A Novel Mutation in the AGXT Gene Causing Primary Hyperoxaluria Type I: Genotype–Phenotype Correlation

c Indian Academy of Sciences

RESEARCH ARTICLE

A novel mutation in the AGXT gene causing primary hyperoxaluria type I: genotype–phenotype correlation

SAOUSSEN M’DIMEGH1∗, CÉCILE AQUAVIVA-BOURDAIN2, ASMA OMEZZINE1, IBTIHEL M’BAREK1, GENEVIÉVE SOUCHE2, DORSAF ZELLAMA4, KAMEL ABIDI3, ABDELATTIF ACHOUR4, TAHAR GARGAH3, SAOUSSEN ABROUG3 and ALI BOUSLAMA1

1Biochemistry Department, LR12SP11, Sahloul University Hospital, 4054 Sousse, Tunisia 2Laboratory of Inborn Metabolic Diseases, Centre de Biologie Est, Hospices Civils de Lyon, Lyon, 69677 Bron Cedex, France 3Pediatric Department, Charles Nicolle University Hospital, 1002-2 Tunis, Tunisia 4Nephrology Department, Sahloul University Hospital, 4054 Sousse, Tunisia

Abstract Primary hyperoxaluria type I (PH1) is an autosomal recessive metabolic disorder caused by inherited mutations in the AGXT gene encoding liver peroxisomal alanine : glyoxylate aminotransferase (AGT) which is deficient or mistargeted to mitochondria. PH1 shows considerable phenotypic and genotypic heterogeneity. The incidence and severity of PH1 varies in different geographic regions. DNA samples of the affected members from two unrelated Tunisian families were tested by amplifying and sequencing each of the AGXT exons and intron–exon junctions. We identified a novel frameshift mutation in the AGXT gene, the c.406_410dupACTGC resulting in a truncated protein (p.Gln137Hisfs*19). It is found in homozygous state in two nonconsanguineous unrelated families from Tunisia. These molecular findings provide genotype/phenotype correlations in the intrafamilial phenotypic and permit accurate carrier detection, and prenatal diagnosis. The novel p.Gln137Hisfs*19 mutation detected in our study extend the spectrum of known AGXT gene mutations in Tunisia.

[M’Dimegh S., Aquaviva-Bourdain C., Omezzine A., M’Barek I., Souche G., Zellama D., Abidi K., Achour A., Gargah T., Abroug S. and Bouslama A. 2016 A novel mutation in the AGXT gene causing primary hyperoxaluria type I: genotype–phenotype correlation. J. Genet. 95, 659–666]

Introduction Most of the PH1-causing mutations identiﬁed on the AGXT gene are single base pair substitutions leading to the Primary hyperoxaluria type1 (PH1) is an autosomal reces- synthesis of an aberrant gene product (Williams et al. 2009). sive metabolic disorder caused by inherited mutations in the Wild-type AGXT gene comes in two polymorphic variants, AGXT gene encoding liver peroxisomal alanine : glyoxylate the most frequent major haplotype (Maj) and the less fre- aminotransferase (AGT) (Danpure and Jennings 1986). AGT quent minor haplotype (Min), carrying two single amino acid is a pyridoxal5 -phosphate (PLP)-dependent enzyme that cat- substitutions (p.Pro11Leu and p.Ile340Met) among other alyzes the transamination between L-alanine and glyoxylate genomic changes in strong linkage disequilibrium. This fact to pyruvate and glycine, respectively. The absence of func- can help in the clinic, when searching for mutations in new tional AGT leads to glyoxylate accumulation peroxisomes. PH1 cases (Williams and Rumsby 2007). The Min haplo- Glyoxylate is then transported into the hepatocyte cytosol to type (p.Pro11Leu) allows a small portion of AGT (about be oxidized to oxalate by cytosolic L-lactate dehydrogenase. 5%) to be rerouted from peroxisomes to mitochondria (Lumb Oxalate overproduction leads to the formation and deposition and Danpure 2000). The p.Pro11Leu does not cause PH1 by of insoluble calcium oxalate crystals, expand in nephrocal- itself, but it is known to act synergistically with the delete- cinosis and urolithiasis. This ultimately causes renal failure rious effects of several common mutations that leads to sen- and the consequent systemic oxalosis with calcium oxalate sitization of the protein (Santana et al. 2003; Danpure and precipitation in the whole body (Hoppe et al. 2009). Rumsby 2004). Most of the mutations described are typi- cally found in either Maj or Min haplotypes, but rarely in ∗ For correspondence. E-mail: [email protected]. both (Williams and Rumsby 2007). AGXT mutations result in

Keywords. primary hyperoxaluria type 1; AGXT gene; genotype–phenotype correlations.

Journal of Genetics, DOI 10.1007/s12041-016-0676-4, Vol. 95, No. 3, September 2016 659 Saoussen M’Dimegh et al. different phenotypic expressions of the disease due to various Methods molecular mechanisms. Four basic mechanisms have been reported to explain the consequences of mutations: mito- Mutational analysis chondrial mistargeting, protein aggregation, abolition of cat- Genomic DNA was extracted from peripheral blood leuco- alytic activity (catalytic defects) and synthesis defect (Salido cytes. Initially, the seven most frequent mutations (c.33dupC et al. 2012). (p.Lys12GlnfsX156), c.508G>A (p.G170R), c.731T>C In the present study, we describe a novel mutation in AGXT (p.I244T), c.454T>A (p.F152I), c.322T>C (p.W108R), identified in two unrelated Tunisian PH1 families. We have c.976delG (p.Val326fs) and c.588G>A (p.G156R)) were also analysed the clinical features to investigate a possible tested by amplification of genomic DNA and restriction relationship between genotype and phenotype. enzyme digestion using primers and conditions that were previously documented (Purdue et al. 1990; Rinat et al. 1999; Coulter-Mackie et al. 2005). The PCR test designed to detect Materials and methods c.33dupC mutation was used simultaneously to distinguish Subjects and data collection the Maj and Min alleles by the research of the IVS1+74 bp in intron 1, which is a characteristic duplication for the Min PH1 was diagnosed in two patients from two unrelated haplotype (Coulter-Mackie et al. 2005). Tunisian families. Informed consent was obtained from all Next, the 11 exons and exon–intron boundaries of the patients for the collection of blood samples. Familial screen- AGXT gene were amplified from genomic DNA using stan- ing in family 2 revealed another affected patient. dard PCR procedures. Direct sequencing of purified PCR products was performed using the BigDye Terminator v3.1 Family 1: Patient II-5, a 8-year-old boy who was diagnosed sequencing kit (Applied Biosystems, Foster City, USA) and with nephrolithiasis at the age of 5, and had been maintained an ABI 3730 genetic analyser. The sequencing of all 11 on dialysis therapy for 7 years prior to the study. exons and flanking intronic sequences of the AGXT gene was performed at the reference laboratory in Lyon, France. Family 2: Other samples were obtained from members of Sequences were analysed using Seqscape Analysis Soft- another family, including a 20-year-old girl (patient V-3) and ware (Applied Biosystems) in comparison with GenBank a 9-year-old boy (patient V-6), who were the case index and reference genomic sequence (NM 000030.2). were diagnosed with nephrolithiasis and her sister (patient Assessment of pathogenicity V-4), 16 years old, who was found by family screening. The families pedigree are shown in figure 1. To investigate the pathogenicity of variants, we performed An information sheet was prepared including the follow- in silico analysis using the AlamutR software (Interactive ing data: clinical presentation, biochemical and radiological Biosoftware, member of the Human Genome Variation data, treatment and outcomes. Society and EBI SME Support Forum).

Figure 1. Pedigree of the families under study.

660 Journal of Genetics, Vol. 95, No. 3, September 2016 Novel mutation in the AGXT gene and outcome

Biochemical analysis recurrent kidney stones. She had a family history of renal failure (patient III-1), but not tested for PH1, which would The urinary oxalate excretion was assayed by a colorimet- be most likely affected by PH1. Physical examination was ric enzymatic method in 24-h urine collections. The plasma normal. Laboratory studies showed: urea = 6.8 mmol/L, cre- oxalate levels were measured by HPLC method in a spe- atinine = 133 μmol/L, Na = 135 mmol/L, K = 5 mmol/L, cialized French laboratory of clinical pathology. In children, Ca = 2.17 mmol/L, Hb = 11.7g/dL, CBEU was positive of the renal function was estimated by the Schwartz formula Enteroccus faecalis germ. Renal ultrasound showed a nor- (Schwartz et al. 1987). mal sized kidneys and multiple bilateral lithiasis. She was Equations of Schwartz: eGFR (glomerular filtration rate) treated with high fluid intake, strict dietary restrictions on 2 = k × height (cm) oxalate-containing foods and a trial of pyridoxine for many (mL/min/1.73 m ) serum creatinine (mg/dL) (the considered k values were 0.55 for girls and for boys <12). months. Then, she had a rise in the creatinine concentration i.e. 172 μmol/L. Patient V-6, a 9-year-old boy was admitted to hospital to manage familial nephropathy. He is sibling of patient V-3. Results He presented with diagnosis of urolithiasis and nephrocalcinosis. In his past history, he had repeated episodes of kidney Clinical report stone and urinary tract infection of Proteus and Entero- Family 1: Patient II-5 is an 8 years boy, native to central bacter germs at the age of 4 years. He had been receiving western Tunisia. He was apparently healthy until 5 years of an ureterostomy with stone extraction, several ureteroscopy age when he was admitted with symptoms of a urinary tract with fragmentation of calculi and a lithotripsy. Stone anal- infection. In his past history, he had a recurrent urinary tract ysis showed whewellite. Physical examination was normal. infection. At the time of admission, the child appeared critically Laboratory studies showed: clearance = 72 mL/min per ill with generalized oedema and pallor. Blood pressure 1.73 m2,urea= 7.1 mmol/L, creatinine = 84 μmol/L, Na = was elevated. Renal ultrasonography revealed multiple bilat- 136 mmol/L, K = 3.7 mmol/L, Ca = 2.41 mmol/L, Hb = eral nephrocalcinosis and urolithiasis with the suspicion 9.3 g/dL, CBEU was positive. Renal ultrasound showed an of PH1. The level of 24-h urinary oxalate excretion was asymmetric size of kidneys and bilateral medullary nephro- very high (1828 mmol/L), oxalate : creatinine ratio was calcinosis. Urinary oxalate excretion was 0.02 mmol/24 h 0.712 mmol/mmol (normal value 0.085 at 10 years of age) which is normal and (normal 0.14–0.42 mmol/24 h). Plasma and hyperglycoluria (>0.5 mmol/1.73 m2 per 24 h). Crystal- oxalate level also was in the normal range of 19 μmol/L luria was positive: presence of calcium oxalate monohydrate (normal < 33 μmol/L). The patient was present with renal stones and with a number of crystals higher than 200/mm3. impairment at follow-up. He was treated conservatively Lab investigations were consistent with acute renal fail- with high fluid intake, avoiding high oxalate foods and ure, with clearance = 70 mL/min per 1.73 m2,urea= 7 pyridoxine. mmol/L, creatinine = 60 μmol/L, Na = 135 mmol/L, K = Patient V-4, a 16-year-old girl, is the sibling of patient 4.2 mmol/L, Ca = 2.6 mmol/L, Hb = 10 g/dL, cytobacteri- V-3 and V-6. She was diagnosed with PH1 upon screening ological examination of the urine (CBEU) was positive for of the entire family. She has not consulted until the time of Proteus germ. The patient progressed to end stage renal dis- diagnosis. She reported in her past history a single episode ease (ESRD) quickly. First, he had been receiving peritoneal of urolithiasis, when she had received a lithotripsy at age of dialysis, followed by a chronic hemodialysis and oral pyri- 6 years. She presented with normal renal function at the time doxine therapy. Pyridoxine therapy was used to reduce uri- of diagnosis. nary oxalate excretion to 0.234 mmol/mmol. After 2 years of hemodialysis, he presented multiorgan failure due to systemic oxalosis (ocular, cardiac and bone). The only remark- AGXT gene analysis able family history was his brother (patient II-2) who died at No they mutation was detected by screening the seven mutations the age of 10 of unknown renal disease. The parents denied (c.33dupC (p.Lys12GlnfsX156), c.508G>A (p.G170R), consanguinity (third degree of consanguinity), and they have c.731T>C (p.I244T), c.454T>A (p.F152I), c.322T>C three other healthy daughters (figure 1). (p.W108R), c.976delG (p.Val326fs) and c.588G>A (p.G156R)). This screening allows in identifying the Maj Family 2: Patient V-3, a 20-year-old girl was referred to allele in homozygous state for all patients. nephrologists for evaluation of her renal insufficiency. She Next, sequencing of exons and intron–exon boundaries of was the index case. She was born from a second degree of AGXT gene revealed a novel duplication of five nucleotides consanguinity. Also she is native to central western Tunisia. in exon 3, c.406_410dupACTGC responsible for a frameshift In her past history, at the department of urology, she was with the substitution of glutamine at position 137 by an diagnosed with chronic kidney disease due to bilateral, mul- histidine and a premature STOP codon 19 aminoacids tiple stones in the kidneys at the age of 2. The patient had downstream (p.Gln137Hisfs*19). The three index cases are repeated surgery to extracted lithiasis and lithotripsy due to homozygous for this mutation. Mutational analysis of the

Journal of Genetics, Vol. 95, No. 3, September 2016 661 Saoussen M’Dimegh et al.

AGXT gene showed heterozygosity in the parents of the two Impact of c.406_410dupACTGC on protein families. Electrophoregrams of the patient, one of the parents The c.406_410dupACTGC duplication of five nucleotides and a control are shown in figure 2. in exon 3 leads to a frameshift with the substitution of Familial testing was performed for family 2 and revealed glutamine at position 137 by an histidine and a premature that one asymptomatic sister is homozygous for this muta- STOP codon 19 amino acids downstream (p.Gln137Hisfs*19). tion and the other one is heterozygous. This novel duplica- Analysis with Alamut R software showed that the tion detected was screened in 80 control individuals and was c.406_410dupACTGC was predicted to produce an mRNA not detected in all of them. which might be targeted for nonsense mediated decay. The research of the IVS1+74 bp in intron 1 by PCR At the amino acid level, this duplication led to create revealed that all patients had the Maj haplotype. The a frameshift from codon Gln137 and ended with a stop sequencing results showed six other single-nucleotide poly- codon 18 positions downstream. The initial 136 amino acids morphisms (SNPs) (table 1), which showed a genotypic were unaffected by the frameshift; however, the follow- combination for the Maj allele. Thus, we noted that the ing 18 residues were predicted to be mutated, and termi- c.406_410dupACTGC mutation cosegregated with the Maj nated by a premature stop codon 18 positions downstream allele. Moreover, we found an additional variation, the (p.Gln137Hisfs*19; figure 3). IVS7-44 A>G in intron 7 in homozygous state. All these polymorphic variants were revealed in total linkage disequilibrium. Haplotype was found in homozygous state for all the Genotype–phenotype correlation three patients. We detected the c.406_410dupACTGC mutation in homozygous state in all patients. In addition, haplotype analysis showed that this mutation cosegregates with a specific haplotype in the two unrelated families from the same region. Consanguinity was reported in the two families. The preva- lence of males was equal to females in our patients (2M/2F).

Interfamilial genotype–phenotype correlation: We found that all patients from the two families presented the same initial symptoms, which were associated to nephrocalcinosis and/or recurrent urolithiasis. The median age at onset was 3.7 years (range 2–5 years). Further, there was a correlation between the clinical outcomes and the male sex in the two families. Patient II-5 and patient V-6 were diagnosed at the same age, same age at onset of symptoms (before the age of 5) and with the same clinical symptoms. Crystalluria was positive in both cases. However, there was obviously no correlation between the biochemical proﬁles. Patient II-5 had high level of oxalate excretion, higer oxalate : creatinine ratio and hyperglycoluria besides patient V-6 had normal values. Moreover, systemic oxalosis was extremely severe in patient II-5, who evolved with a cardiomyopathy, retinopathy and a bone disease. There was a marked phenotypic variability which ranged Figure 2. Electrophoregrams of the c.406_410dupACTGC muta- from ESRD to slightly impaired renal function (table 2). tion in (a) homozygous index case, (b) one of heterozygous parents Patients II-2 and II-5 had been reached ESRD at age of and (c) a control showing the results of AGXT gene sequencing. 10 and 12, respectively. Patients V-3 and V-6 already have

Table 1. Results of haplotype and polymorphisms analysis.

Exon 1 Intron 1 Exon 2 Intron 2 Intron 2 Intron 7 Exon 10 Alleles Patient c.32C>TIVS1+74 bp c.264C>TIVS2+13C>T c.358+56_358+64del IVS7-44 A>G c.1020 A>G Maj/Min

1 CC DD CC CC dd GG AA Maj/Maj 2 CC DD CC CC dd GG AA Maj/Maj 3 CC DD CC CC dd GG AA Maj/Maj

D, allele without 74-bp insertion; d, allele + duplication 9 bp.

662 Journal of Genetics, Vol. 95, No. 3, September 2016 Novel mutation in the AGXT gene and outcome

Figure 3. Effect of mutation on protein. Duplication mutation c.406_410dupACTGC (p.Gln137Hisfs*19) affecting the ﬁrst nucleotide (shaded in red) in the codon for amino acid residue 137 (Gln, Q) of AGXT, and resulting in a change in reading frame and a premature stop codon. Upper sequence, wild-type; lower-sequence, mutant.

Table 2. Clinical characteristics and outcome of the four patients with c.406_410dupACTGC mutation in AGXT gene.

Patient II-5 Patient V-3 Patient V-6 Patient V-4

Sex (M, F) M F F M Origin Central Tunisia Central Tunisia Central Tunisia Central Tunisia Age at diagnosis, years 8 20 9 16 Age at onset of symptoms, years 5 2 4 6 Actual age (survivors), years 15 25 14 16 Age of ESRD, years 10 – – – Consanguinity 3rd degree 2nd degree 2nd degree 2nd degree Symptoms Urolithiasis and Urolithiasis Urolithiasis Asymptomatic nephrocalcinosis and nephrocalcinosis and nephrocalcinosis Renal function at diagnosis Insufficient Preserved Preserved Preserved Renal function at follow-up ESRD Insufficient Insufficient Not available Dialysis Yes No No No Evolution ESRD, systemic Urolithiasis, Urolithiasis, Preserved oxalosis (bones, retina, renal impaired renal renal function heart and vessels) insufficiency function

M: male; F: female; ESRD: end stage renal disease; – : absent ESRD. renal insufﬁciency at age 20 and 14, respectively. Patient V-4 have been PH1. Patient II-5 had presented symptoms at the had preserved renal function. Also, the age of diagnosis was age of 4, developing ESRD associated with severe systemic variable from childhood in male patients to adolescence in oxalosis. female cases, with a median age equal to 14.2 years (range On the other hand, family 2 had three affected children 8–20 years). suffering from PH1 but with milder form. One of them was We noted a decrease in urinary oxalate excretion in patient diagnosed with familial screening. Patients V-3 and V-6 have II-5 after pyridoxine treatment. However, this does not pre- developed clinical symptoms associated with nephrocalcinosis vent him to reach ESRD and to develop oxalosis complica- at the age of 2 and 4 years, respectively. Both have renal tions. In other patients, pyridoxine treatment was used but insufﬁciency and patient V-4 had preserved renal function. responsiveness was not measured. In fact, patients V-3 and They required interventions such as surgery or lithotripsy. V-6 were presented with preserved renal function at the time Patients V-3 and V-6 were treated conservatively. of diagnosis but they showed a slight decline in renal function during follow-up. Patient V-4 had a preserved renal function. Discussion The clinical outcome of PH1 is variable, ranging from early Intrafamilial genotype–phenotype correlation: We noted that death in infancy to the late-onset form with occasional stones family 1 demonstrated a phenotypic severity. It presents the diagnosed in adulthood (Cochat et al. 2010). PH1 is equally most severe form with early onset and progression to ESRD heterogeneous at the molecular level. There is no mutational before the age of 15 years. In fact, we suspect that patient hot spot, but various mutations have been found throughout II-2 had died at the age of 10 due to ESRD but it could the 11 exons of the AGXT gene. More than 150 mutations

Journal of Genetics, Vol. 95, No. 3, September 2016 663 Saoussen M’Dimegh et al. have been described and they have been summarized recently defining genotype–phenotype correlation, we noted a corre- (Williams et al. 2009). Missense mutations are common fol- lation between the clinical symptoms and the male sex in lowed by small insertion/deletions (indels), most notably the two families and no correlation between the biochem- c.33dupC (Lorenzo et al. 2014). ical profiles. However, we marked an interfamilial clinical In the present study, mutational analysis of the AGXT gene in heterogeneity which ranged from ESRD to slightly impaired two unrelated Tunisian families with PH1 revealed a novel renal function. Also, the age of diagnosis was variable from mutation, the c.406_410dupACTGC (p.Gln137Hisfs*19). It childhood in male patients to adolescence in female cases. is a five nucleotide duplication (ACTGC) in exon 3, this The phenotypic heterogeneity of PH1 observed in these fam- duplication leads to secondary shift in the reading frame ilies, on the term of evolution, can be explained by the pres- of the protein after 137 (of 392) amino acids and early ence of the other modifier factors. The other genetic factors stop codon. Analysis with Alamut software showed that the might control the catalytic activity and the subcellular target- c.406_410dupACTGC was predicted to produce an mRNA ing of glyoxylate metabolism (D-amino acid oxidase, glyco- which might be targeted for nonsense mediated decay, which late oxidase, gloxylate aminotransferase (GGT), glyoxylate expected to produce a nonfunctional protein (Frischmeyer reductase (GR), lactic acid dehydrogenase (LDH)) (Hoppe and Dietz 1999). The p.Gln137Hisfs*19 mutation belongs to et al. 1997). Moreover, other factors are not of genetic ori- the large-domain residue conserved in the mammalian AGTs gin and might influence the clinical course of the disease, and it is a part of the active site. The mutation is located including diet, infection and climate (VonSchnakenburg between the side-chains of Trp108 and Val185 (Zhang et al. et al. 1998). 2003). Further, frameshifts have an increased incidence in On the other hand, we noted a genotype–phenotype intra- sequences of repeated single bases (Antonarakis et al. 2001), familial association. In family 1, the c.406_410dupACTGC probably because of a tendency towards slippage and realign- mutation was reported with a very severe disease, from ment during DNA replication. Thus, c.406_410dupACTGC onset at 5 years, rapidly progressed to ESRD in child- may be included in the same category of c.33dupC. hood with many complications of systemic oxalosis and an The p.Gln137Hisfs*19 showed a specific geographical infantile death. These patients displayed serious clinical fea- location, since it is identified in two unrelated families, orig- tures given that they had frameshift mutation, which led inating from the same area in central Tunisia. The historical to hepatic AGT deficiency. The clear sex difference was migratory/invasive flows which took place in north Africa observed in this family, which was previously reported for can be attributed to the founder nature of this mutation. In the homozygous of p.G170R and c.33dupC (Hoppe et al. fact, the central Tunisia is a region which is recognized to be 1997; Mandrile et al. 2008). It is not clear at which level the stronghold of both Arab settlers during Arab expansion the mechanism(s) of the sex limited or sex-related expres- into north Africa in the seventh century AC and migration sions of PH1 are acting. In the KO mouse model of PH1, waves of Arab Bedouin tribes during the eleventh century Salido et al. (2006) found increased levels of urine oxalate AC (El Moncer et al. 2010). We, based on the approach of in both sexes (higher in males), but renal stones were only in homozygosity haplotype, a haplotype defined only by the males, suggesting that sex difference may affect both oxalate homozygous SNPs obtained following omission of the het- production and stone formation in the urinary tract. How- erozygous SNPs (Miyazawa et al. 2007; Jiang et al. 2009). ever, in family 2, the c.406_410dupACTGC mutation was In fact, sequencing results revealed a common haplotype car- reported in three affected members suffering from PH1 with ried by the patients sharing other previously known cod- milder form. A patient was discovered by family screening, ing and intronic variants. These variants were previously who is not consulted until diagnosis. The two other patients described to form a Maj allele (Amoroso et al. 2001; Coulter experienced impaired glomecular filtration rate (GFR). And Mackie et al. 2005; Williams et al. 2009). We revealed addi- they have received conservative treatment, including a high tional polymorphic variations, the IVS7-44 A>G, previously fluid intake, oral pyridoxine and required many surgical reported with a frequency equal to 0.71 in controls (Monico managements of urolithiasis. et al. 2007). This haplotype was found in homozygous state Pyridoxine as an enzyme cofactor may reduce oxalate in all patients. This supports the hypothesis of a central production and therefore, excretion, particularly in cases Tunisia origin of the c.406_410dupACTGC mutation and of with residual enzyme activity (Yendt and Cohanim 1985). an Arab founder effect. Pyridoxine decreases urinary excretion of oxalate in patient The p.Gln137Hisfs*19, which belonged exclusively to our II-5 but does not prevent progression to ESRD. Indeed, an patients in homozygous form, dramatically changed the gene extended phase of advanced renal failure and prolonged dia- function by shifting the reading frame of 19 codon after lysis worsens the prognosis due to insufficient clearance of amino acid 137 and creating a stop codon in amino acid oxalate. The oxalate comes not only from overproduction 156, which is remarkable for the early age at onset and clin- in the liver but probably more from mobilization of oxalate ical symptoms. The first clinical manifestations were present deposits (mainly from skeleton, heart and retina), which in all patients at infantile age and with specific symptoms, cannot be influenced by pyridoxine. such as recurrent urolithiasis and urinary infection. When we Besides in the other cases, pyridoxine was introduced at an used the interfamilial phenotypic consistency as a mode of early stage, which may be effective. They remained without

664 Journal of Genetics, Vol. 95, No. 3, September 2016 Novel mutation in the AGXT gene and outcome

ESRD with renal function being decreased slowly. Pyridox- Frischmeyer P. A. and Dietz H. C. 1999 Nonsense-mediated mRNA ine is also associated with dietary restriction of oxalate-rich decay in health and disease. Hum. Mol. Genet. 8, 1893–1900. foods, high fluid intake and many urological procedures. Jiang H., Orr A., Guernsey D. L., Robitaille J., Asselin G., Samuels M. E. et al. 2009 Application of homozygosity haplotype anal- We can suggest that the p.Gln137Hisfs*19 can be associ- ysis to genetic mapping with high density SNP genotype data. ated with pyridoxine responsiveness when it is taken early PLoS One 4, e5280. and before ESRD. A previous study has reported a mild Hoppe B., Danpure C. J., Rumsby G., Fryer P., Jennings P. R., Blau form detected in patient with B6 treatment responsiveness N. et al. 1997 A vertical (pseudodominant) pattern of inheritance in the autosomal recessive disease primary hyperoxaluria type 1: and carrying the c.33dupC (Amoroso et al. 2001), a muta- lack of relationship between genotype, enzymic phenotype, and tion in the same class with the p.Gln137Hisfs*19. Therefore, disease severity. Am. J. Kidney Dis. 29, 36–44. early timing of diagnosis and treatment at a stage of pre- Hoppe B., Beck B. B. and Milliner D. S. 2009 The primary served renal function may modify the course of the disease. hyperoxalurias. Kidney Int. 75, 1264–1271. However, pyridoxine treatment and responsiveness were not Lorenzo V., Torres A. and Salido E. 2014 Primary hyperoxaluria. Nefrologia 34, 398–412. systematically recorded in our patients, these remains to be Lumb M. J. and Danpure C. J. 2000 Functional synergism between analysed. the most common polymorphism in human alanine : glyoxylate In conclusion, this study showed the first cases of a novel aminotransferase and four of the most common disease-causing mutation, not been previously described. These findings mutations. J. Biol. Chem. 275, 36415–36422. may have importance for the management of Tunisian PH1 Mandrile G., Robbiano A., Giachino D., Sebastiano R., Dondi E., Fenoglio R. et al. 2008 Primary hyperoxaluria: report of an Ital- patients because it is part of health problems of the country. ian family with clear sex conditioned penetrance. Urol. Res. 36, Also direct detection of this mutation allows genetic coun- 309–312. selling in relatives of affected families as well as prenatal Miyazawa H., Kato M., Awata T., Kohda M., Iwasa H., Koyama diagnosis. The c.406_410dupACTGC mutation may be spe- N. et al. 2007 Homozygosity haplotype allows a genome wide cific to Tunisian patients and we must introduce it into the list search for the autosomal segments shared among patients. Am.J.Hum.Genet.80, 1090–1102. of mutations searched on the first line for molecular diagnosis Monico C. G., Rossetti S., Schwanz H. A., Olson J. B., Lundquist of PH1. P.A.,DawsonD.B.et al. 2007 Comprehensive mutation screening in 55 probands with type 1 primary hyperoxaluria shows Acknowledgements feasibility of a gene-based diagnosis. J. Am. Soc. Nephrol. 18, 1905–1914. This work was supported by Tunisian Ministry of Public Health Purdue P. E., Takada Y. and Danpure C. J. 1990 Identification of and the Ministry of Higher Education and Scientific Research. mutations associated with peroxisome-to-mitochondrion mistar- We would like to thank our patients and their families for their geting of alanine/glyoxylate aminotransferase in primary hyper- collaboration to this work. oxaluria type 1. J. Cell Biol. 111, 2341–2351. Rinat C., Wanders R. J., Drukker A., Halle D. and Frishberg Y. 1999 Primary hyperoxaluria type I: a model for multiple mutations in a monogenic disease within a distinct ethnic group. J. Am. Soc. References Nephrol. 10, 2352–2358. Salido E. C., Li X. M., Lu Y., Wang X., Santana A., Roy- Amoroso A., Pirulli D., Florian F., Puzzer D., Boniotto M., Crovella Chowdhury N. et al. 2006 Alanine-glyoxylate aminotransferase- S. et al. 2001 AGXT gene mutations and their influence on clin- deficient mice, a model for primary hyperoxaluria that responds ical heterogeneity of type 1 primary hyperoxaluria. J. Am. Soc. to adenoviral gene transfer. Proc. Natl. Acad. Sci. USA 103, Nephrol. 12, 2072–2079. 18249–18254. Antonarakis S. E., Krawczak M. and Cooper D. N. 2001 The Salido E., Pey A. L., Rodriguez R. and Lorenzo V. 2012 Primary nature and mechanisms of human gene mutation. In: The hyperoxalurias: disorders of glyoxylate detoxification. Biochim. metabolic and molecular bases of inherited disease, 8th edition, Biophys. Acta 1822, 1453–1464. pp. 343–377. Santana A., Salido E., Torres A. and Shapiro L. J. 2003 Primary Cochat P., Fargue S. and Harambat J. 2010 Primary hyperoxaluria hyperoxaluria type 1 in the Canary Islands: a conformational type 1: strategy for organ transplantation. Curr. Opin. Organ disease due to I244T mutation in the P11L-containing alanine: Transplant. 15, 590–593. glyoxylate aminotransferase. Proc. Natl. Acad. Sci. USA 100, Coulter-Mackie M. B., Lian Q., Applegarth D. and Toone J. 7277–7282. 2005 The major allele of the alanine : glyoxylate aminotrans- Schwartz G. J., Brion L. P. and Spitzer A. 1987 The use of plasma ferase gene: nine novel mutations and polymorphisms associ- creatinine concentration for estimating glomerular filtration rate ated with primary hyperoxaluria type 1. Mol. Genet. Metab. 86, in infants, children, and adolescents. Pediatr. Clin. North Am. 34, 172–178. 571–590. Danpure C. J. and Jennings P. R. 1986 Peroxisomal alanine: glyoxy- Von Schnakenburg C., Hulton S. A., Milford D. V., Roper H. P. and late aminotransferase deficiency in primary hyperoxaluria type I. Rumsby G. 1998 Variable presentation of primary hyperoxaluria FEBS Lett. 201, 20–24. type 1 in 2 patients homozygous for a novel combined deletion Danpure C. J. and Rumsby G. 2004 Molecular aetiology of pri- and insertion mutation in exon 8 of the AGXT gene. Nephron 78, mary hyperoxaluria and its implications for clinical management. 485–488. Expert. Rev. Mol. Med. 6, 1–16. Williams E. and Rumsby G. 2007 Selected exonic sequencing of the El Moncer W., Esteban E., Bahri R., Gaya-Vidal M., Carreras- AGXT gene provides a genetic diagnosis in 50% of patients with Torres R., Athanasiadis G. et al. 2010 Mixed origin of the cur- primary hyperoxaluria type 1. Clin. Chem. 53, 1216–1221. rent Tunisian population from the analysis of Alu and Alu/STR Williams E. L., Acquaviva C., Amoroso A., Chevalier F., Coulter- compound systems. J. Hum. Genet. 55, 827–833. Mackie M. B., Monico C. J. et al. 2009 Primary hyperoxaluria

Journal of Genetics, Vol. 95, No. 3, September 2016 665 Saoussen M’Dimegh et al.

type 1: update and additional mutation analysis of the AGXT Zhang X., Mark Roe S., Hou Y., Bartlam M., Rao Z., Pearl L. gene. Hum. Mutat. 30, 910–917. H. et al. 2003 Crystal structure of alanine : glyoxylate amino- Yendt E. R. and Cohanim M. 1985 Response to a physiologic dose transferase and the relationship between genotype and enzymatic of pyridoxine in type I primary hyperoxaluria. N.Engl.J.Med. phenotype in primary hyperoxaluria type 1. J. Mol. Biol. 331, 312, 953–957. 643–652.

Received 7 September 2015, in ﬁnal revised form 6 January 2016; accepted 11 January 2016 Unedited version published online: 14 January 2016 Final version published online: 12 August 2016

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