YMGME-06001; No. of pages: 4; 4C: Molecular Genetics and Metabolism xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Forty-eight novel mutations causing biotinidase deficiency Melinda Procter a, Barry Wolf b,⁎, Rong Mao a,c a b c
ARUP Institute for Clinical and Experimental Pathology, University of Utah, Salt Lake City, UT, USA Department of Research Administration, Henry Ford Hospital and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA Department of Pathology, University of Utah, Salt Lake City, UT, USA
a r t i c l e
i n f o
Article history: Received 13 November 2015 Received in revised form 7 January 2016 Accepted 7 January 2016 Available online xxxx Keywords: Biotinidase deficiency Biotinidase Mutation Biotin-responsive Mutation database
a b s t r a c t Biotinidase deficiency is an autosomal recessively inherited disorder that results in the inability to recycle the vitamin biotin and is characterized by neurological and cutaneous symptoms. The symptoms can be ameliorated or prevented by administering pharmacological doses of biotin. Since 2008, approximately 300 samples have been submitted to ARUP's Molecular Sequencing Laboratory for biotinidase mutation analysis. Of these, 48 novel alterations in the biotinidase gene have been identified. Correlating the individual's serum enzymatic activity with the genotype, we have been able to determine the effect of the novel alteration on enzyme activity and, thereby, determine its likelihood of being pathogenic in 44 of these individuals. The novel mutations and uncertain alterations have been added to the database established by ARUP (http://arup.utah.edu/database/ BTD/BTD_welcome.phps) to help clinicians make decisions about management and to better counsel their patients based on their genotypes. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Biotinidase (EC 3.5.1.12) is the enzyme that catalyzes the recycling of biotin from biocytin, the product of the proteolytic degradation of biotin-dependent carboxylases [1,2]. Biotinidase deficiency (OMIM #609019) is an autosomal recessively inherited disorder that if untreated can result in neurological and cutaneous symptoms [1]. If individuals with biotinidase deficiency are treated with pharmacological doses of biotin before symptoms develop, symptoms can be prevented. If they are treated after symptoms occur, the symptoms will markedly improve, although some symptoms, such as optic atrophy, hearing loss and developmental delay, are likely to be irreversible [1]. Biotinidase deficiency is included in mandatory newborn screening programs in all states in the United States and in many countries. Individuals with biotinidase deficiency either have profound biotinidase deficiency, less than 10% of mean normal serum enzyme activity, or partial biotinidase deficiency, between 10% and 30% of mean normal serum activity [3]. Over 150 mutations have previously been reported that cause biotinidase deficiency [4]. Definitive diagnosis of biotinidase deficiency can be challenging because the biotinidase enzyme is sensitive to suboptimal handling conditions, such as failure to separate the serum from whole blood or storing the serum at non-freezing temperatures, resulting in erroneously low activities and misdiagnosis. For this reason, confirmation of the
⁎ Corresponding author at: Department of Research Administration, Henry Ford Hospital, Detroit, MI 38202, USA. E-mail address: [email protected] (B. Wolf).
diagnosis can necessitate sequencing of BTD in some instances to help determine definitively an individual's status. Since BTD sequencing was incorporated at ARUP's Molecular Sequencing Laboratory in 2008, approximately 300 samples have been submitted for mutation analysis. Of these, we have identified 48 novel alterations and mutations, which are reported here.
2. Materials and methods Blood samples from 300 individuals from the United States were submitted to ARUP, a large clinical diagnostic laboratory (Salt Lake City, UT) for BTD gene sequencing. The vast majority of these samples are from children identified by newborn screening. Serum enzyme activities either indicated enzyme deficiency or indicated ambiguously low-normal activity. For clinical purposes, DNA sequencing was used to confirm the enzymatic activity status. Human BTD consists of four exons. We developed a protocol to sequence the coding regions of the BTD gene as well as intron/exon boundaries. BTD coding regions and intron/exon boundaries of the BTD gene were amplified and sequenced using the Sanger method. DNA extracted from whole blood in a MagNAPure Compact instrument (Roche, Indianapolis, IN). PCR was performed on each of the four exons of BTD using M13-tailed primers Premix D (Epicentre, Madison, WI), and Platinum Taq (Invitrogen, Carlsbad, CA) using a “touchdown” PCR protocol. PCR consisted of a 95 °C denaturation proceeding 10 cycles of 94 °C for 30 s, 62 °C for 45 s with a 0.5 °C decrease in temperature per cycle and 72 °C for 1 min. This procedure is followed by 25 cycles of 94 °C for 30 s, 57 °C for 45 s and 72 °C for 1 min.
http://dx.doi.org/10.1016/j.ymgme.2016.01.002 1096-7192/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
2
M. Procter et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx
Because of the size of the coding region of exon 4, about 1200 bp, this exon was amplified in three overlapping sequences to ensure complete coverage. Next, unused PCR primers and unincorporated nucleotides were inactivated by incubation with ExoSAP (USB Corporation, Cleveland, OH). Bidirectional DNA sequencing was performed with BigDye Terminator chemistry (ABI, Foster City, CA) and M13 primers (IDT, Coralville, IA) and the product was analyzed on the ABI 3100. Data analysis was performed using Mutation Surveyor software (SoftGenetics, State College, PA). In those individuals in whom three alterations are identified, one is the D444H mutation. We have assigned the second mutation to be allelic with the D444H mutation if this combination has been reported previously. Parental testing was not available to determine allelic distribution of the alterations. It is not usually possible to determine whether any two mutations observed by Sanger Sequencing are in cis or trans. However, the allelic phase status can usually be determined by our prior knowledge about the second known mutation, especially when the second mutation includes the D444H mutation. We did not perform copy number analyses on any of the individuals, because the syntony and knowledge of previously established single or combination mutations was sufficient to assign allelic designations. In those cases where enzymatic information was not available, we used the following predictive programs: SIFT [5], MutationTaster [6] and PolyPhen [7]. The effects of the alteration as being pathogenic or pathogenicpartial are based on the strong functional criteria delineated in the guidelines recommended by the American College of Medical Genetics and Genomics and the Association of Molecular Pathology [8].
3. Results Of the 300 samples submitted for mutation analysis, 89% were found to have at least one alteration/mutation. We are now presenting 48 novel alterations in the BTD gene (Tables 1 and 2) that have not previously been reported. They are listed in the order of their location in the BTD gene sequence. Most of the individuals studied were having mutation analysis to determine their genotype to confirm their degree of biotinidase deficiency or whether they actually have biotinidase deficiency. None of the individuals reported was symptomatic. In the United States where testing for biotinidase deficiency is included in newborn screening assays, a diagnosis of biotinidase deficiency is made and biotin supplementation is initiated before a child can become symptomatic. In all cases, the novel alteration was found to be heterozygous. In almost all cases, the second heterozygous mutation had been reported previously and the serum biotinidase activity of the individual was provided with the sample. Therefore, we could attribute the effect of the novel alteration on biotinidase activity. If the alteration was found to be heterozygous with a mutation on the other allele known to cause profound biotinidase deficiency, then the novel alteration was estimated to result in less than 10% of mean normal activity or profound biotinidase deficiency. These alterations were determined to be mutations and are classified as pathogenic (Table 1). If the enzyme activity is calculated to result in more than 10% of mean normal activity, but less than that of normal activity, it is classified as pathogenic-partial? (Table 1). Additional enzymatic information from other individuals having these identical alterations will be required to classify definitively these mutations. Thirty-two of 48 individuals (67%) of novel alterations were found in combination with the D444H mutation on the other allele. If the child's enzymatic activity is consistent with partial deficiency biotinidase activity and the second mutation is the D444H mutation, which results in about 50% of mean normal activity, we can predict that the novel variant results in less than 10% of mean normal activity, causes profound deficiency and is pathogenic.
Table 1 includes those novel alterations in BTD in which their contribution to enzymatic activity can be estimated based on the individual's serum activity and our knowledge of the effect of the second mutation on enzymatic activity or the lack of a second mutation. In six of the novel alterations, multiple individuals were identified with the same novel variant allowing us to assign their classifications as a pathogenic. Two of the individuals (Subjects 21 and 43) with novel alterations have a second previously reported mutation (c.310-15delT) that has been shown to cause partial biotinidase deficiency due to decreased transcription of the BTD gene [9]. The enzymatic activity of Subject 21 is likely erroneous low. Nevertheless, both novel mutations appear to result in essentially complete loss of activity and can be classified as pathogenic. Table 2 includes four novel alterations that could not be assigned a classification based on their effect on enzymatic activity for various reasons. In each of these cases, we have used prediction programs to ascertain whether the alteration is likely to be deleterious to enzyme activity. We did not have biotinidase activity information from two of individuals (Subjects 45 and 47). Therefore, we could not estimate the effects of these novel alterations on enzymatic activity. In these two cases, we have included the predictive mutation data from three programs. However, the definitive effects of these alterations remain to be determined. Subject 46 has a missense alteration on one allele and an alteration within the first intron on the other. Although the alteration within the intron does not likely affect enzymatic activity, because the individual's enzymatic activity is within the low normal range, we still cannot estimate the effect of the novel alteration on enzymatic activity. The three predictive programs all indicate that this alteration is likely pathogenic. Only a single alteration was identified for Subject 48. However, the individual's enzymatic activity was consistent with profound deficiency. It is likely that the enzymatic activity is actually higher than reported, but unfortunately, we were unable to confirm this because follow-up enzymatic testing on this individual was not available. Two of the prediction programs indicate that this novel alteration is pathogenic, whereas one program indicates that the alteration is tolerated. 4. Discussion We are fortunate that in the evaluating individuals for biotinidase deficiency we usually have their biochemical phenotype or enzymatic activity and genotype. This allows us to determine the effect of a novel mutation on enzymatic activity in almost all individuals. Because enzyme activity determines if an individual has profound or partial biotinidase deficiency, we can determine if the novel mutation is pathogenic. In those few cases where enzymatic information was not available, we used predictive programs. Because the laboratory receives enzymatic activity information from various institutions, we do not know if the activities were determined using a newborn screening sample or by confirmatory testing of serum activity. Therefore, as stated above, it is not usually possible to know if the activities were optimally determined and/or are correct. Our estimates of the contributions of the novel alterations on enzyme activities are based on the single activity submitted with the sample. When the activity is inconsistent with the genotype, we have attempted to contact the submitting physicians and/or institutions to clarify the activities, but we were not always successful. Therefore, our conclusions about pathogenicity are likely correct, but may require modification when other individuals with identical alterations and more confidently determined enzymatic activities are reported. We identified 48 novel variants. These variants can be characterized as mutations if we could demonstrate that the novel variant causes marked reduction in biotinidase activity. We were able to categorize 44 of these variants as “pathogenic” or “pathogenic-partial?” based on available biotinidase activities. The remaining four novel mutations were categorized as “uncertain” because of the lack of information
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
M. Procter et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx
3
Table 1 Novel mutations causing biotinidase deficiency. Subject
Exon location
Novel mutation nucleotide change
Novel mutation protein change
Mutation type
Predicted classification
Mutation #2
Serum biotinidase activity (U/L)
1 2 3 4 5 6 7 8 9
2 2 2 3 3 3 3 4 4
c.192GNC c.245CNT c.299CNT c.321TNG c.326dupT c.356ANG c.406delC c.582CNG c.625CNT
p.E64D p.A82V p.A100V p.I107M p.F110Vfs*42 p.N119S p.Q136Rfs*23 p.F194L p.R209C
Missense Missense Missense Missense Insertion Missense Deletion Missense Missense
Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic-partial? Pathogenic-partial? Pathogenic Pathogenic Pathogenic-partial?
10 11 12 13 14 15 16
4 4 4 4 4 4 4
c.631CNT c.646TNA c.664GNC c.692delC 695 TNC c.709GNA c.743TNC
p.R211C p.Y216N p.D222H p.F232Lfs*32 p.F232S p.A237T p.I248T
Missense Missense Missense Deletion Missense Missense Missense
Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic
17 18 19 20
4 4 4 4
c.758CNT c.770TNA c.783CNG c.814TNG
p.P253L p.V257D p.Y261X p.W272G
Missense Missense Nonsense Missense
Pathogenic Pathogenic Pathogenic Pathogenic-partial?
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
c.836TNA c.856ANG c.865GNC c.880ANG c.895GNC c.1001TNA c.1046A NC c.1096-1097dupTC c.1126C NT c.1241_1252del12bp c.1252TNC c.1309C NG c.1334GNA c.1352_1353delGC c.1432GNA c.1455C NG c.1458delG c.1459delT
p.L279X p.K286E p.A289P p.I294V p.A299P p.I334N p.N349T p.G367Qfs*23 p.Q376X p.Y414_Val417del p.C418R p.L437V p.G445E p.G451Dfs*32 p.A478T p.H485Q p.W487Gfs*14 p.W487Gfs*14
Nonsense Missense Missense Missense Missense Missense Missense Duplication Nonsense Deletion Missense Missense Missense Deletion Missense Missense Deletion Deletion
Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic-partial? Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic
39 40 41
4 4 4
c.1481A NG c.1526C NG c.1612C NA
p.Y494C p.P509R p.R538S
Missense Missense Missense
Pathogenic Pathogenic Pathogenic
42 43 44
4 4 4
c.1613GNA c.1628A NT c.1629C NA
p.R538H p.D543V p.D543E
Missense Missense Missense
Pathogenic Pathogenic-partial? Pathogenic
c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. Second mutation not identified. c.1459delT c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1368ANC, p.Q456H. c.528CNT, p.K176N;c.1595CNT, p.T532M c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.528GNT, p.K176N. c.528GNT, p.K176N. c.1489CNT, p.P497S. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H;c.511G NA, p.A171T c.310-15delT. c.1330GNC, p.D444H. c.1330GNC, p.D444H;c.133G NA, p.G45R; Second mutation not identified. c.1413TNC, p.C471C(polymorphism) c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1612CNT, p.R538C. c.1330GNC, p.D444H;c.1171C NT, p.P391S. c.1330GNC, p.D444H;c.1413T NC, p.C471C. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.755ANG, p.D252G. c.1330GNC, p.D444H;c.511G NA, p.A171T c.278ANG, p.Y93C. c.356ANG, p.N119S c.1330GNC, p.D444H;c.511G NA, p.A171T Secondmutationnotidentified. c.1330GNC, p.D444H. c.1126CNT, p.Q376X c.1361ANG, p.Y454C c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. Secondmutationnotidentified. Secondmutationnotidentified. c.98_104delinsTCC c.1330GNC, p.D444H c.1062GNA, p.T354T(polymorphism) c.310-15delT c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. Second mutation not identified. Second mutation not identified.
1.7 0.5 2.0 3.1 4.8 1.1 0.8 2.1 2.1 b0.4 b0.4 2.0 1.0 1.4 1.4 2.6 b0.4 0.8 0.1 1.5 2.3 1.6 1.6 b0.4 0.6 0.6 3.3 1.9 1.2 1.9 0.9 b0.4 0.5 1.9 3.3 2.3 1.4 2.4 0.7 0.2 3.3 b0.4 1.3 0.5 b0.4 0.6 2.2 1.0 1.8 1.1 1.6 b0.4 NA NA 1.5 1.9 2.4 1.7 2.0 0.8 1.4 1.8
NA: not available.
about the individual's serum biotinidase activities or failure to identify a second causative mutation in the BTD gene. Most of the novel mutations were found in exon 4. This exon is the largest exon and contains the active site of the enzyme [10,11]. Therefore, it is not surprising that most of the novel variants that affect enzymatic activity are located in this exon. This information about the novel mutations has been added to the ARUP biotinidase deficiency database (http://arup.utah.edu/database/
BTD/BTD_welcome.phps) so that other laboratories and/or clinicians can use the information to make decisions about management and counsel their patients based on their genotypes. It is also important for physicians and/or laboratories to notify the database if a particular mutation is found in a symptomatic individual, because most of the mutations in the databases were identified in asymptomatic children identified by newborn screening, or if there are discrepancies or confirmations in interpreting the classification of rare mutations.
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
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M. Procter et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx
Table 2 Characterization of novel alterations of uncertain effect on enzymatic activity with predictions. Subject
Exon Novel mutation Novel mutation Mutation location nucleotide protein change type change
Classification
Mutation #2
Serum SIFT MutationTaster PolyPhen biotinidase prediction activity (U/L)
45 46 47 48
2 4 4 4
Uncertain Uncertain Uncertain Likely pathogenic
c.1330GNC, p.D444H. c.-34CNT. c.1330GNC, p.D444H. Second mutation not identified.
NA 5.6 NA b0.4
c.128ANG c.566ANG c.815G NA c.1328TNC
p.H43R p.D189G p.W272Ter p.F443S
Missense Missense Nonsense Missense
Tolerated Damaging Tolerated Tolerated
Polymorphism Disease causing Disease causing Disease causing
Benign Probably damaging NA Probably damaging
NA: Not available.
Acknowledgments This work was supported in part by the Safra Research Fund of the Henry Ford Health System. References [1] B. Wolf, Biotinidase Deficiency, in: R.A. Pagon, T.D. Bird, C.R. Dolan, K. Stephens, M.P. Adam (Eds.), GeneReviews (Internet), University of Washington, Seattle, WA, 2012. [2] B. Wolf, Biotinidase deficiency: if you have to have an inherited metabolic disease, this is the one to have, Genet. Med. 14 (2012) 565–575. [3] B. Wolf, Clinical issues and frequent questions about biotinidase deficiency, Mol. Genet. Metab. 100 (2010) 6–13 20130. [4] M. Procter, B. Wolf, D.K. Crockett, R. Mao, The biotinidase variants registry: a paradigm public database, Genes Genomics Genet. (2013)http://dx.doi.org/10. 1534/g3.113.005835 (pii: g3.113.005835v1). [5] P. Kumar, S. Henikoff, P.C. Ng, Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm, Nat. Protoc. 4 (2009) 1073–1081.
[6] J.M. Schwarz, C. Rodelsperger, M. Schuelke, D. Seelow, MutationTaster evaluates disease causing potential of sequence alterations, Nat. Methods 7 (2010) 575–576. [7] V. Ramensky, P. Bork, S. Sunyaev, Human non-synonymous SNPs: server and survey, Nucleic Acids Res. 30 (2002) 3894–3900. [8] R.S. Aziz, S. Bale, D. Bick, S. Das, J. Gastier-Foster, W.W. Grody, M. Hegde, E. Lyon, E. Spector, K. Voelkerding, H.L. Rehm, ACMG Laboratory Qulaity Assurance Committee, Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and the Association for Molecular Pathology, Genet. Med. 17 (2015) 405–424. [9] H. Li, L. Spencer, F. Nahhas, J. Miller, A. Fribley, G. Feldman, R. Conway, B. Wolf, Novel mutations causing biotinidase deficiency in individuals identified by newborn screening in Michigan including a unique intronic mutation that alters mRNA expression of the biotinidase gene, Mol. Genet. Metab. 112 (2014) 242–246. [10] K. Pindolia, K. Jensen, B. Wolf, Three dimensional structure of human biotinidase: computer modeling and functional correlations, Mol. Genet. Metab. 92 (2013) 13–22. [11] 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.
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Forty-eight novel mutations causing biotinidase deficiency Melinda Procter a, Barry Wolf b,⁎, Rong Mao a,c a b c
ARUP Institute for Clinical and Experimental Pathology, University of Utah, Salt Lake City, UT, USA Department of Research Administration, Henry Ford Hospital and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA Department of Pathology, University of Utah, Salt Lake City, UT, USA
a r t i c l e
i n f o
Article history: Received 13 November 2015 Received in revised form 7 January 2016 Accepted 7 January 2016 Available online xxxx Keywords: Biotinidase deficiency Biotinidase Mutation Biotin-responsive Mutation database
a b s t r a c t Biotinidase deficiency is an autosomal recessively inherited disorder that results in the inability to recycle the vitamin biotin and is characterized by neurological and cutaneous symptoms. The symptoms can be ameliorated or prevented by administering pharmacological doses of biotin. Since 2008, approximately 300 samples have been submitted to ARUP's Molecular Sequencing Laboratory for biotinidase mutation analysis. Of these, 48 novel alterations in the biotinidase gene have been identified. Correlating the individual's serum enzymatic activity with the genotype, we have been able to determine the effect of the novel alteration on enzyme activity and, thereby, determine its likelihood of being pathogenic in 44 of these individuals. The novel mutations and uncertain alterations have been added to the database established by ARUP (http://arup.utah.edu/database/ BTD/BTD_welcome.phps) to help clinicians make decisions about management and to better counsel their patients based on their genotypes. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Biotinidase (EC 3.5.1.12) is the enzyme that catalyzes the recycling of biotin from biocytin, the product of the proteolytic degradation of biotin-dependent carboxylases [1,2]. Biotinidase deficiency (OMIM #609019) is an autosomal recessively inherited disorder that if untreated can result in neurological and cutaneous symptoms [1]. If individuals with biotinidase deficiency are treated with pharmacological doses of biotin before symptoms develop, symptoms can be prevented. If they are treated after symptoms occur, the symptoms will markedly improve, although some symptoms, such as optic atrophy, hearing loss and developmental delay, are likely to be irreversible [1]. Biotinidase deficiency is included in mandatory newborn screening programs in all states in the United States and in many countries. Individuals with biotinidase deficiency either have profound biotinidase deficiency, less than 10% of mean normal serum enzyme activity, or partial biotinidase deficiency, between 10% and 30% of mean normal serum activity [3]. Over 150 mutations have previously been reported that cause biotinidase deficiency [4]. Definitive diagnosis of biotinidase deficiency can be challenging because the biotinidase enzyme is sensitive to suboptimal handling conditions, such as failure to separate the serum from whole blood or storing the serum at non-freezing temperatures, resulting in erroneously low activities and misdiagnosis. For this reason, confirmation of the
⁎ Corresponding author at: Department of Research Administration, Henry Ford Hospital, Detroit, MI 38202, USA. E-mail address: [email protected] (B. Wolf).
diagnosis can necessitate sequencing of BTD in some instances to help determine definitively an individual's status. Since BTD sequencing was incorporated at ARUP's Molecular Sequencing Laboratory in 2008, approximately 300 samples have been submitted for mutation analysis. Of these, we have identified 48 novel alterations and mutations, which are reported here.
2. Materials and methods Blood samples from 300 individuals from the United States were submitted to ARUP, a large clinical diagnostic laboratory (Salt Lake City, UT) for BTD gene sequencing. The vast majority of these samples are from children identified by newborn screening. Serum enzyme activities either indicated enzyme deficiency or indicated ambiguously low-normal activity. For clinical purposes, DNA sequencing was used to confirm the enzymatic activity status. Human BTD consists of four exons. We developed a protocol to sequence the coding regions of the BTD gene as well as intron/exon boundaries. BTD coding regions and intron/exon boundaries of the BTD gene were amplified and sequenced using the Sanger method. DNA extracted from whole blood in a MagNAPure Compact instrument (Roche, Indianapolis, IN). PCR was performed on each of the four exons of BTD using M13-tailed primers Premix D (Epicentre, Madison, WI), and Platinum Taq (Invitrogen, Carlsbad, CA) using a “touchdown” PCR protocol. PCR consisted of a 95 °C denaturation proceeding 10 cycles of 94 °C for 30 s, 62 °C for 45 s with a 0.5 °C decrease in temperature per cycle and 72 °C for 1 min. This procedure is followed by 25 cycles of 94 °C for 30 s, 57 °C for 45 s and 72 °C for 1 min.
http://dx.doi.org/10.1016/j.ymgme.2016.01.002 1096-7192/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
2
M. Procter et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx
Because of the size of the coding region of exon 4, about 1200 bp, this exon was amplified in three overlapping sequences to ensure complete coverage. Next, unused PCR primers and unincorporated nucleotides were inactivated by incubation with ExoSAP (USB Corporation, Cleveland, OH). Bidirectional DNA sequencing was performed with BigDye Terminator chemistry (ABI, Foster City, CA) and M13 primers (IDT, Coralville, IA) and the product was analyzed on the ABI 3100. Data analysis was performed using Mutation Surveyor software (SoftGenetics, State College, PA). In those individuals in whom three alterations are identified, one is the D444H mutation. We have assigned the second mutation to be allelic with the D444H mutation if this combination has been reported previously. Parental testing was not available to determine allelic distribution of the alterations. It is not usually possible to determine whether any two mutations observed by Sanger Sequencing are in cis or trans. However, the allelic phase status can usually be determined by our prior knowledge about the second known mutation, especially when the second mutation includes the D444H mutation. We did not perform copy number analyses on any of the individuals, because the syntony and knowledge of previously established single or combination mutations was sufficient to assign allelic designations. In those cases where enzymatic information was not available, we used the following predictive programs: SIFT [5], MutationTaster [6] and PolyPhen [7]. The effects of the alteration as being pathogenic or pathogenicpartial are based on the strong functional criteria delineated in the guidelines recommended by the American College of Medical Genetics and Genomics and the Association of Molecular Pathology [8].
3. Results Of the 300 samples submitted for mutation analysis, 89% were found to have at least one alteration/mutation. We are now presenting 48 novel alterations in the BTD gene (Tables 1 and 2) that have not previously been reported. They are listed in the order of their location in the BTD gene sequence. Most of the individuals studied were having mutation analysis to determine their genotype to confirm their degree of biotinidase deficiency or whether they actually have biotinidase deficiency. None of the individuals reported was symptomatic. In the United States where testing for biotinidase deficiency is included in newborn screening assays, a diagnosis of biotinidase deficiency is made and biotin supplementation is initiated before a child can become symptomatic. In all cases, the novel alteration was found to be heterozygous. In almost all cases, the second heterozygous mutation had been reported previously and the serum biotinidase activity of the individual was provided with the sample. Therefore, we could attribute the effect of the novel alteration on biotinidase activity. If the alteration was found to be heterozygous with a mutation on the other allele known to cause profound biotinidase deficiency, then the novel alteration was estimated to result in less than 10% of mean normal activity or profound biotinidase deficiency. These alterations were determined to be mutations and are classified as pathogenic (Table 1). If the enzyme activity is calculated to result in more than 10% of mean normal activity, but less than that of normal activity, it is classified as pathogenic-partial? (Table 1). Additional enzymatic information from other individuals having these identical alterations will be required to classify definitively these mutations. Thirty-two of 48 individuals (67%) of novel alterations were found in combination with the D444H mutation on the other allele. If the child's enzymatic activity is consistent with partial deficiency biotinidase activity and the second mutation is the D444H mutation, which results in about 50% of mean normal activity, we can predict that the novel variant results in less than 10% of mean normal activity, causes profound deficiency and is pathogenic.
Table 1 includes those novel alterations in BTD in which their contribution to enzymatic activity can be estimated based on the individual's serum activity and our knowledge of the effect of the second mutation on enzymatic activity or the lack of a second mutation. In six of the novel alterations, multiple individuals were identified with the same novel variant allowing us to assign their classifications as a pathogenic. Two of the individuals (Subjects 21 and 43) with novel alterations have a second previously reported mutation (c.310-15delT) that has been shown to cause partial biotinidase deficiency due to decreased transcription of the BTD gene [9]. The enzymatic activity of Subject 21 is likely erroneous low. Nevertheless, both novel mutations appear to result in essentially complete loss of activity and can be classified as pathogenic. Table 2 includes four novel alterations that could not be assigned a classification based on their effect on enzymatic activity for various reasons. In each of these cases, we have used prediction programs to ascertain whether the alteration is likely to be deleterious to enzyme activity. We did not have biotinidase activity information from two of individuals (Subjects 45 and 47). Therefore, we could not estimate the effects of these novel alterations on enzymatic activity. In these two cases, we have included the predictive mutation data from three programs. However, the definitive effects of these alterations remain to be determined. Subject 46 has a missense alteration on one allele and an alteration within the first intron on the other. Although the alteration within the intron does not likely affect enzymatic activity, because the individual's enzymatic activity is within the low normal range, we still cannot estimate the effect of the novel alteration on enzymatic activity. The three predictive programs all indicate that this alteration is likely pathogenic. Only a single alteration was identified for Subject 48. However, the individual's enzymatic activity was consistent with profound deficiency. It is likely that the enzymatic activity is actually higher than reported, but unfortunately, we were unable to confirm this because follow-up enzymatic testing on this individual was not available. Two of the prediction programs indicate that this novel alteration is pathogenic, whereas one program indicates that the alteration is tolerated. 4. Discussion We are fortunate that in the evaluating individuals for biotinidase deficiency we usually have their biochemical phenotype or enzymatic activity and genotype. This allows us to determine the effect of a novel mutation on enzymatic activity in almost all individuals. Because enzyme activity determines if an individual has profound or partial biotinidase deficiency, we can determine if the novel mutation is pathogenic. In those few cases where enzymatic information was not available, we used predictive programs. Because the laboratory receives enzymatic activity information from various institutions, we do not know if the activities were determined using a newborn screening sample or by confirmatory testing of serum activity. Therefore, as stated above, it is not usually possible to know if the activities were optimally determined and/or are correct. Our estimates of the contributions of the novel alterations on enzyme activities are based on the single activity submitted with the sample. When the activity is inconsistent with the genotype, we have attempted to contact the submitting physicians and/or institutions to clarify the activities, but we were not always successful. Therefore, our conclusions about pathogenicity are likely correct, but may require modification when other individuals with identical alterations and more confidently determined enzymatic activities are reported. We identified 48 novel variants. These variants can be characterized as mutations if we could demonstrate that the novel variant causes marked reduction in biotinidase activity. We were able to categorize 44 of these variants as “pathogenic” or “pathogenic-partial?” based on available biotinidase activities. The remaining four novel mutations were categorized as “uncertain” because of the lack of information
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
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Table 1 Novel mutations causing biotinidase deficiency. Subject
Exon location
Novel mutation nucleotide change
Novel mutation protein change
Mutation type
Predicted classification
Mutation #2
Serum biotinidase activity (U/L)
1 2 3 4 5 6 7 8 9
2 2 2 3 3 3 3 4 4
c.192GNC c.245CNT c.299CNT c.321TNG c.326dupT c.356ANG c.406delC c.582CNG c.625CNT
p.E64D p.A82V p.A100V p.I107M p.F110Vfs*42 p.N119S p.Q136Rfs*23 p.F194L p.R209C
Missense Missense Missense Missense Insertion Missense Deletion Missense Missense
Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic-partial? Pathogenic-partial? Pathogenic Pathogenic Pathogenic-partial?
10 11 12 13 14 15 16
4 4 4 4 4 4 4
c.631CNT c.646TNA c.664GNC c.692delC 695 TNC c.709GNA c.743TNC
p.R211C p.Y216N p.D222H p.F232Lfs*32 p.F232S p.A237T p.I248T
Missense Missense Missense Deletion Missense Missense Missense
Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic
17 18 19 20
4 4 4 4
c.758CNT c.770TNA c.783CNG c.814TNG
p.P253L p.V257D p.Y261X p.W272G
Missense Missense Nonsense Missense
Pathogenic Pathogenic Pathogenic Pathogenic-partial?
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
c.836TNA c.856ANG c.865GNC c.880ANG c.895GNC c.1001TNA c.1046A NC c.1096-1097dupTC c.1126C NT c.1241_1252del12bp c.1252TNC c.1309C NG c.1334GNA c.1352_1353delGC c.1432GNA c.1455C NG c.1458delG c.1459delT
p.L279X p.K286E p.A289P p.I294V p.A299P p.I334N p.N349T p.G367Qfs*23 p.Q376X p.Y414_Val417del p.C418R p.L437V p.G445E p.G451Dfs*32 p.A478T p.H485Q p.W487Gfs*14 p.W487Gfs*14
Nonsense Missense Missense Missense Missense Missense Missense Duplication Nonsense Deletion Missense Missense Missense Deletion Missense Missense Deletion Deletion
Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic-partial? Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic
39 40 41
4 4 4
c.1481A NG c.1526C NG c.1612C NA
p.Y494C p.P509R p.R538S
Missense Missense Missense
Pathogenic Pathogenic Pathogenic
42 43 44
4 4 4
c.1613GNA c.1628A NT c.1629C NA
p.R538H p.D543V p.D543E
Missense Missense Missense
Pathogenic Pathogenic-partial? Pathogenic
c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. Second mutation not identified. c.1459delT c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1368ANC, p.Q456H. c.528CNT, p.K176N;c.1595CNT, p.T532M c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.528GNT, p.K176N. c.528GNT, p.K176N. c.1489CNT, p.P497S. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H;c.511G NA, p.A171T c.310-15delT. c.1330GNC, p.D444H. c.1330GNC, p.D444H;c.133G NA, p.G45R; Second mutation not identified. c.1413TNC, p.C471C(polymorphism) c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1612CNT, p.R538C. c.1330GNC, p.D444H;c.1171C NT, p.P391S. c.1330GNC, p.D444H;c.1413T NC, p.C471C. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.755ANG, p.D252G. c.1330GNC, p.D444H;c.511G NA, p.A171T c.278ANG, p.Y93C. c.356ANG, p.N119S c.1330GNC, p.D444H;c.511G NA, p.A171T Secondmutationnotidentified. c.1330GNC, p.D444H. c.1126CNT, p.Q376X c.1361ANG, p.Y454C c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. Secondmutationnotidentified. Secondmutationnotidentified. c.98_104delinsTCC c.1330GNC, p.D444H c.1062GNA, p.T354T(polymorphism) c.310-15delT c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. c.1330GNC, p.D444H. Second mutation not identified. Second mutation not identified.
1.7 0.5 2.0 3.1 4.8 1.1 0.8 2.1 2.1 b0.4 b0.4 2.0 1.0 1.4 1.4 2.6 b0.4 0.8 0.1 1.5 2.3 1.6 1.6 b0.4 0.6 0.6 3.3 1.9 1.2 1.9 0.9 b0.4 0.5 1.9 3.3 2.3 1.4 2.4 0.7 0.2 3.3 b0.4 1.3 0.5 b0.4 0.6 2.2 1.0 1.8 1.1 1.6 b0.4 NA NA 1.5 1.9 2.4 1.7 2.0 0.8 1.4 1.8
NA: not available.
about the individual's serum biotinidase activities or failure to identify a second causative mutation in the BTD gene. Most of the novel mutations were found in exon 4. This exon is the largest exon and contains the active site of the enzyme [10,11]. Therefore, it is not surprising that most of the novel variants that affect enzymatic activity are located in this exon. This information about the novel mutations has been added to the ARUP biotinidase deficiency database (http://arup.utah.edu/database/
BTD/BTD_welcome.phps) so that other laboratories and/or clinicians can use the information to make decisions about management and counsel their patients based on their genotypes. It is also important for physicians and/or laboratories to notify the database if a particular mutation is found in a symptomatic individual, because most of the mutations in the databases were identified in asymptomatic children identified by newborn screening, or if there are discrepancies or confirmations in interpreting the classification of rare mutations.
Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
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M. Procter et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx
Table 2 Characterization of novel alterations of uncertain effect on enzymatic activity with predictions. Subject
Exon Novel mutation Novel mutation Mutation location nucleotide protein change type change
Classification
Mutation #2
Serum SIFT MutationTaster PolyPhen biotinidase prediction activity (U/L)
45 46 47 48
2 4 4 4
Uncertain Uncertain Uncertain Likely pathogenic
c.1330GNC, p.D444H. c.-34CNT. c.1330GNC, p.D444H. Second mutation not identified.
NA 5.6 NA b0.4
c.128ANG c.566ANG c.815G NA c.1328TNC
p.H43R p.D189G p.W272Ter p.F443S
Missense Missense Nonsense Missense
Tolerated Damaging Tolerated Tolerated
Polymorphism Disease causing Disease causing Disease causing
Benign Probably damaging NA Probably damaging
NA: Not available.
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Please cite this article as: M. Procter, et al., Forty-eight novel mutations causing biotinidase deficiency, Mol. Genet. Metab. (2016), http:// dx.doi.org/10.1016/j.ymgme.2016.01.002
