**2. Genetic screening and gene therapy in patients with craniofacial malformations**

Genetic screening by a clinical geneticist or genetic counselor plays a pivotal role in determining the patients with isolated craniofacial anomalies and syndromes. The identification of specific syndrome is important for the overall care of the patient as it identifies the risks of other medical problems that will have to be taken into account for the overall well being of the infant as well as in the management of the craniofacial malformation/syndrome. This helps the parents and family members to understand the cause and recurrence risk with future child births. Various methods of genetic testing such as karyotype, fluorescence in situ hybridization, chromosomal microarrays and next generation sequencing can be utilized in doing the genetic screening and mapping.

Karyotype analysis finds the chromosome number and searches for deletions and duplications. The flouroscent in situ hybridisation locates specific minor deletions in genome. The chromosomal microarrays/comparative ggenomic hybridisation focusses on complete genetics at finer detail as compared with other two. Advanced technologies are based on next generation sequencing that throws lighton individual base pairs of DNA encoding proteins [1]. This contains either doing a panel of genes of a specific disorder or whole genome sequencing which is available with genetic testing companies and laboratories that offer panels of genes for specific disorders as well as whole genomic sequencing.

The clinical features of the craniofacial anomalies are peculiar as they can occur as isolated, non syndromic or as a part of Mendelian syndromes. The medical geneticist and genetic counselor determine the type of cases that which are syndromic or isolated. Studies have shown linkage of non-syndromic cleft at region of 9q21, which after subsequent fine mapping revealed the significance of forkhead box protein E1 (FOXE1). It is genetically expressed at the point of fusion between maxillary and nasal process during palate formation whose mutations resulted in cleft palate, in mice studies. The other genes are interferon regulatory factor 6(IRF 6), transforming growth factor –alfa (TGFA). GWASs have confirmed the significance of IRF6 and FOXE1, 8Q24, 10Q25 AND 17Q22 in non- syndromic cleft palate cases.

*Perspective Chapter: Role of Genetics, Stem Cells in Reconstructive Surgery—Their Perspectives… DOI: http://dx.doi.org/10.5772/intechopen.109514*

The syndromes associated with cleft lip and palate include chromosomal abnormalities like trisomy 21, 18, 13, microdeletion syndromes (22q11 deletion syndrome), autosomal dominant disorder like VanderWoude syndrome and single gene disorders. The syndromes of cleft lip alone without cleft palate result from single gene defect which account for 75% and are associated with Mendelian disorders. The cleft lip with palate and cleft palate alone are embyologically separate entity with cleft lip and palate associated with syndromes are 50% and cleft lip alone are up to 75%. The cleft palate phenotype have been identified with chromosomes like Xq21 with TBX22 gene, TBX1gene (Di George/velocardiofacial syndromes). The SATB2 gene was identified as the cause of isolated cleft palate through its role in transcriptional regulation and disruption.

On the other side of the coin, the commonest craniosynostosis syndromes are due to mutation in fibroblast growth factor receptor 2 gene (FGFR2) which alters the protein to prolong signaling such that immature embryonic cell become bone embedded cells which can promote the premature fusion of bones in the skull, hands and feet. The eight known FGFR related craniosynostosis include Crouzon syndrome (with and without acanthosis nigricans), Apert syndrome, Pfeiffer syndrome, Jakson –Weiss syndrome, Beare-Stevenson syndrome, Muenke syndrome etc. Meunke syndrome is caused by gene mutation in FGFR3 gene.

Microtia is associated with gene mutations in syndromes such as Treacher Collin's syndrome (TCOF1, POLR 1C, POLR 1D gene), Nager syndrome (SF3B4, PRX1, PRX2 gene).

The causative gene of these craniofacial disorders/syndromes can be identified by DNA sequencing. The craniofacial malformations that are isolated, as well as that which are associated with genetic syndromes help the subject to be aware of the possible diseases and complications associated with other body systems. Majority of the craniosynostosis and syndromes can be diagnosed based on clinical findings, however, identification of genetic mutations is beneficial to the patients, family members and their next generations [2, 3].

#### **2.1 Gene therapy in craniofacial deformities and regeneration**

Conceptually, gene therapy involves insertion of new genetic material into cell to manipulate the endogenous proteins inside the cell. The methods of gene transfer are using DNA either in solution, conjugated to a biomaterial (polymers) or by viruses. In both of these, genes are transferred with in a plasmid that contains the genetic information necessary for the cell to begin making the protein product of that gene once the plasmid enters the nucleus. Viral transduction is the most effective method for gene transfer. The three main classes of viruses used for gene therapy are retroviruses, adeniviruses and adeno associated viruses. Even though the viral transduction is by far thee most effective method for gene transfer, it holds the insidious risk of insertional oncogenesis, toxic immune response, viral replication and dissemination.

Retroviruses are ideal for long term gene therapy, where in, the current human genome contains up to 5–8 of endogenous retroviral sequences that have been acquired over an evolutionary period. Adenoviruses are more suited for short term gene delivery and are used for tissue engineering that require a protein production over several weeks as they are non toxic and self limiting.

Gene therapy has been used to reprogram the fate of cells to generate induced pluripotent stem cells which change to a state of pluripotency and can differentiate to multiple different cell types. Through the insertion of specific genes involved in pluripotency of embryonic stem cells, somatic cells can be reprogrammed into cells that have self renewal and differentiation capabilities and hence are named induced pluripotent stem cells. These cells can regenerate cells from all three germ layers and can be made patient specific that overcomes the ethical issues unlike embryonic stem cells.

Adult stem cells are an excellent choice for reprogramming them into iPS stem cells due to their relative ease of isolation from different tissues, craniofacial region especially such as gingival fibroblasts, dental pulp stem cells. These cells have been successfully reprogrammed into iPS cells since these are ideal donors from the aspect of obtaining their precursor tissue from donor sites such as mandible and third molar teeth [4]. The craniofacially derived stem cells possess epigenetic memory can enhance their differentiating capability towards their tissue of origin and enhance their use for craniofacial reconstruction.

Gene therapy is used to repair and regenerate the complex tissues in craniofacial region as well as in treatment of tumor itself with genetic transfer which has made a significant progress in the last decade. This includes approval of H101 oncolytic adenovirus for treatment of head and neck cancer. Oncolytic viruses work by specifically targetting and replicating in tumor cells to result in cell death and dicrease in overall tumor size. In addition, Onco VEXR, works to regulate the immune response to the tumor by induction of antigen specific T cell responses. Genetic treatments of the tumor itself may limit the subsequent amount of reconstruction required.

In correction of craniofacial deformities, gene therapy can transform cells at the site of injury into protein synthesizing portals towards correcting them [3]. Reterovirus can be delivered directly to the desired site in which host tissue is transduced ex vivo and implanted at the site requiring tissue regeneration. Retrovirus regenerated femoral defects in rats with adenovirus transduced adipose tissue. Like plasmid DNA, viruses can be delivered on biocompatible scaffolds to generate desired protein production and tissue growth through spatial coordination of cells.

Till to date all strategies for whole tooth bioengineering have relied on the use of stem cells derived from dental pulp, periodontal ligament and/or developing tooth germ with very little emphasis on gene delivery. Further studies using recombinant adeno associated virus (AAV) is needed for bone repair due to qualities ofsuperior safety, tissue engineering and in vivo transduction. In vivo AAV mediated expression of constituently active activin receptor like kinase-2 and BMP-7 has enhanced the healing of bone defects in rodent models [5].
