Hum Genet (1996) 97:209-213

© Springer-Verlag 1996

Elisabeth Oppliger Leibundgut • Bendicht Wermuth Jean-Pierre Colombo • Sabina Liechti-Gallati

Identification of four novel splice site mutations in the ornithine transcarbamylase gene

Received: 6 June 1995 / Revised: 17 July 1995

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows Xlinked inheritance with frequent new mutations. Using polymerase chain reaction (PCR) amplification of the individual exons including adjacent intron sequences followed by direct sequencing of the amplimers we identified four new mutations affecting donor splice sites of introns 2, 5, 6, and 8. The mutation at the first position of intron 2 was a G to A exchange associated with acute neonatal hyperammonemia in a male patient at the age of 5 months. A G to C substitution in intron 5 was detected in a boy who developed 2 days after birth hypotonia, and respiratory distress, followed by severe hyperammonemia and terminal coma. The intron 6 mutation, a G to T substitution, was detected in a girl presenting with first episodes of vomiting and agitation at the age of 2 months. The mutation in intron 8, also a G to T transition, caused fatal hyperammonemia and early death at the age of 15 days in a male patient. We present four donor splice site mutations resulting in severe neonatal or very early onset of the disease in three boys and in one female patient. As the GT dinucleotide of the 5" donor splice site is invariant and required for correct splicing the described mutations may lead to improperly spliced mRNAs and aberrant gene products. Abstract

Introduction Omithine transcarbamylase (OTC; EC 2.1.3.3) is a mitochondrial matrix enzyme that is encoded by a gene mapped to the short arm of the X chromosome on band Xp21.1. Within mitochondria, OTC catalyzes the conden-

E. Oppliger Leibundgut - B. Wermuth - J.-P. Colombo Department of Clinical Chemistry, Inselspital, University of Berne, CH-3010 Berne, Switzerland S. Liechti-Gallati ([g~) Department of Clinical ResearCh, University of Berne, CH-3010 Berne, Switzerland

sation of omithine and carbamyl phosphate to form citrulline, the second step of the urea cycle. OTC deficiency is the most common inborn error of ureagenesis in humans. As expected for X-linked disorders, clinical manifestations are generally most severe in hemizygous males, who present with protein intolerance and marked hyperammonemia, often leading to coma and death in the neonatal period. Heterozygous (carrier) females show variable clinical expression of their disease ranging from asymptomatic illness or mild protein intolerance to hyperammonemic coma in the newborn period. A substantial number of patients with OTC deficiency develop later in life, often not until adulthood, clinical symptoms like behavioral changes or seizures associated with hyperammonemia. These patients present with partial enzyme activities that may be inhibited by catabolic stress, infections, high protein intake, and possibly dehydration. The OTC gene has been cloned and characterized (Horwich et al. 1984; Hata et al. 1986, 1988) and found to span 73 kb of genomic DNA. It contains 10 exons and 9 introns, which form a 1700-base mRNA with a coding region of 1062 bases. There is a wide spectrum of mutations in the OTC gene including deletions (Old et al. 1985; Rozen et al. 1985 a, b; Maddalena et al. 1988 a; Grompe et al. 1991; Liechti-Gallati et al. 1991; Suess et al. 1992), point mutations in exons (Maddalena et al. 1988b; Grompe et al. 1989; Hata et al. 1989; Finkelstein et al. 1990; Hata et al. 1991; Hentzen et al. 1991; Tuchman et al. 1992; Matsuura et al. 1993, 1994a; Tuchman et al. 1994a, b; Oppliger Leibundgut et al. 1995) and point mutations in introns resulting in splicing abnormalities (Carstens et al. 1991; Tuchman et al. 1992; Hoshide et al, 1993). Except for a small number of recurrent mutations (Maddalena et al. 1988a; Grompe et al. 1991; Suess et al. 1992; Grompe et al. 1989; Hata et al. 1989; Finkelstein et al. 1990; Hata et al. 1991; Strautnieks et al. 1991; Satoh et al. 1992; Matsuura et al. 1993; Reish et al. 1993; GilbertDussardier et al. 1994; Matsuura et al. 1994 a; Tuchman et al. 1994b; Yoshino et al. 1994; Oppliger Leibundgut et al. 1995), the majority of patients with OTC deficiency carry rare mutations (Matsuura et al. 1993; Strautnieks 1993;

210 T u c h m a n 1993; Matsuura et al. 1994b; T u c h m a n et al. 1994a, b; Yoshino et al. 1994). In order to improve carrier detection and prenatal diagnosis as well as to increase the understanding of the molecular m e c h a n i s m s involved in the pathogenesis of the disease, we systematically screened our OTC patients for mutations within the O T C gene. Polymerase chain reaction (PCR) amplification of all ten e x o n s ( i n c l u d i n g e x o n / i n t r o n b o u n d a r i e s ) f o l l o w e d by single-strand conformational p o l y m o r p h i s m (SSCP) and direct sequencing allowed detection of mutations in most of the patients tested (Oppliger L e i b u n d g u t et al. 1995). We report here on four new splice site mutations found in one female and three male patients.

Materials and methods Patients Patient 1701, the first son of healthy parents and brother of a healthy sister, developed fatal hyperammonemia during the first days of life. Despite hemodialysis and normalization of the hyperammonemia, the boy died at the age of 15 days. Liver biopsy confirmed OTC deficiency presenting with no detectable residual enzyme activity. The male proband 1801 presented with muscle hypotonia, vomiting, loss of consciousness, and hyperammonemia after breastfeeding was stopped at the age of 5 months. The OTC activity in liver tissue was less than 5% of normal values (normal range 9.533 btmol/h per milligram protein), and urinary orotic acid demonstrated an increase to 263 gmot/mmol creatinine (normal < 0.5). The patient's older brother had died as the result of an encephalitic attack of unknown etiology at the age of 7 months. Patient 5401, also a male, was born after a term pregnancy by normal delivery. After the first 2 days of life he developed hypotonia, and respiratory distress, followed by severe hyperamlnonemia

Fig. 1A-C Single-strand con-

1

formational polymorphism (SSCP) gel results. A Exon/intron 2. Abnormal migration pattern in male proband 1801 (lane 5) and in his heterozygnus mother 1803 (lane 4) Control DNAs present either with AA at nucleotide 137 (lane 1), AG (lane 2), or GG (lane 3), owing to a polymorphism (Grompe et al. 1989). B Exon/intron 5. Abnormal migration pattern in male patient 5401 (lane 4) and in his heterozygous mother 5403 (lane 3) lanes I and 2 show normal patterns of one male and one female control. C Exon/intron 6. Migration patterns of female patient 4901 (lane 3) as well as of one male (lane 1) and one female control (lane 2). The bands at the bottom of the gel represent double-stranded DNA

A

2

3

4

and terminal coma. Liver biopsy confirmed OTC deficiency with no detectable residual enzyme activity. The female patient 4901 is the second child of healthy parents. Vomiting, agitation and abnormal movements occurred at 2 months of life. The OTC enzyme activity was found to be 4.73 btmo[/h per milligram protein (50% of the lower reference limit) and orotic acid was 1408 btmol/mmol creatinine confirming the diagnosis of OTC deficiency.

Methods Genomic DNA from patients and their family members was isolated fiom peripheral blood leucocytes or cultured skin fibroblasts using proteinase K, sodium dodecyl sulfate treatment and phenol, chloroform extraction (Herrmann and Frischauf 1987). All ten exons as well as the exon/intron boundaries of the OTC gene were amplified by PCR (Saiki et al. I988) using 200-400 ng of genomic DNA. Amplification was performed according to conditions reported elsewhere (Oppliger Leibundgut et al. 1995), and primers were designed from published sequences (Hata et al. 1988: Matsuura et al. 1993). SSCP analysis of the amplified DNA fi'agments as described by Orita et al. (1989) was modified and optimized for gel composition, etectrophoresis conditions and staining procedures (S, Liechti-Gallati et al., in preparation). Sequencing was perlbrmed using asymmetric PCR followed by direct sequencing of single-stranded DNA (Oppliger Leibundgut et al. 1995) or by direct fluorescence sequencing of double-stranded PCR products using a 373A system (Applied Biosystems).

Results In order to analyze the entire coding region of the OTC gene, the nucleotide sequences containing each exon plus adjacent intron sequences were amplified. SSCP analyses demonstrated for three patients (1801, 4901, 5401) an ab-

5

1

B

2

3

4

1

C

2

3

211 Table 1 Splice site mutations found in four patients with or-

nithine transcarbamylase (OTC) deficiency Patient

Sex

1801 5401 4901 1701 G

Male Male Female Male G

A

G

tntron Site (nucleotide)

Nucleotide change

Onset of disease

2 5 6 8

G--~A G ---)C G-~T G--~T

5 months Neonatal 2 months Neonatal

216 + 540 + 663 + 867 + ,='~0

G

A

1 1 1 1 T

A

T

Discussion

G

T

A

i

) A G

G

A f_~

G

T

A

T

mutation was also detectable in the patient's mother and sister (Fig. 3 C).

T

A

i

B Fig.2A, B Sequence around the site of point mutations in intron 2. The G to A substitution at position 216 + 1 is indicated by an a r r o w in the sequence of the proband 1801 (A) and his mother 1803 (B) normal migration pattern in one amplification product (Fig. 1), whereas the remaining nine exons did not differ from control amplimers. Sequencing of these D N A variants revealed three novel base substitutions in introns 2, 5, and 6, respectively. These mutations, summarized in Table 1, alter the invariant GT dinucleotide at the 5" donor splice sites of the corresponding exon/intron boundaries. In patient 1801 and his mother 1803 a G to A exchange at the donor splice site 216 + 1 of intron 2 was found (Fig. 2). Patient 5401 and his mother 5403 presented with a G to C substitution at nucleotide 540 + 1 of intron 5 (Fig. 3 A), whereas patient 4901, but not her mother, was heterozygous for a G to T exchange at the donor splice site 663 + 1 of intron 6 (Fig. 3 B). No further mutations were detectable in these patients by sequence analysis of the remaining exons. However, an additional novel mutation of intron 8, a G to T transition at position 867 + 1, was detected by sequencing all ten exons of patient 1701. The

There are many different mutations causing OTC deficiency, most of which are specific for an individual family. Using PCR amplification followed by direct sequencing of the amplification products, we were able to detect four novel mutations affecting splice donor sites in introns 2, 5, 6, and 8, respectively. Abnormal m R N A splicing represents a relatively common mechanism in the pathogenesis of OTC deficiency. Several mutations in the splice donor and splice acceptor sites of the OTC gene have been reported, all of them correlating with a neonatal form of the disease (Carstens et al. 1991; Tuchman et al. 1992; Hoshide et al. 1993). Two of our patients with mutations in introns 5 and 8 presented with a neonatal form of the disease and no detectable enzyme activity. The mutation at the donor splice site in intron 2, identified in patient 1801, correlates with a later onset of disease (Table 1) and less than 5% OTC activity in liver tissue. Tuchman et al. (1992) reported also on a mutation in intron 2 altering the AG dinucleotide of the acceptor splice site in a patient with a neonatal form of OTC deficiency. This 2171G---~A mutation is likely to result in an m R N A missing all or part of the exon 3 sequence, whereas the 2 1 6 + l G - - ) A mutation in our patient is expected to skip or affect exon 2. The mutation in intron 6 goes together with early episodes of vomiting and agitation in a female patient whose relatively high residual enzyme activity (50%) is explained by random inactivation of one of the OTC genes. Females who are heterozygous for OTC deficiency show a mosaic pattern in the liver consisting of cells with normal OTC and cells with deficient OTC function. Thus, the risk for carrier females of developing symptoms depends on the fraction of liver cells with deficient OTC activity. Biochemical carrier testing such as protein loading and allopurinol challenge often gives ambiguous results (Becroft et al. 1984; Spence et al. 1989; Hauser et al. 1990; Pelet et al. 1990; Tsai et al. 1993). Carrier detection by restriction fragment length polymorphism (RFLP) analyses may be useful in some cases; however, this approach is time consuming and limited by the high incidence of new mutations of about one-third observed in X-linked lethal 'disorders (Haldane 1935), which has recently been confirmed for OTC deficiency by Tuchman et al. (1995). Based on our own experience, SSCP analysis of all ten exons of the OTC gene proved to be a rapid and reliable method for mutation screening in OTC patients as well as for carrier detection and prenatal diagnosis. Carrier testing in our four families identified the mothers of the three affected boys as well as the sister of patient 1701 as carriers of the splice site mutations found in the probands. The mutation detected in the female patient turned out to have occurred spontaneously. The mutations identified in our patients all affect donor splice sites altering the highly conserved GT dinucleotide

212 Fig.3A-C Partial sequence of amplified DNA for exon/intron boundaries. A The G to C substitution (arrow head) in intron 5 detected in patient 5401 and his mother 5403. B The G to T transversion (arrow head) in intron 6 detected in female patient 4901 but not in her mother 4903. C The G to T exchange (arrow head) in intron 8 detected in patient 1701 as well as in his mother 1703 and in his sister 1704

B

A s4o3

4903

l~llll4901

G A

T

C

G A T C

G

C

A

T C

G A

T

C

~ 1703 ~1704

1701

l

#

G

at the 5" intron b o u n d a r y that is required for correct splicing. As a consequence, aberrant splicing of the O T C prem R N A s would result in abnormal transcripts. O n the one hand, activating a cryptic splice site within the affected intron would generate elongated transcripts that m a y alter the reading frame. Alternatively, skipping of the preceding exon caused by donor splice site mutations has been demonstrated in two cases by Carstens et al. (1991). At the protein level, the aberrant m R N A s are expected to lead to elongated or truncated translation products. Insertions or deletions in the protein may disrupt the functional domains of the enzyme, prevent its mitochondrial processing or alter the three-dimensional structure required for its catalytic function. However, in the complex pathway from R N A transcript to mature protein, the steps responsible for the e n z y m e deficiency in our patients remain to be defined. Acknowledgements We wish to thank Drs. Mallmann, St6ckler, Strobl, and R6schinger, for providing samples and clinical data from their patients. This work was supported by a grant from the

A

T

C

G

A

T

C

G

A

T

C

Swiss National Foundation (32-32472,91) and by a grant from Sandoz Pharma AG.

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