الفهرس 
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Abstract
Branchial arch anomalies representing the embryological precursors of face, neck and pharynx are the second most common head and neck congenital malformations in children with second arch anomalies the most common.
The syndromes involving structures derived from the first and second branchial arches include ; Oculoauriculovertebral spectrum (OAVS), Treacher Collins, Auriculocondylar, Acrofacial Dysostosis (Nager), Miller, Branchi-oto-renal and Townes Brocks syndromes.
Oculoauriculovertebral spectrum (OAVS) is the second most frequent malformative disorder of head and neck with clinically heterogeneous phenotype. The etiology and pathogenesis of OAVS is highly heterogeneous. Genetic causes have been brought up in the literature due to the existence of familial cases and numerous chromosomal abnormalities have been associated with this spectrum. Furthermore several non genetic factors have been described as possible environmental causes. Retinoic acid (RA) signaling pathway has been implicated in various developmental processes and is essential for neural differentiation and craniofacial development. Interestingly, MYT1; the first described candidate gene for OAVS belongs to RA induced transcriptome and functional analysis confirmed this link between MYT1 and craniofacial cartilage alterations through RA signaling pathway.
The aim of this work was to study developmental syndromes with first and second branchial arch anomalies in a group of Egyptian patients and molecular analysis of clinically suspected cases with Oculoauriculovertebral (OAVS) spectrum with an attempt to reveal the genetic etiology in the studied cohort.
The study was carried out on 45 Egyptian patients with features suggestive of first and second branchial arch syndromes recruited from Human Genetics Department, Medical Research Institute, Department of Maxillofacial and Plastic Surgery, Faculty of Dentistry and Department of Ophthalmology, Faculty of Medicine, Alexandria University.
All the patients were subjected to detailed genetic history taking, pregnancy and delivery history, complete clinical genetic examination with special emphasis on craniofacial phenotype, pedigree analysis, clinical photography, chromosomal analysis and various investigations according to individual cases. The clinically suspected OAVS cases were included in the molecular study using array CGH, NGS panel testing, WES for a selected trio and Sanger sequencing of ALX1, ALX3 and ALX4 genes in clinically suspected cases with OAFNS.
The results of this study revealed the following:
After a thorough clinical evaluation and complete work up of the studied cases, the cases were classified into two major groups:
group I: First and second Branchial arch syndromes other than OAVS (9 cases, 20%).
This group of patients included:
- Treacher Collins syndrome (4 cases)
- Auriculocondylar syndrome ACS2 (1 family with 4 affected cases)
- Branchio-oculo-facial syndrome (BOFs) (1 case)
group II: OAVS cases (36 cases, 80%).
This group was sub-classified into:
group IIa: group with unknown etiology (31 cases, 86.1%)
- Suspected OAVS (27 cases; 4 of which with Goldenhar syndrome)
- Suspected OAFNS (3 cases).
- Suspected Delleman Oorthuys syndrome (1 case)
group IIb: group with teratogenic insult (2 cases, 5.6%)
- Methotrexate embryopathy (1 case)
- Retinoic acid embryopathy (1 case)
group IIc: group with chromosomal etiology (3cases, 8.3%)
- Down syndrome (1 case)
- Klinefelter syndrome (1 case)
- 46XY, add (8) (p23) (1 case)
Molecular study:
A-Molecular results of OAVS: Patients with OAVS phenotype with irrelevant teratogenic or numerical chromosomal etiology and who fulfill the minimal selection criteria were included in the molecular study (32 from the total of 36 cases with OAVS).
Array CGH results:
- Dosage anomalies comprising variants of unknown significance (VUS), probably pathogenic and pathogenic variants) was revealed in 6 OAVS patients out of 26 studied (23%).
- De novo 43.3 kb deletion in Xq26.1 (Case12) (VUS)
- De novo 87.2 kb deletion in Xq22.2 (Case13) (VUS)
- De novo 133.3 kb duplication in 4q34.3 (Case13) (VUS)
- De novo large deletion of 532.7 kb in pseudo-autosomal region at Xp22.23 involving the SHOX gene (Case14) (Probably pathogenic)
- De novo 346.3 kb duplication in 8p21.3 (Case 22) (VUS)
- De novo 237.87 kb de novo duplication in 14q22.3 (Case 35) (VUS)
- A very large deletion (6692.46 kb) in 8p23.1-p23.3 and a massive duplication (30.2 Mb) in 12p13.33-p11.22 due to maternal balanced reciprocal translocation between chromosomes 8 and 12 at bands p23 and p11.2 respectively (46, XX, t (8; 12) (p23; p11.2) (Case 45) (Pathogenic)
NGS panel results:
- The present study revealed many probably benign variants in MYT1, HMX1 and ZYG11B genes and rare missense variants of unknown significance (VUS) in ZYG11B and HMX1 genes in 2 patients, those variants were not reported before in UCSC, gnomAD or EXAC databases ; hence with unknown exact minor allele frequencies and unknown effect or protein alteration. All variants were presented in heterozygozity. No variants of interest were found in any of the 4 EYA genes.
- Two missense intronic variants in ZYG11B gene (g.5325993 T>A and g.53271293 C>T) and one missence variant in 3’UTR of HMX1 gene (g.8868930 C>A) (Case 26) (VUS)
- One intronic missense VUS in ZYG11B gene (g.53221999 C>T) (Case 27) (VUS)
- No pathogenic or probably pathogenic variants were revealed in our panel testing.
Whole exome sequencing (WES) results:
- One sporadic clinically suspected Goldenhar syndrome case (Case 10) with the full blown phenotype was selected among the OAVS cohort and genomic DNA samples from the patient and his healthy parents were processed for WES trio study.
- Hundreds of variants were detected in the studied family, and in depth bioinformatics analysis using variable tools and databases was performed to filter the data and categorize the variants of interest.
- One missense de novo heterozygous probably pathogenic variant was identified in DGKD gene located on chromosome 2 (NM_003648.2:c1748 C>T, T583I / NM_152879:c.1880 C>T, T627I).This variant was not reported before in data bases and it is annotated probably damaging in different tools through our data analysis processes.
- Another de novo heterozygous frame shift insertion with unknown protein effect in CAMKK2 gene on chromosome 12, was harbored by the patient (NM_001270486.1:c.1611_1614 C>CTT, rs7787701848). This variant was not also reported before in databases and it is considered according to ACMG guidelines as VUS. - Probably benign missense variant with a protein effect inherited from the healthy father in FKBP15 gene (rs142775124, NM_015258.1:c.2126 T>C, K709R) was also detected; with MAF 0.00002 globally /0.00006 in Africans.
Results of Sanger sequencing of ALX1, ALX2, ALX4 genes :
- Deletions in the 3 ALX genes previously reported in Frontonasal dysplasia patients were screened in 3 patients with clinically suspected OAFNS (Cases 27, 33and 37) by Sanger sequencing. Results were negative in the 3 screened genes in the 3 patients.
B-WES results of ACS family:
- Pathogenic known missense for ACS2 in PLCB4 gene with full penetrance was detected in a heterozygous pattern in the 4 family members (NC_000020.10:g.9389727G>A, NM_000933.3, c.1862G>A, Arg621His). It is considered pathogenic in HGMD; CM123396.