Alkaptonuria

Alkaptonuria JA Gallagher and LR Ranganath, University of Liverpool, Liverpool, UK

A Zatkova, Slovak Academy of Sciences, Bratislava, Slovakia

© 2013 Elsevier Inc. All rights reserved.

This article is a revision of the previous edition article by BN La Du, volume 1, pp 35–36, © 2001, Elsevier Inc.

Glossary Ethyl N-nitrosourea (ENU) A highly potent mutagen used to induce point mutations in experimental mice. Hereditary tyrosinemia type 1 (HT1) An autosomal recessive disease caused by a deficiency of the enzyme fumarylacetoacetate hydrolase. It is the most severe form of tyrosinemia. Nitisinone A drug originally developed as an herbicide which is now used in the treatment of HT1. The systematic chemical name is 2-(2-nitro-4-(trifluoromethyl)benzoyl) cyclohexane-1,3-dione, which is abbreviated to NTBC.

Introduction Alkaptonuria (AKU; MIM 203500) is an iconic disease which holds an important position in the history of genetic disease. It was the first human disorder that was recognized to conform to the principles of Mendelian autosomal recessive inheritance by Sir Archibald Garrod over 100 years ago. It was in his Croonian Lecture at the Royal College of Physicians in 1908 that Garrod introduced the concept of ‘inborn errors of metabolism’ to describe AKU and three other inherited disorders, albinism, cystinuria, and pentosuria. Fifty years later, Bert La Du and colleagues identified the enzymatic defect as deficiency of homogentisate 1,2-dioxygenase (HGD; E.C.1.13.11.5), an enzyme in the metabolism of tyrosine and phenylalanine (Figure 1). AKU is an ultra-rare disease with a low prevalence of 1:100 000–250 000 in most ethnic groups. However, several hot spots have been identified including the Dominican Republic and north-west Slovakia, where the incidence is greater than 1:20 000. Recently, a high incidence of AKU was also discovered in specific regions of Jordan, and it is likely that there is a vast reservoir of undiagnosed AKU worldwide.

HGD Gene The HGD gene was first cloned from Aspergillus nidulans by Fernández-Cañón and coworkers, and they went on to sequence the human HGD gene. The gene maps to the human chromo­ some 3q13.33. It is a single-copy gene spanning 54 363 bp of genomic sequence, with 14 exons coding for a protein of 445 amino acids (Figure 2). AKU arises from homozygous or com­ pound heterozygous mutations in the HGD gene and many AKU-causing mutations have been described. Liver and kidneys are the major sites of HGD activity, but low-level expression has been reported in other tissues. HGD is organized as a complex hexameric protein, a dimer of two disklike trimers. Spatial stability is dependent on

Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1

Ochronosis Deposition of dark pigment in tissues. It is usually a symptom of alkaptonuria but can be caused by exogenous agents. Schmorl’s stain A histological stain for reducing substances in tissues that is widely used to identify melanin. In the staining reaction, ferricyanide is converted into ferrocyanide which is converted to insoluble Prussian blue in the presence of ferric ions. Ultra-rare disease A disease which affects fewer than 20 patients per 1 million of population.

noncovalent bonds between the subunits, and the HGD enzyme function is easily disrupted by mutation. The gene shows remarkable allelic heterogeneity, with more than 115 different AKU mutations and 33 polymorphisms identified (see Figure 2). Haplotype analysis has been used to trace the migration of specific AKU alleles through human history. The three most widespread AKU mutations in Europe, M368V, V300G, and P230S (20%, 5%, and 5% of European AKU mutations, respectively) appear to be ancient mutations that were introduced into Europe with the founder populations. Remarkably 10 different HGD mutations have been identified, in Slovakia, clustering in a small region in the north west of the country. As yet, there is no explanation for this massive increase in the incidence of AKU-causing mutations in this region, which cannot be explained by a classical founder effect. In contrast, the high incidence of AKU in the Dominican Republic arose through a classical founder effect with C120W as the mutation. AKU mutations are distributed throughout the entire HGD gene with some prevalence in exons 6, 8, 10, and 13. Over 66% are missense mutations. As yet, no relationship between speci­ fic mutations and clinical manifestations has been established, although several AKU mutations have been identified, which have been shown to have residual catalytic activities in func­ tional assays.

Metabolic Consequences of HGD Deficiency Deficiency of HGD results in a failure to break down homo­ gentisic acid (HGA; see Figure 1) which is the culprit molecule in pathology of AKU. HGA is a highly soluble molecule and several grams, hundreds of times the normal amount, can be excreted daily in the urine. The level of excretion is greater than that accounted for by glomerular filtration as there is active renal tubular secretion through organic anion transporters. The presence of HGA in the urine leads to characteristic darkening on exposure to air, a reaction which is indicative of AKU but

doi:10.1016/B978-0-12-374984-0.00030-9

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72

Alkaptonuria

O

Phenylalanine

OH

Tyrosine Tyrosine aminotransferase

Thyroid hormones Catecholamine Melanin

NH2

HO 4-Hydroxyphenylpyruvate

4-Hydroxyphenylpyruvate dioxygenase

Benzoquinones

NTBC HO O

Homogentisic acid HO

Homogentisate 1,2-dioxygenase

AKU

OH

Ochronotic pigment

Maleylacetoacetate

Fumarylacetoacetate Fumarylacetoacetate

hydrolase

Succinylacetoacetic acid

Succinylacetone

Fumarate and acetoacetate Figure 1 The metabolism of tyrosine showing the location of the metabolic block in AKU and the site of action of NTBC (nitisinone).

not definitively diagnostic. Darkening can be accelerated by addition of alkali. The excretion of black urine by a healthy schoolboy was reported by G. A. Scribonius as early as 1584, but it was Carl Bödeker in 1857 who partially isolated the chemical responsible and introduced the term ‘alkapton’ to describe the black substance that formed on addition of alkali to the urine of AKU patients. The identity of ‘alkapton’ as HGA was subsequently confirmed by Wolkow and Baumann in 1891. In childhood, the only manifestation of AKU is darkening of the urine and cerumen. The disease frequently goes undiag­ nosed into middle age, and there are accounts of initial diagnosis in patients over 60 years age.

Ochronosis Despite the extensive renal clearance of HGA, there is a rise in plasma HGA above 5 µg ml−1 in AKU. Over time, the high level of HGA present in extracellular fluid leads to the deposition of pigmented polymers in tissues, leading to the characteristic appearance of darkened ear lobes and pigmentation of skin and sclera that appears in AKU patients. This process of pig­ ment deposition is described as ‘ochronosis’, a term first used by Virchow in 1866 to describe the pigmentation observed in histological sections of postmortem connective tissue, espe­ cially cartilage, skin, and sclera. The exact chemical nature of this pigmentation has still not been elucidated, but it has some structural similarities to melanin and can be stained by Schmorl’s stain. It is likely that benzoquinone and its oxidation products are intermediates in the formation of the pigmented polymer and there may be a role for autoxidation. The prefer­ ential deposition of pigment in certain tissues illustrates that HGA is necessary but not sufficient for ochronosis. Tissues initially appear to be resistant to pigmentation and may only become susceptible following oxidative or mechanical damage and/or local metabolic changes. Although most pigment is

deposited extracellularly, there is strong evidence for a role of cells in the process of ochronosis. Some authors have proposed the involvement of enzymes including polyphenol oxidases. Extracellular pigmentation is usually, but not exclusively, associated with fibrocollagens, but the distribution does not correspond directly to any specific collagen types. Pigmentation can be granular or more diffuse, but differences in the chemical structure of the pigment at different sites remain to be elucidated. In the initial stages of tissue ochronosis, there is distinctive association of pigment with the periodicity of collagen cross-banding, suggesting a nucleation effect by specific residues in matrix macromolecules. The deposition of ochronotic pig­ ment can be reproduced in vitro in tissue explants or monolayer cell cultures incubated with HGA. Bone collagen is largely unaffected probably because bone mineral inhibits access of pigment. However, in nonmineralized collagen, there is an increase in calcium content in the tissue associated with pigmen­ tation. Pigment has been identified in many cell types including macrophages, osteoclasts, chondrocytes, osteocytes, osteoblasts, fibroblasts, and glandular epithelial tissue. Presence of pigment in cells can arise through de novo formation or through phago­ cytosis. In hyaline cartilage, pigmentation is initially laid down at the interface with calcified cartilage, spreading through the deep layers of cartilage and eventually to the articular surface (Figure 3). Displaced fragments of ochronotic cartilage are found deep in the subchondral bone and embedded in syno­ vium, where they evoke foreign body giant cells. The potential importance of mechanical loading and load-induced damage in the initiation and progression of ochronosis is highlighted by the anatomical distribution of pigment. In the musculoskeletal system, pigmentation is con­ centrated in tissues subjected to high compressive loading but also in traction tendons and entheses. In the cardiovascular system, pigmentation is observed in regions of high intravas­ cular pressure and turbulent flow, including the aortic root, the aortic and mitral valves, and the carotid sinus regions. Pigment

Alkaptonuria

73

q28 q29

q26.2 q26.31 q26.32 q26.33

q26.1

q24 q25.1 q25.2

q22.3 q23

q21.3 q22.1

q13.31 q13.33

q13.13

q13.11

p12.3 p12.2 p12.1 p11.1 q11.1 q11.2

p13

p14.1

p21.31

p22.3 p22.2 p22.1

p24.1

p24.3

p26.1 p25.3 p25.1

Chr 3

p14.3 p14.2

HGD gene. chromosomal location 3q13.33

HGD gene. 54.363 bp of genomic DNA

1

2 3

4

5

6

7 8 9 10

11

12

13

14

HGD transcript. 17.715 nt

AAAAAA ATG

TGA HGD protein. 445 aa (hexameric)

HGD gene mutations Europe

Europe

Europe

Czech Republic (4) Unknown 12.5% ivs1−1G>A 25.0% ivs5+1G>A 12.5% G161R 50.0%

Portugal (5) R197fs (T196fs)20.0% D291E 20.0% V300G 20.0% M368V 40.0%

Poland (2) ivs1−1G>A G161R ivs13+1G>T

Spain (10) Unknown I216T R225H F227S P230S P230T D291E V300G W322R M368V

25.0% 50.0% 25.0%

Slovakia (54) ivs1−1G>A 4.7% S47L 0.9% S59fs (R58fs) 1.9% ivs5+1G>A 3.8% D153fs (G152fs)15.1% G161R 43.4% E178G 0.9% P230S 4.7% G270R 7.5% V300G 3.8% M368V 1.9% H371fs (P370fs) 11.3%

20.0% 5.0% 10.0% 10.0% 15.0% 5.0% 5.0% 5.0% 10.0% 15.0%

France (17) Unknown 21.2% G11fs (F10fs) 3.0% Y62C 6.1% W97G 6.1% D153fs (G152fs) 9.1% D153G 6.1% Italy (12) ivs7+2T>C 3.0% Unknown 25.0% V157fs 3.0% W60X 8.3% G161R 3.0% D153fs (G152fs) 8.3% V181F 6.1% ivs7+5G>A 4.2% A218fs (G217fs) 3.0% G198D 8.3% G270R 3.0% ivs9−56G>A 8.3% H292R 3.0% ivs9−17G>A 4.2% V300G 3.0% K248R 4.2% G360A 6.1% G270R 8.3% M368V 15.2% G360R 12.5% E401Q 8.3% Finland (8) S59fs (R58fs) 12.5% The Netherlands (2) R330S 12.5% ivs5+1G>T 50.0% M368V 37.5% A122D 25.0% H371R 37.5% M368V 25.0%

UK (22) Unknown E42A S59fs (R58fs) ivs5+1G>T ivs7+ 2T>C C120F G123R G115R G161R F169L V157fs (?) G217W R197G R225H G270R K276N V300G G361R G362E M368V D374H K431fs X446ext

North America

USA (87) 10.0% Unknown 2.9% 10.0% E3A 0.6% 5.0% L4S 1.1% 5.0% L4X 0.6% 2.5% ivs1−1G>A 1.1% 5.0% E42A 3.4% 2.5% R53W 0.6% 5.0% S59fs (R58fs) 6.3% 10.0% S59X 0.6% 2.5% ivs3-2A>G 0.6% 5.0% W60G 1.7% 2.5% L61P 1.1% 5.0% Y62C 0.6% 2.5% F73L 0.6% 2.5% P92T 0.6% 2.5% W97R 1.1% 2.5% ivs5+1G>T 3.4% 5.0% ivs5+1G>A 0.6% 2.5% C120fs 0.6% 7.5% C120F 5.7% 2.5% C120W 1.1% 2.5% G123R 1.1% 10.0% K126fs 1.1% F136Y 0.6% Switzerland (2) L137P 1.7% Unknown 25.0% G115fs (?) 1.1% W97G 25.0% E143D 0.6% R321X 25.0% R145X 0.6% M368V 25.0% N149K 0.6% D153fs (G152fs) 1.7% Germany (8) ivs7+2T>C 0.6% L25P 25.0% P158R 0.6% G161R 6.3% P158L 0.6% V300G 12.5% Q159H 0.6% M368V 56.3% G161R 6.3% E168K 2.9% Macedonia (1) E168X 0.6% P158L 50.0% K171N 0.6% P274L 50.0% E178D 0.6% Q183R 0.6% ivs8+1G>A 0.6% ivs8−2A>C 0.6% R187G 0.6%

Rest of the world

USA (87) cont. G217W 0.6% R225L 0.6% P230S 1.1% Q258P 1.1% H269R 0.6% G270R 1.1% V300G 2.3% S305F 0.6% R321P 0.6% ivs12+2T>A 0.6% ivs12−2A>T 1.1% M339fs 0.6% M339fs 2.9% P359L 2.9% G360R 4.0% G362E 0.6% M368V 16.0% H371fs (P370fs) 1.1% P373L 0.6% E401Q 0.6% X446ext 1.1% Africa

Australia (1) K57N G360R Japan (1) E168K

50.0% 50.0%

100.0%

South Korea (1) Q33R 50.0% G152A 50.0% UAE (1) S59fs (R58fs) 100.0% Turkey (9) S59fs (R58fs) N219S R225H P230S G270R

33.3% 11.1% 33.3% 11.1% 11.1%

India (3) S59fs (R58fs) L116P A122V

12.5% 25.0% 37.5%

Canary Islands (1) P230S 100.0% USA/India (4) A122V 37.5% Algeria (9) E168D 50.0% Unknown 11.1% G360R 12.5% ivs1−1G>A 11.1% L44F 5.6% Jordan (7) G123A 33.3% A122V 92.9% S189I 11.1% ivs1−1G>A 7.1% A218fs (G217fs) 5.6% Latin America S287X 16.7% A407A 5.6% Dominican Republic (8) La Réunion (2) C120W 87.5% S59fs (R58fs) 25.0% G270R 12.5% V300G 75.0%

Figure 2 Diagram of the chromosomal location and structure of the HGD gene, the HGD protein structure, and the position and geographical prevalence of identified mutations in the HGD gene. Selected mutations are marked by color to highlight their distribution.

74

Alkaptonuria

Figure 3 Knee joint of patient with AKU at surgery showing extensive ochronosis. In addition to the hyaline cartilage, pigmentation can also be seen in meniscus, fibrous tissue, and periosteum. Cartilage has been lost from the lateral condyle exposing the underlying subchondral bone. Courtesy of Dr. Adam Taylor, findAKUre Consortium.

locates in the intima, inner media, and adventitia of systemic arteries and fibrolipid components of atheromatous plaques, whereas minimal pigmentation is observed in the venous circulation. Ochronosis increases the stiffness of extracellular matrix leading to altered stress distribution, further mechanical damage, proliferation of the pigmentation, and a downward spiral of tissue destruction.

Clinical Manifestations Eventually, ochronosis leads to severe clinical manifestation including early onset osteoarthopathy. Chronic joint pain fre­ quently develops in the third decade, and there is destruction of the large weight-bearing joints, resembling severe osteoarthritis (Figure 3), and scoliosis and fusion of the vertebrae, resembling ankylosing spondylitis. Tendon and ligament ruptures are com­ mon and there is cardiac valve deterioration and aortic valve stenosis. Although bone collagen appears to be protected from pigmentation, the severe changes in articular cartilage lead to aggressive bone resorption of subchondral bone and localized osteoporosis. AKU patients also frequently suffer renal and pros­ tate stones, and salivary stones have also been reported.

Therapy Currently, there is no effective treatment for AKU and manage­ ment remains symptomatic. Patients have to undergo multiple joint replacements. Dietary protein restriction has been used to reduce plasma HGA levels, but this is not an attractive or effective long-term therapeutic strategy for AKU. Some authors have suggested that antioxidants, including ascorbic acid and N-acetylcysteine, might be useful, and although there is some experimental evidence from in vitro studies, there is as yet no evidence of clinical benefit. Furthermore, ascorbic acid is a cofactor for 4-hydroxyphenylpyruvic acid dioxygenase and

could possibly increase the production of HGA. Research is under way to develop gene and enzyme replacement therapies, but these are long-term projects. Therapeutic strategies invol­ ving gene or enzyme replacement must ensure that the accumulation of the downstream metabolites maleylacetoace­ tate and fumarylacetoacetate is avoided (see Figure 1), as these are the toxic molecules responsible for the severe pathology in hereditary tyrosinemia 1 (HT1). Currently, the most promising treatment for AKU is NTBC or nitisinone, a drug originally developed as an herbicide, which has been very successful in the treatment of HT1. NTBC is a potent inhibitor of p-hydroxyphenylpyruvate dioxy­ genase, and thus of HGA production. Clinical trials have shown that NTBC is very effective at reducing plasma levels of HGA and lowering urinary excretion of HGA (by over 95%). Although there is no evidence yet of a beneficial effect of NTBC in ochronosis or joint disease, the biochemical efficacy of nitisinone in reducing the production of HGA, the culprit molecule, coupled with the tolerability of the drug, strongly suggests that this will be a useful therapeutic agent, and further clinical trials of NTBC in AKU are planned.

Misdiagnosis of AKU Dark urine can arise in non-AKU subjects as a result of porphyrias, myoglobinuria, hemoglobinuria, bilirubinuria, and following ingestion of specific compounds. Exogenous ochronosis of skin and other tissues can result from topical exposure to phenol and hydroquinone, injections of quinine, and ingestions of amiodarone, minocycline, antimalarials, and levodopa and methyldopa used in the treatment of Parkinson’s disease.

Animal Models A mouse model of AKU was developed at the Pasteur Institute in 1994 by N-ethyl N-nitrosourea (ENU)-induced mutagenesis. The mice have a truncated HGD protein resulting from a splice mutation in the HGD gene. Initially, reports indicated that these mice do not exhibit ochronosis, despite excreting suffi­ cient HGA to cause darkening of urine. However, it is now recognized that these animals will develop ochronosis predic­ tably. This model will be an important resource in the further study of the pathophysiology of ochronosis and the develop­ ment and evaluation of new therapies.

See also: Phenylalanine, Chemical Structure; Tyrosine.

Further Reading Bödeker CHD (1859) Über das alcapton: ein beitrag zur frage: welche stoffe des harns können kupferreduction bewirken? (About the alcapton: A beitrag to ask – What substances in the urine can cause copper reduction?) Zeitschrift für Rationelle Medicin 7: 130–145. Fernández-Cañón JM, Granadino B, Beltrán-Valero de Bernabé D, et al. (1996) The molecular basis of alkaptonuria. Nature Genetics 14: 19–24. Garrod AE (1996) The incidence of alkaptonuria: A study in chemical individuality. 1902. Molecular Medicine 2: 274–282.

Alkaptonuria Helliwell TR, Gallagher JA, and Ranganath L (2008) Alkaptonuria – A review of surgical and autopsy pathology. Histopathology 53: 503–512. Introne WJ, Perry MB, Troendle J, et al. (2011) A 3-year randomized therapeutic trial of nitisinone in alkaptonuria. Molecular Genetics and Metabolism. 103: 307–314. Kraus VB (2011) Rare osteoarthritis: Ochronosis, Kashin–Beck disease and Mseleni joint disease. In: Hochberg MC, Smolen SJ, Weinblatt ME, and Weisman MH (eds.) Rheumatology, 5th edn., pp. 1825–1837. Philadelphia, PA: Mosby. La Du BN, Zannoni VG, Laster L, and Seegmiller JE (1958) The nature of the defect in tyrosine metabolism in alcaptonuria. Journal of Biological Chemistry 230: 251–260. Phornphutkul C, Introne WJ, Perry MB, et al. (2002) Natural history of alkaptonuria. The New England Journal of Medicine 347: 2111–2121. Taylor AM, Wlodarski B, Prior IA, et al. (2010) Ultrastructural examination of tissue in a patient with alkaptonuric arthropathy reveals a distinct pattern of binding of ochronotic pigment. Rheumatology (Oxford) 49: 1412–1414.

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Tinti L, Taylor AM, Santucci A, et al. (2011) Development of an in vitro model to investigate joint ochronosis in alkaptonuria. Rheumatology (Oxford) 50: 271–277. Zatkova A (2011) An update on molecular genetics of alkaptonuria (AKU). Journal of Inherited Metabolic Disease 34(6): 1127–1136.

Relevant Websites http://www.alkaptonuria.info – AKU Society. http://findakure.org – findAKUre Research Consortium. http://www.alcap.fr – French ALCAP. http://hgddatabase.cvtisr.sk – HGD mutation database. http://www.aimaku.it – Italian AIMAKU.