PH1 is considered as a rare genetic disorder characterised by allelic and clinical heterogeneity. We reported 57 patients belonging to 40 families, with one or more affected members (Additional file 3, Figure S1). It was reported that PH1 is particularly frequent in Tunisia . The prevalence of PH1 was estimated by Chemli et al. to be 5.5/106 population . It remains underreported with an estimated prevalence ranging from 1 to 3/106 population in Europe and North America, respectively . An increased frequency of PH1 has been also reported in Middle East countries [19–21] as a result of the high rate of consanguinity in these populations. In our cohort, consanguinity was reported in 75% of families, mainly originated from the centres and the south of the country (Table 1), where the consanguineous marriage is still a frequent custom.
Similar to most Tunisian reports [8, 17, 22], we showed a high frequency of PH1 among children referred to our centre of Pediatric Nephrology, that recruits all cases from central and southern regions of the country. However, we believe that the incidence of PH1 may be higher in adults than we reported, because our results represent adult cases referred only to the two centres of adult nephrology in the region of Sahel. The adolescent form of the disease was described in only 17.4% (8/46) of patients. By comparison, European studies reported approximately 10% of affected individuals presented with severe disease before the age of six months and 80%-90% of affected individuals present in late childhood or early adolescence .
We noted that all the index cases were in CRF and ESRD at diagnosis with a wide variability in clinical presentation. The most severe form of the disease was observed in patients before the age of eight months (17.4% (8/46)). In this group, symptoms of PH1 included nephrocalcinosis (17.4% (8/46)) with or without nephrolithiasis (37% (17/46) and 45.6% (21/46) respectively), failure to thrive (37%), and urinary tract infection (50%). Early death was common 6/46 (13%). The youngest infantile patients were presented with non specific symptoms such as failure to thrive. Older patients had symptoms that are often related to the urinary tract and systemic oxalosis in 39%.
In the absence of the liver biopsy, that provides a definitive diagnosis of hyperoxaluria , molecular genetics has the potential to offer a rapid and non invasive method to establish the diagnosis of PH1. There are now more than 145 polymorphisms and mutations identified in the AGXT gene . Whole gene sequence analysis is feasible, but the cost is not insignificant in our country. For this reason, and as first line test, we decided to search I244T, (known to be frequent in Tunisia) and G170R, F152I and 33_34insC mutations that recur frequently and form the basis of DNA screening panels in European countries . We decide to start by studying those mutations because there is not available information about the others mutations frequent in Tunisia.
We have detected two mutations causing disease, I244T and 33_34insC, in 13 of the 46 index cases (28.2%). After families investigations the number of affected patients increased to 24 patients (42% (24/57)), belong to 13 of the 40 families (32.5%). I244T and 33_34insC mutations have alleles frequencies of 68% and 32% respectively, in patients with detected mutations. These frequencies were more elevated than those reported by Rumsby et al, in the mutational screening of the three common mutations (33_34insC, G170R and I244T) which avoid liver biopsy in 34.5% of patients .
Homozygous I244T and 33_34insC mutations were the most frequent among identified mutations in our patients. This phenomenon could be explained by the high inbreeding rate in the regions to which these families belong. Actually, consanguinity can play an important role in the inheritance of the disease and can deepen the molecular diversity and the heterogeneity of PH. But taking into account the consanguinity, we should expected much more patients carrying these mutations alones or in compound heterozygotes for both. This may be explained by the possibility of existing of other frequent mutations (not identified) in our study.
I244T, also called 'mutation Maghrebin' , occurs on the minor allele of AGXT . It leads to AGT misfolding, which produces functionally inactive aggregates. This mutation seems to be the most detected in our population. Its frequency was considerably higher than frequencies reported in previous studies among Tunisians and other populations such as Spanish and North African background [12, 17, 22, 24–26].
I244T appears to constitute the only PH1 mutation associated with a founder effect. A North African origin could be speculated, various patients, natives of the Canary Islands where I224T mutation was frequent (92%), are thought to be originated from Northwest Africa.
In addition, we detected a main incidence 41.1%, among childhood and adolescent patients, and only 17.6% were presented in adulthood. However, in Canary Island, all carriers of I244T mutation were diagnosed in adulthood, with severe renal stone disease and ESRF .
The age of onset and the symptoms of the disease seem to be variable and can be influenced by other factors. We have detected two uncommon cases with pseudo-dominant inheritance in two consanguineous families F17 and F40. Within the same mutation, the clinical progression was quite different between patients and their fathers, although they have the same genotype (I244T/I244T). In fact, at presentation, the two children were in ESRD, at 4 years and 8 months respectively. But their fathers maintained normal renal function at the age of 40 till 36. Clinical analysis detected urolithiasis in the father of F17. This great molecular heterogeneity of PH1 can be explained by differences in activity level of other enzymes important in oxalate synthesis, modifier genes, the quantity of oxalate precursors in the diet, renal oxalate handling, absorption of dietary oxalate, hydration status, infections, and urinary crystallization factors .
The 33_34insC mutation, was the second mutation detected in our patients with alleles frequency of 32%, more elevated than other reports (12 to 13%) [12, 14, 29]. It was first described in Italian patients . This microinsertion occurs on major or minor allele of AGXT and it was considered as the most common PH1 mutation on the major allele (31%) . It has been reported that homozygous, would be expected to have no immunoreactive protein and no catalytic activity . In our study, seven of the 8 detected patients were homozygous for the mutation and associated with ESRD. 87.5% of detected patients carrying 33_34insC mutation were female, with a median age of 3 years, and only one case in adulthood in the sixth decade of life. Clinically, children carrying this mutation suffered a very severe form of PH1, 62.5% (5/8) of them died of their disease. Nevertheless a mild form of disease with B6 treatment responsiveness was exceptionally observed in the adulthood . Curiously, our adult patient preserved her renal function until the age of 60 without B6 treatment. She progressed rapidly to ESRF, because she was diagnosed late after the onset of renal insufficiency and had no medical follow-up. This age and clinical variation may be explained by interactions of 33_34insC with other genes and/or environmental factors.
G170R, F152I were absent in our cohort, in spite of their high frequencies in Caucasians populations, respectively 40 and 7% of disease alleles [16, 32, 33].
In Summary, I244T and 33_34insC mutations were associated with a molecular and phenotypic variation in our cohort. They were identified in 42% of patients in which 28.2% were index case and 11 patients were discovered by family screening. Five of the 11 patients were presymptomatic and prenatal diagnosis was performed in two families. So a preliminary detection of these limited mutations in the AGXT gene can serve as useful tool in the families screening of patients with demonstrated PH1. If a mutation is known, direct detection of the mutation can be used for diagnosis of presymptomatic patients as well as for prenatal diagnosis . 71.7% (33/46) of our index case patients were negative for the common tested mutations and in whom a diagnosis of PH could not be excluded, other frequent mutations screening followed by whole gene sequencing will be done forthcoming for these patients if no known frequent mutation is found.
Concerning treatment, our patients systematically received vitamin B6, but analysis of pyridoxine responsiveness was not possible in patients with ESRF. It was reported that in pyridoxine sensitive patients an improvement in renal function and a decrease in plasma and urine oxalate with high-dose vitamin B6 therapy were noted . Pyridoxine responsiveness seems to be genotypes dependent. It has clearly been demonstrated in the G170R mutation, and even been observed in a patient with F152I and 33_34insC .
Early treatment of presymptomatic patients is possible, and may prevent further loss of renal function. However, patients with early renal failure, classic conservative measures are often insufficient and patients require renal replacement therapy.
Unfortunately, compared to European children, we noted a high rate of mortality in our patients (41.6%), 66% of them were in childhood. This can be explained by the absence of rapid kidney-liver transplantation, which has excellent outcome according to European and the US Registries [36, 37]. In fact, it showed greater graft-survival than in isolated kidney transplantation .
The limitation of our study is the number of tested mutations. We admit that our data can not exactly evaluate the PH mutations frequency in Tunisia, and the existence of other frequent mutations is possible. But with our limited means, we can not deny the usefulness of mutation-based diagnosis testing that allowed us to identify mutations in considered number of patients. In addition we have identified presymptomatic analyzed patients, thereby providing a targeted prenatal diagnosis.
For more general conclusions, our results need to identify the PH mutations in the rest of analysed patients.