research article a novel splicing mutation of kit results...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 689756 6 pageshttpdxdoiorg1011552013689756

Research ArticleA Novel Splicing Mutation of KIT Results in Piebaldism andAuburn Hair Color in a Chinese Family

Yong-jia Yang1 Rui Zhao1 Xin-yu He1 Li-ping Li2 Ke-wei Wang3 Liu Zhao1 Ming Tu1

Jin-song Tang4 Zhi-guo Xie5 and Yi-min Zhu16

1 The Laboratory of Genetics and Metabolism Hunan Childrenrsquos Research Institute (HCRI) Hunan Childrenrsquos HospitalThe Paediatric Academy of University of South China Changsha 410008 China

2The Laboratory of Basic Medicine Hunan Childrenrsquos Research Institute (HCRI) Hunan Childrenrsquos HospitalThe Paediatric Academy of University of South China Changsha 410008 China

3The Department of Research Hunan Childrenrsquos Research Institute (HCRI) Hunan Childrenrsquos HospitalThe Paediatric Academy of University of South China Changsha 410008 China

4 Institute of Mental Health Second Xiangya Hospital Central South University Changsha 410008 China5 Institute of Endocrinology Mental Health Second Xiangya Hospital Central South University Changsha 410008 China6Department of Emergency Hunan Childrenrsquos Hospital The Paediatric Academy of University of South ChinaChangsha 410008 China

Correspondence should be addressed to Yi-min Zhu xiangeryiymyahoocomcn

Received 30 April 2013 Revised 8 July 2013 Accepted 10 July 2013

Academic Editor Davinder Parsad

Copyright copy 2013 Yong-jia Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Piebaldism is a rare autosomal dominant disorder of melanocyte development which is mostly caused by KIT gene The keycharacteristics of piebaldism include localized poliosis congenital leukoderma and other variable manifestations The previousstudy has illustrated that the homogeneousMC1R (a genewhich is associatedwith the hair color) variant (pI120T) coordinatingwithKIT mutation may lead to auburn hair color and piebaldism In this study we have investigated a Chinese family with piebaldismand auburn hair color the mutation screening of KIT andMC1R genes identified that only a splicing mutation (c 2484+1GgtA) ofKIT gene cosegregated with the auburn hair color and piebaldismThe data of this study and others suggests that the KITmutationmay causes of the auburn hair color in the piebaldism patients

1 Introduction

Piebaldism (OMIM 172800) is rare autosomal dominantdisorder of melanocyte development which is mostly causedby KIT gene (stem cell growth factor receptor gene alsoknown as a protooncogene NM 0002222) mutations [1]

The key characteristics of piebaldism include localizedpoliosis and congenital leukoderma (principally affectingthe forehead ventral trunk and limb extremities) and thepatches are usually stable throughout life [1]

Other variable manifestations of KIT-mutated piebald-ism include the following (1) cafe spots or hyperpigmentedspots exist in the depigmented skin area and can develop atthe margins or within the macules [1] (2) in mild form ofpiebaldism the leukoderma was very small a white forelock

cannot be seen and in certain patients even the main featureof leukoderma can be an incomplete penetration [2] (3) KITmutation correlates with other modifier genes in a specificpatient leading to the hair colour change [3]

Recently Oiso et al described a 1-year-old Japanese girlwho presented with piebaldism and auburn hair color [3]The mutation screening of KIT andMC1R genes (MC1R wasa previously identified gene which was associated with redhair in recessive mode NM 0023863 [4]) disclosed a novelmutation pP832L in KIT and coexistence of a homozygousvariant pI120T in MC1R gene [3] The pI120T variant waspreviously deemed as a polymorphism and the incidence ofthe homozygous pI120T of MC1R was one person per 2268Japanese people [5] It is then raising the possibility that thehomozygous pI120T ofMC1R (modifier gene [6]) correlating

2 BioMed Research International

with KIT pP832L mutation results in new phenotype ofauburn hair colour

In this study we have investigated a Chinese family withpiebaldism and auburn hair colour themutation screening ofKIT andMC1R genes has identified only a splicing mutation(c 2484+1GgtA) in KIT gene that cosegregated with thepiebaldism and auburn hair color in the family

2 Materials and Methods

AChinese Han family with fivemembers affected by piebald-ism and auburn hair color (Figure 1(a)) and 60 healthycontrols (31 males and 29 females) were included in thisstudy All adult individuals and the parents of the minorswho participated in this study gave written informed consentwhich was approved by the Ethics Committee of the HunanChildrenrsquos Hospital Changsha China The procedures of thecommittee conformed to the principles of the declaration ofHelsinki 2008 edition

Genomic DNA was extracted from peripheral blood(2mL in heparin sodium tubes) using the phenoltrichloro-methane method prescribed by standard protocol All spec-imens were quantified by spectrophotometry and diluted to50 ng120583L for polymerase chain reaction (PCR)

The coding regions and the intronexon junctions ofthe KIT and MC1R genes were amplified by PCR using theprimers synthesized by local biotech company and designedusing the software Primer3 (httpfrodowimitedu primersand PCR conditions available on request)

Sequencing reaction (BigDye 31 Kit Applied BiosystemsUSA) of the purified PCR products was carried out accordingto the recommended procedures The labeled PCR frag-ments were purified through 70 alcohol precipitation andelectrophoresed on an ABI-A3500 genetic analyzer (AppliedBiosystems USA)

All the results were compared with the reference(KIT NM 0002222 MC1R NM 0023863 httpgenomeucsceducgi-binhgGateway) using SEQMAN software(DNA Star Package WI USA)

For splicing analysis total RNAwas isolated from periph-eral blood of a patient (III2) and a control (II4) usingRNApure Blood Kit (CWBIOTECH Beijing China) Firststrand cDNA was produced by HiFi-MMLV cDNA Kit(CWBIOTECH Beijing China) according to the manufac-turerrsquos recommendations

RT-PCR primers (F 51015840-TGACGAGTTGGCCCTAGA-CT-31015840 R 51015840-GAAGCCTTCCTTGATCATCTTG-31015840 thepredicted product size was 386 bp) were designed accordingto the cDNA sequence of KIT gene (NM 0002222) whichflanked exons 17 and 18 of KIT The PCR products wereelectrophoresed on a 6 polyacrylamide gel The dissectedbands were purified by standard methods and sequenced ona 3500 genetic analyzer which is mentioned previously

3 Results

The proband (Figure 2(a) III2) was a 10-year-old girlwho came to our laboratory for chromosome analysis (due

to her multiple malformations as her parents described)Physical examination revealed that (1) there is a prominentleucoderma on the ventral trunk (Figure 1(a)) knees elbowsand forehead (2) multiple hyperpigmented spots exist in thepatches of her skin (Figure 1(a)) (3) the most of her hairwas auburn color which was mixed with a few of whitehair and a very few of black hair (Figure 1(b)) and (4) aprominent short stature exists (1177 cm normal 1372 cm)Her result for chromosome G band analysis was 46 XX Herfather (Figure 2(a) II3 at the age of 35 years old) also hadleucoderma on the ventral anterior trunk knees elbows anda prominent poliosis of hair eyebrows and eyelashes (Figures1(c) and 1(d)) As he described a small but obvious patch ofhis posterior hair was also auburn color (unfortunately dueto his long-term hair dyeing the picture of his auburn colorhair was unavailable)

The III1 (Figure 2(a)) was a 9-year-old girl who presentedwith leucoderma on the ventral trunk knees elbows aprominent white forelock and poliosis of eyebrows andeyelashes (Figure 1(e)) Except for her frontal forelock anda very small number of black hairs in the middle scalp theremaining hair color was auburn (Figure 1(f))

Her father (Figure 1(a) II1 as she described had deafnessdetailed clinic data was unavailable) and her grandmother(Figure 2(a) I2) also had typical piebaldism phenotype butthe detail of hair color was unavailable

A heterozygous splicing mutation in intron 17 (c2484+1GgtA or IVS17+1GgtA Figures 2(a) and 2(b)) wasfirstly detected in the three affected family members andit was not detected in II4 (Figure 2(a)) and 60 ethnicallymatched control samples by direct sequencing

Themutation screening ofMC1R gene in all available fam-ily individuals (II3 II4 III1 and III2 Figure 1(a)) detected4 heterozygous variants of MC1R including c274GgtAc359TgtC c488 GgtA and c942 AgtG and identified neitherheterogeneous nor homogeneous variant co-segregated withauburn color hair in the family (for detailed variants distri-bution see Figure 2(a))

The PCR products of RT-PCR were electrophoresed ona 6 polyacrylamide gel (Figure 2(c)) In patients (III2Figure 2(a)) two bands were visualized (Figure 2(c)) while incontrol (Figure 2(a)) only one band detected

These prompted us was dissect to these two bands andsequence them on a 3500 genetic analyzer respectively Theresults revealed a heterogenous deletion of 123 base pairs(Figure 2(d) which coincides with all sequences of exon17 of KIT) This result indicated that the splicing mutation(c 2484+1GgtA) skipped exon 17 of KIT gene (Figure 2(d))which leads to a 41 amino acids deletion from the amino acids788 to 828 of KIT protein in the family

4 Discussion

The KIT receptor contains seven domains including a sig-nal sequence (SS amino acids 1ndash22) an amino-terminalextracellular ligand-binding domain (EC amino acids 23ndash520) a transmembrane domain (TM amino acids 521ndash543)a juxtamembrane domain (JM amino acids 544ndash581) and

BioMed Research International 3

(a) (b) (c)

(d) (e)

(f)

Figure 1The clinical manifestation of the Chinese family with piebaldism and auburn hair color ((a) (b))The individual III2 ((c) (d))Theindividual II3 ((e) (f)) The individual III1


II3II2II1MC1Rc 359TgtCI120Tc 488GgtAR163Qc 942AgtGT314TKITc 2484+1GgtA

III1MC1R noneKITc 2484+1GgtA

III2MC1Rc 274GgtAV92Mc 359TgtCI120TKITc 2484+1GgtA

MC1Rc 274GgtAV92Mc 488GgtAR163QKITnone

I1 I2

II4 II5

(a) The pedigree theMC1R polymorphism and KIT mutation identified inthis study

A A A A A A A AA AC C C C CRT G G G G G G GT T T T T T TT

A A A A A A A AA AC C C C CT G G G G G G G GT T T T T T TT

A A A A A A A A AA AC C C C CT G G G G G G GT T T T T T TT

Normal

c 2484+1GgtA

(b) The identified splicing mutation c 2484+1GgtA of KIT gene

II4 III2500bp

400bp

300bp

(c) The effect of c 2484+1GgtA of KIT gene in mRNA leverThe PCR amplifcation using the primers flanked exon 17 ofKIT illustrated two bands in the proband (III2) and one bandin control (II4)

T T T T T TTTC C C C C C C C C CCA A A A A A A AC G G G G G G G


T T T T T T T T TTC C C C C C C C CA A A A A A A A AC G G G G G


Normal allele

Del exon 17

(d) The illustration of the deletion of exon 17 when these two bands weredissected and sequenced

Figure 2 The genetic finding of the Chinese family with piebaldism and auburn hair color

two TK domains (TK1 amino acids 582ndash684 and TK2 aminoacids 762ndash973) separated by a kinase insert domain (KIamino acids 685ndash761) [7 8] The last four domains also wereknown as the cytoplasmic domains or intracellular domains[7] Within TK2 domain the amino acid residue 810ndash839(an activation loop) was a highly conserved enzymatic site[6] The binding of KIT ligand (KITLG) to the extracellulardomain of KIT leads to the receptor dimerization theintracellular autophosphorylation and then tyrosine kinaseactivation (TKA) [9]

The TKA was the final executed step in the KIT bandingprocess which triggers the regulations of the migrationof melanocytes cell proliferation differentiation survivalmelanogenesis and melanosome transfer [10]

Patients with the mutation that occurred in extracellulardomain of KIT usually manifested relatively mild form ofpiebaldism as those of heterogeneous mutations (missenseor a mutation of complete elimination of the production ofKIT by the defective allele) preserved 50 ormore of the KITfunction [7]

In contrast the majority (87) of the most severe formsof KIT-mutated piebaldism (which is well illustrated inFigure 1 of the paper by Murakami et al [7]) were caused bymutations that occurred in intracellular domain especially inTK domains as this kind of mutations tends to be levyingdominant negative effects and haploinsufficiency effectswhich disrupts the TKA process and leads to only 25 or lessof KIT function [7]


The splicing mutation (c 2484+1GgtA) identified in thisstudy was at the donor splice sites of exon 17 of KIT whichleads to the deletion of amino acids 788 to 828 of KIT protein(which was deduced by the mRNA analysis of the propositusin here) This deletion was 19 amino acids that overlappedwith the highly conserved enzymatic site (amino acids 810ndash839 activation loop) of the TK2 domain of KIT

Recently Oiso et al described a 1-year-old Japanesegirl who presented with the severe form of piebaldism andauburn hair color [3] The authors proposed that the severepiebaldismwas caused by themissensemutation (pP832L) ofKIT and the auburn hair color phenotype was caused by thecoordinating effects of theKITmutation and the homozygousvariant pI120T of MC1R Of note the KIT pP832L mutationwas also located in the area of the highly conserved enzymaticsite (amino acids 810ndash839 activation loop) of theTK2domainof KIT

The MC1R gene encoded a melanocortin-1 receptorwhich was located on the plasma membrane of melanocytesand participated in the production of the pigment melaninthrough a process referred to as melanogenesis The MC1Rprotein is a seven transmembraneG-protein coupled receptorand identified as one of the key proteins involved in regulatingmammalian skin and hair color [11]

In human population the MC1R gene (MIM155555) hasmany polymorphisms some of them have been identifiedwith association with red hair fair skin freckling andincreased skin cancer risk [12 13]

The previous study has clearly shown that the majority ofpersons with red hair are either homozygous or compoundheterozygous for a combination of the MC1R variants suchas the variants R151C R160W and D294H [14] Also other10ndash20 of individuals who had red hair (having lighter red-colored hair than those harboring two diminished functionalleles) showed only a heterogeneous change ofMC1R [15]

In this study we have performed the sequencing analysisof MC1R gene in a Chinese Han family and identifiedsuccessfully four polymorphisms (Figure 2(a)) which werepreviously reported in general population [5 12] In thefamily the auburn hair color seems to be cosegregated withthe piebaldism and the splicing mutation (c 2484+1GgtA) ofKIT but not with any of the 4 polymorphisms of the MC1Rgene To our knowledge a previous study of KIT mutationscreening in Chinese population recently has also disclosedthat a KIT mutation (Ala621Asp) leads to severe form ofpiebaldism aswell as auburn hair colour in a female child [16]It is unfortunately that theMC1Rmutation screening was notperformed in that patient [16]

It is interesting that the deafness was occurred in a familymember (the individual II1 (Figure 2(a)) who most probablycarried the KIT mutation (c 2484+1GgtA) ) in this studyHowever the previously study by Spritz andBeighton [17] hadillustrated a heterogeneous KIT mutation (R796G) in a spo-radically case associated with piebaldism and deafness It isthen probably that such kind of deafness with low-penetrancein the KIT heterogeneous-mutated patients may attributesto the different gene background of each individual Furtherstudy for delineation of the deafness and KIT mutation isneeded in the future

In this study we have successfully identified a splicingmutation which results in the deletion of exon 17 of KITand cosegregated it with two phenotypes of the severe formof piebaldism and the auburn hair color in a Chinese Hanfamily This data would expand the knowledge of the KIT-related phenotype-genotype correlations

Acknowledgments

The authors are grateful to the family members who par-ticipated in this study This work was supported by theNational Natural Science Foundation of China (81271946)and Hunan Nature Science Foundation (2012SK3258) andpartly supported by the National Science and TechnologySupport Program (2012BAI04B00)

References

[1] L B Giebel and R A Spritz ldquoMutation of the KIT (maststemcell growth factor receptor) protooncogene in human piebald-ismrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 88 no 19 pp 8696ndash8699 1991

[2] T Narita N Oiso K Fukai et al ldquoTwo children with amild or moderate piebaldism phenotype and a father withoutleukoderma in a family with the same recurrent missensemutation in the kinase domain of KITrdquo European Journal ofDermatology vol 21 no 3 pp 446ndash447 2011

[3] N Oiso K Kishida K Fukai et al ldquoA Japanese piebald patientwith auburn hair colour associated with a novel mutationpP832L in the KIT gene and a homozygous variant pI120T intheMC1R generdquoTheBritish Journal of Dermatology vol 161 no2 pp 468ndash469 2009

[4] K A Beaumont S N Shekar A L Cook D L Duffy and RA Sturm ldquoRed hair is the null phenotype of MC1Rrdquo HumanMutation vol 29 no 8 pp E88ndashE94 2008

[5] T Motokawa T Kato Y Hashimoto et al ldquoCharacteristicMC1R polymorphism in the Japanese populationrdquo Journal ofDermatological Science vol 41 no 2 pp 143ndash145 2006

[6] N Oiso K Fukai A Kawada and T Suzuki ldquoPiebaldismrdquoTheJournal of Dermatology vol 40 no 5 pp 330ndash335 2013

[7] T Murakami K Fukai N Oiso et al ldquoNew KIT mutationsin patients with piebaldismrdquo Journal of Dermatological Sciencevol 35 no 1 pp 29ndash33 2004

[8] S Bondanza M Bellini G Roversi et al ldquoPiebald traitimplication of kit mutation on in vitro melanocyte survival andon the clinical application of cultured epidermal autograftsrdquoJournal of Investigative Dermatology vol 127 no 3 pp 676ndash6862007

[9] S Yuzawa Y Opatowsky Z Zhang V Mandiyan I Lax andJ Schlessinger ldquoStructural basis for activation of the receptortyrosine kinase KIT by stem cell factorrdquo Cell vol 130 no 2 pp323ndash334 2007

[10] J M Grichnik ldquoKIT and melanocyte migrationrdquo Journal ofInvestigative Dermatology vol 126 no 5 pp 945ndash947 2006

[11] D W Roberts R A Newton K A Beaumont J HelenLeonard and R A Sturm ldquoQuantitative analysis of MC1R geneexpression in human skin cell culturesrdquo Pigment Cell Researchvol 19 no 1 pp 76ndash89 2006

[12] K Makova and H Norton ldquoWorldwide polymorphism at theMC1R locus and normal pigmentation variation in humansrdquoPeptides vol 26 no 10 pp 1901ndash1908 2005


[13] B L Sanchez-Laorden C Jimenez-Cervantes and J C Garcıa-Borron ldquoRegulation of human melanocortin 1 receptor signal-ing and trafficking by Thr-308 and Ser-316 and its alteration invariant alleles associated with red hair and skin cancerrdquo Journalof Biological Chemistry vol 282 no 5 pp 3241ndash3251 2007

[14] J L Rees ldquoGenetics of hair and skin colorrdquo Annual Review ofGenetics vol 37 pp 67ndash90 2003

[15] N Flanagan E Healy A Ray et al ldquoPleiotropic effects of themelanocortin 1 receptor (MC1R) gene on human pigmentationrdquoHuman Molecular Genetics vol 9 no 17 pp 2531ndash2537 2000

[16] Z M Lin Z Xu D F Bu and Y Yang ldquoNew mutations of KITgene in two Chinese patients with piebaldismrdquo British Journalof Dermatology vol 155 no 6 pp 1303ndash1304 2006

[17] R A Spritz and P Beighton ldquoPiebaldism with deafness molec-ular evidence for an expanded syndromerdquo American Journal ofMedical Genetics vol 75 no 1 pp 101ndash103 1998

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Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Zoology


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BioMed Research International

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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


with KIT pP832L mutation results in new phenotype ofauburn hair colour

In this study we have investigated a Chinese family withpiebaldism and auburn hair colour themutation screening ofKIT andMC1R genes has identified only a splicing mutation(c 2484+1GgtA) in KIT gene that cosegregated with thepiebaldism and auburn hair color in the family

2 Materials and Methods

AChinese Han family with fivemembers affected by piebald-ism and auburn hair color (Figure 1(a)) and 60 healthycontrols (31 males and 29 females) were included in thisstudy All adult individuals and the parents of the minorswho participated in this study gave written informed consentwhich was approved by the Ethics Committee of the HunanChildrenrsquos Hospital Changsha China The procedures of thecommittee conformed to the principles of the declaration ofHelsinki 2008 edition

Genomic DNA was extracted from peripheral blood(2mL in heparin sodium tubes) using the phenoltrichloro-methane method prescribed by standard protocol All spec-imens were quantified by spectrophotometry and diluted to50 ng120583L for polymerase chain reaction (PCR)

The coding regions and the intronexon junctions ofthe KIT and MC1R genes were amplified by PCR using theprimers synthesized by local biotech company and designedusing the software Primer3 (httpfrodowimitedu primersand PCR conditions available on request)

Sequencing reaction (BigDye 31 Kit Applied BiosystemsUSA) of the purified PCR products was carried out accordingto the recommended procedures The labeled PCR frag-ments were purified through 70 alcohol precipitation andelectrophoresed on an ABI-A3500 genetic analyzer (AppliedBiosystems USA)

All the results were compared with the reference(KIT NM 0002222 MC1R NM 0023863 httpgenomeucsceducgi-binhgGateway) using SEQMAN software(DNA Star Package WI USA)

For splicing analysis total RNAwas isolated from periph-eral blood of a patient (III2) and a control (II4) usingRNApure Blood Kit (CWBIOTECH Beijing China) Firststrand cDNA was produced by HiFi-MMLV cDNA Kit(CWBIOTECH Beijing China) according to the manufac-turerrsquos recommendations

RT-PCR primers (F 51015840-TGACGAGTTGGCCCTAGA-CT-31015840 R 51015840-GAAGCCTTCCTTGATCATCTTG-31015840 thepredicted product size was 386 bp) were designed accordingto the cDNA sequence of KIT gene (NM 0002222) whichflanked exons 17 and 18 of KIT The PCR products wereelectrophoresed on a 6 polyacrylamide gel The dissectedbands were purified by standard methods and sequenced ona 3500 genetic analyzer which is mentioned previously

3 Results

The proband (Figure 2(a) III2) was a 10-year-old girlwho came to our laboratory for chromosome analysis (due

to her multiple malformations as her parents described)Physical examination revealed that (1) there is a prominentleucoderma on the ventral trunk (Figure 1(a)) knees elbowsand forehead (2) multiple hyperpigmented spots exist in thepatches of her skin (Figure 1(a)) (3) the most of her hairwas auburn color which was mixed with a few of whitehair and a very few of black hair (Figure 1(b)) and (4) aprominent short stature exists (1177 cm normal 1372 cm)Her result for chromosome G band analysis was 46 XX Herfather (Figure 2(a) II3 at the age of 35 years old) also hadleucoderma on the ventral anterior trunk knees elbows anda prominent poliosis of hair eyebrows and eyelashes (Figures1(c) and 1(d)) As he described a small but obvious patch ofhis posterior hair was also auburn color (unfortunately dueto his long-term hair dyeing the picture of his auburn colorhair was unavailable)

The III1 (Figure 2(a)) was a 9-year-old girl who presentedwith leucoderma on the ventral trunk knees elbows aprominent white forelock and poliosis of eyebrows andeyelashes (Figure 1(e)) Except for her frontal forelock anda very small number of black hairs in the middle scalp theremaining hair color was auburn (Figure 1(f))

Her father (Figure 1(a) II1 as she described had deafnessdetailed clinic data was unavailable) and her grandmother(Figure 2(a) I2) also had typical piebaldism phenotype butthe detail of hair color was unavailable

A heterozygous splicing mutation in intron 17 (c2484+1GgtA or IVS17+1GgtA Figures 2(a) and 2(b)) wasfirstly detected in the three affected family members andit was not detected in II4 (Figure 2(a)) and 60 ethnicallymatched control samples by direct sequencing

Themutation screening ofMC1R gene in all available fam-ily individuals (II3 II4 III1 and III2 Figure 1(a)) detected4 heterozygous variants of MC1R including c274GgtAc359TgtC c488 GgtA and c942 AgtG and identified neitherheterogeneous nor homogeneous variant co-segregated withauburn color hair in the family (for detailed variants distri-bution see Figure 2(a))

The PCR products of RT-PCR were electrophoresed ona 6 polyacrylamide gel (Figure 2(c)) In patients (III2Figure 2(a)) two bands were visualized (Figure 2(c)) while incontrol (Figure 2(a)) only one band detected

These prompted us was dissect to these two bands andsequence them on a 3500 genetic analyzer respectively Theresults revealed a heterogenous deletion of 123 base pairs(Figure 2(d) which coincides with all sequences of exon17 of KIT) This result indicated that the splicing mutation(c 2484+1GgtA) skipped exon 17 of KIT gene (Figure 2(d))which leads to a 41 amino acids deletion from the amino acids788 to 828 of KIT protein in the family

4 Discussion

The KIT receptor contains seven domains including a sig-nal sequence (SS amino acids 1ndash22) an amino-terminalextracellular ligand-binding domain (EC amino acids 23ndash520) a transmembrane domain (TM amino acids 521ndash543)a juxtamembrane domain (JM amino acids 544ndash581) and


(a) (b) (c)

(d) (e)

(f)







I1 I2

II4 II5





Normal

c 2484+1GgtA


II4 III2500bp

400bp

300bp






Normal allele

Del exon 17
















Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e)

(f)







I1 I2

II4 II5





Normal

c 2484+1GgtA


II4 III2500bp

400bp

300bp






Normal allele

Del exon 17
















Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






I1 I2

II4 II5





Normal

c 2484+1GgtA


II4 III2500bp

400bp

300bp






Normal allele

Del exon 17
















Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Acknowledgments


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

research article a novel splicing mutation of kit results...

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