WO2014019108A1 - Nmnat1 mutant gene, primers, kit, and method for detecting same and use thereof - Google Patents

Description

 V A 7 mutant gene, primers, kits and methods for detecting same, and uses thereof

TECHNICAL FIELD The present invention relates to disease-associated mutant genes, and in particular to Lieber congenital amaurosis-associated mutation genes. Background technique

Leber congenital amaurosis (LCA) is an autosomal recessive retinal dystrophy that manifests as genetic heterogeneity. Leber Congenital Amaurosis is one of the most common inherited cause of blindness in children ^1, is a severe retinal dystrophy, it showed at birth or infancy low vision and nystagmus. To date, LCA has been found to be associated with at least 17 genes. The molecular cause of the lack of identifiable in 20-30% of cases screened to the LCA, the LCA shows that there are not now causative genes ^2--4. Current clinical trials usually look for molecular defects by replacing them, or bypassing metabolic blockade by oral 9-cis retinal in the absence of RPE65 and LRAT.

 Therefore, there is still a need in the art to discover mutations in LCA-related genes, as well as primers, kits and methods for detecting related mutations. Summary of the invention

 The inventors sequenced the exome of individuals with LCA and identified nonsense mutations (c.507G>A, p.Trpl69*) and missense mutations (c.769G>A, p.Glu257Lys) in the NMNAT1 gene, This mutant gene was found to be involved in LCA.

 Thus, in a first aspect, the invention relates to a biomarker of LCA, i.e., a mutated NMNAT1 gene or a NMNAT1 protein, the biomarker being a NMNAT1 gene or a NMNAT1 protein having a mutation selected from:

 Nonsense mutation (c.507G>A, p.Trpl69*);

 Missense mutation (c.769G>A, p.Glu257Lys).

 In one embodiment, the sequence of the mutant Λ3⁄4ίΛ 7 gene of the invention is SEQ D) NO:l having the following mutations:

 Nonsense mutation c.507G>A; and / or

Missense mutation c.769G>A. In another embodiment, the sequence of the mutated NMNAT1 protein of the invention is SEQ D) NO: 2 with the following mutation:

 Nonsense mutation p.Trpl69*; and / or

 Missense mutation p.Glu257Lys.

 In a second aspect, the invention relates to a method of detecting LCA, the method comprising detecting whether a mutation site is present in a NMNAT1 gene or a NMNAT1 protein of a subject, and if there is a mutation site, the subject is: It is identified as having LCA or susceptible to LCA, or its offspring may have LCA or are susceptible to LCA, and the mutation site is selected from any one of the following or a combination thereof:

 Nonsense mutation (c.507G>A, p.Trpl69*); and

 Missense mutation (c.769G>A, p.Glu257Lys).

 In one embodiment, the method of detecting an LCA comprises the steps of:

 Extract the DNA sample of the subject,

 The DNA sample is subjected to PCR amplification to obtain an amplification product, and the NMNAT1 gene exon and exon/intron boundary are obtained.

 The PCR amplification amplification product is sequenced to obtain a sequencing result.

 The sequencing result is analyzed to determine whether a mutation site exists in the NMNAT1 gene, and if there is a mutation site, the subject is identified as having LCA or susceptible to LCA, or the offspring may have LCA or susceptibility LCA, the mutation site is selected from any one of the following or a combination thereof:

 Nonsense mutation (c.507G>A, p.Trpl69*); and

 Missense mutation (c.769G>A, p.Glu257Lys).

 In a more preferred embodiment, PCR uses KAPA2G Robust HotStart

(KAPABIOSYSTEMS, Woburn, MA).

 In a more preferred embodiment, the PCR amplified primers are designed with reference to the human genome, such as at least one set of primers as follows:

 SEQ ID NO: 3 and SEQ ID NO: 4;

 SEQ ID NO: 5 and SEQ ID NO: 6;

 SEQ ID NO: 7 and SEQ ID NO: 8;

 SEQ ID NO: 9 and SEQ ID NO: 10.

 In a more preferred embodiment, the sequencing is a Sanger sequence determination.

 In a more preferred embodiment, the sequence analysis is performed using a Mutation Surveyor program (State College, PA).

In one embodiment, the NMNAT1 protein is a sequence representation of SEQ D) NO:2. In another embodiment, the V3⁄4J gene is represented by the sequence of SEQ ID NO: 1. In yet another embodiment, the method of detecting LCA of the present invention comprises the step of amplifying at least one set of primers as follows:

 SEQ ID NO: 3 and SEQ ID NO: 4;

 SEQ ID NO: 5 and SEQ ID NO: 6;

 SEQ ID NO: 7 and SEQ ID NO: 8;

 SEQ ID NO: 9 and SEQ ID NO: 10.

 In still another embodiment, the method for detecting a mutation in the method for detecting LCA of the present invention is carried out by a technique selected from the group consisting of sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction, and denaturing high performance liquid phase. Chromatography.

 In the method of the second aspect of the invention, it is preferred to detect the homozygous mutant Λ3⁄4ΓΛ 7 gene. In a third aspect, the present invention relates to a method for detecting a mutant NMNAT1 gene or NMNAT1 protein, the method comprising detecting whether a mutation site is present in a NMNAT1 gene or a NMNAT1 protein of a subject, wherein the mutation site is selected from the following One or a combination:

 Nonsense mutation (c.507G>A, p.Trpl69*); and

 Missense mutation (c.769G>A, p.Glu257Lys).

 In one embodiment, the method of detecting a mutant V A3⁄47 gene of the present invention comprises the steps of: extracting a DNA sample of a subject,

 The DNA sample is subjected to PCR amplification to obtain an amplification product, and the NMNAT1 gene exon and exon/intron boundary are obtained.

 More PCR amplification products are sequenced to obtain sequencing results.

 The sequencing result is analyzed to determine whether a mutation site exists in the NMNAT1 gene, and the mutation site is selected from any one of the following or a combination thereof:

 Nonsense mutation (c.507G>A, p.Trpl69*); and

 Missense mutation (c.769G>A, p.Glu257Lys).

 In a more preferred embodiment, PCR uses KAPA2G Robust HotStart

(KAPABIOSYSTEMS, Woburn, MA).

 In a more preferred embodiment, the PCR amplified primers are designed with reference to the human genome, such as at least one set of primers as follows:

 SEQ ID NO: 3 and SEQ ID NO: 4;

 SEQ ID NO: 5 and SEQ ID NO: 6;

SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10.

 In a more preferred embodiment, the sequencing is a Sanger sequence determination.

 In a more preferred embodiment, the sequence analysis is performed using a Mutation Surveyor program (State College, PA). In one embodiment, the NMNAT1 protein is represented by the sequence of SEQ ID NO: 2.

 In another embodiment, the V3⁄4J gene is represented by the sequence of SEQ ID NO: 1. In still another embodiment, the method of detecting a mutant NMNAT1 gene or NMNAT1 protein of the invention comprises the step of amplifying at least one set of primers as follows:

 SEQ ID NO: 3 and SEQ ID NO: 4;

 SEQ ID NO: 5 and SEQ ID NO: 6;

 SEQ ID NO: 7 and SEQ ID NO: 8;

 SEQ ID NO: 9 and SEQ ID NO: 10.

 In still another embodiment, the method for detecting a mutation site in the method for detecting a mutant NMNAT1 gene or NMNAT1 protein of the present invention is carried out by the following techniques: sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction And denaturing high performance liquid chromatography.

 The method for detecting a mutant NMNAT1 gene or NMNAT1 protein according to the third aspect of the present invention can be used for the purpose of diagnosing LCA, and can also be used for the purpose of non-diagnostic disease. In some embodiments, the non-diagnostic diseases described in the present invention include, but are not limited to, studying SNP distribution and polypeptideity for use in family evolution studies. Such applications are understood by those skilled in the art.

 As can be seen from the description herein, some individuals carry the mutant 本3⁄4ίΛ3⁄47 gene of the present invention but do not suffer from LCA, such as a heterozygous genotype in which only one chromosome carries the mutation. Testing of this population may not involve any diagnosis of the disease, as these individuals are not themselves ill. However, the results of their testing can be used as useful information, for example as an important indicator of prenatal testing, for guiding fertility, or for screening for mutation carriers, or as a tool for SNP distribution and polymorphism studies or for tracking genetic mutations. Or family evolution. Such applications are also understood by those skilled in the art. Thus, the method of detecting a mutant 基因3⁄4ίΛ3⁄4 /gene or NMNAT1 protein according to the third aspect of the invention involves detecting a heterozygous mutation.

 However, the method of detecting a mutant NMNAT1 gene or NMNAT1 protein according to the third aspect of the present invention also includes detecting a homozygous mutation.

 In a fourth aspect, the invention relates to a primer pair for use in detecting a mutant NMNAT1 gene or NMNAT1 protein by PCR, the mutation being selected from any one or combination of the following:

Nonsense mutation (c.507G>A, p.Trpl69*); and/or Missense mutation (c.769G>A, p.Glu257Lys),

 Wherein the primer pair is designed before and after the genomic sequence or the cDNA sequence based on a position selected from the following positions such that the position is amplified: position 507 and position 769 of the cDNA sequence.

 In one embodiment, the primer pair is:

 SEQ ID NO: 3 and SEQ ID NO: 4;

 SEQ ID NO: 5 and SEQ ID NO: 6;

 SEQ ID NO: 7 and SEQ ID NO: 8; or

 SEQ ID NO: 9 and SEQ ID NO: 10.

 In a fifth aspect, the invention relates to a nucleic acid probe complementary to a mutant Λ3⁄4ΓΛ7 gene, said mutation being selected from any one or combination of the following:

 Nonsense mutation c.507G>A; and / or

 Missense mutation c.769G>A, position on the cDNA sequence: position 507 and position 769 of the cDNA sequence.

 In a sixth aspect, the invention relates to a kit for detecting a mutant NMNAT1 gene or NMNAT1 protein, comprising one or more sets of primer pairs, wherein the mutation is selected from any one of the following or a combination thereof: nonsense mutation (c.507G) >A,p.Trpl69* ); and

 Missense mutation (c.769G>A, p.Glu257Lys),

 Wherein the primer pair is designed on the genomic sequence or the cDNA sequence based on a position selected from the following, such that the amplification product thereof covers the position: position 507 and position 769 of the cDNA sequence.

 In one embodiment, the kit for detecting a mutant NMNAT1 gene or NMNAT1 protein comprises at least one set of primers selected from the group consisting of:

 SEQ ID NO: 3 and SEQ ID NO: 4;

 SEQ ID NO: 5 and SEQ ID NO: 6;

 SEQ ID NO: 7 and SEQ ID NO: 8;

 SEQ ID NO: 9 and SEQ ID NO: 10.

 In a seventh aspect, the invention relates to a kit for detecting a mutant Λ3⁄4ΓΛ 7 gene comprising one or more nucleic acid probes selected from any one or a combination of the following:

 Nonsense mutation c.507G>A; and

 Missense mutation c.769G>A,

The probe is complementary to a region of the mutated NMNAT1 gene comprising a genomic sequence or a cDNA sequence selected from the group consisting of: 507th and 769th sequences of the cDNA sequence. In an eighth aspect, the invention relates to a method for detecting a mutation in a gene exon and an exon/intron boundary, comprising the steps of:

 1. Extract DNA samples from the subject,

 2. Sequencing the exon subset of the above DNA sample and all exon/intron boundary sequences to obtain sequencing fragments.

 3. Align the above-mentioned sequenced fragment with the reference sequence to obtain a gene exon and an exon/intron boundary mutation.

 In one embodiment, the above sample is subjected to exome capture prior to the second step to collect the CDS region of human genomic DNA, and the exon-enriched DNA library is subjected to a second library construction for sequence determination.

 In one embodiment, the sequencing in the second step is performed as follows:

 1) Randomly break the sample DNA into fragments of about 200-300 bp, and then connect the adaptors at both ends of the fragment to prepare a hybrid library;

 2) After purification, the library is subjected to ligation-mediated PCR (LM-PCR) linear amplification and biotin-labeled DNA library for hybridization and enrichment, and then linearly amplified by LM-PCR, then sequenced on the machine, read Take a length of 90 bp and an average sequencing depth of at least 50 per sample.

 In one embodiment, the repeated sequencing fragments are removed in the third step. DRAWINGS

 Figure 1. Retinal appearance of patient 1 showing retinal vascular reduction, macular pigmentation of the retina, and atrophic macular damage.

 Figure 2. Schematic diagram of the mutations identified in the NMNAT1 gene. LCA patients without previously identified mutations were screened for Λ3⁄4ΓΛ 7 gene mutations by Sanger sequencing. Isolation and analysis of genomic DNA is also performed for available family members. Parents from Patient 6 are not available. Patients 9 and 10 are only available for current samples.

 Figure 3. Screening of NMNAT1 gene mutations by Sanger sequence determination in LCA patients without previously identified mutations. Isolation and analysis of genomic DNA is also performed for available family members. Parent samples from patient 6 were not available. Patients 9 and 10 are only available for current samples.

Figure 4. Image representation showing residues associated with LCA mutations identified on the human NMNAT1 promer structure. The overall structure is shown as a gray ribbon and the combined NAD is an orange rod. All 8 mutation sites were shown as rods (nonsense blue-green, missense green). Red dotted line shows three detailed description

 In the present invention, gene mutations and protein mutations are indicated by the general expressions in the art. In the present invention, c.507G>A indicates that the nucleotide at position 507 of the cDNA changes from G to A; both p.Trpl69* and p.Trpl69stop indicate that the codon at position 169 of the encoded protein is changed from a codon encoding Trp. Stop codon; c.769G>A indicates that the nucleotide of position 769 of the cDNA changes from G to A; p.Glu257Lys indicates that the codon 169 of the encoded protein is changed from the codon encoding Glu to the codon encoding Lys, or The 169th position of the protein changed from Glu to Lys.

 Thus, in one embodiment, the codon of the mutant Λ3⁄4ΓΛ7 gene encoding the amino acid 169 of the present invention may be AAA or AAG.

 The cDNA sequence of the wild type UV 3⁄47 gene is shown in SEQ ID NO: 1. The ^ column of the wild type human NMNAT1 protein is shown in SEQ ID NO: 2. The wild type mRNA3⁄4ίΛ3⁄4 / gene mRNA sequence is shown in SEQ ID NO: 11.

 In the description and claims of the present invention, it is understood by those skilled in the art that the sequence includes any one or two of the complementary strands. For convenience, in the present specification and claims, although only one chain is given in most cases, another chain complementary thereto is actually disclosed. For example, the cDNA sequence of the SJV 7 gene is actually included in the sequence and its complementary sequence. For example, reference is made to SEQ ID NO: 1, which actually includes its complementary sequence. Those skilled in the art will also appreciate that one chain can be used to detect another chain, and vice versa.

 The gene sequence in the present application includes either the DNA form or the RNA form, and one of them is disclosed, meaning that the other is also disclosed. For example, the cDNA sequence of the SJV 7 gene is actually included in the corresponding RNA sequence.

 In the present invention, the exon/intron boundary means 1-200 from the exon-intron boundary, for example 10-100, such as 20-80, such as 30-70, such as 40, 50, 60. Intron nucleotides.

In order to discover mutations in known LCA-related genes, the inventors mapped the coding region of 17 known LCA-related genes and all exon/intron boundaries (at least 50 inclusive from the exon-intron boundary). Sequencing, the 17 genes are PZJ, CABP4, CEP290, CRB1, CRX, GUCY2D. IQCB1, LCA5. LRAT, RD3, RDH12, RPE65, RPGRIPU SPATA7. TULP IMPDH1 and OTX2, these genes Relevant information is available from the Mendelian Human Genetic Database (OMIM). Among the 220 LCA individuals studied, mutant alleles were found on two chromosomes in 160 cases, and one mutant was found in 10 cases. Gene.

Since the known LCA factor was not identified in Patient 1 (see Table 5 below), a full exome sequence analysis was performed (see Tables 1 and 2 and Examples). A total of 2,460 unreported variants were identified. The inventors preferred 10 genes, each with previously unreported nonsense variations, which are expected to be truncated and further analyzed. GeneCards showed that 5 of these 10 genes were expressed in the retina, including V 3⁄47 , and the genetic map of the gene was in the 1ρ36 region. The genetic map previously in the Pakistani family of the same family was on chromosome 1ρ36 ⁵ .

 The gene Λ3⁄4ίΛ3⁄4 / an enzyme encoding the nicotinamide adenine dinucleotide (NAD) biosynthetic pathway, which is considered to be resistant to axonal degeneration.

 Nonsense mutations (c.507G>A, p.Trpl69*) and missense mutations (c.769G>A, p.Glu257Lys) were identified in the Λ3⁄4ίΛ/gene by exome sequencing and passed through the Sanger sequence. The assay (PCR primers provided in ^3) was confirmed. The parental test determined that the male parent carries the heterozygote of the nonsense variation, and the female parent carries the heterozygote of the missense variation, and the LCA caused by the mutation of the NMNAT1 gene is an autosomal recessive genetic disease, so the parents do not cause disease. . However, the offspring also passed on these two mutations, which caused the NMNAT1 gene to function abnormally and cause disease. Analysis shows that unaffected cell members do not carry mutations. Together, these results support that the 7Λ3⁄4ίΛ3⁄41 gene is an LCA candidate gene. Table 1. Screening of LCA genes by exome sequencing

表 2.通过外显子组测序鉴定的新无义突变。 RNA表达模式通过 GeneCards 分析。

Table 2. New nonsense mutations identified by exome sequencing. RNA expression patterns were analyzed by GeneCards.

 Information about these genes can be obtained from the Mendelian Human Genetic Database (OMIM www.omim.or).

 X indicates the termination of the translation.

 ^ . PCR Primer Sequence

为了进一步评估V ¾J基因在 LCA中作用，通过 Sanger序列测定分析 了在已知 LCA相关基因中无以前鉴定突变的 50个不相干 LCA病例。 另外 10 名患者被鉴定在 Λ¾ίΛ¾1基因中有复合的杂合突变（图 2和表 4 )。 值得注意 地， 所有 11名患者携带共同错义突变突变 c.769G>A (p.Glu257Lys), 这是 所鉴定的两个变异等位基因中的一个。如谱系分析所示， 所述鉴定的突变都 与 LCA表型共分离 （图 3 )。 表 4.鉴定的突变 #患者的国家来源和种族总结。

To further assess the role of the V3⁄4J gene in LCA, 50 unrelated LCA cases with no previously identified mutations in known LCA-related genes were analyzed by Sanger sequence analysis. Another 10 patients were identified as having a heterozygous mutation in the Λ3⁄4ίΛ3⁄41 gene (Figure 2 and Table 4). Notably, all 11 patients carried the common missense mutation c.769G>A (p.Glu257Lys), which is one of the two variant alleles identified. As indicated by lineage analysis, the identified mutations were co-segregated with the LCA phenotype (Fig. 3). Table 4. Identification of mutations # National source and ethnicity summary of patients.

 # provides a PolyPhen-2 prediction of the identified missense variation. The maximum value of the predicted damage variation is 1. 0. However, attention should be paid to the interpretation of the data. Variant P. Glu257Lys was initially predicted to be injurious with a score of 0. 903. However, the final predictions indicate that the variation is benign with a score of 0.089. In contrast, the variant p. Met35Thr was initially predicted to be benign with a score of 0. 059. The final prediction is possible damage with a score of 0. 601.

NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) contains four coding exons, encoding a 279 residue protein, which plays an important role in NAD+ biosynthesis, in which it catalyzes a single nucleotide from nicotinamide (NMN) ) and ΑΊ form NAD+. The VMA3⁄47 gene was not previously associated with LCA but was associated with axonal degeneration ⁶ . TVw homozygous knockout mice to not live birth, homozygous mutations nmnat Drosophila (DrosophUa melanogaster) is lethal; These results support the Vw «a gene essential function in some species views ⁷ . The genetic map of the Λ3⁄4ίΛ3⁄4/gene is at 1ρ36.22, near the LCA9 locus where the genetic map was previously identified. The affected individuals in the LCA9 family have congenital non-progressive severe visual impairments ⁵ . Similarly, the 11 LCD cases reported herein have severe early visual loss of vision (Table 5).

By finding disease-associated mutations in 9 unrelated families and 11 cases from 4 different countries, W provides evidence that the gene is a gene that induces LCA (Table 4). Clinically, all 11 cases had severe appearance (Table 5). Although the data is small, the severity of the disease may be related to the nature of the mutation. The term "allele clamping" as used herein relates to a process by which a phenotype of an individual having the same variation (eg, p.Glu257Lys) is examined in the presence of a second variant allele to infer the The effect of the second allele. All four individuals carrying the nonsense mutation p.Trpl69* were blind at birth and still had varying degrees of light perception. For 5 individuals who only carried the missense variation, the visual reduction was reduced within a few years after birth. The retina of all affected individuals has a marginal macula appearance and atrophic macular degeneration-like lesions

(Fig. 1; description of macular scar formation in patient 8). The enlargement of macular lesions was recorded during follow-up examinations at patients 2 years 6-9 months and patients 4 years 5-7 years old. Patient 4 has two variant alleles on the second chromosome. At 8 years old, there are 20/200 OD (right eye) and 20/400 OS (left eye) visual acuity, which is the only retinal current map (ERG). A case where the primary cone is dysfunctional rather than a deep loss of all responses; the individual's diagnosis is corrected for cone dystrophy. Patient 5 developed left eye exudative retinitis (Coats' disease) at age 3 - patient 6 was the only one carrying c.451G>T

Individuals with the (p.Vall51Phe) allele continue to lose most of the light-related vision at 18-24 years of age.

 The allele frequency of c.769G>A (p.Glu257Lys) is estimated to be 0.001 (dbSNP: rsl50726175). Herein, the inventors have not found that the individual is a homozygous c.769G>A (p.Glu257Lys) allele. None of the other variants identified by the inventors have been reported in any public database. The p.Trpl69* variant occurs in four of the nine families; the cause of such a high incidence is unknown.

Several studies involving several different organisms have linked the function of the NMNAT1 gene to axonal degeneration ⁶ . In the mouse model, the role of the hybrid protein formed by fusing the Ube4b sequence with the Nmnat1 protein is to protect the exon ^{9 1ϋ} . At the same time, loss of Nmnat gene function in Drosophila eyes leads to a severe neurodegenerative phenotype, but the catalytic function is not required for photoreceptor differentiation or development. In contrast, Nmnat proteins are involved in the maintenance and integrity of natural neurons. Drosophila photoreceptor Nmnat gene function leads to neuronal cell degeneration has been developed - of which may be considered to be retinal dystrophy phenotype ^u. Prior to this application, VMA3⁄47 was not associated with human disease. Therefore, the inventors have identified the eye as a biologically relevant organ that studies the function of NMNAT1 in vivo.

The NMNAT1 gene is Puda and shows the highest specific activity in the final step of catalyzing NAD+ biosynthesis. Human NMNAT1 is a homohexamer, each of which contains a central six-parallel beta-sheet with a number of helices ^{12 13} (Fig. 4). In this article, all 11 individuals with LCA carry the p.Glu257Lys variant. According to the crystal structure of human NMNAT1, Glu257 is located in short The outer surface in the C-terminal helix. It is worth noting that adjacent Ser256 is predicted to be a plate acidification site and may be involved in physical interaction with other related proteins such as poly (ADP-ribose) polymerase or protein kinase ¹² ' ¹³ . Displacement of a negatively charged glutamic acid with a positively charged lysine may alter the electrostatic properties of the surface, thereby affecting the proposed physical interaction. Asn273 is located in the last short helix (residues 267-274) and is involved in coordinating the active site water molecule ^{12 i3} . Displacement of this residue with an acidic residue (aspartic acid) will likely affect enzyme activity. Met35 and Vall51 are located in the center of the protein, while Val98 and Leul53 are very close to the ligand binding site (distance of about 6-7 A). The four variants (p.Met35Thr, p.Val98Gly.p.Vall51Phe and p.Leul53Val) are the most It is possible to interfere with local interactions, with the consequence of altering the active site and thus affecting enzyme activity. Can provide a new direction. Because Λ3⁄4ίΛ 7 is not large, it is a promising target for gene replacement therapy. Because NMNAT1 has a role in neuroproteins and is an enzyme, drug intervention is also possible. Limiting the clinical manifestations of the retina can be beneficial to these treatments. Recent advances in retinal function found in gene therapy mediated ^{14 '15} highlights the importance of the individual behind all the suffering genetic cause of LCA found.

 Table 5. Summary of patient phenotype details.

患者详细说明

Patient details

 The diagnosis of LCA is based on clinical evaluation (lack of limited imaging, nystagmus and infantile pupillary weak response, fundus examination and diagnosis), and suspected cases are re-examined by electroretinogram (ERG). The patient was diagnosed with a macular defect, which is atrophic damage, not a true congenital defect. Patients 1 - 6 and patient 11 were single cases. Patients 7 and 8 are cell relatives. Patients 9 and 10 are cell relatives. Patients 1, 7 and 8 were from USA; patients 2, 3 and 5 were from Brazil; patients 4, 6, 9 and 10 were from. Patient 11 is from Australia. Written informed consent was signed by their parents or guardians and the study was approved by the BGI and OHSU (P-WC) Institutional Ethnics Committee.

 Patient 1 from Children's Hospital of Los Angeles

 The male infant, whose mother is a descendant of Cherokee Indians, Spaniards and Caucasians, whose father is a descendant of Asians (Chinese), Hispanic and Caucasians, produces a full-term pregnancy, and has a ruptured subdural hematoma. , need to stay in bed until 7 months. The mother noticed that the child lacked significant vision within hours of birth. Within a few months, Claremont Eye Associates pediatric ophthalmologists and Children's Hospital Los Angeles retina specialists found that the electroretinogram (ERG) was not recorded above the noise. Based on the lack of visual response, retinal appearance (Fig. 1) and ERG testing, the patient's skin was diagnosed as LCA. Patient 1 may still have a light sensation. Patient 1 is the only patient in the family. His brother is performing normally.

 Patient 2 provided by JS

 The male is a Brazilian Caucasian ancestor whose maternal ancestors are from Poland, Portugal, Lebanon and Italy. The paternal ancestors are from Turkey, Germany, Italy and Portugal, born for normal pregnancy and cesarean section. Parents noticed the child's eye tremor in the first 3 months. At 6 months, due to nystagmus and no imaging consultation with Sao Paulo's eye genetics expert, the expert found that the child's eyes were light, the macula atrophy (so-called macular defect) and retinal macular pigmentation, hence the LCA. By the age of 9 months, the photo shows an increase in the area of macular atrophy. Patient 2 has no cell relatives and is the only patient with LCA in the family. During the first year of life, there is no light.

 Patient 3 provided by LG

Patient 3 was a Brazilian baby girl whose mother noticed that she was pressing at 4 months. Examination revealed that the Teller card test was visually invisible, had Franceschetti eye-catching signs, nystagmus, eye socket depression, pigmentation of the retina, and the appearance of a yellowed hammered copper. When she was a child, she was already blind. She is the only patient in the family. Patient 4 provided by EH

 Patient 4 is a male whose ancestor is west, has a cell relative, and is the only family member who is ill. A bloodless Western European ancestor. Horizontal tremor and weak vision were found at 2 months. A 6-month-old examination showed pigmentation to reduce macular damage and the patient was diagnosed with LCA. At the age of 5, adjust the chin's head position to see the color clearly. The lateral border hyperpigmentation and lateral atrophic macular damage (defect). At 7 years old, night vision weakens. Examination showed an increase in hypertrophic macular damage and atrophy of the retinal vasculature. The field of view is about 145 degrees. The ERG shows the main functional obstacles of the cone system, with a slight rod-focal b-wave latency delay, all the amplitudes of the reaction and the b-wave separation of the cone are reduced and the 30Hz flicker response is delayed. At 8 years old, distant vision

The 20/200 OD and 20/400 OS are 20/100 on both sides and the field of view is reduced to 95 degrees. The diagnosis was changed to cone-rod malnutrition, not LCA. You can still see the color. Parents ERG is normal. One parent has an indeterminate significant mid-peripheral pigment spot.

 Patient 5 provided by JS

 Patient 5 is a Brazilian male whose maternal ancestors are Spaniards, Caucasians, and the paternal background is African and Portuguese. He ^ L month pregnancy and normal birth. At 3 months of age, his mother observed that he did not see her and any objects. At the age of 1 he was sent to Sao Paulo Hospital for diagnosis of LCA. Initially, the omentum showed macular atrophy and fine retinal pigmentation. At the age of 4, he had exudative retinitis in his left eye. Atrophic macular degeneration-like lesions in Patient 5 were similar to Patient 2. Patient 5 is the only family member with LCA.

 Patient 6 provided by EH

 It was diagnosed with retinitis pigmentosa at 6 months and later changed to LCA. Lost most of the vision at the age of 18. At the age of 20, only the color and shape can be seen, and the patient begins to use the guide dog.

At the age of 22, he lost the ability to see color, and at the age of 24 he lost the ability to see shapes. At the age of 26, only light and dark can be distinguished. Eye tests showed swimming eye movement, macular atrophic damage, vascular atrophy, and pigmentation with bone needles. No family history. The patient is the only member of the LCA family. The patient has 5 cell members. The patient has Greek ancestors, not close relatives. The father has 2 cell friends and the mother has 8 cell friends. The relatives of one of her grandparents have visual impairments.

 Patients 7 and 8: Provided by DW

Patient 7 was a female, aged 10 years old. After her brother's skin was diagnosed with LCA, she was sent to the Retina Foundation for visual function testing and evaluation. At the time of testing, binocular sensitivity was evaluated as LP. The full-field ERG showed no response to the single-flash blue light, and the cone's response to 30 Hz scintillation light decreased by 99% with a delay. These symptoms are consistent with LCA. The clinician's record of her visit Ming, at the age of 2 years and a half, the retinal vessels in the eyes were highly atrophied, the large chorioretinal atrophic lesions in the macula (deteriorated compared with the previous visit) and the peripheral retina were normal, and the initial diagnosis was macular degeneration.

 Patient 7's brother (patient 8) was diagnosed at the Retina Foundation when he was 1 year old. He was discovered by the pediatrician at 4.5 months due to the H3⁄4 movement and lack of a dermatologist. The visual acuity is LP. The left eye full field of view ERG did not respond to the rod response test, and the cone response reduced the 30 Hz flash amplitude by 99% and the b wave latency time was significantly delayed.

 Patients 9 and 10: Provided by EH

 Patient 9 is a Caucasian male who has a weak visual appearance from infancy and is diagnosed with Lieber congenital amaurosis (LCA) during childhood. He was only able to identify bright colors when he was learning to walk, but soon lost that ability. At the age of 21, retinal findings revealed scarring of the macula in both eyes, pale optic nerve head, and retinal vascular atrophy. At the age of 22, the visual acuity report has a light perception. When genetically assessed at the age of 25, it was found that the pupillary response was sluggish, and the reaction was sometimes abnormal, with the pupil becoming larger as the light came in. There are mixed vertical and horizontal high frequency, small amplitude symmetrical eyeball tremors. Retinal enlargement examination revealed macular degeneration-like damage, reduced retinal blood vessels, small and pale papilla, and surrounding pigmentation. The intraocular pressure is normal. Double darkness and lightness ERG has no test results for all stimuli above noise. He could not complete the Goldmann field of view test. Other conveniences such as health and intelligence are normal. A brother, 30 years old, has a suspected LCA; a sister is not sick. Parents and ancestors were British and Dutch. Mother has 1 sister and 2 brothers. The father is adopted, with one brother and one sister.

 Patient 11: Provided by CB

 The female infant, whose mother is a descendant of British origin, whose father is of Australian descent, produces full-term pregnancy without complications. At 8 weeks, she could not watch and follow, and observed nystagmus and patrolling eye movements. The pediatric ophthalmologist then diagnosed as suspected LCA. Other aspects of the baby are healthy. ERG testing showed severe lateral retinal dysfunction with light perception but no graphic perception. Brain MRI is normal. At 2 years and 7 months, there is a certain sense of light. The patient had two unaffected brothers who did not have a family history of LCA. Similar to the clinical appearance of patients 1, 2 and 3, patient 11 presented with severe LCA. Importantly, macular atrophy (macular scar formation or macular degeneration) was also observed in their parents. In fact, suspected Λ3⁄4ίΛ3⁄4 / gene mutations, based on analysis of fundus imaging, suggest testing the V 3⁄47 gene.

 Example

 Method

Sample preparation Collect peripheral blood of patients and related relatives, and use the kit to extract the genome of peripheral blood leukocytes

DNA ( Relax Gene Blood DNA System DP319-02. TIANGEN, China ), using NanoDrop 2000 to measure the concentration and purity of DNA (Science Scientific, USA), the OD260/OD280 values of each specimen genomic DNA are between 1.7-2.0. , the concentration is not less than 100 ng / μ Ι, the total amount is not less than 30 μ1.

 2. Sequencing

 Then, the exome sequence of the above sample was sequenced.

 The above samples were subjected to exome capture using an Agilent 38M (Agilent) chip to collect the CDS region of human genomic DNA. The exon-encoding DNA library was then subjected to a second library construction for Illumina GA sequencing. The sequencing platform is the Illumina Genome Analyzer, which is sequenced according to the Illumina/Solexa standard library instructions (see http://www.illumina.com/), which is briefly described as follows:

 1) Randomly break the sample DNA into fragments of about 200-300 bp, and then connect the adaptors at both ends of the fragment to prepare a hybrid library;

 2) After purification, the library is subjected to ligation-mediated PCR (LM-PCR) linear amplification and biotin-labeled DNA library for hybridization and enrichment, and then linearly amplified by LM-PCR, then sequenced on the machine, read Take a length of 90 bp and an average sequencing depth of at least 50 per sample.

 3. Information analysis

The human reference genome and its gene annotations were downloaded from the UCSC database (http://genome.ucsc.edu/), version hgl9 (build 37). The sequence from the LCA patient sample was aligned with the reference sequence using BWA (Burrows-Wheeler Alignment) and then the duplicated reads were read. The SNP was transferred to the default SOAPsnp component (Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genome resequencing. Genome Res 2009, 19(6): 1124-1132). Transfer the indel (Indel) to Dindel (Albers CA, Lunter Q MacArthur DQ McVean G, Ouwehand WH, Durbin R., Dindel: accurate indel calls from short-read data. Genome Res. 2011 Jun; 21(6):961 -73). The threshold value for filtering SNPs: ^) The consistent quality value must be >= 20 (the quality value is the Phred value, generated by the program SOAPsnp, and the quality value 20 represents 99% of the basic call accuracy); 2) Support The number of unique matching sequencing fragments of the SNP must be >= 4; 3) The estimated copy number is not greater than 2; 4) The distance between the two SNPs should be greater than 5. For indel, only those that have the "PASS" tag provided by Dindel are extracted. High reliability.

 4. Methods for confirming exon sequencing results and identifying NMNAT1 gene mutations in other LCA patients:

(>50 nt) Two-way Sanger DNA sequencing was performed. PCR was performed using KAPA2G Robust HotStart (KAPABIOSYSTEMS, Woburn, MA). The primer design was referenced to the human genome, and the PCR primers for the NMNAT1 gene are shown in Table 3. Sanger Sequence Determination Reaction at SeqWright

(Houston, TX). Sequence analysis was performed using the Mutation Surveyor program (State College, PA).

 PCR amplification system:

 10* Ex Taq Buffer 3 μ1

 dNTP ( 2.5 mM ) 2 μ1

 Forward primer (10 mM) 1 μΐ

 Reverse primer (10 mM) 1 μΐ

 DNA 2 μ1

 Ex Taq 0.2 μΐ

Η ₂ 0 20.8 μΐ

 30 μΐ in total

 PCR ^ should be conditional:

 94 °C: 5 minutes;

 30 cycles: 94 °C 30 seconds, 55 °C 30 seconds, 72 °C 45 seconds;

 72 "C: 10 minutes;

 12 ° C: lasts.

 Sequence table description

 SEQ ID NO: 1: cDNA sequence of wild type AV 3⁄47 gene (where underlined indicates that the position may be mutated) (http: 〃 www.ncbi.nlm.nih.gov/nuccore/95113669) ATGGAA AATTCCGAGA AGACTGAAGT GGTTCTCCTT GCTTGTGGTT CATTCAATCC CATCACCAAC ATGCACCTCA GGTTGTTTGA

GCTGGCCAAG GACTACATGA ATGGAACAGG AAGGTACACA GTTGTCAAAG GCATCATCTC TCCTGTTGGT GATGCCTACA AGAAGAAAGG ACTCATTCCT GCCTATCACC GGGTCATCAT GGCAGAACTT GCTACCAAGA ATTCTAAATG GGTGGAAGTT 81

V0WV310V3 313W33V3V V33VV33100 33VVV3V101 0V100V010V V3V03V00V0 18

 1V31310V31 3V1033V331 丄 WV3V3W0 丄 V00V0V3W 103133VV31 010V3V3VVV IZ

 V011013VVV 3331V0V1V3 113VV33133 31VVV1011V V3VV00V103 19 £

 丄丄 0W3V033 1V01V01333 00V01V1003 10011V010V 33VVV3VV3V V0V10031V3 10 £

 1331131001 0101V01V03 3VVV013113 V0V0V133VV 33V0VV331V V31V0V10V3

 3VV0033103 V3丄丄丄 3丄丄 33 V0100V031V 0VV00V01V0 OOIVVOIIVO 1133131103 L8 L

 1100101133 13VV310V3V V3V300丄丄 W VV331V00V1 10113VV011 0W0VV01V3 IZ l

 Vmvoo丄丄丄 13V0V01313 3V333VV0VV 0V3030333V 313V0V3303 300333010V 19

 03V1330010 1V31331030 0333001303 3103010V00 1131V3V300 V30313V3V3 I

 囟 UVMWM Ύ ^ ^ : Π:ΟΝ QI 03S

 x¾vavxNHOidviiAOVNHaasIssAiNH¾aiAaOAia dAiAHisOoiraivirara Shang ssiawi NAAHI SHHM plexus ΊΛ (Κ3ΛΜΜθνα NOVHXA3nOANVAl0liaaS¾ lNdAV ^ SanaVO31 HA¾dAVMX¾d aiSXHOSSaO Shang 3Ι ΜΗΜ¾Κ) «ΙΗ3Ί Shang dS OOHCOaSVS HaOHHHlA l Shang plexus 3¾0lSa plexus Shang OAaA MSNCH Shang Via ring IAHHAWIlOXSMAVCK dS oz Πί ΗΛΛ Shang A¾K) Shang i) deleted Λ

^^土 ψ Π

： z:ON ai 0as

^^土ψ Π

: z:ON ai 0as

 OVXV 3VOWX30W 丄 ) 丄丄丄 si 丄 ) )丄 Y31DDDD13D XWOOV3VOV

丄: ^ VIO丄丄 XWXV30VW VOXXV3VXW o > )丄 o丄丄 )

XVOV33VXOO 丄丄 ) V丄 ) 丄丄 Y3DYDY33DD OOWi)V3丄 33

 丄 丄 丄 3丄 丄 WX303XVOO

XWOXWOXO 3丄 丄丄 V3 W30V0033V 3VWi )丄 O丄 3 01

DIDIYDDDIY 丄 V丄 3IV丄丄 丄 丄 3D 丄 丄 VWOO丄

 30003丄 3V丄丄 3丄 O丄 丄 ) OOOXVX3W3 300X03XVW

 333V3XV3VO WOXOVOWO 3丄 O丄丄丄

 33丄 OVOO丄丄 V 丄丄丄 VOV OO 3丄 O丄 O丄: )丄 ) OW3XOOVW

 330丄 O丄: )W W3VWW33 OVOVX333XV 丄 O s

V丄 3丄丄 丄 DIDYYODYDY VOOWOO丄 33

OOVWOV丄 3丄 3V丄 ) 3V3丄 3V VOV30V33V3 丄 丄 O丄 丄

 丄 DOOVOi) 丄 XVWOVOW3 XV33V3VOW 丄 ) O丄 丄

 3丄 DDIDYDDYYD 丄丄 3丄 OVW DDDIYDYIYD

ITC100/Z10ZN3/X3d 80Ϊ6Ϊ0/ 0Ζ OAV 61

 101003VV00 0V11001101 VO丄丄丄 0333V V031V03V01 33V100031V Z£

 33W33VV3V 13V3V0VV0V V101V33V31 V01V003311 111111031V V3VVV1133V IQ I£ i

111331V333 31V003V311 V3V3V13333 VV31V31331 1001111033 13VV3V3VV1 IZ I£

 V3V0丄丄丄 003 3V03V0VV31 31V丄丄 03丄丄丄 313V133131 丄丄 003V3V33 1V31V33011 190ε

 V01101V033 11V3110013 VVVV13V031 OOVVVOVVVO 3313VVV311 1W131311V LOOS

 0V110000V1 丄丄 000V13丄丄 3131V11113 33331031V3 1113111310 0331113111 mz

 131331031V 313131VVV3 3V3V丄丄丄 WO 1310V13111 丄 03丄丄丄 WIV 00丄丄丄 V01V0 L882 Of

3100100011 V丄丄丄 3丄丄 VOO 31V1VVVV3V V3130V310V 31VV11V1VV V1VV3333V0

 V0111VV31V V0VV3V113V V331VV1VV3 丄丄丄 OVOOllO W3V0V33VV 0100131333 L9Z.2

 丄丄丄丄 VV1V03 V1VV130V00 31031V3113 1031V33V30 030VV00010 1111103311 iOLZ

 03W0VV3V3 V0V13丄丄丄 03 VV0VV3VVV0 V131110VV0 W03V331V1 丄丄 V10V03丄丄 z

 311V1001V3 丄 003V000V0 310V003V31 V033V0V11V 33310313VV VOO丄丄 00330 i£

100V000310 1V3133V010 0V310010VV 3110133103 3V0V3V1131 V0030丄丄丄 33

 331V3V3V13 V11111V131 丄丄丄 VV10330 0131V00V10 V000V033V0 V11V333103 mz

 V13V310310 03V0100310 01011VV03V VO丄丄 333000 100300100V V0310V0103

 00101V3031 3313V0313V 331033V000 10131010V0 1013V331V3 V3丄丄丄丄丄 3丄丄 mz

 丄丄丄丄3丄丄 V10 01V31VV0V1 333VV13310 003V33V031 1000V33331 31V033VV0V 0£

3310V0100V 33V0100V31 0V10V31310 3V01031000 313100313V V3V010V100 iZZZ

 110031301V 10V1V01010 110VVV3310 31VV00丄丄丄 3 WV3V0310V VV130V0131 mz

 3VV3101V01 13V100V0V1 0VVV333101 00丄丄 0V33丄丄 1VV310VV3V 01V103VV31 z

 V3W03V3V1 V31W13VVV V310013V33 VV3113V3VV 1113V33110 丄丄 03V丄丄 001

 0010丄丄丄丄 VO 1333V31V3V OVIVOIVVVO 1003V33103 3VVV313V11 3W003101V Yang ςζ

001V33V011 133V03V103 V0V31003VV VVVVVVVVVV W0101V001 0V3V303V3V

 0V3丄 333100 3V0010V031 1V0031301V 3VV003VV13 V031133V3V 033V333100 Hidden

 W3丄丄 0301V V3V33V033V 31033V3331 10V103V000 13V1310010 Hidden

 V03V3103V1 1VVWVV0V1 VV3VV10V10 101V0000VV V31331V1VV 00331003V0

 0V3VV0113V 3VV0133V31 30V01V3313 3313VV1003 丄丄丄 OV 03V0001VV1 隐 oz

3100V0V010 300V3VVVVV VVVVVVVVVVV WV0101V00 丄 0WW03VV

 VVW033310 03V0010V03 丄 0V00313丄丄 33VV003V31 3V001133V0 V033V33300 L9S L

 0VV3110301 VV3V33V033 V31033V333 丄丄 0V103V00 OIVVIVIOVV 1V03003133 LOS L

 1V033V003V 丄丄 VWWVOV 丄 VVVVVIOVI 01013V331V 3V3丄丄丄丄丄 31 丄丄 V丄丄丄 3丄丄丄 i

 3111113VV1 WWIOSVV丄 331VV01VVV VV13V111VV VVVVVVVVVV VW0101310 ς\

10V3VV03V3 V0V3033100 3V0010V031 丄 V00V03丄丄 V 3VV003V313 V031133V33 IZ£ 1

 133133V000 0V3110V01V V3V33V033V 31033V3331 10V103V000 13V1310031 L92 L

 30V0331331 31313103V1 1VVVVV0V1V VVVV10V101 01V0000VVV 31331V1VV0 L02 L

 0331001V00 V3V301V3V3 VV0133330V 01V33133V0 33V31033V3 33丄丄丄 0V03V

 0001VV1310 030V010331 3V0V033310 1301VVVVV1 3V1113VVVV 丄丄 03VW10V ■ 01

VV10031131 01V313113V V3VVV33331 OVVIVVIISV 333101VV0V VV3101V333

 3111V00011 0V3V0丄丄丄 VI V31V03V0V1 011VV33V1V 0V3VV103VV 3V0310V0VV L96

 V3V3V03丄丄丄 0000331001 V013333103 1VV33V0V3V V313V3V310 丄 03V0V13丄丄 L06

 1VV1V03VVV V311V0V1VV 3VV0013110 1V3V00V133 丄丄 0V1030丄丄 V03V3V0033

 33W3V0100 03V3V33001 VVVVV0V001 V0101V0V31 W10301V33 1W31VV313 IQL ς

3130V011V0 VV03V3300V 0VVV331310 3131V3301V V31V101V11 13VV3V0103 IZL

 1V31VVV331 03330丄 OV丄丄 3131V1V010 3331V10VV0 0331301VVV 000V01V0V3 L99

 VV313V3VV3 313丄丄丄 WOO 0113103111 0013V3311V 111V3V0333 3131310310 L09

 3VV0133VVV 00313103VV VVOVVVVVOO 3V3V10001V W3VVVV013 V1011V3VV0

ITC100/Z10ZN3/X3d 80Ϊ6Ϊ0/ 0Ζ OAV 3301 CTTTTCACTT TTCTAGAAGT CCAGAAGTTG TTATATGATG AAATAGCCTC CTTTAACGTT

3361 TATTTCTGGG TGCCAACGGA GGCCCATTCC TCTAACATTC TCATAATTTT TCTCAAAGGC

3421 CTATGATCTA AACATTTCAC CACGGCATCC ACTCAGCTGT GAGGCTGCGT ACACAGTCTC

3481 CACCTCTGAA ATCTGAACTT CGTTTACCAG TGGTGCTGTT TGAACTTCAT AATGTCAGCA

3541 CTTCCTGAAC ACTTACTGTG TGCTTGGCTT GTGCTCCTGA GTGCCTTATA TCATAAGGAA

3601 ACGGCAAAAT CAGGGGACTG GTATAAATGG TGAGCTGAGC TTGAATCTAA GCTTTGTCTT

3661 CAGAGCCAGT ACCCCTAATC TCTCTTTCTG TAAAATATTA CTTTTCAAAG AATGAAGTTG

3721 TAGCCAAATC TTGAAATTTT TCATTTACCC TAAGTGAGGA CAAATAAAGC TTTCAACAAC

3781 A

 references

 1. Weleber, R.G, Francis, P.J. & Trzupek, K.M. GeneReviews (eds Pagon, R.A., Bird, T.D., Dolan, C.R. & Stephens, K.) (University of Washington, Seattle, Washington, USA, 1993-2010).

 2. Stone, E.M. Am. J. Ophthalmol. 144, 791-811 (2007)

 3. Berger, W" Kloecknner-Gruissem, B. & Neidhardt, J. Prog. Retin. Eye

Res. 29, 335-375 ( 2010 ) .

 4. Den Hollander, A.I, Black, A., Bennett, J. & Cremers, EE. J Clin Invest 120, 3042-3053 ( 2010 ) .

 5. Keen, T.J. et al. Eur. J. Hum. Genet. 11, 420-423 ( 2003 ) .

 6. Coleman, M.P. & Freeman, M.R. Annu. Rev. Neurosci. 33, 245-267

( 2010 ) .

 7. Conforti, L. et al. FEBS J. 278, 2666-2679 ( 2011 ) .

 8. Schindler, E.I. et al. Hum. Mol. Genet. 19, 3693-3701 ( 2010 ) .

 9. Conforti, L. et al. Proc. Natl. Acad Set USA 97, 11377-11382 (2000) 10. Mack, T.G et al. Nat. Neurosci. 4, 1199-1206 ( 2001 ) .

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Claims

Claim A V 3⁄47 gene or NMNAT1 protein having a mutation selected from the group consisting of: a nonsense mutation (c.507G>A, p.Trpl69*);  Missense mutation (c.769G>A, p.Glu257Lys).  2. The NMNAT1 gene of claim 1, which has the following mutations in the sequence of SEQ Π) ΝΟ:1:  Nonsense mutation (c.507G>A, p.Trpl69*); and  Missense mutation (c.769G>A, p.Glu257Lys).  3. The NMNAT1 protein of claim 1, which has the following mutations in the sequence of SEQ D) NO: 2:  Nonsense mutation (c.507G>A, p.Trpl69*); and  Missense mutation (c.769G>A, p.Glu257Lys).  A method for detecting a mutant V 3⁄47 gene or a NMNAT1 protein, the method comprising detecting the presence or absence of a mutation site in the NMNAT1 gene or the NMNAT1 protein, the mutation site being selected from any one of the following or a combination thereof:  Nonsense mutation (c.507G>A, p.Trpl69*); and  Missense mutation (c.769G>A, p.Glu257Lys).  5. The method of claim 4, comprising the step of amplifying at least one set of primers as follows:  SEQ ID NO: 3 and SEQ ID NO: 4;  SEQ ID NO: 5 and SEQ ID NO: 6;  SEQ ID NO: 7 and SEQ ID NO: 8;  SEQ ID NO: 9 and SEQ ID NO: 10.  6. Detection of a primer pair used in the mutant ^^^\3⁄47 gene or NMNAT1 protein, the mutation being selected from any one of the following or a combination thereof:  Nonsense mutation (c.507G>A, p.Trpl69*); and/or  Missense mutation (c.769G>A, p.Glu257Lys),  Wherein the primer pair is designed before and after the genomic sequence or the cDNA sequence based on a position selected from the following positions such that the position is amplified: position 507 and position 769 of the cDNA sequence.  7. A nucleic acid probe complementary to a mutant NMNAT1 gene, the mutation being selected from any one of the following or a combination thereof: Nonsense mutation c.507G>A; and / or Missense mutation c.769G>A, position on cDNA sequence: 507th and 769th cDNA sequences.  8. A kit for detecting a mutation Λ3⁄4ΓΛ 7 gene or NMNAT1 protein, comprising one or more sets of primer pairs, wherein the mutation is selected from any one of the following or a combination thereof:  Nonsense mutation (c.507G>A, p.Trpl69*); and  Missense mutation (c.769G>A, p.Glu257Lys),  Wherein the primer pair is designed on the genomic sequence or the cDNA sequence based on a position selected from the following, such that the amplification product thereof covers the position: position 507 and position 769 of the cDNA sequence.  9. The kit of claim 8 comprising at least one set of primers selected from the group consisting of:  SEQ ID NO: 3 and SEQ ID NO: 4;  SEQ ID NO: 5 and SEQ ID NO: 6;  SEQ ID NO: 7 and SEQ ID NO: 8;  SEQ ID NO: 9 and SEQ ID NO: 10.  10. A kit for detecting a mutant V 3⁄47 gene comprising one or more nucleic acid probes, the mutation being selected from any one or a combination of the following:  Nonsense mutation c.507G>A; and  Missense mutation c.769G>A,  The probe is complementary to a region of the mutated NMNAT1 gene comprising a genomic sequence or a cDNA sequence selected from the group consisting of positions 507 and 769 of the cDNA sequence.