Submission Details

The gene ALAD is moderately associated with the disease ALAD-related porphyria according to the gene-disease association criteria of the ACMG (SOP version 10). ALAD catalyzes an early step in the tetrapyrrole biosynthesis pathway. The enzyme is primarily octameric but exists in equilibrium with some hexamers (lower activity) and dimers (inactive). The octameric structure is built from a properly assembled dimer. Dimers with a different domain orientation result in hexamers. pH, metals (particularly lead), and drugs can all affect the dimer domain orientation and thereby the quaternary structure of the enzyme (PMID: 31952692). The enzyme binds two molecules of 5-aminolevulinate per subunit and condenses them to form porphobilinogen.

The porphyria associated with mutations in this gene is extremely rare and is characterized by neuro-visceral attacks without cutaneous manifestations. Neurotoxicity without cutaneous manifestations is typically observed because loss of ALAD activity causes primarily the accumulation of the neurotoxic porphyrin precursor 5-aminolevulinic acid (ALA), which is a linear non-fluorescent molecule (PMID: 29516370). In ALAD-related porphyria, there is little accumulation of the circular protoporphyrin molecules, which cause skin photosensitivity and are produced later in the pathway.

ALAD has been extensively characterized in vitro, and its role in tetrapyrrole synthesis is well established. However, since the disease is exceedingly rare, with only a total of 8 published probands (only six were scored in this curation for reasons discussed below), this has prevented a higher score for genetic evidence from being reached. Due to the sufficient biochemical characterization and the known role of this enzyme in tetrapyrrole synthesis, the experts have agreed that several of the variant scores should be upgraded to reach a score of moderate. It is expected that additional published probands will take this classification to definitive. Note that although inheritance is typically autosomal recessive, in one case a heterozygote did present with acute hepatic porphyria late in life (we did not score this individual, PMID:11071662). Heterozygotes also have an increased susceptibility to lead poisoning. Further, towards this point, there was one case of porphyria when one pathogenic variant in ALAD was combined with a CPOX defect in the tetrapyrrole pathway (we did score this individual). Together, this likely means that heterozygosity in the presence of a compounding environmental factor or a second hit in the tetrapyrrole pathway is sufficient to cause disease. Similar to other porphyrias, incomplete penetrance is a characteristic of this disease, as a healthy heterozygote was reported to have exceedingly low levels of ALAD activity <12% of normal (we also did not score this individual).

Summary of Case Level Data (8.3 points): 6 probands were scored, one nonsense, four missense, and one putative splice site mutation.

Summary of Experimental Evidence (1.25 points): Biochemical characterization of ALAD’s enzymatic activity was scored (.5 points). ALA’s structural similarity to GABA and thereby associated neurotoxicity was scored (.5 points). Evidence from a 2019 review that looked at how pathogenic variants affect quaternary structure was scored (.25).

Gene Clinical Validity Standard Operating Procedures (SOP) - SOP10