# Differential impact of Crouzon and Apert syndromes on upper airways morphology: implications for Obstructive Sleep Apnoea

Floriane Remy, Maxime Taverne, Roman Hossein Khonsari, Brigitte Fauroux, Sonia Khirani, Neus Martínez Abadías, Yann Heuzé

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## To cite this version:

Floriane Remy, Maxime Taverne, Roman Hossein Khonsari, Brigitte Fauroux, Sonia Khirani, et al. Differential impact of Crouzon and Apert syndromes on upper airways morphology: implications for Obstructive Sleep Apnoea. Journal of Cranio-Maxillofacial Surgery, 2025, 53 (9), pp.1577-1591. 10.1016/j.jcms.2025.06.014. hal-05343296

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# Differential impact of Crouzon and Apert syndromes on upper airways morphology: implications for Obstructive Sleep Apnoea

Floriane Remy<sup>a, *, D</sup>, Maxime Taverne<sup>b, D</sup>, Roman Hossein Khonsari<sup>b, c, d</sup>, Brigitte Fauroux<sup>e</sup>, Sonia Khirani<sup>e, f, D</sup>, Neus Martínez Abadías<sup>g, D</sup>, Yann Heuzé<sup>a, ** ID</sup>

## ARTICLE INFO

### Keywords:

Syndromic craniosynostosis

Pharynx

Nasal airway

Obstructive respiratory disorders

FGFR

## ABSTRACT

Children with syndromic craniosynostoses face high risk of obstructive sleep apnoea (OSA), but the underlying mechanisms remain unclear. This study investigated how Crouzon and Apert syndromes affect airway morphology and contribute to OSA, controlling for age and posture. We also examined the covariation between the upper airways and the craniofacial region (skeleton and soft tissues). We analysed 37 patients with syndromic craniosynostoses, and 53 control children aged from 0 to 49 months old. A set of 52 landmarks was placed on the 3D models of the upper airways and collected, when available, the apnoea-hypopnoea index (AHI). Our geometric morphometric analyses demonstrated that age, mouth openness and craniocervical posture significantly influenced airway shape, emphasizing the need for standardised imaging. After controlling these factors, we highlighted oropharynx changes in patients with Crouzon syndrome, while Apert syndrome altered the nasopharynx. No direct link between airway shape and OSA was found. Upper airways growth and its morphological covariation with the craniofacial region were not impacted by the diagnosis of syndromic craniosynostosis. Finally, these findings highlight syndrome-specific airway differences, FGFR mutations impacting both bone and surrounding soft tissues. This study provides valuable insights for more precise respiratory diagnoses and personalised treatment plans.

## 1. Introduction

Obstructive sleep apnoea (OSA) is caused by an anatomical narrowing of the upper airways, impairing or reducing the airflow (referred to as apnoea or hypopnoea, respectively). OSA is associated with oxygen desaturation, sleep fragmentation, and various signs and symptoms such as behavioural and cognitive issues, respiratory and cardiac comorbidities, and growth impairment (Guilleminault et al., 1976; Fagundes et al., 2022). Specific craniofacial features have been highlighted in paediatric patients with upper airway obstruction, including a higher frequency of mandibular hypoplasia (i.e. a mandible with reduced dimensions), a narrower and ogival-shaped palate, reduced nasal cavities,

greater facial height and relative macroglossia or glossoptosis (respectively, a tongue that is too large for the oral space or positioned backward in the pharynx) compared to children with no upper airway anomalies (Caron et al., 2015; Finke et al., 2024; Flores-Mir et al., 2013). Usually, OSA is diagnosed and characterised based on a polysomnography which allows the computation of the apnoea/hypopnoea index (AHI). The AHI measures the number of apnoea and hypopnoea events identified per hour of recorded sleep time and constitutes the main parameter to diagnose OSA and its severity (Aurora et al., 2011).

While the prevalence of OSA ranges between 1.2 and 5.8 % in the general paediatric population, it affects approximately 50 % of patients with syndromic craniosynostosis (Bannink et al., 2010; Bixler et al.,

<footnote><sup>a</sup> Univ. Bordeaux, CNRS, Ministère de la Culture, PACEA, UMR 5199, 33615, Pessac, France</footnote>

<footnote><sup>b</sup> Laboratoire 'Forme et Croissance du Crâne', AP-HP Hôpital Necker-Enfants Malades, Paris, France</footnote>

<footnote><sup>c</sup> Université de Paris, Institut Imagine, INSERM UMR1163, Paris, France</footnote>

<footnote><sup>d</sup> Service de Chirurgie Maxillo-Faciale et Chirurgie Plastique, AP-HP, Hôpital Necker-Enfants Malades, CRMR MAFACE, Filière Maladies Rares TeteCou, Université de Paris, Paris, France</footnote>

<footnote><sup>e</sup> Unité de Ventilation Non invasive et du Sommeil, AP-HP, Hôpital Necker-Enfants Malades, Université de Paris EA 7330 VIFASOM, Paris, France</footnote>

<footnote><sup>f</sup> ASV Santé, Gennevilliers, France</footnote>

<footnote><sup>g</sup> Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals (BEECA), Facultat de Biologia, Universitat de Barcelona (UB), Barcelona, Spain</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: Floriane.remy.ymd@gmail.com (F. Remy), yann.heuze@u-bordeaux.fr (Y. Heuzé).</footnote>
2009; Marcus et al., 2012). Craniosynostosis is defined as the premature fusion of one or more cranial sutures, and is associated with skull growth disturbances (Johnson and Wilkie, 2011). It affects approximately 1:2100 to 1:2500 live births, and 40 % of cases are syndromic (Johnson and Wilkie, 2011; Mathijssen, 2015).

Over 150 syndromes are associated with craniosynostosis (Cohen, 1995; Rasmussen et al., 2003). The majority of cases of syndromic craniosynostoses is caused by gain-of-function mutations in the Fibroblast Growth Factor Receptor (FGFR) genes which, among other functions, play an important role in bone development (Azoury et al., 2017; Britto et al., 2001). Among FGFR-related craniosynostoses, Apert and Crouzon syndromes have respective prevalence of approximately 1:100,000 and 1:25,000 live births (Buchanan et al., 2014; Wilkie et al., 2017). More than 50 mutations have been associated with Crouzon syndrome, while only the Ser252Trp, Ser252Phe and Pro253Arg mutations have been reported in Apert syndrome (Azoury et al., 2017; Galvin et al., 1996; Wilkie et al., 1995). The majority of Apert and Crouzon syndrome patients exhibit premature fusion of the coronal suture and restricted growth of the skull in its anteroposterior dimension, counterbalanced by an increased bitemporal width to accommodate brain growth (brachycephaly). Craniofacial anomalies include proptosis (bulging of the eye anteriorly out of the orbit), hypertelorism (increased distance between the orbits), cleft lip and/or palate, and midface hypoplasia, with the frontal, zygomatic and maxillomandibular regions displaying the most marked shape differences (Alam et al., 2023; Azoury et al., 2017; Heuzé et al., 2014; Martínez-Abadías et al., 2010; Wenger et al., 1993). Despite these characteristic features, phenotypic expression in these syndromes is highly variable, with multiple tissues and organs affected, indicating the pleiotropic effect of these FGFR mutations (Britto et al., 2001; McIntosh et al., 2000). The literature also highlights that patients with Apert syndrome tend to exhibit more severe phenotypes when carrying the Ser252Trp mutation compared to the Pro253Arg mutation (Heuzé et al., 2014; Morice et al., 2020; Pinto et al., 2023; Kreiborg and Cohen, 1998).

While it is well known that FGFR overactivation causes abnormalities in different tissues, organs and systems, including the craniofacial region (Alam et al., 2022; Alam et al., 2023; Heuzé et al., 2014; Martínez-Abadías et al., 2018; McIntosh et al., 2000), the direct connection between this mutation and upper airway morphology has not been clearly demonstrated (Nash et al., 2015; Fagundes et al., 2022). Few studies have explored the anatomical basis of respiratory obstruction in children affected with syndromic craniosynostosis, and most have focused solely on dimensional differences (Morice et al., 2023). Some authors have reported increased upper airway length, decreased posterior airway space and a smaller minimum retropalatal cross-sectional area in young patients with syndromic craniosynostosis and OSA (Dentino et al., 2015; Resnick et al., 2020). Still, the mechanistic relationship between these changes in the craniofacial structure and respiratory obstruction remains poorly understood.

This study aimed to assess differences in the shape of upper airways in patients with Crouzon and Apert syndromes, and their relationship with OSA. Given the pleiotropic and specific effects of FGFR mutations, we hypothesised that the upper airways shape would differ significantly between unaffected children and patients with craniosynostosis syndrome, as well as between patients with Crouzon and Apert syndromes. Moreover, considering the anatomical connections between the upper airways and the surrounding soft and hard tissues of the craniofacial region, we anticipated that a child's posture during medical imaging could influence the morphological variation of the upper airways. We aimed to determine whether these posture-related differences should be considered when investigating upper airways shape variability. The same hypothesis was made regarding the age and sex of the patients. Third, we hypothesised that, once relevant confounding variables were accounted for, upper airways would exhibit morphological differences in patients diagnosed with OSA. Finally, we evaluated the morphological covariation between upper airways and the craniofacial region,

testing whether this covariation differed depending on the anatomical area considered and/or the syndrome. Understanding these interactions should improve OSA prediction and guide more tailored therapeutic strategies for affected children.

# 2. Materials and methods

## 2.1. Cohort

Pre-operative computed tomography scans (CT-scans) of 37 children aged from 0 to 49 months, diagnosed with Apert or Crouzon syndrome, were obtained from the Hôpital Necker-Enfants Malades (Paris, France). Only patients who had not undergone any treatment (such as upper airway surgery, tracheotomy, maxillofacial surgery, neurosurgery or continuous positive airway pressure - CPAP) at the time of image acquisition were included in the study. The CT-scans of an age-matched sample of 53 unaffected children was also collected as controls within the databases of Hôpital Necker-Enfants Malades, along with four additional French medical institutions (CHU de Caen Normandie, CHU de Montpellier, Hospices Civils de Lyon, Assistance Publique-Hôpitaux de Marseille). This unaffected sample consisted of children who underwent CT-scans shortly after experiencing minor trauma or infections that did not impact their craniofacial anatomy. All these images were collected under the ethics clearance received from the Institutional Review Board and Committee of Hôpital Necker-Enfants Malades and the Institutional Review Board of Aix-Marseille University.

For each individual, we recorded the genetic diagnosis when available together with sex and age at the time of exam. To account for the rapid growth rates observed during the first years of life, which gradually slow down (Lo et al., 2022; Tutkuviene et al., 2016), and to ensure homogeneous groups size, we defined the five following arbitrary age groups: <3 months, 3-6 months, 6-12 months, 12-24 months, and >24 months (Fig. 1).

## 2.2. Sleep parameters

Twenty-seven patients had a respiratory polygraph (PG): 21 patients with Crouzon syndrome and 6 patients with Apert syndrome. Based on the AHI, four categories of OSA severity were defined: <1 (no OSA), [1-5] (mild OSA), [5-10] (moderate OSA) and ≥10 (severe OSA) (Marcus et al., 2013). Consequently, analyses considering the presence/absence of OSA and its severity were performed only on the 27 patients who had a PG. The mean duration between the CT-scan and the PG was 6 months ±7 months (min - max = 1 day - 29 months).

## 2.3. Other potential confounding parameters

CT-scans were included when the craniofacial anatomy and cervical spine (at least to the 3rd cervical vertebra) were fully visible. Since these images were retrospectively collected, the posture of the child during CT-scan acquisition could not be controlled. Thus, we recorded the craniocervical inclination both qualitatively and quantitatively, categorised as neutral or inclined, without specifying the direction of inclination. The lateral and anteroposterior inclinations of the head were also measured, computed as the angles between the glabella, the 3rd cervical vertebra and, respectively, the anterior nasal spine or the occiput (Fig. 2). We also recorded mouth openness defined as wide open, slightly open or fully closed, and also assessed it with the computation of the opening angle, measured between the right porion, homolateral gonion and gnathion (Fig. 2). Finally, we recorded whether there was any object in the child's mouth (e.g. thumb, pacifier). Patients with nasal or oral intubation were not included in the study.

The distribution of these parameters within each sample (Apert, Crouzon and unaffected) is summarised in Table 1.
<figure><img src="image_5.png" /><figcaption>Fig. 1. Age and sex distribution of the Apert and Crouzon syndrome cohorts (left, from top to bottom) and unaffected sample (right).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 2. Measurements of the angle openness (left) and inclination of the head either on the anteroposterior axis (middle) or on the side (right). Abbreviations: ANS – Anterior Nasal Spine; 3rd CVT – third cervical vertebra.</figcaption></figure>

## 2.4. Digitization of the upper airways volume and of the skeletal components

From the collected CT-scans, 3D models of the upper airways were reconstructed using Avizo® software (Avizo® Standard Edition 9.5.0, Thermo Fisher Scientific, MA, USA).

The Label Analysis module was used to isolate the lumen representing the upper airway tract. With the brush tool, we only segmented the parts with the highest grey-value intensity enclosed within the craniofacial bones, spanning from the nares to the transverse slice of the CT-scan located at the level of the 3rd cervical vertebra body. The oral space, that is the space between the posterior tip of the soft palate and the exit of the mouth, was not included in this reconstruction.

3D volumes of the skull, mandible and upper cervical spine (from the 3rd cervical vertebra to the atlas, including the hyoid bone) were also digitised using Avizo's Isosurface module, selecting a single grey-value threshold optimised for bone detection.

## 2.5. Landmarking

To account for the complex shape of the upper airways, we initially defined 20 slices distributed along their length and parallel to either a coronal plane or an axial reference plane (Fig. 3).

Regarding the nasal tract, 7 slices representing both nares (hence a total of 14 slices), were positioned in alignment with a coronal plane defined by the extremities of the vertebral pedicles and the vertex and at

the levels of the subspinale, nasion, anterior nasal spine, alare, crista galli, and anterior and posterior borders of the clinoid process.

Additional slices were placed through the pharynx along the axial plane defined by the interincisor point and the maxillary tuberosities, at the levels of the choanal bifurcation (one slice per nasal tract), posterior nasal spine, inferior border of the sphenoid, superior tip of the epiglottis and body of the 3rd cervical vertebra.

On each of these 20 slices, we placed a landmark on their foremost, rearmost and outermost aspects, resulting in a set of 80 landmarks.

For some patients, potentially due to bony deviation or increased mucosa volume, the lumen in the area of the crista galli, anterior border of the clinoid process, choanal bifurcation and posterior nasal spine could not be reconstructed, leading to missing landmarks. Consequently, these landmarks were discarded, and our study was ultimately conducted on 52 landmarks, and the presence/absence of an interruption in the upper airway segmentation was recorded. This interruption in the upper airways' segmentation was present in 8 % of Crouzon syndrome patients, 27 % of Apert syndrome patients and 4 % of unaffected children.

To fully characterise the morphology of the craniofacial region (both hard and soft tissues), 71 landmarks were defined (Supporting Information Data S1). Regarding the characterization of soft tissues (i.e. the tongue, epiglottis and soft palate), we extracted the midsagittal slice, defined as the plane passing through the nasion (the junction between the frontal bone and the two nasal bones), vertex (the highest point on the skull along the sagittal suture when viewed laterally) and lambda
<table><caption>**Table 1**

Characteristics of the Crouzon syndrome, Apert syndrome and unaffected co-horts. Abbreviations: AHI – Apnoea hypopnoea index; OSA – Obstructive sleep apnoea; SD – standard deviation.</caption><thead><tr><th></th><th>Crouzon patients</th><th>Apert patients</th><th>Unaffected children</th></tr></thead><tbody><tr><td><strong>Sample size (n)</strong></td><td></td><td></td><td></td></tr><tr><td>Total</td><td>26</td><td>11</td><td>53</td></tr><tr><td>Girls</td><td>13</td><td>6</td><td>22</td></tr><tr><td>Boys</td><td>13</td><td>5</td><td>31</td></tr><tr><td><strong>Age (months)</strong></td><td></td><td></td><td></td></tr><tr><td>Min – Max</td><td>0 – 48.9</td><td>1.4 – 11.0</td><td>0.1 – 47.5</td></tr><tr><td>Mean ± SD</td><td>12.3 ± 12.1</td><td>6.4 ± 3.1</td><td>11.3 ± 11.4</td></tr><tr><td><strong>AHI (events/hour)</strong></td><td></td><td></td><td></td></tr><tr><td>Sample size (n - %)</td><td>21 (81 %)</td><td>6 (54 %)</td><td>0 (0 %)</td></tr><tr><td>Mean ± SD</td><td>11.9 ± 21.0</td><td>2.9 ± 1.8</td><td>–</td></tr><tr><td><strong>OSA (on 27 patients) (n - %)</strong></td><td></td><td></td><td></td></tr><tr><td>None</td><td>5 (19 %)</td><td>1 (9 %)</td><td>–</td></tr><tr><td>Mild</td><td>6 (23 %)</td><td>4 (36 %)</td><td>–</td></tr><tr><td>Moderate</td><td>3 (11 %)</td><td>1 (9 %)</td><td>–</td></tr><tr><td>Severe</td><td>7 (27 %)</td><td>0 (0 %)</td><td>–</td></tr><tr><td><strong>Mouth posture</strong></td><td></td><td></td><td></td></tr><tr><td>Slightly open (n - %)</td><td>13 (50 %)</td><td>6 (54 %)</td><td>27 (51 %)</td></tr><tr><td>Wide-open (n - %)</td><td>8 (31 %)</td><td>5 (46 %)</td><td>6 (11 %)</td></tr><tr><td>Closed (n - %)</td><td>5 (19 %)</td><td>0 (0 %)</td><td>20 (38 %)</td></tr><tr><td>Opening angle (Mean ± SD - °)</td><td>45.26 ± 3.9</td><td>51.57 ± 4.5</td><td>42.98 ± 3.3</td></tr><tr><td><strong>Presence of an object in the mouth (n - %)</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>16 (61 %)</td><td>7 (64 %)</td><td>17 (81 %)</td></tr><tr><td>No</td><td>10 (39 %)</td><td>4 (36 %)</td><td>36 (81 %)</td></tr><tr><td><strong>Craniocervical posture</strong></td><td></td><td></td><td></td></tr><tr><td>Inclined (n - %)</td><td>5 (19 %)</td><td>0 (0 %)</td><td>11 (21 %)</td></tr><tr><td>Neutral (n - %)</td><td>21 (81 %)</td><td>11 (100 %)</td><td>42 (79 %)</td></tr><tr><td>Lateral inclination angle (Mean ± SD - °)</td><td>24.06 ± 3.6</td><td>24.64 ± 4.1</td><td>22.34 ± 2.9</td></tr><tr><td>Anteroposterior inclination angle (Mean ± SD - °)</td><td>30.37 ± 16.6</td><td>41.25 ± 16</td><td>29.79 ± 12.6</td></tr></tbody></table>

(the intersection of the lambdoid and sagittal sutures).

## 2.6. Data analysis

All statistical and geometric morphometric analyses detailed below were carried out with R version 4.2.3 software (R Core Team, 2023) and a significance threshold set at 5 %.

### 2.6.1. Intra-observer measurements reliability

Intra-observer reliability in landmark placement was assessed by the principal observer (F.R.), who repeated the landmark annotation for 15 individuals randomly selected within the total study sample (8 syndromic patients and 7 unaffected) at two separate times. The agreement between the two acquisitions was assessed using the Lin's Concordance Correlation Coefficient (LCC), as implemented by the R package epiR version 2.0.61, using the epi.ccc function (Lin, 1989; Stevenson et al., 2013). Following the literature, repeatability was defined as "poor" (LCC <|0.50|), "moderate" (|0.50|≤ LCC <|0.70|), "substantial" (|0.70|≤ LCC <|0.85|) or "high" (LCC ≥ |0.85|) (Landis and Koch, 1977; Partik et al., 2002).

### 2.6.2. Generalised procrustes superimposition

Shape variation and covariation between craniofacial and upper airways morphologies were investigated using Procrustes coordinates (Goodall, 1991; Rohlf and Slice, 1990). For each anatomical structure (i. e. skull, mandible, soft tissues and upper airways), we independently performed a Procrustes superimposition using the gpagen function of the R package geomorph version 4.0.5 (Adams et al., 2022; Baken et al., 2021). This method performs size normalization, as well as translation and rotation of each configuration of landmarks until an optimal superimposition is achieved, minimising the distances between homologous landmarks according to a least-squares criterion. The coordinates of the resulting scaled, centred, and rotated landmark configurations constitute the Procrustes shape coordinates used in the following analyses.

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Left: slices defined at the level of the subspinale (slice number 1, one slice per side; 1\*2), nasion (2\*2), anterior nasal spine (3\*2), alare (4\*2), crista galli (5\*2), anterior (6\*2) and posterior borders of the clinoid process (7\*2), choana bifurcation (8\*2), posterior nasal spine (9), inferior border of the sphenoid (10), superior tip of the epiglottis (11) and body of the 3rd cervical vertebra (12). Red crosses: regions where the lumen could not be reconstructed, suppressed from the analysis. Right: example of the landmarks set on one slice, used for the characterization of the shape of the nasal airways (upper) and pharynx (lower).</figcaption></figure>
### 2.6.3. Evaluation of the overall upper airways size

Given that previous literature has reported size differences in the upper airways, we analysed whether the overall dimensions of the upper airways varied according to craniosynostosis syndrome, OSA, age and sex. We used centroid size as a proxy for airway dimensions, computed as the square root of the sum of squared distances of all the landmarks of an object from their centroid (Mitteroecker and Gunz, 2009). Kruskal-Wallis' or Mann-Whitney's tests were applied to identify significant differences across groups. When a significant difference was highlighted with the Kruskal-Wallis' test, a Dunn's test was used for post-hoc pairwise comparisons, with p values adjusted using Bonferroni correction.

To investigate how shape was impacted by size across patient groups, we conducted a multivariate analysis of covariance (MANCOVA) using the procD.lm function of the R package geomorph. To visualise this relationship, we used the plotAllometry function from the same package and logarithmic regression lines were computed for the unaffected children, Crouzon or Apert patients and compared using ANOVA. The same analyses were performed to assess the relationship between size and age for each patients' group, that is whether the growth trajectory varied according to craniosynostosis syndrome.

### 2.6.4. Identifying potential confounding factors on the upper airways shape variation

We assessed whether any parameters reflecting the diagnosis (syndromic craniosynostosis or OSA), biological profile (age and sex), or the position of the child during image acquisition (mouth openness, craniocervical posture and presence/absence of an object within the child's mouth) played a role in inter-individual morphological variation of the upper airways. To answer this question, we performed permutation tests (10,000 permutations) using the procD.lm function of the R package geomorph. This test aimed to compare the Procrustes distance between each group mean, defined by a specific categorical variable (i.e. either age, sex, mouth openness, craniocervical posture or presence/absence of an object in the mouth) to the distances obtained by random assignment of observations to groups, assessing whether the average upper airway shape significantly differed according to these variables. These Procrustes distances were computed with procdist function of shapes package version 1.2.7 (Dryden and Mardia, 2016).

The same objective was achieved by computing the R² and p values obtained from the multivariate regression of shape on the quantitative expression of the same parameters (age expressed in months, mouth opening angle, and head inclination angles in the lateral and anteroposterior directions), using the same procD.lm function.

### 2.6.5. Assessment of shape variation of the upper airways according to the child's diagnosis after removing the effect of the significant confounding factors

We also investigated whether significant differences existed in the shape of the upper airways according to the diagnosis of syndromic craniosynostosis or OSA after removing the effect of variables identified earlier as being significantly associated with upper airways shape variation. To achieve this, we first computed the residuals from the multivariate regression of shape on the relevant variables with the procD.lm function and then performed new PCA on these residuals with their associated permutation tests to assess the significance of the Procrustes distance between each group mean. Based on these results, we compared the extreme shapes associated with these PCA using the shapeروع function of the Morpho package version 2.8.

Finally, we performed additional statistical analyses to assess whether any of these variables were significantly associated with the diagnosis of either syndromic craniosynostosis or OSA, using Kruskal-Wallis' or Mann-Whitney's tests for the quantitative parameters (age, AHI, mouth opening and both craniocervical inclination angles) and Chi² tests for the qualitative ones (sex, presence/absence of an interruption in the segmentation of the upper airways).

### 2.6.6. Evaluation of the morphological covariation between the upper airways and the craniocervical region

Finally, we assessed the level of morphological covariation between the nasal airway and the pharynx on one hand, and between the global upper airways and each craniocervical structure (i.e. skull, mandible and soft tissues) on the other. This was done by performing 2-blocks Partial Least Square (PLS) analyses with the two.b.pls function (R package geomorph). For the comparison between the shape of the upper airways and soft tissues, only the landmarks set on the anterior and posterior borders of the upper airways were analysed to balance the size of each landmark configuration.

## 3. Results

### 3.1. Repeatability of landmark annotation

In all anatomical regions (upper airways, skull, mandible and soft tissues), Lin's coefficients assessing measurement agreement were systematically superior to 0.60, and superior to 0.95 for 96 % of the landmarks, indicating an excellent intra-observer reliability of their placement (Supporting Information Data S2) (McBride, 2005).

### 3.2. Evolution of size of the upper airways

Centroid size did not significantly differ with respect to the diagnosis of syndromic craniosynostosis or sex (Fig. 4a and b). Patients with OSA had significantly smaller upper airways than those without OSA, without any difference between the OSA severity levels (Fig. 4c). Centroid size also significantly increased with age (Fig. 4d).

Since the p values resulting from the MANCOVA were systematically superior to 0.05, we concluded that the relationship between centroid size and shape or age did not depend on Crouzon or Apert syndrome. Thus, distinct shape patterns emerged independently of size and age. We also noticed that the growth trajectories of both shape and centroid size did not vary considerably with craniosynostosis syndrome: the ANOVA highlighted no significant differences between the logarithmic regression lines associated with each group (Fig. 5).

Considering all these results, we decided not to remove the influence of size on shape and further analyses were performed on the Procrustes shape coordinates.

### 3.3. Identifying the parameters affecting the morphological variations of the upper airways

The variables for which we tested a potential association with upper airways shape variation included age, sex, mouth openness, craniocervical position, and the presence/absence of an object in the child's mouth. The results of the analyses performed on the qualitative expression of these variables are presented in Supporting Information Data S3. These analyses demonstrated that only age, mouth opening (more especially wide-open vs fully closed mouth) and craniocervical posture significantly influenced the morphological variation of the shape of the upper airways. Conversely, sex and the presence/absence of an object in the child's mouth did not affect upper airways morphology.

Quantitative multivariate linear regression analyses confirmed that age, mouth opening angle, and lateral head inclination angles are potential confounding factors for upper airway shape variation (Table 2).

It should be noted that the mouth opening angle and both craniocervical inclination angles varied significantly according to the diagnosis of syndromic craniosynostosis but not that of OSA. Besides, we noticed that most syndromic patients showed a wide or slightly open mouth: no Apert patients and only 19 % of Crouzon patients presented a closed mouth, as compared to 28 % of children within the unaffected sample. Likewise, there was a majority of individuals having something in their mouth in the syndromic samples compared to the unaffected one and the majority of the individuals had a neutral craniocervical posture.
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Distribution of the centroid size according to a) the diagnosis of syndromic craniosynostosis, b) sex, c) OSA and d) age group (the number of asterisks associated with the brackets indicate the significance of the differences: \*p value < 0.05, \*\*p value < 0.01, \*\*\*p value < 0.001).</figcaption></figure>

These differences in the distribution of each diagnosis were confirmed by statistical tests (Chi² tests' p value < 0.05). The presence of an interruption in the segmentation of the upper airways was not significantly associated with neither of these variables. These detailed results are presented in *Supporting Information Data S4*.

### 3.4. Exploring upper airways shape variation according to the child's diagnosis after removing the effect of the posture

We investigated the morphological variation of the upper airways according to craniosynostosis syndrome and OSA after removing the influence of age, mouth openness and lateral head inclination. Consequently, we ran PCAs on the residuals of the multivariate regression of shape vs. age, mouth openness and lateral inclination.

These PCAs highlighted significant morphological differences of the upper airways' shape between the patients and unaffected children, and between patients with Apert and Crouzon syndromes (Fig. 6 and Table 3). Conversely, no significant differences were found in the shape of the upper airways according to the presence of OSA and its severity (Fig. 7, Table 3).

Considering these results highlighting significant shape differences between unaffected children and both craniosynostosis syndromes, we compared the extreme shapes along the 1st and 3rd PC of the PCA (2D plots on Figs. 8 and 3D plots in Supporting Information Data S5 and S6 respectively). Patients with Apert syndrome displayed a less pronounced angulation of the junction between the nasal airway and pharynx and

maybe a narrower nasopharynx than patients with Crouzon syndrome who also seem to have smaller dimensions than unaffected children, especially in the oropharynx region.

### 3.5. Is there any significant covariation between the upper airways and the craniofacial region?

PLS analyses indicate a significant and strong covariation of the shape of the upper airways with that of the craniofacial region (the skull, mandible and soft tissues): Fig. 9 and Table 4. We also demonstrated significant and strong shape covariation between the nasal airway and the pharynx. However, we noticed that this anatomical integration between the shape of the upper airways and that of the mandible and soft tissues was no longer significant for the patients with Apert syndrome. Likewise, there was no significant morphological integration between the nasal airway and pharynx in these patients. These results may be explained by the small size of the Apert sample (n=11).

## 4. Discussion

### 4.1. Objectives and main results

The objective of this paper was to investigate the shape differences in the upper airways among children with Crouzon or Apert syndromes and their potential links to OSA. We also explored whether factors such as the patient's position during imaging, age, and sex influence the
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Correlation between the shape and either the age (upper) or the centroid size (lower), with the distinction of each diagnosis of syndromic craniosynostosis and the logarithmic regression lines associated to each of them.</figcaption></figure>

airways' morphology. Additionally, this study evaluated the morphological covariation between the upper airways and the associated craniofacial anatomical structures.

We highlighted significant shape differences of the upper airways between patients with syndromic craniosynostosis and unaffected children, and between patients with Apert and Crouzon syndrome patients.

Interestingly, the specific regions of airway constriction appear to differ between the two syndromes: predominantly in the nasopharynx region for patients with Apert syndrome, whereas in the oropharyngeal region for patients with Crouzon syndrome. However, we did not find any significant association between the shape of the upper airways and OSA, regardless of its severity. Our results demonstrated that age, mouth
<table><caption>**Table 2**

Results of the multivariate regression model. Bold values indicate significant p values.</caption><thead><tr><th></th><th>Rho</th><th>P value</th></tr></thead><tbody><tr><td>Age (in months)</td><td>0.081</td><td>9.99e-04</td></tr><tr><td>Mouth opening angle (in °, angle between the right porion, homolateral gonion and gnathion)</td><td>0.035</td><td>0.004</td></tr><tr><td>Lateral inclination (in °, angle between the glabella, the 3rd cervical vertebra and the anterior nasal spine)</td><td>0.082</td><td>0.007</td></tr><tr><td>Anteroposterior inclination (in °, angle between the glabella, the 3rd cervical vertebra and the occiput)</td><td>0.015</td><td>0.209</td></tr></tbody></table>

opening and lateral head inclination are factors to consider when investigating the morphological variation of the upper airways. Despite this morphological variation, the growth trajectory of the upper airways and their covariation with the craniofacial region was not impacted by the syndrome: the relation between either the shape or the centroid size and the age did not vary considerably depending on the diagnosis of syndromic craniosynostosis. Additionally, we observed a strong morphological covariation between the upper airways and the craniofacial region, here again regardless of the diagnosis of syndromic craniosynostosis. This relation was no longer statistically significant for patients with Apert syndrome, most likely due to the small sample size.

## 4.2. A methodological focus: the child's posture as confounding factors of the upper airways' shape variation

We identified a significant effect of mouth openness and craniocervical posture on the shape of the upper airways. Indeed, the medical images analysed in this study were collected retrospectively, so we did not have control on the position and posture of the child during image acquisition: some children had either a closed or open mouth, with various degrees of openness, sometimes related to the presence of an object into the mouth, such as a pacifier. Besides, it is interesting to note that the syndromic patients of our sample were more inclined to present an open mouth compared to unaffected children. Indeed, one may hypothesise that an open mouth is an indirect marker of upper airway obstruction.

Likewise, there was no standardization in the position of the head, with various angles of inclination, in different directions (to one side, backward or forward). Yet, these factors may affect the position and shape of the anatomical structures surrounding the upper airways, such as the tongue (Castilho and Rocha, 2009; Gurani et al., 2016; Hu et al., 2018; Miller et al., 2012). It is interesting to note that only the lateral inclination of the head had a significant effect on the morphological variation of the upper airways: most likely because the anteroposterior inclination was restricted due to the supine position of children during CT-scanning.

These results are also consistent with the strong and significant shape

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** PCA illustrating the inter-individual variation of the shape of the upper airways after removing the effect of the age, mouth openness and lateral inclination of the head, along the first four PCs, with the distinction between pathological and unaffected children (upper), and between patients with Apert and Crouzon syndromes (lower).</figcaption></figure>
<caption>**Table 3**</caption>

Procrustes distances between groups and associated p values (between brackets) based on permutation test (10,000 rounds). Significant p values, in bold, are interpreted as evidence for significant shape differences between the two groups compared.

<table><thead><tr><th colspan="2">Craniosynostosis syndrome</th><th colspan="3">Patients with syndromic craniosynostosis (n=37)</th></tr><tr><th>Unaffected (n=53)</th><th>Crouzon (n=26)</th><th>Apert (n=11)</th><th>Unaffected (n=53)</th></tr></thead><tbody><tr><td>Crouzon</td><td>1</td><td></td><td></td></tr><tr><td>Apert</td><td>0.553 (0.001)</td><td>1</td><td></td></tr><tr><td>Unaffected</td><td>0.438 (0.001)</td><td>0.484 (0.036)</td><td>1</td></tr></tbody></table><table><thead><tr><th colspan="2">Presence of OSA</th><th colspan="4">With OSA (n=21)</th></tr><tr><th>Without OSA (n=6)</th><th>No OSA (n=6)</th><th>Mild OSA (n=10)</th><th>Moderate OSA (n=4)</th><th>Severe OSA (n=7)</th></tr></thead><tbody><tr><td>No OSA</td><td>1</td><td></td><td></td><td></td></tr><tr><td>Mild OSA</td><td>0.542 (0.468)</td><td>1</td><td></td><td></td></tr><tr><td>Moderate OSA</td><td>0.647 (0.424)</td><td>0.367 (0.301)</td><td>1</td><td></td></tr><tr><td>Severe OSA</td><td>0.828 (0.860)</td><td>0.693 (0.379)</td><td>0.643 (0.645)</td><td>1</td></tr></tbody></table>

covariation that we highlighted between the upper airways and the craniofacial region (de Bataille et al., 2023). The fact that this morphological integration was no longer significant (though still strong) in Apert syndrome patients was most probably related to the small number of individuals in this sample. This should be confirmed in future studies.

### 4.3. Syndrome-specific variations in the patterns of morphological variation of the upper airways

Even when considering the significant effect of age, mouth and head posture on the upper airways shape variation, we identified significant differences between patients with Crouzon or Apert syndromes and unaffected children. Moreover, it is interesting to note that we observed more cases of interruption in the upper airways' segmentation, occurring mostly in the regions of the crista galli, anterior border of the clivoid process, choanal bifurcation and posterior nasal spine (i.e. either in the nasal airway or the nasopharynx) in the Apert syndrome sample. This is consistent with the literature, as Calandrelli et al. and Lu et al. identified volume differences according to the pattern of cranial vault suture synostosis, both in Crouzon and Apert syndromes, impacting either the nasal and/or pharyngeal airway (Calandrelli et al., 2018; Lu et al., 2021; Lu et al., 2021). Finally, some authors have reported nasopharyngeal narrowing, secondary to septal deviation, midnasal and choanal abnormalities, as well as soft palate obstruction at the level of the oro/hypopharynx (Forte et al., 2019; Sirotnak et al., 1995). Regarding Apert syndrome, a narrowing of the nasal airway or in the choanal region have been observed, even though tongue-based airway obstruction may also occur (Cohen and Kreiberg, 1992; Lu et al., 2021).

We did not find any significant association between the morphological variation of the upper airways and the diagnosis or severity of OSA. Yet, we detected that the centroid size (i.e. overall size) of upper airways were significantly smaller in patients with OSA than patients without OSA, independently from the severity of the respiratory obstruction. The few studies comparing upper airways of young patients with or without syndromic craniosynostosis also highlighted differences in their dimensions. Dentino et al. and Resnick et al. reported that OSA can be diagnosed in patients with syndromic craniosynostosis according to, respectively, the upper airway length and posterior airway space, or

the minimum retropalatal cross-sectional area (Dentino et al., 2015; Resnick et al., 2020). It should be noted that these studies did not consider the position of the child during image acquisition. Although the association between upper airways shape and OSA did not reach statistical significance in our study, we identified altered upper airways morphology in patients with syndromic craniosynostosis, affecting either the oropharynx or nasopharynx depending on the syndrome. It is likely that the morphological differences observed in these patients may induce a reduction in its volume, thereby increasing the risk of airway obstruction. Further analyses with larger samples need to be performed to confirm this hypothesis.

For all groups (unaffected children or patients with either Crouzon or Apert syndrome), we highlighted significant upper airways shape differences with age, even though we demonstrated that these shape differences were not due to allometric size changes alone. This result is not surprising considering that the upper airways are surrounded by anatomical structures that also evolve during growth (Lo et al., 2022; Tutkuviene et al., 2016). This modification in shape of the upper airways will accompany the progressive development of the functions they are dedicated to: breathing, but also mastication and swallowing, as well as vocalization and speech (Pohunek, 2004). This result is in agreement with previous studies that also demonstrated an important structural evolution of the upper airways, especially in the first years of life (Chuang et al., 2022; Di Cicco et al., 2021; Pohunek, 2004). While some studies have demonstrated a sex differences in the craniofacial growth pattern (Syutkina et al., 2023; Tutkuviene et al., 2016), no sexual dimorphism of the upper airways was identified in our sample. Once again, this result is consistent with the literature (Chuang et al., 2022; Ronen et al., 2007).

### 4.4. Limitations and perspectives

The primary limitation of this study is the relatively small sample size, particularly for subgroup analyses (e.g., Apert vs. Crouzon syndromes, OSA severity stratification). This constraint may have reduced the statistical power to detect subtle but clinically relevant morphological differences, such as the non-significant covariation between upper airways and craniofacial shape in Apert syndrome patients. Similarly, the lack of association between airway morphology and OSA could stem from insufficient power to resolve syndrome-specific patterns, especially given the phenotypic variability inherent to FGFR-related craniosynostoses. Future studies with larger cohorts are needed to validate these exploratory findings. Likewise, the present study focused on children under 5 years old. Future research should explore whether our conclusions hold true during later stages of the child's growth or development of the pathology.

In the current study, we developed an innovative protocol based on the collection of landmarks on various planes running through the length of the upper airways, taking into consideration the different orientations of the nasal and pharyngeal airways. This method took its inspiration from previous studies as that of Di Carlo et al. or Chuang et al. (Chuang et al., 2022; Di Carlo et al., 2015). The landmarks showed an excellent intra-observer reliability. However, we had to deal with missing data when the complete segmentation of the upper airway lumen was impossible, potentially explained by bony deviation or increased mucosa volume. We cannot also exclude the possibility that this interruption in the upper airways segmentation could be linked to the mouth posture, since the associated Chi² test' p value was quite close to the significance threshold of 5 %, though it was not significant (p value = 0.052). Anyway, considering these missing data, we had to exclude some landmarks from the analysis, resulting in a partial representation of the upper airways' morphology. To overcome this difficulty, further research may consider to study the global shape of the upper airways through, for example, the use of semi-landmarks, landmarks-free methods or AI models (e.g. Durrleman et al., 2014; Klatzow et al., 2022; Matava et al., 2020). However, with these more global
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** PCA illustrating the inter-individual variation of the shape of the upper airways after removing the effect of the age, mouth openness and lateral inclination of the head, along the first four PCs, with the distinction between children with or without a diagnosed OSA (upper), and between the different severity classes of OSA (lower).</figcaption></figure>

approaches, a heightened focus on the upper airways' segmentation is needed, especially in the nasal area, with the exclusion of the ducts connecting to the paranasal sinuses or the ethmoidal air cells, or the intricate pathways of the Eustachian tubes and choana which can be difficult to distinguish on medical CT images of paediatric patients (Maréchal et al., 2022; Zimmerman et al., 2019).

Considering that size dimensions seem to provide more insight into the risk of OSA than morphology, one might question the relevance of morphological information when investigating the relationship between the anatomy of upper airways and OSA. Yet, with 3D images such as CT-scan or MRI, the complex morphology of the upper airway can be captured: they can be analysed in both their axial, sagittal and coronal planes, allowing the measurement of cross-sectional areas and volumes, and, most of the time, providing sufficient details to distinct soft tissues (Partinen et al., 1988; Slaats et al., 2015; Vig and Hall, 1980). However, their use is limited by induced radiation, cost and restricted accessibility (Schwab, 1998). More recently, Cone Beam CT (CBCT) has been proposed as an alternative considering its low effective radiation dose but are not available in all hospitals yet (Aboudara et al., 2009; McCrillis et al., 2009).

To better understand the connection of syndromic craniosynostosis with obstructive respiratory disorders, either related to skeletal or soft tissues, further studies should carefully collect data representing the respiratory status of the patient. Unfortunately, due to the retrospective nature of this study, this information was not systematically available as

only 26 patients had a PG. It should also be mentioned that most of the time some delay occurred between the polygraph and the CT-scans acquisition (median time = 4 months). Moreover, according to Petrykowski et al. (2022), who found no correlation between pharynx anomalies and OSA, static images may not accurately represent the dynamic and dysfunctional nature of this pathology. Additionally, if imaging is performed while patients are awake, they may not fully characterise OSA, which occurs during sleep. During sleep there is reduction in muscle tone, which increases the risk of airway collapsibility and obstruction (Petrykowski et al., 2022). An alternative imaging method would be drug-induced sleep endoscopy, where the upper airways are assessed under controlled sedation that simulates natural sleep conditions, and dynamic images can be acquired while the patient is lying.

Finally, even though polysomnography and the measurement of AHI are the gold standard for confirming OSA, they are costly, time-consuming and stressful for the young patient and its family (Asghari and Mohammadi, 2013; McNicholas et al., 2016; Solano-Pérez et al., 2023; Soori et al., 2022). Instead, other approaches could be favoured such as questionnaires assessing, for instance, snoring, sleep quality, breathing mode (nasal vs oral) (Incerti Parenti et al., 2021; Patel et al., 2020; Solow and Kreiborg, 1977). However, we should pay attention to the sensibility and sensitivity of these questionnaires and symptoms when assessing a respiratory obstruction. It should also be noted that most of the previous studies interested in assessing respiratory disorder
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** 2D comparison of the extreme shapes along the 1st and 3rd PC of the PCA (from top to bottom, respectively) representing the shape differences between patients with either Crouzon or Apert syndrome (upper, respectively in red and blue) or between Crouzon syndrome patients and unaffected children (lower, respectively in blue and red): lateral and anterior view of the upper airways (from left to right).</figcaption></figure>

in syndromic craniosynostosis only used clinical outcomes such as the AHI or endoscopy findings (Doerga et al., 2016; Lo and Chen, 1999) or were interested in evaluating treatment effects rather than the aetiology of these disorders (de Water et al., 2014; Mannelli et al., 2019; Nout et al., 2012).

4.5. Broader insights and implications

FGFR mutations causing syndromic craniosynostosis not only affect bone development of the craniofacial structures, resulting in the characteristic craniofacial anomalies mentioned earlier (proptosis, hypertelorism, cleft palate, midface hypoplasia), but may also affect the soft and hard tissues surrounding the upper airways, supporting the

pleiotropic effect of these genes (Alam et al., 2023; Britto et al., 2001; Heuzé et al., 2014; McIntosh et al., 2000). However, further research is needed to distinguish between upper airways deformations that arise secondarily from craniofacial structural deformities and those that result directly from genetic mutations (Martínez-Abadías et al., 2010; Purushothaman et al., 2011).

In order to identify potential differences in the shape of the upper airways, it is essential for future studies to control for age, mouth opening and craniocervical posture during imaging. This could be done with the exclusion of individuals with an open mouth and/or an inclination of their head, but this would inevitably reduce the sample size that is already limited by the prevalence of syndromic craniosynostosis. Moreover, excluding patients with an open mouth during CT scan
<figure><img src="image_13.png" /><figcaption>**Fig. 9.** PLS analyses assessing the level of shape integration between the upper airways and the skull (upper left), the mandible (upper right) and the soft tissues (lower left), and between the nasal airway and the pharynx (lower right), for each diagnosis. The oblique red line represents the covariation computed on the total sample.</figcaption></figure>

<caption>**Table 4**</caption>

PLS analyses assessing the level of integration between the shape of the upper airways and of the craniofacial region (skull, mandible and soft tissues), and between the shape of the nasal airway and of the pharynx. Bold values indicate significant p values.

<table><thead><tr><th></th><th>R</th><th>P value</th></tr></thead><tbody><tr><td colspan="3"><strong>Shape covariation between the upper airways and the skull</strong></td></tr><tr><td>Total sample</td><td>0.80</td><td>0.001</td></tr><tr><td>Unaffected sample</td><td>0.78</td><td>0.001</td></tr><tr><td>Crouzon sample</td><td>0.81</td><td>0.002</td></tr><tr><td>Apert sample</td><td>0.91</td><td>0.022</td></tr><tr><td colspan="3"><strong>Shape covariation between the upper airways and the mandible</strong></td></tr><tr><td>Total sample</td><td>0.77</td><td>0.001</td></tr><tr><td>Unaffected sample</td><td>0.81</td><td>0.001</td></tr><tr><td>Crouzon sample</td><td>0.87</td><td>0.002</td></tr><tr><td>Apert sample</td><td>0.79</td><td>0.503</td></tr><tr><td colspan="3"><strong>Shape covariation between the upper airways and the soft tissues</strong></td></tr><tr><td>Total sample</td><td>0.61</td><td>0.001</td></tr><tr><td>Unaffected sample</td><td>0.64</td><td>0.006</td></tr><tr><td>Crouzon sample</td><td>0.75</td><td>0.02</td></tr><tr><td>Apert sample</td><td>0.92</td><td>0.071</td></tr><tr><td colspan="3"><strong>Shape covariation between the nasal airway and the pharynx</strong></td></tr><tr><td>Total sample</td><td>0.56</td><td>0.002</td></tr><tr><td>Unaffected sample</td><td>0.62</td><td>0.006</td></tr><tr><td>Crouzon sample</td><td>0.75</td><td>0.02</td></tr><tr><td>Apert sample</td><td>0.84</td><td>0.333</td></tr></tbody></table>

acquisition may be inappropriate, as oral breathing has been demonstrated to be more prevalent in children with OSA (Bokov et al., 2022). Besides, it is interesting to note that the syndromic patients of our sample were more inclined to present an open mouth compared to unaffected children. Another solution to control the effect of these

confounding variables could be to lead a prospective study to control those factors, which, for ethical and practical reasons, may be also complicated considering the age of the patients. In the present study, we decided to statistically remove the effect of these parameters in a quantitative way by multivariate linear regression. When removing the
effect of these variables, we were still able to identify significant differences in the shape of the upper airways depending on the diagnosis of syndromic craniosynostosis.

Finally, studying the morphological variation in the upper airways in children with syndromic craniosynostosis can contribute to understand how specific genetic mutation and/or fusion patterns associated with these syndromes lead to particular airway changes and provide insights into the mechanisms of OSA (Bastir et al., 2020; Chuang et al., 2022; Inverso et al., 2016; Lu et al., 2021; Lu et al., 2021; Pohunek, 2004; Wagner et al., 2023). This knowledge can, in turn, participate in developing appropriate treatment strategies with increased success rate, and early diagnostic approaches.

## 5. Conclusion

Using geometric morphometric analysis on 3D models reconstructed from CT-scans, we were able to highlight significant differences in the shape of the upper airways between patients with craniosynostosis and unaffected children, and between patients with Crouzon and Apert syndromes, after controlling for confounding factors such as age, mouth openness and lateral head inclination. Even though we did not find a significant association between the morphology of the upper airways and OSA, we detected upper airway deformations in syndromic patients that likely induce a sufficient narrowing of the airway to increase the risk of occurrence of obstructive events. Despite these shape variations, the growth trajectory of the upper airways and their covariation with the craniofacial region (both hard and soft tissues) were largely not affected by syndromic craniosynostosis.

## Authors contributions

All authors made significant contributions to the preparation of this article. F. Remy drafted the initial version of this manuscript while M. Taverne, R. H. Khonsari, B. Fauroux, S. Khirani, N. Martinez-Abadias and Y. Heuzé participated equally to its critical revision and all consented to the submission of the final draft. This study was designed by all authors and data were acquired, analysed and interpreted by F. Remy, with the support of the co-authors.

## Data availability statement

The full R codes and data used for the present study are available on Zenodo (10.5281/zenodo.14192990).

## Funding

This study received financial support from the French government in the framework of the University of Bordeaux's IdEx "Investments for the Future" program/GPR "Human Past" and from the Foundation Princess Grace.

## Acknowledgments

The authors would like to thank the patient families for giving us access to the data analysed in this study. Finally, we would also like to thank Christian Peter Klingenberg and Frédéric Santos for their invaluable advice on the statistical methodology of this paper.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcms.2025.06.014.

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