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dimerization domain of NFIX causes Sotos-like overgrowth syndrome: A new case
Gene 511 (2012) 103–105
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A peculiar mutation in the DNA-binding/dimerization domain of NFIX causes Sotos-like overgrowth syndrome: A new case Manuela Priolo a,⁎, Enrico Grosso b, Corrado Mammì a, Claudia Labate a, Valeria Giorgia Naretto b, Caterina Vacalebre a, Paola Caridi a, Carmelo Laganà a a b
Unità Operativa di Genetica medica, Azienda Ospedaliera Bianchi-Melacrino-Morelli, Reggio Calabria, Italy SCDU Genetica Medica, Azienda Ospedaliero-Universitaria S. Giovanni Battista di Torino, Italy
a r t i c l e
i n f o
Article history: Accepted 25 August 2012 Available online 13 September 2012 Keywords: DNA-binding/dimerization domain NFIX MH1 domain Deletion Sotos-like overgrowth syndrome
a b s t r a c t The Nuclear Factor I-X (NFIX) is a member of the nuclear factor I (NFI) family proteins, which are implicated as site-specific DNA-binding proteins and is deleted or mutated in a subset of patients with Sotos-like overgrowth syndrome and in patients with Marshall–Smith syndrome. We evaluated an additional patient with clinical features of Sotos-like syndrome by sequencing analysis of the NFIX gene and identified a 21 nucleotide in frame deletion predicting loss of 7 amino acids in the DNA-binding/dimerization domain of the NFIX protein. The deleted residues are all evolutionally conserved amino acids. The present report further confirms that mutations in DNA-binding/dimerization domain cause haploinsufficiency of the NFIX protein and strongly suggests that in individuals with Sotos-like features unrelated to NSD1 changes genetic testing of NFIX should be considered. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Overgrowth syndromes are defined on a height and weight more than + 2 standard deviations (SD) often present at birth and persistent in postnatal life (Malan et al., 2010). They tend to share several characteristics including macrocephaly, mental retardation, similar facial dysmorphic features, advanced bone age, hemihyperplasia, vascular malformations, and predisposition to neoplasia but are clinically and molecularly distinct by the specific combination of these features and the different molecular mechanisms underlying them (Malan et al., 2010). Beckwith–Wiedemann syndrome (BWS [MIM 130650], Sotos syndrome (SS [MIM 117550]), and Simpson–Golabi– Behmel syndrome (SGBS [MIM 312870]) are some examples of overgorwth syndromes caused by mutations at different loci (Neri et al., 1998; Visser and Matsumoto, 2003; Weksberg et al., 2003). Sotos syndrome (MIM 117550) is an overgrowth syndrome characterized by tall stature and/or macrocephaly, distinctive facial appearance and mental retardation (Leventopoulos et al., 2009). The diagnosis of Sotos syndrome is established by confirming NSD1 deletion or point mutations, that causes up to 90% of Sotos syndrome cases. However, a part of patients with suspected Sotos syndrome are known to show no abnormalities in NSD1 (Tatton-Brown and
Rahman, 2007). Recently two patients with Sotos-like overgrowth features presenting microdeletions encompassing Nuclear Factor I-X gene (NFIX) at 19p13.2 have been reported. In addition, a nonsense mutation in NFIX was identified in another unrelated patient with the same Sotos-like features (Malan et al., 2010). Frameshift and donor-splice site mutations at the same gene were also identified in Marshall–Smith syndrome (MIM 602535) an overgrowth syndrome with osteochondrodysplasia characterized by accelerated skeletal maturation, relative postnatal failure to thrive, respiratory difficulties, mental retardation and unusual facial features (Adam et al., 2005). The authors suggested that haploinsufficiency of NFIX leads to Sotos-like features while dominant-negative effects of the truncated NFIX proteins cause Marshall–Smith syndrome (Malan et al., 2010). Very recently, two other mutations of the NFIX gene have been described: both of them are located in the N-terminal DNAbinding/dimerization domain (Yoneda et al., 2012). We report of an additional patient presenting a small in frame deletion of the N terminal portion of the NFIX protein. Detailed genetic and clinical data are provided. 2. Materials and methods 2.1. Patient's history
Abbreviations: NFIX, Nuclear Factor I-X gene; MH1, MAD homology 1; BWS, Beckwith– Wiedemann syndrome; SS, Sotos syndrome; SGBS, Simpson–Golabi–Behmel syndrome; NSD1, NUclear receptor-binding Su-var enhancer of zeste, and trithorax domain protein 1; CGH-array, Comparative genomic hybridization‐array; PCR, Polymerase chain reaction. ⁎ Corresponding author. Tel.: +39 0965 397295; fax: +39 0965 397350. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.08.040
The proband is the first daughter of unrelated healthy parents. Her mother refers three miscarriages before the proband's pregnancy. A threatened abortion during the first trimester was pharmacologically treated. The patient was born after 36 weeks of gestation: at birth,
104
M. Priolo et al. / Gene 511 (2012) 103–105
Fig. 1. Deleted amino acid residues of the NFIX protein. (1a) amino‐acid sequence alignments of NFIX protein from amino‐acid position 51 to 69. The seven amino acids residues are red boxed. Protein sequences were obtained through the NCBI protein database and multiple sequence alignment was performed by CLUSTALW web site (http://clustalw.ddbj.nig. ac.jp/). (1b) Partial schematic representation of the N terminal NFIX protein modified from Pfam web site (http://pfam.sanger.ac.uk/). The NfI_DNAbd pre N domain is depicted in green with its extension numbered (4–46). The MH1 domain is depicted in red with its extension numbered (68–172). A partial section of the CTF_NFI domain is depicted in blue with its complete extension numbered (213–502). The deleted string is highlighted.
body weight was 3250 (> 97° centile), height 52 cm (>97° centile) and OFC 35 cm (90° centile.). The APGAR scores were 6 and 9 at the first and the fifth minutes, respectively. Neonatal hypotonia was recognized. At 6 months, overgrowth, squint with nistagmus and psychomotor retardation have been noticed; a cerebral MRI revealed hypoplastic chiasma, optic nerves and corpus callosum hypoplasia with perculiar involvement of the rostrum. Increased bone age was reported as well (5 years and 6 months at 3 years and 4 months of chronological age). Because of the overgrowth, Sotos syndrome has been suspected and both NSD1 FISH and sequencing analysis performed with standard methods have been performed, giving negative results. In particular sequencing analysis
was carried out by PCR amplification of exons and splicing junctions with specific primers as previously reported (Visser and Matsumoto, 2003). At our first examination (4 years) the following features have been noticed: long/narrow and triangular face with frontal bossing, high forehead, midface hypoplasia, mild proptosis, prominent ears, divergent strabismus, down-slanting palpebral fissures, blue sclerae, short nose with anteverted nares, prominent long philtrum, everted lower lip. The patient showed hypotonia and slender habitus with large hands/feet, prominent fingertips, and pectus excavatum. She had slight ataxic gait, open mouth with hypomimic face. Bullet-shaped phalanges, which are typical features of Marshall–Smith syndrome, were noticed at
Table 1 Clinical features of the proband and review of the other reported patients. Malan et al. 1
Prenatal growth
Postnatal growth Development
Brain MRI Craniofacial
Eyes
Skeletal
Yoneda et al6
NFIX deletion/mutation
Present case Del aa53-59
Patient A Del19p13.3
Patient B Del19p13.3
Patient C Q190X
Patient 1 L60P
Patient 2 R121P
Sex Birth weight (g)
F
M 4500 (>95)
M 3110 (10–50)
F 3600 (50–90)
M 2938 (10–50)
53 (95) 38 (>95) + + + + + + + −/+
49 (50) 33.5 (10) + + + + − + + −/−
52 (95) 37.5 (495) + + + + − + +/−
F 2816 (10–50) 48.8 (50) 33.5 (50) + na + − + + na na
+ + + + + na na + + − − − na + + +
+ + − − − na na + − − + − na +
+ + + + + na na − + + − − na − + +
+ + + na + + na − + − na na na + + +
+ − + − − − − − − na − − − − na
Birth height (cm) OFC (cm) Height > 98° centile OFC > 98° centile Mental retardation Autistic traits Motor retardation Hypotonia Behavioral anomalies Ventricular dilatation/corpus callosum hypoplasia Long/narrow face High forehead Down-slanting palpebral fissures Small mouth Everted lower lip Short nose Proptosis Hypermotropia Strabismus Nystagmus Astigmatism Myopia Blue sclerae Pectus excavatum Scoliosis Advanced bone age
Abbreviations: F, female; M, male; NA, not ascertained.
35( 95° cent) + + + + + + + +/+ + + + + + + − + + − na + + + +
+
51 (50) 35.5 (50–90) + na + − + + − na
M. Priolo et al. / Gene 511 (2012) 103–105
hand X rays. There was moderate-to-severe mental retardation. Her weight was 21 kg (97° centile), height 112.5 cm (>97° centile), OFC 53.8 cm (90° centile). She also presented few sleep apnea. On the basis of the clinical features we suspected a mild form of Marshall–Smith syndrome. We re-evaluated the proband at 8 years and confirmed the same objective features. Her weight was 41 kg (>97° centile) and height 147.5 cm (>97° centile), OFC 56.5 cm (>97° centile). In the meantime, scoliosis and an unusual behavior characterized by anxiety and tendency to repetitive activities have been reported. The patient underwent the following negative genetic analyses: CGH-array (60 K) which helped us to eventually exclude intragenic small deletions of the NSD1 gene, methylation analysis for Angelman/Prader–Willi region and subtelomeric microdeletion screening. 2.2. Mutation analysis Genomic DNA was isolated from peripheral blood leukocytes according to standard methods. Specific primers for PCR amplification of exons and splicing junctions of NFIX were designed as previously reported (Malan et al., 2010). PCR products were purified with QiaQuick purification kit method and directly sequenced on an automated sequencer (ABI 3500, Applied Biosystems) with the Dye Terminator method. 3. Results and discussion An in frame deletion of the NFIX gene has been identified. The deletion of 21 nucleotides in exon 2 (c. 157_177delGAGCGGGCGGTGAAGGA CGAG) is predicted to cause a protein lacking of 7 amino acids (from residue 53 to residue 59 [delERAVKDE]) proximal to the NFIX α-helical region of DNA-binding domain (the lysine helix). Both parent are negative for the same variation that has not been detected in 100 normal controls. These residues are evolutionally conserved amino acids (Stefancsik and Sarkar, 2003). NFI proteins form homo- or heterodimers and bind to the palindromic DNA consensus sequence through its N-terminal DNA-binding/ dimerization domain. Point mutations in DNA-binding/dimerization domain of NFI proteins have been shown to cause loss of dimerization, DNA-binding and replication activities (Stefancsik and Sarkar, 2003). Specifically, the N-terminal 80 amino acids have also been implicated in physically contacting DNA (Dekker et al., 1996). The deleted residues are located at the proximal end of the putative lysine helix forming the MAD homology 1 (MH1) domain (Osada et al., 1999) and are conserved in both human and other species NFI proteins (Stefancsik and Sarkar, 2003) (Fig. 1). This region presents a high homology with the SMAD proteins MH1 domain. Previous studies have proved that somatic mutations of SMAD4 gene located in this same region and nearby the lysine α-helix region completely abolish its capacity to bind DNA (Jones and Kern, 2000). The deleted sequence (ERAVKDE) represents an alternatively positive and negative charged sequence of amino acids which are likely to influence tertiary structure of the protein. Moreover, one of the missense mutation (L60P) reported by Yoneda et al. (Stefancsik and Sarkar, 2003) is immediately subsequent to the observed deleted strand. Yoneda et al. have addressed the question whether patients with NFIX mutations should have been classified as Sotos-Like or Marshall–
105
Smith syndrome (Yoneda et al., 2012). Both syndromes share many common features but they are different in many aspect; in particular Marshall–Smith syndrome has proptosis, underdeveloped midface and prominent premaxilla (Adam et al., 2005; Shaw et al., 2010). The same authors state that one of their patients has some characteristic features of Marshall–Smith syndrome such as everted lower lip, short nose and midface hypoplasia. In this patient the L60P missense mutation was found. The authors concluded that missense mutations in the DNA binding/ dimerization domain, are more likely to cause Sotos-like syndrome, while according to Malan et al., Marshall–Smith syndrome is due to mutations with dominant negative effect. Malan et al. reported two patients with Sotos-like syndrome with deletion of the entire NFIX gene and one with a nonsense mutation (Q190X) in the N terminal domain of the protein whose clinical features included behavioral anomalies such as anxiety and autistic traits (Malan et al., 2010). Our patient seems to be more similar to Malan's patients with respect to the other two reported by Yoneda et al. These latter authors suggested that the autistic traits are more likely to be caused by haploinsufficiency of NFIX, but not by missense mutations. Moreover, our proband shows blue sclerae, mild proptosis, slight bullet-shaped phalanges and few sleep apneae spontaneously resolved with age (Table 1). These features are more specific of the Marshall–Smith syndrome than the Sotos-like syndrome: this might be explained by the fact that the mutation observed is strongly likely to cause a real haploinsufficiency of NFIX protein abolishing its property to bind DNA and determining a phenotype sharing Sotos-like features and very mild characteristics of the Marshall–Smith syndrome. In conclusion this report further confirms that there is a high genotype-phenotype correlation in NFIX mutations, being patients with deletion or non sense mutations more severely affected. Moreover, it might further help in elucidating NFIX different domains functions. References Adam, M.P., et al., 2005. Marshall–Smith syndrome: natural history and evidence of an osteochondrodysplasia with connective tissue abnormalities. Am. J. Med. Genet. A. 137, 117–124. Dekker, J., Van Oosterhout, J.A.W.M., Van der Vliet, P.C., 1996. Two regions within the dna binding domain of nuclear Factor i interact with DNA and stimulate adenovirus DNA replication independently. Mol. Cell. Biol. 16, 4073–4080. Jones, J.B., Kern, S.E., 2000. Functional mapping of the MH1 DNA-binding domain of DPC4/SMAD4. Nucleic Acids Res. 28, 2363–2368. Leventopoulos, G., et al., 2009. A clinical study of Sotos syndrome patients with review of the literature. Pediatr. Neurol. 40, 357–364. Malan, V., et al., 2010. Distinct effects of allelic NFIX mutations on nonsense-mediated mRNA decay engender either a Sotos-like or a Marshall–Smith syndrome. Am. J. Hum. Genet. 87, 189–198. Neri, G., Gurrieri, F., Zanni, G., Lin, A., 1998. Clinical and molecular aspects of the Simpson–Golabi–Behmel syndrome. Am. J. Med. Genet. 79, 279–283. Osada, S., et al., 1999. Expression, DNA-binding specificity and transcriptional regulation of nuclear factor 1 family proteins from rat. Biochem. J. 342, 189–198. Shaw, A.C., et al., 2010. Phenotype and natural history in Marshall–Smith syndrome. Am. J. Med. Genet. A 152, 2714–2726. Stefancsik, R., Sarkar, S., 2003. Relationship between the DNA binding domains of SMAD and NFI/CTF transcription factors defines a new superfamily of genes. DNA Seq. 14, 233–239. Tatton-Brown, K., Rahman, N., 2007. Sotos syndrome. Eur. J. Hum. Genet. 15, 264–271. Visser, R., Matsumoto, N., 2003. Genetics of Sotos syndrome. Curr. Opin. Pediatr. 15, 598–606. Weksberg, R., Smith, A.C., Squire, J., Sadowski, P., 2003. Beckwith–Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum. Mol. Genet. 12, R61–R68. Yoneda, Y., et al., 2012. Missense mutations in the DNA-binding/dimerization domain of NFIX cause Sotos-like features. J. Hum. Genet. 57, 207–211.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A peculiar mutation in the DNA-binding/dimerization domain of NFIX causes Sotos-like overgrowth syndrome: A new case Manuela Priolo a,⁎, Enrico Grosso b, Corrado Mammì a, Claudia Labate a, Valeria Giorgia Naretto b, Caterina Vacalebre a, Paola Caridi a, Carmelo Laganà a a b
Unità Operativa di Genetica medica, Azienda Ospedaliera Bianchi-Melacrino-Morelli, Reggio Calabria, Italy SCDU Genetica Medica, Azienda Ospedaliero-Universitaria S. Giovanni Battista di Torino, Italy
a r t i c l e
i n f o
Article history: Accepted 25 August 2012 Available online 13 September 2012 Keywords: DNA-binding/dimerization domain NFIX MH1 domain Deletion Sotos-like overgrowth syndrome
a b s t r a c t The Nuclear Factor I-X (NFIX) is a member of the nuclear factor I (NFI) family proteins, which are implicated as site-specific DNA-binding proteins and is deleted or mutated in a subset of patients with Sotos-like overgrowth syndrome and in patients with Marshall–Smith syndrome. We evaluated an additional patient with clinical features of Sotos-like syndrome by sequencing analysis of the NFIX gene and identified a 21 nucleotide in frame deletion predicting loss of 7 amino acids in the DNA-binding/dimerization domain of the NFIX protein. The deleted residues are all evolutionally conserved amino acids. The present report further confirms that mutations in DNA-binding/dimerization domain cause haploinsufficiency of the NFIX protein and strongly suggests that in individuals with Sotos-like features unrelated to NSD1 changes genetic testing of NFIX should be considered. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Overgrowth syndromes are defined on a height and weight more than + 2 standard deviations (SD) often present at birth and persistent in postnatal life (Malan et al., 2010). They tend to share several characteristics including macrocephaly, mental retardation, similar facial dysmorphic features, advanced bone age, hemihyperplasia, vascular malformations, and predisposition to neoplasia but are clinically and molecularly distinct by the specific combination of these features and the different molecular mechanisms underlying them (Malan et al., 2010). Beckwith–Wiedemann syndrome (BWS [MIM 130650], Sotos syndrome (SS [MIM 117550]), and Simpson–Golabi– Behmel syndrome (SGBS [MIM 312870]) are some examples of overgorwth syndromes caused by mutations at different loci (Neri et al., 1998; Visser and Matsumoto, 2003; Weksberg et al., 2003). Sotos syndrome (MIM 117550) is an overgrowth syndrome characterized by tall stature and/or macrocephaly, distinctive facial appearance and mental retardation (Leventopoulos et al., 2009). The diagnosis of Sotos syndrome is established by confirming NSD1 deletion or point mutations, that causes up to 90% of Sotos syndrome cases. However, a part of patients with suspected Sotos syndrome are known to show no abnormalities in NSD1 (Tatton-Brown and
Rahman, 2007). Recently two patients with Sotos-like overgrowth features presenting microdeletions encompassing Nuclear Factor I-X gene (NFIX) at 19p13.2 have been reported. In addition, a nonsense mutation in NFIX was identified in another unrelated patient with the same Sotos-like features (Malan et al., 2010). Frameshift and donor-splice site mutations at the same gene were also identified in Marshall–Smith syndrome (MIM 602535) an overgrowth syndrome with osteochondrodysplasia characterized by accelerated skeletal maturation, relative postnatal failure to thrive, respiratory difficulties, mental retardation and unusual facial features (Adam et al., 2005). The authors suggested that haploinsufficiency of NFIX leads to Sotos-like features while dominant-negative effects of the truncated NFIX proteins cause Marshall–Smith syndrome (Malan et al., 2010). Very recently, two other mutations of the NFIX gene have been described: both of them are located in the N-terminal DNAbinding/dimerization domain (Yoneda et al., 2012). We report of an additional patient presenting a small in frame deletion of the N terminal portion of the NFIX protein. Detailed genetic and clinical data are provided. 2. Materials and methods 2.1. Patient's history
Abbreviations: NFIX, Nuclear Factor I-X gene; MH1, MAD homology 1; BWS, Beckwith– Wiedemann syndrome; SS, Sotos syndrome; SGBS, Simpson–Golabi–Behmel syndrome; NSD1, NUclear receptor-binding Su-var enhancer of zeste, and trithorax domain protein 1; CGH-array, Comparative genomic hybridization‐array; PCR, Polymerase chain reaction. ⁎ Corresponding author. Tel.: +39 0965 397295; fax: +39 0965 397350. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.08.040
The proband is the first daughter of unrelated healthy parents. Her mother refers three miscarriages before the proband's pregnancy. A threatened abortion during the first trimester was pharmacologically treated. The patient was born after 36 weeks of gestation: at birth,
104
M. Priolo et al. / Gene 511 (2012) 103–105
Fig. 1. Deleted amino acid residues of the NFIX protein. (1a) amino‐acid sequence alignments of NFIX protein from amino‐acid position 51 to 69. The seven amino acids residues are red boxed. Protein sequences were obtained through the NCBI protein database and multiple sequence alignment was performed by CLUSTALW web site (http://clustalw.ddbj.nig. ac.jp/). (1b) Partial schematic representation of the N terminal NFIX protein modified from Pfam web site (http://pfam.sanger.ac.uk/). The NfI_DNAbd pre N domain is depicted in green with its extension numbered (4–46). The MH1 domain is depicted in red with its extension numbered (68–172). A partial section of the CTF_NFI domain is depicted in blue with its complete extension numbered (213–502). The deleted string is highlighted.
body weight was 3250 (> 97° centile), height 52 cm (>97° centile) and OFC 35 cm (90° centile.). The APGAR scores were 6 and 9 at the first and the fifth minutes, respectively. Neonatal hypotonia was recognized. At 6 months, overgrowth, squint with nistagmus and psychomotor retardation have been noticed; a cerebral MRI revealed hypoplastic chiasma, optic nerves and corpus callosum hypoplasia with perculiar involvement of the rostrum. Increased bone age was reported as well (5 years and 6 months at 3 years and 4 months of chronological age). Because of the overgrowth, Sotos syndrome has been suspected and both NSD1 FISH and sequencing analysis performed with standard methods have been performed, giving negative results. In particular sequencing analysis
was carried out by PCR amplification of exons and splicing junctions with specific primers as previously reported (Visser and Matsumoto, 2003). At our first examination (4 years) the following features have been noticed: long/narrow and triangular face with frontal bossing, high forehead, midface hypoplasia, mild proptosis, prominent ears, divergent strabismus, down-slanting palpebral fissures, blue sclerae, short nose with anteverted nares, prominent long philtrum, everted lower lip. The patient showed hypotonia and slender habitus with large hands/feet, prominent fingertips, and pectus excavatum. She had slight ataxic gait, open mouth with hypomimic face. Bullet-shaped phalanges, which are typical features of Marshall–Smith syndrome, were noticed at
Table 1 Clinical features of the proband and review of the other reported patients. Malan et al. 1
Prenatal growth
Postnatal growth Development
Brain MRI Craniofacial
Eyes
Skeletal
Yoneda et al6
NFIX deletion/mutation
Present case Del aa53-59
Patient A Del19p13.3
Patient B Del19p13.3
Patient C Q190X
Patient 1 L60P
Patient 2 R121P
Sex Birth weight (g)
F
M 4500 (>95)
M 3110 (10–50)
F 3600 (50–90)
M 2938 (10–50)
53 (95) 38 (>95) + + + + + + + −/+
49 (50) 33.5 (10) + + + + − + + −/−
52 (95) 37.5 (495) + + + + − + +/−
F 2816 (10–50) 48.8 (50) 33.5 (50) + na + − + + na na
+ + + + + na na + + − − − na + + +
+ + − − − na na + − − + − na +
+ + + + + na na − + + − − na − + +
+ + + na + + na − + − na na na + + +
+ − + − − − − − − na − − − − na
Birth height (cm) OFC (cm) Height > 98° centile OFC > 98° centile Mental retardation Autistic traits Motor retardation Hypotonia Behavioral anomalies Ventricular dilatation/corpus callosum hypoplasia Long/narrow face High forehead Down-slanting palpebral fissures Small mouth Everted lower lip Short nose Proptosis Hypermotropia Strabismus Nystagmus Astigmatism Myopia Blue sclerae Pectus excavatum Scoliosis Advanced bone age
Abbreviations: F, female; M, male; NA, not ascertained.
35( 95° cent) + + + + + + + +/+ + + + + + + − + + − na + + + +
+
51 (50) 35.5 (50–90) + na + − + + − na
M. Priolo et al. / Gene 511 (2012) 103–105
hand X rays. There was moderate-to-severe mental retardation. Her weight was 21 kg (97° centile), height 112.5 cm (>97° centile), OFC 53.8 cm (90° centile). She also presented few sleep apnea. On the basis of the clinical features we suspected a mild form of Marshall–Smith syndrome. We re-evaluated the proband at 8 years and confirmed the same objective features. Her weight was 41 kg (>97° centile) and height 147.5 cm (>97° centile), OFC 56.5 cm (>97° centile). In the meantime, scoliosis and an unusual behavior characterized by anxiety and tendency to repetitive activities have been reported. The patient underwent the following negative genetic analyses: CGH-array (60 K) which helped us to eventually exclude intragenic small deletions of the NSD1 gene, methylation analysis for Angelman/Prader–Willi region and subtelomeric microdeletion screening. 2.2. Mutation analysis Genomic DNA was isolated from peripheral blood leukocytes according to standard methods. Specific primers for PCR amplification of exons and splicing junctions of NFIX were designed as previously reported (Malan et al., 2010). PCR products were purified with QiaQuick purification kit method and directly sequenced on an automated sequencer (ABI 3500, Applied Biosystems) with the Dye Terminator method. 3. Results and discussion An in frame deletion of the NFIX gene has been identified. The deletion of 21 nucleotides in exon 2 (c. 157_177delGAGCGGGCGGTGAAGGA CGAG) is predicted to cause a protein lacking of 7 amino acids (from residue 53 to residue 59 [delERAVKDE]) proximal to the NFIX α-helical region of DNA-binding domain (the lysine helix). Both parent are negative for the same variation that has not been detected in 100 normal controls. These residues are evolutionally conserved amino acids (Stefancsik and Sarkar, 2003). NFI proteins form homo- or heterodimers and bind to the palindromic DNA consensus sequence through its N-terminal DNA-binding/ dimerization domain. Point mutations in DNA-binding/dimerization domain of NFI proteins have been shown to cause loss of dimerization, DNA-binding and replication activities (Stefancsik and Sarkar, 2003). Specifically, the N-terminal 80 amino acids have also been implicated in physically contacting DNA (Dekker et al., 1996). The deleted residues are located at the proximal end of the putative lysine helix forming the MAD homology 1 (MH1) domain (Osada et al., 1999) and are conserved in both human and other species NFI proteins (Stefancsik and Sarkar, 2003) (Fig. 1). This region presents a high homology with the SMAD proteins MH1 domain. Previous studies have proved that somatic mutations of SMAD4 gene located in this same region and nearby the lysine α-helix region completely abolish its capacity to bind DNA (Jones and Kern, 2000). The deleted sequence (ERAVKDE) represents an alternatively positive and negative charged sequence of amino acids which are likely to influence tertiary structure of the protein. Moreover, one of the missense mutation (L60P) reported by Yoneda et al. (Stefancsik and Sarkar, 2003) is immediately subsequent to the observed deleted strand. Yoneda et al. have addressed the question whether patients with NFIX mutations should have been classified as Sotos-Like or Marshall–
105
Smith syndrome (Yoneda et al., 2012). Both syndromes share many common features but they are different in many aspect; in particular Marshall–Smith syndrome has proptosis, underdeveloped midface and prominent premaxilla (Adam et al., 2005; Shaw et al., 2010). The same authors state that one of their patients has some characteristic features of Marshall–Smith syndrome such as everted lower lip, short nose and midface hypoplasia. In this patient the L60P missense mutation was found. The authors concluded that missense mutations in the DNA binding/ dimerization domain, are more likely to cause Sotos-like syndrome, while according to Malan et al., Marshall–Smith syndrome is due to mutations with dominant negative effect. Malan et al. reported two patients with Sotos-like syndrome with deletion of the entire NFIX gene and one with a nonsense mutation (Q190X) in the N terminal domain of the protein whose clinical features included behavioral anomalies such as anxiety and autistic traits (Malan et al., 2010). Our patient seems to be more similar to Malan's patients with respect to the other two reported by Yoneda et al. These latter authors suggested that the autistic traits are more likely to be caused by haploinsufficiency of NFIX, but not by missense mutations. Moreover, our proband shows blue sclerae, mild proptosis, slight bullet-shaped phalanges and few sleep apneae spontaneously resolved with age (Table 1). These features are more specific of the Marshall–Smith syndrome than the Sotos-like syndrome: this might be explained by the fact that the mutation observed is strongly likely to cause a real haploinsufficiency of NFIX protein abolishing its property to bind DNA and determining a phenotype sharing Sotos-like features and very mild characteristics of the Marshall–Smith syndrome. In conclusion this report further confirms that there is a high genotype-phenotype correlation in NFIX mutations, being patients with deletion or non sense mutations more severely affected. Moreover, it might further help in elucidating NFIX different domains functions. References Adam, M.P., et al., 2005. Marshall–Smith syndrome: natural history and evidence of an osteochondrodysplasia with connective tissue abnormalities. Am. J. Med. Genet. A. 137, 117–124. Dekker, J., Van Oosterhout, J.A.W.M., Van der Vliet, P.C., 1996. Two regions within the dna binding domain of nuclear Factor i interact with DNA and stimulate adenovirus DNA replication independently. Mol. Cell. Biol. 16, 4073–4080. Jones, J.B., Kern, S.E., 2000. Functional mapping of the MH1 DNA-binding domain of DPC4/SMAD4. Nucleic Acids Res. 28, 2363–2368. Leventopoulos, G., et al., 2009. A clinical study of Sotos syndrome patients with review of the literature. Pediatr. Neurol. 40, 357–364. Malan, V., et al., 2010. Distinct effects of allelic NFIX mutations on nonsense-mediated mRNA decay engender either a Sotos-like or a Marshall–Smith syndrome. Am. J. Hum. Genet. 87, 189–198. Neri, G., Gurrieri, F., Zanni, G., Lin, A., 1998. Clinical and molecular aspects of the Simpson–Golabi–Behmel syndrome. Am. J. Med. Genet. 79, 279–283. Osada, S., et al., 1999. Expression, DNA-binding specificity and transcriptional regulation of nuclear factor 1 family proteins from rat. Biochem. J. 342, 189–198. Shaw, A.C., et al., 2010. Phenotype and natural history in Marshall–Smith syndrome. Am. J. Med. Genet. A 152, 2714–2726. Stefancsik, R., Sarkar, S., 2003. Relationship between the DNA binding domains of SMAD and NFI/CTF transcription factors defines a new superfamily of genes. DNA Seq. 14, 233–239. Tatton-Brown, K., Rahman, N., 2007. Sotos syndrome. Eur. J. Hum. Genet. 15, 264–271. Visser, R., Matsumoto, N., 2003. Genetics of Sotos syndrome. Curr. Opin. Pediatr. 15, 598–606. Weksberg, R., Smith, A.C., Squire, J., Sadowski, P., 2003. Beckwith–Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum. Mol. Genet. 12, R61–R68. Yoneda, Y., et al., 2012. Missense mutations in the DNA-binding/dimerization domain of NFIX cause Sotos-like features. J. Hum. Genet. 57, 207–211.