of FEF in the air. Compared with other CFD numerical simulation methods like level set method [START_REF] Hashemi | An enriched finite element/level-set method for simulating two-phase incompressible fluid flows with surface tension[END_REF], phase field method [START_REF] Yang | A phase-field method for two-phase fluid flow in arbitrary domains[END_REF], lattice-Boltzmann method [START_REF] Zheng | Reduction-consistent axisymmetric lattice Boltzmann equation method for N-phase fluids[END_REF] and direct interface tracking method [START_REF] Li | The evaporation and condensation model with interface tracking[END_REF], VOF method has been proven to be a priority choice for the simulation of two-phase flows due to its simplicity, robustness and straightforward implementation and been widely used in numerical simulation for extrusion-based AM [START_REF] Comminal | Modelling of 3D concrete printing based on computational fluid dynamics[END_REF].

In VOF method, the ink and the air were treated as a single continuum and the phase fraction  was defined to distinguish the interface between the two-phase fluid as follows:

i m V V  = . ( 3
)
Where  is the phase fraction for a mesh; i V is the volume of the ink in the mesh; m V is the total volume of the mesh. Values of  are between 0 to 1 and  =0.5 is defined as the cutoff value to capture the sharp front of interface between the ink and the air. Fluid properties of the single continuum were weighted with the phase fraction as follows:

( ) (
)

1 1 sa sa         = + - = + - . (4)
Where s  and s  is density and viscosity of the single continuum;  and a  is density of the ink and the air, respectively;  and a  is viscosity of the ink and the air, respectively. Governing equations for FEF modeling include continuity equation, momentum equilibrium equation and phase fraction equation. Each equation of the two-phase incompressible laminar flow is written as follows [START_REF] De Sousa | Modeling and simulation using OpenFOAM of biodiesel synthesis in structured microreactor[END_REF]: Continuity equation:

0   = U . ( 5
)
Where U is velocity field of the fluid. Momentum equilibrium equation:

( ) ( ) s s s s s p t       +   = - +    + +  σ U UU U g F . ( 6
)
Where p is the pressure in the fluid; g is the gravitational acceleration vector; σ F is surface tension. σ F is defined as follows:

  = σ F . ( 7
)
Where  is the surface tension coefficient;  is the surface curvature which is depended on the shape of FEF. Phase fraction equation:

( ) ( ) ( ) 10 t      +   +   - =  r UU . ( 8
)
Where r U is velocity vector compressing two-phase free surface, which represents the velocity difference between two-phase fluids and can be calculated as follows:

( )

min ,max c    =  r U U U . ( 9
)
Where c is controllable compression factor and it is selected as c =1 in the numerical simulation.

Numerical simulation with VOF was implemented based on the solvers in OpenFOAM. The movement of the piston (B1) along axis direction was implemented using the motion solver, velocityComponentLaplacian. The governing equations for flow were solved based on the solver for two incompressible, isothermal immiscible fluids using VOF method with optional mesh motion and mesh topology changes including adaptive re-meshing, interFoam. PIMPLE algorithm, a combined algorithm of the pressure-implicit split-operator (PISO) and the semiimplicit method for pressure-linked equations (SIMPLE) algorithms, was chosen as the numerical algorithm for interFoam solver.

Evaluation indicators

Evaluation indicators of FEF include mean diameter and stability as continuous and tubular FEF with diameters close to the actual nozzle inner diameter is the basic requirement for a successful extrusion-based AM [START_REF] Geng | Effects of extrusion speed and printing speed on the 3D printing stability of extruded PEEK filament[END_REF]. N measuring points were set along the filament length uniformly from nozzle bottom at fixed sampling intervals of 1 mm and diameter on each measuring point was measured as ( )

1,2,..., i d i N =
. Mean diameter and stability of FEF were expressed using average and standard deviation of ( )

1,2,..., i d N =
, respectively.

Results and discussion

Material properties tests results

The density and surface tension coefficient of the prepared cellulose ink and the Nivea Crème were obtained by density and surface tension tests and the results were listed in Table 2. 3. As shown in Fig. 4, both cellulose ink and Nivea Crème demonstrated shear thinning behavior, characterized by the decrease in viscosity with increasing shear rate. Shear thinning parameters, K and n , were obtained by fitting Eq. ( 2) with the viscosity-shear rate data using data regression. The results were listed in Table 4. The coefficient of determination ( 2 R ) and p-value of fitting results for cellulose ink was 0.9906 and 0.0000<0.05, and 2 R and pvalue of fitting results for Nivea Crème was 0.9947 and 0.0000<0.05, verified the fitting results for the two inks.

Simulation results and experimental verification

In order to validate the proposed model, the prepared cellulose ink and Nivea Crème was used as inks for FEF extrusion tests in a DIW 3D printer. As shown in Fig. 5a, a piston driven DIW 3D printer TM-081 (Tobeca Company, France) was used to extrude ink in the syringe into FEF in the air; and a camera (Canon LEGRIA HF R86 Noir, Canon Inc., Japan) was used to take photos of FEF. As shown in Fig. 5b, the extrusion tool in the DIW 3D printer was composed of a 30mL syringe (Braun Melsungen AG, Germany) and a precision engineered fluid dispensing nozzle (Nordson EFD, USA). Using the symbols in Fig. 1a, dimensions of extrusion tool were: Disregarding ink compressibility, the mass conservation for the FEF producing process is expressed as:

( ) 2 // p e n s v v D D = , (10)
Where p v is the piston velocity, e v is average velocity of FEF. To verify the proposed model considering the universality for different process parameters, 3 levels of p v were set by setting e v from 5 mm/s to 15 mm/s at fixed intervals of 5 mm/s, which were on the printable range. The 3 levels setting for p v was summarized in Table 5. Simulation and experimental results for the cellulose ink and Nivea Crème under 3 levels of p v were plotted in Fig. 6a and Fig. 6b, respectively. Here, all images were plotted using the same graphical scale in order to provide a direct comparison of simulation and experimental results. Modeling was verified by comparing simulation and experimental results for shape and diameter accuracy. For shape, all simulation results had continuous extruded profiles in accordance with experimental results by observing images in Fig. 6. Diameter accuracy described in Section 3.3 was used to verify the prediction accuracy of the model. Diameter accuracy for the cellulose ink and Nivea Crème under 3 levels of p v were summarized in Table 6, where the maximum error was 0.09 mm and the maximum relative error was 10.22%, verified the model had a high diameter accuracy. Thus, the model was verified both from shape and diameter accuracy.

Usages of the model

After experimental verification, the simulation model was used to evaluate the effect of the piston velocity on the FEF, characterize the ink printability and find suitable printable windows for process parameters with the two evaluation indicators. Firstly, p v were set by setting e v from 1 mm/s to 20 mm/s with Eq. ( 10) and used as input conditions in the proposed model. Then, the two evaluation indicators were used to evaluate FEF under different piston velocities based on the model. As shown in Fig. 7a, there was no continuous and stable FEF when setting of e v was below 3 mm/s and 2 mm/s for the cellulose ink and Nivea Crème, respectively, as piston velocity should be large enough to let FEF overcome yield stress and surface tension to produce continuous and stable filaments. The minimum setting of e v to produce stable FEF for the cellulose ink was higher than that for Nivea Crème because surface tension coefficient of the cellulose ink was higher than that of Nivea Crème. When setting of e v was higher than minimum setting of e v to produce stable FEF, mean diameters of FEF decreased with increasing setting of e v both for the cellulose ink and Nivea Crème. For the cellulose ink, mean diameters of FEF got closer to the nozzle inner diameter as setting of e v increased ranging from 1 mm/s to 20 mm/s. For Nivea Crème, mean diameters of FEF got closer to the nozzle inner diameter as setting of e v increased ranging from 1 mm/s to 10 mm/s and was equal to nozzle inner diameter when setting of e v was 10 mm/s. Then, it moved further away from the nozzle inner diameter as the setting of e v was higher than 10 mm/s. As shown in Fig. 7b, stability decreased firstly and then increased as setting of e v increased ranging from 1 mm/s to 20 mm/s both for the cellulose ink and Nivea Crème. From Fig. 7, it is concluded that the printability of Nivea Crème was better than the cellulose ink as mean diameter got closer to the nozzle inner diameter and stability was much smaller for Nivea Crème compared with the cellulose ink. Meanwhile, considering the requirement that FEF should have mean diameter close to nozzle inner diameter and good stability, printable window of setting of

Conclusions

In this study, a model of FEF was established with numerical simulation method and then the FEF was evaluated based on this model. First, ink properties were determined by properties tests. Then, model of FEF was established using numerical simulation method and two evaluation indicators, mean diameter and stability, were proposed to evaluate the FEF. Finally, two inks, a cellulose ink and a Nivea Crème, were used to verify the method. The established model was verified as the shape in simulation was in accordance with experimental results and diameter accuracy was better than 0.09 mm. After experimental verification, the model and evaluation indicators were used to deal with these three questions in DIW as: [START_REF] Cesarano | ROBOCASTING PROVIDES MOLDLESS FABRICATION FROM SLURRY DEPOSITION[END_REF] Analyze effect of the piston velocity on FEF: there was a minimum piston velocity, 3 mm/s for the cellulose ink and 2 mm/s for Nivea Crème for setting of decreased firstly and then increased as piston velocity increased, (2) Characterize the ink printability: the printability of Nivea Crème was better than the cellulose ink as mean diameter got closer to the nozzle inner diameter and stability was much smaller for Nivea Crème compared with the cellulose ink, (3) Find suitable printable windows for process parameters: printable window of setting of e v was 15mm/s-20 mm/s for the cellulose ink and 5 mm/s-15 mm/s for Nivea Crème. Future work could establish a simulation model of the filaments deposited on the substrate considering the ink properties and process parameters. The printing quality could then be improved by using the established simulation model in order to extend the FEF contribution in this study to deposited filaments.

 and 0 

 0 1 s -),  (unit: Pa s  ) are variables named as shear stress, shear rate, dynamic viscosity for inks in flow; n (unit: dimensionless) are rheological properties called as limiting dynamic viscosity, yield stress, consistency index, flow index for inks. If stress< 0  , the ink behaves as a rigid solid, otherwise it behaves as a fluid. For n <1, the ink is shear-thinning, whereas the ink is shearthickening. A large 0  means that the ink will only flow in response to a large applied force. A rheometer ARES (TA Instruments, USA) with a 25 mm plate-plate geometry at a gap distance of 1 mm was used to obtain parameters in Eq. (1) and Eq. (2). by shear stress ramp test ranging from 0.01 to 100 Pa: abscissa and ordinate of intersection point between two linear regressions at the plateau-region and viscosity-drop regions of the viscosity-shear stress diagrams was 0 , respectively. K , n were determined by shear rate sweep test ranging from 0.0125 to 10001

sDL

  ; the ink filled length s L ; the nozzle inner diameter n D ; the nozzle length n L ; the gap between nozzle bottom and substrate h . In this study, h was set as 26 mm to avoid FEF touching the substrate. and the initial s L were set to match the actual device dimensions: 21.6 mm, 0.84 mm, 18 mm, 30 mm, respectively. Fig.1bpresents the 3D model established in OpenFOAM for FEF at the initial time, where red regime represents the ink and blue regime represents the air. As shown in Fig.1c, the 3D model was simplified into a 2D axisymmetric model by capturing 1/120 of the 3D model in the circumferential direction to reduce computation cost under the axisymmetric laminar flow condition for the ink extrusion.

Fig. 1

 1 Fig. 1 Geometrical model of FEF in numerical simulation for DIW: (a) dimensions of the geometrical model; (b) established 3D model at the initial time; (c) simplified 2D axisymmetric model at the initial time.

Fig. 2

 2 Fig. 2 Meshes and boundary conditions for model of FEF in numerical simulation for DIW: (a) meshes; (b) boundary conditions.

 and 0 

 0 were determined using the intersection point of two tangents in the diagram of shear stress ramp test data, one was stable platform line (red dotted line) in the plateau-region and one is the quickly-drop line (purple dotted line) in the viscosity-drop region. Abscissa and ordinate of the intersection point was 0 , respectively. The results were listed in Table

Fig. 3

 3 Fig. 3 Shear stress ramp data for (a) cellulose ink and (b) Nivea Crème.

Fig. 4

 4 Fig. 4 Experimental data and fitting curve of shear rate sweep test for (a) cellulose ink and (b) Nivea Crème.

Fig. 5

 5 Fig. 5 Experimental validation setup for the proposed model: (a) producing FEF using DIW 3D printer and observing FEF using camera; (b) extrusion tool in the DIW 3D printer.

Fig. 6

 6 Fig. 6 Simulation and experimental results of FEF under 3 levels of piston velocities for: (a) cellulose ink; (b) Nivea Crème.

  e v for FEF was summarized from Fig. 7: 15mm/s-20 mm/s for the cellulose ink; 5 mm/s-15 mm/s for Nivea Crème.

Fig. 7

 7 Fig. 7 Evaluation of the FEF for cellulose ink and Nivea Crème using the proposed two evaluation indicators as: (a) mean diameter; (b) stability.

Table 1

 1 Definition of boundary conditions

	Name	Physical meaning	Type	Setting
	B1	Piston	Moving boundary	Velocity is equal to piston velocity
	B2	Inner walls of syringe; walls of nozzle and substrate	No-slip boundary	No-slip
	B3	Air	InletOutlet boundary	Depended on dynamic calculation
	B4	Symmetrical axis	Symmetrical boundary	Empty condition

Table 2

 2 Results of density and surface tension coefficient for cellulose ink and Nivea Crème.

Table 3

 3 Results of yield stress and limiting dynamic viscosity for cellulose ink and Nivea Crème.

Table 4

 4 Results of shear thinning parameters for cellulose ink and Nivea Crème.

Table 5

 5 Piston velocity setting in the experimental validation.

Table 6

 6 Results of diameter accuracy of the proposed model for cellulose ink and Nivea Crème.

		Diameter accuracy under
	Ink	3 levels of

e v , to produce continuous and stable FEF; mean diameters of FEF decreased with increasing piston velocity. Stability

Funding

This work has been supported by the China Scholarship Council (No. 201906020135).

Data availability

The authors confirm that all data and materials reported in this paper are available.

Authors contributions

YT: Methodology, Writing-original draft. AH: Conceptualization, Methodology, Writingreview & editing. JA: Test, Writing-original draft. UKZ: Writing -review & editing. AS: Supervision. GY: Supervision.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Ethical approval