Advertisement

A heterozygous mutation in GOT1 is associated with familial macro-aspartate aminotransferase

  • Author Footnotes
    † These authors contributed equally to this work.
    Maria Kulecka
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 01-813, Poland
    Search for articles by this author
  • Author Footnotes
    † These authors contributed equally to this work.
    Aldona Wierzbicka
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Department of Biochemistry, Radioimmunology and Experimental Medicine, Children’s Memorial Health Institute, Warsaw 04-730, Poland
    Search for articles by this author
  • Agnieszka Paziewska
    Affiliations
    Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 01-813, Poland
    Search for articles by this author
  • Michal Mikula
    Affiliations
    Department of Genetics, Cancer Center-Institute, Warsaw 02-781, Poland
    Search for articles by this author
  • Andrzej Habior
    Affiliations
    Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 01-813, Poland
    Search for articles by this author
  • Wojciech Janczyk
    Affiliations
    Department of Gastroenterology, Hepatology and Feeding Disorders, Children’s Memorial Health Institute, Warsaw 04-730, Poland
    Search for articles by this author
  • Michalina Dabrowska
    Affiliations
    Department of Genetics, Cancer Center-Institute, Warsaw 02-781, Poland
    Search for articles by this author
  • Jakub Karczmarski
    Affiliations
    Department of Genetics, Cancer Center-Institute, Warsaw 02-781, Poland
    Search for articles by this author
  • Michal Lazniewski
    Affiliations
    Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Warsaw 02-089, Poland

    Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Warsaw 02-097, Poland
    Search for articles by this author
  • Krzysztof Ginalski
    Affiliations
    Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Warsaw 02-089, Poland
    Search for articles by this author
  • Anna Czlonkowska
    Affiliations
    Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland
    Search for articles by this author
  • Piotr Socha
    Correspondence
    Corresponding authors. Addresses: Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 01-813, Poland. Tel.: +48 225462575; fax: +48 225463191, or Department of Gastroenterology, Hepatology and Feeding Disorders, Children’s Memorial Health Institute, Warsaw 04-730, Poland.
    Affiliations
    Department of Gastroenterology, Hepatology and Feeding Disorders, Children’s Memorial Health Institute, Warsaw 04-730, Poland
    Search for articles by this author
  • Jerzy Ostrowski
    Correspondence
    Corresponding authors. Addresses: Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 01-813, Poland. Tel.: +48 225462575; fax: +48 225463191, or Department of Gastroenterology, Hepatology and Feeding Disorders, Children’s Memorial Health Institute, Warsaw 04-730, Poland.
    Affiliations
    Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education, Warsaw 01-813, Poland

    Department of Genetics, Cancer Center-Institute, Warsaw 02-781, Poland
    Search for articles by this author
  • Author Footnotes
    † These authors contributed equally to this work.
Open AccessPublished:July 14, 2017DOI:https://doi.org/10.1016/j.jhep.2017.07.003

      Highlights

      • Individuals with macro-AST demonstrate fluctuating, not persistent, elevation of AST levels in blood.
      • The causative gene of macro-AST has not yet been identified.
      • A genetic variant associated with macro-AST was found in theGOT1 gene.
      • Testing for this variant could aid diagnosis of macro-AST.

      Background & Aims

      Macro-aspartate aminotransferase (macro-AST) manifests as a persistent elevation of AST levels, because of association of the protein with immunoglobulins in the circulation. Macro-AST is a rare, benign condition without a previously confirmed genetic basis.

      Methods

      Whole exome sequencing (WES)-based screening was performed on 32 participants with suspected familial macro-AST, while validation of variants was performed on an extended cohort of 92 probands and 1,644 healthy controls using Taqman genotyping.

      Results

      A missense variant (p.Gln208Glu, rs374966349) in glutamate oxaloacetate transaminase 1 (GOT1) was found, as a putative causal variant predisposing to familial macro-AST. The GOT1 p.Gln208Glu mutation was detected in 50 (54.3%) of 92 probands from 20 of 29 (69%) families, while its prevalence in healthy controls was only 0.18%. In silico analysis demonstrated that the amino acid at this position is not conserved among different species and that, functionally, a negatively charged glutamate on the GOT1 surface could strongly anchor serum immunoglobulins.

      Conclusions

      Our data highlight that testing for the p.Gln208Glu genetic variant may be useful in diagnosis of macro-AST.

      Lay summary

      Higher than normal levels of aspartate aminotransferase (AST) in the bloodstream may be a sign of a health problem. Individuals with macro-AST have elevated blood AST levels, without ongoing disease and often undergo unnecessary medical tests before the diagnosis of macro-AST is established. We found a genetic variant in the GOT1 gene associated with macro-AST. Genetic testing for this variant may aid diagnosis of macro-AST.

      Graphical abstract

      Keywords

      Introduction

      Macroenzymes, serum high-molecular-weight compounds that are formed by polymerization or association with other serum constituents (primarily immunoglobulins
      • Moriyama T.
      • Tamura S.
      • Nakano K.
      • Otsuka K.
      • Shigemura M.
      • Honma N.
      Laboratory and clinical features of abnormal macroenzymes found in human sera.
      ), are rare in the general healthy population.
      • Lee M.
      • Vajro P.
      • Keeffe E.B.
      Isolated aspartate aminotransferase elevation: think macro-AST.
      There is evidence that they may be associated with hepatitis C;
      • Collins J.
      • Ritter D.
      • Bacon B.R.
      • Landt M.
      • Creer M.H.
      Macro-aspartate aminotransferase in a female with antibodies to hepatitis C virus.
      autoimmune conditions, including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, ulcerative colitis, and celiac disease;
      • Galasso P.J.
      • Litin S.C.
      • O’Brien J.F.
      The macroenzymes: a clinical review.
      • Asanuma K.
      • Yagihashi A.
      • Watanabe N.
      Characterization of the aspartate aminotransferase-immunoglobulin M complex in a Japanese woman.
      • Triester S.L.
      • Douglas D.D.
      Development of macro-aspartate aminotransferase in a patient undergoing specific allergen injection immunotherapy.
      • Werner T.
      • Vargas H.E.
      • Chalasani N.
      Macro-aspartate aminotransferase and monoclonal gammopathy: a review of two cases.
      • Zaman Z.
      • Van Orshoven A.
      • Mariën G.
      • Fevery J.
      • Blanckaert N.
      Simultaneous macroamylasemia and macrolipasemia.
      and neoplastic disorders.
      • Moriyama T.
      • Tamura S.
      • Nakano K.
      • Otsuka K.
      • Shigemura M.
      • Honma N.
      Laboratory and clinical features of abnormal macroenzymes found in human sera.
      • Konttinen A.
      • Murros J.
      • Ojala K.
      • Salaspuro M.
      • Somer H.
      • Räsänen J.
      A new cause of increased serum aspartate aminotransferase activity.
      A typical macroenzyme is the macroenzyme form of aspartate aminotransferase (macro-AST), the presence of which is generally characterized by increased serum AST activity.
      AST is highly conserved and expressed in many cell types, including hepatocytes, erythrocytes, and skeletal muscle cells. There are two similar isoenzymes of AST that localize to the cytoplasm and mitochondria and are encoded by GOT1 and GOT2 on chromosomes 10q24 and 16q12, respectively. Serum AST activity is largely of cytosolic origin,
      • Shen H.
      • Damcott C.
      • Shuldiner S.R.
      • Chai S.
      • Yang R.
      • Hu H.
      • et al.
      Genome-wide association study identifies genetic variants in GOT1 determining serum aspartate aminotransferase levels.
      and, while elevated serum AST activity is considered a basic biochemical marker of hepatic, cardiac, muscle, endocrine, and metabolic disorders, macro-AST is generally a benign condition. However, the diagnosis of macro-AST is usually delayed because of confusion about the reason for elevated AST activity, and many unnecessary medical tests and procedures are frequently performed, including invasive liver biopsies, before the final diagnosis is established.
      • Moriyama T.
      • Tamura S.
      • Nakano K.
      • Otsuka K.
      • Shigemura M.
      • Honma N.
      Laboratory and clinical features of abnormal macroenzymes found in human sera.
      No specific treatment is required for macro-AST.
      Macro-AST, which is a type I macroenzyme,
      • Lee M.
      • Vajro P.
      • Keeffe E.B.
      Isolated aspartate aminotransferase elevation: think macro-AST.
      is produced by formation of an ∼250 kDa complex of AST with serum immunoglobulins (IgA, IgG, or both), and the specific enzyme binding site is mostly on the Fab and F(ab1)2 fragment of the immunoglobulin molecule,
      • Moriyama T.
      • Tamura S.
      • Nakano K.
      • Otsuka K.
      • Shigemura M.
      • Honma N.
      Laboratory and clinical features of abnormal macroenzymes found in human sera.
      leading to altered renal clearance and excretion of the enzyme and, ultimately, to higher levels of activity. The mechanisms of the immune complex formation are unclear, but may be due to autoimmunity, with immunoglobulins targeting enzymes as antigens via molecular mimicry.
      • Remaley A.T.
      • Wilding P.
      Macroenzymes: biochemical characterization, clinical significance, and laboratory detection.
      Macro-AST was first reported by Konttinen et al.
      • Konttinen A.
      • Murros J.
      • Ojala K.
      • Salaspuro M.
      • Somer H.
      • Räsänen J.
      A new cause of increased serum aspartate aminotransferase activity.
      in 1978 in two healthy women with an unexplained persistent elevation of AST activity. Their serum AST isoenzymes contained a fraction that migrated between cytoplasmic and mitochondrial AST on electrophoresis, and an AST-IgG complex was confirmed in one of them.
      • Konttinen A.
      • Murros J.
      • Ojala K.
      • Salaspuro M.
      • Somer H.
      • Räsänen J.
      A new cause of increased serum aspartate aminotransferase activity.
      Subsequently, several reports on macro-AST have been published (summarized in
      • Moriyama T.
      • Tamura S.
      • Nakano K.
      • Otsuka K.
      • Shigemura M.
      • Honma N.
      Laboratory and clinical features of abnormal macroenzymes found in human sera.
      ), wherein diagnosis was performed using different methods, including electrophoresis, gel permeation chromatography, polyethylene glycol precipitation, and heat stability measurements.
      • Stasia M.J.
      • Surla A.
      • Renversez J.C.
      • Pene F.
      • Morel-Femelez A.
      • Morel F.
      Aspartate aminotransferase macroenzyme complex in serum identified and characterized.
      Macro-AST is rare in the general population, with a prevalence of 0.014% and 9.09% among a general population of gastroenterological patients and those with isolated increased AST activity without liver abnormalities, respectively.
      • Patteet L.
      • Simoens M.
      • Piqueur M.
      • Wauters A.
      Laboratory detection of macro-aspartate aminotransferase: case report and evaluation of the PEG-precipitation method.
      While in adult patients macro-AST is often accompanied by other diseases, including neoplasms and autoimmune diseases, isolated macro-AST is more frequently identified in children.
      • Lee M.
      • Vajro P.
      • Keeffe E.B.
      Isolated aspartate aminotransferase elevation: think macro-AST.
      • Triester S.L.
      • Douglas D.D.
      Development of macro-aspartate aminotransferase in a patient undergoing specific allergen injection immunotherapy.
      To date, no genetic tendency has been described in either adult or pediatric macro-AST, probably because of the rarity of the condition, particularly in children. Only a single case of suspected familial macro-AST has previously been reported in a mother and her 2-month-old baby with macro-AST, who were otherwise healthy.
      • Orlando R.
      • Lirussi F.
      Macro-aspartate aminotransferase and pregnancy: any influence?.

      Patients and methods

      Patients

      Between 2005 and 2016, a clinical diagnosis of macro-AST was established in 69 out of 744 pediatric patients admitted to the Departments of Gastroenterology, Hepatology, Nutritional Disorders, or Pediatrics, Children’s Health Memorial Institute, Warsaw for diagnosis of an unexplained increase in AST activity levels. The patients affected by macro-AST exhibited asymptomatic, fluctuating (rather than persistent) increased AST activity observed over months or years. None of these children had suffered from any major medical problems, except one who was diagnosed with neuroblastoma during clinical follow-up. None were treated with hepatotoxic drugs or with drugs that could affect liver enzyme activities, none had symptoms of liver disease or cholelithasis, and neuromuscular status was normal. Except for elevated serum AST activity, the results of the following tests were normal: serum alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, cholinesterase, creatine kinase and its isoenzymes, lactate dehydrogenase and its isoenzymes, aldolase, amylase, total bilirubin, conjugated bilirubin, and prothrombin time. Tests for viral (hepatitis A, B, and C viruses, cytomegalovirus, and Epstein-Barr virus), metabolic (α-1-antitrypsin, ceruloplasmin, and urinary copper before and after penicillamine load), and autoimmune (antinuclear, anti-smooth muscle, antimitochondrial, and anti-liver-kidney microsomal and anti-transglutaminase antibodies) conditions also revealed no abnormalities. Liver ultrasound was normal. Additionally, hemolytic, renal, and pancreatic causes of elevated AST activity were excluded.

      Laboratory tests for diagnosis of macro-AST

      Polyethylene glycol (PEG) precipitation tests. Equal volumes (100 μl) of a 24% solution of PEG 6000 and patients’ sera were mixed, equilibrated for 10 min. at 37 °C and centrifuged at 1,500g for 20 min at 22 °C. To calculate the PEG precipitable activity (PPA), AST activity was measured in the supernatant, and after correction for dilution was compared with samples obtained from unprecipitated serum.
      AST isoenzyme electrophoresis. Electrophoresis on 1% agarose gel was carried out in a barbiturate buffer (pH 8.6) at 100 Volts, then gels were soaked in a staining reagent mixture (10 µM alpha-ketoglutarate, 200 µM L-cysteine sulfinate, 0.1 mg m-PMS, 0.8 mg MTT, 2 µM EDTA, 20 mg dextran and 100 µM imidazole buffer [pH 7.5]) for 20 min. at 37 °C, immersed in 10% acetic acid for 5 min, washed with water and dried in an oven at 65 °C. Purple bands indicated the presence of free-AST-molecules or AST complexes.

      DNA isolation, exome sequencing, and genotyping

      The study protocol was approved by the local Bioethical Committee, and all participants, or their parents, provided written informed consent. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Genomic DNA was extracted from whole blood treated with EDTA as previously described.
      • Gaj P.
      • Kluska A.
      • Nowakowska D.
      • Bałabas A.
      • Piątkowska M.
      • Dabrowska M.
      • et al.
      High frequency of BRCA1 founder mutations in Polish women with nonfamilial breast cancer.
      Human exome sequencing libraries were constructed using AmpliSeq™ Exome technology (Thermo Fisher Scientific), and generated amplicons were sequenced using the Ion Proton™ platform (Thermo Fisher Scientific) as previously described.
      • Ostrowski J.
      • Paziewska A.
      • Lazowska I.
      • Ambrozkiewicz F.
      • Goryca K.
      • Kulecka M.
      • et al.
      Genetic architecture differences between pediatric and adult-onset inflammatory bowel diseases in the Polish population.
      Raw reads were processed using the Torrent Suite version 5.0 analysis pipeline and mapped to human genome assembly hg19 with TMAP Aligner (Thermo Fisher Scientific). Variant calls were made using Torrent Variant Caller version 5.0-13

      Torrent Variant Caller. GitHub https://github.com/iontorrent/TS​ ​(accessed July 21, 2015).

      with the default parameters for germline variant calling. Variant calls were filtered with parameters detailed by Damiati et al.
      • Damiati E.
      • Borsani G.
      • Giacopuzzi E.
      Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.
      with high stringency for base insertions/deletions (indels), and low stringency for single nucleotide polymorphisms (SNPs). If multiple alleles were reported in one position, they were separated using the vcflib script, vcfbreakmulti.

      Garrison, Erik. vcflib : a C++ library for parsing and manipulating VCF files. GitHub https://github.com/ekg/vcflib (accessed July 21, 2015).

      Variants were annotated using Variant Effect Predictor
      • McLaren W.
      • Pritchard B.
      • Rios D.
      • Chen Y.
      • Flicek P.
      • Cunningham F.
      Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor.
      release 86. Statistical tests were conducted using rvtests version 20150104.
      • Zhan X.
      • Hu Y.
      • Li B.
      • Abecasis G.R.
      • Liu D.J.
      RVTESTS: An efficient and comprehensive tool for rare variant association analysis using sequence data.
      software with imputation based on Hardy-Weinberg equilibrium. For very rare (the Exome Aggregation Consortium [ExAC] and Kaviar
      • Glusman G.
      • Caballero J.
      • Mauldin D.E.
      • Hood L.
      • Roach J.C.
      Kaviar: an accessible system for testing SNV novelty.
      overall minor allele frequency [MAF] <1%), non-synonymous variants, the Fisher’s exact test for allele over-representation was conducted using WES data obtained from a cohort of 77 pediatric patients with Wilson’s disease (manuscript in preparation) as controls. Variants that reached genome-wide statistical significance were additionally checked using a variant-filter script.

      Kandoth, Cyriac, Koboldt, Dan, Larson, David. variant-filter. GitHub https://github.com/ckandoth/variant-filter (accessed July 17, 2015).

      Genotyping of the rs374966349 variant was performed with TaqMan SNP Genotyping Assays (Thermo) on an ABI 7900HT qPCR system (Thermo), as previously described.
      • Gaj P.
      • Kluska A.
      • Nowakowska D.
      • Bałabas A.
      • Piątkowska M.
      • Dabrowska M.
      • et al.
      High frequency of BRCA1 founder mutations in Polish women with nonfamilial breast cancer.

      Data availability

      Sequencing data (fragments of BAM files containing variant rs374966349) have been deposited in The European Nucleotide Archive under the accession number PRJEB20091.
      For further details regarding the materials used, please refer to the Supplementary material and the CTAT table.

      Results

      Pediatric patients admitted with unexplained elevated levels of AST activity and no evidence of associated diseases, underwent PEG screening test and AST isoenzyme electrophoresis examination to confirm or exclude the presence of macro-AST. Although the PEG precipitation technique has been considered a gold standard for the diagnosis of macro-AST, PPA reference ranges are still not defined.
      • Caropreso M.
      • Fortunato G.
      • Lenta S.
      • Palmieri D.
      • Esposito M.
      • Vitale D.F.
      • et al.
      Prevalence and long-term course of macro-aspartate aminotransferase in children.
      In our study, gel electrophoresis of the sera with PPA of <52% exhibited typical electrophoretic patterns of isoenzymes, composed of an anionic and a cationic band, which represent the cystolic or supernatant (cAST/sAST) and the mitochondrial (mAST) isozymes, respectively. In turn, in 69 patients whose PPA ranged between 59% and 78%, electrophoresis revealed a single band with an abnormal migration, either between the sAST and the mAST bands or near to the sAST position. As suggested by Moriyama et al., the abnormal bands may indicate the type, amount and isoenzyme specificity of bound immunoglobulin and may differentiate between IgG and IgA complexed-AST.
      • Moriyama T.
      • Tamura S.
      • Nakano K.
      • Otsuka K.
      • Shigemura M.
      • Honma N.
      Laboratory and clinical features of abnormal macroenzymes found in human sera.
      Once macro-AST was diagnosed, the diagnostic procedure for macro-AST was also performed in parents and siblings. In 37 families, at least one other family member was also macro-AST-positive (familial macro-AST), while all relatives from the other 32 families were macro-AST-negative (sporadic macro-AST). To investigate the genetic background of patients with suspected familial macro-AST, whole exome sequencing (WES)-based screening was performed on 32 participants, including 15 male children, eight female children, six male adults, and three female adults.
      On average, 21,514,330 reads (median = 21,415,862) that mapped to human genome assembly hg19 were generated per sequencing run. An average of 74% of bases exhibited coverage of ⩾20×. In patient samples, 65,472 variants were discovered in coding regions: 1,423 indels and 64,244 SNPs. Each patient had a mean of 18,444 variants, of which 18,257 were SNPs and 177 were indels. Among these, four rare non-synonymous variants passed all filters and reached the genome-wide significance level (p <5×e−8) by Fisher’s exact test, including a rare (MAF in ExAC database as low as 0.02%) missense variant in GOT1, encoding p.Gln208Glu (rs374966349; two-tailed p = 1.73×e−11). Other variants were located in the genes MUC4 (two variants) and NBPF10; however, according to their Gene Damage Index Scores, both of these genes are highly tolerant to mutations.
      • Itan Y.
      • Shang L.
      • Boisson B.
      • Patin E.
      • Bolze A.
      • Moncada-Vélez M.
      • et al.
      The human gene damage index as a gene-level approach to prioritizing exome variants.
      The rs374966349 variant was further genotyped in extended cohorts of probands and healthy controls. Among 1,644 controls, only three individuals were carriers of the heterozygous GOT1 mutation, indicating a prevalence of 0.18%. The mutated genotype was detected in 38 and 6 macro-AST–positive and macro-AST–negative probands, respectively, recruited from families with familial macro-AST (Fig. 1). Out of 12 patients who were macro-AST–positive, but only had macro-AST–negative relatives (sporadic macro-AST), three children were carriers of the heterozygous mutations; among their family members one mutation carrier was found in each of the three families who were otherwise macro-AST–negative (Fig. 1, family #3, #7 and #20). The only homozygous GOT1 mutation was found in a father of two sons; all three of them were affected by macro-AST (Fig. 1, family #16). Altogether, the GOT1 mutation was present in 50 probands from 20 (69%) families studied; 41 carriers of the mutation were macro-AST–positive and nine were macro-AST–negative (Fig. 1). While this mutation has less than 100% penetrance, and in nine (31%) families with familial macro-AST no mutation was detected, we cannot exclude the possibility that familial macro-AST has polygenic inheritance, in which the same clinical phenotype arises from different impact loci and/or from additive interactions between them.
      Figure thumbnail gr1
      Fig. 1Prevalence of the glutamate oxaloacetate transaminase 1 (GOT1) p.Gln208Glu mutation in families with at least two occurrences of macro-aspartate aminotransferase.
      The serum AST activity levels are summarized in Table 1. As expected, elevated AST activity was determined in macro-AST–positive patients; normal or slightly over the upper limit levels were found in the macro-AST–negative carriers of GOT1 mutations; and normal levels were detected in macro-AST–negative relatives exhibiting the wild-type GOT1 genotype. Interestingly, in subjects diagnosed with macro-AST who were the mutation carriers, serum AST levels were significantly higher compared to non-carriers (p value = 0.043; Welch Two Sample t test) (Fig. 2).
      Table 1Relation between the GOT1 genotype and serum AST activity levels in patients with a clinically diagnosed familial macro-AST and their unaffected relatives.
      GOT1 genotype/clinical diagnosis of macro-AST
      19 families with mutated genotype9 families with wild-type genotype
      Mutated/Macro-AST positiveMutated/Macro-AST negativeWild type/Macro-AST negativeWild type/Macro-AST positiveWild type/Macro-AST negative
      Proband number419131811
      AST; min–max55–22826–5413–4045–17313–35
      AST; median9137317017
      AST, aspartate aminotransferase; normal range, 10–40 U/L.
      Figure thumbnail gr2
      Fig. 2The serum aspartate aminotransferase (AST) activity levels in subjects diagnosed with macro-AST who were either carriers or non-carries of glutamate oxaloacetate transaminase 1 p.Gln208Glu mutation. The statistical difference was assessed with Welch Two Sample t test.
      To characterize the potential influence of the identified missense mutation on protein function at the structural level, we mapped the mutation onto the three-dimensional structure of the human GOT1 homodimer (pdb|3ii0) (Fig. 3). This analysis demonstrated that GOT1 p.Gln208Glu is located away from the protein’s active site, on the surface of a helix exposed to the solution, and on the opposite side from the site that mediates dimerization. Further bioinformatic analysis revealed that the mutated amino acid at this position is not conserved in different species. Overall, the results indicate that this mutation, which generates a surface exposed, negatively charged glutamate, may have a causative function in macro-AST by inducing a stronger association between AST and serum components, likely immunoglobulins. Although this rare variant is incompletely penetrant and probably not the only factor causing familial macro-AST, testing for this genetic variant could be useful in diagnosis of macro-AST, at least in some families.
      Figure thumbnail gr3
      Fig. 3Location of the p.Gln208Glu mutation, identified in patients with macro-aspartate aminotransferase (AST), in the structure of the human glutamate oxaloacetate transaminase 1 homodimer. The amino acid mutated in macro-AST is shown in red. Monomers are colored blue and gray, while key residues from both active sites are indicated in yellow and magenta. (This figure appears in colour on the web.)
      While genetic diseases or rare pathogenic mutations are frequently reported, many rare diseases remain undiagnosed.
      • Shashi V.
      • McConkie-Rosell A.
      • Rosell B.
      • Schoch K.
      • Vellore K.
      • McDonald M.
      • et al.
      The utility of the traditional medical genetics diagnostic evaluation in the context of next-generation sequencing for undiagnosed genetic disorders.
      Up to 25–30 million people and 25% of pediatric patients in the United States may be affected by a rare genetic disease.
      • Farwell K.D.
      • Shahmirzadi L.
      • El-Khechen D.
      • Powis Z.
      • Chao E.C.
      • Tippin Davis B.
      • et al.
      Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.
      WES is a cost-effective technology for detection of disease variants underlying Mendelian disorders, as well as for cataloguing common and rare disease-related genomic alterations;
      • Auer P.L.
      • Lettre G.
      Rare variant association studies: considerations, challenges and opportunities.
      • Biesecker L.G.
      • Green R.C.
      Diagnostic clinical genome and exome sequencing.
      however, its diagnostic yield depends on age of disease onset, the presence of a positive family history, and specific clinical phenotypes. The diagnostic success rate for WES-based identification of rare causative variants is only 15–30%.
      • Farwell K.D.
      • Shahmirzadi L.
      • El-Khechen D.
      • Powis Z.
      • Chao E.C.
      • Tippin Davis B.
      • et al.
      Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.
      • Biesecker L.G.
      • Green R.C.
      Diagnostic clinical genome and exome sequencing.
      • Zhu X.
      • Petrovski S.
      • Xie P.
      • Ruzzo E.K.
      • Lu Y.-F.
      • McSweeney K.M.
      • et al.
      Whole-exome sequencing in undiagnosed genetic diseases: interpreting 119 trios.
      Identification of macro-AST causative variants from existing incidental findings and variants of unknown/uncertain significance was possible because, over 11 years, we have assembled the largest cohort of macro-AST patients published to date. Furthermore, we describe familial macro-AST for the first time. Selection of such a large number of patients was possible because staff in our Departments of Gastroenterology, Hepatology, Nutritional Disorders, and Pediatrics at the Children’s Health Memorial Institute are trained in diagnosis of macro-AST, and 744 pediatric patients with unexplained AST elevation have been directed to our diagnostic center from hospitals across Poland. Furthermore, when well-defined macro-AST was established in a pediatric patient, the diagnostic procedure was extended to other members of the immediate family. Finally, WES-based genetic screening was conducted in an academic laboratory to identify a non-synonymous change in the gene encoding cytoplasmic GOT1, which co-segregates convincingly with the macro-AST phenotype and has functional plausibility as the causative agent of this phenomenon.

      Conclusions

      The AST protein is conserved in all prokaryotic and eukaryotic organisms.
      • Shen H.
      • Damcott C.
      • Shuldiner S.R.
      • Chai S.
      • Yang R.
      • Hu H.
      • et al.
      Genome-wide association study identifies genetic variants in GOT1 determining serum aspartate aminotransferase levels.
      While serum AST activity levels are affected by additive genetic and environmental factors,
      • Whitfield J.B.
      • Zhu G.
      • Nestler J.E.
      • Heath A.C.
      • Martin N.G.
      Genetic covariation between serum gamma-glutamyltransferase activity and cardiovascular risk factors.
      only one report has revealed a direct impact of a rare in-frame deletion of three nucleotides in GOT1, encoding asparagine at position 389 (p.Asn389del), and leading to decreased GOT1 serum enzyme activity.
      • Shen H.
      • Damcott C.
      • Shuldiner S.R.
      • Chai S.
      • Yang R.
      • Hu H.
      • et al.
      Genome-wide association study identifies genetic variants in GOT1 determining serum aspartate aminotransferase levels.
      Using WES and strict filtering criteria we uncovered, for the first time, a very rare amino acid substitution in GOT1, which co-segregated with familial macro-AST. Additionally, by performing in silico analysis we identified a potential relationship between AST structure and function, underlying the formation of high-molecular-weight serum macro-AST. Finally, screening for this alteration in cases of unexplained elevation of AST activity has the potential to prevent unnecessary and intrusive tests.

      Financial support

      This work was supported by: the National Science Centre [ 2013/11/B/NZ2/00130 ] and the Children’s Memorial Health Institute [ S131/2013 ]. JO was supported by the National Science Centre [ 2011/02/A/NZ5/00339 ]. KG and ML were supported by the Foundation for Polish Science (TEAM).

      Conflict of interest

      Prof. Czlonkowska and Prof. Ostrowski report grants from The National Science Centre, during the conduct of the study.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      Conception and design of the study: J.O., P.S.; Patients recruitment and clinical data compilation: P.S., A.W., A.H., W.J., A.C.; DNA isolation, exome sequencing, Taqman genotyping: A.P., J.K., M.D.; Exome-Seq dataset analyses and interpretation: M.K., M.M.; In silico GOT1 analysis and interpretation: K.G., M.L.; Drafting of the manuscript: J.O., M.K., M.M.

      References

      Author names in bold designate shared co-first authorship

        • Moriyama T.
        • Tamura S.
        • Nakano K.
        • Otsuka K.
        • Shigemura M.
        • Honma N.
        Laboratory and clinical features of abnormal macroenzymes found in human sera.
        Biochim Biophys Acta. 2015; 1854: 658-667
        • Lee M.
        • Vajro P.
        • Keeffe E.B.
        Isolated aspartate aminotransferase elevation: think macro-AST.
        Dig Dis Sci. 2011; 56: 311-313
        • Collins J.
        • Ritter D.
        • Bacon B.R.
        • Landt M.
        • Creer M.H.
        Macro-aspartate aminotransferase in a female with antibodies to hepatitis C virus.
        Liver. 2002; 22: 501-506
        • Galasso P.J.
        • Litin S.C.
        • O’Brien J.F.
        The macroenzymes: a clinical review.
        Mayo Clin Proc. 1993; 68: 349-354
        • Asanuma K.
        • Yagihashi A.
        • Watanabe N.
        Characterization of the aspartate aminotransferase-immunoglobulin M complex in a Japanese woman.
        Clin Chem. 1997; 43: 686-687
        • Triester S.L.
        • Douglas D.D.
        Development of macro-aspartate aminotransferase in a patient undergoing specific allergen injection immunotherapy.
        Am J Gastroenterol. 2005; 100: 243-245
        • Werner T.
        • Vargas H.E.
        • Chalasani N.
        Macro-aspartate aminotransferase and monoclonal gammopathy: a review of two cases.
        Dig Dis Sci. 2007; 52: 1197-1198
        • Zaman Z.
        • Van Orshoven A.
        • Mariën G.
        • Fevery J.
        • Blanckaert N.
        Simultaneous macroamylasemia and macrolipasemia.
        Clin Chem. 1994; 40: 939-942
        • Konttinen A.
        • Murros J.
        • Ojala K.
        • Salaspuro M.
        • Somer H.
        • Räsänen J.
        A new cause of increased serum aspartate aminotransferase activity.
        Clin Chim Acta. 1978; 84: 145-147
        • Shen H.
        • Damcott C.
        • Shuldiner S.R.
        • Chai S.
        • Yang R.
        • Hu H.
        • et al.
        Genome-wide association study identifies genetic variants in GOT1 determining serum aspartate aminotransferase levels.
        J Hum Genet. 2011; 56: 801-805
        • Remaley A.T.
        • Wilding P.
        Macroenzymes: biochemical characterization, clinical significance, and laboratory detection.
        Clin Chem. 1989; 35: 2261-2270
        • Stasia M.J.
        • Surla A.
        • Renversez J.C.
        • Pene F.
        • Morel-Femelez A.
        • Morel F.
        Aspartate aminotransferase macroenzyme complex in serum identified and characterized.
        Clin Chem. 1994; 40: 1340-1343
        • Patteet L.
        • Simoens M.
        • Piqueur M.
        • Wauters A.
        Laboratory detection of macro-aspartate aminotransferase: case report and evaluation of the PEG-precipitation method.
        Clin Biochem. 2012; 45: 691-693
        • Orlando R.
        • Lirussi F.
        Macro-aspartate aminotransferase and pregnancy: any influence?.
        Eur J Gastroenterol Hepatol. 2004; 16: 717-718
        • Gaj P.
        • Kluska A.
        • Nowakowska D.
        • Bałabas A.
        • Piątkowska M.
        • Dabrowska M.
        • et al.
        High frequency of BRCA1 founder mutations in Polish women with nonfamilial breast cancer.
        Fam Cancer. 2012; 11: 623-628
        • Ostrowski J.
        • Paziewska A.
        • Lazowska I.
        • Ambrozkiewicz F.
        • Goryca K.
        • Kulecka M.
        • et al.
        Genetic architecture differences between pediatric and adult-onset inflammatory bowel diseases in the Polish population.
        Sci Rep. 2016; 6: 39831
      1. Torrent Variant Caller. GitHub https://github.com/iontorrent/TS​ ​(accessed July 21, 2015).

        • Damiati E.
        • Borsani G.
        • Giacopuzzi E.
        Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.
        Hum Genet. 2016; 135: 499-511
      2. Garrison, Erik. vcflib : a C++ library for parsing and manipulating VCF files. GitHub https://github.com/ekg/vcflib (accessed July 21, 2015).

        • McLaren W.
        • Pritchard B.
        • Rios D.
        • Chen Y.
        • Flicek P.
        • Cunningham F.
        Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor.
        Bioinformatics. 2010; 26: 2069-2070
        • Zhan X.
        • Hu Y.
        • Li B.
        • Abecasis G.R.
        • Liu D.J.
        RVTESTS: An efficient and comprehensive tool for rare variant association analysis using sequence data.
        Bioinformatics. 2016; 32: 1423-1426
        • Glusman G.
        • Caballero J.
        • Mauldin D.E.
        • Hood L.
        • Roach J.C.
        Kaviar: an accessible system for testing SNV novelty.
        Bioinformatics. 2011; 27: 3216-3217
      3. Kandoth, Cyriac, Koboldt, Dan, Larson, David. variant-filter. GitHub https://github.com/ckandoth/variant-filter (accessed July 17, 2015).

        • Caropreso M.
        • Fortunato G.
        • Lenta S.
        • Palmieri D.
        • Esposito M.
        • Vitale D.F.
        • et al.
        Prevalence and long-term course of macro-aspartate aminotransferase in children.
        J Pediatr. 2009; 154: 744-748
        • Itan Y.
        • Shang L.
        • Boisson B.
        • Patin E.
        • Bolze A.
        • Moncada-Vélez M.
        • et al.
        The human gene damage index as a gene-level approach to prioritizing exome variants.
        Proc Natl Acad Sci U S A. 2015; 112: 13615-13620
        • Shashi V.
        • McConkie-Rosell A.
        • Rosell B.
        • Schoch K.
        • Vellore K.
        • McDonald M.
        • et al.
        The utility of the traditional medical genetics diagnostic evaluation in the context of next-generation sequencing for undiagnosed genetic disorders.
        Genet Med. 2014; 16: 176-182
        • Farwell K.D.
        • Shahmirzadi L.
        • El-Khechen D.
        • Powis Z.
        • Chao E.C.
        • Tippin Davis B.
        • et al.
        Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions.
        Genet Med. 2015; 17: 578-586
        • Auer P.L.
        • Lettre G.
        Rare variant association studies: considerations, challenges and opportunities.
        Genome Med. 2015; 7: 16
        • Biesecker L.G.
        • Green R.C.
        Diagnostic clinical genome and exome sequencing.
        N Engl J Med. 2014; 370: 2418-2425
        • Zhu X.
        • Petrovski S.
        • Xie P.
        • Ruzzo E.K.
        • Lu Y.-F.
        • McSweeney K.M.
        • et al.
        Whole-exome sequencing in undiagnosed genetic diseases: interpreting 119 trios.
        Genet Med. 2015; 17: 774-781
        • Whitfield J.B.
        • Zhu G.
        • Nestler J.E.
        • Heath A.C.
        • Martin N.G.
        Genetic covariation between serum gamma-glutamyltransferase activity and cardiovascular risk factors.
        Clin Chem. 2002; 48: 1426-1431