Toshinobu Matsuura

Genetic mutations in exons 3 and 4 of the pancreatic secretory trypsin inhibitor in patients with pancreatitis.

Purpose. We hypothesized that mutations in the pancreatic secretory trypsin inhibitor (PSTI) gene could promote autodigestion, leading to acute or chronic pancreatitis. Our investigation involved mutation analysis of the PSTI gene in patients with acute or chronic pancreatitis. Methods. Mutation analysis for the PSTI gene was performed in patients with acute or chronic pancreatitis. Unrelated healthy volunteers and family members of a chronic pancreatitis patient with point mutations in the PSTI gene were also analyzed. Results. Two types of single-point mutation in the PSTI gene were observed in one patient with chronic pancreatitis: 34Asn (AAT)-to-Ser (AGT) (101 A > G N34S: N34S) in exon 3, and 67Arg (CGC)-to-Cys (TGC) (199 C > T R67C: R67C) in exon 4. No mutations with amino-acid substitution were found in other patients or in the volunteer group. In the patient with the PSTI gene mutations, no additional mutations were observed in the cationic trypsinogen gene. The family study revealed that the mother and a maternal uncle were homozygotes for the N34S mutation, while the father and brother were compound heterozygotes for the N34S and R67C mutations. The uncle (N34S/N34S) showed clinical manifestations of pancreatitis, but the other family members did not. Conclusions. The N34S mutation may cause a predisposition to pancreatitis, with incomplete penetrance. However, with the limited information available, it is not known whether the R67C mutation promotes pancreatitis.

For novel gene mutations in five Japanese male patients with neonatal or late onset OTC deficiency: application of PCR-single-strand conformation polymorphisms for all exons and adjacent introns

Ornithine transcarbamylase deficiency (OTC), the most common inborn error of the urea cycle, shows an X-linked inheritance with frequent new mutations. Southern blots reveal only a small percent of the mutation, but amplification of cDNA or genomic DNA using the polymerase chain reaction (PCR) followed by DNA sequencing, has contributed greatly to overcoming this difficulty. Problems remaining are the limited availability of fresh liver samples for preparation of intact mRNA in the former case, and there are primer sequences for PCR for only some exons in the latter case. Here, we report the structures of intron sequences which are long enough to analyze all exons and adjacent introns of the OTC gene using PCR and PCR single-strand conformation polymorphisms (PCR-SSCP). We carried out a DNA analysis of findings in five Japanese male patients with neonatal or late onset form. Five patients had mutations in the protein coding region. C to G (S192R), A to T (D196V), A to G (T264A), T to C (M268T), and C to T (R277W) substitutions. The first four of these were novel missense mutations and the presence of the mutation was confirmed in the corresponding families.

Phenotypic variability in male patients carrying the mutant ornithine transcarbamylase (OTC) allele, Arg40His, ranging from a child with an unfavourable prognosis to an asymptomatic older adult.

In five different Japanese families, we identified six male hemizygotes (aged 6, 9, 15, 17, 56, and 65 years) and a putative candidate (aged 48 years), carrying a mutant allele of the ornithine transcarbamylase (OTC) gene, a G to A substitution at nucleotide 119 in exon 2 generating histidine in place of arginine. OTC activity in the necropsied liver tissue was reduced to approximately 12% of the control and that of COS 1 cells transfected with Arg40His OTC cDNA was 10.2 +/- 1.8% of the control transfected with wild type OTC cDNA. Clinical features ranged from death during a hyperammonaemic attack (a 9 year old) to a 65 year old asymptomatic man. We consider that the amount of protein ingested by these subjects may be one predisposing factor leading to the manifestation of this disease.

A novel missense mutation in exon 8 of the ornithine transcarbamylase gene in two unrelated male patients with mild ornithine transcarbamylase deficiency

SummaryWe studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.G.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the OTC gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family.

Carbamyl phosphate synthetase I deficiency. One base substitution in an exon of the CPS I gene causes a 9-basepair deletion due to aberrant splicing.

Carbamyl phosphate synthetase I (CPS I; EC6,3,4,16) is an autosomal recessive disorder characterized by hyperammonemia. We studied the molecular bases of CPS I deficiency in a newborn Japanese girl with consanguineous parents. Northern and Western blots revealed a marked decrease in CPS I mRNA and enzyme protein but with a size similar to that of the control, respectively. Sequencing of the patients cDNA revealed a nine-nucleotide deletion at position 832-840. Sequencing analysis of the genomic DNA revealed a G to C transversion at position 840, the last nucleotide of an exon in the splice donor site. This substitution altered the consensus sequence of the splice donor site and the newly cryptical donor site in the exon caused the 9-bp in-frame deletion. This report seems to be the first complete definition of CPS I deficiency, at the molecular level.

Clinical Manifestations of Inborn Errors of the Urea Cycle and Related Metabolic Disorders during Childhood

Various disorders cause hyperammonemia during childhood. Among them are those caused by inherited defects in urea synthesis and related metabolic pathways. These disorders can be grouped into two types: disorders of the enzymes that comprise the urea cycle, and disorders of the transporters or metabolites of the amino acids related to the urea cycle. Principal clinical features of these disorders are caused by elevated levels of blood ammonium. Additional disease-specific symptoms are related to the particular metabolic defect. These specific clinical manifestations are often due to an excess or lack of specific amino acids. Treatment of urea cycle disorders and related metabolic diseases consists of nutritional restriction of proteins, administration of specific amino acids, and use of alternative pathways for discarding excess nitrogen. Although combinations of these treatments are extensively employed, the prognosis of severe cases remains unsatisfactory. Liver transplantation is one alternative for which a better prognosis is reported.

The R40H mutation in a late onset type of human ornithine transcarbamylase deficiency in male patients

Abstract Ornithine transcarbamylase (OTC) deficiency is an X-linked trait and is one of the most frequent of the inherited urea cycle enzyme deficiencies. Most male patients with OTC deficiency develop a hyperammonemic crisis and die in the neonatal period or in early infancy. In contrast to those patients, in some male patients the disease first becomes overt in adolescence or during the reproductive age period. In the present report, we describe six such male patients who first developed clinical signs at ages ranging from 6 to 58 years, all of whom came from a limited area of the northern part of Kyushu Island in southern Japan. The mutation analysis disclosed a R40H mutation in exon 2 of the OTC gene in each of these patients. Transmission of this mutant gene through paternal lineage as well as through maternal lineage was documented in one family. The levels of mRNA of the mutant OTC gene expressed in transfected Cos 1 cells and in the liver tissue obtained by biopsy in one patient were both similar to those of the wild-type gene. The activity of the mutant OTC was, however, decreased to a level of 28% of the wild-type OTC, and the levels of the mutant OTC protein expressed in Cos 1 cells were decreased, as assessed by western blot analysis. Apparent Km values of the mutant enzyme for ornithine (1.1 mM) and carbamylophosphate (2.0 mM) were similar to those of the wild-type enzyme. Both enzymes gave similar pH-dependency profiles, giving a maximal activity at pH 7.8–7.9. Activity of wild-type OTC expressed in Cos 1 cells did not change after five cycles of freezing and thawing, whereas that of the mutant OTC decreased to 17% by this treatment. These results suggest that deficiency is due to inactivation of the mutant OTC under certain conditions.

Familial lethal inheritance of a mutated paternal gene in females causing X-linked ornithine transcarbamylase (OTC) deficiency

A Leu148Phe substitution of the ornithine transcarbamylase (OTC) gene was identified in a 2-year-old girl with OTC deficiency (14% of control). Her two elder sisters died in childhood of hyperammonemia, and the patient also died of OTC deficiency. Enzyme activity in Cos1 cells transfected by the mutant cDNA was undetectable, thereby indicating a definite pathogenic mutation. Familial gene analysis showed that the mother had wild-type OTC alleles on both X-chromosomes and the father was a mosaic for the mutant allele in his lymphocytes and spermatozoa. This clinical case shows that a somatic and germline mosaicism for a single-gene disorder led to an unusual pattern of X-linked inheritance in the family, and all three daughters in the family died of OTC deficiency. The possibility that inherited factors will lead to skewed X-inactivation needs to be considered.

Specificity of PCR-SSCP for detection of the mutant ornithine transcarbamylase (OTC) gene in patients with OTC deficiency

Ornithine transcarbamylase (OTC) (EC 2.1.3.3), the second enzyme of the hepatic urea cycle, is required for detoxication of ammonia and for biosynthesis of urea. The enzyme is apparently expressed only in the liver and small intestine. OTC deficiency is an X-linked genetic disease and the most common of the urea cycle disorders • (Brusilow and Horwich 1989; Nagata et al 1991a). Approximately one-third of male patients fall in the neonatal-onset group, and most die in early infancy of hyperammonaemic coma (Matsuda et al 1991). Those who do survive are severely mentally retarded and have an abnormal EEG and a severely damaged brain (Msall et al 1984; Nagata et al 1991b). Most male patients belonging to the late-onset group are normal or have moderate signs and symptoms with adequate treatment (Finkelstein et al 1990a; Matsuda et al 1991). Female heterozygotes have manifestations ranging from asymptomatic to mild or severe because of the allelic heterogeneity (Rowe et al 1986; Matsuda et al 1984) and random inactivation of the Xchromosome in hepatocytes (Lyone 1961). The human OTC cDNA was cloned by Horwich et al (1984) and by Hata et al (1988). Structural organization of the human OTC gene was reported by Hata et al (1988). There is a wide spectrum of mutations of the OTC gene, including deletions (Old et al 1985; Rozen et al 1985, 1986; Maddalena et al 1988a; Grompe et al 1991), point mutations in exons (Grompe et al 1991; Maddalena et al 1988b; Finkelstein et al 1990b; Hata et al 1989, 1991; Hentzen et al 1991; Matsuura et al 1993a,b) and point mutations in introns that resulted in splicing abnormalities (Carstens et al 1991). We examined the reliability of polymerase chain reaction single-strand conformation polymorphisms (PCRSSCP) for detecting involved exons of the OTC gene, using seven known and four unknown mutant alleles.

Identification of two new aberrant splicings in the ornithine carbamoyltransferase (OCT) gene in two patients with early and late onset OCT deficiency

SummaryOrnithine carbamoyltransferase (OCT) is a liver-specific enzyme located in the mitochondrial matrix. OCT deficiency is an X-linked disease with a heterogeneous phenotype, even in affected males. We studied two male patients (K.M., K.G.) with early and late onset, respectively. OCT activity was zero in the autopsied liver of patient K.M. and was 6% of control in the biopsied liver of K.G. Sequencing of OCT cDNAs revealed exon 5 skipping in K.M., resulting from a T-to-C transition of the initial dinucleotide of the 5′ splicing donor site of intron 5, and a G-to-T transversion at position +45 in exon 9 (L304F) in K.G., providing three OCT mRNAs of different lengths: a normally spliced transcript, 23 bp insertion of intron 8 and the first 50bp missing within exon 9. Exon 5 skipping and two other aberrant splicings produced stop codons early downstream in mature OCT mRNAs. Expression study of a missense allele, L304F, transfected to cultured Cos 1 cells revealed a 34.4% value of the control. The difference of OCT activities between the patient liver and transfected cells (6% vs. 34%) can be explained by this splicing abnormality.