- Email: [email protected]
Novel mutation in the TCOF1 gene in a patient with Treacher Collins syndrome
pediatria polska 89 (2014) 462–465
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pepo
Case report/Kazuistyka
Novel mutation in the TCOF1 gene in a patient with Treacher Collins syndrome Nowa mutacja w genie TCOF1 u pacjenta z zespołem Treachera Collinsa Bożena A. Marszałek-Kruk 1,*, Robert Śmigiel 2, Maria M. Sąsiadek 3 1
Department of Genetics, Wroclaw University of Environmental and Life Sciences, Poland Department of Social Pediatrics, Wroclaw Medical University, Poland 3 Department of Genetics, Wroclaw Medical University, Poland 2
article info
abstract
Article history:
Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial deve-
Received: 21.08.2014
lopment. The syndrome is characterized by a similar phenotype: micrognathia, microtia,
Accepted: 23.09.2014
midface hypoplasia, cleft lip and palate. The estimated incidence rate is 1/50 000 live
Available online: 06.10.2014
births. Treacher Collins syndrome is mostly caused by mutations in the TCOF1 gene, which encodes the serine/alanine-rich protein Treacle. TCS can also be caused by mutations in the POLR1C and POLR1D genes. About 70% of recognized mutations of the TCOF1
Keywords: TCOF1
gene in TCS patients are deletions, which lead to formation of a termination codon and
Novel deletion Treacher Collins syndrome
gene causing a Treacher Collins syndrome. Examined DNA was isolated from peripheral
shortening of the protein. The aim of the study was to search for mutations in TCOF1 blood leukocytes of the male patient born at 38 weeks of gestation and his healthy
Słowa kluczowe: TCOF1 Nowa delecja Zespół Treachera Collinsa
parents. The PCR products were subjected to MSSCP analysis. The PCR products were purified on the DNA GelOut columns followed by direct sequencing. A novel, heterozygous deletion c.1978delC was detected in one patient with typical facial symptoms of Treacher Collins syndrome. The patient's parents were tested and no mutation was observed, indicating de novo origin of the examined mutation. The c.1978delC deletion causes premature termination of translation at 677aa. The finding will facilitate precise diagnosis of the patient and will extend knowledge on the pathogenesis of TCS. © 2014 Polish Pediatric Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
* Corresponding author at: Katedra Genetyki Uniwersytet Przyrodniczy we Wrocławiu, ul. Kożuchowska 7, 51-631 Wrocław, Poland. Tel.: +48 71 3205926; fax: +48 71 3205758. E-mail address: [email protected] (B.A. Marszałek-Kruk). http://dx.doi.org/10.1016/j.pepo.2014.09.006 0031-3939/© 2014 Polish Pediatric Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
pediatria polska 89 (2014) 462–465
Introduction Treacher Collins syndrome (TCS) [1] is an autosomal dominant disorder affecting differentiation of the first and the second pharyngeal arches [2]. This syndrome occurs with an estimated prevalence of 1 in 50 000 live births. In 60% of cases the disease is caused by de novo mutations; however, family history was confirmed in 40% of patients [3]. Patients with Treacher Collins syndrome are characterized by a typical phenotype: downslanting palpebral fissures, coloboma of the lateral part of the lower lid, hypoplasia of the mandible and the zygomatic bones, auricular malformations, hearing loss as well as, cleft lip and palate [4]. Treacher Collins syndrome is caused mainly by mutations in the TCOF1 gene (Treacher Collins-Franceschetti 1), which encodes a low complexity serine/alanine-rich nucleolar phosphoprotein called Treacle. Treacle participates in the formation of pre-rRNA by interacting with UBF (upstream binding factor) and binding to the UCE sequence (upstream control element) as well as the main promoter region, causing DNA looping during the transcription process [5]. Dauwerse et al. [6] detected
463
mutations in genes encoding subunits of RNA polymerases I and III (POLR1C and POLR1D) in patients with Treacher Collins syndrome. That study confirmed, that TCS is a ribosomopathy and is genetically heterogeneous. Most mutations identified in the TCOF1 gene are deletions resulting in a truncated protein [7–13]. No genotype/ phenotype correlations have been found. We have identified a novel mutation in exon 13 of the TCOF1 gene in a patient with typical facial symptoms of Treacher Collins syndrome.
Materials and methods A male infant was born at 38 weeks of gestation. At birth his weight was 2720 g (3–10 centile), length was 55 cm (25–50 centile) and skull circumference was 35 cm (3–10 centile). Physical and diagnostic examinations after birth revealed asymmetrical facial abnormalities, including absence of the right auditory canal, bilateral microtia, malar hypoplasia as well as downslanting palpebral fissures, bilateral partial coloboma of the lateral part of the lower lids, sparse eyelashes and mandibular hypoplasia (micro- and retrognathia). Moreover, hearing loss was diagnosed. The external
Fig. 1 – Phenotype and analysis of the amplified fragment of exon 13 of the TCOF1 gene in the patient with TCS and in his parents. (A) Phenotype of the patient with TCS. (B) Sequence analysis of the amplified fragment of exon 13 of the TCOF1 gene: 1, patient; 2, mother; 3, father; C, control. Arrow indicates location of the deletion Ryc. 1 – Fenotyp i analiza amplifikowanego fragmentu eksonu 13 genu TCOF1 pacjenta z TCS i rodziców. (A) Fenotyp pacjenta z TCS. (B) Analiza sekwencji amplifikowanego fragmentu eksonu 13 genu TCOF1: 1 pacjent; 2, matka; 3, ojciec; C, kontrola. Strzałka wskazuje lokalizację delecji
464
pediatria polska 89 (2014) 462–465
genitalia were normal. Further examinations at the age of 3 and 5 months showed normal psychomotor and somatic development (Fig. 1a). DNA was isolated from peripheral blood leukocytes of the patient and his healthy parents. Exons 1 through 27 of TCOF1, including exon–intron borders, were amplified by PCR under optimal conditions, using specific primers. The PCR products were subjected to multitemperature single-stranded conformation polymorphism (MSSCP) analysis at 5 8C, 15 8C and 25 8C, using the DNA Pointer Mutation Detection System. The electrophoresis was followed by silver staining. The PCR products were purified on the DNA GelOut columns (A&A Biotechnology, Poland) followed by direct sequencing with the use of a BigDye ver.3.0 dye terminator cycle sequencing kit and specific primers. The dideoxyterminated fragments were identified by capillary gel-electrophoresis based on the ABI 310 DNA Analysis System. The MSSCP analysis of the amplified fragments of exon 13 of the TCOF1 gene demonstrated changes in the electrophoretic mobility in this patient, while the changes were not observed in the patient's parents. In order to confirm the results obtained in MSSCP analysis a direct sequence analysis was performed.
Results, discussion and conclusions Sequence analysis demonstrated a novel, heterozygotic c.1978delC mutation in exon 13 of TCOF1. In the case of the patient's parents direct DNA sequencing showed normal sequences (Fig. 1b). A majority of mutations responsible for Treacher Collins syndrome are localized in exons, mainly in the hot spots in exons 10, 13, 15, 16, 23 and 24 [9]. The most commonly occurring mutations of the TCOF1 gene include deletions, which cause a shift of the reading frame, formation of the termination codon and shortening of the protein product. The next most common mutations of the TCOF1 gene are insertions, the longest insertion localized on exon 5 [14]. In the presented patient a novel, heterozygotic deletion c.1978delC was detected in the TCOF1 gene. This mutation was absent in the patient's parents which probably indicates a de novo origin. Analysis of the novel c.1978delC deletion with the use of the OMIGA 2.0 system indicates that it causes a premature termination of translation at 677aa, which results in the formation of a protein product of the gene devoid of the nuclear localization signal. We believe that these findings will facilitate precise diagnosis of the patient and will extend our knowledge on the pathogenesis of TCS. Molecular diagnosis of TCS is essential in prenatal and postnatal screening, being of great importance for genetic counseling as well.
Authors' contributions/Wkład autorów BAM-K – study design, data collection and interpretation, acceptance of final manuscript version, literature search. RS
– data collection, acceptance of final manuscript version. MMS – acceptance of final manuscript version.
Conflict of interest/Konflikt interesu None declared.
Financial support/Finansowanie None declared.
Ethics/Etyka The work described in this article has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals. The patient gave his consent to submit his images for publication purposes.
references/pi smiennictwo
[1] Online Mendelian Inheritance in Man, OMIM. 154500 [10/31/ 2000]. Baltimore, MD: Johns Hopkins University. http:// www.ncbi.nlm.nih.gov/omim/. [2] Gorlin RJ, Cohen MM, Hennekam RCM. Syndromes of the head and neck, 4th ed., New York: Oxford University Press; 2001. [3] Jones KL, Smith DW, Harvey MA, Hall BD, Quan L. Older paternal age and fresh gene mutation: data on additional disorders. J Pediatr 1975;86:84–88. [4] Dixon MJ. Treacher Collins syndrome. Hum Mol Genet 1996;5:1391–1396. [5] Valdez BC, Henning D, So RB, Dixon J, Dixon MJ. The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor. Proc Natl Acad Sci U S A 2004;101:10709–10714. [6] Dauwerse JG, Dixon J, Seland S, Ruivenkamp CAL, Haeringen A, Hoefsloot LH, et al. Mutations in genes encoding subunits of RNA polumerases I and III cause Treacher Collins syndrome. Nat Genet 2011;43(1): 20–22. [7] Gladwin AJ, Dixon J, Loftus SK, Edwards S, Wasmuth JJ, Hennekam RCM, et al. Treacher Collins syndrome may result from insertions, deletions or splicing mutations, which introduce a termination codon into the gene. Hum Mol Genet 1996;5:1533–1538. [8] Wise CA, Chiang LC, Paznekas WA, Sharma M, Musy MM, Ashley JA, et al. TCOF1 gene encodes a putative nucleolar phosphorylation that exhibits mutation in Treacher Collins syndrome throughout its coding region. Proc Natl Acad Sci U S A 1997;94:3110–3115. [9] Splendore A, Silva EO, Alonso LG, Richieri-Costa A, Alonso N, Rosa A, et al. High mutation detection rate in TCOF1 among Treacher Collins syndrome patients reveals clustering of mutations and 16 novel pathogenic changes. Hum Mutat 2000;16:315–322.
pediatria polska 89 (2014) 462–465
[10] Ellis PE, Dawson M, Dixon MJ. Mutation testing in Treacher Collins syndrome. J Orthod 2002;29:293–297. [11] Marszałek B, Wiśniewski SA, Wójcicki P, Kobus K, Trzeciak WH. Novel mutation in the 50 splice site of exon 4 of TCOF1 gene in the patient with Treacher Collins syndrome. Am J Med Genet 2003;123A:169–171. [12] Dixon J, Ellis I, Bottani A, Temple K, Dixon MJ. Identification of mutations in TCOF1: use of molecular analysis in the
465
pre- and postnatal diagnosis of Treacher Collins syndrome. Am J Med Genet 2004;127A:244–248. [13] Su PH, Chen JY, Chen SJ, Yu JS. Treacher Collins syndrome with a de Novo 5-bp deletion in the TCOF1 gene. J Formos Med Assoc 2006;105(6):518–521. [14] Marszalek-Kruk BA, Wojcicki P, Smigiel R, Trzeciak WH. Novel insertion in exon 5 of the TCOF1 gene in twin sisters with Treacher Collins syndrome. J Appl Genet 2012;53:279–282.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pepo
Case report/Kazuistyka
Novel mutation in the TCOF1 gene in a patient with Treacher Collins syndrome Nowa mutacja w genie TCOF1 u pacjenta z zespołem Treachera Collinsa Bożena A. Marszałek-Kruk 1,*, Robert Śmigiel 2, Maria M. Sąsiadek 3 1
Department of Genetics, Wroclaw University of Environmental and Life Sciences, Poland Department of Social Pediatrics, Wroclaw Medical University, Poland 3 Department of Genetics, Wroclaw Medical University, Poland 2
article info
abstract
Article history:
Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial deve-
Received: 21.08.2014
lopment. The syndrome is characterized by a similar phenotype: micrognathia, microtia,
Accepted: 23.09.2014
midface hypoplasia, cleft lip and palate. The estimated incidence rate is 1/50 000 live
Available online: 06.10.2014
births. Treacher Collins syndrome is mostly caused by mutations in the TCOF1 gene, which encodes the serine/alanine-rich protein Treacle. TCS can also be caused by mutations in the POLR1C and POLR1D genes. About 70% of recognized mutations of the TCOF1
Keywords: TCOF1
gene in TCS patients are deletions, which lead to formation of a termination codon and
Novel deletion Treacher Collins syndrome
gene causing a Treacher Collins syndrome. Examined DNA was isolated from peripheral
shortening of the protein. The aim of the study was to search for mutations in TCOF1 blood leukocytes of the male patient born at 38 weeks of gestation and his healthy
Słowa kluczowe: TCOF1 Nowa delecja Zespół Treachera Collinsa
parents. The PCR products were subjected to MSSCP analysis. The PCR products were purified on the DNA GelOut columns followed by direct sequencing. A novel, heterozygous deletion c.1978delC was detected in one patient with typical facial symptoms of Treacher Collins syndrome. The patient's parents were tested and no mutation was observed, indicating de novo origin of the examined mutation. The c.1978delC deletion causes premature termination of translation at 677aa. The finding will facilitate precise diagnosis of the patient and will extend knowledge on the pathogenesis of TCS. © 2014 Polish Pediatric Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
* Corresponding author at: Katedra Genetyki Uniwersytet Przyrodniczy we Wrocławiu, ul. Kożuchowska 7, 51-631 Wrocław, Poland. Tel.: +48 71 3205926; fax: +48 71 3205758. E-mail address: [email protected] (B.A. Marszałek-Kruk). http://dx.doi.org/10.1016/j.pepo.2014.09.006 0031-3939/© 2014 Polish Pediatric Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
pediatria polska 89 (2014) 462–465
Introduction Treacher Collins syndrome (TCS) [1] is an autosomal dominant disorder affecting differentiation of the first and the second pharyngeal arches [2]. This syndrome occurs with an estimated prevalence of 1 in 50 000 live births. In 60% of cases the disease is caused by de novo mutations; however, family history was confirmed in 40% of patients [3]. Patients with Treacher Collins syndrome are characterized by a typical phenotype: downslanting palpebral fissures, coloboma of the lateral part of the lower lid, hypoplasia of the mandible and the zygomatic bones, auricular malformations, hearing loss as well as, cleft lip and palate [4]. Treacher Collins syndrome is caused mainly by mutations in the TCOF1 gene (Treacher Collins-Franceschetti 1), which encodes a low complexity serine/alanine-rich nucleolar phosphoprotein called Treacle. Treacle participates in the formation of pre-rRNA by interacting with UBF (upstream binding factor) and binding to the UCE sequence (upstream control element) as well as the main promoter region, causing DNA looping during the transcription process [5]. Dauwerse et al. [6] detected
463
mutations in genes encoding subunits of RNA polymerases I and III (POLR1C and POLR1D) in patients with Treacher Collins syndrome. That study confirmed, that TCS is a ribosomopathy and is genetically heterogeneous. Most mutations identified in the TCOF1 gene are deletions resulting in a truncated protein [7–13]. No genotype/ phenotype correlations have been found. We have identified a novel mutation in exon 13 of the TCOF1 gene in a patient with typical facial symptoms of Treacher Collins syndrome.
Materials and methods A male infant was born at 38 weeks of gestation. At birth his weight was 2720 g (3–10 centile), length was 55 cm (25–50 centile) and skull circumference was 35 cm (3–10 centile). Physical and diagnostic examinations after birth revealed asymmetrical facial abnormalities, including absence of the right auditory canal, bilateral microtia, malar hypoplasia as well as downslanting palpebral fissures, bilateral partial coloboma of the lateral part of the lower lids, sparse eyelashes and mandibular hypoplasia (micro- and retrognathia). Moreover, hearing loss was diagnosed. The external
Fig. 1 – Phenotype and analysis of the amplified fragment of exon 13 of the TCOF1 gene in the patient with TCS and in his parents. (A) Phenotype of the patient with TCS. (B) Sequence analysis of the amplified fragment of exon 13 of the TCOF1 gene: 1, patient; 2, mother; 3, father; C, control. Arrow indicates location of the deletion Ryc. 1 – Fenotyp i analiza amplifikowanego fragmentu eksonu 13 genu TCOF1 pacjenta z TCS i rodziców. (A) Fenotyp pacjenta z TCS. (B) Analiza sekwencji amplifikowanego fragmentu eksonu 13 genu TCOF1: 1 pacjent; 2, matka; 3, ojciec; C, kontrola. Strzałka wskazuje lokalizację delecji
464
pediatria polska 89 (2014) 462–465
genitalia were normal. Further examinations at the age of 3 and 5 months showed normal psychomotor and somatic development (Fig. 1a). DNA was isolated from peripheral blood leukocytes of the patient and his healthy parents. Exons 1 through 27 of TCOF1, including exon–intron borders, were amplified by PCR under optimal conditions, using specific primers. The PCR products were subjected to multitemperature single-stranded conformation polymorphism (MSSCP) analysis at 5 8C, 15 8C and 25 8C, using the DNA Pointer Mutation Detection System. The electrophoresis was followed by silver staining. The PCR products were purified on the DNA GelOut columns (A&A Biotechnology, Poland) followed by direct sequencing with the use of a BigDye ver.3.0 dye terminator cycle sequencing kit and specific primers. The dideoxyterminated fragments were identified by capillary gel-electrophoresis based on the ABI 310 DNA Analysis System. The MSSCP analysis of the amplified fragments of exon 13 of the TCOF1 gene demonstrated changes in the electrophoretic mobility in this patient, while the changes were not observed in the patient's parents. In order to confirm the results obtained in MSSCP analysis a direct sequence analysis was performed.
Results, discussion and conclusions Sequence analysis demonstrated a novel, heterozygotic c.1978delC mutation in exon 13 of TCOF1. In the case of the patient's parents direct DNA sequencing showed normal sequences (Fig. 1b). A majority of mutations responsible for Treacher Collins syndrome are localized in exons, mainly in the hot spots in exons 10, 13, 15, 16, 23 and 24 [9]. The most commonly occurring mutations of the TCOF1 gene include deletions, which cause a shift of the reading frame, formation of the termination codon and shortening of the protein product. The next most common mutations of the TCOF1 gene are insertions, the longest insertion localized on exon 5 [14]. In the presented patient a novel, heterozygotic deletion c.1978delC was detected in the TCOF1 gene. This mutation was absent in the patient's parents which probably indicates a de novo origin. Analysis of the novel c.1978delC deletion with the use of the OMIGA 2.0 system indicates that it causes a premature termination of translation at 677aa, which results in the formation of a protein product of the gene devoid of the nuclear localization signal. We believe that these findings will facilitate precise diagnosis of the patient and will extend our knowledge on the pathogenesis of TCS. Molecular diagnosis of TCS is essential in prenatal and postnatal screening, being of great importance for genetic counseling as well.
Authors' contributions/Wkład autorów BAM-K – study design, data collection and interpretation, acceptance of final manuscript version, literature search. RS
– data collection, acceptance of final manuscript version. MMS – acceptance of final manuscript version.
Conflict of interest/Konflikt interesu None declared.
Financial support/Finansowanie None declared.
Ethics/Etyka The work described in this article has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals. The patient gave his consent to submit his images for publication purposes.
references/pi smiennictwo
[1] Online Mendelian Inheritance in Man, OMIM. 154500 [10/31/ 2000]. Baltimore, MD: Johns Hopkins University. http:// www.ncbi.nlm.nih.gov/omim/. [2] Gorlin RJ, Cohen MM, Hennekam RCM. Syndromes of the head and neck, 4th ed., New York: Oxford University Press; 2001. [3] Jones KL, Smith DW, Harvey MA, Hall BD, Quan L. Older paternal age and fresh gene mutation: data on additional disorders. J Pediatr 1975;86:84–88. [4] Dixon MJ. Treacher Collins syndrome. Hum Mol Genet 1996;5:1391–1396. [5] Valdez BC, Henning D, So RB, Dixon J, Dixon MJ. The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor. Proc Natl Acad Sci U S A 2004;101:10709–10714. [6] Dauwerse JG, Dixon J, Seland S, Ruivenkamp CAL, Haeringen A, Hoefsloot LH, et al. Mutations in genes encoding subunits of RNA polumerases I and III cause Treacher Collins syndrome. Nat Genet 2011;43(1): 20–22. [7] Gladwin AJ, Dixon J, Loftus SK, Edwards S, Wasmuth JJ, Hennekam RCM, et al. Treacher Collins syndrome may result from insertions, deletions or splicing mutations, which introduce a termination codon into the gene. Hum Mol Genet 1996;5:1533–1538. [8] Wise CA, Chiang LC, Paznekas WA, Sharma M, Musy MM, Ashley JA, et al. TCOF1 gene encodes a putative nucleolar phosphorylation that exhibits mutation in Treacher Collins syndrome throughout its coding region. Proc Natl Acad Sci U S A 1997;94:3110–3115. [9] Splendore A, Silva EO, Alonso LG, Richieri-Costa A, Alonso N, Rosa A, et al. High mutation detection rate in TCOF1 among Treacher Collins syndrome patients reveals clustering of mutations and 16 novel pathogenic changes. Hum Mutat 2000;16:315–322.
pediatria polska 89 (2014) 462–465
[10] Ellis PE, Dawson M, Dixon MJ. Mutation testing in Treacher Collins syndrome. J Orthod 2002;29:293–297. [11] Marszałek B, Wiśniewski SA, Wójcicki P, Kobus K, Trzeciak WH. Novel mutation in the 50 splice site of exon 4 of TCOF1 gene in the patient with Treacher Collins syndrome. Am J Med Genet 2003;123A:169–171. [12] Dixon J, Ellis I, Bottani A, Temple K, Dixon MJ. Identification of mutations in TCOF1: use of molecular analysis in the
465
pre- and postnatal diagnosis of Treacher Collins syndrome. Am J Med Genet 2004;127A:244–248. [13] Su PH, Chen JY, Chen SJ, Yu JS. Treacher Collins syndrome with a de Novo 5-bp deletion in the TCOF1 gene. J Formos Med Assoc 2006;105(6):518–521. [14] Marszalek-Kruk BA, Wojcicki P, Smigiel R, Trzeciak WH. Novel insertion in exon 5 of the TCOF1 gene in twin sisters with Treacher Collins syndrome. J Appl Genet 2012;53:279–282.