- Email: [email protected]
Mutations causing biotinidase deficiency in children ascertained by newborn screening in Western Hungary
Molecular Genetics and Metabolism 90 (2007) 345–348 www.elsevier.com/locate/ymgme
Brief Communication
Mutations causing biotinidase deWciency in children ascertained by newborn screening in Western Hungary Ilona Milánkovics a,¤, Enikö Kámory b, Béla Csókay b, Flóra Fodor b, Csilla Somogyi a, Ágnes Schuler a a
Metabolic Screening Centre, Buda Children’s Hospital, Budapest H-1023 Bolyai u. 5-7., Hungary b LabOrigo Diagnostic Ltd., Budapest, Hungary
Received 11 September 2006; received in revised form 7 November 2006; accepted 7 November 2006 Available online 20 December 2006
Abstract In Hungary the national newborn screening programme for the detection of biotinidase deWciency was launched in 1989. In this study, we determined the genotypes of all patients identiWed at the Budapest Screening Centre that covers half of the country. The incidence of the disorder in Western Hungary is about three times the worldwide incidence. Overall, 21 diVerent mutations were identiWed in 49 patients, including four novel mutations. Ten mutations proved to be unique to the Hungarian population. © 2006 Elsevier Inc. All rights reserved. Keywords: Biotinidase deWciency; Biotinidase activity; Mutation detection; Incidence; Newborn screening; Population genetics
Introduction Biotin is an essential water-soluble vitamin that serves as a coenzyme for four carboxylases in humans. Its serum level depends on dietary biotin intake and the recycling of endogenous biotin. Biotinidase (BTD) is the enzyme that catalyzes the cleavage of biotin from biocytin or biotinylpeptides, the products of carboxylase degradation [1,2]. Biotinidase deWciency (MIM# 253260) is an autosomal recessive inherited disorder [3]. Untreated patients develop various clinical symptoms [4,5]. Many symptoms can be reversed by biotin treatment, however, some symptoms such as eye and hearing problems and developmental delay may be irreversible. Early recognition and biotin supplementation result in rapid clinical improvement. Newborn screening allows early presymptomatic treatment that can prevent neurological decay [6]. Biotinidase deWciency is classiWed as either profound or partial based on the serum biotinyl-hydrolase enzyme activity (0–10% and 10–30%, respectively). *
Corresponding author. Fax: +36 06 1 326 0571. E-mail address: [email protected] (I. Milánkovics).
1096-7192/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2006.11.005
Biotinidase deWciency can theoretically be detected in 100% of aVected infants who participate in the newborn screening programme. The national programme for biotinidase screening was launched in 1989 in Hungary [7] with two screening centers. The BTD gene, located on chromosome 3p25 [8,9], is the only gene known to be associated with biotinidase deWciency. It consists of four exons designated as 1–4. Exon 2 contains the N-terminal methionine of the mature enzyme [10]. Our aim was to apply mutation analysis for biotinidase deWciency in the Hungarian setting and to identify the genetic cause of the profound or partial biotinidase deWciency, both retrospectively and prospectively, in all Hungarian patients screened at the Budapest Screening Centre, as well as to identify carriers among family members. Materials and methods Patients Forty-nine patients from 41 families were identiWed at the Budapest Screening Centre, covering Western Hungary, between 1989 and June 2006. All patients were ascertained by routine newborn screening except for patient 23-02, who is the father of a patient. The diagnosis of
346
I. Milánkovics et al. / Molecular Genetics and Metabolism 90 (2007) 345–348
biotinidase deWciency was declared when the patient had less than 30% of the mean normal biotinyl-hydrolase activity. Nearly all children were treated with oral biotin supplementation immediately after biotinidase deWciency was diagnosed. In addition, 121 family members were examined from 31 families. Consents for the mutation analysis were obtained from the parents of all patients.
Biotinyl-hydrolase activity Biotinyl-hydrolase activity was determined quantitatively by a colorimetric assay that uses biotinyl-p-aminobenzoate (Sigma, Saint Louis, MO) as substrate [3]. Mean normal biotinyl-hydrolase activity was 7.05 nmol/ min/ml serum (range, 4.21–11.26 nmol/min/ml). The results were given as percentages of the normal mean enzyme activity.
DNA isolation, polymerase chain reaction (PCR), and direct sequencing DNA was extracted from Guthrie card samples according to the protocol of the High Pure PCR Template PuriWcation kit (Roche Diagnostics GmbH, Mannheim, Germany). Five pairs of primers were designed to amplify Wve fragments in separate PCR including exons 2, 3, and 4. Standard PCR conditions were used. Direct DNA sequencing was performed on an ABI-PRISM 310 genetic analyser using the BigDye terminator cycle sequencing kit v.3.1 (Applied Biosystems, Foster City, CA).
Results Twenty-one diVerent germline mutations were identiWed in 49 patients. Eleven patients from 10 families proved to have profound biotinidase deWciency having 0.17 § 0.4 nmol/min/ml (2.4%) mean normal biotinidase activity in serum (Table 1). In this group of patients, the p.Q456H was the most common mutation occurring in Wve alleles in four patients, followed in frequency by the p.R538C and the p.E112K. The c.98:d7i3 mutation, which is a deletion of seven and insertion of three bases (c.98_104delinsTCC) within the putative signal sequence encoded by exon 2, was found in only one patient with profound biotinidase deWciency. There was one patient (38-01) in which no mutation on the second allele could be detected. Thirty-eight patients from 31 families had partial biotinidase deWciency with 1.38 § 0.67 nmol/min/ml (19.5%) mean normal serum activity (Table 1). All of these patients were found to carry the missense mutation c.G1330>C (p.D444H) at least in one allele. The most frequent mutation in the other allele was the p.Q456H, which was found in nine patients. The c.98:d7i3 mutation was identiWed in eight patients, followed in frequency by the p.[A171T; D444H] double mutation in seven patients. We identiWed three novel missense changes and one novel nonsense alteration (p.T152P, p.R157C, p.N195S, and p.E46X) in patients with partial enzyme deWciency. Two silent alterations were also observed: the c.645C>T was detected in one patient and the c.1413T>C was found in six individuals from three families. From the 121 family members studied, 84 individuals were found to carry at least one alteration.
Discussion Over 950,000 neonates from Western Hungary were screened for biotinidase deWciency in the Budapest Screening Centre between 1989 and June 2006. In this period, 48 patients with profound or partial biotinidase deWciency from 41 families were identiWed through routine newborn screening and one additional patient (23-02) by family examination. The incidence of the disease in Hungary is one in 85,000 for profound biotinidase deWciency, one in 26,000 for partial biotinidase deWciency, and one in 20,000 for profound and partial biotinidase deWciency combined. Based on these data, the incidence of biotinidase deWciency in our population is approximately three times higher then the worldwide incidence [11]. About 90 mutations have been described worldwide to date. We found 21 diVerent mutations in our patients, including four novel mutations (p.E46X, p.T152P, p.R157C, and p.N195S). The p.E46X (c.136G>T) nonsense mutation results in a termination codon and truncation of the enzyme near the beginning of the translated protein. Two brothers with partial biotinidase deWciency (21-01, 21-03) were found to be compound heterozygotes for the p.R157C (c.469C>T) novel mutation and the p.D444H mutation. We ascertained by newborn screening one patient (23-01) who was a compound heterozygote for the third novel missense mutation (p.N195S) and for the p.D444H mutation. Interestingly, his father (23-02), who was the only patient identiWed by family screening, had the same genotype. Threonine at codon 152 is a conserved amino acid in mammals and birds, arginine at codon 157 is also conserved in mammals, birds and in amphibians [12]. Asparagine at codon 195 is conserved in mammals, birds, amphibians, Wsh, Drosophila and also in fungi [12]. Hence, the three missense novel mutations are localized in highly conserved regions of the gene, supporting the disease-causing nature of these novel alterations. In patients with profound biotinidase deWciency the p.Q456H, p.R538C and p.E112K were the most frequent mutations. The mutation p.Q456H was also the most common [13] and the p.R538C was the second most common mutation among newborn children with profound biotinidase deWciency in the United States [14]. The p.E112K, the p.E218Q, the p.L278V, the p.A82D mutations and the p.[L71P; R79H] double mutation have been described earlier in the Hungarian population of biotinidase deWciency patients [15]. There was only one patient (38-01) in whom we did not Wnd the mutation on the second allele. Considering that the analysis in this study was limited to the coding sequence of the biotinidase gene, it is reasonable to suppose that patient 38-01 may harbour a regulatory mutation [16]. All patients with partial biotinidase deWciency were found to be compound heterozygotes for the p.D444H mutation on one allele. Swango et al. [17] has reported that this combination of mutations was the most common cause of partial biotinidase deWciency, which is supported by the results of the present study. The p.D444H is similar to the
Table 1 Genotypes and biotinidase activities in the serum of newborns with profound and partial biotinidase deWciency, respectively Mutation 1 based on the cDNA
Protein change 1
Mutation 2 based on the cDNA
Protein change 2
Biotinyl-hydrolase activity (nmol/min/ml)
Percent of normal mean biotinyl-hydrolase activity (%)
05-01 06-01 07-01 19-01 28-01, 28-02 30-01 36-01 37-01 38-01 39-01
1368A>C 652G>C 334G>A 933delT 334G>A 1368A>C 98:d7i3 184G>A 245C>A 595G>A
Q456H E218Q E112K Frameshift E112K Q456H Frameshift V62M A82D V199M
1368A>C 832C>G 1368A>C 1368A>C 1612C>T 1612C>T 212T>C 236G>A 310-1G>T ND 631C>T
Q456H L278V Q456H Q456H R538C R538C L71P R79H Splice site ND R211C
0.14 0.07 0.07 0.21 0.07 0.14 0.14 0.07 0.28 0.57
2 1 1 3 1 2 2 1 4 8
01-02, 01-03, 10-01, 24-01, 27-01, 35-01, 44-01 02-01,11-01, 14-01, 17-01, 17-02, 22-01, 25-01, 33-01 03-01 04-01, 09-01, 09-02, 13-01, 13-02, 15-01, 26-01, 31-01, 45-01 12-01 16-01 20-01, 41-01 21-01, 21-03 23-01, 23-02 32-01 34-01, 34-02, 42-01 43-01
511G>A 1330G>C 98:d7i3 454A>C 1368A>C 1253G>C 136G>T 832C>G 469C>T 584A>G 643C>T 1612C>T 933delT
A171T D444H Frameshift T152P Q456H C418S E46X L278V R157C N195S L215F R538C Frameshift
1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C
D444H D444H D444H D444H D444H D444H D444H D444H D444H D444H D444H D444H
1.07–1.92 0.71–1.21 1.42 1.28–2.06 1.70 1.42 0.99–1.42 1.28-1.63 1.49 1.21 1.14–1.76 1.85
15–27 10–17 20 18–29 24 20 14–20 18–23 21 17 16–25 26
I. Milánkovics et al. / Molecular Genetics and Metabolism 90 (2007) 345–348
Patient number¤
Novel mutations are in bold. ND: not detected. ¤ The Wrst number refers to the family, and the second number indicates the member within that family.
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Duarte variant in galactosaemia in that it causes an approximately 50% reduction of enzyme activity in the aVected allele [17]. Of all the mutations detected in this group of patients, six have only been found in the Hungarian population. Four were novel mutations (p.E46X, p.T152P, p.R157C, and p.N195S), and two (p.C418S, p.L278V) have been described earlier by László et al. [15]. The p.D444H, p.Q456H, c.98:d7i3, p.[A171T; D444H], and the p.R538C mutations are the most frequent mutations worldwide [18]. These Wve mutations were found to account for 76% of all abnormal alleles in Western Hungary. Ten out of the 21 diVerent mutations detected have only been observed in Hungarian patients, suggesting that these mutations are unique to the Hungarian population. In our study, these population-speciWc unique mutations accounted for 16% of all abnormal alleles and 27% of the mutations causing profound biotinidase deWciency. In conclusion, we determined the mutation spectra causing partial and profound biotinidase deWciency in patients from Western Hungary. The results of this work add four more mutations (p.E46X, p.T152P, p.R157C, and p.N195S) to the total number of mutations described so far as causing profound biotinidase deWcieny. Mutation analysis does not only provide clues about phenotype/genotype correlation, but also serves with valuable information on DNA variation among populations and ethnic groups. Acknowledgment We thank Judit Bali for the excellent technical assistance. References [1] R.W. Thoma, The enzymatic degradation of soluble bound biotin, J. Biol. Chem. 210 (1954) 569–579. [2] J. Pispa, Animal biotinidase, Ann. Med. Exp. Biol. Fenn. 43 (1965) 1–39. [3] B. Wolf, R.E. Grier, R.J. Allen, S.I. Goodman, C.L. Kien, Biotinidase deWciency: the enzymatic defect in late-onset multiple carboxylase deWciency, Clin. Chim. Acta 131 (1983) 273–281. [4] B. Wolf, R.E. Grier, R.J. Allen, S.I. Goodman, C.L. Kien, W.D. Parker, D.M. Howell, D.L. Hurst, Phenotypic variation in biotinidase deWciency, J. Pediatr. 103 (1983) 233–237.
[5] B. Wolf, G.S. Heard, K.A. Weissbecker, J.R. Secor McVoy, R.E. Grier, R.T. Leshner, Biotinidase deWciency: initial clinical features and rapid diagnosis, Ann. Neurol. 18 (1985) 614–617. [6] G.S. Heard, B. Wolf, R.G. JeVerson, K.A. Weissbecker, V.E. Nance, J.R. Secor McVoy, A. Napolitano, P.L. Mitchell, A.L. Linyear, Neonatal screening for biotinidase deWciency: results of a 1-year pilot study, J. Pediatr. 108 (1986) 40–46. [7] Z. Havass, Neonatal Screening for biotinidase deWciency in EastHungary, J. Inher. Metab. Dis. 14 (1991) 928–931. [8] H. Cole, T.R. Reynolds, G.B. Buck, J.M. Lockyer, T. Denson, J.E. Spence, J. Hymes, B. Wolf, Human serum biotinidase: cDNA cloning, sequence, and characterization, J. Biol. Chem. 269 (1994) 6566–6570. [9] H. Cole, H. Weremowicz, C.C. Morton, B. Wolf, Localization of serum biotinidase (BTD to human chromosome 3 in band p25), Genomics 22 (1994) 662–663. [10] H.C. Knight, T.R. Reynolds, G.A. Meyers, R.J. Pomponio, G.A. Buck, B. Wolf, Structure of the human biotinidase gene, Mamm. Genome 9 (1998) 327–330. [11] B. Wolf, Worldwide survey of neonatal screening for biotinidase deWciency, J. Inherit. Metab. Dis. 14 (1991) 923–927. [12] B. Wolf, K. Jensen, Evolutionary conservation of biotinidase: Implications for the enzyme’s structure and subcellular localization, Mol. Genet. Met. 86 (2005) 44–50. [13] K.J. Norrgard, R.J. Pomponio, K.L. Swango, J. Hymes, T.R. Reynolds, G.A. Buck, B. Wolf, Mutation (Q456H) is the most common cause of prodound biotinidase deWciency in children ascertained by newborn screening in the United States, Biochem. Molecul. Med. 61 (1997) 22–27. [14] R.J. Pomponio, K.J. Norrgard, J. Hymes, T.R. Reynolds, G.A. Buck, R. Baumgartner, T. Suormala, B. Wolf, Arg538 to Cys mutation in a CpG dinucleotide of the human biotindase gene is the second most common cause of profound biotinidase deWciency in symptomatic children, Hum. Genet. 99 (1997) 506–512. [15] A. Laszlo, A. Schuler, E. Sallay, E. EndreVy, Cs. Somogyi, A. Varkonyi, Z. Havass, K.P. Jansen, B. Wolf, Neonatal screening for biotinidase deWciency in Hungary: clinical, biochemical and molecular studies, J. Inherit. Metab. Dis. 26 (2003) 693–698. [16] A. Muhl, D. Moslinger, C.B. Item, S. Stockler-Ipsiroglu, Molecular characterisation of 34 patients with biotinidase deWciency ascertained by newborn screening and family investigation, Eur. J. Hum. Genet. 9 (2001) 237–243. [17] K.L. Swango, M. Demirkol, G. Hüner, E. Pronicka, J. SykutCegielska, A. Schulze, B. Wolf, Partial biotinidase deWciency is usually due to the D444H mutation in the biotinidase gene, Hum. Genet. 102 (1998) 571–575. [18] S.F. Dobrowolski, J. Angeletti, R.A. Banas, E.W. Naylor, Real time PCR assays to detect common mutations the biotinidase gene and application of mutational analysis to newborn screening for biotinidase deWciency, Mol. Genet. Metab. 78 (2003) 100–107.
Brief Communication
Mutations causing biotinidase deWciency in children ascertained by newborn screening in Western Hungary Ilona Milánkovics a,¤, Enikö Kámory b, Béla Csókay b, Flóra Fodor b, Csilla Somogyi a, Ágnes Schuler a a
Metabolic Screening Centre, Buda Children’s Hospital, Budapest H-1023 Bolyai u. 5-7., Hungary b LabOrigo Diagnostic Ltd., Budapest, Hungary
Received 11 September 2006; received in revised form 7 November 2006; accepted 7 November 2006 Available online 20 December 2006
Abstract In Hungary the national newborn screening programme for the detection of biotinidase deWciency was launched in 1989. In this study, we determined the genotypes of all patients identiWed at the Budapest Screening Centre that covers half of the country. The incidence of the disorder in Western Hungary is about three times the worldwide incidence. Overall, 21 diVerent mutations were identiWed in 49 patients, including four novel mutations. Ten mutations proved to be unique to the Hungarian population. © 2006 Elsevier Inc. All rights reserved. Keywords: Biotinidase deWciency; Biotinidase activity; Mutation detection; Incidence; Newborn screening; Population genetics
Introduction Biotin is an essential water-soluble vitamin that serves as a coenzyme for four carboxylases in humans. Its serum level depends on dietary biotin intake and the recycling of endogenous biotin. Biotinidase (BTD) is the enzyme that catalyzes the cleavage of biotin from biocytin or biotinylpeptides, the products of carboxylase degradation [1,2]. Biotinidase deWciency (MIM# 253260) is an autosomal recessive inherited disorder [3]. Untreated patients develop various clinical symptoms [4,5]. Many symptoms can be reversed by biotin treatment, however, some symptoms such as eye and hearing problems and developmental delay may be irreversible. Early recognition and biotin supplementation result in rapid clinical improvement. Newborn screening allows early presymptomatic treatment that can prevent neurological decay [6]. Biotinidase deWciency is classiWed as either profound or partial based on the serum biotinyl-hydrolase enzyme activity (0–10% and 10–30%, respectively). *
Corresponding author. Fax: +36 06 1 326 0571. E-mail address: [email protected] (I. Milánkovics).
1096-7192/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2006.11.005
Biotinidase deWciency can theoretically be detected in 100% of aVected infants who participate in the newborn screening programme. The national programme for biotinidase screening was launched in 1989 in Hungary [7] with two screening centers. The BTD gene, located on chromosome 3p25 [8,9], is the only gene known to be associated with biotinidase deWciency. It consists of four exons designated as 1–4. Exon 2 contains the N-terminal methionine of the mature enzyme [10]. Our aim was to apply mutation analysis for biotinidase deWciency in the Hungarian setting and to identify the genetic cause of the profound or partial biotinidase deWciency, both retrospectively and prospectively, in all Hungarian patients screened at the Budapest Screening Centre, as well as to identify carriers among family members. Materials and methods Patients Forty-nine patients from 41 families were identiWed at the Budapest Screening Centre, covering Western Hungary, between 1989 and June 2006. All patients were ascertained by routine newborn screening except for patient 23-02, who is the father of a patient. The diagnosis of
346
I. Milánkovics et al. / Molecular Genetics and Metabolism 90 (2007) 345–348
biotinidase deWciency was declared when the patient had less than 30% of the mean normal biotinyl-hydrolase activity. Nearly all children were treated with oral biotin supplementation immediately after biotinidase deWciency was diagnosed. In addition, 121 family members were examined from 31 families. Consents for the mutation analysis were obtained from the parents of all patients.
Biotinyl-hydrolase activity Biotinyl-hydrolase activity was determined quantitatively by a colorimetric assay that uses biotinyl-p-aminobenzoate (Sigma, Saint Louis, MO) as substrate [3]. Mean normal biotinyl-hydrolase activity was 7.05 nmol/ min/ml serum (range, 4.21–11.26 nmol/min/ml). The results were given as percentages of the normal mean enzyme activity.
DNA isolation, polymerase chain reaction (PCR), and direct sequencing DNA was extracted from Guthrie card samples according to the protocol of the High Pure PCR Template PuriWcation kit (Roche Diagnostics GmbH, Mannheim, Germany). Five pairs of primers were designed to amplify Wve fragments in separate PCR including exons 2, 3, and 4. Standard PCR conditions were used. Direct DNA sequencing was performed on an ABI-PRISM 310 genetic analyser using the BigDye terminator cycle sequencing kit v.3.1 (Applied Biosystems, Foster City, CA).
Results Twenty-one diVerent germline mutations were identiWed in 49 patients. Eleven patients from 10 families proved to have profound biotinidase deWciency having 0.17 § 0.4 nmol/min/ml (2.4%) mean normal biotinidase activity in serum (Table 1). In this group of patients, the p.Q456H was the most common mutation occurring in Wve alleles in four patients, followed in frequency by the p.R538C and the p.E112K. The c.98:d7i3 mutation, which is a deletion of seven and insertion of three bases (c.98_104delinsTCC) within the putative signal sequence encoded by exon 2, was found in only one patient with profound biotinidase deWciency. There was one patient (38-01) in which no mutation on the second allele could be detected. Thirty-eight patients from 31 families had partial biotinidase deWciency with 1.38 § 0.67 nmol/min/ml (19.5%) mean normal serum activity (Table 1). All of these patients were found to carry the missense mutation c.G1330>C (p.D444H) at least in one allele. The most frequent mutation in the other allele was the p.Q456H, which was found in nine patients. The c.98:d7i3 mutation was identiWed in eight patients, followed in frequency by the p.[A171T; D444H] double mutation in seven patients. We identiWed three novel missense changes and one novel nonsense alteration (p.T152P, p.R157C, p.N195S, and p.E46X) in patients with partial enzyme deWciency. Two silent alterations were also observed: the c.645C>T was detected in one patient and the c.1413T>C was found in six individuals from three families. From the 121 family members studied, 84 individuals were found to carry at least one alteration.
Discussion Over 950,000 neonates from Western Hungary were screened for biotinidase deWciency in the Budapest Screening Centre between 1989 and June 2006. In this period, 48 patients with profound or partial biotinidase deWciency from 41 families were identiWed through routine newborn screening and one additional patient (23-02) by family examination. The incidence of the disease in Hungary is one in 85,000 for profound biotinidase deWciency, one in 26,000 for partial biotinidase deWciency, and one in 20,000 for profound and partial biotinidase deWciency combined. Based on these data, the incidence of biotinidase deWciency in our population is approximately three times higher then the worldwide incidence [11]. About 90 mutations have been described worldwide to date. We found 21 diVerent mutations in our patients, including four novel mutations (p.E46X, p.T152P, p.R157C, and p.N195S). The p.E46X (c.136G>T) nonsense mutation results in a termination codon and truncation of the enzyme near the beginning of the translated protein. Two brothers with partial biotinidase deWciency (21-01, 21-03) were found to be compound heterozygotes for the p.R157C (c.469C>T) novel mutation and the p.D444H mutation. We ascertained by newborn screening one patient (23-01) who was a compound heterozygote for the third novel missense mutation (p.N195S) and for the p.D444H mutation. Interestingly, his father (23-02), who was the only patient identiWed by family screening, had the same genotype. Threonine at codon 152 is a conserved amino acid in mammals and birds, arginine at codon 157 is also conserved in mammals, birds and in amphibians [12]. Asparagine at codon 195 is conserved in mammals, birds, amphibians, Wsh, Drosophila and also in fungi [12]. Hence, the three missense novel mutations are localized in highly conserved regions of the gene, supporting the disease-causing nature of these novel alterations. In patients with profound biotinidase deWciency the p.Q456H, p.R538C and p.E112K were the most frequent mutations. The mutation p.Q456H was also the most common [13] and the p.R538C was the second most common mutation among newborn children with profound biotinidase deWciency in the United States [14]. The p.E112K, the p.E218Q, the p.L278V, the p.A82D mutations and the p.[L71P; R79H] double mutation have been described earlier in the Hungarian population of biotinidase deWciency patients [15]. There was only one patient (38-01) in whom we did not Wnd the mutation on the second allele. Considering that the analysis in this study was limited to the coding sequence of the biotinidase gene, it is reasonable to suppose that patient 38-01 may harbour a regulatory mutation [16]. All patients with partial biotinidase deWciency were found to be compound heterozygotes for the p.D444H mutation on one allele. Swango et al. [17] has reported that this combination of mutations was the most common cause of partial biotinidase deWciency, which is supported by the results of the present study. The p.D444H is similar to the
Table 1 Genotypes and biotinidase activities in the serum of newborns with profound and partial biotinidase deWciency, respectively Mutation 1 based on the cDNA
Protein change 1
Mutation 2 based on the cDNA
Protein change 2
Biotinyl-hydrolase activity (nmol/min/ml)
Percent of normal mean biotinyl-hydrolase activity (%)
05-01 06-01 07-01 19-01 28-01, 28-02 30-01 36-01 37-01 38-01 39-01
1368A>C 652G>C 334G>A 933delT 334G>A 1368A>C 98:d7i3 184G>A 245C>A 595G>A
Q456H E218Q E112K Frameshift E112K Q456H Frameshift V62M A82D V199M
1368A>C 832C>G 1368A>C 1368A>C 1612C>T 1612C>T 212T>C 236G>A 310-1G>T ND 631C>T
Q456H L278V Q456H Q456H R538C R538C L71P R79H Splice site ND R211C
0.14 0.07 0.07 0.21 0.07 0.14 0.14 0.07 0.28 0.57
2 1 1 3 1 2 2 1 4 8
01-02, 01-03, 10-01, 24-01, 27-01, 35-01, 44-01 02-01,11-01, 14-01, 17-01, 17-02, 22-01, 25-01, 33-01 03-01 04-01, 09-01, 09-02, 13-01, 13-02, 15-01, 26-01, 31-01, 45-01 12-01 16-01 20-01, 41-01 21-01, 21-03 23-01, 23-02 32-01 34-01, 34-02, 42-01 43-01
511G>A 1330G>C 98:d7i3 454A>C 1368A>C 1253G>C 136G>T 832C>G 469C>T 584A>G 643C>T 1612C>T 933delT
A171T D444H Frameshift T152P Q456H C418S E46X L278V R157C N195S L215F R538C Frameshift
1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C 1330G>C
D444H D444H D444H D444H D444H D444H D444H D444H D444H D444H D444H D444H
1.07–1.92 0.71–1.21 1.42 1.28–2.06 1.70 1.42 0.99–1.42 1.28-1.63 1.49 1.21 1.14–1.76 1.85
15–27 10–17 20 18–29 24 20 14–20 18–23 21 17 16–25 26
I. Milánkovics et al. / Molecular Genetics and Metabolism 90 (2007) 345–348
Patient number¤
Novel mutations are in bold. ND: not detected. ¤ The Wrst number refers to the family, and the second number indicates the member within that family.
347
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Duarte variant in galactosaemia in that it causes an approximately 50% reduction of enzyme activity in the aVected allele [17]. Of all the mutations detected in this group of patients, six have only been found in the Hungarian population. Four were novel mutations (p.E46X, p.T152P, p.R157C, and p.N195S), and two (p.C418S, p.L278V) have been described earlier by László et al. [15]. The p.D444H, p.Q456H, c.98:d7i3, p.[A171T; D444H], and the p.R538C mutations are the most frequent mutations worldwide [18]. These Wve mutations were found to account for 76% of all abnormal alleles in Western Hungary. Ten out of the 21 diVerent mutations detected have only been observed in Hungarian patients, suggesting that these mutations are unique to the Hungarian population. In our study, these population-speciWc unique mutations accounted for 16% of all abnormal alleles and 27% of the mutations causing profound biotinidase deWciency. In conclusion, we determined the mutation spectra causing partial and profound biotinidase deWciency in patients from Western Hungary. The results of this work add four more mutations (p.E46X, p.T152P, p.R157C, and p.N195S) to the total number of mutations described so far as causing profound biotinidase deWcieny. Mutation analysis does not only provide clues about phenotype/genotype correlation, but also serves with valuable information on DNA variation among populations and ethnic groups. Acknowledgment We thank Judit Bali for the excellent technical assistance. References [1] R.W. Thoma, The enzymatic degradation of soluble bound biotin, J. Biol. Chem. 210 (1954) 569–579. [2] J. Pispa, Animal biotinidase, Ann. Med. Exp. Biol. Fenn. 43 (1965) 1–39. [3] B. Wolf, R.E. Grier, R.J. Allen, S.I. Goodman, C.L. Kien, Biotinidase deWciency: the enzymatic defect in late-onset multiple carboxylase deWciency, Clin. Chim. Acta 131 (1983) 273–281. [4] B. Wolf, R.E. Grier, R.J. Allen, S.I. Goodman, C.L. Kien, W.D. Parker, D.M. Howell, D.L. Hurst, Phenotypic variation in biotinidase deWciency, J. Pediatr. 103 (1983) 233–237.
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