Hereditary Fructose Intolerance due to a Mutation in Aldolase B gene in Various Populations Alexandra Smith Senior Seminar February 23, 2012 Table of Contents Introduction 3 Methods of Diagnosis 4 Mutations 5 Significance of Research 6 Review of Literature 7 Critical Analysis of Literature 10 Future Research 12 Conclusion 13 References 15 2 Many people suffer from adverse reactions to food. An adverse food reaction may or may not require the immune system to create antibodies. These categories are known as immune-mediated and non-immune-mediated. Within the non-immunemediated reaction are metabolic, pharmacologic, toxic, and undefined causes (Skypala 1877-1878). One metabolic, adverse food reaction is becoming more prevalent throughout the world. This paper focuses on the genetic mutations that impair the aldolase B enzyme and cause hereditary fructose intolerance in French, Spanish, Central European, Italian, Polish, and American populations. INTRODUCTION: Hereditary fructose intolerance (HFI) is a metabolic, genetic disease that causes a deficiency in aldolase B. Aldolase B, also called liver-type aldolase or fructosebisphosphate aldolase B, is an enzyme found in the intestine, liver, and kidneys. In HFI, a mutation on chromosome nine causes a defect in aldolase B when transcription occurs. The aldolase B gene contains nine exons and is 14,500 base pairs long (DavitSpraul et al. 443). Aldolase B’s purpose is to cleave fructose-1, 6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate and fructose-1-phosphate into glyceraldehyde and dihydroxyacetone phosphate in the liver (Wong 165). When there is a deficiency of aldolase B, fructose 1-phosphate rapidly accrues in tissues due to fructose 1-phosphate not being broken down. Not only is fructose 1-phosphate toxic to cells, but it is an unusable form of phosphate. This results in a drop in both ATP and phosphate stores. When there is not enough ATP, protein synthesis is stopped and the 3 liver and kidneys lose their function. Without readily available phosphate, glycogenolysis in the liver is stopped causing hypoglycemia. In hypoglycemia, gluconeogenesis is active to increase blood glucose. Gluconeogenesis is inhibited by fructose 1phosphate, thus decreasing the quantity of glucose available (Wong 165). Many adults are thought to have undiagnosed HFI, though typically infants are diagnosed when they start to wean off formula or breast milk (Yasawy et al. 2412). This disease is believed to affect 1:20,000 people in American populations, but could have a carrier frequency of 1:50 (Coffee and Tolan 715). Symptoms vary, but the most notable ones are strong aversion to sweet foods, cirrhosis of the liver, and renal failure. The only way to treat HFI is to completely remove fructose from the diet. If fructose is not removed completely out of the diet, liver and kidney failure could cause death (Coffee et al. 34). Fructose in foods is found attached to glucose as a disaccharide, by itself as a monosaccharide, or as a fructooligosaccharide. Fructose is present in many consumables; the most notable sources being fruits, vegetables, table sugar, honey, high-fructose corn syrup, maple syrup, and fruit juice (Marcason 1). Often times, HFI is confused with fructose malabsorption. Fructose is very similar to glucose in that both are hexose sugars, but the main difference is in metabolism. Fructose is absorbed in the small intestine through facilitated diffusion. When large amounts of fructose are eaten, not all of the fructose is absorbed in fructose malabsorption. Leftover fructose enters the intestinal lumen causing an influx of fluid due to a change in osmolarity of the intestinal contents. When the large intestine is 4 reached, normal bacteria in the colon further break down fructose into many gases, including methane and hydrogen (Choi et al. 1348). Symptoms of fructose malabsorption include vomiting, diarrhea, and stomach pains after the consumption of fructose. Symptoms last only a few hours and do not typically cause permanent problems. For HFI and fructose malabsorption, the only treatment is a lifelong fructose-free diet (Choi et al. 1348). METHODS OF DIAGNOSIS: There are two main ways to diagnose HFI. The first and most risky method is an intravenous fructose load test. In an intravenous fructose load test, fructose is intravenously injected into the patient. Over a set amount of time, fructose, phosphate, and glucose levels are observed (Kullberg-Lindh et al. 572). The main concern with this method is the damage it can cause, especially in newborns. The second method is an aldolase B assay on a liver biopsy. In this method, a biopsy is obtained from the liver and aldolase B activity is measured (Coffee et al. 34). Since both methods are invasive, DNA analysis is becoming increasingly accepted as a method to determine HFI. This method requires lymphocytes filtered from a blood sample. The DNA in the lymphocytes then undergoes polymerase chain reaction (PCR) technique. PCR allows a few strands of DNA to be duplicated, producing thousands of copies. After PCR, DNA is screened for aldolase B mutations (Esposito et al. “Structural and Functional Analysis”; Santer, et al. 2). MUTATIONS: 5 HFI is caused by a mutation in the aldolase B gene. To date, at least thirty-five mutations, (Santer et al. 2) but as many as forty-five mutations, have been identified (Davit-Spraul et al. 443). The four most common mutant alleles are the A149P, A174D, L288ΔC, and N334K. A149P is a missense mutation in exon 5 that causes a guanine to cytosine transversion. The change from a purine to pyrimidine causes proline to be produced instead of alanine (Esposito et al. 153). This particular mutation accounts for 44% of the total disease-causing alleles in the world population (Coffee et al. 33). A174D, making up 9% of mutant alleles, is a missense mutation that codes for aspartate instead of alanine (Coffee et al. 33; Esposito et al. 153). L288ΔC, a deletion mutation, deletes a base pair and causes a frameshift which codes for a stop codon. N334K, a missense mutation, causes lysine to be coded instead of asparagine (Coffee et al. 33-34; Esposito et al. 153). Δ4E4 and R59Op are two other mutations worth mentioning. Both are nonsense mutations that cause a stop codon to code. Each mutation makes up 4% of alleles (Coffee et al. 33). SIGNIFICANCE OF RESEARCH: Since HFI is becoming more prevalent, research on this topic is needed to help those affected. The symptoms of HFI can be very severe and even fatal. Within hours of fructose consumption, the liver and kidneys can have a global breakdown, which manifest as coagulopathy and anuria, respectively (Fauth and Halmágyi 213). Due to the risks involved with intravenous fructose load test and liver biopsy, there is a greater need for DNA analysis to be used as a diagnostic test. DNA analysis does not harm the patient and allows doctors to better understand what is affecting their 6 patient (Coffee et al. 34). With each new study, novel disease-causing mutations are discovered along with more efficient methods to identify known mutations. Even research on common mutations adds insight into the disease. The more knowledge there is about HFI and the mutations that cause HFI, the better the outcome patients have. REVIEW OF LITERATURE: Davit-Spraul et al. conducted a study on aldolase B mutation frequencies in France. 160 patients from ninety-two unrelated families were diagnosed with HFI based on DNA analysis, positive intravenous fructose load test, and positive aldolase B assay on liver biopsy. The majority of the families were French, while the rest were a mixture of Belgian and Mediterranean immigrants. In this study, the A149P, A174D, N334K, A338V, R60X, Δ4E4, K108K, and c.625-1G>A mutations were observed in this population (443-447). Eight new mutations were also found. In intron two, the c.112+1G>A mutation affected the splicing site. Exon three contained two new mutations: p.V49GfsX27 (c.146delT), where a thymine was deleted and p.R57P (c.170G>C), where a cytosine replaced a guanine. In exon five, the p.W148X (c.444G>A) nonsense mutation replaced a guanine with an adenine. In exon seven, the p.K230MfsX136 (c.689-690insTGCTAA) mutation inserted six extra bases. In exon eight, there were three new mutations: p.A280P (c.839C>A), p.L311P (c.932T>C), and p.A318-A332del (c.953-994del42bp). These mutations replace cytosine with adenine, thymine with cytosine, and delete forty- 7 two bases (fourteen amino acid residues not coded), respectively (Davit-Spraul et al. 444-446). Sanchez-Gutierrez et al. conducted a similar study on aldolase B mutations in Spain. Twenty-eight patients with HFI were used. Clinical symptoms and positive aldolase B assay on liver biopsy were used to diagnosis HFI. In this study, the A149P, A174D, N334K, and Δ4E4 mutations were observed in this population (1). The study identified two new mutations, g.4271C>G and g.1133G>A. The g.4271C>G (P184R) mutation causes guanine to replace cytosine, so an arginine is produced instead of proline. This mutation also causes a deletion of a certain restriction site needed for mutation detection. g.1133G>A (V104_K107del) causes a deletion of twelve bases from the aldolase B gene. Removing twelve bases would cause four amino acids to be left out of the final protein (Sanchez-Gutierrez et al. 4-5). Sebastio et al. conducted a study on aldolase B mutations in Italy. Eleven patients of Italian ethnicity were used in the study. HFI was diagnosed by positive intravenous fructose load test or aldolase B assay on liver biopsy. Again, the A149P, A174D, and N334K mutations were identified in the study. In this study, no rearrangements, deletions, or any new mutations were noted of the aldolase B gene (Sebastio et al. 241-243) Gruchota et al. studied the aldolase B mutation spectrum of Polish patients. Their goals were to establish a carrier rate of certain mutations and estimate disease frequency. Twenty-eight patients were studied. HFI was diagnosed through a fructose load test and clinical symptoms (Gruchota et al. 376). 8 Six mutations were noted: A149P, A174D, c.313-314ins12nt, g.922-925delgGTA, c.360-363delCAAA, and p.Y204X. Two new mutations were also found: c.250delC and c.522C>G. These new mutations have been found to delete the active center of the enzyme, which suggests they are pathogenic. This study was able to determine two new mutations and estimate HFI frequency in Poland (Gruchota et al. 377). Santer et al. conducted a study on the range and frequency of aldolase B mutations in Central Europe (1). Eighty patients from seventy-two different families were studied. German was the major ethnicity with the remaining subjects being Mediterranean immigrants. HFI was diagnosed by presence of clinical symptoms, positive aldolase B assay on liver biopsy, positive intravenous fructose load test, and DNA analysis. In this study, fifteen different mutations were identified. The common mutations A149P, A174D, N334K, c.360-363delCAAA, p.R60X, c.865delC, and p.Y204X (c.612T>A) were noted in this study. Eight new mutations were also noted: c.113-1G>A in intervening sequence two, c.345-72del28 in exon four, c.532T>C in exon five, c.612T>A in exon six, c.799+2T>A in intervening sequence seven, c.841-842delAC and c.1005C>G in exon eight, and c.1044-1049delTTCTGGinsACACT in exon nine (Santer et al. 4-5). Coffee et al. performed a study on the incidence of aldolase B mutations in an American population. 153 patients from 131 different families were studied. The ethnic origins of the subjects included Argentina, Brazil, Canada, and the United States (84% of subjects) (Coffee et al. 34-35). 9 In this study, mutations A149P, A174D, N334K, Δ4E4, R59Op, L256P, and A337V were found. The American population differed from the European populations in that the most common mutations were A149P, A174D, Δ4E4 and R59Op not A149P, A174D, N334K (Coffee et al. 35-37). In this study, Coffee et al. were able to determine that aldolase B is not needed for metabolic preservation or proper development (33, 39). CRITICAL ANALYSIS OF LITERATURE: Davit-Spraul et al. studied spectrum and frequencies of mutations in a French population. They used 160 individuals from ninety-two unrelated families. They collected DNA samples and screened for the three most recurrent mutations. With the screening, they were able to confirm HFI in 75% of the subjects. Of the three common mutations, they found A149P with a prevalence of 64%, A174D with 16%, and N334K with 5% (Davit-Spraul et al. 443-447). This study did well in obtaining a relatively large test group. Their mutation frequencies did reflect the results of many other studies. Though this study is very thorough in describing known and novel mutations, more information could have been given regarding their method of DNA analysis. Sanchez-Gutierrez et al. aimed to identify mutations in the aldolase B gene in subjects from Spain. Twenty-eight subjects with HFI were studied. DNA samples were obtained from the subjects and were screened for A149P, A174D, and Δ4E4. The three previous mutations were found at frequencies of 67.4%, 9.3%, and 16.3%, respectively (Sanchez-Gutierrez et al. 6-7). This study did not have quite as large of a population, but was sound in statistics. The study was very difficult to follow due to too much 10 irrelevant information. The study also attempted to correlate genotypes and aldolase B activity, but was unsuccessful for the reason that there was not enough data. Future research on a correlation between genotypes and aldolase B activity should be attempted again, due to the significance of HFI. Sebastio et al. investigated the prevalence of four common mutations. Eleven unrelated, HFI patients from Italy were observed. DNA samples were taken and screened for A149P, A174D, N334K, and L288ΔC. The frequencies in this population were found to be 50%, and 35%, with the last two frequencies not being recorded (Sebastio et al. 241-243). This study had a very small population, and did not explain the results clearly. Instead of stating findings, the paper hid behind its overly complex method section. The study attempted to relate clinical symptoms to genotype but was unable, probably due to the small population and too little data. If this study were to be conducted again, a larger population should be used. Gruchota et al. targeted mutation profiles of Polish patients. They aimed to conclude frequency rate of common mutations and prevalence of HFI in Poland. DNA samples were taken from twenty-eight patients and screened for mutations. A149P was the only mutation that was noted with a frequency (67.3%). The prevalence of disease was determined to be 1:31,000 (Gruchota et al. 377). Despite the study’s objective of reporting the frequency rate of common mutations, only the most recurring mutation was noted. After correction for numbers of comparison, the values that were noted were not statistically significant. The only accomplishment of this study seemed to be in that two new mutations were discovered and an estimate of HFI prevalence was determined. 11 Santer et al. examined HFI mutation spectrum and the prevalence of disease in Central Europe. DNA samples from eighty subjects were screened for aldolase B gene mutations. Fifteen different mutations were noted, and by screening for A149P, A174D, and N334K, HFI was confirmed in 72% of subjects. In the overall study, HFI was confirmed in 93% of subjects by DNA analysis. The prevalence of HFI in Central Europe was also determined to be 1:26,100 (Santer et al. 1). The population size of this study was appropriate and the objectives were met. Results were similar to the other studies, and statistics were found to be significant. Coffee et al. aimed to determine profile and prevalence of mutant null alleles in an American population. DNA samples were taken from 153 patients and screened for A149P, A174D, and N334K. The prevalence of these mutations was found to be 44%, 9%, and 2%, respectively (Coffee et al. 36). This study had a large population, and mutation frequencies reflected worldwide values. The study was very thorough in describing mutation profile and frequency and took into account many other studies. FUTURE RESEARCH: More research should be done due to how potentially deadly this disease can be. Since Sanchez-Gutierrez et al. found a lack of determinants of aldolase B activity in the liver of affected patients, the effects of g.4271C>G on aldolase activity would need more study. Also, since g.1133G>A deleted four amino acids, protein stability and enzymatic properties were affected and would apparently add to the phenotype, but more evidence would be needed to support this (Sanchez-Gutierrez et al. 7). 12 Santer et al. found eight new mutations in their study. The effects of these mutations can be predicted due to the amino acids they affect, though more studies would be needed to prove they actually cause HFI. Since there is a high rate of the common mutations in the central European population, there is a possibility that mutations can be screened for in all neonates. Further studies would be able to make this procedure even more widely accepted and more efficient (Santer et al. 7-8) Davit-Spraul et al. used long range PCR in their study for DNA analysis. This allowed them to check for abnormalities in the aldolase B gene. LR-PCR would be beneficial for studies at the RNA level so that abnormal splicing could be found. This would help give a better understanding of the mutations that cause HFI (Davit-Spraul, et al. 447). Celiac disease has been known to have strong associations with other gastrointestinal disorders. In a study on genetic association between Celiac disease (CD) and HFI, Ciacci et al. noted that CD and HFI were more associated than CD and other disorders were (636). More research should be done on associations between HFI and other gastrointestinal disorders so there would be better treatment outcomes for afflicted individuals. CONCLUSION: HFI is an increasing genetic disease that causes many severe and occasionally fatal symptoms. To date, more than thirty-five mutations that impair the aldolase B enzyme have been discovered. The most common mutations worldwide are A149P, A174D, L288ΔC, and N334K. In new studies, novel mutations are found, allowing for 13 more comprehensive profile of HFI. 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