Genotype–Phenotype Correlations in 2q37-Deletion Syndrome: An Update of the Clinical Spectrum and Literature Review

2q37 microdeletion/deletion syndrome (2q37DS) is one of the most common subtelomeric deletion disorders, caused by a 2q37 deletion of variable size. The syndrome is characterized by a broad and diverse spectrum of clinical findings: characteristic facial dysmorphism, developmental delay/intellectual disability (ID), brachydactyly type E, short stature, obesity, hypotonia in infancy, and abnormal behavior with autism spectrum disorder. Although numerous cases have been described so far, the exact mapping of the genotype and phenotype have not yet been achieved. Materials and Methods: In this study we analyzed nine newly diagnosed cases with 2q37 deletion (3 male/6 female, aged between 2 and 30 years old), and followed up at the Iasi Regional Medical Genetics Centre. All patients were tested first with MLPA using combined kits P036/P070 subtelomeric screening mix and follow-up mix P264; after, the deletion size and location were confirmed via CGH-array. We compared our findings with the data of other cases reported in the literature. Results: From nine cases, four had pure 2q37 deletions of variable sizes, and five presented deletion/duplication rearrangements (with chromosomes 2q, 9q, and 11p). In most cases, characteristic phenotypic aspects were observed: 9/9 facial dysmorphism, 8/9 global developmental delay and ID, 6/9 hypotonia, 5/9 behavior disorders, and 8/9 skeletal anomalies—especially brachydactyly type E. Two cases had obesity, one case had craniosynostosis, and four had heart defects. Other features found in our cases included translucent skin and telangiectasias (6/9), and a hump of fat on the upper thorax (5/9). Conclusions: Our study enriches the literature data by describing new clinical features associated with 2q37 deletion, and possible genotype–phenotype correlations.


Introduction
2q37 microdeletion/deletion syndrome (2q37DS), also known as Albright hereditary osteodystrophy-like syndrome or brachydactyly-intellectual disability syndrome, is a rare genetic disorder caused by a deletion of variable size in the long (q) arm of chromosome 2, in region 2q37. The syndrome is characterized by a broad and diverse spectrum of clinical findings. The most common phenotypic features include mild to moderate developmental delay/intellectual disability (ID), brachymetaphalangy of digits 3-5 (brachydactyly type E) [1][2][3], short stature, obesity, hypotonia in infancy, abnormal behavior with autism spectrum disorder (24-35%) [3][4][5], joint hypermobility, and scoliosis [6]. Most individuals with 2q37DS have a typical dysmorphic face: broad or rounded facies (41%), frontal bossing Genes 2023, 14,465 3 of 14 between 2 and 30 years; 6 were females and 3 were males. In only 2 cases were the patients related (siblings), while in the other 7 cases, no relationship was established.
Before enrolment in the study, each individual provided their informed consent for inclusion. The protocol was accepted by the Ethical Committee of the University of Medicine and Pharmacy "Grigore T. Popa" Iasi (approved at 7 July 2019, No. 14629), and all the requirements of the Declaration of Helsinki were followed. Anonymization of patients' personal data was carried out by assigning code numbers to each patient, and no personal information was disclosed unless it was necessary for the study. Before the start of the study, patients (or their legal guardians) gave their informed consent. Every patient voluntarily participated in the study.
For the clinical assessment of our patients, echocardiography, MRI, abdominal and pelvic ultrasound were used in all cases to establish the phenotypic defects. For the investigation of skeletal anomalies, Rx of the limbs were taken. The degree of intellectual disability and developmental delay was established by the Bayles Scale for Infant Development IV (0-42 months), DF-MOT (Motor developmental scales, 0-4 years), Neuropsychomotor Function Evaluation Battery-NP-MOT (4-12 years), and the Stanford-Binet and Wechsler Intelligence Scale V (children-6 years and adults); for autism spectrum disorders, the Autism Spectrum Rating Scales (ASRS) were used.
DNA extraction from peripheral blood was performed using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA).
In this study, we initially detected 237 deletions using the MLPA (Multiplex ligationdependent probe amplification) approach, utilizing P036/P070 subtelomeric screening probe mixes; subsequently, the 2q changes were confirmed by the follow-up mix P264 (MRC Holland, Amsterdam, The Netherlands), which contained 13 probes in the terminal 5.0 Mb of 2q37.3.
MLPA analysis was performed, as previously described [14]. Briefly, 100 ng of genomic DNA was denatured and hybridized with selected subtelomeric probe mixes at 60 • C for 20 h. After a ligation step at 54 • C for 15 min and a PCR protocol using Cy5-labeled universal primers contained in the kit, the fluorescent amplification products were separated by capillary electrophoresis in a CEQ 8000 GeXP Genetic Analysis System sequencer (Beckman Coulter, Brea, CA. USA). The DNA copy number was estimated using the Coffalyser.net program. Heterozygous deletions/duplications were established when recognition sequences had a 35-50% reduced/increased relative peak area of the amplification product of that probe.
Furthermore, CGH-array was performed in all cases to describe the deletion size better, and to identify other associated CNVs.
CGH-array analysis was performed using the SurePrint G3 Human ISCA CGH+SNP Microarray slides 4×180K (Agilent Technologies ® , Santa Clara, CA, USA). The microarray analysis was conducted according to manufacturer's recommendations. Briefly, 1 µg of genomic DNA from patients and from a reference sex-matched DNA (Agilent Technologies ® , Santa Clara, CA, USA) were fragmented with enzymatic digestion, using restriction enzymes AluI and RsaI. Patient and reference DNA were fluorescently labelled with Cy5 and Cy3, respectively. After columns purification of the labeled DNA, patients and matched reference DNA were combined and hybridized with Cot-1 DNA (1.0 mg/mL) to SurePrint G3 Human ISCA CGH+SNP 4×180K (Agilent Technologies) array slides. After hybridization and washing, microarrays slides were scanned using a SureScan Microarray Scanner (Agilent Technologies). Genomic CytoGenomics software (Agilent Technologies) was used for data extraction and quality control evaluation, and aberration reports. The ADM-2 algorithm was used to call aberrations (filtering option of a minimum 5 probes in the region, ≥100 kb size and minimum average log2 ratio > 0. 3 15 October 2022), which provide cytogenetic and clinical information on large series of patients. CNVs are considered pathogenic if they are reported in publicly available databases and in the medical literature as being associated with known disease, and are likely to be clinically significant and considered as benign variants if the CNVs are recorded in publicly available genomic databases as polymorphic variants among control individuals.
Parental DNA samples were available from 3/9 patients tested with CGH-array. We also tested patient's parents with the karyotype and MLPA test, kit P264; the results were normal.

Results
The MLPA tests performed initially identified the 2q37 deletion in all nine analyzed cases. Using CGH-array, we identified different deletion sizes in our cases, from 1.84 Mb to 8.14 Mb, as well as other associated CNVs ( Figure 1). the region, ≥100 kb size and minimum average log2 ratio > 0.3). Genomic positions were defined using GRCh38. Homozygosity regions were considered DNA sequences free of heterozygosity, with dimensions of at least 1 Mb. For the interpretation and classification of CNVs, all detected CNVs were systematically searched in the public databases, Database of Genomic Variants (http://dgvbeta.tcag.ca/dgv/app/ home?ref= GRCh38 accessed on 15 October 2022), the Database of Chromosomal Imbalance and Phenotype in Humans using Ensemble Resources (DECIPHER) (https://decipher.sanger.ac.uk/ accessed on 15 October 2022), and the Database of Genomic Variants OMIM (https://www.ncbi.nlm. nih.gov/omim/ accessed on 15 October 2022), which provide cytogenetic and clinical information on large series of patients. CNVs are considered pathogenic if they are reported in publicly available databases and in the medical literature as being associated with known disease, and are likely to be clinically significant and considered as benign variants if the CNVs are recorded in publicly available genomic databases as polymorphic variants among control individuals.
Parental DNA samples were available from 3/9 patients tested with CGH-array. We also tested patient's parents with the karyotype and MLPA test, kit P264; the results were normal.

Results
The MLPA tests performed initially identified the 2q37 deletion in all nine analyzed cases. Using CGH-array, we identified different deletion sizes in our cases, from 1.84 Mb to 8.14 Mb, as well as other associated CNVs ( Figure 1).  (Figures 2A and 3A) was observed that corresponded to the 2q duplication syndrome spectrum in which the most common clinical findings are similar to the 2q37 deletion: developmental delay, ID, behavioral problems, autism spectrum disorder, dysmorphic face, microcephaly, hypotonia, short stature, and underweightness [18][19][20].  (Figures 2A and 3A) was observed that corresponded to the 2q duplication syndrome spectrum in which the most common clinical findings are similar to the 2q37 deletion: developmental delay, ID, behavioral problems, autism spectrum disorder, dysmorphic face, microcephaly, hypotonia, short stature, and underweightness [18][19][20].
Case P6 presented smaller 2q37.3 duplication ( Figures 2B and 3B) of 1.01 Mb (including 16 genes). No genes were reported as having triplosensitivity, and there are no reported cases of pathogenic phenotypes that overlap these duplications.
ing 16 genes). No genes were reported as having triplosensitivity, and there are no reported cases of pathogenic phenotypes that overlap these duplications.
Case P9 ( Figure 4D) presented a duplication of 1.06 Mb in 11p15.5-p15.4 ( Figure 2B) region that contains 37 genes). Case P6 presented smaller 2q37.3 duplication (Figures 2B and 3B) of 1.01 Mb (including 16 genes). No genes were reported as having triplosensitivity, and there are no reported cases of pathogenic phenotypes that overlap these duplications.
Case P9 ( Figure 4D) presented a duplication of 1.06 Mb in 11p15.5-p15.4 ( Figure 2B) region that contains 37 genes).  delay and ID, of varying degrees of severity, were reported in eight out of the nine cases, and hypotonia was noted in only four cases. Only one patient presented autism spectrum disorder, whereas other behavioral problems were noticed in four cases: stereotypies, over friendly behavior, laughter crises, aggressivity, and ADHD. Short stature was noted in seven out of the nine cases, and obesity/overweightness only in two cases, with four other subjects being actually underweight. Craniofacial dysmorphism was suggestive for 2q37 DS in most subjects ( Figure 4). Skeletal anomalies were observed in eight out of the nine patients, especially malformations of the hands or feet, brachydactyly type E, asymmetric limbs or malpositioning of fingers and toes, and hypoplastic toes/fingers ( Figure 5). Case P9 ( Figure 4D) presented a duplication of 1.06 Mb in 11p15.5-p15.4 ( Figure 2B) region that contains 37 genes).
Regarding the clinical pictures, in most of our cases we observed phenotypic aspects similar to those described for 2q37DS in the literature (Table 1). Global developmental delay and ID, of varying degrees of severity, were reported in eight out of the nine cases, and hypotonia was noted in only four cases. Only one patient presented autism spectrum disorder, whereas other behavioral problems were noticed in four cases: stereotypies, over friendly behavior, laughter crises, aggressivity, and ADHD.
Short stature was noted in seven out of the nine cases, and obesity/overweightness only in two cases, with four other subjects being actually underweight. Craniofacial dysmorphism was suggestive for 2q37 DS in most subjects (Figure 4).
Skeletal anomalies were observed in eight out of the nine patients, especially malformations of the hands or feet, brachydactyly type E, asymmetric limbs or malpositioning of fingers and toes, and hypoplastic toes/fingers ( Figure 5). Regarding the clinical pictures, in most of our cases we observed phenotypic aspe similar to those described for 2q37DS in the literature (Table 1). Global developmen delay and ID, of varying degrees of severity, were reported in eight out of the nine ca and hypotonia was noted in only four cases. Only one patient presented autism spectr disorder, whereas other behavioral problems were noticed in four cases: stereotypies, o friendly behavior, laughter crises, aggressivity, and ADHD.
Short stature was noted in seven out of the nine cases, and obesity/overweightn only in two cases, with four other subjects being actually underweight. Craniofacial d morphism was suggestive for 2q37 DS in most subjects ( Figure 4). Skeletal anomalies were observed in eight out of the nine patients, especially malf mations of the hands or feet, brachydactyly type E, asymmetric limbs or malposition of fingers and toes, and hypoplastic toes/fingers ( Figure 5).   Heart defects were observed in four out of the nine cases, especially represented by septal defects; anomalies of the gastrointestinal system varied from mild hernia (inguinal and/or umbilical) to more severe phenotypes: intestinal malrotation, duodenal stenosis, or anorectal malformation. Moreover, in two cases, renal abnormalities were noticed; however, Wilms tumor was not identified in any case.

Discussion
Our study evaluated four new cases with pure 2q37 deletion (cases P1-P4), and five cases with deletion/duplication arrangements (cases P5-P9). In the literature, although microdeletions are better defined than microduplications, the expressions of many duplications/microduplications still remain unclear, with incomplete penetrance constantly observed. Moreover, pure duplications are quite rarely reported, which increases the difficulty of their clinical characterization. The general agreement is that the phenotypes of microduplications/partial trisomies are milder and better tolerated (in many cases no phenotypic defects are present) than deletions/microdeletions; thus, in a deletion/duplication rearrangement, the phenotype is determined mostly by the haploinsufficiency of some genes [23][24][25][26].
In our study, case P5 had a large duplication of 42 Mb on chromosome 2q32.1-q37.3, which corresponded to the 2q34 duplication syndrome. Partial 2q duplication is a very rare chromosomal abnormality usually found in chromosomal rearrangements that also involve a deletion of another partner chromosome, extremely rarely being described as pure 2q duplication (five cases reported so far). In our patient's case, the phenotypic aspects can be attributed in part to 2q34 duplication: intellectual disability (present in all reported cases with 2q34 duplication), prominent forehead, broad nasal bridge, hypertelorism (reported in three cases in the literature with 2q duplication), thin upper lip, dental defects, clinodactyly of finger 5, and genital anomalies [27].
However, the presence of type E brachydactyly, septal defects, characteristic craniofacial dysmorphia for 2q37 deletion-aspects that are not reported in cases with 2q34 duplication-underlines the contribution of the deletion to the clinical picture.
In case P6, the associated 2q37duplication was smaller, and did not contain genes with triplosensitivity, so we can consider that the phenotype was caused only by the 2.48 Mb microdeletion in 2q37. The same situation can be applied to case P9, with a deletion of 4.99 Mb in 2q37 and a duplication of 1.06 Mb in 11 (p15.5-p15.4).
For case P7 and case P8, although they had the same rearrangement, 5.71 Mb deletion in 2q37 and 6.94 Mb duplication in 9 (q34.11-q34.3), the phenotypes were slightly different. In the literature, patients with 9q34 duplication have moderate developmental delay, limited vocabulary, hyperactivity, low birth weight, normal birth length, initial poor feeding and thriving, dolichocephaly, facial asymmetry, narrow horizontal palpebral fissures, microphthalmia, small mouth, thin upper lip with down-turned corners, musculo-skeletal defects with joint contractures, long thin limbs, and arachnodactyly. They can also present abnormal implantation of the thumb, increased space between the first and second fingers, and cardiovascular and ocular defects. Although some of these aspects were found in our cases, the phenotypes of the two siblings seemed to be more suggestive of 2q37DS [28,29].
A special case was P2, in which the chromosomal analysis identified the following mosaic: 46,XX,1qh+/46,XX,1qh+,del(2q37.3). To our knowledge, cases of mosaicism are very rare. The presence of the mosaic could be responsible for a milder phenotype; however, that was not true in our patient's case, where the phenotypic aspects were quite characteristic for 2q37DS.
Although a syndrome with contiguous genes deletion, in 2q37DS the size of the deletion cannot be correlated with the severity of the abnormal phenotype. In some previous studies, the discordance between large deletions of 9.5-10 Mb with mild phenotype was highlighted, as well as small deletions of 828 Kb with a more complex and severe phenotype [6,30,31]. Our study underlines this aspect, not observing major phenotypic differences between small deletions of~2 Mb (P5, P6) and large deletions of~8 Mb (P4); the differences between the cases was given rather by the dosage sensitivity of the lost genes than by the size of the deletion. However, in the 2q37 region, not many genes have been reported as having a known dosage sensitivity.
The HDAC4 gene has been proposed as being mainly responsible in producing the phenotypic aspects of the 2q37 microdeletion, especially brachydactyly type E, behavioral disorders, and ID-the marker clinical features in this syndrome [7,32]. The gene encodes for histone deacetylase 4, which is a class IIa HDAC that shuttles between the cytoplasm and nucleus. The nuclear localization and activity of HDAC4 are dependent on the binding of 14-3-3 proteins. HDAC4 acts as a scaffold for other co-repressor systems, such as the N-CoR/HDAC3 complex, and represses gene expressions. Furthermore, HDAC4 represses the activity of runt-related transcription factor 2 (RUNX2) and myocyte enhancer factors (MEF2A, MEF2C). Animal studies showed that HDAC4 deficiency alone induces neurological consequences similar to 2q37 DS in humans [25,26]. Williams et al. reported two cases of intragenic HDAC4 deletion/insertion with phenotypes similar to 2q37DS [7]. Morris et al. postulated that the severity of 2q37 DS may be due to the HDAC4 dosage effect [31]. Moretti et al. reported three related patients with 2q37 deletions downstream of HDAC4 who presented intellectual disability, facial dysmorphisms, microcephaly, and congenital heart defects, but no brachydactyly type E [33]. Wakeling et al. reported seven unrelated patients with heterozygous de novo missense variants HDAC4 that impair 14-3-3 binding, and lead to a gain-of-function effect. The phenotype included significant DD/ID, seizures, distinctive facial features (hypertelorism, a full lower lip, long palpebral fissures, frontal upsweep of hair, widely spaced teeth, and large ears), scoliosis, delayed closure of the anterior fontanelle, but none of the patients had brachydactyly type E [34].
In our study, eight of the nine patients had haploinsufficiency for this gene, but only six of them had brachydactyly. Moreover, in the subject without the HDAC4 gene loss (P5), brachydactyly type E was noted. In literature data, only 50-62% of cases had brachydactyly [32]; therefore, it was suggested that this was a variable expressivity or incomplete penetrance of the gene, as well as the existence of other genes possibly responsible for the occurrence of type E brachydactyly. In this sense, PER2, TWIST2, GPC1 [11,12], GPR35 [35], FARP2, STK25, and PDC1 [36] were proposed as candidates. In case P5, the 1.84 Mb deletion included GPR35, FARP2, STK25, and PDC1 genes, which could have been potential causes of this feature in our patient.
Mild to moderate developmental delay (DD) and intellectual disability (ID) was reported in 79% of the cases with 2q37DS by Le et al. (2019), in an extensive study of 103 cases. [6]. Other previous studies reported intellectual disability and developmental delay in 100% of cases [6]. All but one of our cases presented DD and subsequently ID; however, in two cases, severe ID was noted. Some cases of 2q37 deletion with severe ID have been reported [6,30], but in two of our cases (P5, P7), the severity could also have been attributed to the associated CNVs.
Le et al. also reported abnormal behavior in 79% of cases: autism spectrum disorder in 30% of cases, repetitive behavior in 24%, hyperactivity in 15%, aggressive behavior in 12%, delayed social skills in 10%, attention deficit disorder in 9%, and friendly disposition in 12% [6,32]. In our study, cases 5 and 9 had abnormal behavior. Contrary to other studies where a haploinsufficiency of KIF1A [37], FARP2, HDLBP [10], and AGAP1 genes [13,38] was cited as a possible cause for autism in 30-35% of 2q37DS [1,3], in our patients, autistic spectrum disorder was found in just one case. This could be explained by the small number of cases in our study. Although autism was rare, other behavioral disorders (extremely friendly behavior, laughter crises, aggressiveness, and ADHD) were noted in four of the cases.
Characteristic facial dysmorphism, noted in 86% of the cases in the literature, is also a key factor for the recognition of this syndrome. The main features found are frontal bossing (41%), broad face (35%), special arched appearance of the eyebrows (32%), shortened palpebral fissures (27% of cases), deep-set eyes (19% of subjects), a thin upper lip (39%), depressed nasal bridge (25%), prominence of the columella (18%), and hypoplastic alae (15%) [6,32]. Our findings support the statistics reported in previous studies, in that eight out of nine of our patients presented a characteristic dysmorphic face. However, we noticed in our cases some particularities or clinical features that are less often reported. Arched eyebrows were observed in seven out of nine of the patients, but five cases presented bushy eyebrows; (three other cases with bushy eyebrows were reported in other studies) [14,39]. Four cases presented a low frontal hairline, an aspect frequently described in 2q37DS (46% cases [6,38]); however, two cases presented a high frontal hairline, which has not been reported thus far in 2q37DS. Microcephaly, reported more frequently in recent studies of 2q37DS [6,[39][40][41], was observed in six cases from our study. Furthermore, one case presented craniosynostosis; in the literature, another case with craniosynostosis was reported in a patient with del 2q37 associated with a duplication of 5q34 [42].
Another difference from the literature noted in our cases regarded growth parameters. While short stature was reported in the literature in 22% cases [1,32], it was noted in more than half of our cases (5/9). 2q37 DS is usually associated with overweightness or obesity (34-75% of cases), especially in deletions larger than 4.8 Mb [3,6,32]. The genes HDAC4, CAPN10, and HDLBP have been proposed as being responsible for the weight gain manifested since childhood (after 2 years old) [2,7], as well as the AGAP1 and SH3BP4 genes in larger or proximal deletions [13]. In our study, only one patient presented obesity, and one was overweight, but in four subjects, underweightness was observed (with standard deviations between −3 and −2.5 SD). Two of these underweight patients were young, and overweightness may not have set in yet. The other two had a deletion/duplication arrangement, and it is possible that their low weight was a consequence of the associated CNV or gastrointestinal malformations. As for the cases with normal weight, most of our patients were diagnosed early and kept under observation; therefore, excessive weight gain prevention was possible by maintaining a healthy diet and good management.
Heart defects are not a consistent sign in 2q37 DS, being presented in the literature in 16-20% cases [32,43]. The defect is usually mild, with mostly septal defects, less often coarctation of the aorta, or arch anomalies [2,32]. Severe cardiac anomalies are very rare in 2q37DS [1,32]. Four of our patients presented cardiac abnormalities: three had VSD, and one had a more severe cardiac malformation, with chronic heart failure, ASD, and a bicuspid aortic valve. In a study on 20 patients, Aldred et al. [1] proposed that a gene from the 3.4 Mb overlapping region between the RAMP1 and AC005237CA genes is responsible for the cardiac abnormalities. Two of our cases covered this region, but the other two had a smaller deletion, suggesting a restriction of the overlapping area for cardiac defects to a region of 0.66 Mb between the GPC1 and STICK25 genes.
Regarding neurological aspects, hypotonia was reported by Le et al. in 27% of cases, seizures in 16%, and brain malformations in 10% (agenesis of the corpus callosum, Dandy-Walker malformation, cerebral atrophy, holoprosencephaly) [6,32]. Although in five out of nine of our cases, hypotonia in infancy was observed, and seizures in four out of nine; only one of these cases had associated brain malformations.
Other clinical aspects, described less often in previous studies but observed in our patients, were translucent skin and telangiectasias (6/9 cases). Low set nipples were also observed in seven out of nine patients (reported in literature in 13% of cases [32]). A more peculiar phenotypic feature that was found in five of our cases was a particular disposition of the adipose tissue as a hump of fat on the upper thorax. To our knowledge, this sign has not yet been described in the literature in patients with 2q37 DS.
Besides the great phenotypic variability, 2q37DS also has great molecular variability. Thus far, no common breakpoints have been established, and the minimum critical deleted region was reduced by 2-3 Mb, including several genes, but especially the HDAC4 gene [6,7,44]. In the identification of 2q37DS, as in the rest of the syndromes with deletions and microdeletions, CGH-array is considered the gold standard. In our study, we first used MLPA as a diagnostic test, and later, for a more detailed analysis, we used CGH-array. MLPA is a feasible diagnostic test in cases with the classic phenotype, considering its low cost, and the follow-up kit allows for confirmation of deletions. In cases with a complex phenotype that would suggest the involvement of other anomalies, it is useful to perform CGH-array for the detection of other involved CNVs. Moreover, array-CGH allows determination of the size of the deletion, which is extremely useful for establishing future genotype-phenotype correlations.

Conclusions
The present study focused on finding new clinical signs that could improve the recognition of the syndrome, based on correlations between clinical molecular diagnostic traits.
Even in the case that SNP+CGH-array technique is not available, being more expensive and complex, MLPA has the advantage to be an easy, fast, and unexpensive technique, and is a very reliable tool for the diagnosis of 2q37DS syndrome; clinicians should be confident in the diagnosis established by this technique. In this sense, we propose the use of MLPA as a screening test in all patients with a slightly suggestive phenotype for 2q37DS, as well as in their relatives, and later analysis of the deletion through a SNP+CGH-array technique to establish a more complex management. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical reasons.