The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein.

Citrullinaemia (CTLN) is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase (ASS). Adult-onset type II citrullinaemia (CTLN2) is characterized by a liver-specific ASS deficiency with no abnormalities in hepatic ASS mRNA or the gene ASS (refs 1-17). CTLN2 patients (1/100,000 in Japan) suffer from a disturbance of consciousness and coma, and most die with cerebral edema within a few years of onset. CTLN2 differs from classical citrullinaemia (CTLN1, OMIM 215700) in that CTLN1 is neonatal or infantile in onset, with ASS enzyme defects (in all tissues) arising due to mutations in ASS on chromosome 9q34 (refs 18-21). We collected 118 CTLN2 families, and localized the CTLN2 locus to chromosome 7q21.3 by homozygosity mapping analysis of individuals from 18 consanguineous unions. Using positional cloning we identified a novel gene, SLC25A13, and found five different DNA sequence alterations that account for mutations in all consanguineous patients examined. SLC25A13 encodes a 3.4-kb transcript expressed most abundantly in liver. The protein encoded by SLC25A13, named citrin, is bipartite in structure, containing a mitochondrial carrier motif and four EF-hand domains, suggesting it is a calcium-dependent mitochondrial solute transporter with a role in urea cycle function.

Pancreatic secretory trypsin inhibitor gene is highly expressed in the liver of adult-onset type II citrullinemia.

Deficiency of argininosuccinate synthetase (ASS) causes citrullinemia. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS is deficient specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there are no apparent abnormalities in the amount, translational activity, and nucleotide sequence of hepatic ASS mRNA. Recent results of homozygosity testing indicate that the primary defect of type II citrullinemia is not within the ASS gene locus. In this present work, to understand the pathogenesis and pathophysiology of type II citrullinemia, we have characterized the alterations of gene expression in the liver of type II patients using the recently developed mRNA differential display method. Some cDNA bands expressed differently in type II citrullinemia patients and control were selected, cloned, and sequenced. Nucleotide sequence analysis and homology searching revealed an interesting clone which has 99% homology with the human pancreatic secretory trypsin inhibitor (hPSTI). Northern blot and RT-PCR analyses showed that the expression of hPSTI mRNA increased significantly in the liver of all type II patients tested. Furthermore, the concentration of hPSTI protein was found to be higher in the liver of type II citrullinemia than in control. These results suggest that hPSTI may be related to the primary defect of type II citrullinemia and may be useful as a diagnostic marker, although the detailed mechanism of the high expression of hPSTI mRNA in type II liver is not yet known.

A novel gene suppressed in the ventricle of carnitine-deficient juvenile visceral steatosis mice.

In order to clarify the pathogenesis and pathophysiology of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice, we performed mRNA differential display analysis with total RNA extracted from the ventricles of control and JVS mice at 14 days of age. We identified four up-regulated genes, two known and two unknown, and a novel down-regulated gene. Northern blot analysis with a novel cDNA probe derived from the down-regulated gene fragment 8A2 revealed three mRNA species of 1.1-, 1.3-, and 2.6-kb. The 1.1- and 1.3-kb mRNA species were found only in the heart, and the 2.6-kb species was found in the heart, kidney and brain, but not in skeletal muscle or liver. The 1.1- and 1.3-kb species were down-regulated in the ventricles of JVS mice, but not in the auricles, and increased to the control level with carnitine treatment. We isolated cDNA clones from ventricle RNA, termed CDV-1 (carnitine deficiency-associated gene expressed in ventricle) and from brain RNA, termed CDV-1R (CDV-1-related gene) by 5'- and 3'-RACE analyses. The entire nucleotide sequence except the 5'-terminal 64 bp of CDV-1 cDNA was completely identical to the 992 bp sequence from the 3'-end of CDV-1R cDNA. The CDV-1 cDNA contained an open reading frame predicting a peptide of 107 amino acids, which composed the C-terminal portion of CDV-1R peptide consisting of 414 amino acids.

Pyruvate dehydrogenase kinase 4 mRNA is increased in the hypertrophied ventricles of carnitine-deficient juvenile visceral steatosis (JVS) mice.

We isolated a mouse homologue cDNA of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) with differential mRNA display as an up-regulated gene in the hypertrophied ventricles of juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency. The PDK4 mRNA level was 5 times higher in JVS mice than in control mice under fed conditions. After 24 h starvation, this level increased to 20 times in JVS and 7 times in control, compared with the control fed level. On the other hand, carnitine administration reduced the high level of PDK4 mRNA in JVS mice to the control fed level. In control mice, the change in PDK4 mRNA was inversely correlated with the change in PDH activity. In JVS mice, however, the PDK4 mRNA level was not always correlated with the active-form PDH level.

Mutations in argininosuccinate synthetase mRNA of Japanese patients, causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia.

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6(-2) (a transition of A to G at the second nucleotide position within the 3' splice cleavage site of intron 6) and IVS-13(+5) (a transition of G to A at the fifth nucleotide position within the 5' splice cleavage site of intron 13), respectively. The IVS-6(-2) mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6(-2) mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.

Characterization of human wild-type and mutant argininosuccinate synthetase proteins expressed in bacterial cells.

Argininosuccinate synthetase (ASS) is a urea cycle enzyme with a tetrameric structure composed of identical subunits. Citrullinemia is an autosomal recessive disease caused by a deficiency of ASS. We have previously identified 20 mutations in ASS mRNA of human classical citrullinemia. However, it is difficult to evaluate the effects of each mutation on the enzyme structure and function, since most of the patients are compound heterozygotes. In the present study, wild-type ASS and 12 mutant ASSs were expressed with a bacterial expression system and analyzed enzymologically and immunochemically. The properties of the purified recombinant protein with wild-type human ASS showed good agreement with native enzyme purified from human liver. Mutant ASS proteins with an expected molecular mass, except for delta 7b/Ex16, were highly expressed in the bacterial cells. It was difficult to extract ASS proteins with some mutations (A118T, delta Ex7, R157H, R363W, R363L, G390R and ins37b/Ex15&16) from cells by freezing and thawing. Extractable mutant proteins were as follows: G280R mutant was extracted with an amount of ASS protein similar to wild-type but with no ASS activity, and A192V, R272C and R304W mutants detected various amounts of ASS protein (13, 110 and 33% of wild-type, respectively) with a low ASS activity and abnormal kinetics. Higher Km values for citrulline were obtained in mutant ASSs with A192V (15 mmol/1), R272C (4.2 mmol/l) and R304W. (190 mmol/l) than in wild-type ASS (0.056 mmol/l). The results confirm that these mutations are responsible for ASS deficiency and also indicate that these amino acid residues are important for the function and structure of ASS protein.

Mutations and DNA diagnoses of classical citrullinemia.

Classical citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase (ASS). We have previously identified 20 mutations in ASS mRNA of human classical citrullinemia and already established the DNA diagnosis of seven mutations as follows. By Southern blot analysis, each of the alleles with exon 5 or 6 deletion in mRNA appears to involve deletion of genomic DNA from this region. Five mutations involving R304W, G324S, IVS-6(-2) (delta Ex7), IVS-13(+5) (delta Ex13), and delta 13 bp/Ex15&IVS-15 (ins37 b/Ex15&16) are diagnosed by a combination of PCR (or modified PCR) and restriction enzyme digestion. It is important to identify the mutation in genomic DNA for prenatal diagnosis and carrier detection. In the present study, we report a novel missense mutation (R279Q) and a new abnormality in the ASS gene (delta 11 bp/IVS-15). As three missense mutations (R272C, R279Q, and G280R) were found in exon 12, we isolated and sequenced the intron regions surrounding exon 12 to establish a DNA diagnostic test. Although a mutation with a deletion of the first seven bases in exon 16 of mRNA (delta 7 b/Ex16) was found in both Japanese and American patients, the abnormality on the ASS gene was different between the Japanese allele (delta 11 bp/IVS-15) and American allele (IVS-15(-1)). The DNA diagnosis of 47 Japanese alleles with classical citrullinemia showed that the IVS-6(-2) and R304W mutations were found in 49% and 17% of the mutated alleles, respectively. We now have DNA diagnosis systems to detect 14 out of 22 mutations and are performing prenatal diagnosis and carrier detection using genomic DNA on classical citrullinemia.