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Human Molecular Genetics Pages 1657-1662
Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure
Introduction
Results
   Proposed mechanisms
   Mechanism 1
   Mechanism 2
Discussion
Materials And Methods
   Subjects
   Materials
   Amplification of genomic BTD DNA
   Screening for mutations using single strand conformation analysis (SSCA)
   Sequencing analysis
Acknowledgements
References

Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure

Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure Robert J. Pomponio1, Vasant Narasimhan1, Thomas R. Reynolds2, Gregory A. Buck2, Lawrence F. Povirk3 and Barry Wolf1,4,*

Departments of 1Human Genetics, 2Microbiology and Immunology, 3Pharmacology and Toxicology, and 4Pediatrics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA, USA

Received June 3, 1996; Revised and Accepted July 8, 1996

Biotinidase is responsible for recycling the vitamin biotin from biocytin that is formed after the proteolytic degradation of the biotin-dependent carboxylases. We have identified a deletion/insertion mutation within exon D of the human biotinidase gene in a child with biotinidase deficiency. The mutation causes a frame shift and premature termination which are predicted to result in a truncated protein. We propose that the mutation occurred during DNA replication by either of two mechanisms. Both mechanisms involve formation of a quasipalindromic hairpin loop in the template and dissociation of DNA polymerase [alpha]. This mutation supports the formation of palindromic structures as a possible cause of deletions in eukaryotes, and supports the proposal, derived from in vitro studies, that polymerase [alpha] may preferentially arrest or dissociate at specific template sequences.

INTRODUCTION

Biotin, an essential water-soluble B vitamin, is the coenzyme for four carboxylases in humans (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 the holocarboxylases (3 ). The holocarboxylases are ultimately degraded to biocytin (biotin-[epsilon]-lysine) which are then hydrolyzed by biotinidase (biotin-amide amidohydrolase, EC 3.5.1.12) to yield biotin for reutilization (3 ,4 ).

Biotinidase deficiency is an autosomal recessively inherited trait (3 ,5 ). Children with biotinidase deficiency cannot hydrolyze biocytin and thus cannot recycle biotin. If these children are not treated with biotin supplementation, they become biotin deficient, resulting in multiple carboxylase deficiency and the accumulation of toxic metabolites (6 ). Untreated individuals with biotinidase deficiency may exhibit seizures, hypotonia, ataxia, developmental delay, hearing loss, ophthalmologic abnormalities, alopecia, skin rash, ketolactic acidosis, and organic aciduria, which may result in coma (7 ,8 ). Treatment with pharmacologic doses of biotin resolves and reverses most or all of the clinical symptoms (3 ). Early diagnosis and treatment of biotinidase deficiency are important to prevent permanent neurological damage. Many states and countries perform newborn screening for biotinidase deficiency (9 ).

We have characterized the cDNA for normal human serum biotinidase (BTD) (10 ), and localized the gene to chromosome 3p25 (11 ). We have also described a common 7 bp deletion/3 bp insertion (G98:d7i3) in about half of the symptomatic children with profound biotinidase deficiency (12 ). We now describe a child with profound biotinidase deficiency who is homozygous for a deletion/insertion mutation within exon D of the biotinidase gene (13 ). We propose that the mutation resulted from the formation of a quasipalindromic stem-loop structure and the dissociation and reassociation of polymerase [alpha] during replication.

RESULTS

The oligonucleotide primers amplified a 300 bp PCR product. PCR amplification of this region of the BTD DNA did not reveal any obvious differences in the size of the PCR product of the patient when compared to that of the individuals with normal biotinidase activity by non-denaturing polyacrylamide gel electrophoresis (data not shown).

Single stranded conformational analysis (SSCA) of the PCR product from 16 normal individuals, as exemplified by patients 93, 209, 113, 114 and 115, exhibited four bands (the upper two bands migrating as a doublet) (Fig. 1 ). The SSCA patterns of patient 20 revealed three bands (the upper two bands as a doublet) that were different from any of the normal bands. Patient 20's mother, 68, and his father, 69, each had bands that corresponded to those of the normals and to those of their son. These results suggested that patient 20 was homozygous for a mutation and each of his parents were heterozygotes.


Figure 1. Single stranded conformation analysis of PCR-amplified DNA. ID numbers refer to the individuals tested. PCR-amplified allelic conformations are from several normal individuals (open symbol) , and patient 20, an individual with profound biotinidase deficiency (blackened symbol). The heterozygous parents are designated by the half-blackened symbols. Patient 10 is a child with profound biotinidase deficiency confirmed to be homozygous for the G98:d7i3 mutation.

Sequences were analyzed from normal individuals, the patient and his parents. The normal individuals all had sequences that were identical to the normal cDNA sequence (10 ). In contrast, the sequence of patient 20 revealed that he is homozygous for a deletion of 15 bp and an insertion of 11 bp (Fig. 2 ). This abnormality is predicted to result in a frame shift and premature termination at a stop codon producing a truncated protein with an aberrant amino acid sequence. The parents are both heterozygous for this mutation and the normal sequence.

Proposed mechanisms

We have identified a child with biotinidase deficiency with a mutation with a net 15 bp deletion and an 11 bp insertion. The normal double-stranded sequence of the exon involved in the mutation is shown in Figures 2 A and 3 A. The net deleted sequence (dashed underline), the net inserted sequence (underline) and the repeated flanking sequences (bolded) are also shown in these figures. We propose that either of the following two mechanisms caused the mutation.

Mechanism 1

In this mechanism, the deletion resulted from the formation of a quasipalindromic stem-loop structure in the sequence of the normal DNA (Fig. 2 B). This stem-loop structure is similar to those reported for other deletions suspected of involving formation of palindromic and quasipalindromic structures (14 -16 ). The free energy of the quasipalindromic structure is -4.2 kcal/mol (17 -19 ).

Following the formation of the quasipalindrome, replication proceeded along the template (3' -> 5'), bypassing the stem-loop structure and resulting in the deletion of the 27 nucleotides of the quasipalindrome (boxed in Fig. 2 B). Replication then continued along the template incorporating the sequence of the insertion (Fig. 2 B).

We predict that the polymerase dissociated at either the second -GAG or -AGA (bold) of the template (Fig. 2 B). In fact, studies have shown that polymerase [alpha] arrest or dissociation sites are GC-rich regions, with the best site being -GAG (20 ). The trinucleotide AGA, although not as good a dissociation site, is high on the list of sequences that can result in dissociation. We propose that the polymerase dissociated from the template at this point. The stem-loop structure then relaxed and the template slipped forward. The polymerase, with the newly synthesized strand ending in -TCT (assuming dissociation occurred at -AGA), reassociated with the first -AGA (bold), which was previously located within the quasipalindromic structure (Fig. 2 B,C). Replication then continued (Fig. 2 D), ultimately producing the sequence of the mutation shown in Figure 2 E.

Mechanism 2

In the second mechanism, we propose that transcription proceeded through the first -CAGAC (bold) of the template (Fig. 3 B). The polymerase then dissociated from the template at this -GAC, a trinucleotide that is also favorable for dissociation (20 ). The -GTCTG (bold) of the newly synthesized strand then reannealed to the downstream complementary -CAGAC sequence (bold in Fig. 3 B), which is further stabilized by the formation of the quasipalindromic structure (Fig. 3 C) with a free energy of -4.7 kcal/mol (17 -19 ). Replication continued to the second -AGA and then as described in mechanism 1 (Fig. 3 D). The stem-loop relaxed, the polymerase and the newly synthesized strand dissociated, then reassociated, and resulted in the sequence of the mutation shown in Figure 3 E.


Figure 2. Proposed mechanism 1 for the formation of the deletion/insertion mutation. (A) Normal double-stranded sequence of exon D of the BTD gene that is involved in the mutation. The net deleted sequence (dashed underline), the net inserted sequence (underline), and the repeated flanking sequences (bold) are indicated. (B) The quasipalindromic stem-loop structure is formed. Replication proceeded along the template until DNA polymerase [alpha] (DNA POL [alpha]; represented above the newly replicated strand), paused and dissociated at the second -AGA (bold). (C) The hairpin loop relaxed and the template slipped forward allowing the polymerase to reanneal to the first -AGA (bold). (D) Replication then continued along the template. (E) The resulting 5' -> 3' sequence of the strand with the mutation of patient 20. The repeated sequences are double underlined.


Figure 3. Proposed mechanism 2 for the formation of the deletion/insertion mutation. (A) Normal double-stranded sequence of exon D of the BTD gene that is involved in the mutation. The net deleted sequence (dashed underline), the net inserted sequence (underline), and the repeated flanking sequences (bold) are indicated. (B) Replication proceeded to the first -CAGAC (bold), where DNA polymerase [alpha] (DNA POL [alpha]) arrested, dissociated and reannealed to the downstream CAGAC (bold). (C) Reannealing is concomitantly stabilized by the formation of the quasipalindromic stem-loop structure. Replication continued along the template until the second -AGA (bold). The polymerase again arrested and dissociated. (D) The hairpin loop relaxed, the template slipped forward and is stabilized by pairing with the first -CTCTG in the template. The polymerase then reassociated to the first -AGA (bold). Replication then continued along the template as described in mechanism 1 (Fig. 2). (E) The resulting 5' -> 3'sequence of the strand with the mutation of patient 20. The repeated sequences are double underlined.

DISCUSSION

Deletions, insertions, and complex deletions/insertions occur with surprising frequency in human genetic disease (14 ,15 ,21 -25 ). Various mechanisms have been proposed to explain how these mutations arose. In general, they involve either slipped mispairing through the formation of various secondary structural intermediates or strand-switching or base misincorporation (21 ). Some deletions have been hypothesized to have resulted from the formation of palindromic or quasipalindromic cruciform (hairpin or stem-loop) structures (14 ,15 ,26 ). In the case of deletion/insertion mutations, the origin of the insertion is usually unknown and the mechanism of its formation is unexplained.

Although the quasipalindromic structure has a negative free energy in both mechanisms, it is not usually expected to form a stable structure. This is supported by our findings that this mutation has only been observed in one of 58 patients with profound biotinidase deficiency studied (Pomponio, Norrgard and Wolf, unpublished data). It is, however, important that the hypothesized conformation existed for only a short time and at one moment in time. The nucleotide loop at the 3'-base of the stem-loop structure is mildly strained and would not be expected to result in a stable conformation. If the structure did produce a stable conformation, such as occur in tRNAs, then the chances of mutation would be greatly increased and would likely have occurred often.

Because there are repeated nucleotide sequences at the 5'-end (GTCTG) and at the 3'-end (TCT) of the deleted sequence and surrounding the downstream sequence that is copied in the insertion, the actual size of the deletion and/or insertion is impossible to determine. The proposed mechanisms allow us to predict the origin of the bases in the mutation.

Formation of the secondary structure in the lagging strand is more likely than in the leading strand, because the lagging strand template becomes transiently single-stranded at the replication fork (27 ). As suggested by Bissler et al. (15 ), the cruciform structure may be stabilized when the RNA segment at the Okazaki fragment is replaced by the polymerase. The locations of the replication origins in the BTD gene, and thus the identity of leading and lagging strands in this region are not known.

We cannot definitively exclude other possibilities, such as a double crossover or gene conversion, as mechanisms that resulted in the mutation described here. However, the above mechanisms precisely encompass available experimental data supporting palindromic formation and predict the site of polymerase [alpha] dissociation. Undoubtedly, other mutations that occur in eukaryotes and cause diseases in humans will be found to occur through similar mechanisms.

MATERIALS AND METHODS

Subjects

The child (designated patient 20) is an 18 month old Hispanic male. At 1 month of age he exhibited shaking of the extremities, fluttering eyelids, foaming at the mouth, and staring. He subsequently had multiple similar intermittent episodes which were controlled with phenobarbital. Serum bicarbonate concentrations were 15 and 16 mg/dl. An EEG was abnormal with high amplitude sharp and slow wave activity bilaterally beginning in the right central region. There was also intermittent asynchrony of activity between the hemispheres. Magnetic resonance imaging of the brain was normal. Newborn screening for biotinidase deficiency indicated that the child was affected. Multiple attempts to locate the child were unsuccessful. At 2 months of age, he was admitted to the hospital for seizures and biotinidase deficiency testing was performed. His serum biotinidase activity is 0.17 nmol/min per ml (mean normal activity is 7.1 nmol/min per ml; range is 4.4-12) (5 ), confirming that he had profound biotinidase deficiency (less than 10 % of mean normal serum biotinidase activity) (28 ). The child's serum contained no cross-reacting material to antibodies prepared against normal serum biotinidase purified to homogeneity (28 ). There was also no biotinyl-transferase activity detected in his serum (29 ). He was subsequently treated with biotin and his seizures stopped within hours. With continued oral biotin supplementation, he has had no more seizure activity and is developmentally normal.

The biotinidase activity of the father (designated 69) is 2.29 nmol/min per ml serum and the activity of the mother (designated 68) is 2.47 nmol/min per ml serum. The family history reveals that he is the only child of parents whose families were both from the same town or region in Puerto Rico. The mother was adopted at 3 years of age. The parents are unaware of consanguinity.

Materials

Whole blood was obtained from the child with biotinidase deficiency, his parents, and 33 individuals with normal serum biotinidase activity. Lymphoblast cultures were established as described previously (12 ). Genomic DNA was isolated from lymphoblast cultures and whole blood samples using the Gentra (Minneapolis, MN) Pure Gene DNA isolation kit according to the manufacturer's recommendations. The concentration of each DNA sample was calculated from the optical density at 260 nm and diluted to a concentration of 0.2 [mu]g/[mu]l.

Amplification of genomic BTD DNA

One [mu]g of gDNA from each subject was amplified in a 50 [mu]l polymerase chain reaction (PCR) (30 ) using the previously described amplification parameters (12 ) and oligonucleotide primers 277.S (5'-AACGGACCCATCCCATAGT-3') and 267.A (5'-GTAAGTGCCATGTACTGT-3') based on the cDNA sequence of human biotinidase (10 ). The annealing temperature used for this oligonucleotide pair was 58oC.

Screening for mutations using single strand conformation analysis (SSCA)

SSCA was performed on PCR-amplified products from the patient, his parents, and individuals with normal biotinidase activity for this region of exon D of the genomic biotinidase gene using previously published methods (12 ,31 ,32 ).

Sequencing analysis

Templates for sequence analysis of patient and normal gDNA were prepared by non-radioactive PCR amplification of this region of exon D [nucleotides 1059-1359 based on the published cDNA sequence (10 )] on using primers 277.S and 267.A using the method described previously (12 ). Briefly, six separate 50 [mu]l PCR reactions and a negative control lacking DNA template were prepared for each individual. Sequencing reactions were performed using at least 200 ng of purified PCR product, 20 ng of primer (either 277.S and 267.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).

Sequences of interest were screened for potential secondary structure using the STEMLOOP function of the University of Wisconsin/GCG program.

ACKNOWLEDGEMENTS

This work was supported by NIH grant HD48258 to B.W. We thank Karen Norrgard, Kristin Fleischhauer, Jeanne Hymes, and Greg Meyers for technical assistance and advice, and Dr Marilyn Cowger and Cheryl Clow, RN, at the Children's Hospital at Albany Medical Center for providing samples and medical history from this patient and his family.

REFERENCES

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13 Cole,H. (1994) Cloning and characterization of the human biotinidase gene. Doctoral Thesis, Medical College of Virginia/Virginia Commonwealth University. Richmond, VA.

14 Yamakawa-Kobayashi,K., Kobayashi,T., Yanagi,H., Shimakura,Y., Satoh,J. and Hamaguchi,H. (1994) A novel complex in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region. Hum. Genet., 93, 625-628. MEDLINE Abstract

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20 Weaver,D.T. and DePamphilis,M.L. (1982) Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha. J. Biol. Chem., 257, 2075-2086. MEDLINE Abstract

21 Krawczak,M. and Cooper,D.N. (1991) Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment. Hum. Genet., 86, 425-441. MEDLINE Abstract

22 Tahara,T., Kraus,J.P. and Rosenberg,L.E. (1990) An unusual insertion/deletion in the gene encoding the beta-subunit of propionyl-CoA carboxylase is a frequent mutation in Caucasian propionic acidemia. Proc. Natl Acad. Sci. USA, 87, 1372-1376. MEDLINE Abstract

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32 Orita,M., Iwahana,H., Kanazawa,H. and Hayashi,K. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-stranded conformation polymorphisms. Proc. Natl Acad. Sci. USA, 86, 2766-2770. MEDLINE Abstract

*To whom correspondence should be addressed

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