# Patent

## Title
Nucleic acid, probe comprising the nucleic acid and screening method using the probe

## Abstract

A nucleic acid is (a) a nucleic acid comprising a base sequence shown in base numbers 1–39726 of SEQ ID NO: 1, or (b) a nucleic acid wherein a part of the bases 1–39726 of SEQ ID NO: 1 is deleted, substituted or added, and having a homology of 80% for the base sequence. Also, a probe comprises the above nucleic acid, and a screening is carried out by using such a probe.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a nucleic acid, a probe and a screening method using such a probe, and more particularly to a nucleic acid, a probe comprising the nucleic acid and a screening method using the probe for a gene diagnosis.
2. Description of Related Art
As a gene mapping method of positioning a human genome on a chromosome to make a chromosomal map, there have been popularly practiced a method wherein a remaining specific human chromosome or a part of the chromosome is analyzed by using a clone panel of a somatic cell hybrid or a partially deletion chromosome between a human and a rodent, and a method wherein a cloned gene is used as a probe in a Southern blotting.
Lately, there is used a so-called in situ hybridization wherein a gene or DNA marker cloned and labeled from genome DNA is used as a probe and a molecule hybrid is directly formed in a chromosome sample on a slide glass to detect a gene existing part in order to efficiently obtain information on specified sites of many genes such as human genes and a mutual sequence order.
As the in situ hybridization, there are a method wherein DNA labeled with a radioisotope (mainly 3H) is used as a probe and its site is detected by an autoradiography, and a method wherein a fluorescence signal of a labeled DNA probe is detected by means of a fluorescence microscope.
The latter fluorescence in situ hybridization method (hereinafter referred to as FISH method) is excellent in points that an RI equipment is useless, and an operating procedure is simple, and a minute mapping is exactly carried out on a chromosome band for a short time (2 days).
At present, probes useful for various diseases/syndromes are developed for using these methods to make a clinic diagnosis. For example, there is known to be an inspection probe suitable for the FISH method on various chromosomes aimed at a syndrome having a microdeletion of a congenital anomaly syndrome. Such a probe inspects a gene of a target disease, and if an inspection result is positive (i.e. a deletion or the like is existent), the confirmed diagnosis is made.
For example, the above probe is effective for a Prader Willi syndrome because about 60% of patients have a deletion.
As mentioned above, useful probes have been found in a part of cloning of genes for the diseases. However, there are still idiopathic diseases, and inspection and diagnosis methods effective thereto are not found except for physical findings and symptoms of the disease. If a probe can be found by using a gene and/or a homolog resulted in these diseases, it becomes very beneficial in the filed of a gene diagnosis.
SUMMARY OF THE INVENTION
It is, therefore, an object of the invention to provide a nucleic acid, a useful probe using the nucleic acid and a screening method using such a probe.
In order to achieve the above object, the inventors have made various studies with respect to a relationship between a congenital anomaly syndrome and a gene and found out a probe and a screening method using such a probe according to the invention.
The nucleic acid according to a first aspect of the invention is (a) a nucleic acid comprising a base sequence shown in base numbers 1–39726 of SEQ ID NO: 1, or (b) a nucleic acid wherein a part of the bases 1–39726 of SEQ ID NO: 1 is deleted, substituted or added, and the nucleic acid has a homology of 80% for the base sequence.
Furthermore, the nucleic acid according to a second aspect of the invention is (a) a nucleic acid comprising a base sequence shown in base numbers 1–8511 of SEQ ID NO: 2, or (b) a nucleic acid wherein a part of the bases 1–8511 of SEQ ID NO: 2 is deleted, substituted or added, and the nucleic acid has a homology of 80% for the base sequence.
The probe according to a third aspect of the invention comprises the nucleic acid as defined in the first or second aspect of the invention.
In a preferable embodiment, the probe according to the invention is used as a diagnosis for Sotos syndrome.
The peptide fragment according to a fourth aspect of the invention is (a) a peptide fragment comprising an amino acid sequence shown in amino acid numbers 1–309 of SEQ ID NO: 3, or (b) a peptide fragment wherein a part of the amino acid sequence shown in SEQ ID NO: 3 is deleted, substituted or added, and the peptide fragment has a homology of 80% for the base sequence.
The peptide fragment according to a fifth aspect of the invention is (a) a peptide fragment comprising an amino acid sequence shown in amino acid numbers 1–2696 of SEQ ID NO: 4, or (b) a peptide fragment wherein a part of the amino acid sequence shown in SEQ ID NO: 4 is deleted, substituted or added, and the peptide fragment has a homology of 80% for the base sequence.
The probe according to a sixth aspect of the invention comprises the peptide fragment as defined in the fourth or fifth aspect of the invention.
A probe for using as a diagnosis for Sotos syndrome according to the invention, is characterized in that the probe comprises the following (a) or (b) on the human chromosome 5:

    
    
        (a) a nucleic acid comprising a base sequence shown in base numbers 1–190 of SEQ ID NO: 5, or
        (b) a nucleic acid wherein a part of the bases 1–190 of SEQ ID NO: 5 is deleted, substituted or added, and having a homology of 80% for the base sequence.
    
    


A probe for using as a diagnosis for Sotos syndrome according to the invention, is characterized in that the probe comprises the following (a) or (b) on the human chromosome 5:

    
    
        (a) a nucleic acid comprising a base sequence shown in base numbers 1–275 of SEQ ID NO: 6,or
        (b) a nucleic acid wherein a part of the bases 1–275 of SEQ ID NO: 6 is deleted, substituted or added, and having a homology of 80% for the base sequence.
    
    


A probe for using as a diagnosis for Sotos syndrome according to the invention, is characterized in that the probe comprises any sequence existed in between the following (a) and (b) on the human chromosome 5:

    
    
        (a) a nucleic acid comprising a base sequence shown in base numbers 1–190 of SEQ ID NO: 5, and
        (b) a nucleic acid comprising a base sequence shown in bases 1–275 of SEQ ID NO: 6.
    
    


The screening method according to a seventh aspect of the invention is characterized by using the probe as defined in the third, forth or seventh to tenth aspect of the invention.
In a preferable embodiment of the screening method according to the invention, the screening is carried out by using at least one selected from the group consisting of an in situ hybridization method, a Southern blotting method, a macroarray-based hybridization method and a base sequence determination method (Dideoxy chain termination method and the like).
In a preferable embodiment of the screening method according to the invention, the in situ hybridization method is a fluorescence in situ hybridization method.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The invention will be described with reference to the accompanying drawings, wherein:
FIG. 1 is a view showing a physical mapping;
FIG. 2 is a view showing various mutations of NSD1;
FIG. 3 is a view showing results on a direct sequence determination in an immortalizing lymphoblast cell line of a patient;
FIG. 4 is a view showing a result of FISH analysis.



DETAILED DESCRIPTION OF THE INVENTION
First of all, the nucleic acid according to the invention is explained. The nucleic acid according to the invention is (a) a nucleic acid comprising a base sequence shown in base numbers 1–39726 of SEQ ID NO: 1, or (b) a nucleic acid wherein a part of base numbers 1–39726 of SEQ ID NO: 1 is deleted, substituted or added, and the nucleic acid has a homology of 80%, preferably 90%, more preferably 95% for the base sequence. Such a nucleic acid is derived from NSD1 of a human chromosome 5 and is complementary with a part of the NSD1. Concretely, it is a nucleic acid complementary with a genomic DNA including an exon 1, an exon 2 and an intron of the NSD1. Also, the nucleic acid according to the invention includes a nucleic acid wherein a part of base numbers 1–39726 of SEQ ID NO: 1 is deleted, substituted or added, and the nucleic acid has a homology of 80%, preferably 90%, more preferably 95% for the base sequence. Even if the part is deleted, substituted or added, the latter nucleic acid can be utilized as a probe as mentioned later.
Further, the nucleic acid according to the invention comprises a base sequence shown in base numbers 1–8511 of SEQ ID NO: 2. Such a nucleic acid is derived from NSD1 of a human chromosome 5, and is complementary with a part of the NSD1. Concretely, this is a nucleic acid complementary with a cDNA including exons 1–23 of the NSD1. Also, the nucleic acid according to the invention includes a nucleic acid wherein a part of the base numbers 1–8511 of SEQ ID NO: 2 is deleted, substituted or added, and the nucleic acid has a homology of 80%, preferably 90%, more preferably 95% for the base sequence.
In addition, an amino acid sequence corresponding to a base sequence shown in SEQ ID NO: 2 is as follows. Such an amino acid sequence is shown in SEQ ID NO: 4. A translation region of the base sequence in SEQ ID NO: 1, i.e. an amino acid sequence corresponding to the second exon of the NSD1 is shown in SEQ ID NO: 3.
The peptide fragment according to the invention is (a) a peptide fragment comprising an amino acid sequence shown in amino acid numbers 1–309 of SEQ ID NO: 3, or (b) a peptide fragment wherein a part of the amino acids in SEQ ID NO: 3 is deleted, substituted or added, and the amino acid sequence has a homology of 80%, preferably 90%, more preferably 95% for the amino acid sequence. Also, the peptide fragment according to the invention is (a) a peptide fragment comprising an amino acid sequence shown in amino acid numbers 1–2696 of SEQ ID NO: 4, or (b) a peptide fragment wherein a part of the amino acids in SEQ ID NO: 4 is deleted, substituted or added, and the amino acid sequence has a homology of 80%, preferably 90%, more preferably 95% for the amino acid sequence. Such amino acid sequences can be used for an immunologic test utilizing an antigen antibody reaction.
With respect to the aforementioned nucleic acids, a purification and isolation method will be described below. The above nucleic acid, not particularly limited, can be purified and isolated by the following procedure. This nucleic acid includes exons 1 and 2 of NSD1 and is obtained by subcloning a PAC clone called a RP1-118m12 according to the following method. At first, a PAC DNA is purified. Then, the purified PAC DNA is isolated by using a Midi-Prep column (Qiagen, Chatsworth, Calif.) and partially digested by using a restriction enzyme such as Sau3AI. Next, a SuperCos1 cosmid vector is prepared according to manufacturer's instructions (Stratagene, La Jolla, Calif.) and ligated to the digested PAC DNA using T4DNA ligase. The ligation reaction products are packaged using the Gigapack III gold extract (Stratagene) and transfected into XL1-Blue MR host cells. A genomic sequence is constructed by means of STS content mapping using a PCR. Thus, a nucleic acid according to the invention can be obtained.
Moreover, a base sequence of DNA can be determined by any method well known in those skilled in the art. For example, the base sequence can be determined by using a terminator method or the like.
As a method of using the probe according to the invention, the above nucleic acid is amplified directly or by a PCR method and immobilized by blotting on a polymer membrane and then subjected to a hybridization. The hybridization is not particularly limited according to usual manner, but may include, for example, a Southern blotting method, an in situ hybridization method, a microarray-based method and a base sequence determination method. The in situ hybridization method is preferable from a viewpoint of a quick and accurate screening. As the in situ hybridization method, there are a fluorescence in situ hybridization method (hereinafter referred to as FISH method), a radioisotope in situ hybridization method and the like. The FISH method is preferable from a viewpoint that an RI equipment is not required. In the FISH method, it is general that a chromosome sample is prepared on a slide glass, hybridized with a labelled probe, and directly investigated by a microscope.
As a support medium used in the hybridization of the probe according to the invention, mention may be made of a thin film, a powder, a particulate matter, a gel, a bead, a fiber, a glass, a dispersion liquid, an emulsion and so on. They may be used by filling into an adequate column. Among them, the thin film such as a nitrocellulose film or a nylon film is preferable.
An example of the label used in the probe according to the invention is explained. As the label, use may be made of ones well known by persons skilled in the art. The label is not particularly limited, but includes, for example, a radioactive atom such as 32P, 35S or the like, a biotin group, an avidin group, an enzyme, a fluorescence label and so on. In the case of utilizing an antigen-antibody system, the label may contain an antigen, which is included within the scope of the invention.
The nucleic acid according to the invention complementarily bonds with a part of a normal chromosome 5. However, if the chromosome 5 has an abnormality, that is, a deletion abnormality, the nucleic acid according to the invention does not bond with such an abnormal chromosome 5. When the nucleic acid according to the invention is used as a probe by utilizing the above property, the abnormality of the chromosome 5 can be confirmed. Therefore, the prove according to the invention can also be used as a diagnostic probe for a Sotos syndrome having a deletion of the chromosome 5 in 60% of patients with Sotos syndrome.
A probe for using as a diagnosis for Sotos syndrome according to the invention, comprises the following (a) or (b) on the human chromosome 5:

    
    
        (a) a nucleic acid comprising a base sequence shown in base numbers 1–190 of SEQ ID NO: 5, or
        (b) a nucleic acid wherein a part of the bases 1–190 of SEQ ID NO: 5 is deleted, substituted or added, and having a homology of 80% for the base sequence.
    
    


A probe for using as a diagnosis for Sotos syndrome according to the invention, comprises the following (a) or (b) on the human chromosome 5:

    
    
        (a) a nucleic acid comprising a base sequence shown in base numbers 1–275 of SEQ ID NO: 6, or
        (b) a nucleic acid wherein a part of the bases 1–275 of SEQ ID NO: 6 is deleted, substituted or added, and having a homology of 80% for the base sequence.
    
    


A probe for using as a diagnosis for Sotos syndrome according to the invention, comprises any sequence existing between the following (a) and (b) on the human chromosome 5:

    
    
        (a) a nucleic acid comprising a base sequence shown in base numbers 1–190 of SEQ ID NO: 5, and
        (b) a nucleic acid comprising a base sequence shown in base numbers 1–275 of SEQ ID NO: 6.
    
    


These probes can be used for this is dependent on a finding from the inventors in that the case sequence is deleted, which existed in between a sequence as in SEQ ID NO: 5 and a sequence as in SEQ ID NO: 6 on the chromosome 5 of a patient suffered from Sotos syndrome. Therefore, the base sequence of SEQ ID NO: 5 may be used by itself as a probe, the base sequence of SEQ ID NO: 6 may be used by itself as a probe. Also, since a sequence existed in a normal human at between the above SEQ ID NO: 5 and SEQ ID NO: 6 does not exist in a patient suffering from Sotos syndrome, any sequences of them may be used for a diagnosis of Sotos syndrome. A base length of the probe, depending on the method of screening, is not particularly limited, but 5 kb or more than 5 kb. Particularly, 5–15 kb is preferable, 7–12 kb is more preferable.
Although the sequence of the above SEQ ID NO: 5 and SEQ ID NO: 6 exists in the normal human, since 1 copy of the chromosome of a patient suffered from Sotos syndrome is deleted, a gene diagnosis may be carried out by these fact.
The following examples are given in illustration of the invention and are not intended as limitations thereof.
The following examples are intended to illustrate an embodiment of the invention, and it is naturally possible to properly change the invention without departing from the spirit and the scope of the invention claimed in claims.
EXAMPLE 1
Sotos syndrome (SS, OMIM 117550), also known as cerebral gigantism, is a neurological disorder characterized by prenatal-childhood overgrowth with advanced bone age, a peculiar face with large skull, acromegalic features and pointed chin, occasional brain anomalies and seizures, and mental retardation.
Although most cases are sporadic, occasional parent-child transmissions of the disease are known. SS patients are estimated to have a 3.9% risk of benign/malignant tumors. Firstly, NSD1 gene is isolated by positional cloning from the 5q35 breakpoint of a patient with a de novo t(5;8)(q35;q24.1). The gene encodes 2,696 amino acids with SET, PHD finger and PWWP domains, and interacts with nuclear receptors (NRs). Among 38 SS patients examined by direct sequencing, four de novo point mutations including a nonsense mutation (1310C->A, S437X), a one-bp deletion (3536delA), a one-bp insertion (5998insT) and a splice-donor-site mutation (6135+1G->A) are detected in NSD1 (10.5%). Also, 20 submicroscopic deletions (66.7%) involving NSD1 are identified in 30 SS patients whose metaphase and/or interphase cells are available for FISH studies. Overall, 77% of SS patients are estimated to have NSD1 mutations. These results indicate that haploinsufficiency of NSD1 causes Sotos syndrome and NSD1 plays a significant role in growth and brain development in human.
There is adopted a girl with Sotos syndrome (SS) associated with de novo reciprocal translocation, 46, XX, t(5;8)(q35;q24.1) (patient BP). A 5q35 region is likely to harbor a gene locus for SS, because there are another translocation [t(5;15)(q35;q24.1)] and a deletion involving 5q35[del(5)(q35.1qter)] associated with SS or SS-like features, respectively. Therefore, the 5q35 breakpoint is cloned. At first, FGFR4 is focused, as it is mapped to 5q35-qter and mutations in its gene family are known to be associated with some craniofacial and growth abnormalities. Then, a PAC clone (RP1-251c21) covering FGFR4 is isolated, and it is confirmed that it covers the 5q35 breakpoint by FISH analysis. Thereafter, a BAC/PAC/cosmid-based physical map covering the breakpoint is constructed to validate additional genes around the breakpoint (FIG. 1a). The FISH analysis shows that RP1-118m12 and its cosmid subclones (c2b,c4D,c6A, and c6B) span the breakpoint (FIG. 1b). Another gene, JAZ (Genbank Accession No. XM 012279) is identified through draft/complete sequences (AC027314 for CTC-286c20 and AC008570 for CTC-549a4) within the physical map. The coding regions of JAZ are sequenced in the 12 patients, but no nucleotide change is found, either. BlastN searches using a partial genomic sequence (AC02314) corresponding to c6B identified sequences homologous to the mouse Nsd1 gene. Since Nsd1 encoding a nuclear receptor (NR) binding protein is possibly related to transcriptional regulation, its human ortholog, NSD1 becomes an attractive candidate gene, and is isolated. The isolated NSD1 has an 8,088-bp open reading frame and consists of at least 23 exons (Genbank accession No. AF395588).
The breakpoint is located within NSD1, because the FISH analysis using c6B gives split signals on both der(5) and der(8) chromosomes of the patient and each signal intensity is almost even (FIGS. 1a and b). FIG. 1a shows a physical map, wherein a heavy black line indicates BAC/PAC cosmid clones and red and blue lines indicate a clone covering the 5q35 breakpoint by FISH analysis, and complete genome sequences, respectively. Arrows indicate candidate genes. FIG. 1b shows a FISH analysis using a cosmid, c6B on chromosomes of the patient BP, wherein red and yellow arrows indicate signals on normal chromosome 5, and derivative chromosomes 5 and 8, respectively. Forty sets of primers are designed to amplify the entire coding region of the gene. A genomic DNA from a total of 38 unrelated normal patients with SS is analyzed by direct sequencing.
Then, four different point mutations of NSD1 in four of 39 patients including patient BP is identified (FIG. 2, Table 1). FIG. 2 shows a genomic structure of NSD1 and mutations detected in the patients with Sotos syndrome. A particular region is shown as a colored box, wherein light blue is an NR-interaction domain (NID−L), pink is an NR-interaction domain (NID+L), blue is PHD finger domains, green is PWWP domains, and red is a SET domain. The mutations include a nonsense mutation (1310C->A, S437X) in exon 5, a one-base deletion (3536delA) in exon 5, a one-base insertion (5998insT) in exon 19, and a base substitution (6151+1G->A) at the splice donor site in intron 20. As their respective parents confirmed for paternity have no such changes, the four mutations are all de novo types. The TCA(Ser)->TAA(stop) nonsense mutation is predicted to lead a truncation of NSD1 protein(S437X) at amino acid position 437. The deletion (3536delA) leads to a premature stop codon at nt 3651–3653, and the insertion (5998insT) also results in a premature stop codon at nt 6022–6024. The splice site mutation (6151+1G->A) is predicted to skip exon 20, producing a smaller cDNA and resulting in a truncated protein added with only 9 amino acids after exon 19. The shorter cDNA deleting exon 20 is confirmed by RT-PCR and direct sequencing in the patient's immortalized lymphoblastoid cell line (FIG. 3). FIG. 3a shows results of RT-PCR analysis, wherein black and red arrows indicate a normal 471-bp product and an abnormal 329-bp product, respectively. Also, symbol Pt is a patient 24, symbol Wt is a normal control, symbol + is the use of a reverse transcriptase, and symbol − is no use of a reverse transcriptase. In FIG. 3b, an upper line shows exons 19, 20 and 21, and a small red circle indicates a mutation at a splice-donor site. A sequence analysis of the 329 bp product shows a skipping of exon 20, resulting in a frame-shift and a new stop codon at 10th codon of exon 21. Although five other base-substitutions (three synonymous and two non-synonymous changes) are identified in either exon 5 or exon 23, they are commonly observed in normal controls and are likely to be polymorphic, i.e. single nucleotide polymorphisms (SNPs)(Table 1). Surprisingly, 29 of the 39 patients show a homozygous pattern at all these five SNP locations. These data strongly suggest the presence of deletion involving NSD1, while the data in 12 normal control individuals are consistent with the Hardy-Weinberg equilibrium. The FISH analysis using RP1-118m12 as a probe detects a deletion in 20 (66.7%) of 30 patients whose metaphase and/or interphase cells are available (FIG. 4), wherein red and yellow arrows indicate a signal on normal chromosome 5 and no signal on the deleted chromosome 5, respectively. The FISH analysis suggests together with the data(4/38) for point mutations that 77% of SS patients in this series have NSD1 mutations (Table 1). All the changes identified are deletions (hemizygous) or heterozygous mutations causing protein truncation. Thus, it is most likely that haploinsufficency of NSD1 is the major cause of Sotos syndrome.
NSD1 is a bifunctional nuclear protein, acting as a nuclear receptor corepressor and as a coactivator by interacting with the ligand-binding domain of NRs. Human NSD1 has two distinct NR-interaction domains called NID−L and NID+L, and other conserved domains such as SET (su(var)3-9, enhancer-of-zestem trutgirax), PWWP (proline-tryptophan-proline), and PHD (plant homeodomain protein) finger domains. The two protein truncation (PT) mutations (S437X and 3536delA) observed in SS patients may result in loss of PHD-I, PHD-II, PWWP-II, SET, and PHD-II domains. The one-bp insertion (5998insT) and the splice-site mutation (6151+1G>A) may lose SET and PHD-III domains (FIG. 2). These findings suggest that the minimal deletion of the SET and PHD-III domains involved in the protein truncations in SS patients are functionally critical in this protein.
The PHD finger domain may interact with other proteins and be involved in chromatin regulation.
Recently, an NSD1 -derived chimeric protein is isolated from a childhood acute myeloid leukemia (AML) associated with t(5;11)(q35;p15.5) in which NSD1 is fused with NUP98 at NSD1 intron 5. In the patient BP, the chromosome break is presumed to be located at intron 2. Therefore, this may suggest that mutation types are different between the two conditions, i.e. constitutional protein truncation mutation versus somatic mutation with fusion-gene formation.
NSD1 shows 75% homology at nucleotide level to WHSC1 (NSD2) isolated from a minimum deletion region of the 165-kc Wolf-Hirschhorn syndrome (WHS). The WHS (OMIM 194190) is characterized by multiple anomalies, severe growth retardation, mental defect, and hemizygous deletion (haploinsufficiency) of 4 p. Further, NSD2 cause a type of multiple myeloma when being fused with the IgH gene by t(4;14). It is likely that both genes (NSD1 and NSD2) are important for human growth and brain development as well as for cell growth.
Since SS is thought to be genetically heterogeneous, as SS is described in a mother and two daughters, two brothers, and three sibs (monozygotic twin girls and their brother), autosomal recessive inheritance is postulated. Also, the other patients with de novo t(3;6)(p21;p21), mos dup (20)(p11.2–p12.1)[12/66] and t(2;12)(q33.3;q15) mat are described. Thus, either chromosomal breakpoints or segments are suggested to harbor another SS locus.
However, the aforementioned data indicate that the majority (up to 77%) of SS patients have an NSD1 mutation. This finding of NSD1 defects in Sotos syndrome will facilitate the diagnosis of SS and shed light in understanding the mechanisms of mental retardation and growth disorders in the human.
Physical Map Construction
The RPCI-1, -3, human PAC libraries are screened by PCR using STSs as described previously. Cosmid subclones are prepared from a PAC spanning the breakpoint. A purified PAC DNA is isolated by using Midi-PrepTM columns (Qiagen, Chatsworth, Calif.) and partially digested with Sau3AI. The SuperCos1 cosmid vector is prepared according to the manufacture's instructions (Stratagene, La Jolla, Calif.) and ligated to the digested PAC DNA by using T4DNA ligase. This ligation reaction products are packaged by using the Gigapack III gold extract (Stratagene) and transfected into XL1-Blue MR host cells (Stratagene). Contigs are constructed by means of STS content mapping using PCR with reference to draft/complete genomic sequences. BAC/PAC/cosmmid DNA is extracted with Midi-PrepTM columns. New STSs are generated from clone-end sequences using T7/SP6 primers for BAC or T7/T3 for cosmid. Sequencing reactions are carried out with ABI PrismTM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster, Calif.) using 2 μg of BAC/PAC DNA or 1 μg of cosmid DNA as a template and 40 pmol primer.
Cycle sequencing is performed for 50 cycles at 96° C. for 10 seconds, 50° C. for 5 seconds, and 60° C. for 4 minutes.
FISH Analysis
The FISH analysis using BAC/PAC/cosmid DNA is performed on metaphase chromosomes and/or interphase cells from SS patients and a normal control. A cloned DNA is labeled with SpectrumGreenTM-11-dUTP or SpectrumOrangeTM-11-dUTP (Vysis, Downers Grove, Ill.) by a nick translation, and denatured at 76° C. for 10 minutes. Probe-hybridization mixtures (10 μl) are applied on the chromosomes, incubated at 37° C. for 16 hours, and then washed as described previously. A fluorescence photomicroscopy is performed under a Zeiss Axioskop microscope equipped with a quad filter set with single band excitation filters (84000, Chroma Technology Corp., Brattleboro, Vt.). Images are collected and merged by using a cooled CCD camera (TEA/CCD-1317-G1, Princeton Instruments, Trenton, N.J.) and IPLab/MAC software (Scanalytics, Inc., Fairfax, Va.).
Patient and Parent Samples
DNA is extracted from peripheral blood leukocytes or lymphoblastoid cell lines of sporadic patients with Sotos syndrome after an informed consent is obtained. In several cases, the samples are also collected from their parents. All but one patient (a Japanese-Pakistani) is of Japanese origin. Experimental protocols are approved by the Ethical Committee for Gene Research of Nagasaki University School of Medicine.
Mutation Analysis
Twenty-two NSD1 exons (exons 2–23) covering the coding region are amplified by PCR. The PCR is cycled 35 times at 95° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 1 minute in a volume of 50 μl, containing 1×PCR buffer with 1.5 mM MgCl2, 0.2 mM each dNTP, 1 μM each primer and 2.5U TaqGold polymerase (PE Applied Biosystems).
PCR products are purified by using the QIAquick PCR purification kit (Qiagen, Chatsworth, Calif.) and sequenced on both strands with BigDye Terminator chemistry by a standard protocol (PE Applied Biosystems) as described preciously.
RT-PCR Analysis
A total RNA is prepared from lymphoclastoid cell lines established from a patient and a normal control using the Trizol reagent (Gibco-BRL, Gaithersburg, Md.).
A reverse transcription is carried out by using random hexamers and SuperscriptTM first-strand synthesis system for RT-PCR (Gibco-BRL) according to the manufacturer's protocol. The cDNAs are amplified by PCR with primers, NSD1PT1F, 5′-ATTTGTGAATGAGTATGTGG-3′ (SEQ ID NO: 7) (nt 5898–5917) and NSD1RT1R, 5′-CTAAAACACTCATCTTCTCG-3′ (SEQ ID NO: 8) (nt 6349–6368) for 35 cycles at annealing temperature of 48° C. The PCR product is directly sequenced.
Genbank Accession Numbers
Human NSD1, AF395588; NSD2, XM 055926; FGFR4, XM 030308; JAZ, XM 012279; mouse Nsd1, NM 008739; BAC clone CTC-286c20, AC027314, CTC-549a4, AC008570.







TABLE 1







NSD1 mutations identified in patients with


Sotos syndrome and SNPs of NSDI











Patient or
Exon or
Type of




SNP
intron
mutation
Inheritance
Consequence





Patient BP
intron 2*
translocation
de novo
gene disruption


Patients

deletion
de novo
missing gene


1–20


Patient 21
exon 5
1310C −> A
de novo
S437X


Patient 22
exon 5
3536delA
de novo
Protein






truncation


Patient 23
exon 19
5989insT
de novo
Protein






truncation


Patient 24
Intron 20
6165 + 1G −> A
de novo
Protein






truncation


SNP 1
exon 5
1482C −> T

Synonymous


SNP 2
exon 5
1749G −> A

Synonymous


SNP 3
exon 5
1840G −> T

V614L


SNP 4
exon 5
2176T −> C

S727P


SNP 5
exon 23
6829C −> T

Synonymous





SNP, single nucleotide polymorphism;


*estimated by FISH data






EXAMPLE 2 
Next, an object is examined by using the probe comprising sequence number 5 and 6 of the sequence table as to whether or not they are a patient suffered from Sotos syndrome. The FISH analysis is carried out by the same manner as the example 1.
Specifically, FISH analysis is carried out by using RPCI-11 147K7 and RPCI-11 1006E8. RPCI-11 147K7 and RPCI-11 1006E8 can be easily available from genome databases.
As a result of this, a deletion is confirmed at 60% of a patient suffered from Sotos syndrome. Therefore, it is found that a gene diagnosis can be carried out by using the present probe.
Conclusively, a confirmed diagnosis whether or not a patient is suffered from Sotos syndrome may be carried out by the FISH analysis in a similar manner wherein a nucleic acid concerning NSD1 region of the present invention is used as a probe.
The nucleic acid according to the invention and the probe using such a nucleic acid have an advantageous effect that they can be used as a gene diagnosis and a gene therapy.
Particularly, according to the invention using a fluorescence in situ hybridization, the RI equipment is useless, and the operating procedure is simple, and it is excellent in a point that in-depth mapping is exactly carried out on a chromosome band for a short time (2 days), so that it is also useful for a rapid and safety gene diagnosis.

## Claims

1. A screening method for diagnosing Sotos Syndrome comprising contacting a probe with a sample which is being evaluated for indication of Sotos Syndrome, said probe being derived from a genomic DNA sequence including an exon 1, an exon 2 and an intron of NSD1;
(a) a nucleic acid comprising a base sequence shown in base numbers 1–39726 of SEQ ID NO: 1, or
(b) a modified nucleic acid wherein a part of the bases 1–39726 of SEQ ID NO: 1 has been deleted or substituted, or to which at least one base has been added, said modified nucleic acid having a homology of 80% for said base sequence shown in base numbers 1–39726 of SEQ ID NO: 1.

2. The method according to claim 1, wherein the screening is carried out by using at least one selected from the group consisting of in situ hybridization method, Southern blotting method, and a base sequence determination method.

3. The method according to claim 2, wherein the in situ hybridization method is a fluorescence in situ hybridization method.