Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3' splice acceptor site within an exon of the human biotinidase gene
Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3 ' splice acceptor site within an exon of the human biotinidase geneRobert J. Pomponio1, Thomas R. Reynolds2, Hanna Mandel4, Osnat Admoni4, Pamela D. Melone1, Gregory A. Buck2and Barry Wolf1,3,*
1Department of Human Genetics, 2Department of Microbiology and Immunology and 3Department of Pediatrics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA and 4Department of Pediatrics, Section of Inherited Disorders of Metabolism, Rambam Medical Center, Technion, Israel Institute of Technology, Haifa, Israel
Received December 17, 1996;Revised and Accepted February 25, 1997
Biotinidase recycles the vitamin biotin from biocytin upon the degradation of the biotin-dependent carboxylases. We have identified a novel point mutation within the biotinidase gene that encodes the signal peptide in two unrelated individuals with profound biotinidase deficiency. Sequence analysis of genomic DNA from these individuals revealed a G to A transition (G100 -> A) located 57 bases downstream of the authentic splice acceptor site in exon B. Although this mutation predicts a G34S substitution, it also generates a 3' splice acceptor site. Sequence of the PCR-amplified cDNA from the homozygous child revealed that all the product was shorter than that of normal individuals and was the result of aberrant splicing. The aberrantly spliced transcript lacked 57 bases, including a second in-frame ATG, that encode most of the putative signal peptide and results in an in-frame deletion of 19 amino acids. The mutation results in failure to secrete the aberrant protein into the blood. This is the first reported example in which a point mutation creates a cryptic 3' splice acceptor site motif that is used preferentially over the upstream authentic splice site. The preferential usage of the downstream splice site is not consistent with the 5' -> 3' scanning model, but is consistent with the exon definition model of RNA splicing.
Biotin, an essential water-soluble B vitamin, is the coenzyme for four carboxylases in humans: propionyl-CoA carboxylase, [beta]-methylcrotonyl-CoA carboxylase, acetyl-CoA carboxylase, and pyruvate carboxylase (1 ,2 ). These carboxylases are involved in amino acid catabolism, fatty acid synthesis and gluconeogenesis. The carboxyl group of biotin is covalently attached through an amide bond to an [epsilon]-amino group of a lysyl residue of the apocarboxylases by holocarboxylase synthetase, thereby forming holocarboxylases (3 ). The holocarboxylases are eventually degraded proteolytically to biocytin (biotin-[epsilon]-lysine) which is then cleaved by biotinidase (biotin-amide amidohydrolase, EC 3.5.1.12) releasing biotin for reutilization, thereby completing the biotin cycle (3 ,4 ).
Biotinidase deficiency is inherited as an autosomal recessive trait (3 ,5 ). Children with biotinidase deficiency cannot cleave biocytin and, therefore, cannot recycle biotin. If these children are not supplemented with free biotin, they become secondarily biotin deficient which, in turn, results in decreased activities of the biotin-dependent carboxylases and the subsequent accumulation of toxic metabolites causing clinical symptoms (6 ). Untreated individuals with biotinidase deficiency may exhibit seizures, hypotonia, ataxia, developmental delay, hearing loss, optic abnormalities, alopecia, and skin rash as well as ketolactic acidosis and organic aciduria (7 ,8 ). Some affected children have lapsed into coma and died before they were diagnosed and treated. There is considerable variability in the age of onset of symptoms and in the severity of the clinical features (7 ). Initiation of treatment with pharmacologic doses of biotin resolves and reverses many of the clinical symptoms (3 ). If treatment is delayed, deficits in hearing, sight or development may not resolve after biotin therapy (9 -11 ). Therefore, it is important that biotinidase deficiency is diagnosed early so that biotin therapy can be initiated before permanent neurological damage occurs. More than 20 states in the USA and over 20 other countries perform newborn screening for biotinidase deficiency (12 ).
We have isolated and characterized the cDNA for normal human serum biotinidase (BTD) (13 ), and localized the gene to chromosome 3p25 (14 ). We have also identified a common deletion/insertion mutation (designated delG98-G104:ins TCC, or G98:d7i3) in the biotinidase gene that results in profound biotinidase activity (<10% of mean normal serum enzyme activity) in symptomatic children (15 ).
We now describe a novel mutation in two unrelated patients with profound biotinidase deficiency. One child exhibited symptoms and is homozygous for this mutation, whereas the other child was identified by newborn screening and is a compound heterozygote. This mutation activates a cryptic 3' acceptor splice site that is located downstream of the authentic 3' splice acceptor site and is used preferentially over the authentic site.
PCR amplification of the complete 269 bp exon B and adjacent intron-exon boundaries (362 bp total) of BTD DNA did not reveal any differences in the size of the PCR product of the patients when compared to that of the individuals with normal biotinidase activity by non-denaturing polyacrylamide gel electrophoresis (data not shown).
SSCA of the 362 bp product from 42 normal individuals, as exemplified by individual P87, exhibited a tight doublet or an intense single band after longer exposure (Fig. 1 ). P19 had two aberrantly migrating bands, one slower and one faster than normal. P17 and P18 (parents of P19), P170 (not shown) and her father, P134 (not shown), had bands which corresponded to those of P19 and a band that migrated identically to that of the normal individuals, suggesting that they are heterozygous for a mutation in this exon. P125, the mother of P170 (not shown), had band conformations that were similar to those of the normals. P19 is presumed to be homozygous for the abnormal allele based on the SSCA patterns of her parents.
Because of the potential competition for use of this cryptic 3' splice acceptor site rather than the authentic site, we examined the mRNA produced in lymphocytes from these patients to determine if this mutation affects RNA splicing. End-labeled RT-PCR products from each individual, as well as a RT-PCR product from RNA isolated from human liver, were generated as described above and electrophoresed on a 6% polyacrylamide gel (Fig. 5 ). The normal controls (P87 and human liver) exhibit only one intense band at ~200 bp, the expected size of the product if the authentic 3' acceptor splice site is used. RT-PCR analysis of RNA from P19 revealed that almost the entire product was faster migrating and of a lower molecular weight than normal. A very small quantity of product corresponding to the normal size was detected. This suggested that the cryptic splice acceptor site is used preferentially to the upstream authentic 3' splice acceptor site. Although it was possible that the shorter RNA template caused by the mutation was amplified by PCR more efficiently than the slightly longer normal template, this is not substantiated by the nearly equal distribution of normal to abnormal end-labeled PCR products in heterozygotes for this mutation (the parents of P19 and P170), where there is an equal distribution of amplified cDNA templates (Fig. 5 ).
We have previously reported two common mutations that cause biotinidase deficiency. The first is a deletion/insertion (designated delG98-G104:ins TCC, or G98:d7i3) which results in a frameshift and the premature termination of the enzyme protein (15 ), and the second is a substitution of C1612 -> T, which results in a Cys for Arg538 (designated R538C) and appears to cause inappropriate inter- or intramolecular disulfide bond formation, increased intracellular degradation and failure of the enzyme to be secreted from the cell (17 ). We now report another mutation (G100 -> A) in the coding sequence of the putative signal peptide in two other children with profound biotinidase deficiency. P19 is homozygous for this novel mutation, whereas P170 is heterozygous and must be a compound heterozygote with a second as yet unidentified mutation that results in profound biotinidase deficiency. The G100 -> A mutation was not found in 24 other children with profound biotinidase deficiency.
Sequencing of this region revealed a G to A transition at nucleotide position 100 within the coding sequence of a putative secretory signal peptide of the BTD gene. This mutation predicts a change of a glycine to serine at amino acid position 34. This amino acid change is conservative even in the homozygous state and would not be expected to alter the signal peptide sufficiently to result in profound biotinidase deficiency. Close examination of the sequence revealed that the G to A transition at this position creates a consensus sequence for a 3' splice acceptor site within the exon. This cryptic splice acceptor site is 57 nt downstream from the authentic splice acceptor site.
RT-PCR analysis of RNA from P19 revealed that essentially all of the cDNA product was of a smaller molecular weight than that corresponding to mRNA from normal individuals. This observation suggests that the cryptic splice acceptor site is used preferentially over the upstream authentic 3' splice acceptor site. The use of the cryptic site results in an in-frame deletion of 57 nt in the mRNA and the subsequent deletion of 19 amino acids from the secretory signal peptide.
The nucleotide sequence that encodes the leader sequence contains two in-frame ATG codons, either of which may be the site of translation initiation (13 ). Biotinidase, with the help of the signal peptide, is normally secreted from hepatocytes and other tissues into the blood (4 ,18 ). Similar to other secretory proteins, the signal peptide is normally removed before secretion resulting in the presence of mature biotinidase in blood with Ala42 at its N-terminus. The mutation reported here results in an in-frame deletion of almost half of the putative secretory signal peptide of biotinidase. If the first in-frame ATG is used for translation initiation, then this deletion of the signal peptide is expected to produce a truncated, ineffective signal peptide and result in the failure to secrete the enzyme. If the second in-frame ATG is normally used, then the mutation would eliminate this codon and translation initiation could not occur. Whichever possibility occurs, the effect is substantiated by our finding that biotinidase activity and biotinidase protein are undetectable in the serum of P19. It is possible that the protein produced by this child accumulates in low abundance in cells and is ultimately degraded proteolytically. Fibroblasts from this patient were not available to determine whether the enzyme is secreted or accumulates in the cell. Instead, we only had access to lymphoblasts which normally have very low enzyme activity. However, because P19 was symptomatic, our results further support the physiological importance of having active biotinidase in blood.
The mutation is novel because it activates a cryptic 3' splice acceptor site that is used preferentially over the authentic site. There are only a small number of reports of mutations that cause preferential usage of a cryptic splice site and a majority of these have involved the 5' splice donor site (16 ,19 ). In their review, Nakai and Sakamoto (20 ), state that aberrant splicing patterns based on the creation of a new 3' splice site exclusively occur upstream of the authentic 3' splice acceptor site, hence, within the intron. There have been, however, several reports that are exceptions to this statement, in which downstream exonic cryptic 3' splice acceptor sites are preferentially used over the authentic site (21 -26 ). Three of these mutations involve single nucleotide substitutions in the polypyrimidine tract of the cryptic splice acceptor sites (24 -26 ). These mutations indicate the importance of the polypyrimidine tract in splice site selection. Our mutation is novel in that it is the first described that creates a splice site motif within an exon, adjacent to an apparently optimal 12 nt polypyrimidine tract.
Shapiro and Senapathy (16 ) described a scoring system that assigns a numerical value to 5' splice donor and 3' splice acceptor sequences. Comparison of the sequence of the authentic 3' splice acceptor site (Fig. 4 b) to that of the downstream activated cryptic 3' splice acceptor site revealed that the sequence of the cryptic splice site had a higher value, 0.920, than that of the authentic site, 0.856 (a perfect consensus splice site has a value of unity). The difference in the values is due specifically to the presence of purines in the polypyrimidine tract of the authentic site, whereas there are none in the polypyrimidine tract of the cryptic splice site created by the mutation. This suggests that the polypyrimidine tract of the cryptic splice acceptor site is a critical determining factor for its preferential use.
The effects of this mutation on mRNA splicing and those reported by others of the preferential use of a downstream 3' splice acceptor site (26 ) are inconsistent with the 5' -> 3' scanning process. In contrast, these mutations suggest that the splice acceptor dinucleotide AG and polypyrimidine tract are the determinants of splice site selection (27 ). Our results support the exon definition model, where there is pairing of defined splice sites across an exon (28 ). Generally, internal exons in vertebrates have a maximum size of 300 nt. Activated cryptic splice sites usually result in smaller exons and are occasionally used over the normal sites (29 ,30 ). Our cryptic acceptor splice site is not only selected preferentially over the authentic site based on the strength of the adjacent polypyrimidine tract, but the distance between the cryptic site and the downstream 5' donor site is now closer than the authentic site and this donor site.
In conclusion, we have described a novel mutation that causes profound biotinidase deficiency that creates and preferentially uses a 3' splice acceptor site in an exon that is downstream of the authentic 3' splice site. This mutation supports the exon definition model of RNA splicing in that snRNPs and various protein factors recognize and bind to specific nucleotide sequences. The apparent preferential usage of the cryptic 3' splice acceptor site indicates the importance of the content of the polypyrimidine tract for splicing and further questions the validity of the 5' -> 3' scanning model of RNA splice site selection. This mutation results in the deletion of 19 amino acids of the signal peptide sequence, prevents the secretion of the enzyme into serum and causes profound biotinidase deficiency.
P19 is an Arab-Israeli female who at 2 months of age developed suspected seizures, jerking movement of her limbs and roving eye movements. The child was the product of an uneventful pregnancy. Family history disclosed that the mother, P17, and father, P18, are first-cousins. An electroencephalogram reportedly showed multifocal epileptiform abnormalities. Infantile spasms were diagnosed and the patient was treated with corticotropin. Computerized tomography of the brain was normal. The seizures subsided and the child appeared again to develop normally. At 8 months of age she had generalized and right focal seizures that were treated with valproic acid. A month later, she exhibited hyperpnea, hypotonia and a generalized maculopapular rash. She could sit at 7 months, but now was unable to sit unassisted. Blood pH was 7.21 and the bicarbonate concentration was 12 mmol/l. Serum lactic acid concentration was increased (6 mmol/l; normal <2 mmol/l). Gas-liquid chromatography of urinary organic acids revealed elevated concentrations of lactate, [beta]-hydroxyisovalerate and methylcitrate. Biotinidase activity in the plasma was 0.12 nmol/min/ml (5 ). Plasma biotinidase activities of the father and mother were 2.55 and 2.70 nmol/min/ml, respectively (mean normal activity is 7.1 nmol/min/ml; range 4.5-10). The child's condition improved promptly following oral biotin administration (10 mg/kg/day). Blood pH and bicarbonate concentration returned to normal within several hours. The skin rash disappeared in several days. Examination at 1 year of age showed that the child was doing well and had not had any more seizures. At 2 years of age the child's physical and neurological examination were normal. Bilateral brainstem auditory evoked potential studies were normal.
Immunochemical studies of serum from this child indicate that her serum had no detectable cross-reacting material to polyclonal antibodies prepared to normal human serum biotinidase (31 ,32 ).
P170 is a 4 year old female with profound biotinidase deficiency who was identified by newborn screening at one month of age. She has been treated with 10 mg biotin since the time of diagnosis and has remained asymptomatic and has developed normally. Her serum biotinidase activity was 0.36 nmol/min/ml serum. Her parents had serum activities in the heterozygous range. The father, P134, has Irish ancestry and the mother, P125, has Irish, English and German ancestry. The parents are not known to be consanguineous. P19 and P170 are not related and are of different ethnic backgrounds.
P10, a symptomatic child with profound biotinidase deficiency who was previously shown to be homozygous for the G98:d7i3 mutation, and P2, a symptomatic child with profound biotinidase deficiency who was previously shown to be heterozygous for the G98:d7i3 mutation (15 ), were included in our studies as controls.
Whole blood was obtained from the above patients, their parents and 42 individuals with normal serum biotinidase activity to establish lymphoblast cultures. DNA and RNA were isolated from the lymphoblastic cells as described below. This study was approved by the institutional review board at the Medical College of Virginia/Virginia Commonwealth University.
Genomic DNA was isolated from lymphoblast cultures (15 ) and whole blood samples using the Gentra (Minneapolis, MN) Pure Gene DNA isolation kit, according to the manufacturer's recommendations. Total RNA was isolated from cultured lymphoblasts by acid guanidinium thiocyanate-phenol-chloroform extraction (33 ) from the patients, relatives, individuals with normal biotinidase activity, and from human liver from an individual with normal biotinidase activity.
One [mu]g of gDNA from each subject was amplified in a 50 [mu]l PCR (34 ) using the previously described amplification parameters and 21mer oligonucleotide primers 161.S and 156.A that flank the intron-exon boundaries and include the entire B exon of the human biotinidase gene (13 ). Exon B encodes for the putative signal peptide as well as the N-terminus of the mature biotinidase enzyme (15 ).
SSCA was carried out on PCR-amplified products from patients, relatives, and individuals with normal biotinidase activity for this region of the genomic biotinidase gene as described previously (15 ,35 ,36 ).
Templates for sequence analysis of patient and normal genomic DNA were prepared by PCR amplification using primers 161.S and 156.A as described previously (15 ), gel purified on a 1% Seakem GTG agarose gel containing ethidium bromide stain, excised from the gel and the DNA electroeluted to recover the product. Electroeluted samples were desalted and concentrated in Centricon 30 Microconcentrators (Ambion Inc, Beverly, MA) according to the manufacturer's instructions. The purified samples were lyophilized and dissolved in 25 [mu]l of distilled water. Five [mu]l of this purified product were electrophoresed in a 1% agarose gel in parallel with standards to estimate yields. Sequencing reactions were performed using at least 200 ng of purified PCR product, 20 ng of primer (either 161.S or 156.A), and fluorescently labeled Taq DyeDeoxyTM terminator reaction mix (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Reactions were performed in a Perkin Elmer 9600 thermocycler and DNA sequence was determined using a 373A Automated DNA Sequencer (Applied Biosystems). Both strands were sequenced on at least three separate occasions for verification. Sequence editing was performed using SequencherTM software, version 3.0 (Gene Codes Corporation, Ann Arbor, MI).
First strand cDNA was synthesized by reverse transcription of 5 [mu]g of total RNA from lymphoblasts from patients, normals, and from liver using the First-Strand cDNA Synthesis Kit (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's recommendations. Ten pmol of the gene-specific antisense primer 1150.A (5'-GACTTCCTGAGCATCCTTCT-3') was included in the 20 [mu]l reaction volume. The entire cDNA product was amplified by PCR using the oligonucleotides 1.S (5'-GCCAGCTGGAGCGTTTTC-3') and 273.A (5'-CGGCATGAAGTCCAAAAATG-3'), under the following conditions: initial denaturation at 94oC for 5 min followed by 30 cycles consisting of denaturation at 94oC for 30 s, annealing at 55oC for 30 s, and extension at 72oC for 1 min, followed by a final extension at 72oC for 10 min. Five [mu]l of each product was used as template for a secondary amplification using the nested oligonucleotide primers 28.S (5'-AAGGGAGAATGGCGCATGCGCA-3') and 337.A (5'-CTCATACACGGCAGCCACA-3'), which are derived from the sequence of the normal BTD cDNA (13 ) for 30 cycles as described previously (15 ). Products from the secondary amplification were 5' end-labeled with [[gamma]-32P]dATP (800 Ci/mM) (DuPont NEN, Boston, MA) using standard procedures (37 ). Five [mu]l of each product was electrophoresed on a 6% non-denaturing polyacrylamide gel (37:1, acryl:bisacrylamide) in 1* TBE buffer in parallel with an end-labeled [Phi]X174 HaeIII molecular weight marker.
Six non-radioactive secondary PCR reactions were prepared for each individual by using the oligonucleotide primers 28.S and 337.A as described above. PCR products were gel purified and concentrated as described above. Automated sequencing was performed using 28.S as the forward primer and 337.A as the reverse primer.
Primers AB0.S (5'-CTCTGCGGCTGTTACGTG-3') and AB19.S (5'-CTCTGCAGCTGTTACGTG-3') were synthesized to detect normal and mutant sequences, respectively, in genomic DNA. Each primer was 5' end-labeled with [[gamma]-32P]dATP (800 Ci/mM) (DuPont NEN, Boston,MA) as described above. A non-radioactive PCR reaction was prepared for each individual using the primers 161.S and 156.A and the PCR conditions described above. Samples were prepared for dot blotting and hybridization by methods described previously (15 ). Membranes were then subjected to autoradiography at -70oC for 1-3 days.
This work was supported by NIH grant HD48258 to B.W. We thank Karen Norrgard, Kristin Fleischhauer, Vasant Narasimhan, Jeanne Hymes, and Greg Meyers for technical assistance and advice. The authors would also like to thank Joyce Lloyd for her patient review of this manuscript.
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*To whom correspondence should be addressed. Tel: +1 804 828 9632; Fax: +1 804 828 3760; Email: bwolf@gems.vcu.edu
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