ALX4-Related Disorders via the ALX4 Gene

Pathogenic variants in ALX4 have been associated with parietal foramina type 2 (PFM2). The hallmark symptom of PFM2 is the enlarged openings of the two bones that form the top and side of the skull. Other PFM2 clinical features include cranium bifidum, hypertelorism, a wide and depressed nasal ridge, and some focal alopecia. The prevalence of enlarged parietal foramina is estimated to be between 1:15,000 – 1:50,000 (Lodge. 1975. PubMed ID: 776315; Reddy et al. 2000. PubMed ID: 10720293).

Pathogenic variants in ALX4 have also been associated with frontonasal dysplasia type 2 (FND2). Patients with FND2 present with many overlapping symptoms with PFM2 such as parietal foramina, cranium bifidum, hypertelorism, and a wide nasal bridge. However, there are additional and more severe symptoms such as intellectual disability, facial clefts, hypoplasia or complete aplasia of the corpus callosum, cerebellar vermis hypoplasia, and complete alopecia. Almost all cases thus far have been in consanguineous families, indicating this disorder is very rare (El-Ruby et al. 2018. PubMed ID: 29681084).

It is currently unclear if PFM2 and FND2 represent distinct disorders or simply a dosage effect that scales with disease severity, with PFM2 being milder and FND2 being more severe. Consistent with a dosage effect, multiple patients have been reported with the more severe FND2 who inherited a variant from each parent; upon close examination both parents displayed parietal foramina consistent with the milder PFM2 (El-Ruby et al. 2018. PubMed ID: 29681084; Kariminejad et al. 2014. PubMed ID: 24668755).

A third disorder called Potaki-Shaffer syndrome (PSS) is caused by a chromosomal deletion, including the ALX4 gene. The clinical features of PSS include the hallmark symptom of parietal foramina seen from disturbances of the ALX4 gene. Additional symptoms include exostoses, or outgrowths of the bone; hearing loss; developmental delay and impaired intellectual development; hypotonia; and seizures.

By testing for variants in ALX4, a definitive diagnosis can be made between the various related disorders of PFM2, FND2, and PSS. Each disorder has a unique prognosis and set of early interventions. Treatments include surgery for facial clefts seen in FND2 and special education interventions for intellectual disability seen in PSS. Cranioplasty with bone grafts or a mesh plating system can be performed for patients at risk of injury from parietal foramina.

Parietal foramina 2 (PFM2) is inherited in an autosomal dominant manner with high, but not complete, penetrance. It is suspected that a pathogenic variant in a single copy of the ALX4 gene causes disease because the protein product is dosage sensitive (ie. haploinsufficient; Mavrogiannis et al. 2001. PubMed ID: 11137991). Parietal foramina can also be caused by variants in the gene MSX2 (PFM1); the two disorders have a similar incidence and are clinically indistinguishable (Mavrogiannis et al. 2006. PubMed ID: 16319823).

Frontonasal dysplasia type 2 (FND2) is inherited in an autosomal recessive manner. Given the dosage sensitivity seen in monoallelic cases for PFM2, the biallelic cases of FND2 are typically more severe. Frontonasal dysplasia can also be caused by biallelic variants in ALX3 (FND1) or ALX1 (FND3).

Potaki-Shaffer syndrome (PSS) is caused by a heterozygous deletion of a portion of the short arm of chromosome 11 (11p11.2), including the ALX4 gene. Other genes in this region include EXT2, which likely explains the symptoms of exostoses (Hall et al. 2001. PubMed ID: 11903336), and PHF21A, which likely explains the intellectual disability seen in PSS patients (Kim et al. 2012. PubMed ID: 22770980).

The ALX4 gene encodes the aristaless homeobox 4 protein that functions as a critical transcription factor during embryonic development of bones (especially the skull), hair, teeth, and mammary tissue. Amino acid residues 214-273 comprise the homeobox domain which is responsible for recognizing and binding to relevant DNA sequences. Mouse studies knocking out ALX4 have recapitulated the skull defects seen in patients as well as verified that severity of symptoms is worse in a homozygous state than a heterozygous state (Antonopoulou et al. 2004. PubMed ID: 15198690). ALX4 has been cited as a nonessential gene for growth of human tissue culture cells (Online Gene Essentiality).

The majority of causative variants documented thus far for ALX4 are loss of function, either nonsense or frameshift. There are, however, a few reported missense variants, all clustered around the DNA-binding homeobox domain (Mavrogiannis et al. 2006. PubMed ID: 16319823). The only ALX4 pathogenic variant that has been documented in a large population database was present at a globally frequency of 0.0012%, indicating that all disease-causing variants in ALX4 are very rare (https://gnomad.broadinstitute.org/variant/11-44297022-C-T?dataset=gnomad_r2_1). There is not clear documentation on the frequency of de novo variants causing disease. A few variants have been identified that likely cause a gain-of-function in the DNA-binding domain leading to increased ossification of the skull or craniosynostosis. Craniosynostosis can be caused by variants in many different genes, and it is likely that each variant simply increases risk by a small percentage. The opposite phenotypes from loss-of-function versus gain-of-function variants lends even more evidence to the idea that ALX4 is very sensitive to dosage effects.

When a patient presents with parietal foramina, the causative variant is found in ALX4 about 70% of the time. For the remaining 30% of cases, a variant is found in the MSX2 gene. For patients only presenting with craniosynostosis, a causative variant is found in ALX4 only ~1% of the time (Mavrogiannis et al. 2006. PubMed ID: 16319823).

This test is performed using Next-Generation sequencing with additional Sanger sequencing as necessary.

This test provides full coverage of all coding exons of the ALX4 gene plus 10 bases flanking noncoding DNA in all available transcripts in addition to non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).