Alkaptonuria via the HGD Gene

Alkaptonuria (AKU) is a condition diagnosed when excess homogentisic acid (HGA) is found in the urine, but other tyrosine metabolic products are not elevated and urinary amino acids are normal (Vilboux et al. 2009). In some affected individuals, the urine may turn dark upon standing due to the oxidation of the homogentisic acid (Zatkova 2011; Introne and Gahl 2013). Clinically, patients develop ochronosis (the accumulation of bluish-black pigment in the connective tissues, which is often visible in the sclera of the eyes and cartilage of the ears) and arthritis of the spine and larger joints. Flattening and calcification of intervertebral discs is observed when the spine is examined radiologically (Introne and Gahl 2013). The spine and joint symptoms often resemble ankylosing spondylitis, except in alkaptonuria patients the sacroiliac joint is not involved (Phornphutkul et al. 2002). While increased HGA has been found in the urine from an early age, ochronosis does not occur until after 30 years of age. Arthritis most often begins in the third decade of life, and generally begins earlier and progresses more rapidly in males than in females. As a result, approximately fifty percent of alkaptonuria patients need some kind of joint replacement by 55 years of age (Zatkova 2011; Introne and Gahl 2013). Other clinical symptoms can include renal stones, prostate stones, and tendon-related findings, such as tendonitis or tendon rupture. Deterioration of aortic valves may occur due to pigment deposition, and aortic stenosis is common in beginning in the sixth or seventh decade (Zatkova 2011; Introne and Gahl 2013). AKU patients do not appear to have a reduced lifespan, although the rate of disability is high in affected individuals, particularly in later life (Vilboux et al. 2009).

Currently, treatment is aimed at managing and alleviating symptoms in AKU patients, with physical and occupational therapy recommended. Joint replacement may be performed to manage pain associated with specific joints, and surgical treatment may be required for renal and prostate stones. Valve replacement may be required in patients that develop aortic stenosis (Introne and Gahl 2013). In an attempt to reduce the severity of clinical symptoms, dietary protein restriction as well as treatment with high doses of vitamin C may be recommended, although neither has been shown to have a dramatic effect (Introne and Gahl 2013). Clinical trials have been performed to assess the efficacy of nitisinone (approved for treatment of tyrosinemia type I) as an AKU therapeutic, although to date it has not been approved for treatment of AKU (www.clinicaltrials.gov; identifier: NCT00107783).

Alkaptonuria is an autosomal recessive disorder, and HGD is the only gene involved. The estimated worldwide prevalence of AKU is 1:250,000 to 1:1,000,000 worldwide, though this is thought to be an underestimate (Introne and Gahl 2013). The prevalence is higher in the Dominican Republic and Slovakia, where it is estimated to be 1:19,000 (Zatkova 2011; Introne and Gahl 2013). To date, over 115 causative variants have been reported in the HGD gene. The majority of reported pathogenic variants are missense, although nonsense, frameshift, splice site, small deletions, small insertions, indels and gross deletions have all been reported (Human Gene Mutation Database; Vilboux et al. 2009). More pathogenic variants are concentrated in exons 3, 6, 8 and 13, although pathogenic variants have been reported throughout the gene (Vilboux et al. 2009). Four founder mutations account for approximately 80% of HGD pathogenic alleles in the Slovak population (c.481G>A, c.457dup, c.808G>A, c.1111dup), whereas six different mutations are common in other populations but not in the Slovak population (c.688C>T, c.899T>G, c.174delA, c.16-1G>A, c.342+1G>A, c.140C>T) (Introne and Gahl 2013). To date, no genotype-phenotype correlations have been made (Vilboux et al. 2009; Zatkova 2011).

Alkaptonuria is caused by defects in the homogentisate 1,2-dioxygenase enzyme, which is part of the phenylalanine and tyrosine degradation pathway (Rodriguez et al. 2000; Phornphutkul et al. 2002; Introne and Gahl 2013). The homogentisate 1,2-dioxygenase enzyme is responsible for the conversion of homogentisic acid to maleylacetoacetic acid, and is active primarily in the liver and kidneys (Kayser, Introne and Gahl, 2014). The connection between molecular defects in the HGD gene and the clinical symptoms exhibited by AKU patients are not yet fully understood (Kayser, Introne and Gahl, 2014).

Clinical sensitivity of this test is expected to be high as AKU can be diagnosed unequivocally based on biochemical analysis, clinical features are often distinctive, and HGD is the only gene involved. Overall, the analytical sensitivity of this test is also expected to be high because the great majority of variants reported to date in the HGD gene are detectable via DNA sequencing. In one study of 58 patients with biochemically confirmed alkaptonuria, pathogenic variants were identified in 104 of 116 alleles, giving an overall detection rate of 90% (Phornphutkul et al. 2002). This is consistent with the rate of detection of causative mutations in the HGD gene by direct DNA sequencing listed by Introne and Gahl (2013). A second, more recent study reported detection of pathogenic variants in 154 of 158 alleles from 79 probands, giving an overall detection rate of ~97.5% (Vilboux et al. 2009).

At least one large deletion, unlikely to be detected by direct DNA sequencing, has been reported in the HGD gene (Zouheir Habbal et al. 2014). Such deletions may account for the remaining unidentified causative variants in these studies.

This test provides full coverage of all coding exons of the HGD gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other 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).