Unusual clinical outcome of primary Hyperoxaluria type 1 in Tunisian patients carrying 33_34InsC mutation

Primary hyperoxaluria type 1 (PH1) is the common and severe form of hyperoxaluria. This rare autosomal recessive inborn disease is a result of glyoxylate metabolism defect, caused by an absence, deficiency or mislocalization of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGXT). The kidney is the first organ affected by the massive rise in urinary oxalate through the genesis of recurrent stones and / or progressive nephrocalcinosis to finish in an early end-stage renal disease (ESRD) [1]. The increased plasma oxalate levels along the disease progress, and calcium oxalate deposition in various tissues leads to the systemic oxalosis, engendering serious complications as well as a fatal outcomes [2]. A combined liver-kidney transplantation is the unique cure of this defect. Isolated kidney transplantation leads ultimately to the recurrence of the disease [3].

PH1 has heterogeneous appearances and can affect patients whatever their age. The presentation varies from infantile nephrocalcinosis and failure to thrive, as a result of renal impairment, to a recurrent or only occasional urolithiasis [4] usually one half of patients experience ESRD at the time of diagnosis and 80% develop ESRD by the age of 3 years. Recurrent urolithiasis and progressive renal failure are mainly described in childhood or adolescence onset of PH1. However, the diagnosis of patients affected during adulthood is often altered, over 50% of them reach ESRD at the time of diagnosis, and are characterized by occasional stone passage [5, 6].

More than 178 pathological mutations in AGXT gene have been documented [7]. The 33-34InsC mutation is classified as severe by the fact that this insertion leads to a truncated protein without catalytic activity [8]. Clinically, 33-34InsC is known to be responsible for severe presentation and early death in childhood [9], although, it has also been described more rarely in adult patients.

In this report, we describe a controversial effect of the 33_34InsC mutation in Tunisian patients. We reported a pediatric cases with severe clinical forms and adults patients with a mild and unusual clinical course of PH1 toward ESRD.

In this study, a molecular genetic diagnosis was performed in 111 index cases with suspected PH1. We reported 16 cases (16/118, 13.5%) identified with 33-34InsC mutation, nine of them (four children and five adult) were index cases and the seven others (four children and three adult) were diagnosed during familial investigations. The median age of patients was 24 years (range 0.5–73 years) with a sex-ratio of 0.6 (ten female and six male) and they were originated from central and south of Tunisia. Only one patient was in heterozygote state (I244T/ 33-34InsC), with Mi/Ma alleles. All the rest were homozygotes for 33-34InsC mutation and have the major alleles (Ma/Ma).

Clinically, PH1 has a variable presentation in our cohort and patients presented several symptoms at the time of diagnosis (Table 1). The detection of PH1 was dictated by unspecific symptoms of nephrolithiasis and renal insufficiency (RI). In fact, some signs corresponded mainly to manifestations of CRF, but other signs were not specific such as anemia, diarrhea, vomiting and general alteration.

PH1, is a very rare inherited disease with a high prevalence in Mediterranean countries and Arabic nations due to genetic make-up and a higher rate of consanguineous marriages [11]. Tunisia is one of the countries with the highest frequencies of PH1 worldwide [12, 13]. Indeed, PH1 is responsible for more than 13% and 10% of ESRD in children in Tunisia and Kuwait versus 0.3% and 0.7% in, Europe and North America, respectively [12, 14].

Molecular genetic testing is the efficient and non-invasive approach, approved to confirm the definitive diagnosis of PH1. We identified the 33_34 InsC mutation in 13.5% (16/118) of our patients and in our knowledge, it is the highest frequency of patients carrying this mutation and having the same ethnicity reported until now. In particular interest, the 33_34InsC mutation was mainly described in pediatric cases (12%) and was very rarely reported in late onset particularly in the homozygote’s states [1, 15‐17]. Compared to the others reports, we noted that the 33_34InsC mutation frequency in the Tunisian cohort is on par with that reported by Coulter-Mackie’s et al. (13.5%) [9], however the age of patients was mainly different. In fact the majority of patients in Coulter-Mackie’s cohort, were pediatric cases and only one adult women (54 years) was reported. In this report, we described 33_34InsC mutation in 17% (8/47) of pediatric cases and interestingly in 11.26% (8/71) of our adults. Otherwise, our results were slightly higher than those reported by Rumsby’s et al. (12%) [15], we have some similitudes for pediatric cases, but a difference in the number and the age of onset of the disease was mainly noted for adults. In fact, we reported more patients over 30 years- old.

This micro-insertion was considered as the most common PH1 mutation on the major allele (31%) of AGXT [9]. It has been reported that the severity of this micro-insertion, especially in homozygous state, is due to the production of no immune-reactive mature protein with no catalytic activity [8]. 33_34InsC mutation, was described firstly in the Italian population (of Roman origin) [18], then in other populations [19]. Given to her strategic geographical position and the historical invasive flows that took place in North Africa, Tunisia is considered as a crossroads of civilizations [20]. We hypothesize that 33_34InsC variation, is associated with a specific population or ethnic group and might have been introduced in Tunisia, as many other variations [21], during the Roman settlement up to the fifth century AC. Interestingly, 75% of our patients are originated from the cities (Kasserine, Mahdia, and Sfax), known for a long Roman colonization.

In our observation, the clinical manifestations were entirely different between children and adults patients and we noted the absence of the adolescent form. Interestingly, the pediatric patients suffered from a very severe clinical form caracterised by a renal symptoms ranged from urolithiasis, nephrocalcinosis or even a combined form, associated to rapid evolution to ESRD. Systemic oxalosis as ocular and digestive damaging were also present in two patients. Unfortunately, 75% of children carrying this mutation died with a median age of 2.5 years. Contrary to this instance, we noted the absence of the expected severity attributed to 33_34InsC mutation in adults’. Indeed, all of our adult patients have a slow development of the clinical symptoms of the PH1 characterised by urolithiasis. Owing to the lack of a strict medical follow-up and the delay of the PH1 diagnosis, five adults reached ESRD at the median age of 54 ± 8.7 years-old. Curiously, 37.5% (3/8) of our adult patients preserved a normal renal function until the age of 40 without B6 treatment. Only two of our patients, cases 13 and 15, have initial symptoms as recurrent lithiasis from the age of 9 and 7 years respectively. The patient 15 had preserved a normal renal function at last follow-up; however case 13 developed CRF at 43 years-old.

Strangely enough, a marked intra-familial clinical heterogenicity was noted, although the patients harbor the same AGXT mutations. Some are present with early and severe clinical manifestations, whereas others may be asymptomatic for long time. In fact, the case 11, was asymptomatic for many years and even with a near-normal urinary oxalate excretion, has first symptoms’ disease in the age of 48. However, her niece (case 12) died at the age of 12 years, after a history of urolithiasis and ESRD. For note, we have detected an uncommon case with pseudo-dominant inheritance in a consanguineous family (F6). With the same mutation and the same genotype (33-34insC/33-34insC), the clinical progression was different between patient and his father. In fact, at presentation, the case 9 started the CRF at the age of 50; however, his father preserved until the age of 70 a normal renal function. We have reported previously a cases of pseudo-dominant inheritance in families with I244T mutation [22], we suggested that consanguinity, strongly present in our population study (≈89%), has a serious effect in this great molecular heterogeneity of PH1.

Interestingly, this variation in the age of diagnosis and the reality of a moderately severe outcome for adult patients with no functional AGXT, supports the idea that other enzymes may be involved in oxalate synthesis, or that the metabolism of glyoxylate can be modulated by interactions with modifier genes and/or with environmental factors. These hypotheses have been occasionally mentioned [15], but we suppose that families described in this report could hold the key to further discoveries in PH.

Moreover, to maintain the renal function and avoid complications [23], several hypotheses were proposed to explain the effect of the diet as conservative measure to limit the quantity of oxalate precursors in the patient’s diet [24]. It was reported that oxalate-rich food restriction intakes is recommended for PH patients [25] because of their lower intestinal oxalate absorption, compared to normal subjects [24]. More than, it was appeared that the incidence of calcium lithiasis can be reduced by fiber intake that leads to a change in bowel transit and diminishes the absorption of both calcium and oxalate. For that reason, fiber intake is recommended for patients with recurrent lithiasis [23, 26]. Nevertheless, there is not enough scientific evidence to corroborate the benefits of this measure.

Looking for the diet of our adult cases having the alteration of the CRF stage under the age of 40, raises the question of the influence of the diet in this instance. Interestingly, the majority of adults are originated from West-central of Tunisia, the diet costumers in those regions are based on a rich fiber intake in their food as a whole wheat bread, spices, apples, barley; in addition to nutriment rich in vit B12 as fatty fish, lamb, offal, cereal, …

The limitation of our study was the shortage of information about the metabolic profile in adults. Unfortunately, the majority of them were addressed by different Tunisian nephrology departments for genetic analysis of PH1, but the biological measurement were unaffordable, at recruitment, because the majority of them reached ESRD. That restriction of data, make impossible the effective comparison of the metabolic profile between adults and children, in order to get more information on the causes of the variable clinical course.

The 33_34InsC mutation causing primary hyperoxaluria type 1 is a severe mutation that seems to provide a high morbidity and ruthless infantile PH1. We report that 33_34insC can be present in adults’ forms with a mild clinical features, suggesting the implication of other factors in this slowly progression.