Molecular Analysis of the HGD Gene in 9 Families With Alkaptonuric Ochronosis in Iran and Identification of Two Novel Variants

ABSTRACT

Objectives

Alkaptonuria (AKU) (MIM number 203500) or homogentisic acid oxidase deficiency is a metabolic autosomal recessive disorder caused by mutations in the homogentisate 1, 2‐dioxygenase (HGD) (MIM number 607474) gene. The HGD is located on chromosome 3q13 with 14 exons, and encodes the homogentisate 1, 2‐dioxygenase enzyme, which is composed of 445 amino acids. Although AKU was first described more than 120 years ago, we lack studies investigating its mutational spectrum in Iran.

Materials and Results

This study aimed to investigate the spectrum of homogenetic gene mutations in patients diagnosed with Ochronosis Alkaptonuria in Ardabil, Iran. To achieve this, all individuals affected by this disease, totaling 23 individuals, whose affliction with Ochronosis Alkaptonuria had previously been definitively confirmed, were enrolled in the study. Sanger sequencing identified five unique variants in the HGD gene (NM_000187.4), all of which were homozygous variants. Two of the variants (c.113delA and c.342+5G>A) were novel and have not been reported in variant databases, including gnomAD, ClinVar, and HGMD. Other identified variants were c.175delA, c.334T>G, and c.680T>C, which have been previously reported in variant databases.

Conclusion

In this study, the mutational spectrum of alkaptonuric ochronosis was investigated in Iran. The HGD gene is a well‐studied gene, and hundreds of variants responsible for alkaptonuria have been reported to date. However, we found two novel variants that have not been reported in previous studies.

There has been a wide range of mutations identified in the HGD gene in alkaptonuric patients. Some HGD mutations are spread worldwide, while others are specific to certain countries. Alkaptonuria has not been extensively studied in Iran; it is likely to be high since consanguineous marriages are common.

1. Introduction

Alkaptonuria (AKU) (MIM number 203500) or homogentisic acid oxidase deficiency is a metabolic autosomal recessive disorder caused by mutations in the homogentisate 1, 2‐dioxygenase (HGD) (MIM number 607474) gene (Vilboux et al. 2009). Alkaptonuria has three main clinical features, which usually represent in different stages of the disease course through life: darkened urine on standing, ochronosis, and ochronotic osteoarthropathy, respectively (Aquaron 2013). These manifestations are attributed to the accumulation of homogentisic acid (HGA) due to the deficiency in the homogentisate 1, 2‐dioxygenase activity (Roopnarinesingh et al. 2021). Circulating HGA deposits dark brown pigments in connective tissues (a process known as ochronosis), primarily in skin, sclera, spine, and large joint cartilage, as well as in the valves of the heart. In AKU patients, severe arthropathy usually begins in their early 30s (Zatkova et al. 2022).

While it affects around one in 250,000 people worldwide, it is significantly more common in Slovakia and Dominican Republic populations (Vilboux et al. 2009; Cervenansky et al. 1959). There is no clear indication of disease's prevalence in Iran, and most Iranian studies are only case reports in nature; as an example, in Mazandaran, this disease was reported in one case in 2001 (Torabi zadeh et al. 2001), as well as three cases in another study in 2012 (Yousefghahari et al. 2013), one case in Urmia in 2009 (Abbasi et al. 2009), one case in Tehran in 2012 (Karimi et al. 2020). Azami et al. reported seven patients with alkaptonuric ochronosis in Ardabil, Iran, in 2014 (Azami et al. 2014).

The HGD is located on chromosome 3q13 with 14 exons and encodes the homogentisate 1, 2‐dioxygenase enzyme, which is composed of 445 amino acids. Enzymatic loss in AKU was first confirmed in 1996 in Spanish patients with missense mutations in HGD gene (Fernández‐Cañón et al. 1996). One study showed that nearly two‐thirds of mutations in the HGD gene are missense mutations, followed by frameshift and splicing mutations, which account for around 25% of mutations together, and Nonsense mutations account for only 6% of all mutations (Vilboux et al. 2009). Some HGD mutations are spread worldwide. However, there are mutations that are specific to certain countries and regions, such as Slovakia and the Czech Republic (Vilboux et al. 2009). Although AKU was first described more than 120 years ago, we lack studies investigating its mutational spectrum in Iran. Here, we report molecular analysis of 9 families, including 23 patients of Iranian ethnicity, and report 2 novel mutations for the first time.

2. Material and Methods

2.1. Editorial Policies and Ethical Considerations

The present study received approval from the Research Ethics Committee of Ardabil University of Medical Sciences (Ethics Code: 1400.280.IR.ARUMS.REC). The study was performed in accordance with the Declaration of Helsinki. Participation in this study was voluntary, and individuals entered the study with written informed consent. The collected data were anonymous.

2.2. Sampling and Data Collection

This study aimed to investigate the spectrum of homogenetic gene mutations in patients diagnosed with Ochronosis Alkaptonuria in Ardabil, Iran. To achieve this, all individuals affected by this disease, totaling 23 individuals, whose affliction with Ochronosis Alkaptonuria had previously been definitively confirmed, were enrolled in the study by examining the increased levels of homogenetic acid in urine and the color change to dark brown or bluish‐brown in connective tissues.

2.3. DNA Extraction and Quality Control

Genomic DNA samples for this study were extracted from peripheral blood leukocytes collected from the probands and their family members. The salting‐out method was employed for DNA extraction. Following extraction, the quality and purity of the DNA samples were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to determine the absorbance ratios, and 1% agarose gel electrophoresis was performed to evaluate DNA integrity (Kisa et al. 2021; Beltrán‐Valero de Bernabé et al. 1998).

2.4. The Sequencing of Homogenetic Acid Oxidase (HGD) Gene

Mutation in the homogenetic acid oxidase (HGD) gene coding region was amplified and screened using PCR and direct sequencing. PCR reactions were carried out in a 25 μL volume, which included 35 ng of DNA sample, 1X buffer, 200 μM dNTPs, 10 picomoles of each specific primer, and 1 unit of Taq polymerase enzyme.

2.5. Electrophoresis With Agarose Gel

The agarose gel, prepared with a concentration of 1%–2% in TBE buffer, is utilized for band size estimation. SYBR markers, mixed with 2 μL of PCR dye, are employed for this purpose. These markers include products of 50 and 100 base pairs (bp), along with VIL and V green. Each of these marker mixtures is placed individually in wells on the gel. The gels are subsequently subjected to a voltage of 110 V for 30 to 45 min. Band visualization and gel imaging are accomplished with the assistance of a gel documentation system.

2.6. Bioinformatics

Franklin website was used to assess the pathogenicity of identified variants in the HGD gene based on the American College of Medical Genetics and Genomics (ACMG) guidelines (https://franklin.genoox.com/clinical‐db/home). We also used the CADD tool (Combined Annotation Dependent Depletion) to further investigate the pathogenicity of these variants (https://cadd.gs.washington.edu/score). SplicerAI was applied to splice‐site mutations to predict the probability of splice donor gain or loss (https://spliceailookup.broadinstitute.org).

3. Results

In this study, 23 individuals from 9 families with alkaptonuric ochronosis were investigated. About 56% (n = 13) of the patients were male, and 44% (n = 10) were female, with an age range of 28 to 68 years. However, the majority of patients were in the age groups of 51–60 and 61–70. Consanguinity was present in all of the patients. All patients had positive urine darkening upon exposure to air. The mean age of disease onset was about 42, and the most frequent initiating clinical symptom was lumbar pain (n = 11, 48%), followed by nail pigmentation (n = 8, 35%) and joint pain (n = 4, 17%). At the time of last physical examination, lumbar pain was present in 91.5% (n = 21) of the patients. Ear (n = 18, 78%), hand (n = 17, 74%), eye (n = 16, 70%), and nail (n = 8, 35%) pigmentations, and also joint pain (n = 17, 74%) were the other clinical symptoms observed in these patients. Bone densitometry reported osteopenia/osteoporosis in 43% (n = 10) of these patients. No genotype–phenotype correlation was observed.

The HGD gene mutations were investigated in the affected families. The pedigrees of these families are illustrated in Figure 1. Sanger sequencing identified five unique variants in the HGD gene (NM_000187.4) (Figure 2A), all of which were homozygous variants. Two of the variants (c.113delA and c.342+5G>A) were novel and have not been reported in variant databases, including gnomAD, ClinVar, HGMD, and the HGD mutation database (Zatkova et al. 2012). Other identified variants were c.175delA, c.334T>G, and c.680T>C, which have been previously reported in variant databases. The Sanger sequencing results of the identified variants are illustrated in Figure 2B.

FIGURE 1.

Pedigrees of families I to IX. Families V and VI have similar pedigrees.

FIGURE 2.

(A) Schematic illustration of the HGD gene and location of variants identified in this study. (B) Sanger sequencing results and identification of five unique variants.

The c.113del, p.Asn38IlefsTer73 variant is a frameshift deletion mutation. Based on ACMG guidelines, this variant was categorized as pathogenic. This variant was identified in a single proband in their family and further evaluation was not possible because her parents had passed away years ago. The c.342+5G>A variant was identified in 3 probands from 3 families. This mutation is a splice‐site mutation located at the fifth nucleotide of intron 5. According to ACMG guidelines, this variant was classified as a VUS (Variant of Uncertain Significance). For further evaluation of its pathogenicity, the CADD (Combined Annotation Dependent Depletion) tool was used, and it categorized this variant as pathogenic (score: 22.9). SplicerAI predicted that this variant can disrupt splicing.

The pathogenicity of c.175delA p.(Ser59AlafsTer52)?, c.334T>G p.(Phe112Val), and c.680T>C p.(Phe227Ser) variants was previously examined by other studies. However, ACMG and CADD were used to examine their pathogenicity. Detailed information regarding five unique variants identified in this study is presented in Table 1.

TABLE 1.

Summary of the five unique variants identified in 9 families and their pathogenicity.

Family	Affected patients	Consanguinity	Variant/Protein	Zygosity	Exon	Novelty	Pathogenicity
			NM_000187.4/NP_000178.2				ACMG	CADD
I	1	+	c.113del p.(Asn38IlefsTer73)	Homozygous	Exon 3	Novel	Pathogenic	NA
II, III, IV	3, 4, 1	+, +, +	c.342+5G>A p.?	Homozygous	Intron 5	Novel	VUS	Pathogenic (Score: 22.9)
V, VI	1, 1	+, +	c.175del p.(Ser59AlafsTer52)?	Homozygous	Exon 3	Reported	Pathogenic	NA
VII	7	+	c.334T>G p.(Phe112Val)	Homozygous	Exon 5	Reported	Likely pathogenic	Pathogenic (Score: 27.2)
VIII, IX	2, 3	+, +	c.680T>C p.(Phe227Ser)	Homozygous	Exon 10	Reported	Pathogenic	Pathogenic (Score: 29)

4. Discussion

There has been a wide range of mutations identified in the HGD gene in patients with alkaptonuria, and so far, 252 variants have been reported in 726 patients from different countries (Grasso et al. 2022). The first link between HGD gene mutations and AKU disease was demonstrated by Fernandez‐Canon et al. Their findings included two missense mutations, one in exon 10 (C855T) and the other in exon 12 (T1066G) (Zatkova 2023). Following that, many researchers from different countries attempted to identify genetic mutations in these patients. Although alkaptonuria has not been extensively studied in Iran, it is likely to be high since consanguineous marriages are very common, especially in provinces such as Ardabil. There have been case reports since 2001 reporting the different treatment protocols and clinical manifestations of these patients in Iran. However, molecular analysis was not utilized in previous studies. In the current study, five unique variants were identified in the HGD gene in 9 families with alkaptonuric ochronosis in Iran, and to the best of our knowledge, this study reports the largest cohort of patients with alkaptonuric ochronosis in Iran.

In the current study, the c.175del variant was identified in two probands from two families. This frameshift deletion variant is the most common variant reported in the HGD gene, which has been reported 131 times across a wide range of nations, including Belgium, Canada, Finland, France, India, Italy, Russia, Slovakia, Turkey, the United Arab Emirates, the United Kingdom, the United States, and Wales (Zatkova 2023). It is interesting to note that an Iranian AKU patient who underwent genetic testing in Italy previously had the mutation identified as homozygous (Ascher et al. 2019). The missense variant c.334T>G was identified as homozygous in one of our AKU patients in a family with seven affected individuals. Only one patient from Turkey has previously been reported with this variant as a compound heterozygous c.334T>G/c.808G>A (Kisa et al. 2021). The c.680T>C variant in exon 10 was first reported in 1998 in Spain by Beltran et al. They also identified two additional variants in exon 10 and stated that the short peptide encoded by this exon probably has a substantial role in the enzyme activity (Beltrán‐Valero de Bernabé et al. 1998).

The c.342+5G>A variant substitutes the 5th nucleotide in intron 5 and resides in the 5′ splice site donor sequence. Pre‐mRNA splicing is a vital process for proper gene expression. 5′ splice sites are extremely diverse; however, it has been shown that these sequences are strongly conserved, and alterations in these conserved sequences can cause defective splicing and aberrant expression of the gene. The 5′ splice sites are recognized by a 9‐nucleotide motif: 3 nucleotides from the upstream exon (−3 to −1) followed by the 6 nucleotides of the intron (+1 to +6). This motif is vital for the initial recognition of the splice site by U1 small nuclear ribonucleoprotein (snRNP). The GUAAGU sequence is a canonical 5′ splice site. After the GU sequence, which is the most conserved 5′ splice site position in humans (99%), the −1G and +5G positions are the second highly conserved positions and are responsible for the strong base‐pairing with U1 (Anna and Monika 2018; Roca et al. 2013; Sheth et al. 2006; Wong et al. 2018). The c.342+5G>A is located in the +5 position, and the substitution of nucleotide G with A can disrupt the normal splicing by altering the base‐pairing with U1. SplicerAI predicted that this variant could result in both splice donor loss (Δ score = 0.83, 5 bp) and splice donor gain (Δ score = 0.34, −124 bp). The probability of natural splice donor loss was 0.83 (range: 0 to 1), and the probability of the creation of a new splice site donor was 0.34. Based on ACMG guidelines, this variant was categorized as a variant of uncertain significance (PM2, PP3, and PP4). The CADD (Combined Annotation Dependent Depletion) tool was used and categorized this variant as pathogenic (score: 22.9).

The c.113del, p.Asn38IlefsTer73 variant is a frameshift deletion mutation that removes nucleotide A from the AAT codon in exon 3, leading to the formation of 72 novel amino acids and finally terminating the translation with a TGA stop codon. This creates a non‐functional short protein with a different structure. According to ACMG guidelines, this variant was categorized as pathogenic (PVS1, PM2, and PP4).

5. Conclusion

In this study the mutational spectrum of alkaptonuric ochronosis was investigated in Iran. The HGD gene is a well‐studied gene and hundreds of variants responsible for alkaptonuria have been reported to date. However, we found two novel variants that have not been reported in previous studies.

Author Contributions

B.D. and A.A. conceptualized and supervised the study. A.A., M.J., Y.I.M., and D.M. wrote the original draft, revised the manuscript and prepared tables and figures. B.D., H.M., M.J., and D.M. contributed to the laboratory work. A.A. was responsible for patients' clinical management. All authors have read and approved the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Azami, A. , Jahanpanah M., Marani Y. I., Mokhtari D., Mokaber H., and Davarnia B.. 2025. “Molecular Analysis of the HGD Gene in 9 Families With Alkaptonuric Ochronosis in Iran and Identification of Two Novel Variants.” Molecular Genetics & Genomic Medicine 13, no. 11: e70156. 10.1002/mgg3.70156.

Data Availability Statement

The dataset generated during the current study is available in the NCBI repository (ClinVar): Link: https://www.ncbi.nlm.nih.gov/clinvar/?term=C0002066[trait+identifier]+AND+%22Department%20of%20Medical%20Genetics%20and%20Pathology%2C%20Ardabil%20University%20of%20Medical%20Sciences%22[submitter].