Comprehensive Genetic Evaluation of Children With Syndromic Craniosynostosis by a Combination of Cytogenetics, Multiplex Ligation-dependent Probe Amplication and Array-based Comparative Genomic Hybridisation

Syndromic craniosynostosis (SC) is a genetically determined premature closure of one or more of the cranial sutures, which may result in severe dysmorphism, increased intracranial pressure along with many other clinical manifestations. The considerable risk of complications along with signicant incidence makes these cranial deformations an important medical problem. Despite the efforts to clarify the pathogenesis of SC in recent years, its genetic aspects remain largely unknown. Aiming to elucidate the complex genetic etiology of syndromic craniosynostosis, we conducted an investigation of 39 children, screened systematically with a combination of conventional cytogenetic analysis, multiplex ligation-dependent probe amplication (MLPA) and array-based comparative genomic hybridisation (aCGH). Pathological ndings were established in 15.3% (6/39) of the cases using aCGH, in 7.7% (3/39) using MLPA and 2.5% (1/39) using conventional karyotyping. About 12.8% (5/39) of the patients with normal karyotype carried submicroscopic chromosomal rearrangements. Duplications were found to be more common than deletions. Conclusion: The systematic genetic evaluation of children with SC revealed a high prevalence of submicrosopic chromosomal rearrangements (most commonly duplications and gain-of-function variations). This suggests the leading role of those defects in the pathogenesis of syndromic craniosynostosis. The genetic complexity of SC was rearmed by the discovery of pathological ndings in various chromosomal regions. Certain genes were discussed in conjunction with craniosynostosis. segregation analysis, multiplex ligation-Page


Introduction
Craniosynostosis (CRS) is the process of premature fusion and ossi cation of one or more cranial sutures [1]. It has a cumulative incidence of about 1 in 2500 newborn children [2]. When untreated, craniosynostosis can lead to serious medical complications -increased intracranial pressure, mental retardation, hearing or vision defects, behavioural anomalies, craniofacial asymmetry and dysmorphism, seizures [3]. The considerable risk of complications along with signi cant incidence makes these cranial deformations an important medical problem.
CRS can be classi ed as syndromic -when the cranial synostosis is a part of a malformative syndrome or nonsyndromic -when it presents as an isolated feature. Nonsyndromic craniosynostosis (NSC) constitutes about 80% of all cases [4]. Recent studies have shown that genetic variations play a leading role in the pathogenesis of NSC. Considerably rarer are the syndromic forms of craniosynostosis -20 % of all repoted cases. About 30% of the SC are caused by pathogenic variants in certain genes (FGFR1, FGFR2, FGFR3, TWIST1, EFNB1, MSX2, RAB23, RUNX2) [4]. These are called "monogenic" or "mendelian" forms of craniosynostosis. They are inherited in an autosomal dominant pattern (except for RAB23) with a variable degree of expression and penetration that yields a large array of clinical manifestations. Chromosomal anomalies account for about 16% of syndromic craniosynostosis cases [4]. MLPA and array CGH offer similar diagnostic value in literature and can be used in tandem to con rm a certain nding [4]. Array CGH is however more valuable due to its higher detection rate and whole genome scope.
Despite the serious advancements in the last 2 decades, the genetic basis of craniosynostosis remains rather poorly understood. Trying to clarify the complex factors involved in the pathogenesis of syndromic craniosynostosis we conducted an investigation of 39 children which was carried out by a systematic, combined approach consisting of cytogenetics, MLPA and aCGH.

Subjects
We investigated 39 children with syndromic craniosynostosis referred to our department in the 2016-2020 year period. 29 of them were male and 10 were female resulting in a sex ratio of 2.9:1. Clinical selection was based on the presence of craniosynostosis along with additional dysmorphic features (SC) -documented by imaging studies (cranial radiography and/or computed tomography). The presence of clinical or phenotypic resemblance to any well-known monogenic craniosynostosis disorders was considered an exclusion factor.

Methods
Conventional chromosomal analyses at 550 G-band resolution were performed on peripheral blood lymphocytes on all 39 of our patients.
Multiplex Ligation-dependent Probe Ampli cation (MLPA) is a method to determine the copy number of up to 45 genomic DNA sequences in a single multiplex PCR based reaction. For this study we used MLPA P245 Microdeletion Syndromes for screening of the most common microdeletion syndromes and MLPA P036 Subtelomeres Mix 1 for screening of subtelomeric deletions/duplications. To con rm alternations discovered with MLPA P036 Subtelomeres Mix 1 we used MLPA P070 Subtelomeres Mix 2B.
Array CGHthe whole genome CNVs screening was carried out by oligo array CGH. DNA was isolated from peripheral blood by phenol-chloroform extraction. We used OGT 4x44k format oligonucleotide microarray with targeted CN resolution of 1 probe every 52kb and backbone CN resolution of 1 probe every 81kb. The slides were scanned on a GenePix 4100A two-colour uorescent scanner (Axon Instruments, Union City, CA, U.S.A.). The arrays were analyzed by CytoSure Interpret Software.

Patients' medical reports
The medical history, clinical features and heritage of all patients with pathogenic/probably pathogenic genetic ndings are summarized in Table 1. Further patient information can be provided by the authors if requested.

Results
In 27 (about 70%) of our patients, craniosynostosis was simple (a single cranial suture is obliterated). In 10 cases two sutures were simultaneously fused repesenting a complex craniosynostosis. In the other 2 patients three cranial sutures were prematurely ossi ed.
Array CGH was used to screen for submicroscopic chromosomal rearrangements in all 39 patients. Pathogenic and probably pathogenic submicroscopic defects were found in 6 patients, representing 15.3% of all tested children (Tables 2 and 3). About 12.8% (5/39) of the patients with normal karyotype carried submicroscopic chromosomal rearrangements. Five of those defects were duplications and three were deletions.

Discussion
Craniosynostosis is frequently classi ed as [5]: simple -in cases with a single fused cranial suture (sagittal, coronal, metopic or lambdoid -respectively) and complex or compound when two or more sutures are prematurely and simultaneously closed.
The distribution of suture involvement in syndromic craniosynostosis in literature [4][5][6] is sagittal in about 50-60%, followed by coronal in 20-25%, metopic in 15% and lambdoid in approximately 5% of all cases. This differs from our results, which may indicate that the size of our sample is insu cient to be statistically representative in this regard.
Patient 2 (Table 1) had normal results from conventional chromosome analysis and MLPA. Subsequently we performed aCGH. A pathogenic duplication of the long arm of chromosome 1 (1q21.1) was found (Tables 2 and  3). None of the genes within this region have been associated with CRS so far. Rare, recurrent chromosome 1q21.1 duplications and deletions have been linked with developmental delay, autism, congenital heart anomalies and macrocephaly in children [7]. Microduplications and deletions in the same locus are associated with an increased risk of psychiatric complications (schizophrenia) in adults [8]. Our patient was diagnosed with ASD, which is congruent with the duplication found in 1q21.1. No direct associations with syndromic craniosynostosis were found. This nding rea rms the complex genetic etiology of SC and requires further investigation.
In patient 8 (Table 1), conventional cytogenetics established a normal female karyotype, MLPA also showed a normal result. The patient was screened for submicroscopic rearrangements using aCGH, yielding one probably pathogenic deletion of 14q32.33 (177.93Kb). Several gene sequences have been mapped on this region (Tables 2  and 3). None of these have been connected to premature cranial suture ossi cation in literature. A submicroscopic deletion of the long arm of chromosome 14 is associated with two conditions -Dubowitz syndome [9] and 14q32.3 deletion syndrome [10]. Due to the patient 8 facial dysmorphism and the presence of gastrointestinal symptoms as well as brachydactyly we are inclined towards Dubowitz symdrome. However 14q32.3 deletion syndrome can not be disproved conclusively at this point since these are extremely rare entities with highly variable phenotypic manifestations. As far as we know neither Dubowitz syndrome nor 14q32.3 deletion syndrome have been associated with craniosynostosis. The association of submicroscopic deletion 14q.32 and SC needs further investigation.
In patient 22 (Table 1) aCGH revealed a deletion of the 5q35 region (5q35.2-5q35.3). The deletion was 1.665 Mb in size (Tables 2 and 3), encompassing 40 HGNC and 24 OMIM genes, including NSD1 and FGFR4. The array CGH results were con rmed by MLPA. The patient's parents were unavailable for testing. This result is consistent with Sotos syndrome (SoS). It is a rare but well-known disorder causing overgrowth in childhood. Ten percent of affected individuals have 5q35 microdeletions [11]. The size and mechanism of formation of 5q35 microdeletions differ depending upon the ethnic origin of the patients [12]. The presented features of our patient (Table 1)developmental delay, characteristic facial appearance, and macro-dolichocephaly were typical for SoS, although the overgrowth was absent. Childhood overgrowth is a cardinal symptom of SoS, and is present in over 90% of the cases. Our patient's microdeletion includes the NSD1 and FGFR4 genes. Overall, the individuals with microdeletions have less prominent overgrowth than patients with NSD1 variants [13]. Douglas et al. also described a patient with 5q35 microdeletion involving NSD1 and FGFR4 genes and craniosynostosis [14]. Fibroblast growth factor (FGF) and broblast growth factor receptor (FGFR) signaling pathways play essential roles in the earliest stages of skeletal development, thus mutations in these genes can cause differenent bone diseases, including craniosynostosis syndromes [15]. Nie et al. speculated that FGFR4 is involved in growth regulation of face and head structures, although the effect of FGFR4 on bone development remains unknown and needs further elucidation [16].
The genetic evalutaion of patient 25 (Table 1) began with chromosome analysis and MLPA which both showed normal results. Array CGH however, revealed a probably pathogenic microduplication of chromosme 1 (1q12q21.2) spanning across 4.62 Mb (Tables 2 and 3). None of the genes within this chromosome region have been associated with craniosynostosis. Brisset et al. present a complex nding of paternally inherited duplication 1q12q21.2 (5.8 Mb) in combination with maternally inherited deletion of 16p11.2 of 545 Kb in a child with several malformations (including plagiocephaly), psychomotor delay, seizures and overweight [17]. Brisset's nding clearly differs from our patient 25, most likely due to the additional deletion of 16p. To our knowledge, the evidence of association between microduplication 1q12q21.2 and syndromic craniosynostosis is insu cient to be basis for concrete genotype-phenotype correlations.
Patient 29 (Table 1) presented with a pathological female karyotype − 46,ХХ,t(2;7)(q14;q35). The same translocation was found in her mother (who had mild facial dysmorphism and no other complications). The father had a normal male karyotype (46,XY). MLPA revealed a microduplication of the short arm of chromosome 2 -dupl 2p16.1. Several cases with de-novo interstitial microduplications involving 2p16.1-p15 are reported in literature with facial dysmorphism, intellectual disability, developmental delay, congenital heart defects and various additional nonspeci c features [18]. No associations with craniosynostosis were found. Finally aCGH was performed, which revealed a large pathogenic duplication of 2p -dupl 2p22-3p16.1 (25.19 Mb). This chromosomal region is fairly large, containing a number of genes (Table 3). We found no variations of those sequences that are reported as causative of craniosynostosis with one exception -the SIX2 gene. It encodes a transcription factor associated with cell differentiation and migration, crucial for the development of several organs (stomach, kidneys and skull). The increased dosage of SIX2 could lead to early and pronounced ossi cation of cranial sutures which links with the craniofacial dysmorphism in our patient, making this nding possibly causative. Hufnagel et al. however, report a case with frontonasal dysplasia with sagittal craniosynostosis due to microdeletion of the SIX2 gene [19]. These ndings rea rm the complex genetic etiology of SC.
In patient 34 (Table 1) (Table 3) including the TGFBR3 gene. It encodes the transforming growth factor (TGF)-beta type III receptor. These receptors, along with the FGF receptor family are widely expressed in bone cells and bone matrix and play an important role in premature pathological suture closure [21][22][23][24][25]. Based on this nding we hypothesize that the duplication of 1p22.1 containing the TGFBR3 gene links with the metopic craniosynostosis in our patient, making the nding potentially causative. This particular chromosome region is a promising candidate for further investigation regarding syndromic craniosynostosis. Additionally our patient presented with hypoplasia of corpus callosum which is characteristic of 1p22 duplications. The disparity between MLPA and aCGH ndings found in patient 34 is a result of method limitations.

Conclusion
In an effort to elucidate various genetic factors involved in the pathogenesis of syndromic craniosynostosis we conducted an investigation of 39 children using a combination of cytogenetics, MLPA and array CGH. In total we found 6 patients with pathological genetic variations. This constitutes 15.3% of the children in our sample which corresponds to the data we observed in literature.
In our study, aCGH had the highest detection rate proving that submicroscopic chromosomal rearrangements play an important role in the pathogenesis of syndromic craniosynostosis. MLPA and conventional karyotyping yielded respectively 7.7% and 2.5% pathological ndings. Duplications were found to be more common then the deletions, underlining the importance of increased dosage of certain genes in syndromic craniosynostosis.
Coronal synostosis was the most common anatomical substrate we found, which differs from the established suture involvement distribution in literature, probably due to certain limitations in our sample. No funding was received to assist with the preparation of this manuscript

Con icts of interest/Competing interests:
The authors have no con icts of interest to declare that are relevant to the content of this article Availability of data and material: Additional data and source material will be provided by the authors if requested All procedures performed in this study involving human participants were in accordance with the ethical standarts of the institutional research comitee and with the 1964 Helsinki Decladarion and its later amendments of comparable ethical standarts. The study was approved by the Ethics Commitee of Medical Unversity of So a.

Consent to participate:
Informed consent was obtained from all participants, including their respective family members (and/or legal guardians) before clinical selection was performed.