Recessive pathogenic variants that lead to a loss-of-function in ALX1 have been determined as the genetic cause of ALX1-related FND. All pathogenic variants described until today are in consanguineous families or in families from close localities/villages, and thus are homozygous. However, compound heterozygous variants also can be associated with ALX1-related FND. The heterozygous carriers of the pathogenic variants appear completely normal.
ALX1 is one of the three members of vertebral ALX homeobox gene family. All ALX homeobox genes are transcription factors with DNA-binding homeodomains and C-terminal OAR domains (Meijlink et al. 1999). They play a role in the control of embryonic development, especially in the craniofacial region. All recessively inherited loss-of-function mutations in ALX1, ALX3 and ALX4 have been associated with frontonasal dysplasias in humans (Uz et al. 2010; Twigg et al. 2009; Kayserili et al. 2009). In several animal models, it has been shown that these transcription factors are expressed during several stages of facial primordial structures (Dee et al. 2013; Zhao et al. 1996).
ALX1 (also known as CART1) is a 4-exon gene located on chromosome 12q21.3. Three pathogenic variants in three families have been linked to ALX1-related FND until now. Two of these are splice site mutations (c.531+1G>A, c.661-1G>C) and one is a large deletion spanning 3.7 Mb and deleting the whole ALX1 as well as several other genes.
Sequencing of all exons, including the non-protein-coding exons of ALX1 will help ascertain the diagnosis of ALX1-related FND in suspected individuals. As a homozygous Copy Number Variation (CNV) in at least one family has been identified, absence of PCR amplification would suggest a homozygous deletion. These cases must be handled carefully and suspected CNVs should be confirmed by other methods such as quantitative or breakpoint-specific PCR methods. In individuals for whom pathogenic ALX1 variants cannot be determined, homozygosity mapping (in cases of parental consanguinity), linkage analysis, massively parallel sequencing methods may be useful in indirect genetic diagnosis, determination of non-coding pathogenic variants, determination of CNVs and discovery of other disease-related genes.