Alkaptonuria via the HGD Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
Order Kits


Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1188 HGD$910.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

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.

See More

See Less

Clinical Features

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 (; 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).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the HGD gene plus ~10 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Individuals found to exhibit homogentisic aciduria, as well as those exhibiting clinical symptoms suggestive of alkaptonuria, are good candidates for this test. Family members of patients who have known HGD variants are candidates. We will also sequence the HGD gene to determine carrier status.


Official Gene Symbol OMIM ID
HGD 607474
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Alkaptonuria 203500


Genetic Counselors
  • Human Gene Mutation Database (Bio-base).
  • Introne WJ and Gahl WA. 2013. Alkaptonuria. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301627
  • Kayser MA, Introne W and Gahl WA. 2014. Alkaptonuria. In: Valle D, Beaudet A.L., Vogelstein B, et al., editors. New York, NY: McGraw-Hill. OMMBID.
  • Phornphutkul C. et al. 2002. The New England Journal of Medicine. 347: 2111-21.  PubMed ID: 12501223
  • Rodríguez JM. et al. 2000. Human Molecular Genetics. 9: 2341-50.  PubMed ID: 11001939
  • Vilboux T. et al. 2009. Human Mutation. 30: 1611-9. PubMed ID: 19862842
  •, identifier: NCT00107783
  • Zatkova A. 2011. Journal of Inherited Metabolic Disease. 34: 1127-36. PubMed ID: 21720873
  • Zatkova A. 2011. Journal of Inherited Metabolic Disease. 34: 1127-36. 
  • Zouheir Habbal M. et al. 2014. Plos One. 9: e106948.  PubMed ID: 25233259
Order Kits

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Order Kits

Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
loading Loading... ×

Copy Text to Clipboard