Reviewer's□report 
□Investigation□of□KIT□gene□mutations□in□women□with□46,XX□spontaneous□premature□ovarian□failure Title: 
Authors: 
Kyoko□Shibanuma□( ShibanumaK@aol.com )
Zhi-Bin□Tong□( TongZ@cc1.nichd.nih.gov )
Vien□H□Vanderhoof□( VanderhV@mail.nih.gov )
Konstantina□Vanevski□( Vanevski@mail.nih.gov )
Lawrence□M□Nelson□( Lawrence_Nelson@nih.gov )
□1□ □12□Jun□2002 Version: Date: 
□Dr□Andrew□Shelling Reviewer: 
□A□paper□whose□findings□are□important□to□those□with□closely□related□research□interests Level□of□interest: 
□Accept□after□discretionary□revisions Advice□on□publication: 
This□is□a□report□on□a□study□of□KIT□mutations□as□a□potential□candidate□gene□for□premature□ovarian□failure.□ 
The□authors□provide□a□sensible□report□of□their□study,□whereby□KIT,□a□plausible□candidate□gene□is□ 
screened□in□patients□for□mutations.□They□do□not□find□mutations,□however,□as□it□is□a□plausible□candidate□ 
gene,□it□is□worth□reporting.□We□are□aware□that□premature□ovarian□failure□is□a□heterogeneous□condition,□ 
and□it□is□likely□that□a□large□number□of□genes□will□be□screened□over□the□years,□that□will□provide□both□ 
positive□and□negative□outcomes.□I□have□some□minor□comments□to□make,□which□will□require□minor□ 
changes□in□the□text.□ 
Compulsory□revisions.□ 
1.□I□have□some□concerns□about□their□use□of□SSCP□in□this□study□as□a□primary□screening□method,□and□they□ 
will□need□to□acknowledge□in□the□text□that□they□are□not□likely□to□detect□all□mutations□due□to□the□conditions□ 
they□have□used.□Most□laboratories□would□suggest□that□SSCP□is□at□best□70-80%□likely□to□detect□a□mutation 
for□a□PCR□fragment□under□250□base□pairs□of□length,□and□we□would□routinely□attempt□to□ensure□that□ 
amplicons□to□be□screened□are□150-200□bp.□Most□laboratories□would□also□try□a□number□of□different□gel□ running□conditions□(such□as□+/-□5%□glycerol,□or□being□run□at□different□temperatures).□In□this□report,□all□of□ 
the□amplicons□but□one,□are□>250□bp,□with□the□largest□at□438□bp.□I□would□have□thought□that□the□detection□ 
rate□in□these□larger□amplicons□is□quite□low.□We□would□routinely□break□these□up□into□smaller□overlapping□ PCR□fragments,□or□digest□with□a□suitable□restriction□enzyme.□In□addition,□sensitivity□for□cold□SSCP□is□ enhanced□by□using□silver□staining,□rather□than□ethidium□bromide□staining.□I□would□suggest□that□the□ 
authors□outline□some□of□the□shortcomings□in□their□methodology,□particularly□when□dealing□with□a□small□ 
number□of□patient□samples,□however,□recognising□that□if□mutations□were□common□they□would□have□seen□ 
them.□ 
2.□Discretionary□revisions□ 
I□have□a□few□minor□comments□that□the□authors□may□consider□worth□revising.□Going□through□in□order.□ 
1.□Subjects.□As□women□with□premature□ovarian□failure□with□known□karyotype□abnormalities□were□ 
excluded□from□the□study,□perhaps□it□might□be□best□to□exclude□the□two□showing□partial□monosomy□of□the□ 
X□chromosome,□and□in□fact□not□even□mention□them,□and□assume□that□only□40□women□were□screened.□ 
XO□mosiacism□is□a□known□karyotypic□abnormality□in□premature□ovarian□failure.□ 
2.□Need□missing□details□of□Invitrogen□(under□SSCP□section).□ 
3.□Are□the□clinical□descriptions□of□the□two□women□with□partial□monosomy□worth□describing,□while□they□are□ 
interesting,□it□isnaETMt□really□part□of□the□central□focus□of□this□report□on□KIT.□ 
4.□Its□probably□more□appropriate□to□describe□the□intron□9□polymorphism□as□being□aEoe71□nucleotides□ 
upstreamaE□rather□than□71st.□ 
5.□It□is□worth□stating□that□RFLP□was□not□performed□on□the□intronic□polymorphisms□for□other□reasons□than□ there□not□being□a□suitable□restriction□site.□Other□methods□exist□to□screen□for□such□polymorphisms,□ including□creating□a□forced□RFLP□(FRFLP).□Was□it□possible□to□determine□zygousity□be□examining□the□ 
electropherograms?□For□the□patient□shown□in□figure□3,□presumably□you□can□suggest□from□the□ 
electropherogram□that□she□is□homozygous□for□the□shorter□allele□(unless□that□is□a□cloned□product).□ 
6.□Figure□1,□state□what□the□//□represent□(between□exons□1□and□2,□and□7□and□8),□presumably□they□indicate□ 
a□break□in□sequence□and□the□diagram□is□not□to□scale.□ 
7.□Figure□2,□perhaps□not□necessary,□only□to□state□that□single□bands□of□the□expected□size□were□amplified□in 
each□case.□ 
Competing□interests: 
None□declared.