A Novel Loss-of-Sclerostin Function Mutation in a First Egyptian Family with Sclerosteosis

Research ArticleA Novel Loss-of-Sclerostin Function Mutation ina First Egyptian Family with Sclerosteosis

Alaaeldin Fayez1 Mona Aglan2 Nora Esmaiel1 Taher El Zanaty3

Mohamed Abdel Kader4 and Mona El Ruby2

1Molecular Genetics and Enzymology Department Human Genetics amp Genome Research Division National Research Centre33 El Bohouth Street (Former El Tahrir Street) PO Box 12622 Dokki Giza Egypt2Clinical Genetics Department Human Genetics amp Genome Research Division National Research Centre Egypt3Department of Medicine Faculty of Medicine Cairo University Egypt4Department of Orodental Genetics Medical Research Division National Research Centre Egypt

Correspondence should be addressed to Alaaeldin Fayez afayez nrcyahoocom

Received 4 January 2015 Revised 13 March 2015 Accepted 29 March 2015

Academic Editor Jozef Zustin

Copyright copy 2015 Alaaeldin Fayez et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sclerosteosis is a rare autosomal recessive condition characterized by increased bone density Mutations in SOST gene coding forsclerostin are linked to sclerosteosis Two Egyptian brothers with sclerosteosis and their apparently normal consanguineous parentswere included in this study Clinical evaluation and genomic sequencing of the SOST gene were performed followed by in silicoanalysis of the resulting variation A novel homozygous frameshift mutation in the SOST gene characterized as one nucleotidecytosine insertion that led to premature stop codon and loss of functional sclerostin was identified in the two affected brothersTheir parents were heterozygous for the same mutation To our knowledge this is the first Egyptian study of sclerosteosis and SOSTgene causing mutation

1 Introduction

Sclerosteosis (SOST1 MIM 269500) is an autosomal reces-sive sclerosing skeletal dysplasia in which bone overgrowththroughout life affecting mainly the cranial and tubularbones leads to distortion of facies and entrapment of cranialnerves Syndactyly is a variable manifestation but representsan important diagnostic feature The disorder is rare and themajority of affected individuals have been reported in theAfrikaner population of South Africa A small number ofindividuals and familieswith sclerosteosis have been reportedin other parts of the world including Brazil United StatesGermany Senegal and Turkey [1]

Due to genetic heterogeneity sclerosteosis was given 2MIM numbers despite the same clinical features Sclerosteo-sis-1 (SOST1 MIM 269500) is linked to a genetic defect inthe SOST gene coding for sclerostinThe SOST gene productsclerostin is secreted by osteocytes and transported to thebone surface where it inhibits osteoblastic bone formation

by antagonizing Wnt signaling [2] Five different loss-of-function mutations relevant to sclerosteosis in SOST genehave been reported as pathogenic in ClinVar database to date[3] Heterozygous and homozygous missense mutations inthe LRP4 gene were reported in 2 unrelated families withsclerosteosis-2 (SOST2 MIM 614305) also LRB4 and LRB5interacted with sclerostin by Leupin et al [4]

In this study we sequenced the coding and exon-intronboundaries sequence of the entire SOST gene in two Egyptianbrothers offspring of consanguineous parents with scleros-teosis To our knowledge this is the first Egyptian report ofpatients with sclerosteosis

2 Subjects and Methods

This research complies with the standards established by theMedical Ethical Committee of the National Research CentreA written informed consent was signed by the patients

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 517815 8 pageshttpdxdoiorg1011552015517815

2 BioMed Research International

Table 1 Sequence of designed PCR primers for SOST gene

Name Forward (51015840-31015840) Reverse (51015840-31015840) Ta

SOST1 CTGAGGGAAACATGGGACCAGC CAGGCAAGACTGTTCCTCGACCA 56SOST2 ACAGGGTGCGCAGGAGAGCTT CCCATCGGTCACGTAGCGGG 56SOST3 ACTTCACCCGCTACGTGACCGA CACGCGCAGAGGACAGAAATGT 55Ta means ldquoAnnealing Temperaturerdquo

and their parents The study included a consanguineousEgyptian family from Upper Egypt with 2 affected siblingswith sclerosteosis

21 Clinical Evaluation The referred family was subjectedto history taking pedigree analysis full clinical examina-tion and photography Skeletal survey hearing assessmentcomplete eye evaluation including fundus examination CTscan and MRI brain abdominal ultrasound complete bloodpicture liver and kidney function tests hormonal studiesand cytogenetic studies by G-banding using peripheral bloodleukocytes were carried out for the 2 affected brothers

22 Samples and DNA Extraction After informed consentthree mL of the peripheral blood samples was collectedfrom the patients and their parents using K

2EDTA as anti-

coagulant inside vacutainer sterile tubes Genomic DNAwas isolated from peripheral blood leukocytes by QIAampDNAMini Kit (50 preps) catalog number 51304 Germany(httpswwwqiagencomeg)Theparents deniedmolecularstudies to the normal siblings

23 Primers Designing We designed the primers coveringboth two exons and exon-intron boundaries sequence usingNCBI Primer-BLAST tool (httpwwwncbinlmnihgovtoolsprimer-blast) Primers consist of three sets one setcovered whole exon 1 and exon 1-IVS1 boundary (from 177109900 to 7110409) and two sets covered both IVS1-exon 2boundary (from 17 7107228 to 7107470) and last region ofexon 2 (from 17 7106785 to 7107253) as shown in Table 1

24 Mutation Analysis Mutation analysis of both exons andexon-intron boundaries sequence of the SOST gene wasperformed by conventional PCR using Alliance Bio TaqDNAPolymerase catalog number M001TP20 (httpwwwallian-cebiocom) Amplified fragments were verified by agarosegel electrophoresis containing ethidium bromide The ver-ified amplicons were purified and sequenced directly byABI3730XL sequencer in Macrogen sequencing serviceKorea [5]

25 In Silico Analysis The sequencing data were analyzed byFinchTV version 140 [6] Prediction of the disease causingmutations and putative effect of themutations were identifiedusing the Mutation Taster (MT) tool [7] and SNP annotationtool (SNPnexus) [8] Exploration of the resulting protein wasdone by SWISS-MODEL tool [9] and UniProt database [10]

3 6

SB

SB still birth

Hydrocephalus

Mental subnormality

Figure 1 Family pedigree

3 Results

31 Clinical Data

Patient 1 The proband is a 25-year-old Egyptian male whowas referred to us because of progressive diminution ofvision hearing loss and change in facial features during thelast years The patient is the offspring of first cousin parentsThere was history of two older female siblings who died atthe age of 4 months due to an unknown cause a previous stillbirth at 7 months of gestational age a younger female siblingwho died at 20 years of age from atrial fibrillation 2 normalfemale and male siblings and a younger brother who beganto show changes in facial features (patient 2) There was alsofamily history of a maternal cousin who died at 4 years of agewith hydrocephalus and his sibling withmental subnormalitywho died at 2 years of age their parents are consanguineousFamily pedigree is shown in Figure 1 Pregnancy and deliveryhistories were uneventful

At the age of 6 years the parents noticed facial palsy andthe probandrsquos complaint of recurrent headache A progressivecourse was reported with hearing affection and diminutionof vision By examination the patient was uncooperative andunable to communicate He was tall (height was 183 cm =20 SD above the mean) with increase in relative arm span(arm span was 191 cm) and he had a large head (headcircumference 63 cm = 45 SD above the mean) and hisweight was at the mean Delayed milestones of developmentdelayed speech and poor school performance were reported

BioMed Research International 3

(a) (b)

(c) (d)

Figure 2 Proband (on the right hand side of the photo) and his affected brother (on the left hand side of the photo) showing distorted faciesand strong body build (a) lateral view of the face of the proband showing proptosis and prognathism (b) both hands of the proband showingbilateral radial deviation of index fingers camptodactyly of 5th fingers and soft tissue syndactyly between fingers (c) both feet of the probandshowing bilateral broad big toes with wide space between the 1st and 2nd toes bilateral clinodactyly of 4th and 5th toes and hypoplastic nailsand soft tissue syndactyly between toes (d)

by the parents however it was not possible to perform anintelligence quotient (IQ) test because of difficulty in hearingand vision and the incooperation of the patient

Facial features in the form of frontal bossing highanterior hair line prominent supraorbital ridges proptosisstrabismus depressed nasal root midface hypoplasia andprognathism were noted (Figures 2(a) and 2(b)) Orodentalexamination revealed receding premaxilla macrognathiathick alveolar ridge high arched palate malaligned irregularconical shaped teeth and class II occlusion

Skeletal examination revealed prominence of medialparts of the clavicles and upper ribs bilateral radial deviationof index fingers camptodactyly of 5th fingers bilateral broadbig toeswithwide space between the 1st and 2nd toes bilateralclinodactyly of 4th and 5th toes and hypoplastic nails andsoft tissue syndactyly between fingers and toes (Figures 2(c)and 2(d)) No history of previous bony fractures was reportedand the patient denied further studies to assess bone densityby DEXA scan

X-ray studies showed diffuse thick dense cranial bonesthick calvarium narrow skull base foramina normal sizeand shape of sella turcica hypoplastic maxilla protrudedmandible diffuse sclerotic texture of long bones withmedullary cavity expansion of metacarpals and metatarsalsand phalanges of hands and feet (Figure 3) MRI of the brain

denoted no pituitary gland lesions with patent cavernoussinuses and inferior sagging of the infundibular recess of thirdventricle

Complete blood picture coagulation profile liver func-tion tests and kidney function tests were normal Growthhormone level and thyroid profile were normal Audiogramshowed bilateral profoundmixed hearing loss Eye evaluationand fundus examination revealed hypotropia exotropiaand bilateral optic atrophy Abdominopelvic ultrasound wasnormal Cytogenetic analysis by G-banding using peripheralblood leukocytes denoted normal male karyotype in allstudied metaphases (46 XY)

Patient 2 He was a 17-year-old male patient the youngerbrother of patient 1 He began to show changes of facialfeatures at the age of 15 years By examination he wascooperative and tall (height 188 cm= 30 SD above themean)with increase in relative arm span (arm span 194 cm) andhe had a large head (head circumference 60 cm = 30 SDabove the mean) and his weight was at the mean He hadlong face broad forehead depressed nasal root midfacehypoplasia short philtrum and macrognathia (Figure 2(a))Open mouth with fissured lower lip thick alveolar ridgemacroglossia with median grooved tongue high arched


(a) (b)

(c) (d)

Figure 3 Radiologicalmanifestations in patient 1 Skull X-ray (AP and lateral views) showing diffusely thickened dense calvarial bones of skullbase and prognathism (a) X-ray chest (AP view) showing right clavicle and humerus andX-ray pelvis and upper part of both femora (AP view)with diffuse sclerotic texture and widened clavicles and ribs (b) X-ray of both hands (AP view) showing sclerotic appearance with medullaryexpansion of metacarpals and phalanges and hypoplastic terminal phalanges (c) X-ray of both feet (AP view) showing sclerotic appearancewith medullary expansion of metatarsals and phalanges and hypoplastic terminal phalanges bilateral hallux valgus and overcrowded tarsalbones (d)

palate torus palatines short uvula and enamel hypocalcifica-tion were detected by orodental examination Mild deafnesswas noted Broad chest with medial prominence of claviclesand long neck were other features (Figure 2(a)) Examinationof limbs revealed bilateral camptodactyly of 2nd 3rd and5th fingers broad interphalangeal joints partial syndactylybetween 2nd 3rd 4th and 5th right fingers and completesyndactyly between the left 2nd and 3rd fingers operatedupon bulbous big toes with wide space between 1st and2nd toes and clinodactyly of 4th and 5th toes bilaterallyNo history of previous bony fractures was reported and thepatient and his parents denied further studies to assess bonedensity by DEXA scan

32 Molecular Analysis

321 Sequencing Results Direct sequencing of the SOST genedata in the studied family was analyzed by FinchTV softwareand revealed a novel homozygous c87 88insC mutation (itwas registered by the author under NCBI rs377648601) inexon 1 for the proband and his brother (nucleotide num-bering refers to the cDNA RefSeq using the A of the ATGtranslation initiation codon as nucleotide +1)This nucleotidechange was confirmed in his parents in the heterozygousstate (Figure 4) This mutation is not recorded in the NCBI(dbSNP) Ensembl SNPs 1000 Genomes Project (TGB) theHuman Gene Mutation Database (public HGMD) ClinVar

The Exome Aggregation Consortium (ExAC) browser andNHLBI Exome Sequencing Project (ESP) server databases

322 Effect Prediction of c90 91insC Mutation on SclerostinFunction Using SNP annotation tool (SNPnexus) (httpsnp-nexusorg) to predict possible function of c87 88insCmutation we found that this mutation led to K30Q substi-tution and frameshift stop-gain resulting in acquired earlystop codon (TGA) at codon 32 producing truncated nonfunc-tional sclerostin protein To explore the truncated proteinresulting in this mutation we used SWISS-MODEL tool andUniProt database where it was found that this mutationled to the production of 8-oligopeptide chain only afterremoving the first 23 signal peptide amino acids besides thatthe resulting truncated protein did not contain C-terminalcysteine knot-like (CTCK) domain (Figures 5 and 6)

323 Prediction Disease Causing By using Mutation Taster(MT) tool to predict the extent towhich c87 88insCmutationmay cause the occurrence of disease we found that thismuta-tion led to amino acid change K30Q with further changesdownstream to create premature stop codon (PTC) D32X(Figure 6) Depending on bioinformatics prediction toolswhichwere used in this study we predicted that thismutationled to shortened truncated sclerostin (8 oligopeptides only)and created PTC at a 32 residue Hence because of D32Xlocated away about 125 upstream nucleotides from the firstexon-exon junction complex the mutant mRNA will be


25 26 27 28 29 30 31 32 333 34

G W Q A F K N D A T

Codon number

The affected proband

The affected brother

The father

AAG AAT GAT

GGG TGG CAG GCG TTC GAA TGACAA

GGGTGGCAGGCGTTCAAGAATGATGCCACG wt

GGG TGG TTC GAA TGACAG GCGMtG W Q QA F E lowastaa

Seq

CAA

Figure 4 Paternal partial sequence chromatogram displaying the DNA sequence of affected proband brother and father (the mothershowed the same father sequence chromatogram) The lined nucleotide indicates the position of the homozygous one nucleotide insertion(c87 88insC NCBI rs377648601) resulting in a frameshift mutation and early stop codon (lowast) and red box indicate the position of TGA stopcodon () indicates the substituent nucleotides wt wild type Mt mutant type and aa amino acids

(a) Normal sclerostin protein (b) Truncatedsclerostin protein

Figure 5 Sclerostin model for wild type (a) and mutant sclerostin protein (b) where sclerostin protein was truncated at Asparagine (ASP)amino acid at codon 32 to present premature stop codon (PTC) D32X resulting in the discovered c87 88insC mutation

Q EX

MQLPLALCLV CLLVHTAFRV VEGQGWQAFK NDATEIIPEL GEYPEPPPEL

ENNKTMNRAE NGGRPPHHPF ETKDVSEYSC RELHFTRYVT DGPCRSAKPV TELVCSGQCG

TRFHNQSELK DFGTEAARPQ KGRKPRPRAR SAKANQAELE NAY

lowast

PARLLPNAIG RGKWWRPSGP DFRCIPDRYR AQRVQLLCPG GEAPRARKVR LVASCKCKRL

Figure 6 Sclerostin protein sequence illustrates the first 23 putative secretory signal sequences highlighted in green and CTCK domainhighlighted in yellow Altered amino acids are shadowed with gray highlighted substituents and red star (lowast) indicated to end of the truncatedsclerostin protein

predicted to degrade by NMD pathway to prevent it totranslate this relies on the reported reference stating that PTCwhich is located further than about 50 nucleotides upstreamof the exon-exon junction complex triggers NMD So thismutation has a pathogenic effect and is regarded as a diseasemutation (full data shown in the Appendix)

4 Discussion

Sclerosteosis is a severe sclerosing skeletal genetic disorderThe disease affects bone modeling and remodeling espe-cially in the skull and diaphyseal region of long bonesThe increased rate of bone formation suggests a defect in


osteoblast function [11] Although the condition is rare thevast majority are present in the Afrikaner population ofDutch descent in SouthAfrica A small number of individualsand families with sclerosteosis have been reported in otherparts of the world including Brazil United States GermanySenegal and Turkey [1] The rarity of sclerosteosis led toknowledge gaps in the molecular and cellular basis of thisdisease and resulted in unavailable satisfactory diagnostic andtherapeutic strategies

In this study two Egyptian patients offspring of consan-guineous parents were reported The progressive bone over-growth leading to tall stature distorted facies frontal promi-nence with midface hypoplasia mandibular prognathismdental malocclusion and progressive bony encroachmentupon the middle ear cavities and auditory nerve canalscausing deafness in addition to optic nerve atrophy wasconsistent with sclerosteosis

Sclerosteosis must be differentiated from osteopetrosisand other sclerosing bone dysplasias [12] Autosomal reces-sive van Buchem disease (VBCH MIM 239100) is clinicallysimilar to sclerosteosisHowever themain clinical differencesbetween the 2 diseases are gigantism and hand abnormalitiespresent in sclerosteosis but never in VBCH In our studiedsibs the tall stature skeletal overgrowth presence of syn-dactyly irregular teeth with malocclusion torus palatinesand nail hypoplasia support the diagnosis of sclerosteosis

Balemans et al [13] assigned the locus for sclerosteosis to17q12ndashq21 the same general region as the locus for VBCHbecause of the clinical similarities However the possibilitythat sclerosteosis and VBCH are caused by mutation in thesame gene appeared to have been excluded by the studyof Brunkow et al [14] in which mutations in the codingregion of the SOST gene were found in cases of sclerosteosisbut not in cases of VBCH Further studies by Van Hul etal [15] and Balemans et al [16] revealed that VBCH wascaused by a 52-kb deletion approximately 35 kb downstreamof the SOST gene that removes a SOST-specific regulatoryelement Loots et al [17] concluded that VBCH is caused bydeletion of a SOST-specific regulatory element and is allelicto sclerosteosis

Mutations in human SOST are responsible for sclerosteo-sis-1 SOST is a negative regulator of bone formation [2]According to ClinVar database there are five SOST genemutations that have pathogenic effect causing sclerosteosisThe current study is the first to detect 1 bp cytosine insertionin codon number 30 (c87 88insC) as frameshift mutationresulting in early stop codon at number 32 and by usingbioinformatics prediction tools it likely to be pathogenicmutation to give nonfunctional short sclerostin protein(Figure 7)

In silico analysis of the discovered new frameshift stopgained mutation revealed that it is a pathogenic mutationthat led to nonfunctional sclerostin proteinThis is supportedwith what was mentioned in patients with sclerosteosis inwhich premature termination codons in the SOST gene leadto an inhibitory effect of the gene product sclerostin on boneformation that was supported by the inhibited proliferationand differentiation of mouse and human osteoblastic cells

IVS1 + 3AT

IVS1 minus 67AC

Exon 1 Exon 2

Gln24X

Trp124X

Arg126X

5998400 3

998400

423bp220bp 2758bp

c87_88insC(Lys30Glnfs)

gtat

Figure 7 Schematic representation of sclerosteosis relevantreported ClinVar pathogenic mutations in SOST gene The newmutations are shown in red color

after the addition of exogenous sclerostin to osteogeniccultures [18ndash20]

The clinical manifestations reported in the studied broth-ers in the form of tall stature distorted facies orodentalfindings syndactyly and encroachment of cranial nerves areconsistent with sclerosteosis and mutation in SOST geneHowever the relative increase of arm span compared toheightwas not previously noted in the literature Also of notethe presence of learning difficulties in patient 1 in this studyhas not been reported before in sclerosteosis Although thiscan be an expansion of the phenotypic characteristics of thesyndrome the maternal history of intrauterine fetal deathunexplained infant deaths and other affected family mem-bers with mental subnormality and hydrocephalus stronglysuggest the possibility of another inherited disorder in thisconsanguineous family which calls for further studies in thisfamily

5 Conclusions

Up to our knowledge this is the first study of Egyptianpatients with sclerosteosis describing the associated clinicaltraits and sclerosteosis causing mutations in SOST gene Weidentified a novel mutation in SOST gene in two affectedEgyptian brothers with sclerosteosis characterized by onenucleotide insertion resulting in a frameshift mutation Insilico analysis indicated that this nucleotide sequence changeclassified as severe disruptive mutation resulted in loss-of-sclerostin function The clinical traits are compatible withthe genetic investigations except for learning difficulties inpatient 1 that require further studies to identify their geneticbackground

Appendix

See Table 2

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Table 2 Data of Mutation Taster (MT) tool about prediction of effect of c87 88insC mutation

Summary

◻ NMD◻ Amino acid sequence changed◻ Frameshift◻ Protein features (might be) affected◻ Splice site changes

Analysed issue Analysis resultName of alteration (HGNC) SOSTAlteration (phys location) chr17 41836022 41836023insGAlteration typeregion InsertionCDSAA changes K30Qfslowast3Position(s) of altered AA 30 (frameshift or PTC further changes downstream)Frameshift YesphyloPphastCons 2271 1 (flanking)0445 1 (flanking)Length of protein NMDPosition (AA) of stop codon in wtmu AA sequence 21432Theoretical NMD boundary in CDS 170

Wild type AA sequence

MQLPLALCLVCLLVHTAFRVVEGQGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRAR SAKANQAELE NAYlowast

Mutated AA sequence MQLPLALCLVCLLVHTAFRVVEGQGWQAFQ Elowast

All positions are in base pairs (bp) if not explicitly stated differently AAaa amino acid CDS coding sequence mu mutated NMD nonsense-mediatedmRNA decay nt nucleotide wt wild type TGP 1000 Genomes Project

Funding

Financial fund of this paper was paid by authors only alsowriting monitor and language review were made by authors

Acknowledgment

The authors are very grateful to patients and their family fortheir participation and cooperation during this study

References

[1] S K Bhadada A Rastogi E Steenackers et al ldquoNovel SOSTgene mutation in a sclerosteosis patient and her parentsrdquo Bonevol 52 no 2 pp 707ndash710 2013

[2] E Piters C Culha M Moester et al ldquoFirst missense mutationin the SOST gene causing sclerosteosis by loss of sclerostinfunctionrdquo Human Mutation vol 31 no 7 pp E1526ndashE15432010

[3] httpwwwncbinlmnihgovclinvar[4] O Leupin E Piters C Halleux et al ldquoBone overgrowth-

associated mutations in the LRP4 gene impair sclerostin facili-tator functionrdquoThe Journal of Biological Chemistry vol 286 no22 pp 19489ndash19500 2011

[5] httpdnamacrogencom[6] httpwwwgeospizacom[7] httpwwwmutationtasterorg[8] httpsnp-nexusorg

[9] httpswissmodelexpasyorg[10] httpwwwuniprotorg[11] P J Marie and M Kassem ldquoOsteoblasts in osteoporosis past

emerging and future anabolic targetsrdquo European Journal ofEndocrinology vol 165 no 1 pp 1ndash10 2011

[12] P Beighton ldquoSclerosteosisrdquo Journal of Medical Genetics vol 25no 3 Article ID 3351908 pp 200ndash203 1988

[13] W Balemans J van den Ende A F Paes-Alves et al ldquoLocaliza-tion of the gene for sclerosteosis to the van Buchem diseasemdashgene region on chromosome 17q12-q21rdquo The American Journalof Human Genetics vol 64 no 6 pp 1661ndash1669 1999

[14] M E Brunkow J C Gardner J Van Ness et al ldquoBone dysplasiasclerosteosis results from loss of the SOST gene product a novelcystine knot-containing proteinrdquo American Journal of HumanGenetics vol 68 no 3 pp 577ndash589 2001

[15] W Van Hul W Balemans E Van Hul et al ldquoVan Buchem dis-ease (Hyperostosis corticalis generalisata)maps to chromosome17q12-q21rdquo American Journal of Human Genetics vol 62 no 2pp 391ndash399 1998

[16] W Balemans N Patel M Ebeling et al ldquoIdentification of a 52kb deletion downstream of the SOST gene in patients with vanBuchem diseaserdquo Journal of Medical Genetics vol 39 no 2 pp91ndash97 2002

[17] G G Loots M Kneissel H Keller et al ldquoGenomic deletionof a long-range bone enhancer misregulates sclerostin in VanBuchem diseaserdquo Genome Research vol 15 no 7 pp 928ndash9352005

[18] R L van Bezooijen B A J Roelen A Visser et al ldquoSclerostinis an osteocyte-expressed negative regulator of bone formation


but not a classical BMP antagonistrdquo Journal of ExperimentalMedicine vol 199 no 6 pp 805ndash814 2004

[19] M K Sutherland J C Geoghegan C Yu et al ldquoSclerostinpromotes the apoptosis of human osteoblastic cells a novelregulation of bone formationrdquo Bone vol 35 no 4 pp 828ndash8352004

[20] D G Winkler M K Sutherland J C Geoghegan et alldquoOsteocyte control of bone formation via sclerostin a novelBMP antagonistrdquoThe EMBO Journal vol 22 no 23 pp 6267ndash6276 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Sequence of designed PCR primers for SOST gene

Name Forward (51015840-31015840) Reverse (51015840-31015840) Ta

SOST1 CTGAGGGAAACATGGGACCAGC CAGGCAAGACTGTTCCTCGACCA 56SOST2 ACAGGGTGCGCAGGAGAGCTT CCCATCGGTCACGTAGCGGG 56SOST3 ACTTCACCCGCTACGTGACCGA CACGCGCAGAGGACAGAAATGT 55Ta means ldquoAnnealing Temperaturerdquo

and their parents The study included a consanguineousEgyptian family from Upper Egypt with 2 affected siblingswith sclerosteosis

21 Clinical Evaluation The referred family was subjectedto history taking pedigree analysis full clinical examina-tion and photography Skeletal survey hearing assessmentcomplete eye evaluation including fundus examination CTscan and MRI brain abdominal ultrasound complete bloodpicture liver and kidney function tests hormonal studiesand cytogenetic studies by G-banding using peripheral bloodleukocytes were carried out for the 2 affected brothers

22 Samples and DNA Extraction After informed consentthree mL of the peripheral blood samples was collectedfrom the patients and their parents using K

2EDTA as anti-

coagulant inside vacutainer sterile tubes Genomic DNAwas isolated from peripheral blood leukocytes by QIAampDNAMini Kit (50 preps) catalog number 51304 Germany(httpswwwqiagencomeg)Theparents deniedmolecularstudies to the normal siblings

23 Primers Designing We designed the primers coveringboth two exons and exon-intron boundaries sequence usingNCBI Primer-BLAST tool (httpwwwncbinlmnihgovtoolsprimer-blast) Primers consist of three sets one setcovered whole exon 1 and exon 1-IVS1 boundary (from 177109900 to 7110409) and two sets covered both IVS1-exon 2boundary (from 17 7107228 to 7107470) and last region ofexon 2 (from 17 7106785 to 7107253) as shown in Table 1

24 Mutation Analysis Mutation analysis of both exons andexon-intron boundaries sequence of the SOST gene wasperformed by conventional PCR using Alliance Bio TaqDNAPolymerase catalog number M001TP20 (httpwwwallian-cebiocom) Amplified fragments were verified by agarosegel electrophoresis containing ethidium bromide The ver-ified amplicons were purified and sequenced directly byABI3730XL sequencer in Macrogen sequencing serviceKorea [5]

25 In Silico Analysis The sequencing data were analyzed byFinchTV version 140 [6] Prediction of the disease causingmutations and putative effect of themutations were identifiedusing the Mutation Taster (MT) tool [7] and SNP annotationtool (SNPnexus) [8] Exploration of the resulting protein wasdone by SWISS-MODEL tool [9] and UniProt database [10]

3 6

SB

SB still birth

Hydrocephalus

Mental subnormality

Figure 1 Family pedigree

3 Results

31 Clinical Data

Patient 1 The proband is a 25-year-old Egyptian male whowas referred to us because of progressive diminution ofvision hearing loss and change in facial features during thelast years The patient is the offspring of first cousin parentsThere was history of two older female siblings who died atthe age of 4 months due to an unknown cause a previous stillbirth at 7 months of gestational age a younger female siblingwho died at 20 years of age from atrial fibrillation 2 normalfemale and male siblings and a younger brother who beganto show changes in facial features (patient 2) There was alsofamily history of a maternal cousin who died at 4 years of agewith hydrocephalus and his sibling withmental subnormalitywho died at 2 years of age their parents are consanguineousFamily pedigree is shown in Figure 1 Pregnancy and deliveryhistories were uneventful

At the age of 6 years the parents noticed facial palsy andthe probandrsquos complaint of recurrent headache A progressivecourse was reported with hearing affection and diminutionof vision By examination the patient was uncooperative andunable to communicate He was tall (height was 183 cm =20 SD above the mean) with increase in relative arm span(arm span was 191 cm) and he had a large head (headcircumference 63 cm = 45 SD above the mean) and hisweight was at the mean Delayed milestones of developmentdelayed speech and poor school performance were reported


(a) (b)

(c) (d)










(a) (b)

(c) (d)









25 26 27 28 29 30 31 32 333 34

G W Q A F K N D A T

Codon number



The father

AAG AAT GAT




Seq

CAA




Q EX




lowast




4 Discussion









IVS1 + 3AT

IVS1 minus 67AC

Exon 1 Exon 2

Gln24X

Trp124X

Arg126X

5998400 3

998400

423bp220bp 2758bp


gtat




5 Conclusions


Appendix

See Table 2





Summary







Funding


Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)










(a) (b)

(c) (d)









25 26 27 28 29 30 31 32 333 34

G W Q A F K N D A T

Codon number



The father

AAG AAT GAT




Seq

CAA




Q EX




lowast




4 Discussion









IVS1 + 3AT

IVS1 minus 67AC

Exon 1 Exon 2

Gln24X

Trp124X

Arg126X

5998400 3

998400

423bp220bp 2758bp


gtat




5 Conclusions


Appendix

See Table 2





Summary







Funding


Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)









25 26 27 28 29 30 31 32 333 34

G W Q A F K N D A T

Codon number



The father

AAG AAT GAT




Seq

CAA




Q EX




lowast




4 Discussion









IVS1 + 3AT

IVS1 minus 67AC

Exon 1 Exon 2

Gln24X

Trp124X

Arg126X

5998400 3

998400

423bp220bp 2758bp


gtat




5 Conclusions


Appendix

See Table 2





Summary







Funding


Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


25 26 27 28 29 30 31 32 333 34

G W Q A F K N D A T

Codon number



The father

AAG AAT GAT




Seq

CAA




Q EX




lowast




4 Discussion









IVS1 + 3AT

IVS1 minus 67AC

Exon 1 Exon 2

Gln24X

Trp124X

Arg126X

5998400 3

998400

423bp220bp 2758bp


gtat




5 Conclusions


Appendix

See Table 2





Summary







Funding


Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








IVS1 + 3AT

IVS1 minus 67AC

Exon 1 Exon 2

Gln24X

Trp124X

Arg126X

5998400 3

998400

423bp220bp 2758bp


gtat




5 Conclusions


Appendix

See Table 2





Summary







Funding


Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Summary







Funding


Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

A Novel Loss-of-Sclerostin Function Mutation in a First Egyptian Family with Sclerosteosis

Documents

Transcript of A Novel Loss-of-Sclerostin Function Mutation in a First Egyptian Family with Sclerosteosis