A novel mutation in SMOC1 and variable phenotypic ...

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Aug 12, 2017 - Cryptorchidism. a b s t r a c t. Waardenburg anophthalmia syndrome (WAS) is a rare disorder that mostly affects the eyes and distal limbs.

European Journal of Medical Genetics 60 (2017) 578e582

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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg

A novel mutation in SMOC1 and variable phenotypic expression in two patients with Waardenburg anophthalmia syndrome Javad Jamshidi a, b, Shokoufeh Abdollahi c, Hamid Ghaedi d, Elham Alehabib d, Abbas Tafakhori e, Somayeh Alinaghi d, Marjan Chapi d, Amir Hossein Johari d, Hossein Darvish d, * a

Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran Department of Medical Genetics, Fasa University of Medical Sciences, Fasa, Iran Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Neurology, School of Medicine, Imam Khomeini Hospital and Iranian Center of Neurological Research, Tehran University of Medical Sciences, Tehran, Iran b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 March 2017 Received in revised form 9 August 2017 Accepted 10 August 2017 Available online 12 August 2017

Waardenburg anophthalmia syndrome (WAS) is a rare disorder that mostly affects the eyes and distal limbs. In the current study we reported two Iranian patients with WAS. The first case was a 26-year-old girl with unilateral anophthalmia, bilateral camptodactyly and clinodactyly in her hands, oligodactly in her left foot and syndactyly of the second to fifth toes in her right foot. She also had severe hearing loss in both ears. The second case was a 12-year-old boy with bilateral anophthalmia, camptodactyly in his right hand, oligodactyly in his foot, clubfoot, and cryptorchidism. Both patients were mentally normal. To detect the causative mutation all exons and exon-intron boundaries of SMOC1 gene were sequenced in patients and other normal family members. We found a homozygous missense mutation (NM_001034852.2(SMOC1):c.367T > C) in exon 3 of SMOC1 gene in both patients. As the mutation segregated with the disease in the family, it should be the causative mutation. Our study extended the mutation spectrum of SMOC1 gene related to WAS. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Waardenburg anophthalmia SMOC1 Oligodactyly Iranian Hearing loss Cryptorchidism

1. Introduction Waardenburg anophthalmia syndrome (WAS) is a rare syndrome which mostly affects two unrelated organs, eyes and distal limbs (Tekin et al., 2000). It is also known as microphthalmia with limb anomalies and ophthalmo-acromelic syndrome. The syndrome was first presented by Waardenburg in 1961 by reporting two unrelated families with four affected patients, consisting three females and a male with a rather variable phenotype (Waardenburg, 1961). There are several abnormalities reported in patients with WAS such as anophthalmia, hand and food malformation, especially the presence of only 4 toes as a distinctive foot abnormality, facial abnormalities as well as mental retardation in some cases (Cogulu et al., 2000; Quarrell, 1995; Rainger et al., 2011).

Mutations in SPARC-Related Modular Calcium-Binding Protein 1 Gene, SMOC1, was first reported to be the genetic cause of WAS in 2011 (Abouzeid et al., 2011; Okada et al., 2011). This gene encodes a secreted protein with an important role in ocular and limb development (Okada et al., 2011). Further studies also found SMOC1 mutations in patients with WAS (Rainger et al., 2011), although the genetic heterogeneity of the disease should be also considered. About 54 WAS cases have been reported to date from different ethnicities. In the current study we reported the causative mutation in two Iranian patients with WAS along with their detailed clinical symptoms. This would be the first report of WAS from Iran.

2. Patient data * Corresponding author. Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, Tehran, Iran. E-mail address: [email protected] (H. Darvish). http://dx.doi.org/10.1016/j.ejmg.2017.08.006 1769-7212/© 2017 Elsevier Masson SAS. All rights reserved.

Two patients with WAS from northern cities of Iran were included in our study. The patients were second cousins and their both parents were first cousins (Fig. 1).

J. Jamshidi et al. / European Journal of Medical Genetics 60 (2017) 578e582


Fig. 1. Pedigree of the family and partial sequence of SMOC1 exon 3 where the mutation had occurred. Filled symbols represent individuals with WAS. The arrow above the partial sequence of SMOC1 is the site of mutation.

2.1. Patient 1 Patient IV.2 was a 26-year-old Iranian girl. She was born following an uneventful pregnancy and delivery. Her birthweight was 2.7 kg (z-score: 1.23), her length was 47 cm (z-score: 1.15) and her head circumstance was 33 cm (z-score: 0.74). At the age of 26, she had a relatively short stature with the height of 153 cm (z-score: 1.55). She had unilateral anophthalmia; her right eyeball was absent (Fig. 2A). She had bilateral camptodactyly and clinodactyly in her hands (Fig. 2B). She had oligodactly (absent of the second toe) in her left foot. Her right foot showed syndactyly of second to fifth toes (Fig. 2C). She was diagnosed with severe hearing loss in both ears. This patient was mentally normal.

undescended right testis (cryptorchidism). His intellectual activity was normal with a good verbal contact.

3. Methods Written informed consent was signed by the girl and the parents of the boy before the clinical and laboratory examinations. The ethics committee at Fasa University of Medical Sciences approved this study (IR.FUMS.REC.1395.113). The investigations were conducted according to the codes expressed in the Declaration of Helsinki.

2.2. Patient 2

3.1. Genetic analysis

He was a 12-year-old Iranian boy (IV.1). His mother's pregnancy and the screening tests were both normal. At birth his weight was 2.9 kg (z-score: 0.96), his length was 49 cm (z-score: 0.47) and his head circumstance was 35 cm (z-score: 0.42). His height at the age of 12 was 141 (z-score: 1.5). The patient had bilateral anophthalmia with normally developed eyelashes (Fig. 2D). He had fifth finger camptodactyly in his right hand (Fig. 2E). His gait was not normal due to bilateral clubfoot deformity. He had bilateral oligodactly (absent of the second toe) in his feet. A notable finding was two fold lines in front of his calf (Fig. 2F). He also had

We extracted DNA from peripheral blood of the patients and other family members, using a standard salting out method. As the main candidate gene for the disease is SMOC1, all exons and exonintron boundaries of SMOC1 gene were amplified using PCR. The PCR products then were sequenced on an ABI3130 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The results were analysed using Sequencher 5.0 software (Gene Codes Corporation, Ann Arbor, MI, USA). We searched in the dbSNP, ExAC, and human gene mutation database (HGMD) to exclude the normal variations and confirm the novelty of the mutation.


J. Jamshidi et al. / European Journal of Medical Genetics 60 (2017) 578e582

Fig. 2. Clinical features related to WAS in the two patients. Unilateral anophthalmia (A), hands and finger abnormality (B), and foot and toe abnormality (C) in IV.2. Bilateral anophthalmia (D), hands and fingers abnormality (E) and foot and toe abnormality (F) in IV.1.

3.2. In silico functional analysis of Ser123Pro mutation To obtain a consensus pathogenicity estimate for the p.Ser123Pro change, a number of independent prediction algorithms including SIFT, PolyPhen-2, M-CAP, Mutation taster, CADD score and Grantham score were used. Further, for visualization propose and to obtain SMOC1 tertiary structure, we used the Protein Homology/analogY Recognition Engine V 2.0 (Phyre2, http://www.sbg. bio.ic.ac.uk/phyre2) web tool. Moreover, three dimensional (3D) analyses and p.Ser123Pro modeled on SMOC1 were generated and analysed through project HOPE (http://www.cmbi.ru.nl/hope).

that the variant is deleterious and does adversely disrupt the protein function. It was predicted that the mutated residue to be located within a domain, annotated in UniProt as Thyroglobulin type-1. The mutation introduces an amino acid with different properties, which can disturb this domain and consequently abolish its function. The wildtype and mutant amino acids differ in size. The mutant residue is bigger than the wild-type. The residue is located on the surface of the protein, therefore its mutation can disturb interactions with other molecules or other parts of the protein. Fig. 3 provides graphical representation of the NM_001034852.2:p.(Ser123Pro) mutation modeled on SMOC1 protein.

4. Results 5. Discussion 4.1. Mutation analysis Sequencing analysis revealed a homozygote missense mutation in exon 3 of SMOC1 gene (NM_001034852.2:p.(Ser123Pro)) in both patients (IV.1 and IV.2). The mutation replaces a thymine with cytosine and consequently the substitution of proline for serine. Both parents of affected patients were carrier of the same mutation in heterozygote state. Fig. 1 illustrates the partial sequence of SMOC1 exon 3 where the mutation had occurred along with the pedigree of the family. The mutation segregated with the disease in the family. The variant was submitted in ClinVar (http://www.ncbi. nlm.nih.gov/clinvar) with the accession number SCV000579456 (https://www.ncbi.nlm.nih.gov/clinvar/variation/427815/). 4.2. Bioinformatics analysis NM_001034852.2(SMOC1):c.367T > C was neither found in ExAc nor in SNP databases. The serine to proline change was assessed by different algorithms (Table 1) and the results suggest

In the current study we reported two patients; a girl and a boy with WAS. The patients were second cousins with consanguineous parents. A missenses mutation was detected in exon three of SMOC1 gene in both patients, which was interpreted as the cause of their disease. To the best of our knowledge, this is the first report of WAS from Iran. There was a variable phenotype expression between our two patients despite the same mutation. The variable phenotype has been reported before, even in a single family (Hamanoue et al., 2009; Richieri-Costa et al., 1983). The most obvious dissimilar phenotype in our two cases was the bilateral anophthalmia in one patient and unilateral in the other. As the mutation in both patients was the same, it can be concluded that some other genes can modify the role of SMOC1 in eye development during the embryonic growth in human. The other clinical differences could be due to other modifier genes, which can affect organogenesis. However, the effect of environmental factors during the embryonic growth could not be excluded.

Table 1 Summary of deleteriousness prediction methods analysed in this study. SIFT score (prediction)

PolyPhen-2 score (prediction)

M-CAP score (prediction)


CADD score

Grantham Score


0.63 (Possibly Damaging)

0.04 (Possibly Pathogenic)

Disease Causing



J. Jamshidi et al. / European Journal of Medical Genetics 60 (2017) 578e582


We could not study the function of this mutation in vitro to confirm the destructive effects of the mutation in SMOC1 gene. Nevertheless, bioinformatics analysis showed that this mutation is potentially deleterious and disrupts the protein function. SMOC1 protein has two thyroglobulin domains, which are important in its functions. The mutation in our patients occurred in 123th amino acid of the protein, which is located in the first thyroglobulin domain of the gene. Mutations in the second thyroglobulin domain of SMOC1 have been previously reported to be associated with WAS (Rainger et al., 2011). As the homozygous NM_001034852.2(SMOC1):c.367T > C mutation is segregated with the disease in the family, this mutation most probably caused the disease. Our study provided further evidence for involvement of SMOC1 gene in WAS. Our report also extended the mutation spectrum of this gene related to WAS. Disclosure of interest The authors report no conflicts of interest. Fig. 3. Overview of the protein and mutation. A. representation of smoc1 protein in ribbon-presentation. The protein is colored grey, the side chain of the mutated residue is colored magenta and shown as small balls. B, C and D provide close-up of the mutation. The side chains of both the wild-type and the mutant residue are shown and colored green and red respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Acknowledgements We would like to sincerely thank the patients and their families for their kind participation in the study. Funding

We observed the two rare phenotypes of WAS -hearing loss and cryptorchidism-in our patients. Hearing loss has been reported in a few cases before (Galasso et al., 2007; Pallotta and Dallapiccola, 1984) and our female patient showed a severe form of it. It has been suggested that this could be due to impairment in the cochclear cells and the auditory nerve (Galasso et al., 2007). Alternatively, hearing problems in WAS patients may be related to middle ear bones malfunction, as it is known that SMOC1 has a role in bone development (Choi et al., 2010). Cryptorchidism was reported in two cases with WAS (Garavelli et al., 2006; Hamanoue et al., 2009). Our male patient in the current study also had undescended right testis. It has been shown that SMOC1 participates in cell typespecific differentiation and intercellular signaling during gonad development (Pazin and Albrecht, 2009). This observation can justify the incidence of cryptorchidism in males with WAS. Another notable rare phenotype in our male patient was two fold lines in front of his calf. This phenotype was also evident in patients studied by Ullah et al. in Pakistan (Ullah et al., 2017). This phenotype can be a result of a mild cell migration or differentiation aberration during the embryonic development; although for a more precise conclusion a histopathologic examination is required. Most previous reports of WAS have not described the causative mutations, since the gene defect was not known until 2011. Because of this issue, it is not possible to know that the previous reports or even the original report of Wanderburg were due to the mutations in SMOC1 or other genes. Moreover, genetic heterogeneity is described for WAS. Therefore, some variable phenotypes between the families could be owing to different causative genes (Kondo et al., 2013). The mutation was the same in the two patients, probably inherited to both of them from a common ancestor. As WAS is a rare condition and has an autosomal recessive pattern of inheritance, it is not surprising that most reported cases have consanguineous parents (about 83%). A considerable number of cases of WAS have been reported from Turkey (12 out of 54 reported cases, about 22%) and Arabic countries where the consanguinity of parents is common. The patients in our study also have consanguineous parents.

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