Adami, A

Caciotti, A

Donati, Ma

Guerrini, Renzo

Morrone, Amelia

Zammarchi, Enrico

English

Florence Research

118 Original articleDifferent genotypes in a large Italian family with recurrenthereditary fructose intoleranceAnna Caciottia, Maria Alice Donatia, Andrea Adamib, Renzo Guerrinia,Enrico Zammarchia and Amelia MorroneaObjectives Hereditary fructose intolerance is caused by adeficiency of the aldolase B enzyme, which is expressed inthe liver, small intestine and kidneys. Patients usually showa marked aversion to fruits and sweets; if, however, it is notdiagnosed, persistent or incidental ingestion of fructosemight be lethal. Our paper aims at improving the clinicaland molecular characterizations of these patients, to avoiddangerous misdiagnoses.Methods Here we report the molecular results in an Italiancohort: on the occurrence of aldolase B mutations and, inparticular, on the clinical and molecular characterization ofa large family with recurrent hereditary fructoseintolerance.Results Patients included in our cohort showed the threemost common mutations (p.A150P, p.A175D and p.N335K).Such molecular tests were enough to cover all the mutatedalleles of hereditary fructose intolerance found in ourpatients. The allele frequencies of hereditary fructoseintolerance mutations detected were 69.2% for p.A150P,23.1% for p.A175D and 7.7% for p.N335K. The probandof the family with recurrence of the disease washeterozygous for the known p.A150P and p.A175D mutatedalleles of the aldolase B gene. Molecular characterizationof at-risk family members also identified the p.N335Kmutation. In addition, the oldest affected patients exhibitedmild clinical impairment.Conclusions Our results indicate that the diagnosis ofhereditary fructose intolerance can be complicated byclinical and genetic intrafamilial variability. A knowledgeof the clinical and geographical history of each familymember is thus essential, to reduce potentially lethalmisdiagnoses and to facilitate such patients to receiveappropriate genetic counselling. Eur J GastroenterolHepatol 20:118–121 c 2008 Wolters Kluwer Health |Lippincott Williams & Wilkins.European Journal of Gastroenterology & Hepatology 2008, 20:118–121Keywords: aldolase B, genetic counselling, hereditary fructose intolerance,prevalence of aldolase B mutationsaAOU Meyer, Metabolic and Muscular Unit, Clinic of Pediatric Neurology,Florence and bAOU San Carlo Borromeo, UO of Pediatric and NeonatalPathology, Milan, ItalyCorrespondence to Professor Enrico Zammarchi, Department of Pediatrics,AOU Meyer, Via Luca Giordano 13, 50132 Florence, ItalyTel: + 39 055 5662482; fax: + 39 055 570380;e-mail: neuromet@meyer.itReceived 16 April 2007 Accepted 10 August 2007IntroductionHereditary fructose intolerance (HFI; Online MendelianInheritance in Man +229600), which is caused by theabsence or reduction of the aldolase (EC 4.1.2.13) enzyme,B isoform (ALDOB), has an estimated frequency of 1 in20000 births [1]. Despite eating habits that includeincreased sugar consumption in industrialized countries,patients’ distinct aversion to fruit and sweets is the majorreason for underdiagnoses of HFI [1,2]. Untreatedpatients, being unable to assimilate dietary fructose, candevelop a broad range of gastrointestinal symptoms, lacticacidosis and hypoglycaemia. Persistent ingestion of fruc-tose and related sugars can lead to severe liver and kidneydamage, seizures, coma and risk of death [1].The human ALDOB gene has been cloned and character-ized: the gene consists of nine exons encoding apolypeptide containing 364 amino acids [3]. So far, about35 mutations in the ALDOB gene have been reported[4,5]. The most common ALDOB mutations, p.A150P,p.A175D and p.N335K, account for about 72–76% of HFImutated alleles worldwide [4–6]. The p.A150P mutationhas a greater frequency in Northern Europe, whereas thep.A175D mutation is predicted to be more frequent inSouthern Europe, and the p.N335K in Central andEastern Europe [5,7]. These general population studiesincluded the Italian population. In addition, Italian HFIpatients have been analysed as an exclusive group [8,9].The estimated frequencies of the p.A150P and p.A175Dmutated alleles in Italy are reported to be about 50 and30%, respectively [8,9]. Seven mutations have, however,been proposed to account for about 95% of Italian ALDOBmutated alleles [10].Given the high prevalence of a small number of ALDOBmutations, a rational mutation-screening programmecould be introduced. It seems likely that the frequencyof HFI has been underestimated: hence, such a0954-691X c 2008 Wolters Kluwer Health | Lippincott Williams & WilkinsCopyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.programme could have a dramatic impact for potentialpatients, as dietary treatment can prevent this potentiallylethal disease [5].To increase the number of characterized HFI patientsand to ascertain the prevalence of each mutation in theItalian population better, we report the allele frequenciesof HFI mutations that were detected in the ALDOB geneof 13 index patients. This HFI cohort includes indexpatients from a large Italian family with recurrent HFIand atypical molecular characterization due to thepresence of the three common ALDOB mutations.MethodsPatientsThe clinical presentations of the HFI patients, belongingto the large Italian family here reported, are summarizedin Table 1. The eight additional unrelated Italianpatients, who were included in our cohort, presentedclassic HFI phenotypes.Genomic DNA isolation and analysisConsent statements for genetic analysis were obtainedfrom all patients. Genomic DNA was obtained frompatients’ lymphocytes using a commercial DNA-extrac-tion kit (Qiagen, Hilden, Germany). The oligonucleo-tides and the PCR-amplifying conditions have beendescribed previously [11]. The three most commonALDOB mutations were screened. Three ALDOB frag-ments, encompassing exons 5 and 9 and containing thep.A150P, p.A175D and p.N335K mutations, were ampli-fied and directly sequenced on both strands.The PCR products were checked on a 1.5% agarose gel,and were excised and purified using Nucleospin Extractkit (Macherey-Nagel, Du¨ren, Germany).Sequencing reactions were performed using the ABIPRISM 310 Genetic Analyzer (Applied Biosystems,Foster City, California, USA), as recommended by themanufacturer. The nomenclature of the genetic lesions ofthe ALDOB gene is as designated previously [12–14].The mutation frequencies of the three common HFImutations, which refer to the index patients hereanalysed, are reported.ResultsMolecular analysis of HFI index patients included in ourcohort showed the three most common mutated alleles(resulting in p.A150P, p.A175D and p.N335K mutations).The allele frequencies of the HFI mutations detectedwere 69.2% for p.A150P, 23.1% for p.A175D and 7.7% forp.N335K.As part of our cohort, the index patients of a large Italianfamily with recurrent HFI were further investigated atclinical and molecular levels (Table 1, Fig. 1). Theproband (patient III:6) presented at 13 months withhypoglycaemic seizures and hypotonia, after the unwillingintake of a fruit shake at kindergarten. HFI wassuspected because of mild metabolic acidosis, hepatome-galy, a rapid rescue of the patient’s condition after theinfusion of glucosaline solutions and her aversion tosweet-tasting foods. To provide a confirmatory diagnosis,molecular analysis of the most common ALDOB genemutations was opted for, and the p.A150P and p.A175Dmutated alleles were detected. Two of the proband’scousins (patients III:1 and III:2) presented with a strongaversion to foods containing fructose as the only indicatorof the disease. Mutation analysis again detected thep.A150P and p.A175D mutations in both patients,although patients II:6 and II:20 were reported to benonconsanguineous.Aversion to sweet-tasting foods and abdominal pain alsoprompted patient III:5 to undergo ALDOB-gene mole-cular investigation. Interestingly, the p.N335K mutationwas also detected along with the p.A150P mutation.Table 1 Synopses of the main characteristics of the members of the large family reported herePatient III:6 proband III:7 III:1 III:2 III:5Age 3 years 2 years 23 years 21 years 18 monthsOrigin Northern Italy Northern Italy Northern Italy Northern Italy Northern ItalyGrowth Normal At the 10th centile Normal Normal NormalMetabolic presentationa Hypoglycaemia, mildmetabolic acidosis andvomitingAbsent Absent Vomiting AbsentGastrointestinalabnormalitiesMild hepatomegaly andhepatic steatosisMild hepatomegaly andhepatic steatosisNormal Normal Hepatomegaly andabdominal cramps inthe preceding monthsLaboratory findings Transient hypertransaminasaemiaTransient hypertransaminasaemiaNormal Normal Transient hyper trans-aminasaemia at 11 monthsCrisisa Hypoglycaemic seizuresat 13 monthsNo, diagnosis at birth Lipothymia at 19 years No NoAversion to any sourceof fructoseYes No fructose ingestion Yes Yes YesTherapy Vitamin C, no fructoseingestionVitamin C, no fructoseingestionVitamin C, no fructoseingestionVitamin C, no fructoseingestionNo fructose ingestionaWhen presenting with a crisis, symptoms have followed the ingestion of sweets and/or fruit.Recurrent hereditary fructose intolerance Caciotti et al. 119Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.DiscussionDeficient activity of ALDOB results in the accumulationof fructose 1-phosphate, which can be particularly toxicin early life [1], cause severe liver disease and, ifundiagnosed, can lead to death [15].It has been suggested that establishing the prevalence ofALDOB-gene mutations is a premise for rational mole-cular analyses in patients with suspected HFI, and forthe introduction of a screening programme for newbornbabies [5]. Molecular analyses also replace invasivediagnostic procedures such as the intravenous fructoseload or enzyme assays that are performed on tissue biopsysamples [1]. A method that uses denaturing high-pressure liquid chromatography technology has beenproposed for use in screening programmes for newborninfants [5]; however, projects in this field might becomplicated by the occurrence of private or rare HFImutations.Our molecular results showed a distribution of thep.A150P mutation that was relatively closer to that ofthe pan-European population [16], than to that of Italianpatients [6,9]. The p.A150P mutation has been describedas being more frequent in North Europe than in Italy;however, a frequent mutation reported in the Italianpopulation (c.865delC) has been predicted to be limitedto Sicily [6]. The inclusion of Sicilian patients in a HFIsurvey can thus create an imbalance in the estimates ofthe prevalence of ALDOB mutations in our country.As predicted [6,9], the frequency of the p.A175Dmutation was particularly high, compared with recentlyreported frequencies in Central Europe (15%) or Spain(15.8%) [5,16]. The p.N335K mutation has probablyspread from Eastern and/or Central Europe [4,7]. Thismutated allele, however, also proved to be relativelycommon in Italy, with a frequency close to that in CentralEurope [5,16].A relationship between clinical symptoms and genotypehas not been demonstrated [17]. As the severity of thedisease might vary and as affected patients often imposefructose restrictions on themselves, adult patients mightbe symptom free. Patient III:7, however, despite havingthe same genotype as patients III:1 and III:2, and despitehaving avoided fructose from birth, exhibited hepatome-galy and mild steatosis; so did her sister (patient III:6),suggesting that follow-up through adulthood might beessential to assess the outcome of the disease. It is thuslikely that the young patients reported herein will showan improvement in their clinical presentations as theyage, like their older relatives (patients III:1 and III:2). Inaddition, we stress the consequences of a spread, andcorrect genetic counselling for the diagnoses of patientsIII:1 and III:2, who were essentially free of symptoms.Interestingly, our report showed three independentlysegregating alleles within a family unit. Therefore,clinicians should ideally perform molecular assays of atleast the three common ALDOB mutations. This mightavoid dangerous misdiagnoses, and allow for the earlyintroduction of a diet eliminating fructose-containingfoods and for the provision of oral vitamin C supplementsto offset the imbalances that a diet without fruit mightentail [18]. Large population screening has previouslydemonstrated that HFI patients are heterozygous for atleast one of those three mutations (mutated allelefrequencies 94%) [5]; despite this, seven mutations arenecessary to achieve the 95% prevalence of HFI allelesin Italian patients [10]. Therefore, the negative resultsof the molecular tests performed for the three mostFig. 1I:1II:1 II:2 II:3 II:4III:1 III:2 III:4 III:3 III:4 III:5 III:6 III:7II:5 II:6 II:7 II:8 II:9 II:10 II:11 II:12 II:13 II:14 II:15 II:16 II:17 II:18 II:19 II:20I:2 I:3 I:4 I:5 I:6 I:7Family pedigree. The proband is indicated by an arrow. Horizontal lines indicate the p.A150P mutation; vertical lines indicate the p.N335K mutation;the black filling represents the p.A175D mutated allele.120 European Journal of Gastroenterology & Hepatology 2008, Vol 20 No 2Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.common mutations do not exclude a positive diagnosis.In addition, heterozygous patients should be furtherinvestigated at the molecular level.In conclusion, our data underline the relevance of acritical approach to the molecular analysis of common,and possible new, ALDOB mutations. This informationcould be useful to screen the ALDOB gene more carefullyin suspected patients (i.e. those at risk), starting from thenumerous parents of the large family here reported. Acareful clinical and molecular diagnosis can be particularlyuseful in genetic counselling concerning newborn babiesin affected families, as a very simple dietary therapy canprevent potentially serious damage caused by the disease.AcknowledgementConflict of interest: none declared.References1 Steinman B, Gitzelmann R, van den Berghe G. Disorders of fructosemetabolism. In: Scriver CR, Beaudet AL, Valle D, Sly WS, editors.The metabolic and molecular bases of inherited disease. New York:McGraw-Hill; 2001. pp. 1489–1520.2 Cox TM. The genetic consequences of our sweet tooth. Nat Rev Genet2002; 3:481–487.3 Tolan DR, Penhoet EE. Characterization of the human aldolase B gene.Mol Biol Med 1986; 3:245–264.4 Tolan DR. Molecular basis of hereditary fructose intolerance: mutationsand polymorphisms in the human aldolase B gene. Hum Mutat 1995; 6:210–218.5 Santer R, Rischewski J, von Weihe M, Niederhaus M, Schneppenheim S,Baerlocher K, et al. The spectrum of aldolase B (ALDOB) mutations and theprevalence of hereditary fructose intolerance in Central Europe. Hum Mutat2005; 25:594.6 Cross NC, de Franchis R, Sebastio G, Dazzo C, Tolan DR, Gregori C, et al.Molecular analysis of aldolase B genes in hereditary fructose intolerance.Lancet 1990; 335:306–309.7 Cross NC, Stojanov LM, Cox TM. A new aldolase B variant, N334K, is acommon cause of hereditary fructose intolerance in Yugoslavia. NucleicAcids Res 1990; 18:1925.8 Santamaria R, Scarano MI, Esposito G, Chiandetti L, Izzo P, Salvatore F.The molecular basis of hereditary fructose intolerance in Italian children.Eur J Clin Chem Clin Biochem 1993; 31:675–678.9 Sebastio G, de Franchis R, Strisciuglio P, Andria G, Dionisi Vici C,Sabetta G, et al. Aldolase B mutations in Italian families affected byhereditary fructose intolerance. J Med Genet 1991; 28:241–243.10 Esposito G, Santamaria R, Vitagliano L, Ieno L, Viola A, Fiori L, et al. Six novelalleles identified in Italian hereditary fructose intolerance patients enlarge themutation spectrum of the aldolase B gene. Hum Mutat 2004; 24:534.11 Dazzo C, Tolan R. Molecular evidence for compound heterozygosity inhereditary fructose intolerance. Am J Hum Genet 1990; 46:1194–1199.12 Den Dunnen JT, Antonarakis SE. Nomenclature for the description of humansequence variations. Hum Genet 2001; 109:121–124.13 Den Dunnen JT, Paalman MH. Standardizing mutation nomenclature:why bother? Hum Mutat 2003; 22:181–182.14 Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NS, et al. Genemutation database (HGMD): 2003 update. Hum Mutat 2003; 21:577–581.15 Ali M, Rosien U, Cox TM. DNA diagnosis of fatal fructose intolerance fromarchival tissue. Q J Med 1993; 86:25–30.16 Sanchez-Gutierrez JC, Benlloch T, Leal MA, Samper B, Garcia-Ripoll I,Feliu JE. Molecular analysis of the aldolase B gene in patients withhereditary fructose intolerance from Spain. J Med Genet 2002; 39:e56.17 Ali M, Rellos P, Cox TM. Hereditary fructose intolerance. J Med Genet 1998;35:353–365.18 Novy MA, Schwarz KB. Nutritional considerations and management of thechild with liver disease. Nutrition 1997; 13:177–184.Recurrent hereditary fructose intolerance Caciotti et al. 121Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Different genotypes in a large Italian family with recurrent hereditary fructose intolerance.

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Different genotypes in a large Italian family with recurrent hereditary fructose intolerance.

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