Homogentisate 1,2-dioxygenase (HGD) gene variants in young Egyptian patients with alkaptonuria

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by pathogenic variants in the homogentisate 1,2-dioxygenase (HGD) gene. This leads to a deficient HGD enzyme with the consequent accumulation of homogentisic acid (HGA) in different tissues causing complications in various organs, particularly in joints, heart valves and kidneys. The genetic basis of AKU in Egypt is completely unknown. We evaluated the clinical and genetic spectrum of six pediatric and adolescents AKU patients from four unrelated Egyptian families. All probands had a high level of HGA in urine by qualitative GC/MS before genetic confirmation by Sanger sequencing. Recruited AKU patients were four females and two males (median age 13 years). We identified four different pathogenic missense variants within HGD gene. Detected variants included a novel variant c.1079G > T;p.(Gly360Val) and three recurrent variants; c.1078G > C;p.(Gly360Arg), c.808G > A;p.(Gly270Arg) and c.473C > T;p.(Pro158Leu). All identified variants were properly segregating in the four families consistent with autosomal recessive inheritance. In this study, we reported the phenotypic and genotypic spectrum of alkaptonuria for the first time in Egypt. We further enriched the HGD-variant database with another novel pathogenic variant. The recent availability of nitisinone may promote the need for genetic confirmation at younger ages to start therapy earlier and prevent serious complications.

Genomic DNA extraction and HGD sanger sequencing.EDTA blood was collected from all suspected AKU patients, their parents and all available healthy siblings.Genomic DNA was extracted using QIAamp® DNA Blood Mini Kit (QIAGEN, Germany).The quantity and quality of the extracted DNA were evaluated by the Nan-oDrop2000® (Thermo Scientific, USA) and DNA was stored at − 20 °C until further processing.
The HGD gene of each index case was sequenced using specific primers designed with the help of Integrated DNA Technologies, OligoAnalyzer™ Tool (https:// www.idtdna.com/ calc/ analy zer).The sequences of all used primers are listed in Supplementary Table 1.This was followed by family segregation analysis for the detected variant in each proband in the corresponding exon for both parents and all available siblings.PCR reactions were conducted using Dream-Taq Green PCR Master-Mix (Thermo Scientific, USA).In short, an initial denaturation for 10 min at 95 °C was followed by 35 cycles of (30 s denaturation at 95 °C, 45 s annealing at 56-64.6 °C and extension for 1 min at 72 °C).Final extension at 72 °C was for 10 min.The specific annealing temperatures for different primer pairs are provided in Supplementary Table 1.PCR products were run by electrophoresis using 2% agarose gel to confirm band specificity.DNA was then purified from the amplification reaction with the GeneJET™ Genomic DNA Purification Kit (Applied Biosystems, USA) according to manufacturer's protocol.Purified DNA was stored at − 20 °C until sequenced using the Brillant Dye™ terminator V3.1 Cycle Sequencing kit (NimaGen, the Netherlands) through the ABI 3500 gene analyzer.

Results
Clinical features.Six AKU patients from four unrelated Egyptian families were recruited in the current study (4 females/2 males, 1-20 years).Three families were reported consanguineous (parents were first cousins), while in family II, consanguinity was denied; however, both parents originated from the same small village and a distant common ancestor cannot be excluded.Demographic and clinical features of affected individuals are summarized in Table 1, while family pedigrees are provided in Fig. 1.
The proband of family I (Patient I-1) is currently a 20-year-old male and his sister (I-2) is 17 years old.The family was from Beni-Suef governorate in Central Egypt (Supplementary Fig. 1).Both affected siblings had mild intellectual disability and complained of intractable pains in weight bearing joints starting in mid-adolescence (12-14 years).The older male sibling had congenital anomaly in the kidney, while his sister had severe sensorineural hearing loss, high myopia, strabismus, delayed bone age, short stature and mitral regurgitation (Table 1).The latter could be attributed to rheumatic heart disease as she was receiving long acting penicillin and had persistently increased ESR and ASOT titers.
The other two siblings, although having a normal organic acid profile in urine and only being carriers for the pathogenic variant detected in homozygosity in their AKU siblings, they were also suffering.The third female sibling had bilateral severe to profound sensorineural hearing loss and the fourth male sibling had mild hepatosplenomegaly and right iliac bone lesion that was initially suspected as an alkaptonuric joint lesion before alkaptonuria being excluded by urinary organic acid and genetic analyses.
Family II had two siblings, a female (proband) and an unaffected male.The proband (II-3) was diagnosed with AKU through HGA detection in urine when she was 6-month-old.Her only manifestations so far are darkening of urine upon standing and hyper pigmentation and brown discoloration of buttocks (Table 1).Her sibling was symptom free and was revealed as heterozygous by genetic analysis.
Family III consisted of four siblings, three females including two AKU patients and one male sibling.The proband (III-4) was diagnosed with AKU at 6 months of age.She recently developed hyperpigmentation of buttocks and a long-standing bluish-brownish patch on the arm.Consequently, due to the experience gained by the mother, her sister (III-5) was spotted at one month of age with the same blackish discoloration of urine (Table 1).The other two siblings were completely healthy.
The proband (IV-6) of family IV was the youngest male and the only affected member among three older siblings.He had normal motor and mental development.Parents noticed pink to light red urine and black ear cerumen immediately after birth.At the age of 2 months, he was misdiagnosed as having urinary bleeding, although all his renal functions were normal.At the age of 6 years he started having joint pains and recurrent abdominal pain (Table 1).Alkaptonuria was then suspected and was confirmed by GC/MS.All six AKU patients were confirmed to have massive amounts of homogentisic acid in urine by qualitative organic acid analysis by GC/MS.

Genetic diagnosis.
Sanger sequencing of the HGD gene revealed the existence of four different missense pathogenic/likely pathogenic variants in exons 8, 11 and 13 in the four recruited families including a novel pathogenic variant in exon 13 detected in family III (Table 2).www.nature.com/scientificreports/All detected variants fully segregated in all tested family members (homozygous in the AKU patients, heterozygous in parents and heterozygous or wild type in available unaffected siblings) consistent with the autosomal recessive inheritance nature of the disease (Fig. 1).
The first family had a missense pathogenic variant in HGD exon 13; NM_000187.4:c.1078G> C, NP_000178.2:p.(Gly360Arg).The variant is recurrent and has been reported previously in the HGD variant database and predicted to be pathogenic according to the American College of Medical Genetics (ACMG) criteria for the classification of pathogenic variants 11 (Table 2).The variant has been previously reported as disease causing in several studies 12,15 .
The fourth family (IV) had a missense likely pathogenic variant in exon 8 of HGD gene NM_000187.4:c.473C> T; NP_000178.2:p.(Pro158Leu).This variant was detected in homozygous state in the proband (IV-6) and heterozygous in both parents.It is predicted to be likely pathogenic according to ACMG criteria 11 ; however, it was reported as variant of uncertain significance in ClinVar (Table 2).The variant has been also previously reported as disease causing 14,17 .

Discussion
In the current study, we evaluated the clinical and genetic characteristics of four unrelated Egyptian families suffering from alkaptonuria.All patients presented with isolated dark urine at a median age of 6 months and were confirmed to have a high level of homogentisic acid in urine by qualitative GC/MS evaluation of organic acids in urine.
AKU is a rare metabolic disorder that is almost forgotten in Egypt and in the Arab region, as only a few cases were reported from Jordan 18 , Algeria 19 , UAE 20 , Iraq 21 , Lebanon 22 and Morocco 23 .Furthermore, the prevalence and genetic makeup of the disease are completely unknown in Egypt, although expected to be more prevalent than developed countries due to higher rates of consanguineous marriages that propagate the autosomal recessive inheritance of the disease.We conducted this study to identify the underlying HGD pathogenic variants within Egypt causing AKU and to raise awareness among Egyptian health care professionals about the disease.Furthermore, we wanted to establish the routine genetic confirmation of AKU in Egypt before the authorization of the specific therapeutic agent, nitisinone in Egypt.
In our study, we identified four different variants causing AKU, including three recurrent variants reported in several populations as disease causing: c.1078G > C; p.(Gly360Arg) detected in family I, has been reported in patients from Italy, Australia, UK, France, Spain, India and the USA.The variant c.808G > A; p.(Gly270Arg) detected in family II, has been previously reported in patients from Italy, Slovakia, France, Armenia, Brazil, USA, Dominican republic, Turkey, the UK, Russia, Germany, Peru and India, while c.473C > T; p.(Pro158Leu) detected in family IV in our study, has been previously reported in patients from USA, Canada, Macedonia, and Taiwan 15 .
The last detected variant in our study is a novel missense pathogenic variant c.1079G > T; p.(Gly360Val) that was encountered in family III.This variant is not reported in ClinVar; however, it lies within a mutational hotspot Table 2. HGD pathogenic variants detected in Egyptian AKU families.*Variants nomenclature is according to HGD: NM_000187.4and NP_000178.2Refseq gene transcript and protein, respectively; MAF, minor allele frequency according to gnomAD population database (https:// gnomad.broad insti tute.org/); H, homozygous.All variants segregated properly in both parents and available siblings of the diseased children according to strict autosomal recessive inheritance.in exon 13 of the HGD gene, which contains over ten missense pathogenic variants including the detected variant at the same amino acid location in family I, c.1078G > C;p.(Gly360Arg), indicating that this domain of the HGD protein is very sensitive to amino-acid alterations, and non-synonymous missense variants most likely will result in a deleterious effect on the protein function.All four variants were detected in homozygous state in affected patients and segregated properly in their families.Although many of our patients had arthralgic pain in weight bearing joints, especially adolescents, they did not have evident radiographic abnormalities commonly detected in adult AKU patients, such as spine osteoporosis, calcification of the nucleus pulposus, osteophytosis or reactive sclerosis of the articular surfaces 24 .They also didn't have any noticeable teeth discoloration characteristic of alkaptonuria 25 , and repeated clinical examination didn't pick up any cardiac or renal complications, which is mainly related to the relatively young age of most of our patients.
The recent availability of the new medication nitisinone (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedioneor, NTBC), which inhibits the enzyme 4-OH phenylpyruvate dioxygenase, thus blocking the formation of HGA, enhances the need for genetic confirmation and carrier frequency detection in families before starting this rather expensive therapy.
Low dose nitisinone in the range of 0.2-2 mg daily reduces urinary HGA excretion by > 90%, which may prevent AKU-related complications in the long run 26 .This drug has been used for more than 20 years for the treatment of children with tyrosinemia type I 27 .An earlier start of nitisinone together with a diet restricting phenylalanine and tyrosine intake may lead to an improved long-term functional outcome.At the same time, nitisinone treatment should be cautiously monitored to avoid the development of mental and cognitive disturbances [28][29][30] .The low dose used should also reduce the possibility of increased plasma tyrosine levels, which may cause nitisinone keratopathy.In a recent study conducted by Ranganath et al., to compare the effects of nitisinone 2 mg and 10 mg in the treatment of alkaptonuria, they reported that nitisinone 10 mg was more efficient in slowing the disease progression; however, it simultaneously increased tyrosine levels and the incidence of corneal keratopathy (14.5% vs 4.9%) 31 .Furthermore, ntisinone is still a very expensive drug, so one of the major advantages of the low-dose therapy will be its economic benefit, especially in a resource-limited country, such as Egypt.
The early development of remarkable and progressive joint complaints in the majority of pediatric and adolescent Egyptian AKU patients in our study indicates that diet restriction of phenylalanine and tyrosine and supportive therapeutic strategies are not efficient in controlling HGA deposition in tissues and that nitisinone, which is not yet covered by insurance in Egypt, is needed.
Identifying the underlying genetic variants of families with AKU in Egypt should have an impact on health community policy towards the disease.Since heterozygous variants have a major role as one of the liability genetic determinants in subsequent generations, raising awareness regarding premarital genetic counseling at least in affected families may break the line of inheritance in such disorders.Furthermore, biochemical analysis of HGA in urine and genetic confirmation should be routine screening steps for all family members of affected individuals to detect presymptomatic cases, especially that a specific therapy that can minimize long term complications is currently available.
Concerning family I, we further recommend whole exome or genome sequencing, as their complex genetic trait requires a more extensive genetic sequencing technique to fully elucidate the etiology behind their apparently syndromic intellectual disability and sensorineural hearing loss.
In conclusion, in this study we explored the phenotypic and genotypic spectrum for alkaptonuria for the first time in Egypt.Although we revealed the genetic makeup of only a limited number of Egyptian patients, this study may form a base to be built upon for establishing clinical genetic screening and family counseling of AKU in Egypt and surrounding populations.We further enriched the mutational spectrum of the HGD gene with a novel pathogenic variant detected in two AKU siblings.

Figure 1 .
Figure 1.Family pedigrees and genetic variants of affected AKU patients.All detected variants were fully segregated in all tested family members (homozygous in the AKU patients, heterozygous in parents and heterozygous or wild type in unaffected siblings) consistent with the autosomal recessive inheritance nature of the disease.H, Homozygous; h, heterozygous.In family pedigrees homozygous individuals are marked in black, while confirmed heterozygous carriers are marked with a black dot inside.

Table 1 .
Demographic and clinical features of AKU patients (n = 6).