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Two novel NIPBL gene mutations in Chinese patients with Cornelia de Lange syndrome
Gene 555 (2015) 476–480
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
Two novel NIPBL gene mutations in Chinese patients with Cornelia de Lange syndrome Libin Mei, Desheng Liang, Yanru Huang, Qian Pan, Lingqian Wu ⁎ State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan 410078, China
a r t i c l e
i n f o
Article history: Received 9 October 2014 Received in revised form 9 November 2014 Accepted 13 November 2014 Available online 18 November 2014 Keywords: Cornelia de Lange syndrome NIPBL Frameshift mutation Missense mutation
a b s t r a c t Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder characterized by distinctive facial features, mental retardation, and upper limb defects, with the involvement of multiple organs and systems. To date, mutations have been identified in five genes responsible for CdLS: NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Here, we present a clinical and molecular characterization of five unrelated Chinese patients whose clinical presentation is consistent with that of CdLS. There were no chromosomal abnormalities in the five children. In three patients, DNA sequencing revealed a previously reported frameshift mutation c.2479delA (p.Arg827GlyfsX20), and two novel mutations including a heterozygous mutation c.6272 GNT (p.Cys2091Phe) and a frameshift mutation c.1672delA (p.Thr558LeufsX7) in NIPBL. For the remaining patients, large deletions and/or duplications within the NIPBL gene were excluded as playing a role in the pathogenesis, by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. These findings broaden the mutation spectrum of NIPBL and further our understanding of the diverse and variable effects of NIPBL mutations on CdLS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cornelia de Lange syndrome (CdLS, also called Brachmann de Lange syndrome; OMIM: 122470, 300590, and 610759), which was initially described in 1916 by Brachmann and further characterized by de Lange, is a rare congenital malformation disorder characterized by typical facial features, growth and mental retardation, upper limb malformations, hirsutism, cardiac defects, and a variety of other abnormalities affecting a wide range of tissues and organs (Brachmann, 1916; de Lange, 1933). The prevalence of CdLS is estimated to be about 1:10,000 live births (Opitz, 1985). Although most cases are sporadic, familial cases have also been reported (Russell et al., 2001; Barisic et al., 2008). Mutations in the cohesin regulator NIPBL (5p13.1, MIM: 608667) are present in approximately 60% of classical CdLS patients (Krantz et al., 2004; Tonkin et al., 2004; Gillis et al., 2004). However, CdLS exhibits extensive genetic heterogeneity, and mutations in the SMC1A (Xp11.2, MIM: 300040), SMC3 (10q25, MIM: 606062), RAD21 (8q24, MIM: 614701), and HDAC8 (Xq13.1, MIM: 300882) genes have also been identified in a smaller proportion of individuals with CdLS (Musio et al., 2006; Deardorff et al., 2007, 2012a, 2012b). All of the genes involved in CdLS code for a series of cohesin complex and associated proteins (Liu and Krantz, 2009). Cohesin is responsible for ensuring
correct chromosome segregation during cell division. Beyond this role, cohesin also functions in the regulation of gene expression and DNA repair (Dorsett and Krantz, 2009; Feeney et al., 2010). Consequently, deleterious variation of any of the genes coding for cohesin proteins may affect early stages of embryonic development (Dorsett, 2011). Previous reports have shown that patients with NIPBL mutations are more severely affected in limb anomalies, growth, and cognitive function than those with mutations in SMC1A, SMC3, RAD21, and HDAC8 genes or in those with unknown etiology (Deardorff et al., 2012a, 2012b; Dempsey et al., 2014). Genotype–phenotype correlation studies indicated that patients with truncating mutations in NIPBL have a more severe phenotype than those with missense mutations (Gillis et al., 2004; Bhuiyan et al., 2006; Selicorni et al., 2007). In this study, five Chinese patients diagnosed with CdLS were selected. By direct sequencing of the five candidate genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8, two novel mutations in NIPBL were identified separately in two patients. MLPA analysis was also performed to exclude large deletions/duplications within the NIPBL gene for the remaining two patients without identifiable point mutations in the five causative genes. 2. Materials and methods 2.1. Patients
Abbreviations: CdLS, Cornelia de Lange syndrome; NIPBL, Nipped-B-like; PCR, polymerase chain reaction; MLPA, Multiplex Ligation-dependent Probe Amplification. ⁎ Corresponding author at: State Key Laboratory of Medical Genetics, Xiangya Hospital, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, China. E-mail address: [email protected] (L. Wu).
http://dx.doi.org/10.1016/j.gene.2014.11.033 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Five affected and unrelated children from five Chinese families were referred to our institution for genetic diagnosis and counseling. The clinical symptoms of each case are summarized in Table 1. All patients
L. Mei et al. / Gene 555 (2015) 476–480 Table 1 Summary of clinical findings and NIPBL detected mutations. Clinical data
Gender Weight at birth Age at test Height at test Weight at test Head circumference at test Postnatal growth retardation Mental retardation Language delay Microcephaly Low hair line Synophrys Hirsutism Long curly eyelashes Low set ears Depressed nasal bridge Long philtrum Thin upper lips Cleft palate Finger deformity Single transverse palmar crease Chromosome karyotype NIPBL Mutation Mutation origin
Patient 1
2
3
4
5
M 2.5 kg 7 years 95 cm 11.5 kg 44 cm
F 2.65 kg 6 years 90 cm 11.0 kg 43 cm
F 2.8 kg 2 years 70 cm 6.0 kg 41 cm
M 2.6 k g 7 years 96 cm 13 kg 46 cm
F 3.0 kg 60 days 60 cm 3.5 kg 36.5 cm
+
+
+
+
+
Severe + + + + + + + + + + + + +
Moderate + + + + − + − + + + − + −
Severe + + + + + + + + + + − + +
Severe + + + + + + − + + + − + −
Severe + + + + + + + + + + + − +
46,XY
46,XX
46,XX
46,XY
46,XX
c.2479delA c.6272 GNT c.1672delA NA De novo De novo De novo NA
M, male; F, female; NA, not available.
NA NA
477
presented with typical facial features and limb abnormalities in addition to other characteristic manifestations of CdLS (Fig. 1). None had chromosomal abnormalities. This study fully complied with the Tenets of the Declaration of Helsinki and was approved by the University Ethics Committee. Informed consent was obtained from the subjects' parents before testing. 2.2. Mutation analysis by direct sequencing Genomic DNA was extracted from peripheral blood samples by standard methods. All coding regions and intron/exon boundaries of NIPBL, SMC1A, SMC3, RAD21, and HDAC8 genes were amplified by the polymerase chain reaction (PCR). The PCR primers were designed using Primer5 software (Premier Biosoft International, Palo Alto, CA); the products were verified by polyacrylamide gel electrophoresis (PAGE) and directly sequenced with an ABI PRISM BigDye kit on an ABI 3130 DNA sequencer (Applied Biosystems, Carlsbad, CA). Sequencing results were analyzed using the DNASTAR package (DNASTAR, Madison, WI). DNA sequencing was also performed on the patients' parents and 200 unrelated health controls (105 male individuals and 95 female individuals; aged 2–68 years, with an average age of 30 years) to determine whether the detected nucleotide changes were relevant mutations or genetic polymorphisms. 2.3. MLPA analysis MLPA analysis was implemented using kits P141-A2 and P142-A2 (MRC-Holland, Netherlands) for patients 1 and 2, according to the manufacturer's recommendations. In total, 100 ng of DNA was used
Fig. 1. Facial views of present 5 patients. (A) Facial features and hand defects in patient 1, (B) patient 2, (C) patient 3, (D) patient 4, and (E) patient 5. Their clinical findings are summarized in Table 1.
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L. Mei et al. / Gene 555 (2015) 476–480
Fig. 2. Mutations in NIPBL in three CdLS patients. (A) Pedigree of family 1, sequencing showing the c.2479delA frameshift mutation in exon 10 of NIPBL in patient 1. Arrow denotes the mutation site. (B) Pedigree of family 2, sequencing showing the c.6272 GNT heterozygous mutation in exon 36 of NIPBL in patient 2. Arrow denotes the mutation site. (C) Pedigree of family 3, sequencing showing the c.1672del frameshift mutation in exon 10 of NIPBL in patient 3. Arrow denotes the mutation site.
for DNA denaturation, hybridization, connection, and PCR amplification of each sample. Amplification products were run on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Carlsbad, CA) and examined using Coffalyser software (MRC-Holland, Netherlands). Three healthy males and three healthy females without CdLS served as controls.
Mutation Database (HGMD), Leiden Open Variation Database (LOVD), dbSNP135, or Exome Variant Server (EVS). No point mutations of NIPBL, SMC1A, SMC3, RAD21 and HDAC8, or any large deletions/duplications of NIPBL were identified in patients 4 and 5 (data not shown). All three variants were not found in the patients' parents or the 200 unrelated normal subjects.
3. Results 4. Discussion Direct sequencing of PCR products amplified from the genomic DNA of patient 1 revealed a frameshift mutation, c.2479delA (p.Arg827GlyfsX20) in exon 10 of the NIPBL gene (Fig. 2A), which was previously described (Gillis et al., 2004). In patient 2, a novel heterozygous mutation, c.6272 GNT, was identified in exon 36 of the NIPBL gene (Fig. 2B), resulting in the replacement of cysteine by phenylalanine at codon 2091 (p.C2091F). The p.C2091F mutation occurred within a highly conserved domain of NIPBL (Fig. 3A). In addition, this variant was predicted to be “probably damaging” by PolyPhen-2 (Adzhubei et al., 2010), with a score of 1.00 (sensitivity: 0.00; specificity: 1.00; Fig. 3C), which further demonstrates the pathogenicity of this mutation. In patient 3, sequencing showed a novel single-base deletion mutation (c.1672delA) in exon 10 of the NIPBL gene (Fig. 2C), which is predicted to cause a frameshift at codon 558, with the substitution of a leucine for a threonine, and the introduction of a putative stop codon 7 amino acids downstream in the translated protein (p.Thr558LeufsX7) (Fig. 3B). Both the c.6272 GNT and c.1672delA mutations in NIPBL have not been previously reported in the Human Gene
CdLS is a genetically heterogeneous disorder, related to mutations in the cohesin complex genes. Heterozygous mutations in NIPBL are thought to be major causes of CdLS and were detected in many patients. The NIPBL gene is the human homolog of the Drosophila Nipped-B gene. It is located on chromosome 5p13 and encodes the protein delangin, which is involved in chromatid cohesion processes and enhancer–promoter communications (Krantz et al., 2004; Tonkin et al., 2004; Strachan, 2005). The exact function of delangin is unknown; however, it is strongly expressed in fetal and adult heart and skeletal muscle and thymus (Tonkin et al., 2004), and is essential for the proper development of many organs and tissues in the growing embryo. The types of mutations detected in NIPBL included missense, nonsense, frameshift, splicing mutations, and intragenic deletions. Previously identified NIPBL mutations indicated that reduced expression or activity of delangin protein led to CdLS (Krantz et al., 2004; Hulinsky et al., 2005). In the present study, three different NIPBL mutations
L. Mei et al. / Gene 555 (2015) 476–480
479
Fig. 3. (A) Conservation of the amino acid residues altered by c.6272 GNT, p.C2091F across different species. (B) Conservation of the amino acid residues altered by c.1672delA, p.T558LfsX7 across different species. (C) PolyPhen-2 reports for the pathogenicity of the amino acid substitution p.C2091F in NIPBL.
were observed in three patients with CdLS. In patients 1 and 3, the c.2479delA and c.1672delA mutations were identified respectively, of which c.2479delA had been previously reported (Gillis et al., 2004). These frameshift mutations can produce a prematurely truncated protein, which could result in haploinsufficiency of NIPBL and, as expected, induce more severe phenotypes. Although it is currently unknown whether the mutant proteins are expressed or not, a large deletion that removed the NIPBL region, however, was observed in a severe CdLS case, supporting the idea that haploinsufficiency is a mechanism of CdLS (Krantz et al., 2004). Patient 2, who had a mild phenotype, carried a novel missense mutation c.6272 GNT. This mutated residue p.C2091F is most likely disease causing as it is close to previously identified CdLS-causing mutations c.6269 GNT (p.S2090I) and c.6274 C NG (p.L2092V), affecting highly conserved residues (Pié et al., 2010; Borck, 2004). The 200 control alleles exhibited wild-type sequences at that site, confirming that the mutation is not a single nucleotide polymorphism (SNP). Additionally, a prediction from an online software program (Poly-Phen2) strongly suggested that p.C2091F is a pathogenic mutation. All three variants appear to be de novo mutations in the patients, because they are absent in their parents. The genotype–phenotype correlation in CdLS is unclear. Previous reports suggested that patients with classic CdLS facial features are more likely to have a mutation in the NIPBL gene, especially a truncating mutation (Bhuiyan et al., 2006). Selicorni et al. (2007) concluded that mutation-positive patients were more severely affected than mutation-negative patients with respect to prenatal growth, limb defects, and speech impairment. When compared to patients with missense mutations or non-identifiable mutations, Gillis et al. (2004) found that individuals with truncating mutations presented a more severe phenotype. In this study, patients 1 and 3 had a truncating mutation and presented with more severe mental retardation than did patient 2, who had a missense mutation. All three patients had a phenotype with typical facial features and growth retardation; but patients 1 and 3 had additional clinical features such as hirsutism, low set
ears, and single transverse palmar crease. Since only five affected individuals were studied here, a clear genotype–phenotype correlation could not be established. Although mutations in NIPBL account for about 60% of cases, a smaller proportion of cases have mutations in the SMC1A, SMC3, RAD21, or HDAC8 cohesin subunit genes. However, no mutations in these genes were detected in patients 4 and 5. No abnormalities, i.e., whole exon deletions or duplications, were found by MLPA analysis. It is possible that genes other than the five known ones might be associated with CdLS, or potential alternative mechanisms of alteration in these known genes may be involved during pathogenesis. Mosaicism might be another cause of CdLS. Huisman et al. (2013) found a high frequency of somatic mosaicism for an NIPBL mutation in individuals with CdLS by analyzing buccal cells. Unfortunately, we were unable to obtain buccal cells from patients 4 and 5, and therefore the possibility of mosaic NIPBL mutations cannot be excluded. We plan to perform next generation sequencing studies (NGS) in DNA derived from peripheral blood to detect the possible existence of additional pathogenic genes.
5. Conclusion Three mutations in the NIPBL gene, including two novel ones, were identified in 5 sporadic Chinese CdLS patients. These findings contribute additional information to the kinds of NIPBL mutations that cause CdLS and may provide new insights into the cause and diagnosis of CdLS. More cases need to be reviewed to clarify the genotype–phenotype relationships in CdLS in a Chinese population.
Conflict of interest There is no conflict of interest.
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Acknowledgments We thank the participating family members for their help and support. This study was supported by the National Key Basic Research Program of China (2012CB944600) and the Special Fund on Scientific Research in Health Public Welfare Industry (201302001). References Adzhubei, I.A., et al., 2010. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249. Barisic, I., et al., 2008. Descriptive epidemiology of Cornelia de Lange syndrome in Europe. Am. J. Med. Genet. A 146, 51–59. Bhuiyan, Z.A., et al., 2006. Genotype–phenotype correlations of 39 patients with Cornelia de Lange syndrome: the Dutch experience. J. Med. Genet. 43, 568–575. Borck, G., 2004. NIPBL mutations and genetic heterogeneity in Cornelia de Lange syndrome. J. Med. Genet. 41, e128. Brachmann, W., 1916. Ein fall von symmetrischer monodaktylie durch Ulnadefekt, mit symmetrischer flughautbildung in den ellenbeugen, sowie anderen abnormitaten (zwerghaftogkeit, halsrippen, behaarung). Jarb. Kinder. Phys. Erzie. 84, 225–235. de Lange, C., 1933. Sur un type nouveau de dégénération (typus Amstelodamensis). Arch. Méd. Enfants. 36, 713–719. Deardorff, M.A., et al., 2007. Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of Cornelia de Lange syndrome with predominant mental retardation. Am. J. Hum. Genet. 80, 485–494. Deardorff, M.A., et al., 2012a. RAD21 mutations cause a human cohesinopathy. Am. J. Hum. Genet. 90, 1014–1027. Deardorff, M.A., et al., 2012b. HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle. Nature 489, 313–317. Dempsey, M.A., et al., 2014. Molecular confirmation of nine cases of Cornelia de Lange syndrome diagnosed prenatally. Prenat. Diagn. 34, 163–167.
Dorsett, D., 2011. Cohesin: genomic insights into controlling gene transcription and development. Curr. Opin. Genet. Dev. 21, 199–206. Dorsett, D., Krantz, I.D., 2009. On the molecular etiology of Cornelia de Lange syndrome. Ann. N. Y. Acad. Sci. 1151, 22–37. Feeney, K.M., Wasson, C.W., Parish, J.L., 2010. Cohesin: a regulator of genome integrity and gene expression. Biochem. J. 428, 147–161. Gillis, L.A., et al., 2004. NIPBL mutational analysis in 120 individuals with Cornelia de Lange syndrome and evaluation of genotype–phenotype correlations. Am. J. Hum. Genet. 75, 610–623. Huisman, S.A., Redeker, E.J., Maas, S.M., Mannens, M.M., Hennekam, R.C., 2013. High rate of mosaicism in individuals with Cornelia de Lange syndrome. J. Med. Genet. 50, 339–344. Hulinsky, R., et al., 2005. Fetus with interstitial del(5)(p13.1p14.2) diagnosed postnatally with Cornelia de Lange syndrome. Am. J. Hum. Genet. A 137, 336–338. Krantz, I.D., et al., 2004. Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat. Genet. 36, 631–635. Liu, J., Krantz, I.D., 2009. Cornelia de Lange syndrome, cohesin, and beyond. Clin. Genet. 76, 303–314. Musio, A., et al., 2006. X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations. Nat. Genet. 38, 528–530. Opitz, J.M., 1985. The Brachmann–de Lange syndrome. Am. J. Med. Genet. 22, 89–102. Pié, J., et al., 2010. Mutations and variants in the cohesion factor genes NIPBL, SMC1A, and SMC3 in a cohort of 30 unrelated patients with Cornelia de Lange syndrome. Am. J. Med. Genet. A 152, 924–929. Russell, K.L., Ming, J.E., Patel, K., Jukofsky, L., Magnusson, M., Krantz, I.D., 2001. Dominant paternal transmission of Cornelia de Lange syndrome: a new case and review of 25 previously reported familial recurrences. Am. J. Med. Genet. 104, 267–276. Selicorni, A., et al., 2007. Clinical score of 62 Italian patients with Cornelia de Lange syndrome and correlations with the presence and type of NIPBL mutation. Clin. Genet. 72, 98–108. Strachan, T., 2005. Cornelia de Lange syndrome and the link between chromosomal function, DNA repair and developmental gene regulation. Curr. Opin. Genet. Dev. 15, 258–264. Tonkin, E.T., Wang, T.J., Lisgo, S., Bamshad, M.J., Strachan, T., 2004. NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome. Nat. Genet. 36, 636–641.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
Two novel NIPBL gene mutations in Chinese patients with Cornelia de Lange syndrome Libin Mei, Desheng Liang, Yanru Huang, Qian Pan, Lingqian Wu ⁎ State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan 410078, China
a r t i c l e
i n f o
Article history: Received 9 October 2014 Received in revised form 9 November 2014 Accepted 13 November 2014 Available online 18 November 2014 Keywords: Cornelia de Lange syndrome NIPBL Frameshift mutation Missense mutation
a b s t r a c t Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder characterized by distinctive facial features, mental retardation, and upper limb defects, with the involvement of multiple organs and systems. To date, mutations have been identified in five genes responsible for CdLS: NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Here, we present a clinical and molecular characterization of five unrelated Chinese patients whose clinical presentation is consistent with that of CdLS. There were no chromosomal abnormalities in the five children. In three patients, DNA sequencing revealed a previously reported frameshift mutation c.2479delA (p.Arg827GlyfsX20), and two novel mutations including a heterozygous mutation c.6272 GNT (p.Cys2091Phe) and a frameshift mutation c.1672delA (p.Thr558LeufsX7) in NIPBL. For the remaining patients, large deletions and/or duplications within the NIPBL gene were excluded as playing a role in the pathogenesis, by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. These findings broaden the mutation spectrum of NIPBL and further our understanding of the diverse and variable effects of NIPBL mutations on CdLS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cornelia de Lange syndrome (CdLS, also called Brachmann de Lange syndrome; OMIM: 122470, 300590, and 610759), which was initially described in 1916 by Brachmann and further characterized by de Lange, is a rare congenital malformation disorder characterized by typical facial features, growth and mental retardation, upper limb malformations, hirsutism, cardiac defects, and a variety of other abnormalities affecting a wide range of tissues and organs (Brachmann, 1916; de Lange, 1933). The prevalence of CdLS is estimated to be about 1:10,000 live births (Opitz, 1985). Although most cases are sporadic, familial cases have also been reported (Russell et al., 2001; Barisic et al., 2008). Mutations in the cohesin regulator NIPBL (5p13.1, MIM: 608667) are present in approximately 60% of classical CdLS patients (Krantz et al., 2004; Tonkin et al., 2004; Gillis et al., 2004). However, CdLS exhibits extensive genetic heterogeneity, and mutations in the SMC1A (Xp11.2, MIM: 300040), SMC3 (10q25, MIM: 606062), RAD21 (8q24, MIM: 614701), and HDAC8 (Xq13.1, MIM: 300882) genes have also been identified in a smaller proportion of individuals with CdLS (Musio et al., 2006; Deardorff et al., 2007, 2012a, 2012b). All of the genes involved in CdLS code for a series of cohesin complex and associated proteins (Liu and Krantz, 2009). Cohesin is responsible for ensuring
correct chromosome segregation during cell division. Beyond this role, cohesin also functions in the regulation of gene expression and DNA repair (Dorsett and Krantz, 2009; Feeney et al., 2010). Consequently, deleterious variation of any of the genes coding for cohesin proteins may affect early stages of embryonic development (Dorsett, 2011). Previous reports have shown that patients with NIPBL mutations are more severely affected in limb anomalies, growth, and cognitive function than those with mutations in SMC1A, SMC3, RAD21, and HDAC8 genes or in those with unknown etiology (Deardorff et al., 2012a, 2012b; Dempsey et al., 2014). Genotype–phenotype correlation studies indicated that patients with truncating mutations in NIPBL have a more severe phenotype than those with missense mutations (Gillis et al., 2004; Bhuiyan et al., 2006; Selicorni et al., 2007). In this study, five Chinese patients diagnosed with CdLS were selected. By direct sequencing of the five candidate genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8, two novel mutations in NIPBL were identified separately in two patients. MLPA analysis was also performed to exclude large deletions/duplications within the NIPBL gene for the remaining two patients without identifiable point mutations in the five causative genes. 2. Materials and methods 2.1. Patients
Abbreviations: CdLS, Cornelia de Lange syndrome; NIPBL, Nipped-B-like; PCR, polymerase chain reaction; MLPA, Multiplex Ligation-dependent Probe Amplification. ⁎ Corresponding author at: State Key Laboratory of Medical Genetics, Xiangya Hospital, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, China. E-mail address: [email protected] (L. Wu).
http://dx.doi.org/10.1016/j.gene.2014.11.033 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Five affected and unrelated children from five Chinese families were referred to our institution for genetic diagnosis and counseling. The clinical symptoms of each case are summarized in Table 1. All patients
L. Mei et al. / Gene 555 (2015) 476–480 Table 1 Summary of clinical findings and NIPBL detected mutations. Clinical data
Gender Weight at birth Age at test Height at test Weight at test Head circumference at test Postnatal growth retardation Mental retardation Language delay Microcephaly Low hair line Synophrys Hirsutism Long curly eyelashes Low set ears Depressed nasal bridge Long philtrum Thin upper lips Cleft palate Finger deformity Single transverse palmar crease Chromosome karyotype NIPBL Mutation Mutation origin
Patient 1
2
3
4
5
M 2.5 kg 7 years 95 cm 11.5 kg 44 cm
F 2.65 kg 6 years 90 cm 11.0 kg 43 cm
F 2.8 kg 2 years 70 cm 6.0 kg 41 cm
M 2.6 k g 7 years 96 cm 13 kg 46 cm
F 3.0 kg 60 days 60 cm 3.5 kg 36.5 cm
+
+
+
+
+
Severe + + + + + + + + + + + + +
Moderate + + + + − + − + + + − + −
Severe + + + + + + + + + + − + +
Severe + + + + + + − + + + − + −
Severe + + + + + + + + + + + − +
46,XY
46,XX
46,XX
46,XY
46,XX
c.2479delA c.6272 GNT c.1672delA NA De novo De novo De novo NA
M, male; F, female; NA, not available.
NA NA
477
presented with typical facial features and limb abnormalities in addition to other characteristic manifestations of CdLS (Fig. 1). None had chromosomal abnormalities. This study fully complied with the Tenets of the Declaration of Helsinki and was approved by the University Ethics Committee. Informed consent was obtained from the subjects' parents before testing. 2.2. Mutation analysis by direct sequencing Genomic DNA was extracted from peripheral blood samples by standard methods. All coding regions and intron/exon boundaries of NIPBL, SMC1A, SMC3, RAD21, and HDAC8 genes were amplified by the polymerase chain reaction (PCR). The PCR primers were designed using Primer5 software (Premier Biosoft International, Palo Alto, CA); the products were verified by polyacrylamide gel electrophoresis (PAGE) and directly sequenced with an ABI PRISM BigDye kit on an ABI 3130 DNA sequencer (Applied Biosystems, Carlsbad, CA). Sequencing results were analyzed using the DNASTAR package (DNASTAR, Madison, WI). DNA sequencing was also performed on the patients' parents and 200 unrelated health controls (105 male individuals and 95 female individuals; aged 2–68 years, with an average age of 30 years) to determine whether the detected nucleotide changes were relevant mutations or genetic polymorphisms. 2.3. MLPA analysis MLPA analysis was implemented using kits P141-A2 and P142-A2 (MRC-Holland, Netherlands) for patients 1 and 2, according to the manufacturer's recommendations. In total, 100 ng of DNA was used
Fig. 1. Facial views of present 5 patients. (A) Facial features and hand defects in patient 1, (B) patient 2, (C) patient 3, (D) patient 4, and (E) patient 5. Their clinical findings are summarized in Table 1.
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L. Mei et al. / Gene 555 (2015) 476–480
Fig. 2. Mutations in NIPBL in three CdLS patients. (A) Pedigree of family 1, sequencing showing the c.2479delA frameshift mutation in exon 10 of NIPBL in patient 1. Arrow denotes the mutation site. (B) Pedigree of family 2, sequencing showing the c.6272 GNT heterozygous mutation in exon 36 of NIPBL in patient 2. Arrow denotes the mutation site. (C) Pedigree of family 3, sequencing showing the c.1672del frameshift mutation in exon 10 of NIPBL in patient 3. Arrow denotes the mutation site.
for DNA denaturation, hybridization, connection, and PCR amplification of each sample. Amplification products were run on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Carlsbad, CA) and examined using Coffalyser software (MRC-Holland, Netherlands). Three healthy males and three healthy females without CdLS served as controls.
Mutation Database (HGMD), Leiden Open Variation Database (LOVD), dbSNP135, or Exome Variant Server (EVS). No point mutations of NIPBL, SMC1A, SMC3, RAD21 and HDAC8, or any large deletions/duplications of NIPBL were identified in patients 4 and 5 (data not shown). All three variants were not found in the patients' parents or the 200 unrelated normal subjects.
3. Results 4. Discussion Direct sequencing of PCR products amplified from the genomic DNA of patient 1 revealed a frameshift mutation, c.2479delA (p.Arg827GlyfsX20) in exon 10 of the NIPBL gene (Fig. 2A), which was previously described (Gillis et al., 2004). In patient 2, a novel heterozygous mutation, c.6272 GNT, was identified in exon 36 of the NIPBL gene (Fig. 2B), resulting in the replacement of cysteine by phenylalanine at codon 2091 (p.C2091F). The p.C2091F mutation occurred within a highly conserved domain of NIPBL (Fig. 3A). In addition, this variant was predicted to be “probably damaging” by PolyPhen-2 (Adzhubei et al., 2010), with a score of 1.00 (sensitivity: 0.00; specificity: 1.00; Fig. 3C), which further demonstrates the pathogenicity of this mutation. In patient 3, sequencing showed a novel single-base deletion mutation (c.1672delA) in exon 10 of the NIPBL gene (Fig. 2C), which is predicted to cause a frameshift at codon 558, with the substitution of a leucine for a threonine, and the introduction of a putative stop codon 7 amino acids downstream in the translated protein (p.Thr558LeufsX7) (Fig. 3B). Both the c.6272 GNT and c.1672delA mutations in NIPBL have not been previously reported in the Human Gene
CdLS is a genetically heterogeneous disorder, related to mutations in the cohesin complex genes. Heterozygous mutations in NIPBL are thought to be major causes of CdLS and were detected in many patients. The NIPBL gene is the human homolog of the Drosophila Nipped-B gene. It is located on chromosome 5p13 and encodes the protein delangin, which is involved in chromatid cohesion processes and enhancer–promoter communications (Krantz et al., 2004; Tonkin et al., 2004; Strachan, 2005). The exact function of delangin is unknown; however, it is strongly expressed in fetal and adult heart and skeletal muscle and thymus (Tonkin et al., 2004), and is essential for the proper development of many organs and tissues in the growing embryo. The types of mutations detected in NIPBL included missense, nonsense, frameshift, splicing mutations, and intragenic deletions. Previously identified NIPBL mutations indicated that reduced expression or activity of delangin protein led to CdLS (Krantz et al., 2004; Hulinsky et al., 2005). In the present study, three different NIPBL mutations
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Fig. 3. (A) Conservation of the amino acid residues altered by c.6272 GNT, p.C2091F across different species. (B) Conservation of the amino acid residues altered by c.1672delA, p.T558LfsX7 across different species. (C) PolyPhen-2 reports for the pathogenicity of the amino acid substitution p.C2091F in NIPBL.
were observed in three patients with CdLS. In patients 1 and 3, the c.2479delA and c.1672delA mutations were identified respectively, of which c.2479delA had been previously reported (Gillis et al., 2004). These frameshift mutations can produce a prematurely truncated protein, which could result in haploinsufficiency of NIPBL and, as expected, induce more severe phenotypes. Although it is currently unknown whether the mutant proteins are expressed or not, a large deletion that removed the NIPBL region, however, was observed in a severe CdLS case, supporting the idea that haploinsufficiency is a mechanism of CdLS (Krantz et al., 2004). Patient 2, who had a mild phenotype, carried a novel missense mutation c.6272 GNT. This mutated residue p.C2091F is most likely disease causing as it is close to previously identified CdLS-causing mutations c.6269 GNT (p.S2090I) and c.6274 C NG (p.L2092V), affecting highly conserved residues (Pié et al., 2010; Borck, 2004). The 200 control alleles exhibited wild-type sequences at that site, confirming that the mutation is not a single nucleotide polymorphism (SNP). Additionally, a prediction from an online software program (Poly-Phen2) strongly suggested that p.C2091F is a pathogenic mutation. All three variants appear to be de novo mutations in the patients, because they are absent in their parents. The genotype–phenotype correlation in CdLS is unclear. Previous reports suggested that patients with classic CdLS facial features are more likely to have a mutation in the NIPBL gene, especially a truncating mutation (Bhuiyan et al., 2006). Selicorni et al. (2007) concluded that mutation-positive patients were more severely affected than mutation-negative patients with respect to prenatal growth, limb defects, and speech impairment. When compared to patients with missense mutations or non-identifiable mutations, Gillis et al. (2004) found that individuals with truncating mutations presented a more severe phenotype. In this study, patients 1 and 3 had a truncating mutation and presented with more severe mental retardation than did patient 2, who had a missense mutation. All three patients had a phenotype with typical facial features and growth retardation; but patients 1 and 3 had additional clinical features such as hirsutism, low set
ears, and single transverse palmar crease. Since only five affected individuals were studied here, a clear genotype–phenotype correlation could not be established. Although mutations in NIPBL account for about 60% of cases, a smaller proportion of cases have mutations in the SMC1A, SMC3, RAD21, or HDAC8 cohesin subunit genes. However, no mutations in these genes were detected in patients 4 and 5. No abnormalities, i.e., whole exon deletions or duplications, were found by MLPA analysis. It is possible that genes other than the five known ones might be associated with CdLS, or potential alternative mechanisms of alteration in these known genes may be involved during pathogenesis. Mosaicism might be another cause of CdLS. Huisman et al. (2013) found a high frequency of somatic mosaicism for an NIPBL mutation in individuals with CdLS by analyzing buccal cells. Unfortunately, we were unable to obtain buccal cells from patients 4 and 5, and therefore the possibility of mosaic NIPBL mutations cannot be excluded. We plan to perform next generation sequencing studies (NGS) in DNA derived from peripheral blood to detect the possible existence of additional pathogenic genes.
5. Conclusion Three mutations in the NIPBL gene, including two novel ones, were identified in 5 sporadic Chinese CdLS patients. These findings contribute additional information to the kinds of NIPBL mutations that cause CdLS and may provide new insights into the cause and diagnosis of CdLS. More cases need to be reviewed to clarify the genotype–phenotype relationships in CdLS in a Chinese population.
Conflict of interest There is no conflict of interest.
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