Identification of 11 Novel Homogentisate 1,2 Dioxygenase Variants in Alkaptonuria Patients and Establishment of a Novel LOVD-Based HGD Mutation Database

Abstract

Enzymatic loss in alkaptonuria (AKU), an autosomal recessive disorder, is caused by mutations in the homogentisate 1,2 dioxygenase (HGD) gene, which decrease or completely inactivate the function of the HGD protein to metabolize homogentisic acid (HGA). AKU shows a very low prevalence (1:100,000–250,000) in most ethnic groups, but there are countries with much higher incidence, such as Slovakia and the Dominican Republic. In this work, we report 11 novel HGD mutations identified during analysis of 36 AKU patients and 41 family members from 27 families originating from 9 different countries, mainly from Slovakia and France. In Slovak patients, we identified two additional mutations, thus a total number of HGD mutations identified in this small country is 12. In order to record AKU-causing mutations and variants of the HGD gene, we have created a HGD mutation database that is open for future submissions and is available online (http://hgddatabase.cvtisr.sk/). It is founded on the Leiden Open (source) Variation Database (LOVD) system and includes data from the original AKU database (http://www.alkaptonuria.cib.csic.es) and also all so far reported variants and AKU patients. Where available, HGD-haplotypes associated with the mutations are also presented. Currently, this database contains 148 unique variants, of which 115 are reported pathogenic mutations. It provides a valuable tool for information exchange in AKU research and care fields and certainly presents a useful data source for genotype–phenotype correlations and also for future clinical trials.

Electronic supplementary material The online version of this article (doi:10.1007/8904_2011) contains supplementary material, which is available to authorized users.

Introduction

Alkaptonuria (AKU) [MIM 203500] is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase [E.C.1.13.11.5] (HGD) activity (La Du et al. 1958). The enzymatic defect in AKU is caused by homozygous or compound heterozygous mutations within the HGD gene (Fernández-Cañón et al. 1996), which maps to the human chromosome 3q21–q23 (Pollak et al. 1993). This disease has a very low prevalence (1:100,000–250,000) in most ethnic groups, but it presents a remarkable allelic heterogeneity – about 96 different HGD mutations and 33 polymorphisms have already been reported (summarized in a review article Zatkova (2011) JIMD).

The HGD gene is a single-copy gene that spans 54,363 bp of genomic sequence split into 14 exons and coding for the HGD protein composed of 445 amino acids (Fernández-Cañón et al. 1996; Granadino et al. 1997).

By reexamination of the mutations and polymorphisms reported in HGD by 1999, Beltrán-Valero de Bernabé et al. showed that the “CCC” sequence motif and its inverted complement, “GGG” are preferentially mutated (Beltrán-Valero de Bernabé et al. 1999). Subsequently, nucleotide c.342+1G was also described as a mutational hotspot in HGD (Zatkova et al. 2000a). Therefore, this nucleotide and “CCC” triplets, together with CpGs, are considered to be mutational hotspots in the HGD gene.

The establishment of the crystal structure of the human HGD enzyme provided a framework for understanding the pathogenic effect of the AKU mutations. The active form of the HGD is organized as a hexameric protein, dimer of trimers: two disk-like trimers stacked base-to-base about twofold axes to form hexamers (Titus et al. 2000). Many noncovalent bonds between amino acid residues (hydrogen, salt, and hydrophobic bonds) are required to maintain the spatial structure of the monomer, of the trimer and finally of the hexamer. Thus, intersubunit interactions are important for the activity of the HGD enzyme and the complex structure of the functional HGD protein can be easily disrupted by mutations.

So far, 626 AKU patients have been reported in about 40 countries worldwide (Ranganath et al. 2011). Interestingly, countries such as Slovakia and the Dominican Republic exhibit an increased incidence in this disorder of up to 1:19,000 (Milch 1960; Srsen and Varga 1978). This increase is especially noticeable in Slovakia where 208 patients have been registered, including 110 children (Srsen et al. 2002). Ten different HGD mutations have currently been reported in Slovakia (Gehrig et al. 1997; Muller et al. 1999; Zatkova et al. 2000b; Zatkova et al. 2003; Zatkova et al. 2000c), and therefore it is difficult to explain the increased incidence of AKU in this relatively small country by a classical founder effect.

In this study, we report 11 novel HGD gene mutations identified in patients from different countries and discuss the genetic aspects of AKU in Slovakia. We inform also on a new HGD mutation database (http://hgddatabase.cvtisr.sk/) that we decided to create because of our involvement in the routine genetic screening of AKU patients sent to our laboratory from different countries and also because we saw that centralization of information regarding all reported patients with this rare error of metabolism would be useful. Additionally, the original AKU database (http://www.alkaptonuria.cib.csic.es/) located in Madrid has not been updated since 2001, and there was a need to adapt and correct the nomenclature of all known mutations according to the recommendations of the HGVS.

Material and Methods

Patients

Herein, 36 AKU patients and 41 family members from 27 families were analyzed. Of these, 18 patients were from 13 Slovak families, and the remaining 18 cases from 15 families from different countries were sent to our laboratory for mutation analysis (Supplementary Table 1).

Supplementary Table 1.

List of all patients and family members analyzed in the present study . AKU family code is indicated which enables the reader to identify members of identical family and each AKU chromosome in the HGD mutation database and in Supplementary Table 2. Old names are indicated in brackets for the mutations that have been reclassified according to the HGVS nomenclature recommendations. Relationships between the members of one family are indicated – always referring to the patient=P

AKU family code	Status (P=patient)	Sex	Age (years)	HDG allele 1	HDG allele 2	Country	Notes
AKU_DB_113	P	F	39	G123A	G123A	Algeria
AKU_DB_113	P	M	28	G123A	G123A	Algeria
AKU_DB_117	P	M	62	G123A	G123A	Algeria
AKU_DB_118	P	M	78	G123A	G123A	Algeria
AKU_DB_108	P^	F	57	S287X	S287X	Algeria	Father and mother are cousins
AKU_DB_108	Husband of ^	M	56	L44F	wt	Algeria
AKU_DB_108	P (son of ^)	M	23	L44F	S287X	Algeria
AKU_DB_97	P	F	38	D153fs (G152fs)	A218fs	Algeria/France
AKU_DB_97	Mother	F	58	wt	A218fs	Algeria
AKU_DB_97	Son1	F	5	wt	?	Algeria/France
AKU_DB_97	Son2	F	7	wt	?	Algeria/France
AKU_DB_96	P	M	52	IVS7+2T>C	M368V	France	Father and mother are not consanguineous but come from the same region in north of France, he has affected sister
AKU_DB_111	P	M	61	G360A	G360A	France	Parents are not consanguineous but come from the same region in south–west of France
AKU_DB_109	P	F	54	G270R	M368V	France/Armenia	Father from Armenia, mother from France
AKU_DB_109	Mother	F	91	wt	M368V	France
AKU_DB_110	P	M	46	G161R	V157fs	France/Serbia
AKU_DB_110	Father	M	79	G161R	wt	Serbia
AKU_DB_110	Son1	M	20	G161R	wt	France/Serbia
AKU_DB_110	Son2	M	7	wt	V157fs	France/Serbia
AKU_DB_110	Son3	M	3	G161R	wt	France/Serbia
AKU_DB_110	Son4	M	16	G161R	wt	France/Serbia
AKU_DB_110	Daughter	F	12	G161R	wt	France/Serbia
AKU_DB_110	Daughter	F	9	G161R	wt	France/Serbia
AKU_DB_112	P	F	56	L116P	L116P	India	Parents are not consanguineous
AKU_DB_99	P	M		A122V	A122V	India/Canada
AKU_DB_107	P	F	52	W60X	W60X	Italy	Parents are not consanguineous, she has affected brother
AKU_DB_81	P	M	57	G161R	G161R	Poland
AKU_DB_100	P	M		Q33R	G152A	South Korea
AKU_DB_100	Mother	F		wt	G152A	South Korea
AKU_DB_100	Father	M		Q33R	wt	South Korea
AKU_DB_98	P	F	14	N219S	N219S	Turkey	Parents are not consanguineous, she has affected sister
AKU_DB_98	P	F	10	N219S	N219S	Turkey	Parents are not consanguineous, she has affected sister, also has FMF
AKU_DB_98	Father	M	45	wt	N219S	Turkey
AKU_DB_98	Mother	F	40	N219S	wt	Turkey
AKU_DB_98	Brother	M	3	N219S	wt	Turkey	Symptom free
AKU_DB_98	Sister	F	5	N219S	wt	Turkey	Symptom free
AKU_DB_101	P	F	10	G161R	D153fs (G152fs)	Slovakia
AKU_DB_101	Father	M	45	G161R	wt	Slovakia
AKU_DB_101	Mother	F	37	wt	D153fs (G152fs)	Slovakia
AKU_DB_101	Brother	M	13	G161R	wt	Slovakia
AKU_ZAT_1	P	M	24	G161R	G161R	Slovakia
AKU_DB_102	P	M	24	G161R	V300G	Slovakia
AKU_DB_102	P	M	9	G161R	V300G	Slovakia
AKU_DB_102	Brother	M	11	wt	wt	Slovakia
AKU_DB_102	Father	M	52	G161R	wt	Slovakia
AKU_DB_102	Mother	F	46	wt	V300G	Slovakia
AKU_DB_103	P	F	8	H371fs (P370fs)	G125fs	Slovakia
AKU_DB_103	Grandfather	M	58	H371fs (P370fs)	wt	Slovakia
AKU_DB_103	Mother	F	37	H371fs (P370fs)	wt	Slovakia
AKU_DB_103	Sister	F	15	H371fs (P370fs)	wt	Slovakia
AKU_DB_104	P	F	53	G161R	G161R	Slovakia
AKU_DB_104	P*	M	51	G161R	G161R	Slovakia
AKU_DB_104	Wife of*	F	46	wt	G161R	Slovakia
AKU_DB_104	P (son of*)#	M	10	G161R	G161R	Slovakia
AKU_DB_104	Cousin of#	M	36	wt	G161R	Slovakia
AKU_DB_104	Wife of cousin	F	36	wt	G161R	Slovakia
AKU_DB_104	P (son of cousin)	M	9	G161R	G161R	Slovakia
AKU_DB_95	P	F	6	P230S	M368V	Slovakia
AKU_DB_95	P	F	7	P230S	M368V	Slovakia
AKU_DB_105	P	F	31	G161R	G161R	Slovakia
AKU_DB_106	P	F	32	G161R	G161R	Slovakia	She has affected brother
AKU_DB_93	P	F	27	E178G	G161R	Slovakia
AKU_DB_94	P	F	52	G161R	M368V	Slovakia
AKU_DB_94	Daughter	F	32	wt	M368V	Slovakia
AKU_DB_94	Son	M	27	G161R	wt	Slovakia
AKU_DB_115	P	M	3m	G161R	G270R	Slovakia
AKU_DB_115	Father	M	32	wt	G270R	Slovakia
AKU_DB_115	Mother	F	27	G161R	wt	Slovakia
AKU_DB_127	P	M	11	G161R	G161R	Slovakia
AKU_DB_127	Father	M	39	G161R	wt	Slovakia
AKU_DB_127	Mother	F	40	wt	G161R	Slovakia
AKU_DB_127	Maternal grandfather	M	67	G161R	wt	Slovakia
AKU_DB_127	Maternal grandmother	F	66	wt	G161R	Slovakia
AKU_DB_127	Paternal grandfather	M	76	G161R	wt	Slovakia
AKU_DB_127	Paternal grandmother	F	70	wt	G161R	Slovakia
AKU_DB_127	Step brother (mother side)	M	19	G161R	wt	Slovakia
AKU_DB_128	P	F	52	G161R	G161R	Slovakia	He has affected siblings

Mutation Analysis

Diagnostic tests were employed for 10 known Slovak AKU mutations in the Slovak patients (Zatkova et al. 2003). Concurrently, in 18 foreign patients and in 3 Slovak cases where diagnostic screening revealed only one mutated allele, all individual HGD exons were sequenced using commercial sequencing kits and ABI PRISM^® 3100-Avant Genetic Analyzer (primer sequences are available upon request). Mutations are described according to the Human Genome Variation Society (HGVS) nomenclature additions (den Dunnen and Antonarakis 2000). The cDNA change position is based on coding DNA Reference Sequence NM_000187.3 with the first base of the Met-codon counted as position +1.

Haplotype Analysis

In order to construct HGD-haplotypes where possible, seven single nucleotide polymorphisms were analyzed by sequencing (IVS3-112C/T (c.176-112C/T), H80Q (c.240T/A), IVS4+31A/G (c.282+31A/G), IVS5+25T/C (c.342+25T/C), IVS6+46C/A (c.434+46C/A), IVS11+18A/G (c.879+18A/G)). Additionally, three dinucleotide repeats (HGO-3 /D3S4556, HGO-1 /D3S4496, HGO-2 /D3S4497) were ascertained using PCR with fluorescently labeled primers and subsequent fragment analysis on the ABI PRISM^® 3100-Avant Genetic Analyzer.

Variant Verification

For novel missense mutations, the conservation of the affected amino acid position between Homo sapiens and Mus musculus, Rattus norvegicus, Danio rerio, Drosophila melanogaster, Arabinopsis thaliana and Aspergillus nidulans was checked, using ClustalW2.

Segregation of mutations was followed in the families wherever possible.

PolyPhen-2 (Polymorphism Phenotyping v2, http://genetics.bwh.harvard.edu/pph2/) (Adzhubei et al. 2010) and SNAP (Screening for nonacceptable polymorphisms, http://cubic.bioc.columbia.edu/services/SNAP/) (Bromberg and Rost 2007) programs were used to predict the possible effect of amino acid substitutions on the structure and function of the human HGD protein (NP_000178.2). PolyPhen-2 is a tool which uses straightforward physical and comparative considerations, and for a mutation it calculates the Naïve Bayes posterior probability that this mutation is damaging and reports estimates of false positive (the chance that the mutation is classified as damaging when it is in fact nondamaging) and true positive (the chance that the mutation is classified as damaging when it is indeed damaging) rates. The mutation is also appraised qualitatively, as benign, possibly damaging, or probably damaging based on the model’s false positive rate. We used HumVar-trained PolyPhen-2, which enables distinguishment of mutations with drastic effects from all the remaining human variation, including the abundant mildly deleterious alleles.

SNAP is a neural network-based method. Its reliability index (RI) ranges between 0 and 9, with higher reliability indexes strongly correlating with a higher accuracy of prediction. The expected accuracy at a given reliability index is the number of correctly predicted neutral or nonneutral samples in the SNAP testing set. This measure of accuracy establishes the likelihood that a given prediction is correct.

The effect of the splicing mutation was predicted using Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html).

Database Construction

Data for the novel HGD-mutation database was summarized based on literature review. It includes all HGD variants and AKU patients reported so far, incorporating also the data from original AKU database with the agreement of Prof. Santiago Rodríguez de Córdoba. The new database is founded on the Leiden Open (Source) Variation Database (LOVD) system (Fokkema et al. 2005), and it is located in Bratislava. Submitted data is automatically forwarded to the curator and each variant receives a unique identifier as recommended (Claustres et al. 2002). Provided that there are no publication restrictions, all new variants will be entered in the database.

Results

Mutation Analysis

In all tested cases, two AKU-causing mutations were identified; from which 11 were novel (Table 1). The HGD mutations found in all AKU patients and their family members are summarized in Supplementary Table 1.

Table 1.

Eleven novel HGD mutations identified in our cohort as well as eight novel mutations found recently in the United Kingdom. The mutational hot spot is indicated, as well as segregation in the family, conservation of amino acids and PolyPhen-2 and SNAP predictions for missense mutations (HGD protein sequence NP_000178.2). DNA numbering system is based on cDNA (NM_000187.3), with +1 corresponding to the A of the ATG

Exon	Short name	Nucleotide change	Protein change	Hot-spot	Country of origin	AKU chromosome code	Segregation in family	Conserved amino acid	PolyPhen-2 predictions, score (HumVar)	SNAP predictions (reliability Index; expected accuracy)
03	Q33R	c.98A>G	p.(Gln33Arg)		South Korea	AKU_DB_100a	Yes (from father)	Yes (except Arabidopsis thaliana)	Possibly damaging with a score of 0.804 (sensitivity: 0.74; specificity: 0.82)	Neutral (1;60%)
03	L44F	c.130C>T	p.(Leu44Phe)		Algeria	AKU_DB_108c	Yes (from father)	Yes (except Arabidopsis thaliana)	Possibly damaging with a score of 0.675 (sensitivity: 0.79; specificity: 0.78)	Neutral (3;78%)
04	W60X	c.179G>A	p.(Trp60X)		Italy	AKU_DB_107a,b
06	G115R	c.343G>C	p.(Gly115Arg)	“CCC” triplet	United Kingdom	AKU_DB_124a		Yes	Probably damaging with a score of 0.99 (sensitivity: 0.07; specificity: 0.99)	Non-neutral (5;87%)
06	L116P	c.347T>C	p.(Leu116Pro)		France (Indian origin)	AKU_DB_112a,b		Yes	Probably damaging with a score of 0.995 (sensitivity: 0.33; specificity: 0.96)	Non-neutral (3;78%)
06	G123A	c.368G>C	p.(Gly123Ala)		Algeria	AKU_DB_113a,b	Two affected sibs	Yes	Probably damaging with a score of 0.965 (sensitivity: 0.58; specificity: 0.90)	neutral (0;53%)
07	G152A	c.455G>C	p.(Gly152Ala)	“CCC” triplet	South Korea	AKU_DB_100b	Yes (from mother)	Yes	Probably damaging with a score of 0.980 (sensitivity: 0.52; specificity: 0.92)	Neutral (4;85%)
08	V157fs	c.469_470dupA	p.(Val157AspfsX22)		France	AKU_DB_110b	Yes (from mother)
08	V157fs*	c.470-1_494del25	p.(Val157GlufsX11) (predicted skipping of entire exon)		United Kingdom	AKU_DB_123b
08	F169L	c.507T>G	p.(Phe169Leu)		United Kingdom	AKU_DB_122b		Yes (except Arabidopsis thaliana and Aspergillus nidulans)	Benign with a score of 0.103 (sensitivity: 0.92; specificity: 0.58)	Neutral (6;92%)
08	E178G	c.533A>G	p.(Glu178Gly)		Slovakia	AKU_DB_93b		Yes	Probably damaging with a score of 0.997 (sensitivity: 0.20; specificity: 0.98)	Non-neutral (2;70%)
09	R197G	c.589A>G	p.(Arg197Gly)		United Kingdom	AKU_DB_120a		Yes	Probably damaging with a score of 0.998 (sensitivity: 0.13; specificity: 0.99)	Non-neutral (3;78%)
10	N219S	c.656A>G	p.(Asn219Ser)		Turkey	AKU_DB_98a,b	Yes	Yes	Probably damaging with a score of 0.880 (sensitivity: 0.70; specificity: 0.84)	Non-neutral (2;70%)
11	K276N	c.828G>C	p.(Lys276Asn)		United Kingdom	AKU_DB_121a		Yes	Probably damaging with a score of 0.995 (sensitivity: 0.33; specificity: 0.96)	Non-neutral (1;63%)
11	S287X	c.860C>A	p.(Ser287X)		Algeria	AKU_DB_108a,b
13	G360A	c.1079G>C	p.(Gly360Ala)	“CCC” triplet	France	AKU_DB_111a,b		Yes (except Aspergillus nidulans)	Probably damaging with a score of 0.960 (sensitivity: 0.60; specificity: 0.90)	Neutral (2;69%)
13	G361R	c.1081G>A	p.(Gly361Arg)	“CCC” triplet	United Kingdom	AKU_DB_121b, AKU_DB_126b		Yes	Probably damaging with a score of 0.998 (sensitivity: 0.13; specificity: 0.99)	Non-neutral (5;87%)
13	D374H	c.1120G>C	p.(Asp374His)		United Kingdom	AKU_DB_125b		Yes	Probably damaging with a score of 0.987 (sensitivity: 0.46; specificity: 0.94)	Non-neutral (2;70%)
14	K431fs	c.1282_1292delGAG CCACTCAA	p.(Lys431HisfsX11)		United Kingdom	AKU_DB_124b

^, #, * these are the symbols that refer to the patient from the specific family. the patients are labeled for example P^ and all his relatives are described as mother of patient ^, etc..

The conservation of amino acid positions affected by novel missense mutations is summarized in Table 1, and it can be also viewed in Supplementary Fig. 1. PolyPhen2 predicted that all but one novel missense mutations have a “possibly” or a “probably damaging effect” (Table 1). SNAP predicted Q33R, L44F, G123A, G152A, and G360A to be neutral. F169L found in the patient in United Kingdom was predicted benign by both programs.

Fig. 1.

HGD mutation database home page divided into five sections with corresponding links that are described in the text

We also analyzed 136–140 control chromosomes from the Slovak population by small amplicon-based high-resolution melting (HRM) assays, for the presence of the novel mutations Q33R, L116P, G152A, E178G, and N219S (data not shown). None of the tested variants was identified among healthy individuals, which further indicates that these do not represent rare variants, but rather that they are pathogenic mutations.

Allelic associations (haplotypes) of all novel HGD mutations from this study and also all mutations currently recognized in Slovakia are summarized in Supplementary Table 2.

Supplementary Table 2.

Allelic associations of all HGD mutations found so far in Slovakia (A) and of novel mutations identified in the present study (B). All haplotypes are compared to those described so far. Differences in the haplotypes are indicated by color. The AKU allele code serves for identification of each allele in the HGD mutation database (http://hgddatabase.cvtisr.sk/). An empty box indicates that polymorphism has not been analyzed. Two different alleles in one box show that it was not possible to establish it unambiguously. The position of the mutation within the haplotype is marked by a thick black line. The short name, with the original name in brackets, is indicated for the mutations where the numbering has changed due to nomenclature recommendations. The DNA numbering system is based on cDNA (NM_000187.3), with +1 corresponding to the A of the ATG

Exon/intron	Mutation	Short name (original name)	Nucleotide change	IVS2+35	IVS2-218	IVS3-112	ex4 (c.240)	IVS4+31	HGO-3	HGO-1	IVS5+25	IVS6+46	IVS11+18	HGO-2	Origin	Allele code in AKU database	Reference
(A)
01i	Ivs1-1G>A	INV1-1G>A	c.16-1G>A	A	A	T	T	A	201	161	T	C	A	181	Algeria	AKU_DB_43a,b	Zatkova et al. (2000a)
				A	A	T	T	A	201	161	T	C	A	181	Poland	AKU_DB_22a	Beltrán-Valero de Bernabé et al. (1999)
				A	A	T	T	A	201	161	T	C	A	181	Slovakia	AKU_DB_53c	Zatkova et al. (2000a)
				A	A	T	T	A	201	161	T	C	A	181	Slovakia	AKU_DB_54a	Zatkova et al. (2000a)
				A	A	T/C	T	A	201/189	161	T	C	A	181/187	Slovakia	AKU_DB_71a	Zatkova et al. (2000a)
03	p.Ser47Leu	S47L	c.140C>T	A	T	T	A	A	197	161	T	C	A	197	Slovakia	AKU_DB_57a	Zatkova et al. (2000a)
03	p.Ser59AlafsX52	S59fs (R58fs)	c.175delA	A	A	T	T	A	193	161	T	C	A	181	India	AKU_DB_49a	Zatkova et al. (2000a)
				A	A	T	T	A	197	161	T	C	A	181	Finland	AKU_DB_40a,b	Beltrán-Valero de Bernabé et al. (1999)
				A	A	T	T	A	197	161	T	C	A	181	Slovakia	AKU_DB_66a	Zatkova et al. (2000a)
						T	T	A	197	161	T	C	A	181	Turkey	AKU_DB_89a,b	Uyguner et al. (2003)
						T	T	A	197	161	T	C	A	181	Turkey	AKU_DB_92a,b	Uyguner et al. (2003)
						T	T	A	197	161	T	C	A	181/189	Turkey	AKU_DB_90a	Uyguner et al. (2003)
						T/C	T	A	197/195	161/163	T/C	C/A	A	181/187	Turkey	AKU_DB_90b	Uyguner et al. (2003)
05i	IVS5+1G>A	INV5+1G>A	c.342+1G>A	A	T	T	A	A	193	161	T	C	A	187/191	Slovakia	AKU_DB_41a	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	G	183	Slovakia	AKU_DB_51a,b	Zatkova et al. (2000a)
07	p.Asp153GlyfsX26	A153fs (G152fs)	c.457dupG	T	A	C	T	A	191	161	T	C	A	187	France	AKU_DB_39a,b	Zatkova et al. (2000a)
						C	T	A	191	161	T	C/A	A	187	France	AKU_DB_97a	Present report
				T	A	C	T	A	191	161	T	C	A	187	Italy	AKU_DB_28a,b	Porfirio et al. (2000)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_50a	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_51c	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_55a,b	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_58a	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_60a,b	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_61a	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	187	Slovakia	AKU_DB_70a,b	Zatkova et al. (2000a)
				A	A	C/T	T	A	189/201	161	T	C	A	187/181	Slovakia	AKU_DB_71b	Zatkova et al. (2000a)
						C	T/A	A	193	161	T	C	A	187	Slovakia	AKU_DB_101a	Present report
						C/T	T	A/G	199	161	T	C	A	187	Slovakia	AKU_DB_103a	Present report
08	p.Gly161Arg	G161R	c.481G>A			T	A	A	193/191	161/163	T	C/A	A	191/187	Serbia	AKU_DB_110a	Present report
				A		T	A	A	193	161	T	C	A	191	Poland	AKU_DB_81a,b	Present report
				A	T	T	A/T	A	193	161	T	C	A	191/173	USA	AKU_DB_45b	Zatkova et al. (2000a)
						T	A/T	A	193	161	T	C	A	191	Slovakia	AKU_DB_101b	Present report
				A	T	T	A	A	193	161	T	C	A	191	Slovakia	AKU_DB_58b	Zatkova et al. (2000a)
						T	A	A	193	161	T	C	A	191	Slovakia	AKU_DB_104a,b,c,d	Present report
				A	T	T	A	A	193	161	T	C	A	191	Slovakia	AKU_DB_62a,b	Zatkova et al. (2000a)
						T	A	A	193	161	T	C	A	191	Slovakia	AKU_DB_106a,b	Present report
				A	T	T	A	A	193	161	T	C	A	191/187	Slovakia	AKU_DB_41b	Zatkova et al. (2000a)
				A	T	T	A	A	193	161	T	C	A	191	Slovakia	AKU_DB_63a,b	Zatkova et al. (2000a)
						T/C	A/T	A	193/189	161/163	T/C	C/A	A	191	Slovakia	AKU_DB_93a	Present report
						T	A	A	193	161	T	C	A	191	Slovakia	AKU_DB_94a	Present report
						T	A	A			T	C	A		Slovakia	AKU_DB_127a,b	Present report
						C	T	A			T	C	A		Slovakia	AKU_DB_115a	Present report
						C	T	A	197	161	T	C	A	191	Slovakia	AKU_DB_105a,b	present report
						C	T	A	197/189	161	T	C	A	191	Slovakia	AKU_DB_102a	Present report
				A	A	C	T	A	197	161	T	C	A	191	Slovakia	AKU_DB_53d	Zatkova et al. (2000a)
				A	A	C	T	A	197	161	T	C	A	191	Slovakia	AKU_DB_57b	Zatkova et al. (2000a)
				A	A	C	T	A	197	161	T	C	A	191	Slovakia	AKU_DB_59a	Zatkova et al. (2000a)
				A	A	C	T	A	197	161	T	C	A	191	Slovakia	AKU_DB_61b	Zatkova et al. (2000a)
				A	A	C	T	A	197	161	T	C	A	191	Slovakia	AKU_DB_67a	Zatkova et al. (2000a)
						C	T	A			T	C	A		Slovakia	AKU_DB_128a,b	Present report
08	p.Glu178Gly	E178G	c.533A>G			T/C	T/A	A	189/193	163/161	T/C	C/A	A	185/191	Slovakia	AKU_DB_93b	Present report
10	p.Pro230Ser	P230S	c.688C>T	A	A	C	T	A	189	163	C	A	A	185	Slovakia	AKU_DB_64a,b	Zatkova et al., (2000a)
						C	T	A	189/199	163/161	C	A/C	A	185/183	Slovakia	AKU_DB_95a	Present report
				A	A	C	T	A	189	163	C	A	A	185	Spain	AKU_DB_1a,b	Beltrán-Valero de Bernabé et al. (1998)
				A	A	T	A	A	191	163	C	A	A	185	Spain	AKU_DB_2a	Beltrán-Valero de Bernabé et al. (1998)
				T	A	C	T	A	191	163	C	A	A	185	Turkey	AKU_DB_3a,b	Beltrán-Valero de Bernabé et al. (1998)
11	p.Gly270Arg	G270R	c.808G>A	A		T	A	A	191	163	C	A	A	189	Rep Dom	AKU_DB_77a,b	Goicoechea de Jorge et al. (2002)
						T/C	T	A	195/193	161	T	C	A	183/189	France/Armenia	AKU_DB_109a	Present report
				A	A	T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_52a	Zatkova et al. (2000a)
				A	A	T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_53a	Zatkova et al. (2000a)
				A	A	T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_59b	Zatkova et al. (2000a)
				A	A	T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_61c	Zatkova et al. (2000a)
				A	A	T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_66b	Zatkova et al. (2000a)
				A	A	T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_67b	Zatkova et al. (2000a)
						T	T	A			T	C	A		Slovakia	AKU_DB_115b	Present report
				T	A	C	T	A	195	161	T	C	A	187	Italy	AKU_DB_29a,b	Porfirio et al., (2000)
						T	T	A	201	161	T	C	A	187	Turkey	AKU_DB_91a,b	Uyguner et al. (2003)
				A/T			T				T	C	A		United Kingdom	AKU_DB_125a	Present report
12	p.Val300Gly	V300G	c.899T>G	A	A	C	T	A	189	163	C	A	A	187	France	AKU_DB_5a	Beltrán-Valero de Bernabé et al. (1998)
				A	A	C	T	A	189	163	C	A	A	187	Germany	AKU_DB_6a,b	Beltrán-Valero de Bernabé et al. (1998)
				A		C	T	A	189	163	C	A	A	187	Portugal	AKU_DB_84a,b	AKU database
				A	A	C	T	A	189	163	C	A	A	187	Spain	AKU_DB_2b	Beltrán-Valero de Bernabé et al. (1998)
						C	T	A	189/197	163	C	A	A	187	Slovakia	AKU_DB_102b	present report
				T	A	C	T	A	191	161	T	A	A	187	Slovakia	AKU_DB_50b	Zatkova et al. (2000a)
13	p.Met368Val	M368V	c.1102A>G	A	A	T	T	A	195	161	T	C	A	183	Finland	AKU_DB_36a,b	Beltrán-Valero de Bernabé et al. (1999)
				A	A	T	T	A	195	161	T	C	A	183	Finland	AKU_DB_38a,b	Beltrán-Valero de Bernabé et al. (1999)
				A	A	T/C	T	A	195	161	T	C/A	A	183/181	France	AKU_DB_33c	AKU database
						T/C	T	A	195/189	161/163	T/C	C/A	A	183/189	France	AKU_DB_96b	present report
				A	A	T	T	A	195	161	T	C	A	183	Germany	AKU_DB_7a,b	Beltrán-Valero de Bernabé et al. (1998)
						T	T	A	195/193	161	T	C	A	183/189	France/Armenia	AKU_DB_109b	Present report
				A	A	T	T	A	195	161	T	C	A	181	Portugal	AKU_DB_47a,b	AKU database
				A		T	T	A	195	161	T	C	A	181	Portugal	AKU_DB_85a,b	AKU database
						T	T	A	195	161	T	C	A	181	Slovakia	AKU_DB_94b	Present report
				T	A	T	T	A	195	161	T	C	A	181	France	AKU_DB_5b	Beltrán-Valero de Bernabé et al. (1998)
				A	A/T	T	T/A	A	195	161	T	C	A	181	The Netherlands	AKU_DB_25b	Beltrán-Valero de Bernabé et al. (1999)
				A/T	A	T/C	T	A	195	161	T	C/A	A	181/179	USA	AKU_DB_23a	Beltrán-Valero de Bernabé et al. (1999)
				A	A	T	T	A	195	161	T	C	A	179	Spain	AKU_DB_24a,b	Beltrán-Valero de Bernabé et al. (1999)
				A	A	T	T	A	199	161	T	C	A	181	Spain	AKU_DB_18a	Beltrán-Valero de Bernabé et al. (1999)
				A		T	T	A	193	161	T	A	A	181/179	France	AKU_DB_82a	AKU database
						C	T	A	199/189	161/163	T	C/A	A	183/185	Slovakia	AKU_DB_95b	Present report
13	p.His371ProfsX4	H371fs (P370fs)	c.1111dupC			T/C	T	G/A	199	161	T	C	A	179	Slovakia	AKU_DB_103b	Present report
				A	A	T	T	G	199	161	T	C	A	179	Slovakia	AKU_DB_52b	Zatkova et al. (2000a)
				A	A	T	T	G	199	161	T	C	A	179	Slovakia	AKU_DB_54b	Zatkova et al. (2000a)
				A	A	T	T	G	199	161	T	C	A	179	Slovakia	AKU_DB_69a,b	Zatkova et al. (2000a)
				A	A	C	T	A	189	161	T	C	A	179	Slovakia	AKU_DB_53b	Zatkova et al. (2000a)
(B)
03	p.Glu33Arg	Q33R	c.98G>A			C	T	A	191	161	T	C	A	181	Korea	AKU_DB_100a	Present report
03	p.Leu44Phe	L44F	c.130C>T			C	T	A			T	A	A		Algeria	AKU_DB_108c	Present report
04	p.Trp60X	W60X	c.179G>A			T	A	A	191	161	T	C	A	189	Italy	AKU_DB_107a,b	Present report
06	p.Gly115Arg	G115R	c.343G>C	A			T/A	A			T	C	A/G		United Kingdom	AKU_DB_124a	Present report
															United Kingdom	AKU_DB_126a	Present report
06	p.Leu116Pro	L116P	c.347T>C			T	T	G	191	161	T	A	A	183	France (Indian origin)	AKU_DB_112a,b	Present report
06	p.Cys120Phe	C120F	c.359G>T	A			T	A			T/C	A	A		United Kingdom	AKU_DB_120a	Present report
06	p.Ala122Val	A122V	c.365C>T			T	T	A	197	161	T	C	A	191	Canada (Indian origin)	AKU_DB_99a,b	Present report
				A	A	T	T	A	193	161	T	C	A	185	India	AKU_DB_49b	AKU database
06	p.Gly123Ala	G123A	c.368G>C			T	A	A			T	C	A		Algeria	AKU_DB_113a,b	Present report
						T	A	A			T	C	A		Algeria	AKU_DB_117a,b	Present report
						T	A	A			T	C	A		Algeria	AKU_DB_118a,b	Present report
07	p.Gly152Ala	G152A	c.455G>C			C	T	A	191	161	T	A	A	179	Korea	AKU_DB_100b	Present report
07i	IVS7+2T>C	INV7+2T>C	c.469+2T>C			T/C	T	A	189/195	163/161	C/T	C/A	A	189/183	France	AKU_DB_96a	Present report
				A			T	A/G			C/T	C/A	A		United Kingdom	AKU_DB_122a	Present report
08	p.Val157GlufsX11	V157fs	c.470-1_494del25	A/T			T	A			T	C	A		United Kingdom	AKU_DB_123b	Present report
08	p.Val157AspfsX22	V157fs	c.470-1_470insA			C	T/A	A	191/193	163/161	C	A/C	A	187/191	France	AKU_DB_110b	Present report
08	p.Pro158Leu	P158L	c.473C>T			C	T	A			T	A	A		Macedonia	AKU_DB_116a	Present report
08	p.Phe169Leu	F169L	c.507T>G	A			T	A/G			T/C	C/A	A		United Kingdom	AKU_DB_122b	Present report
09	p.Arg197Gly	R197G	c.589A>G	A			T	A			T/C	A	A		United Kingdom	AKU_DB_120b	Present report
10	p.Ala218ProfsX11	A218fs (G217fs)	c.652delG	A		T	T	A	191	161	C	A	A	179	Algeria	AKU_DB_83a	AKU database
						C	T	A	191	161	T	C/A	A	181	Algeria	AKU_DB_97b	Present report
10	p.Asn219Ser	N219S	c.656A>G			C	T	A	191	161	T	A	A	187	Turkey	AKU_DB_98a,b	Present report
11	p.Pro274Leu	P274L	c.821C>T			T	T	A			C	A	A		Macedonia	AKU_DB_116b	Present report
11	p.Lys276Asn	K276N	c.828G>C	A			T	A			T	A	A		United Kingdom	AKU_DB_121a	Present report
11	p.Ser287X	S287X	c.860C>A			T	A	A	195	163	C	A	A	201	Algeria	AKU_DB_108a,b	Present report
13	p.Gly360Ala	G360A	c.1079G>C			C	T	A	193		T	C	A	197	France	AKU_DB_111a,b	Present report
13	p.Gly361Arg	G361R	c.1081G>A	A			T	A			T	A	A		United Kingdom	AKU_DB_121b	Present report
															United Kingdom	AKU_DB_126b	Present report
13	p.Asp374His	D374H	c.1120G>C	A/T			T				T	C	A		United Kingdom	AKU_DB_125b	Present report
14	p.Lys431HisfsX11	K431fs	c.1282_1292 delGAGCCACTCAA	A			T/A	A			T	C	A/G		United Kingdom	AKU_DB_121a	Present report

Described herein is the first AKU patient from South Korea who is a compound heterozygote for two novel missense mutations, Q33R (c.98A>G) and G152A (c.455G>C). Additionally, one novel haplotype associated with exon 10 mutation A218fs was described in an Algerian patient, and another associated with an A122V mutation was found in a patient from India (Supplementary Table 2). Accordingly, differences in haplotypes may indicate recurrent mutational events in these patients.

A total number of 12 different AKU mutations were established in Slovakia in 104 AKU chromosomes from 50 families currently appearing in the literature and in this report, and this further underscores the allele heterogeneity of AKU in this country (Table 2). Individual allele frequencies observed in Slovakia were also compared with those observed elsewhere.

Table 2.

Summary of 12 HGD mutations identified in 50 Slovak families with Slovak origin (104 AKU chromosomes). Mutational hotspots are indicated. Allele frequencies in Slovakia are compared to those found in an additional 422 AKU chromosomes with mutations identified reported worldwide so far. References for all reported patients carrying specific mutation can be found in Supplementary Table 2. In the column “Where reported” the country of the first reported patient carrying relevant mutation is listed first. Numbers in brackets indicate the numbers of AKU chromosomes reported. Shaded in gray are mutations that most likely have their origin in Slovakia. The DNA numbering system is based on cDNA (NM_000187.3), with +1 corresponding to the A of the ATG. All 526 AKU chromosomes are reported in HGD mutation database (http://hgddatabase.cvtisr.sk/)

HGD exon/intron	Short name (original description)	Nucleotide change (ATG=+1)	Protein change	Hot-spot	# of Slovak AKU chr.	% of 104 Slovak AKU chr.	# of AKU chr. in other countries	% of 422 AKU chr. from other countries	Where reported
01i	IVS1-1G>A	c.16-1G>A	p.(Tyr6_Gln29del)		5	4.8%	7	1.7%	Poland, Algeria(2), Slovakia(5), Czech(2), USA(2)
03	S47L	c.140C>T	p.(Ser47Leu)	CpG	1	1.0%	0	0.0%	Slovakia
03	S59fs (R58fs)	c.174_175delA	p.(Ser59AlafsX52)		2	1.9%	25	5.9%	Finland(2), India, Slovakia(2), Turkey(6), UAE(2), USA(11), La Reunion, UK(2)
05i	IVS5+1G>A	c.342+1G>A	p.(Leu95_Ser114del)	c.342+1	3	2.9%	2	0.5%	Slovakia (3), Czech. rep, USA
07	D153fs (G152fs)	c.454_457insG	p.(Asp153GlyfsX26)	“CCC” triplet	15	14.4%	8	1.9%	Slovakia (15), Italy(2), USA(3), France(3)
08	G161R	c.481G>A	p.(Gly161Arg)	“CCC” triplet	46	44.2%	23	5.5%	Slovakia(44), Czech(4), Germany, USA(11), Slovakia/Hungary(2), Poland(2), France/Serbia, UK(4)
08	E178G	c.533A>G	p.(Glu178Gly)		1	1.0%	0	0%	Slovakia
10	P230S	c.688C>T	p.(Pro230Ser)	“CCC” triplet	5	4.8%	9	2.1%	Spain(3), Turkey(2), Slovakia(5), USA(2), Canary Islands(2)
11	G270R	c.808G>A	p.(Gly270Arg)	“CCC” triplet, CpG	8	7.7%	10	2.5%	Italy(2), Slovakia(8), DomRep(2), Turkey(2), France/Armenia, USA(2), UK
12	V300G	c.899T>G	p.(Val300Gly)		4	3.8%	14	3.5%	France, Spain, Germany(2), Slovakia(4), Portugal(2), USA(4), La Reunion(3), UK
13	M368V	c.1102A>G	p.(Met368Val)		2	1.9%	57	14.2%	Germany(9), France(4), France/Armenia, USA(28), The Netherlands, Finland(4), Portugal(4), Spain(3), Slovakia(2), Switzerland/Belgium, UK(2)
13	H371fs (P370fs)	c.1111insC	p.(His371ProfsX4)	“CCC” triplet	12	11.5%	2	0.5%	Slovakia (12), USA(2)

The HGD Mutation Database: Structure and Content

General Information

The database homepage (Fig. 1) contains the main section in which gene and database “General information” are summarized; e.g., the chromosomal and database location, the curator name and gene reference sequence that can be also downloaded. Using links in the NOTE, all users can access schematic drawings showing the location of the pathogenic variants and polymorphisms within the gene (“HGD variants schematic”), tables summarizing about 240 so far established and published HGD-haplotypes (“HGD haplotypes associated with AKU mutations”), as well as “HGD haplotypes in the normal Spanish and Slovak population”, “Expression and functional characterization of AKU alleles in E. Coli”, “Mutations Aspergillus nidulans” and “Mutations Mus musculus”. Part of this data has already been previously published and/or it was located in the original AKU database (Rodríguez et al. 2000; Zatkova et al. 2000a). In case of so far unpublished mutations, in the figure “HGD variants schematic” only the type of mutation is indicated. HGD haplotypes associated with AKU mutations were constructed based on the analysis of seven single nucleotide polymorphisms and three dinucleotide repeats as reported before (Beltrán-Valero de Bernabé et al. 1999; Zatkova et al. 2000a). Comparison of the haplotypes associated with the same mutations in the AKU patients from different countries enables studying the possible origin of each mutation as well as uncovering possible novel mutational events. General information section contains also links for registration and submission of the variants.

Graphic Displays and Utilities

“Graphic displays and utilities” include links to summary tables, UCSC, and Ensembl genome browsers and NCBI sequence viewer.

Sequence Variant Tables

The HGD mutation database currently in March 2011 contains 148 unique variants; of which 115 were reported to be pathogenic (107 are public at the present) occurring independently in 267 AKU families. It includes also 33 variants which were reported as nondisease-related polymorphisms either in the original AKU database or recently (Vilboux et al. 2009). The pathogenic mutations are distributed as follows: 77 missense, 7 nonsense (stop), 14 small deletions and insertions causing frameshift, 14 affecting splicing, 2 larger deletions, and 1 extension of the protein. For more statistics, see the review article Zatkova 2011 JIMD.

The “Sequence variant tables” section includes variant data set out in tables accessible via hyperlinks. In the “Unique sequence variant table,” the unique mutations are sorted by exon number without patient data being shown (Fig. 2); in the “Complete sequence variant table,” mutations associated with each patient are described in detail.

Fig. 2.

Unique variants view from HGD mutation database. All main columns described in the text are shown

Mutations are named according to the Human Genome Variation Society (HGVS) nomenclature additions (den Dunnen and Antonarakis 2000) and identified by a unique database ID. The cDNA change position is based on coding DNA Reference Sequence NCBI: NM_000187.3 with the first base of the Met-codon counted as position +1. All available data on each mutation is provided. In addition to standard database categories, an Allele code is included, which enables identification of all alleles from the same patients (i.e., patient with the Patient_ID AKU_AQR_11 has alleles AKU_AQR_11a and AKU_AQR_11b). The Database Identifier: AKU_00000 is used for all AKU patients alleles where no mutation was identified so far (unknown). Patients for whom HGD haplotypes associated with AKU mutations are reported can be recognized by their ID which begins with “AKU_DB_”.

In the database was included a column indicating an involvement of the mutation hot-spots (“CCC”triplets, c.342+1G, CpG) that have been identified within HGD gene (Beltrán-Valero de Bernabé et al. 1999; Zatkova et al. 2000a). A creation or abolition of recognition site of some common restriction enzymes is included for easy identification of each mutation (Fig. 2).

In the “Variants with no known pathogenicity table,” all reported polymorphisms are shown. There are also the two variants ivs9-56G>A and ivs9-17G>A listed here. Although these were published as AKU causing mutations (Beltrán-Valero de Bernabé et al. 1998), Vilboux et al. (2009) considered that they most likely represent benign variants based on the negative predictions of their effect on splicing. Predictions of the potential effect of most of the reported missense and splicing mutations have recently been discussed and can be found in the supplementary material of (Vilboux et al. 2009) The full sequence variant table of the HGD database can be downloaded in tab-delimited text format.

Search the Database

The home page also provides a “Search the database” section for browsing data using simple search by type of variant and exon number, or search based on the patient’s origin. Through a more advanced search tool, the user can also mine data using sequence variation description, protein description or reference.

Links to the Other Resources

Furthermore, “Links to the other resources” include links to the three gene-related resources of MIM (http://www.ncbi.nlm.nih.gov/omim), HGMD (http://www.hgmd.cf.ac.uk) and Entrez (http://www.ncbi.nlm.nih.gov/Entrez). Connections to the websites of the AKU Society (http://www.alkaptonuria.info), French ALCAP (http://www.alcap.fr/), Italian AIMAKU (http://www.aimaku.it/) and findAKUre project (http://www.findakure.org) are also available. AKU Societies are support networks for AKU patients that provide them with the best information about the latest news, research and treatments of AKU, while the FindAKUre project is a joint collaborative research project of the AKU Society and the University of Liverpool.

Discussion

In this research, we identified 11 novel AKU causing mutations and we report on our new HGD gene mutation database. Recently, 8 novel mutations were identified in 21 AKU patients from the United Kingdom, which will be published soon (listed in Table 1), bringing the total number of known HGD mutations to 115 worldwide.

Since no functional studies were available, we used PolyPhen2 and SNAP analysis in order to validate the effect of novel missense variants. While PolyPhen2 predicted that all but one novel missense mutations have a “possibly” or a “probably damaging effect”, SNAP predicted Q33R, L44F, G123A, G152A, and G360A to be neutral. F169L found in one AKU patient was predicted benign by both programs. However, this and other novel mutations identified in United Kingdom will be reported separately.

In general, the performances of the prediction tools are estimated between 50% and 80% accurate (Bromberg and Rost 2007; Ng and Henikoff 2006). It is known that the HGD protein functions as a hexamer composed of two trimers (Titus et al. 2000). Although both PolyPhen-2 and SNAP programs use 3D protein structures, they have their limitation in considering the complexity of all inter-subunit interactions between the HGD monomers within the complex hexamer that can be easily affected by single residue change (Rodríguez et al. 2000). It is possible that amino acid substitutions, which would be benign if HGD functioned as a monomer, show deleterious effects due to disturbance to the higher organization of the functional hexamer. We presume that the same holds true for novel mutations predicted as neutral by SNAP. Evidence that all exons of the HGD gene with neighboring intronic sequences have been sequenced in the patients carrying the above-mentioned mutations, and no other pathogenic changes have been identified, also favors the pathogenic effect of these variants. Moreover, amino acids affected by substitutions are highly conserved among species, and mutations segregate in the families. Functional studies, which unfortunately are not currently available, would be required to confirm the functional consequences of these mutations.

Recently, also (Vilboux et al. 2009) assessed the potential effect of all missense variations on protein function; thus, their study and this report, together with the novel HGD mutation database, provide a valuable resource of complete information on the molecular analysis of AKU mutations, their origin and their possible effect on HGD function.

Slovak Aku Genetic Specificities

In Slovakia, a total number of 12 different AKU mutations have been established. This further underscores the allele heterogeneity of AKU in this country.

As already mentioned, the most frequently found were missense mutations, followed by splicing and/or frameshift mutations (Zatkova (2011) JIMD). Although distribution in Slovakia is similar, Slovak patients had more than twice the proportion of frameshift mutations. But this might just reflect the small mutation number and founder effect in this country.

In the previous study by (Zatkova et al. 2000a, b), and also herein, an allelic association was performed for 11 HGD intragenic polymorphisms in a total of 69 AKU chromosomes from 32 Slovak pedigrees. This was then compared to the HGD haplotypes of all AKU chromosomes carrying identical mutations characterized thus far in non-Slovak patients to study the possible origin of these mutations. Based on the analysis and comparison of haplotypes, two groups of HGD mutations were observed in Slovakia.

In the first group are the mutations such as P230S, V300G, S59fs (R58fs), M368V, and IVS1-1G>A which were shared by different populations. These mutations represent only 18/104, which accounts for 17.3% of the Slovak AKU chromosomes and thus provides a marginal contribution to the AKU gene pool in Slovakia. The most frequent European mutation M368V is present in one copy in only two unrelated Slovak families. Mutations of this group have most likely been introduced into Slovakia by the founder populations that spread throughout Europe (Zatkova et al. 2000a).

The second group consists of the remaining seven mutations established in 82.7% of Slovak patients. These include the most prevalent G161R, H371fs (P370fs), D153fs (G152fs), and G270R (Table 2), the splicing mutations IVS5+1G>A, and also the S47L and E178G mutations observed in only one patient and specific for Slovakia.

The Exon 8 mutation, G161R, is the most frequent, and it is found in 46 of 104 Slovak AKU chromosomes (44.2%). Four haplotypes, two of which are prevalent, have so far been shown to be associated with this mutation in Slovakia, exhibiting the differences in the 5′part (HGO-3 and distal polymorphisms), which can be explained by novel mutations events or by recombination (Supplementary Table 2). Patients with G161R reported in the USA, Poland, and France/Serbia share the same haplotype described in Slovakia. Mutation G270R in exon 11 was also found in Italy, Turkey, The Dominican Republic, and France/Armenia. The G270R-associated haplotypes in all these countries, except for France/Armenia, differ from Slovak ones in both the 5′and 3′ parts, indicating either recurrent mutation events in these countries or a high recombination rate. A similar situation is observed in the D153fs (G152fs) mutation, which is also seen in cases in Italy, France, and France/Algeria. The difference in haplotypes in these cases is, however, restricted to the 5′end and it can be explained by recombination (Supplementary Table 2).

The IVS5+1G>A mutation is present on two different haplotypes in Slovakia, indicating recurrent mutation (Zatkova et al. 2000a). This mutation was also found in one case in both the USA and The Czech Republic, but since no haplotypes have been described they cannot be compared.

In one out of five patients, the HGD haplotype associated with H371fs (P370fs) mutation differs from the remaining ones in the distal 5′end, but this can be explained by recombination (Supplementary Table 2). Although this mutation was also identified in two patients in the USA, it otherwise appears to be specific for Slovakia.

It is likely that mutations from this second group originated in Slovakia and spread into other countries with different migrations.

The distribution of the identified mutations within Slovak territory is also interesting. As previously reported, examination of the geographical origin of Slovak AKU mutations shows remarkable clustering in a small area in North-West Slovakia, with these mutations most likely originating in this area and spreading into other regions after the breakdown of genetic isolates in the 1950s (Zatkova et al. 2000a).

Sequence analysis shows that six of the seven prevalent Slovak AKU mutations are associated with hypermutated sequences in the HGD (“CCC” triplet, c.342+1, CpG; Table (1). In addition, as the haplotype analysis shows, one of the P230S, M368V and V300G alleles in Slovak patients may also represent a novel HGD mutational event (haplotypes show differences, Supplementary Table 2). Thus, 7 of the 12 (58.3%) AKU mutations which most likely originated in Slovakia are associated with hyper-mutated sequences in the HGD while worldwide it is 40/115 (34.8%) (HGD mutation database). Therefore, it is possible that an increased mutation rate in the HGD gene in a small geographical region is responsible for the high genetic heterogeneity in Slovak AKU (Zatkova et al. 2000b). However, it remains unclear which mechanism acted specifically on the HGD gene to increase its mutation rate, since similar targets are also present in other genes without evident elevated gene frequency in Slovakia (Srsen et al. 2002; Zatkova et al. 2000a).

It has been discussed that the Valachian colonization during the fourteenth to seventeenth centuries may also have played a role in the increased prevalence of AKU in Slovakia (Srsen et al. 2002; Zatkova et al. 2000a). Valachs were nomadic tribes who did not represent an ethnically defined group since they always mixed with the local populations. They came to Slovakia from The Balkans on the Carpathian Mountain curve through Romania and West Ukraine. No AKU cases from these countries have been reported so far, except for one recent young patient from Macedonia who, however, carries different mutations (P158L, P274L) (Gucev et al. 2011).

The increased number of mutations could also be the result of random accumulation of mutations in the region. The preservation of the most prevalent AKU variants in Slovakia may then be the result of a founder effect and genetic drift, due to the geographic isolation of villages in North-West Slovakia.

Perspectives

Since also some external factors, such as the use of minocycline for treatment of dermatologic or rheumatologic disorders may mimic AKU phenotype (Vilboux et al. 2009), identification of two HGD mutations represents the final confirmation of AKU diagnosis. Distribution on mutations and studying their haplotype background can contribute also to the understanding the genetics of the studied population. The presented HGD mutation database provides a valuable tool for information exchange in AKU research and care fields. It certainly presents a useful data source for genotype–phenotype correlations and also for future clinical trials.

Details of the Contributions of Individual Authors

AZ performed majority of work, including the analysis of patients, haplotype constructions, database construction and writing the manuscript. TS, MN, and HP contributed to the mutation analysis, JR contributed to the CA-repeat analysis for haplotypes, RA, ID provided patients DNA. JLU performed mutation analysis in patients from United Kingdom. All authors approved the content of the final version of the manuscript.

Funding

This research was funded by IMPG SAS and FNS UK Bratislava, Slovakia and the project “Infrastructure for research and development- data center for research and development” with the financial support of European fund for regional development (project code: 26210120001, 26230120001).

Ethics Approval

No special ethic approval was needed. All patients signed informed consent for DNA analysis prior to a peripheral blood sample was taken from them.