Clin Genet 2016 Printed in Singapore. All rights reserved
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12700
Short Report
Exome sequencing revealed a novel biallelic deletion in the DCAF17 gene underlying Woodhouse Sakati syndrome Ali R.H., Shah K., Nasir A., Steyaert W., Coucke P.J., Ahmad W. Exome sequencing revealed a novel biallelic deletion in the DCAF17 gene underlying Woodhouse Sakati syndrome. Clin Genet 2016. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 Woodhouse Sakati syndrome (WSS, MIM 241080) is a rare autosomal recessive genetic condition characterized by alopecia, hypogonadism, hearing impairment, diabetes mellitus, learning disabilities and extrapydamidal manifestations. Sequence variants in the gene DCAF17, encoding nucleolar substrate receptor, were identified as the underlying cause of inherited WSS. Considerable phenotypic heterogeneity exists in WSS with regard to severity, organs involvement and age of onset, both in inter-familial and intra-familial cases. In this study, the genetic characterization of a consanguineous pedigree showing mild features of WSS was performed, followed by structural analysis of truncated protein. Exome sequencing identified a novel single base deletion variant (c.270delA; K90Nfs8*) in third exon of the gene DCAF17 (RefSeq; NM_025000), resulting in a truncated protein. Structural analysis of truncated DCAF17 revealed absence of amino acid residues crucial for interaction with DDB1. Taken together, the data confirmed the single base pair deletion as the underlying cause of this second report of WSS from Pakistan. This signifies the vital yet unexplored role of DCAF17 both in development and maintenance of adult tissues homeostasis. Conflict of interest
None declared.
Woodhouse Sakati syndrome (WSS) is a rare, genetically homogenous multisystemic disorder, invariably presented with frontotemporal alopecia, hypogonadism and decreased levels of IGF-1 (1). A myriad of associated abnormalities including neuroectodermal manifestations, diabetes, endocrine findings, extrapyramidal signs, keratoconus, syndactyly of hands and feet, progeria, thymus and cardiac anomalies, anodontia, polyneuropathy and seizures were reported in various combinations in WSS patients (2–4). Alazami et al. (5) mapped the WSS locus on chromosome 2q22.3–2q35 in several Saudi consanguineous families showing typical features of WSS and identified a biallelic founder variant in gene C2orf37 (now
R.H. Alia,b , K. Shaha , A. Nasirc , W. Steyaertb , P.J. Couckeb and W. Ahmada a Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan, b Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, and c Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Mardan, Pakistan
Key words: alopecia – CUL4-DDB1 – DCAF17 – hypogonadism – IGF-1 – Woodhouse Sakati syndrome Corresponding author: Wasim Ahmad, PhD, Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Tel: +92 51 9064 3003; fax: +92 51 9064 3003; e-mail: [email protected] Received 4 October 2015, revised and accepted for publication 7 November 2015
known as DCAF17) encoding a nucleolar protein. The gene shows expression in brain, skin and liver that correlates with the organ involvement in WSS patients. Since first reported in 1983, nearly 30 families with 76 patients were studied, but the exact pathology is not revealed, owing to poor knowledge of DCAF17 functional aspects. Substantial clues regarding DCAF17 functioning as a substrate co-receptor for one of the largest ubiquitin ligase complex Cul4-DDB1 (6, 7), began to highlight the hidden aspects of nucleolus as a caretaker of normal development and post-development maintenance, as evident from the pleiotropic manifestation in WSS patients with mutant DCAF17. Interestingly, all of the nine variants including nonsense, splicing and frameshifts
1
Ali et al. reported to date in gene DCAF17, results in a truncated non-functional protein product. In this study, a four-generation consanguineous Pakistani family presenting mild features of WSS was genetically evaluated. Exome sequencing identified a novel homozygous single base pair deletion (c.270delA; K90Nfs8*) in exon 3 of the gene DCAF17 in all affected members of the pedigree. Protein modeling of DCAF17 and subsequent docking analysis revealed its interaction with DDB1 in CUL4-DDB1 complex, conferred by specific amino acid residues. Materials and methods
(http://www-genome.wi.mit.edu/genome_software/ other/primer3.html). PCR recipe and thermocycling conditions for exon-specific Sanger dideoxy chain termination sequencing were followed as described earlier (9). The deletion variant was identified by BioEdit sequence alignment editor version 6.0.7 (http://www. mbio.ncsu.edu/BioEdit/bioedit.html), using the standard sequence of DCAF17 from UCSC genome browser. The ClinVar accession number for the pathogenic variant is SCV000246162. Protein modeling
In this study, a four-generation consanguineous pedigree of Pakistani origin, inheriting an autosomal recessive form of WSS, was ascertained from an isolated region in Khyber Pakthtunkhwa (KPK) province of Pakistan. The family members were briefed about objectives of the study. Both affected and unaffected members of the family and normal controls were informed about procedures to conduct genetic investigation and approval of the study from the institutional review board (IRB) of Quaid-i-Azam University, Islamabad, Pakistan. Blood samples were collected from the family members after obtaining written consent on ethical committee approved consent form. Permission to undertake the study was obtained from the IRB of Quaid-i-Azam University, Islamabad, Pakistan.
Homology modeling techniques were used to construct the three-dimensional (3D) structure of human DCAF17. The amino acid sequence of DCAF17 protein was retrieved from the NCBI database (http://www.ncbi. nlm.nih.gov/), imported to pBlast search against Protein Data Bank (PDB). The model was built for normal and mutated DCAF17 protein by Molecular Operating Environment using crystallographic structure of Escherichia coli Pol II (PDB code: 3K5L) as a template. The model was visually inspected using PYMOL viewer (http://www.pymol.org), and Ramachandran plot and ERRAT tools (10) were utilized to validate the predicted 3D models. To investigate interaction between wild and mutant DCAF17 with DDB1–CUL4A ubiquitin ligase complex was investigated using ZDOCK server (11). Docking was carried out using previously explained protocol (12).
DNA extraction and exome sequencing
Results
Research samples and ethical approval
Blood samples from four affected (IV-1, IV-2, IV-5 and IV-6) and five unaffected individuals (III-1, III-3, IV-3, IV-4 and IV-7) of the family were collected in ethylenediaminetetraacetic acid (EDTA) containing vacutainer sets (BD, Franklin Lakes, NJ). Genomic DNA from the blood samples was extracted using GenElute™ Blood Genomic DNA Kit (Sigma-Aldrich, St. Loius, Missouri, MO). To quantify DNA, Nanodrop-1000 spectrophotometer (Thermal Scientific, Wilmington, DC), measuring optical density at 260 nm, was used. DNA of single affected individual (IV-5) was processed for whole exome sequencing. The exome facility available at the Center for Medical Genetics Ghent (CMGG), Belgium was used. The exome capture was carried out using the SureSelectXT Human All Exon V5 Enrichment Kit (Agilent, Santa Clara, CA) and sequencing was performed on a NextSeq 500 platform (Illumina, San Diego, CA) with paired-end 150-bp reads according to accompanying manual. The exome data generated were analyzed using the same procedure described previously (8). Variant analysis
A single primer pair to PCR-amplify a homozygous single base pair deletion variant (c.270delA) was designed using online version of primer3 program
2
Clinical presentation
Clinical examinations of affected members presented sparse scalp hairs, with prominent involvement of frontal and temporal regions (Fig. 1b,d,e). Eyebrows and eyelashes were sparse and less dense. Affected male members were devoid of beard, mustache (Fig. 1b,d), pubic and axillary hairs. Two affected members (IV-1 and IV-5) had underdeveloped genitalia, and in the third affected member (IV-6), a condition of hypogonadism was established through laboratory reports (Table 1). Some of the previously reported features including facial abnormalities, intellectual disability and extrapyramidal signs, associated with WSS patients, were not observed in any affected member of the present family. An affected individual (IV-1) is working as a shopkeeper and two other affected individuals (IV-5 and IV-6) are studying in a local school revealing a normal social life, intellect and ability to respond to daily circumstances and events in a normal manner. In two affected members (IV-1 and IV-5), magnetic resonance imaging (MRI) scans of brain confirmed a healthy normal brain. Other abnormalities including ocular, dental, skeletal, cutaneous and cardiovascular were not identified in the present family. The obligate heterozygous carriers were phenotypically normal and indifferent from genotypically normal individuals.
A biallelic deletion variant in DCAF17, causing familial WSS
Fig. 1. Pedigree of the Pakistani family segregating Woodhouse Sakati syndrome (WSS) in an autosomal recessive manner. Male individuals are represented by squares and females by circles. Double lines are indicative of a consanguineous union. Clear symbols represent unaffected while filled symbols represent affected individuals. Symbols with asterisks indicate individuals who were clinically and genetically analyzed (a). Clinical manifestations of WSS. An affected individual (IV-1) presenting loss of most of the facial hairs. Note the presence of very few eyebrows and a prominent forehead (b), diffuse hypotrichosis on scalp in the same individual (c), lateral view of head and part of face in affected individuals (IV-5, IV-6) depicting characteristic hypotrichosis on right temporal region of skull. Rest of scalp hair is also unusual both in terms of density and length (d, e). Sequence chromatograms of a novel out of frame deleted variant (c.270delA; K90Nfs8*) in affected members (f), heterozygous carriers (g) and normal individuals (h). The exact position of deleted nucleotide is pointed by an arrow in panel (f).
Laboratory tests revealed perturbed levels of glycated hemoglobin (hbA1C), IGF-1, PRL in an affected member (IV-1). High levels of LH and FSH, and low estradiol contents undoubtedly confirmed hypogonadotropic hypogonadism in an affected female member (IV-6). Another affected member (IV-5) was found to have high levels of hbA1C (Table 1).
Variant identification and segregation analysis
Analysis of the exome data identified 25 pure homozygous variants. Scrutiny of the genes having the variants was performed using OMIM. In addition, available genetic, functional and expression data of the genes were analyzed for each variant containing gene. Single base pair deletion (270delA) in the DCAF17 gene
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Ali et al. Table 1. Clinical and laboratory attributes of affected individuals Affected Individuals
Sex Age (years) Height (cm) Weight (kg) Ectodermal appendages Alopecia Adontia Premature aged appearance Ocular findings Keratoconus Neurological Manifestations Deafness MRI brain Cognitive impairment Extrapyramidal features Dystonia Blepharospasm Choreoathetosis Dysarthria Dysphagia Spastic quadriplegia Endocrinal issues Hypogonadism Failure of secondary sexual characters development Facial deformities Triangular elongated lower face Hypertelorism Prominent nasal bridge Laboratory testsa IGF-1 (ng/μl) LH (mIU/ml) FSH (mIU/ml) PRL (ng/dl) TSH (uIU/ml) Estradiol (pg/ml) Testosterone (ng/ml) hbA1C (%)
IV-1
IV-2
IV-5
IV-6
Male 50 164.5 60
Female 48 160.4 55
Male 14 167.64 35
Female 13 164.5 30
+ − +
+ − +
+ − −
+ − −
−
−
−
−
+ − − − − − − − − −
+ − − − − − − − − −
− − − − − − − − − −
− − − − − − − − − −
+ +
+ +
+ +
+ N/A
− − −
− − −
− − −
− − −
53 NA NA 578.8 2.64 NA 3.39 6.2%
NA NA NA NA NA NA NA NA
177 NA NA 80.35 3.98 NA 9.50 6.0%
NA 17.96 99.92 35.02 4.51 55.0 NA 5.7%
+, presence of feature; −, absence of sign; MRI, medical resonance imaging; NA, not available. a Reference range of laboratory tests; IGF-1: males, 50 years (57–246 ng/μl), 14 years (102–520 ng/μl); LH: females, follicular (3.9–12.0 mIU/ml), mid-cycle (2.9–9.0 mIU/ml), luteal (1.5–7.0 mIU/ml); FSH: females, follicular (1.5–8.0 mIU/ml), mid-cycle (2.0–8.0 mIU/ml), luteal (0.2–6.0 mIU/ml); PRL: males (42.5–414 ng/dl), females (51.0–580 ng/dl); TSH: (0.35–5.29 uIU/ml); Estradiol: females, follicular (57–227 pg/ml), mid-cycle (127–476 pg/ml), luteal (77–277 pg/ml); Testosterone, males (1.95–11.38 ng/ml); hbA1C, non-diabetic level (8.5%).
representing an out of frame deletion was considered as the most potential candidate consistent with previous reports of causing the symptoms observed in our family. Sanger sequencing in all available DNA samples revealed homozygous deletion of c.270A in affected (IV-1, IV-2, IV-5 and IV-6) and heterozygous in the parents (IIII-1 and III-3) and two normal siblings (IV-3 and IV-7), and wild type nucleotide sequence in another normal individual (IV-4). Absence of the deletion variant, identified here, in available databases of genetic variations (dbSNP, EVS, EXAC genome browser) and in 80 ethnically matched control individuals of Pakistani origin,
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positively concluded of accepting pathogenicity caused by single base pair deletion in setting up syndromic phenotype in our family. The online available mutation effect prediction tool SIFT MutationTaster (http://www.mutationtaster.org/index.html) scores the out of frame deletion variant as pathogenic with a protein product having 96 amino acids when compared with 520 amino acids of its normal counterpart. Bioinformatics analysis
The 3D models of normal and mutated DCAF17 were predicted using homology modeling and further
A biallelic deletion variant in DCAF17, causing familial WSS
Fig. 2. Visualization of best docked DCAF17-DDB1 complex. Normal DCAF17 illustrated by cyan color ribbons (a). Surface view of normal DCAF17-DDB1 interaction (b), truncated DCAF17 docking with DDB1 protein (c) and mutated DCAF17-DDB1 complex interaction (d). Interacting residues are shown in atomic representations, and H-bonds are shown by dotted black lines.
characterized by online structure analysis tools. The quality of modeled structures was evaluated through Ramachandran plot and ERRAT. The stereochemical evaluation of backbone Psi and Phi dihedral angles for DCAF17 model revealed that 95.12% residues lie in favored regions. ERRAT measures the overall quality factor of non-bonded atomic interactions, and an accepted range of above 50 is considered for a high quality model. ERRAT scores were within the range of 77%. The data indicated that our predicted structures are of good quality and can be used for protein–protein docking studies. Molecular docking analysis revealed that binding of normal DCAF17–DDB1 occurs through 12 amino acids (Val9, Leu102, Trp103, Lys143, Ile144, Leu146, Tyr149,
Phe152, Arg153, Asp158, Val192 and Phe 193), with 10 amino acids (Met1, Ser2, Ile909, Phe949, Asp980, Thr984, Tyr985, Val995, Leu1052 and Asp1116) of DDB1 (Fig. 2a). In case of mutated DCAF17 (c.270delA; K90Nfs8*), only five residues (Arg12, Ser14, Arg16, Val40 and Arg56) were involved in interaction with four residues (Asp962, Asp963, Leu1017 and Asp1116) of DDB1 (Fig. 2c). Discussion
The present report described an inbred family presenting mild features of WSS. Clinical features such as profound alopecia, hypogonadism associated with abnormal levels of IGF-1, estradiol, FSH, PRL and hbA1C established
5
Ali et al. WSS diagnosis in the family. Absence of extrapyramidal features and cognitive impairment presents a unique finding of WSS in the family. Previously reported cases revealed the presence of extrapyramidal features (dystonia) in 42%, cognitive impairment in 87% and deafness in 76% WSS patients (1). Because of mutated DCAF17, involvement of genetic modifiers in influencing the development of phenotypic features in the family cannot be ruled out. Exome sequencing followed by validation by Sanger sequencing detected a novel single base pair deletion in exon 3 of the DCAF17 gene. The variant affected both major gene transcripts (NM_025000, NM_001164821), resulting in a truncated non-functional protein. To date, nine disease-causing truncating sequence variants including three nonsense, one deletion (13, 14), two frameshift deletions (5) and three splice-site variants (5, 9) in the gene DCAF17 have been reported in the families of diverse ethnicities. Length of the truncated proteins do not parallel the phenotypes reported in patients with WSS, suggesting a nonsense-mediated decay (NMD) of mRNAs irrespective of the position of the variant(s) in the gene. The cellular clearance of the truncated DCAF17 version through NMD probably rescues the unexpected outcomes of cellular events, which could be perturbed by inefficient interaction of the truncated protein. Two other anomalies including Shwachman–Diamond syndrome (OMIM; 260400) (15) and alopecia– neurologic defects–endocrinopathy syndrome (OMIM; 612079) (16) caused by variants in the nucleolar genes SBDS and RMB28 respectively showed compromised ribosomal biosynthesis and nucleolar hypersensitivity as reported in WSS. Alazami et al. (5) suggested a distinct mode of pathogenesis for WSS ascertained by assembly defects of nucleoli observed exclusively in this disorder. During analysis of CUL4-DDB1 ubiquitin ligase complex, C2orf37 was identified as a substrate co-receptor along with 60 other proteins containing WD40 repeats (6). The interacting proteins were collectively called DDB1-CUL4 associated factors (DCAF’s). C2orf37 being a member of DCAF family was named DCAF17 (7). Mutations in DDB1 and CUL4 are known to result in xeroderma pigmentosa (17) and various types of cancers (18), respectively, both of which are not characteristic of WSS. The frameshift deletion, identified in this study, is anticipated to form a truncated DCAF17 protein with missing domains and motifs necessary to interact with DDB1-CUL4 ubiquitin ligase complex and ultimate recruitment of substrates. The molecular docking analysis also suggests an atypical interaction between truncated DCAF17 and DDB1 carried out by an unusual profile of 9 amino acids when compared with their normal counterparts with 22 amino acids (Fig. 2a,c). Thus postulating an inaccurate and inefficient association between the truncated DCAF17 (acting as scaffold) and proteins destined to post-translational modification and/or degradation via DDB1-CUL4 for maintaining cellular homeostasis. The escaped proteins could be involved in regulating one of the several prominent
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roles associated with nucleoli including cell cycle, cellular aging, signal recognition particle biosynthesis, small-RNA processing, mRNA transport and even apoptosis (19–21). The combination of different organs and severity of features reported imply a possible disruption of multiple checkpoints needed for optimal cellular functioning, apoptosis being one of them as difference in expression of apoptosis genes was indeed observed between control and patient lymphoblasts (5). The aberrant apoptosis in part is also speculated to occur by impaired ribosomal biogenesis driven by a quantitative imbalance of ribosome precursors (15). However, unless functionally characterized, the underlying pathology is hard to interpret. Moreover, involvement of nucleolar proteins in pleiotropic phenotypes is suggestive of some vital yet unknown functions undertaken by this organelle, rather than the universal ribosome synthesis, that needs focus in near future to get insights of these new group of nucleolus-based pathologies, a mouse model could prove helpful in detailed unmasking of disruptive mechanisms. Acknowledgements We are grateful to the members of the family for their invaluable participation and co-operation. The study was funded by Higher Education Commission (HEC), Islamabad, Pakistan. R. H. A. was supported by International Research Support Initiative Program (IRSIP) to visit CMGG, Belgium from Higher Education Commission (HEC) Islamabad, Pakistan and HEC Indigenous PhD fellowship.
References 1. Agopiantz M, Corbonnois P, Sorlin A et al. Endocrine disorders in Woodhouse-Sakati syndrome: a systematic review of the literature. J Endocrinol Invest 2014: 37: 1–7. 2. Woodhouse NJ, Sakati NA. A syndrome of hypogonadism, alopecia, diabetes mellitus, mental retardation, deafness, and ECG abnormalities. J Med Genet 1983: 20: 216–219. 3. Al-Semari A, Bohlega S. Autosomal-recessive syndrome with alopecia, hypogonadism, progressive extra-pyramidal disorder, white matter disease, sensory neural deafness, diabetes mellitus, and low IGF1. Am J Med Genet A 2007: 143A: 149–160. 4. Al-Swailem SA, Al-Assiri AA, Al-Torbak AA. Woodhouse Sakati syndrome associated with bilateral keratoconus. Br J Ophthalmol 2006: 90: 116–117. 5. Alazami AM, Al-Saif A, Al-Semari A. Mutations in C2orf37, encoding a nucleolar protein, cause hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome. Am J Hum Genet 2008: 83: 684–691. 6. Angers S, Li T, Yi X, MacCoss MJ, Moon RT, Zheng N. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 2006: 443: 590–593. 7. Lee J, Zhou P. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase. Mol Cell 2007: 26: 775–780. 8. Hosen MJ, Van Nieuwerburgh F, Steyaert W. Efficiency of exome sequencing for the molecular diagnosis of pseudoxanthoma elasticum. J Invest Dermatol 2015: 135: 992–998. 9. Habib R, Basit S, Khan S, Khan MN, Ahmad W. A novel splice site mutation in gene C2orf37 underlying Woodhouse-Sakati syndrome (WSS) in a consanguineous family of Pakistani origin. Gene 2011: 490: 26–31. 10. Colovos C, Yeates TO. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 1993: 2: 1511–1519. 11. Chen R, Li L, Weng Z. ZDOCK: an initial-stage protein-docking algorithm. Proteins 2003: 52: 80–87.
A biallelic deletion variant in DCAF17, causing familial WSS 12. Junaid M, Muhseen ZT, Ullah A, Wadood A, Liu J, Zhang H. Molecular modeling and molecular dynamics simulation study of the human Rab9 and RhoBTB3 C-terminus complex. Bioinformation 2014: 10: 757–763. 13. Alazami AM, Schneider SA, Bonneau D et al. C2orf37 mutational spectrum in Woodhouse-Sakati syndrome patients. Clin Genet 2010: 78: 585–590. 14. Steindl K, Alazami AM, Bhatia KP et al. A novel C2orf37 mutation causes the first Italian cases of Woodhouse Sakati syndrome. Clin Genet 2010: 78: 594–597. 15. Ganapathi KA, Austin KM, Lee CS. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA. Blood 2007: 110: 1458–1465. 16. Nousbeck J, Spiegel R, Ishida-Yamamoto A. Alopecia, neurological defects, and endocrinopathy syndrome caused by decreased expression of RBM28, a nucleolar protein associated with ribosome biogenesis. Am J Hum Genet 2008: 82: 1114–1121.
17. Nichols AF, Itoh T, Graham JA, Liu W, Yamaizumi M, Linn S. Human damage-specific DNA-binding protein p48. Characterization of XPE mutations and regulation following UV irradiation. J Biol Chem 2000: 275: 21422–21428. 18. Hung MS, Mao JH, Xu Z. Cul4A is an oncogene in malignant pleural mesothelioma. J Cell Mol Med 2011: 15: 350–358. 19. Dousset T, Wang C, Verheggen C, Chen D, Hernandez-Verdun D, Huang S. Initiation of nucleolar assembly is independent of RNA polymerase I transcription. Mol Biol Cell 2000: 11: 2705–2717. 20. Kerr LE, Birse-Archbold JL, Short DM et al. Nucleophosmin is a novel Bax chaperone that regulates apoptotic cell death. Oncogene 2007: 26: 2554–2562. 21. Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI. The multifunctional nucleolus. Nat Rev Mol Cell Biol 2007: 80: 574–585.
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© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12700
Short Report
Exome sequencing revealed a novel biallelic deletion in the DCAF17 gene underlying Woodhouse Sakati syndrome Ali R.H., Shah K., Nasir A., Steyaert W., Coucke P.J., Ahmad W. Exome sequencing revealed a novel biallelic deletion in the DCAF17 gene underlying Woodhouse Sakati syndrome. Clin Genet 2016. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 Woodhouse Sakati syndrome (WSS, MIM 241080) is a rare autosomal recessive genetic condition characterized by alopecia, hypogonadism, hearing impairment, diabetes mellitus, learning disabilities and extrapydamidal manifestations. Sequence variants in the gene DCAF17, encoding nucleolar substrate receptor, were identified as the underlying cause of inherited WSS. Considerable phenotypic heterogeneity exists in WSS with regard to severity, organs involvement and age of onset, both in inter-familial and intra-familial cases. In this study, the genetic characterization of a consanguineous pedigree showing mild features of WSS was performed, followed by structural analysis of truncated protein. Exome sequencing identified a novel single base deletion variant (c.270delA; K90Nfs8*) in third exon of the gene DCAF17 (RefSeq; NM_025000), resulting in a truncated protein. Structural analysis of truncated DCAF17 revealed absence of amino acid residues crucial for interaction with DDB1. Taken together, the data confirmed the single base pair deletion as the underlying cause of this second report of WSS from Pakistan. This signifies the vital yet unexplored role of DCAF17 both in development and maintenance of adult tissues homeostasis. Conflict of interest
None declared.
Woodhouse Sakati syndrome (WSS) is a rare, genetically homogenous multisystemic disorder, invariably presented with frontotemporal alopecia, hypogonadism and decreased levels of IGF-1 (1). A myriad of associated abnormalities including neuroectodermal manifestations, diabetes, endocrine findings, extrapyramidal signs, keratoconus, syndactyly of hands and feet, progeria, thymus and cardiac anomalies, anodontia, polyneuropathy and seizures were reported in various combinations in WSS patients (2–4). Alazami et al. (5) mapped the WSS locus on chromosome 2q22.3–2q35 in several Saudi consanguineous families showing typical features of WSS and identified a biallelic founder variant in gene C2orf37 (now
R.H. Alia,b , K. Shaha , A. Nasirc , W. Steyaertb , P.J. Couckeb and W. Ahmada a Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan, b Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, and c Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Mardan, Pakistan
Key words: alopecia – CUL4-DDB1 – DCAF17 – hypogonadism – IGF-1 – Woodhouse Sakati syndrome Corresponding author: Wasim Ahmad, PhD, Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Tel: +92 51 9064 3003; fax: +92 51 9064 3003; e-mail: [email protected] Received 4 October 2015, revised and accepted for publication 7 November 2015
known as DCAF17) encoding a nucleolar protein. The gene shows expression in brain, skin and liver that correlates with the organ involvement in WSS patients. Since first reported in 1983, nearly 30 families with 76 patients were studied, but the exact pathology is not revealed, owing to poor knowledge of DCAF17 functional aspects. Substantial clues regarding DCAF17 functioning as a substrate co-receptor for one of the largest ubiquitin ligase complex Cul4-DDB1 (6, 7), began to highlight the hidden aspects of nucleolus as a caretaker of normal development and post-development maintenance, as evident from the pleiotropic manifestation in WSS patients with mutant DCAF17. Interestingly, all of the nine variants including nonsense, splicing and frameshifts
1
Ali et al. reported to date in gene DCAF17, results in a truncated non-functional protein product. In this study, a four-generation consanguineous Pakistani family presenting mild features of WSS was genetically evaluated. Exome sequencing identified a novel homozygous single base pair deletion (c.270delA; K90Nfs8*) in exon 3 of the gene DCAF17 in all affected members of the pedigree. Protein modeling of DCAF17 and subsequent docking analysis revealed its interaction with DDB1 in CUL4-DDB1 complex, conferred by specific amino acid residues. Materials and methods
(http://www-genome.wi.mit.edu/genome_software/ other/primer3.html). PCR recipe and thermocycling conditions for exon-specific Sanger dideoxy chain termination sequencing were followed as described earlier (9). The deletion variant was identified by BioEdit sequence alignment editor version 6.0.7 (http://www. mbio.ncsu.edu/BioEdit/bioedit.html), using the standard sequence of DCAF17 from UCSC genome browser. The ClinVar accession number for the pathogenic variant is SCV000246162. Protein modeling
In this study, a four-generation consanguineous pedigree of Pakistani origin, inheriting an autosomal recessive form of WSS, was ascertained from an isolated region in Khyber Pakthtunkhwa (KPK) province of Pakistan. The family members were briefed about objectives of the study. Both affected and unaffected members of the family and normal controls were informed about procedures to conduct genetic investigation and approval of the study from the institutional review board (IRB) of Quaid-i-Azam University, Islamabad, Pakistan. Blood samples were collected from the family members after obtaining written consent on ethical committee approved consent form. Permission to undertake the study was obtained from the IRB of Quaid-i-Azam University, Islamabad, Pakistan.
Homology modeling techniques were used to construct the three-dimensional (3D) structure of human DCAF17. The amino acid sequence of DCAF17 protein was retrieved from the NCBI database (http://www.ncbi. nlm.nih.gov/), imported to pBlast search against Protein Data Bank (PDB). The model was built for normal and mutated DCAF17 protein by Molecular Operating Environment using crystallographic structure of Escherichia coli Pol II (PDB code: 3K5L) as a template. The model was visually inspected using PYMOL viewer (http://www.pymol.org), and Ramachandran plot and ERRAT tools (10) were utilized to validate the predicted 3D models. To investigate interaction between wild and mutant DCAF17 with DDB1–CUL4A ubiquitin ligase complex was investigated using ZDOCK server (11). Docking was carried out using previously explained protocol (12).
DNA extraction and exome sequencing
Results
Research samples and ethical approval
Blood samples from four affected (IV-1, IV-2, IV-5 and IV-6) and five unaffected individuals (III-1, III-3, IV-3, IV-4 and IV-7) of the family were collected in ethylenediaminetetraacetic acid (EDTA) containing vacutainer sets (BD, Franklin Lakes, NJ). Genomic DNA from the blood samples was extracted using GenElute™ Blood Genomic DNA Kit (Sigma-Aldrich, St. Loius, Missouri, MO). To quantify DNA, Nanodrop-1000 spectrophotometer (Thermal Scientific, Wilmington, DC), measuring optical density at 260 nm, was used. DNA of single affected individual (IV-5) was processed for whole exome sequencing. The exome facility available at the Center for Medical Genetics Ghent (CMGG), Belgium was used. The exome capture was carried out using the SureSelectXT Human All Exon V5 Enrichment Kit (Agilent, Santa Clara, CA) and sequencing was performed on a NextSeq 500 platform (Illumina, San Diego, CA) with paired-end 150-bp reads according to accompanying manual. The exome data generated were analyzed using the same procedure described previously (8). Variant analysis
A single primer pair to PCR-amplify a homozygous single base pair deletion variant (c.270delA) was designed using online version of primer3 program
2
Clinical presentation
Clinical examinations of affected members presented sparse scalp hairs, with prominent involvement of frontal and temporal regions (Fig. 1b,d,e). Eyebrows and eyelashes were sparse and less dense. Affected male members were devoid of beard, mustache (Fig. 1b,d), pubic and axillary hairs. Two affected members (IV-1 and IV-5) had underdeveloped genitalia, and in the third affected member (IV-6), a condition of hypogonadism was established through laboratory reports (Table 1). Some of the previously reported features including facial abnormalities, intellectual disability and extrapyramidal signs, associated with WSS patients, were not observed in any affected member of the present family. An affected individual (IV-1) is working as a shopkeeper and two other affected individuals (IV-5 and IV-6) are studying in a local school revealing a normal social life, intellect and ability to respond to daily circumstances and events in a normal manner. In two affected members (IV-1 and IV-5), magnetic resonance imaging (MRI) scans of brain confirmed a healthy normal brain. Other abnormalities including ocular, dental, skeletal, cutaneous and cardiovascular were not identified in the present family. The obligate heterozygous carriers were phenotypically normal and indifferent from genotypically normal individuals.
A biallelic deletion variant in DCAF17, causing familial WSS
Fig. 1. Pedigree of the Pakistani family segregating Woodhouse Sakati syndrome (WSS) in an autosomal recessive manner. Male individuals are represented by squares and females by circles. Double lines are indicative of a consanguineous union. Clear symbols represent unaffected while filled symbols represent affected individuals. Symbols with asterisks indicate individuals who were clinically and genetically analyzed (a). Clinical manifestations of WSS. An affected individual (IV-1) presenting loss of most of the facial hairs. Note the presence of very few eyebrows and a prominent forehead (b), diffuse hypotrichosis on scalp in the same individual (c), lateral view of head and part of face in affected individuals (IV-5, IV-6) depicting characteristic hypotrichosis on right temporal region of skull. Rest of scalp hair is also unusual both in terms of density and length (d, e). Sequence chromatograms of a novel out of frame deleted variant (c.270delA; K90Nfs8*) in affected members (f), heterozygous carriers (g) and normal individuals (h). The exact position of deleted nucleotide is pointed by an arrow in panel (f).
Laboratory tests revealed perturbed levels of glycated hemoglobin (hbA1C), IGF-1, PRL in an affected member (IV-1). High levels of LH and FSH, and low estradiol contents undoubtedly confirmed hypogonadotropic hypogonadism in an affected female member (IV-6). Another affected member (IV-5) was found to have high levels of hbA1C (Table 1).
Variant identification and segregation analysis
Analysis of the exome data identified 25 pure homozygous variants. Scrutiny of the genes having the variants was performed using OMIM. In addition, available genetic, functional and expression data of the genes were analyzed for each variant containing gene. Single base pair deletion (270delA) in the DCAF17 gene
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Ali et al. Table 1. Clinical and laboratory attributes of affected individuals Affected Individuals
Sex Age (years) Height (cm) Weight (kg) Ectodermal appendages Alopecia Adontia Premature aged appearance Ocular findings Keratoconus Neurological Manifestations Deafness MRI brain Cognitive impairment Extrapyramidal features Dystonia Blepharospasm Choreoathetosis Dysarthria Dysphagia Spastic quadriplegia Endocrinal issues Hypogonadism Failure of secondary sexual characters development Facial deformities Triangular elongated lower face Hypertelorism Prominent nasal bridge Laboratory testsa IGF-1 (ng/μl) LH (mIU/ml) FSH (mIU/ml) PRL (ng/dl) TSH (uIU/ml) Estradiol (pg/ml) Testosterone (ng/ml) hbA1C (%)
IV-1
IV-2
IV-5
IV-6
Male 50 164.5 60
Female 48 160.4 55
Male 14 167.64 35
Female 13 164.5 30
+ − +
+ − +
+ − −
+ − −
−
−
−
−
+ − − − − − − − − −
+ − − − − − − − − −
− − − − − − − − − −
− − − − − − − − − −
+ +
+ +
+ +
+ N/A
− − −
− − −
− − −
− − −
53 NA NA 578.8 2.64 NA 3.39 6.2%
NA NA NA NA NA NA NA NA
177 NA NA 80.35 3.98 NA 9.50 6.0%
NA 17.96 99.92 35.02 4.51 55.0 NA 5.7%
+, presence of feature; −, absence of sign; MRI, medical resonance imaging; NA, not available. a Reference range of laboratory tests; IGF-1: males, 50 years (57–246 ng/μl), 14 years (102–520 ng/μl); LH: females, follicular (3.9–12.0 mIU/ml), mid-cycle (2.9–9.0 mIU/ml), luteal (1.5–7.0 mIU/ml); FSH: females, follicular (1.5–8.0 mIU/ml), mid-cycle (2.0–8.0 mIU/ml), luteal (0.2–6.0 mIU/ml); PRL: males (42.5–414 ng/dl), females (51.0–580 ng/dl); TSH: (0.35–5.29 uIU/ml); Estradiol: females, follicular (57–227 pg/ml), mid-cycle (127–476 pg/ml), luteal (77–277 pg/ml); Testosterone, males (1.95–11.38 ng/ml); hbA1C, non-diabetic level (8.5%).
representing an out of frame deletion was considered as the most potential candidate consistent with previous reports of causing the symptoms observed in our family. Sanger sequencing in all available DNA samples revealed homozygous deletion of c.270A in affected (IV-1, IV-2, IV-5 and IV-6) and heterozygous in the parents (IIII-1 and III-3) and two normal siblings (IV-3 and IV-7), and wild type nucleotide sequence in another normal individual (IV-4). Absence of the deletion variant, identified here, in available databases of genetic variations (dbSNP, EVS, EXAC genome browser) and in 80 ethnically matched control individuals of Pakistani origin,
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positively concluded of accepting pathogenicity caused by single base pair deletion in setting up syndromic phenotype in our family. The online available mutation effect prediction tool SIFT MutationTaster (http://www.mutationtaster.org/index.html) scores the out of frame deletion variant as pathogenic with a protein product having 96 amino acids when compared with 520 amino acids of its normal counterpart. Bioinformatics analysis
The 3D models of normal and mutated DCAF17 were predicted using homology modeling and further
A biallelic deletion variant in DCAF17, causing familial WSS
Fig. 2. Visualization of best docked DCAF17-DDB1 complex. Normal DCAF17 illustrated by cyan color ribbons (a). Surface view of normal DCAF17-DDB1 interaction (b), truncated DCAF17 docking with DDB1 protein (c) and mutated DCAF17-DDB1 complex interaction (d). Interacting residues are shown in atomic representations, and H-bonds are shown by dotted black lines.
characterized by online structure analysis tools. The quality of modeled structures was evaluated through Ramachandran plot and ERRAT. The stereochemical evaluation of backbone Psi and Phi dihedral angles for DCAF17 model revealed that 95.12% residues lie in favored regions. ERRAT measures the overall quality factor of non-bonded atomic interactions, and an accepted range of above 50 is considered for a high quality model. ERRAT scores were within the range of 77%. The data indicated that our predicted structures are of good quality and can be used for protein–protein docking studies. Molecular docking analysis revealed that binding of normal DCAF17–DDB1 occurs through 12 amino acids (Val9, Leu102, Trp103, Lys143, Ile144, Leu146, Tyr149,
Phe152, Arg153, Asp158, Val192 and Phe 193), with 10 amino acids (Met1, Ser2, Ile909, Phe949, Asp980, Thr984, Tyr985, Val995, Leu1052 and Asp1116) of DDB1 (Fig. 2a). In case of mutated DCAF17 (c.270delA; K90Nfs8*), only five residues (Arg12, Ser14, Arg16, Val40 and Arg56) were involved in interaction with four residues (Asp962, Asp963, Leu1017 and Asp1116) of DDB1 (Fig. 2c). Discussion
The present report described an inbred family presenting mild features of WSS. Clinical features such as profound alopecia, hypogonadism associated with abnormal levels of IGF-1, estradiol, FSH, PRL and hbA1C established
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Ali et al. WSS diagnosis in the family. Absence of extrapyramidal features and cognitive impairment presents a unique finding of WSS in the family. Previously reported cases revealed the presence of extrapyramidal features (dystonia) in 42%, cognitive impairment in 87% and deafness in 76% WSS patients (1). Because of mutated DCAF17, involvement of genetic modifiers in influencing the development of phenotypic features in the family cannot be ruled out. Exome sequencing followed by validation by Sanger sequencing detected a novel single base pair deletion in exon 3 of the DCAF17 gene. The variant affected both major gene transcripts (NM_025000, NM_001164821), resulting in a truncated non-functional protein. To date, nine disease-causing truncating sequence variants including three nonsense, one deletion (13, 14), two frameshift deletions (5) and three splice-site variants (5, 9) in the gene DCAF17 have been reported in the families of diverse ethnicities. Length of the truncated proteins do not parallel the phenotypes reported in patients with WSS, suggesting a nonsense-mediated decay (NMD) of mRNAs irrespective of the position of the variant(s) in the gene. The cellular clearance of the truncated DCAF17 version through NMD probably rescues the unexpected outcomes of cellular events, which could be perturbed by inefficient interaction of the truncated protein. Two other anomalies including Shwachman–Diamond syndrome (OMIM; 260400) (15) and alopecia– neurologic defects–endocrinopathy syndrome (OMIM; 612079) (16) caused by variants in the nucleolar genes SBDS and RMB28 respectively showed compromised ribosomal biosynthesis and nucleolar hypersensitivity as reported in WSS. Alazami et al. (5) suggested a distinct mode of pathogenesis for WSS ascertained by assembly defects of nucleoli observed exclusively in this disorder. During analysis of CUL4-DDB1 ubiquitin ligase complex, C2orf37 was identified as a substrate co-receptor along with 60 other proteins containing WD40 repeats (6). The interacting proteins were collectively called DDB1-CUL4 associated factors (DCAF’s). C2orf37 being a member of DCAF family was named DCAF17 (7). Mutations in DDB1 and CUL4 are known to result in xeroderma pigmentosa (17) and various types of cancers (18), respectively, both of which are not characteristic of WSS. The frameshift deletion, identified in this study, is anticipated to form a truncated DCAF17 protein with missing domains and motifs necessary to interact with DDB1-CUL4 ubiquitin ligase complex and ultimate recruitment of substrates. The molecular docking analysis also suggests an atypical interaction between truncated DCAF17 and DDB1 carried out by an unusual profile of 9 amino acids when compared with their normal counterparts with 22 amino acids (Fig. 2a,c). Thus postulating an inaccurate and inefficient association between the truncated DCAF17 (acting as scaffold) and proteins destined to post-translational modification and/or degradation via DDB1-CUL4 for maintaining cellular homeostasis. The escaped proteins could be involved in regulating one of the several prominent
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roles associated with nucleoli including cell cycle, cellular aging, signal recognition particle biosynthesis, small-RNA processing, mRNA transport and even apoptosis (19–21). The combination of different organs and severity of features reported imply a possible disruption of multiple checkpoints needed for optimal cellular functioning, apoptosis being one of them as difference in expression of apoptosis genes was indeed observed between control and patient lymphoblasts (5). The aberrant apoptosis in part is also speculated to occur by impaired ribosomal biogenesis driven by a quantitative imbalance of ribosome precursors (15). However, unless functionally characterized, the underlying pathology is hard to interpret. Moreover, involvement of nucleolar proteins in pleiotropic phenotypes is suggestive of some vital yet unknown functions undertaken by this organelle, rather than the universal ribosome synthesis, that needs focus in near future to get insights of these new group of nucleolus-based pathologies, a mouse model could prove helpful in detailed unmasking of disruptive mechanisms. Acknowledgements We are grateful to the members of the family for their invaluable participation and co-operation. The study was funded by Higher Education Commission (HEC), Islamabad, Pakistan. R. H. A. was supported by International Research Support Initiative Program (IRSIP) to visit CMGG, Belgium from Higher Education Commission (HEC) Islamabad, Pakistan and HEC Indigenous PhD fellowship.
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