JP2011234641A - Detection method for waardenburg anophthalmia syndrome - Google Patents
Detection method for waardenburg anophthalmia syndrome Download PDFInfo
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本発明は、Waardenburg無眼球症候群の検出方法に関する。 The present invention relates to a method for detecting Waardenburg annocular syndrome.
Waardenburg無眼球症候群 （OMIM % 206920、Waardenburg anophthalmia syndrome）は、四肢異常を伴う小眼球症（Microphthalmia with limb anomalies, MLA）あるいは眼肢端症候群（Ophthalmoacromelic syndrome）とも呼ばれ、眼球の無/低形成と四肢末端の異常を主徴とする、非常に稀な常染色体劣性遺伝性疾患である（以下MLAと呼ぶことがある)。1935年にWaardenbergが初めて紹介しこれまでに少なくとも 21家系 35症例 の報告がある。 MLAの多く（90％）は血族婚家系で認めら常染色体劣性遺伝性疾患であることが疑われる。MLA家系の3家系を対象にMLA責任遺伝子座マッピングを行い、10p11.23の422 kb領域が候補領域の一つとして疑われ、そこに唯一存在していたMPP7の変異の有無を確認したが変異は同定されなかった。よって10p11.23以外に責任遺伝子座が存在する可能性が想定された。 Waardenburg anophthalmia syndrome (OMIM% 206920, Waardenburg anophthalmia syndrome), also called microphthalmia with limb anomalies (MLA) or Ophthalmoacromelic syndrome, It is a very rare autosomal recessive disorder that is mainly caused by abnormalities in the extremities of the extremities (hereinafter sometimes referred to as MLA). First introduced by Waardenberg in 1935, so far there have been reports of at least 21 families and 35 cases. Many (90%) of MLA are suspected to be autosomal recessive inherited disorders found in blood family families. MLA responsible locus mapping was performed in three MLA families, and the 422 kb region of 10p11.23 was suspected as one of the candidate regions, and the presence or absence of a mutation in MPP7 that was present only was confirmed. Was not identified. Therefore, it was assumed that there might be a responsible locus other than 10p11.23.
本発明の目的は、遺伝子検査によるWaardenburg無眼球症候群の検出方法を提供することである。 An object of the present invention is to provide a method for detecting Waardenburg anoxia syndrome by genetic testing.
本願発明者は、下記実施例に具体的に記載する方法により、ホモ接合性マッピング及びハプロタイプマッピングを行った結果、Waardenburg無眼球症候群の責任遺伝子が染色体の14q24.1-q24.2にあるSMOC1遺伝子であることを突き止め、本発明を完成した。 As a result of homozygosity mapping and haplotype mapping by the method specifically described in the following examples, the inventor of the present application has found that the SMOC1 gene in which the responsible gene for Waardenburg eyeless syndrome is located on chromosome 14q24.1-q24.2 As a result, the present invention was completed.
すなわち、本発明は、ヒトから採取した細胞中のSMOC1遺伝子に機能喪失型の変異があるかどうかを調べることを含む、Waardenburg無眼球症候群の検出方法を提供する。 That is, the present invention provides a method for detecting Waardenburg anopia syndrome, which comprises examining whether or not there is a loss-of-function mutation in the SMOC1 gene in cells collected from humans.
本発明により、Waardenburg無眼球症候群の遺伝子検査による確定診断が初めて可能になった。 The present invention makes it possible for the first time to make a definitive diagnosis by genetic testing of the Waardenburg eyeless syndrome.
下記実施例に具体的に記載する方法により、ホモ接合性マッピング及びハプロタイプマッピングを行った結果、Waardenburg無眼球症候群（以下、単に「無眼球症候群」）の責任遺伝子が染色体の14q24.1-q24.2にあるSMOC1遺伝子であり、無眼球症候群患者では、SMOC1遺伝子に機能喪失型の変異が生じていることが明らかになった。 As a result of homozygosity mapping and haplotype mapping by the method specifically described in the following examples, the responsible gene for Waardenburg anomaly syndrome (hereinafter simply referred to as “anophthalmia syndrome”) is 14q24.1-q24. The SMOC1 gene found in No. 2 was found to have a loss-of-function mutation in the SMOC1 gene in patients with anophthalmia syndrome.
ヒトSMOC1遺伝子のゲノムＤＮＡの塩基配列及びこれがコードするアミノ酸配列は公知であり、例えば、NC_000014 REGION: 69415896..69568836のaccession No.でGenBankに登録されている。ヒトSMOC1遺伝子のゲノムＤＮＡの全長の塩基配列を配列表に記載すると長大なものになるので、本発明の実施に有用な部分のみ、すなわち、各エクソン及びそれらの前後の領域を取り出して配列番号１〜５に示す。配列番号１は、NC_000014 REGION: 69415896..69568836の5'末端から１番目〜1000番目(以下、これを「1-1000nt」のように示す。他の領域も同様）、配列番号２は72001〜75000nt、配列番号３は960001-99000nt、配列番号４は112001〜116000nt、配列番号５は、131001〜152941nt（3'末端）を示す。NC_000014 REGION: 69415896..69568836中、エクソン１は1-352nt、エクソン２は72713-72877nt、エクソン３は73995-74107nt、エクソン４は96290-96389nt、エクソン５は98493-98540nt、エクソン６は112992-113048nt、エクソン７は114975-115055nt、エクソン８は131329-131521nt、エクソン９は132060-132142、エクソン１０は133961-134066、エクソン１１は133778-144022nt、エクソン１２は150817(150820)-152941nt（括弧内はバリアント２）にある。従って、エクソン１は、配列番号１の1-352nt、エクソン２は配列番号２の713-878nt、エクソン３は配列番号２の1995-2107nt、エクソン４は配列番号３の290-389nt、エクソン５は配列番号３の2493-2540nt、エクソン６は配列番号４の992-1048nt、エクソン７は配列番号４の2975-3055nt、エクソン８は配列番号５の329-521nt、エクソン９は配列番号５の1060-1142nt、エクソン１０は配列番号５の2961-3066nt、エクソン１１は配列番号５の12778-13022nt、エクソン１２は配列番号５の19817 (19820)-21941nt（括弧内はバリアント２）にある。また、ヒトSMOC1遺伝子のcDNAの塩基配列及び該cDNAによりコードされるアミノ酸配列を配列番号６に示す。本発明の方法では、このSMOC1遺伝子に機能喪失型の変異が生じているか否かを調べる。ここで、機能喪失型の変異とは、少なくともいずれかのエクソン領域中にナンセンス突然変異、フレームシフト突然変異若しくはアミノ酸の置換を伴う、若しくは複数の点突然変異が存在すること又は少なくともいずれかのイントロン領域の少なくとも一方の端部の塩基が置換した変異を意味する。イントロン領域の少なくとも一方の端部の塩基が置換している場合、スプライシングに異常が生じ、正常な遺伝子産物が生産されない可能性がある。実施例で具体的に確認された変異は、c.718C>T(p.Gln240X)、c.664+1g>a及びc.378+1g>aである。ここで、c.718C>Tは、配列番号６中のコード領域の第718番目（すなわち、配列番号６の971ntのcがtに置換する点突然変異（すなわち、配列番号５の382ntのcがtに置換する点突然変異）であり、これにより、エクソン８中のGlnをコードするCAGが停止コドンTAGとなるナンセンス突然変異である。c.664+1g>aは、配列番号６中のコード領域の664番目の次の塩基（エクソン７の3'末端に隣接するイントロン中の塩基）、すなわち、配列番号４の3056ntのgがaに置換する点突然変異であり、これにより、エクソン７の下流に隣接するイントロンの3'末端のgがaに変異する。c.378+1g>aは、配列番号６中のコード領域の378番目の次の塩基のgがaに置換する点突然変異であり、これにより、エクソン３の下流に隣接するイントロンの3'末端のgがaに変異する。なお、上記のとおり、SMOC1遺伝子には、エクソン１２中の１つのコドン（配列番号６中の1544-1546ntのgta、すなわち、配列番号５中の19817-19819ntのtag)が欠失したバリアント２が知られているが、いずれのバリアントであっても本発明の方法に供することが可能である。なお、バリアント２のcDNAの塩基配列及びそのコード領域のアミノ酸配列を配列番号８及び９にそれぞれ示す。 The base sequence of the genomic DNA of human SMOC1 gene and the amino acid sequence encoded by it are known, and are registered in GenBank, for example, with an accession number of NC_000014 REGION: 69415896..69568836. Since the full length base sequence of the genomic DNA of the human SMOC1 gene is described in the sequence listing, it becomes long. Therefore, only the portions useful for the practice of the present invention, ie, each exon and the regions before and after them are taken out and SEQ ID NO: 1 Shown in ~ 5. SEQ ID NO: 1 is NC_000014 REGION: 69415896..69568836 1st to 1000th from the 5 ′ end (hereinafter, this is shown as “1-1000nt”, the same applies to other regions), and SEQ ID NO: 2 is 72001 to 75000 nt, SEQ ID NO: 3 is 960001-99000 nt, SEQ ID NO: 4 is 112001-116000 nt, SEQ ID NO: 5 is 131001-152941 nt (3 ′ end). NC_000014 REGION: 69415896..69568836, exon 1 is 1-352nt, exon 2 is 72713-72877nt, exon 3 is 73995-74107nt, exon 4 is 96290-96389nt, exon 5 is 98493-98540nt, exon 6 is 112992-113048nt , Exon 7 is 114975-115055nt, exon 8 is 131329-131521nt, exon 9 is 132060-132142, exon 10 is 133961-134066, exon 11 is 133778-144022nt, exon 12 is 150817 (150820) -152941nt 2). Therefore, exon 1 is 1 to 352 nt of SEQ ID NO: 1, exon 2 is 713 to 878 nt of SEQ ID NO: 2, exon 3 is 1995 to 2107 nt of SEQ ID NO: 2, exon 4 is 290 to 389 nt of SEQ ID NO: 3, and exon 5 is 2493-2540nt of SEQ ID NO: 3, exon 6 is 992-1048nt of SEQ ID NO: 4, exon 7 is 2975-3055nt of SEQ ID NO: 4, exon 8 is 329-521nt of SEQ ID NO: 5, and exon 9 is 1060- of SEQ ID NO: 5. 1142 nt, exon 10 is 2961-3066 nt of SEQ ID NO: 5, exon 11 is 12778-13022 nt of SEQ ID NO: 5, and exon 12 is 19187 (19820) -21941 nt of SEQ ID NO: 5 (variant 2 in parentheses). The nucleotide sequence of cDNA of human SMOC1 gene and the amino acid sequence encoded by the cDNA are shown in SEQ ID NO: 6. In the method of the present invention, it is examined whether or not a loss-of-function mutation has occurred in the SMOC1 gene. Here, the loss-of-function mutation refers to a nonsense mutation, a frameshift mutation or an amino acid substitution in at least any exon region, or a plurality of point mutations, or at least one intron. It means a mutation in which the base at at least one end of the region is substituted. When the base at at least one end of the intron region is substituted, splicing may be abnormal, and a normal gene product may not be produced. The mutations specifically identified in the examples are c.718C> T (p.Gln240X), c.664 + 1g> a and c.378 + 1g> a. Here, c.718C> T is the 718th position of the coding region in SEQ ID NO: 6 (that is, a point mutation in which 971 nt c in SEQ ID NO: 6 is replaced with t (ie, 382 nt c in SEQ ID NO: 5 is This is a nonsense mutation in which CAG encoding Gln in exon 8 becomes a stop codon TAG, and c.664 + 1g> a is the code in SEQ ID NO: 6. A point mutation in which the 664th next base of the region (the base in the intron adjacent to the 3 ′ end of exon 7), that is, 3056 nt g of SEQ ID NO: 4, is replaced by a. G at the 3 ′ end of the adjacent intron adjacent to the downstream is mutated to a. C.378 + 1g> a is a point mutation in which g of the 378th base of the coding region in SEQ ID NO: 6 is replaced with a. As a result, the g at the 3 ′ end of the intron adjacent to the downstream of exon 3 is mutated to a. The SMOC1 gene is known to have variant 2 in which one codon in exon 12 (1544-1546 nt gta in SEQ ID NO: 6, ie, 19817-19819 nt tag in SEQ ID NO: 5) has been deleted. Any variant can be used for the method of the present invention, and the nucleotide sequence of the variant 2 cDNA and the amino acid sequence of its coding region are shown in SEQ ID NOs: 8 and 9, respectively.
これら３種類の変異は、居住地が大きく異なる無関係な３つの家系で独立に見出されたもので、それぞれの家系において偶発的に発生した変異であると考えられる。これらの３種の無関係なSMOC1中の機能喪失型変異により無眼球症候群が生じるので、SMOC1遺伝子中の天然に生じる他の機能喪失型変異であっても無眼球症候群を生じると考えられる。従って、本発明の方法は、上記した３種の変異の検出に限定されず、SMOC1遺伝子中の天然に生じる他の機能喪失型変異を検出することも本発明の範囲に包含される。 These three types of mutations were found independently in three unrelated families that differ greatly in residence, and are considered to have occurred accidentally in each family. Since these three unrelated loss-of-function mutations in SMOC1 cause an eyeless syndrome, even other naturally occurring loss-of-function mutations in the SMOC1 gene are thought to cause an eyeless syndrome. Therefore, the method of the present invention is not limited to the detection of the three types of mutations described above, and detection of other naturally occurring loss-of-function mutations in the SMOC1 gene is also included in the scope of the present invention.
本発明の方法では、ヒトから採取した細胞中のSMOC1遺伝子に機能喪失型の変異があるかどうかを調べる。これらの変異は、ゲノムＤＮＡ中の変異であるので、被検試料となる細胞は、染色体を含む細胞であればいずれの細胞であってもよく、採取の容易性から末梢血白血球等の末梢血中の細胞が好都合であるがこれらに限定されるものではない。また、羊水や絨毛を検体とすることもでき、この場合には胎児の無眼球症候群を検出することができる。なお、細胞からのゲノムＤＮＡの抽出は、そのための市販のキットを用いて周知の常法により行うことができる。 In the method of the present invention, it is examined whether there is a loss-of-function mutation in the SMOC1 gene in cells collected from humans. Since these mutations are mutations in genomic DNA, the cells to be tested may be any cells as long as they contain chromosomes. Peripheral blood such as peripheral blood leukocytes can be collected for ease of collection. Medium cells are convenient, but not limited to. In addition, amniotic fluid and villi can be used as specimens, and in this case, fetal anophthalmia syndrome can be detected. Extraction of genomic DNA from cells can be performed by a well-known conventional method using a commercially available kit for that purpose.
ゲノムＤＮＡにおける、SMOC1遺伝子中の機能喪失型変異の検出は、SMOC1遺伝子の各エクソン及びその隣接領域をＰＣＲ等の核酸増幅法により増幅し、ダイレクトシーケンス法により増幅産物の塩基配列を決定することにより行うことができる。SMOC1遺伝子の各エクソン及びその隣接領域の塩基配列は上記のとおり公知であるので、これらの配列を元に、核酸増幅に用いるプライマーを適宜設定することができる。なお、利用可能なプライマーの具体的な塩基配列は、下記実施例にも記載されている。ダイレクトシークエンス法は、市販の装置及びキットを用いた周知の常法により行うことができる。 Detection of loss-of-function mutations in the SMOC1 gene in genomic DNA is accomplished by amplifying each exon of the SMOC1 gene and its adjacent region by a nucleic acid amplification method such as PCR, and determining the base sequence of the amplified product by the direct sequence method. It can be carried out. Since the base sequences of each exon of SMOC1 gene and its adjacent region are known as described above, primers used for nucleic acid amplification can be appropriately set based on these sequences. In addition, the specific base sequence of the primer which can be utilized is also described in the following Example. The direct sequencing method can be performed by a well-known ordinary method using a commercially available apparatus and kit.
また、実施例で具体的に検出された、特定の１又は複数の変異を検出する場合には、上記したダイレクトシークエンス法の他に、変異部分を含むプローブを用いて被検ＤＮＡがハイブリダイズするか否かを調べる方法や、変異部分を含むプライマーを用いた核酸増幅法により増幅が起きるか否かを調べる方法等を採用することもできる。前者の方法では、変異した点を含む領域をプローブとして基板上に固定化したＤＮＡチップを用いることができる。これらの手法はいずれも周知であり、検出すべき変異及びその周辺の塩基配列がわかっていれば容易に実施可能である。 In addition, in the case of detecting one or more specific mutations specifically detected in the examples, in addition to the direct sequencing method described above, the test DNA is hybridized using a probe containing a mutated portion. It is also possible to employ a method for examining whether or not amplification occurs by a nucleic acid amplification method using a primer containing a mutated portion. In the former method, a DNA chip immobilized on a substrate using a region containing a mutated point as a probe can be used. All of these techniques are well known, and can be easily carried out if the mutation to be detected and the surrounding base sequence are known.
無眼球症候群は、機能喪失型変異を有するSMOC1遺伝子がホモ接合になった場合に生じるので、上記方法により、機能喪失型変異を有するSMOC1遺伝子がホモ接合になっているかどうかを調べることにより無眼球症候群を検出することができる。上記した方法により、機能喪失型変異を有するSMOC1遺伝子が両アリルに検出された場合にはホモ接合（あるいは複合へテロ接合）、機能喪失型変異を有するSMOC1遺伝子と正常型遺伝子の両者が検出された場合にはヘテロ接合である。 Since the eyeless syndrome occurs when the SMOC1 gene having a loss-of-function mutation becomes homozygous, the above method is used to determine whether the SMOC1 gene having a loss-of-function mutation is homozygous. Syndrome can be detected. When the SMOC1 gene having a loss-of-function mutation is detected in both alleles by the above method, both the SMOC1 gene having a loss-of-function mutation and a normal gene are detected. In this case, it is a heterojunction.
以下、本発明を実施例に基づきより具体的に説明する。 Hereinafter, the present invention will be described more specifically based on examples.
１．材料と方法
(1)症例
臨床的にMLAと診断された症例を有する4家系を対象にした（日本人沖縄県家系1、レバノン人家系2、トルコ人家系1）。合計で5名のMLA症例と14名の非罹患者より末梢血白血球からQuickGene 610-L (FUJIFILM)を用いてゲノムDNAを抽出し解析に用いた。
1. Materials and methods
(1) Cases Four families with clinically diagnosed MLA were targeted (Japanese Okinawa family 1, Lebanese family 2, Turkish family 1). Genomic DNA was extracted from peripheral blood leukocytes from 5 MLA cases and 14 unaffected individuals using QuickGene 610-L (FUJIFILM) and used for analysis.
(2) ホモ接合性マッピングおよびハプロタイプ解析
GeneChip Human Mapping 50K Array Xbal (商品名、Affymetrix社)を用いて全ゲノムSNPタイピングを施行した。Affymetrix社推奨プロトコールに従いタイピングを行った。50K Arrayの結果を元にA家系では症例に於いてホモ接合性領域をほとんど認めず複合ヘテロ接合性変異により発症していることが疑われた。A家系では罹患者2例で共通で認められ非罹患者では認めないハプロタイプを有する領域（共通ハプロタイプ領域）と家系B、CおよびXで認めた同祖性領域（IBD region）の共通領域を探索するも全家系で共通する領域は認めなかったためA,B,CとXの中であらゆる3家系の組み合わせでの共通領域を探索し、Bを除く3家系で共通の14q24.1-q24.2の領域に注目、追加マイクロサテライトマーカー（表１）にてタイピング、そして共通責任領域3.0 Mbに絞り込んだ。
(2) Homozygous mapping and haplotype analysis
Whole genome SNP typing was performed using GeneChip Human Mapping 50K Array Xbal (trade name, Affymetrix). Typing was performed according to Affymetrix recommended protocol. Based on the results of the 50K Array, it was suspected that in the A family, there were almost no homozygous regions in the case, and it was suspected to be caused by a complex heterozygous mutation. In A family, search for common areas between haplotypes common to two affected individuals but not afflicted (common haplotype regions) and homozygous regions found in families B, C, and X (IBD regions) However, since no common areas were found in all families, a common area was searched for combinations of all three families in A, B, C, and X, and 14q24.1-q24.2 common to all three families except B Focused on the area, typing with additional microsatellite markers (Table 1), and narrowed down to the common responsibility area of 3.0 Mb.
(3) 変異解析
3.0 Mbの領域内には少なくとも24個の遺伝子がマップされていた。この内RAD51L1, ACTN1, ERH, SFRS5, WDR22, COX16, EXDL2, GALNTL1, SLC39A9, KIAA0247, MED6, TTC9, MAP3K9, SMOC1の変異解析を3家系で行った。全ての遺伝子のタンパク質翻訳領域のエクソンとエクソンイントロン境界領域をPCR法にて増幅し、精製後ダイレクトシーケンス法で塩基配列を決定した。PCR反応液は、1×ExTaq buffer, 0.2 mM each dNTP, 0.2 μM each primer, 0.25 U Ex TaqHS polymerase (TAKARA) もしくは1×GC buffer II, 0.2 mM each dNTP, 0.2 μM each primer, 0.25 U LA Taq polymerase (TAKARA)もしくは1x Buffer for KOD plus, 0.2 mM each dNTP, 1.0 mM MgSO4, 0.30 μM each primer, 0.4U KOD -plus- (商品名、TOYOBO) の組成である。反応条件と用いたプライマーのシーケンスを表２に示す。PCR産物をExoSAP-IT (GE healthcare)で精製後、BigDye Terminator chemistry version 3 (商品名、Applied Biosystems) を用いてサイクルシーケンス反応を行った。反応物はSephadex G-50 (商品名、GE healthcare) とMultiscreen-96 (商品名、Millipore)によるゲル濾過にて精製し、ABI Genetic Analyzer 3100 (商品名、Applied Biosystems) でシーケンスを得た。得られたシーケンスは、Seqscape software ver. 2.1 (商品名、Applied Biosystems)を用いて変異の有無について解析を行った。
(3) Mutation analysis
At least 24 genes were mapped within the 3.0 Mb region. Among them, mutation analysis of RAD51L1, ACTN1, ERH, SFRS5, WDR22, COX16, EXDL2, GALNTL1, SLC39A9, KIAA0247, MED6, TTC9, MAP3K9 and SMOC1 was conducted in three families. Exon and exon intron border regions of protein translation regions of all genes were amplified by PCR, and after purification, nucleotide sequences were determined by direct sequencing. PCR reaction solution is 1 × ExTaq buffer, 0.2 mM each dNTP, 0.2 μM each primer, 0.25 U Ex TaqHS polymerase (TAKARA) or 1 × GC buffer II, 0.2 mM each dNTP, 0.2 μM each primer, 0.25 U LA Taq polymerase (TAKARA) or 1x Buffer for KOD plus, 0.2 mM each dNTP, 1.0 mM MgSO 4 , 0.30 μM each primer, 0.4 U KOD -plus- (trade name, TOYOBO). Table 2 shows the reaction conditions and the primer sequences used. The PCR product was purified by ExoSAP-IT (GE healthcare) and then subjected to cycle sequence reaction using BigDye Terminator chemistry version 3 (trade name, Applied Biosystems). The reaction product was purified by gel filtration using Sephadex G-50 (trade name, GE healthcare) and Multiscreen-96 (trade name, Millipore), and a sequence was obtained using ABI Genetic Analyzer 3100 (trade name, Applied Biosystems). The obtained sequences were analyzed for the presence or absence of mutations using Seqscape software ver. 2.1 (trade name, Applied Biosystems).
２．結果
日本人家系1例（A）、レバノン人家系2例（B, C）、トルコ人家系1例（X）(2)を対象に、ホモ接合性マッピング（あるいは共通ハプロタイプによるマッピング）を行った。マッピングにはGeneChip Human Mapping 50 K Array Xba I （Affymetrix社）を用いた。対象４家系で共通の責任遺伝子座候補領域は存在しなかったため、解析対象の家系で遺伝的異質性が存在する可能性があると考え、４家系中３家系の様々な組み合わせで共通の責任遺伝子座を探索したところ14q24.1-q24.2が、家系A, C, Xに共通責任遺伝子座である可能性が示唆された。さらに蛍光ラベルしたサテライトマーカーを追加し（表１）、詳細なマッピングを行い、AFM114YH10〜ch14-STS6 (UCSC coordinates, GRCh37: chromosome 14: 71347694-71347908 bp)の3.0 Mbを候補領域として絞り込んだ（図１）。この3.0 Mbの領域内には少なくとも24個の遺伝子がマップされていた。この内RAD51L1, ACTN1, ERH, SFRS5, WDR22, COX16, EXDL2, GALNTL1, SLC39A9, KIAA0247, MED6, TTC9, MAP3K9の変異解析を3家系で行うも変異は同定されなかったが、SMOC1において全家系にホモ接合性変異を認めた：c.718C>T (p.Gln240X) （日本人家系 A）, c.664+1g>a （レバノン人家系C）, c.378+1g>a （トルコ人家系X）（図２）。C.718C>T変異は沖縄県人100例を含む日本人289人の正常対照では認めなかった（578アリル）。他の２つの変異は同じ民族背景を持つ正常対照での確認は行っていないが、スプライシングドナーサイトの1番目の塩基異常であり、病的意義は明らかである。他の家系AではSMOC1変異の周辺のハプロタイプは父方アリルと母方アリルで完全に異なるため同祖性（同じ祖先から伝わること）は考えにくく偶然同じ変異が別々に生じた可能性が高い。
2. Results Homozygous mapping (or mapping with a common haplotype) was performed in one Japanese family (A), two Lebanese families (B, C), and one Turkish family (X) (2). . For mapping, GeneChip Human Mapping 50 K Array Xba I (Affymetrix) was used. Since there was no common responsible locus candidate region in the four families, it is considered that there may be genetic heterogeneity in the family to be analyzed. Common responsible genes in various combinations of three families in the four families A search for a locus suggested that 14q24.1-q24.2 might be a common responsible locus for families A, C, and X. Furthermore, satellite markers labeled with fluorescence were added (Table 1), detailed mapping was performed, and 3.0 Mb from AFM114YH10 to ch14-STS6 (UCSC coordinates, GRCh37: chromosome 14: 71347694-71347908 bp) was narrowed down as a candidate region (Fig. 1). 1). At least 24 genes were mapped within this 3.0 Mb region. Among these, RAD51L1, ACTN1, ERH, SFRS5, WDR22, COX16, EXDL2, GALNTL1, SLC39A9, KIAA0247, MED6, TTC9, and MAP3K9 mutation analysis were performed in three families, but no mutation was identified. Conjugative mutations were found: c.718C> T (p.Gln240X) (Japanese family A), c.664 + 1g> a (Lebanese family C), c.378 + 1g> a (Turkish family X) (FIG. 2). The C.718C> T mutation was not observed in normal controls of 289 Japanese including 100 Okinawans (578 alleles). The other two mutations have not been confirmed in normal controls with the same ethnic background, but are the first base abnormality at the splicing donor site, and the pathological significance is clear. In other family A, the haplotypes around the SMOC1 mutation are completely different between the paternal allele and the maternal allele, so it is unlikely that homosexuality (transmitted from the same ancestor) is likely and the same mutation is likely to have occurred by chance.
SMOC1の遺伝子発現を観察するためマウス胎仔whole mount in situ hybridizationを行い、E10.5〜E11.5に眼組織に（E10.5では眼胞と眼茎、E11.5では眼杯腹側）、E10.5-E13.5に肢芽~手に極めて特徴的は発現パターンを認め（図３）、ヒトに於いてもMLAの罹患部位（眼と四肢末端）に同様に発現している可能性が高いためSMOC1遺伝子の変異によりMLAが発症することが支持される。 In order to observe the gene expression of SMOC1, mouse fetus whole mount in situ hybridization was performed, and in E10.5 to E11.5 in the eye tissue (E10.5 in the optic vesicle and eye stem, E11.5 in the eye cup) E10.5-E13.5 has a very characteristic expression pattern in the limb bud to hand (Fig. 3), and in humans, it may be expressed in the affected area (eye and extremity) of MLA as well. Therefore, it is supported that MLA develops due to mutations in the SMOC1 gene.
以上の結果からSMOC1遺伝子変異により無眼球症候群が発症していると考えられる。 Based on the above results, it is considered that the eyeless syndrome has developed due to the SMOC1 gene mutation.
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