Abstract:
A process for detecting mutations in the gene responsible for galactosemia, galactose-1-phosphate uridyl transferase (GALT), is described. In one embodiment, the process can be used to detect over 85% of the mutations known to cause galactosemia in the United States population by using six different oligonucleotide probes, which span single-nucleotide Missense or nonsense mutations in the GALT gene. Hybridization conditions which can distinguish a single nucleotide mismatch are used to detect both the presence and zygosity of mutations in the GALT gene to aid in genetic counseling. A kit for use in detecting mutations in the GALT gene is also disclosed.

Description:
This application claims priority in U.S. Provisional Application No. 60/103,286 filed Oct. 6, 1998. 
    
    
     The government may have certain rights in this invention, which was funded in part by National Institute of Child Health and Human Development Grant PO-1 HD 29847-01 and U.S. Public Health Services Grant M01-RR00039. 
    
    
     FIELD OF THE INVENTION 
     The field of this invention is the diagnosis of galactosemia using nucleic acid-based techniques. 
     BACKGROUND OF THE INVENTION 
     Galactosemia, an inherited metabolic disorder of milk sugar metabolism, is caused by certain mutations in the gene coding for the enzyme, galactose-1-phosphate uridyl transferase (GALT), which result in defective enzyme activity. This disorder is potentially fatal, so newborns throughout the U.S. and developed countries are screened for galactosemia, which when detected and treated early, saves lives and money. Evaluation of outcome in galactosemic patients began in the 1980s, and by 1990, an enigma was revealed (for example, see Kornrower, G M,  J. Inher. Metab. Dis ., 1982, 5:96-104; Waggoner and Buist, 1993 , Internat. Pediatr . 8:97-100). Although neonatal infants were screened and treated within days of life with a galactose-restricted diet, older children had unexpectedly poor outcomes. Dysfunctions included ovarian failure, verbal dyspraxia, growth and developmental delays, and neurological signs of cortical and extrapyramidal tract impairment. Since the GALT enzyme structure is highly conserved throughout evolution, the human GALT cDNA was cloned using probes homologous to known sequences in the bacterial enzyme. When the human cDNA and gene were sequenced (see Flach, Reichardt, and Elsas, 1990, Mol. Biol. Med. 7:365-369; and Leslie et al. 1992, Genomics 14:474-480), the first mutations in the GALT gene were associated with human galactosemia (Reichardt et al. 1991, Am. J. Hum. Genet. 49:860-867; Reichardt et al, 1992, Genomics 12: 596-600) and relationships between the mutations and the resulting phenotype were being reported (Elsas et al. 1993, Internat Pediatr. 8:101). The Q188R mutation, first reported in 1991, is the most common mutation among galactosemic patients of Caucasian ethnicity (Reichert et al. 1991, Ibid.) and causes a complete loss in GALT activity. The S135L mutation is common among African-Americans (Lai et al., J. Pediatr 1996, 128: 89-95), and results in severe reduction in GALT activity. The N314D mutation, also known as the Duarte allele (Elsas et al., 1994, Am J. Hum Genet 54: 1030-1036), causes an instability in the GALT protein that leads to a 50% reduction in activity in homozygous patients. The K285N mutation is the second most common mutation among white galactosemic patients, and is the most prevalent among patients in southern Germany and Austria (Leslie et al., 1992, Ibid.). Other common mutations identified in the white population are R148W and L195P (Reichart et al. 1992, Ibid.). 
     These reports led to the realizations that different GALT mutations can result in different phenotypes and that therapy recommendations may vary depending on the specific mutation. 
     Methods currently used in newborn screening determine only the level of enzymatic activity or the accumulation of precursors. Results from current techniques are altered by the sampling parameters, such as ambient temperature and time of feeding relative to collection of sample. Also, they do not reveal the specific mutations that cause the change in enzyme activity. Certain mutations cause a mild disease, while others cause severe effects. Identifying the mutation is necessary to make a diagnosis, initiate appropriate therapy, estimate prognosis, and provide appropriate genetic counseling for the family. 
     There remains a need for a test with increased sensitivity and specificity for screening and diagnosis of galactosemia and asymptomatic carriers of galactosemia. There is a need for a test that can be run simultaneously on a large number of samples, such as in a micro titer plate format now commonly in use in clinical diagnostic laboratories. There is a need for a testing protocol that can quickly and specifically identify the majority of the alleles that can contribute to galactosemia in the human populations. 
     The claimed invention describes a nucleic acid-based method that can detect and specify the mutation involved in over 85% of individuals with some form of galactosemia rapidly and economically. Such a screen would be applicable to universal newborn screening and should supplement or completely replace current screening strategies. The screen can also be used to detect heterozygous mutations and the presence of multiple mutations in the GALT gene, which are necessary for understanding and counseling heritability and recurrence risks to family members. 
     SUMMARY OF INVENTION 
     The invention as claimed is a nucleic acid-based method that can detect and specify the mutation involved in the great majority of individuals with some form of galactosemia rapidly and economically. The method involves the detection of the mutation in amplified DNA from a patient utilizing probes that span the mutation. Six mutations are identified as being responsible for over 85% of the mutations in the United States population, and a detection kit is described for detecting these six mutations. The methods of this invention can also be used to determine the zygosity of the mutations and to detect the presence of multiple mutations in the GALT gene, both of which are necessary for understanding and counseling heritability and recurrence risks to family members. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions: 
     “GALT” means the enzyme galactose-1-phosphate uridyltransferase. 
     “Galactosemia” is a deficiency in the level or expression of GALT which results in detectable deviations from normalcy in animals. 
     As used in the claims, “a” can mean one or more, depending on the context of the claim. 
     Detailed Description of Invention 
     The invention described herein involves the use of a group or set of DNA probes to detect mutations in the GALT gene. Prior to this invention, only enzyme activity analyses have been used in clinical screening applications. With the practice of this invention, a rapid and economical screening system for detecting galactosemia in human populations, particularly in newborns, is provided. Based on the discovery of the frequency of the K285N mutation and the existence and frequency of the Y209C mutation, a process for detecting specific mutations in the GALT gene that cause over 85% of the mutations leading to galactosemia currently known in the U.S. population is provided. A preferred embodiment of this invention comprises assaying for the presence of six mutations, listed in Table 1A. In a survey of newborns the prevalence of these mutations is shown in Table 1B. If desired, this process can be followed by additional screening for less common mutations, e.g. listed in Table 2. The process comprises the collection of an RNA- or DNA-containing sample from a patient or a newborn infant, amplification of the DNA (following reverse transcription of the RNA, if needed) that encodes either a portion of or the entire GALT gene, hybridization of the amplified DNA, either serially or in parallel, with each member of a set of detectable probes, each probe being at least 10 nucleotides in length, the set comprising probes having sequences complementary to the sequences set forth in Table 1A, under hybridization conditions that allow only exactly complementary strands to remain as hybrids, and detecting the presence or absence of the hybrids. 
     
       
         
               
               
               
             
           
               
                 TABLE 1A 
               
               
                   
               
               
                   
                   
                 Approx % found in 
               
               
                   
                   
                 galactosemic 
               
               
                 Designation# 
                 Sequence* 
                 population 
               
               
                   
               
             
             
               
                 S135L 
                 TGGT T GGAT 
                 62% of African- 
               
               
                   
                   
                 American population 
               
               
                   
               
               
                 Q188R 
                 TGCC G GGTA 
                 70% of 
               
               
                   
                   
                 white population 
               
               
                   
               
               
                 L195P 
                 TTCC C GCCA 
                 8% of all ethnic 
               
               
                   
                   
                 groups 
               
               
                   
               
               
                 K285N 
                 CCAA T TATG 
                 17% of 
               
               
                   
                   
                 white populations 
               
               
                   
               
               
                 Y209C 
                 GCCT G TAAG 
                 6-20% of all 
               
               
                   
                   
                 ethnic groups 
               
               
                   
               
               
                 N314D 
                 CTGG G ACCA 
                 8% of all ethnic 
               
               
                   
                   
                 groups 
               
               
                   
               
               
                 [deletion] 
                 [absence 
                 in Ashkenazi 
               
               
                   
                 of signal 
                 Jewish population 
               
               
                   
                 with any probe] 
               
               
                   
               
               
                 #The number in the designation refers to the amino acid position in the human GALT protein (see, e.g. Leslie et al., ibid., or SEQ ID NO:15). The single lettersor on either side of the number identify the amino acid in the wild-type protein (preceding the amino acid number) and the amino acid present in the mutant protein.  
               
               
                 *underlined nucleotide is the one mutated from the wild-type sequence  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 B 
               
             
             
               
                   
               
               
                 Observed Prevalence In 250 Patients (500 Mutant Alleles) 
               
             
          
           
               
                   
                 Designation 
                 # of Alleles 
                 Percent of Total 
               
               
                   
                   
               
               
                   
                 Q188R 
                 270 
                 54.0 
               
               
                   
                 S135L 
                  42 
                  8.4 
               
               
                   
                 K285N 
                  24 
                  4.8 
               
               
                   
                 L195P 
                  8 
                  1.6 
               
               
                   
                 Y209C 
                  6 
                  1.2 
               
               
                   
                 F171S 
                  5 
                  1.0 
               
               
                   
                 [deletion] 
                  5 
                  1.0 
               
               
                   
                 Private 
                  90 
                 18.0 
               
               
                   
                 Unknown 
                  50 
                 10.0 
               
               
                   
                   
                 500 
                 100.00 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
             
               
               
             
               
               
               
               
               
             
               
               
             
               
               
               
             
               
               
             
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
             
             
               
                   
                 Designation# 
                 Sequence* 
                 Designation# 
                 Sequence* 
               
               
                   
                   
               
             
          
           
               
                   
                 A. Missense/Nonsense mutations 
               
             
          
           
               
                   
                 D28Y 
                   T AC 
                 Q212H 
                 C C G 
               
               
                   
                 I32N 
                 A A C 
                 L217Pn 
                 C C A 
               
               
                   
                 Q38P 
                 C C G 
                 L226Pn 
                 C C A 
               
               
                   
                 V44L 
                   T TG 
                 R231H 
                 C A T 
               
               
                   
                 V44M 
                   A TG 
                 W249R 
                   A GG 
               
               
                   
                 R51L 
                 C T C 
                 Y251C 
                 T G C 
               
               
                   
                 G55C 
                   T GT 
                 Y251Sn 
                 T C C 
               
               
                   
                 L62M 
                   A TG 
                 R258Cn 
                   T GT 
               
               
                   
                 R67C 
                   T GC 
                 R259W 
                   T GG 
               
               
                   
                 L74P 
                 C C G 
                 R262Pn 
                 C C G 
               
               
                   
                 A81T 
                   A CC 
                 R273G 
                   G GT 
               
               
                   
                 N97S 
                 A G C 
                 L282Vn 
                   G TC 
               
               
                   
                 D98N 
                   A AC 
                 L289Rn 
                 C G C 
               
               
                   
                 D113Nn 
                   A AT 
                 E291K 
                   A AG 
               
               
                   
                 H114Ln 
                 C T T 
                 E308K 
                   A AG 
               
               
                   
                 F117Sn 
                 T C C 
                 Q317R 
                 C G G 
               
               
                   
                 Q118Hn 
                 CA C   
                 Q317Hn 
                 CA T   
               
               
                   
                 R123G 
                   G GA 
                 H319Q 
                 CA A   
               
               
                   
                 R123Qn 
                 C A A 
                 A320T 
                   A CT 
               
               
                   
                 V125An 
                 G C C 
                 Y323H 
                   C AC 
               
               
                   
                 K127En 
                   G AG 
                 Y323D 
                   G AC 
               
               
                   
                 C13OYn 
                 T A C 
                 P324S 
                   T CT 
               
               
                   
                 H132Yn 
                   T AC 
                 P325Ln 
                 C T G 
               
               
                   
                 T138M 
                 A T G 
                 R328Hn 
                 C A C 
               
               
                   
                 L139P 
                 C C G 
                 S329F 
                 T T T 
               
               
                   
                 M142V 
                 G T G 
                 A330V 
                 G T C 
               
               
                   
                 M142K 
                 A A G 
                 R333W 
                   T GG 
               
               
                   
                 S143L 
                 T T G 
                 R333G 
                   G GG 
               
               
                   
                 R148W 
                   T GG 
                 R333Q 
                 C A G 
               
               
                   
                 R148Q 
                 C A G 
                 K334R 
                 A G A 
               
               
                   
                 R148Gn 
                   G GG 
                 M336Ln 
                   T TG 
               
               
                   
                 V150L 
                   C TT 
                 Q344Kn 
                   A AG 
               
               
                   
                 V151A 
                 G C T 
                 T350A 
                   G CC 
               
               
                   
                 W154Gn 
                   G GG 
                 Q54Stopn 
                   T AG 
               
               
                   
                 F171S 
                 T C T 
                 R8OStop 
                   T GA 
               
               
                   
                 G179D 
                 G A C 
                 W154Stopn 
                 TG A   
               
               
                   
                 P183T 
                   A CC 
                 R2O4Stop 
                   T GA 
               
               
                   
                 H184Qn 
                 CA A   
                 Q2l2Stop 
                   T AG 
               
               
                   
                 S192N 
                 A A C 
                 W249Stop 
                 TG A   
               
               
                   
                 F194L 
                   C TC 
                 L264Stopn 
                 TA G   
               
               
                   
                 I198Mn 
                 AT G   
                 W3l6Stop 
                 T A G 
               
               
                   
                 I198Tn 
                 A C T 
                 E34OStop 
                   T AA 
               
               
                   
                 A199T 
                   A CC 
                 Q353Stop 
                   T AG 
               
               
                   
                 R2O1H 
                 C A T 
                 Y366Stop 
                 TA A   
               
               
                   
                 E203K 
                   A AG 
                 Q37OStop 
                   T AG 
               
               
                   
                 Y209S 
                 T C T 
               
             
          
           
               
                   
                 B. Small Insertions/Deletions 
               
             
          
           
               
                   
                 528insG 
                 insG at base 528 
               
               
                   
                 S112fs insA 
                 insA at base 333 
               
               
                   
                 Wl34fs delT 
                 del T at base 400 
               
               
                   
                 ΔC(bp1677)n 
                 ΔC(bp1677) 
               
               
                   
                 L256/P257fs 
                 at bases 768-770, 
               
               
                   
                   
                 delete GCC-fs 
               
               
                   
                 Δamino acids n   
                 Δamino acids 
               
               
                   
                 #260-263 
                 #260-263 
               
               
                   
                 ΔT(bp2359) n   
                 ΔT(bp2359) 
               
               
                   
                 ΔC(bp2756) n   
                 ΔC(bp2756) 
               
               
                   
                 ΔC(bp2782) 
                 ΔC(bp2782) 
               
               
                   
                 L327delC 
                 base 979, del C-fs 
               
               
                   
                 P35lfsdelC 
                 base 1051, del C-fs 
               
             
          
           
               
                   
                 1. Splice Site Mutations 
               
             
          
           
               
                   
                 318A → G 
                 base 318A → G 
               
               
                   
                 IVSC 
                 base 956A → C 
               
               
                   
                 IVS3nt + 29G → C 
                 c.328 + 29 G → C 
               
               
                   
                 IVS4nt + 1 
                 c.377 + 1 G → C 
               
               
                   
                 IVS4nt − 27G → C 
                 c.378 − 27 G → C 
               
               
                   
                 IVS5nt + 62G → A 
                 c.507 + 62 G → A 
               
               
                   
                 IVSFnt1 n   
                 base 1472 G → A 
               
               
                   
                 IVSF 
                 base 1631 A → G 
               
               
                   
                 IVS7nt + 2 
                 c. 687 + 2 T → C 
               
               
                   
                 IVS8nt + 13 
                 c. 820 + 13 A → G 
               
               
                   
                 IVS8nt + 32 
                 c.820 + 32 A → G 
               
               
                   
                 IvS8nt + 58 
                 c.820 + 58 G → T 
               
               
                   
                 IVSHnt13 
                 base 2207 Ab → G 
               
               
                   
                 IVSJ 
                 C → G 
               
               
                   
                   
               
               
                   
                 #The number in the designation refers to the amino acid position in the human GALT protein, see Leslie et al., ibid., or SEQ ID NO:15. The single letters on either side of the number identify the amino acid in the wild-type protein (preceding the amino acid number) and the amino acid present in the mutant protein.  
               
               
                   
                 *underlined nucleotide is the one mutated from the wild-type sequence  
               
               
                   
                   n nove1 mutations not previously identified  
               
             
          
         
       
     
     The invention herein described provides the novel mutation, Y209C, in which the amino acid tyrosine is replaced with cysteine at amino acid 209. When a gene carrying the Y209C mutation is analyzed for expression in  E. coli , it is scored as a “null” mutation, i.e. there is no significant GALT activity detected. In one embodiment, the invention comprises a process for detecting this mutation in the GALT gene comprising amplifying the GALT gene, or the portion containing exon VII, hybridizing the amplified DNA with a probe at least 10 nucleotides in length that comprises sequence complementary to GCCTGTAAG, and detecting the presence or absence of this hybrid. In one embodiment, the invention comprises a set of probes comprising a probe for the Y209C mutation, as described above, and a probe for at least one other mutation chosen from the groups of mutations listed in Table 1A and Table 2. In another embodiment, the invention provides a set of probes comprising at least one of the novel mutations listed in Table 2 (i.e. those marked with the superscript“ n ”) and at least one other mutation chosen from the groups of mutations listed in Table 1A and Table 2. 
     The DNA hybrids produced by the processes of this invention can be detected by any of a number of methods known in the art, e.g. radioactive, chemiluminescent, fluorescent or bioluminescent labeling of the probe, antibody-based detection of a ligand attached to the probe, or detection of double-stranded nucleic acid. Specific examples of labels are digoxigenin, fluorescein, luciferases, and aequorin. Alternatively, the probe can be labeled with biotin and detected with avidin or streptavidin conjugates. 
     This process can be used to detect carriers of galactosemia, who may themselves be asymptomatic or mildly symptomatic and therefore undiagnosed. Carriers are heterozygous for a mutation, for example one of those listed in Table 1A. Carriers have not previously been identified by the existing screening methods, since they do not demonstrate a significant change in GALT activity. 
     In a specific embodiment, a portion of the GALT gene comprising exons 5-10 (e.g. nucleotides 1153-2863, GenBank Accession No. M96264; SEQ ID NO: 8) is amplified from the subject&#39;s DNA. Alternatively, the GALT gene can be amplified in sections, and the resulting amplicons can be hybridized only with those oligonucleotides carrying a mutation in the amplified region of the gene. 
     The process of this invention can be used to determine the zygosity of a mutation by hybridizing the amplified DNA to one or more probes comprising the wild-type sequences that correspond, for example, to the mutated sequences listed in Table 1A. 
     In specific embodiments, the probes used to detect the GALT mutations can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. 
     In a specific embodiment, the probes used to detect the set of mutations, such as the set in Table 1A, are all of the same length, between 10 and 25 nucleotides, and the mutated nucleotide (or the wild-type nucleotide corresponding in position to the location of the mutation) is positioned exactly in the middle of each probe, in the case of probes with an odd number of nucleotides, or, in the case of a probe with an even number of nucleotides, n, the mutation would be located at n/2±1. In such an embodiment, identical hybridization conditions can be used for each probe. 
     Alternatively, the mutation can be positioned anywhere in the probe that is at least 3 nucleotides from either end of the probe. In using such an alternative approach for detecting a set of mutations, the mutated nucleotide should be in the same position in each probe. 
     In an alternative embodiment, allele-specific amplification is employed resulting in the amplification of only selected GALT gene alleles. In this embodiment, probes are designed such that the 3′ end of one primer is the nucleotide which is mutated (see Blasczyk R, J Wehling et al. 1997 Allele-specific PCR amplification of factor V Leiden to identify patients at risk for thromboembolism,  Beitr Infusionsther Transfusionsmed  34: 236-41). 
     In a specific embodiment, tetramethyl ammonium chloride (TMAC) is included in the hybridization buffer, and the reaction is carried out at 55 C. Typically, the temperature of the hybridization is used to discriminate between an exact sequence match and a mismatch, and the temperature at which a given hybrid will “melt”, i.e. dissociate into the two complementary single strands, is determined by the length and nucleotide composition of the strands. To minimize the variation in the reaction temperature required to discriminate between the desired exact-match hybrid and the mismatched hybrid, trimethyl ammonium chloride (TMAC) is added to the hybridization buffer. TMAC greatly minimizes the effect of nucleotide composition on the hybridization reaction, so that length of the oligonucleotide becomes the determining factor, which can be easily controlled. Other salts can alternatively be used with similar effects as TMAC (see, for example, Mechior, Jr., WB and P H Von Hippel, 1973, Proc. Natl. Acad. Sci (USA) 70(2): 298-302). In a specific embodiment, probes of 21 nucleotides, with the mutation in the exact middle of the probe, as set forth herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, are used in the process. 
     In another embodiment, a kit is provided for use, for example, by diagnostic laboratories in hospitals, clinics or research centers that contains the necessary components to perform a rapid and economical screen for the six most common mutations in the GALT gene. Such a kit comprises a set of probes comprising sequences complementary to the sequences set forth in Table 1A, and a set of amplification primers, complementary to sequences selected from the known sequence of the human GALT gene (e.g. GenBank Accession No. M96264 or SEQ ID NO:7) to amplify the desired portion of or the entire GALT gene in a patient sample. In a specific embodiment, the primers comprise a single pair which amplify a region of the human GALT gene that comprises at least nucleotides 1153-2748 (numbering as in the above-referenced GenBank accession, SEQ ID NO:8). Amplification primers can be 14 to 30 nucleotides in length, although longer ones can also be used. 
     In another embodiment, the detection method used is an ELISA-based oligonucleotide ligation assay. Probes spanning the various GALT mutations described herein can be divided into a pair of smaller probes or, alternatively, new probe pairs can be designed so that their ends meet within a few base pairs of the mutation (or corresponding wild-type sequence). Such a pair of probes, spanning the sequence of a GALT mutation, are hybridized to the patient&#39;s amplified GALT DNA, then ligated, and the probes are then analyzed to determine the presence of a single large probe, which would be the product of the ligation of the perfectly matched pair of smaller probes (see Nickerson, D A, R Kaiser, S. Lappin, J. Stewart, L. Hood, and U. Landegren, 1990, Automated DNA diagnostics using ELISA-based oligonucleotide ligation assay; Proc. Natl. Acad. Sci. USA 87:8923-8927). The absence of a large product indicates the absence of the GALT mutation represented by the pair of probes. 
     In another embodiment involving a solution-based assay, detection of the GALT gene alleles involves the use of two different labels. For example, the DNA amplification is performed with primers that are labeled with digoxigenin. Multiple alleles can be analyzed by including primers labeled with a different detectable moiety for each allele. The amplified product is melted in solution, and allele-specific probes (as described herein) conjugated with biotin are added to the solution and hybrids are allowed to form. The solution containing these hybrids is added to an ELISA plate coated with avidin or streptavidin to capture the allele-specific hybrids. The hybrids are detected through the presence of digoxigenin. 
     Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. 
    
    
     EXAMPLES 
     Example 1 
     A. Oligonucleotides Useful in Detecting GALT Gene Mutations 
     Oligonucleotides useful in detecting the GALT gene mutation can be synthesized by techniques known in the art. The oligonucleotides can be from nine to twenty-five nucleotides in length; nucleotides shorter than 9 nucleotides will not provide accurate and reproducible results. Nucleotides longer than 25 nucleotides in length for detecting a single mutation in GALT can be used, but they provide no advantages over oligonucleotides 9-25 nucleotides in length. The single nucleotide mutation can be located anywhere in the oligonucleotide, as long as it is at least 3 nucleotides from either end of the oligonucleotide. One set of oligonucleotides of this invention, wherein the mutation is located exactly in the middle of the oligonucleotide, is presented in Table 3. 
     B. Amplification of DNA 
     1. GALT Gene PCR 
     Samples are collected from any tissue or organ of a patient from which RNA or DNA can be amplified. For example, DNA in blood collected and dried onto filter paper can be amplified. Alternatively, a buffy coat fraction from freshly collected blood is preferred for RNA amplification. Patient&#39;s DNA or RNA can be amplified in three sections using the following primers: 
     
       
         
               
               
               
             
           
               
                   
               
               
                 Primer Name 
                 Nucleotide Sequence 
                 SEQ ID NO: 
               
               
                   
               
             
             
               
                 F-S135L 
                 TGT AGT GGC TCT AGC TCT GG 
                 9 
               
               
                 R-S135L 
                 TAT CAG ATA GGG ATA CGG AGC 
                 10 
               
               
                 F-Q188R 
                 GTC AGG AGG GAG TTG ACT TGG 
                 11 
               
               
                 R-Q188R 
                 GCC CTT TTA CAG TTA TCC AGG 
                 12 
               
               
                 F-N314D 
                 TCA AGC AGG GGA TCC TGG GAG 
                 13 
               
               
                 R-N314D 
                 ATC TCT GAA GGT TCA CAC TCC 
                 14 
               
               
                   
               
             
          
         
       
     
     2. GALT Allele-specific Amplification 
     The presence of a specific GALT mutation or wild type can be detected by using allele specific amplification. A synthetic oligonucleotide, designed to end with a nucleotide that creates the mutation, is used as one of the primers in polymerase chain reaction amplification (for example, see Blasczyk R, J. Wehling et al. 1997 Allele-specific PCR amplification of factor V Leiden to identify patients at risk for thromboembolism,  Beitr Infusionsther Transfusionsmed  34: 236-41). One or more adjacent nucleotides of the primer may also be modified to increase the discrimination between allele-specific and non-specific hybridization. In addition, this technique can also be used to simultaneously detect multiple mutations. Primers for each of the different GALT alleles are designed to result in differing lengths of the PCR product, or different detectable moieties (e.g. fluorescent molecules, biotin, digoxigenin, or antibody ligands) are attached to the allele-specific oligonucleotide primers. 
     For example, the following primer sets can be used to detect two specific mutations of this invention. For the mutation S135L, the following primers can be used: 
     Mutated allele primer: 5′ GGT CAT GTG CTT CCA CCC CTG GTT 3′ 
     Wild-type allele primer: 5′ GGT CAT GTG CTT CCA CCC CTG GTC 3′ 
     Common reverse primer: 5′ CTG CAC CCA AGG GTA CTG GGC ACC 3′ 
     PCR Product=127 base pairs 
     For mutation Q188R, the following primers can be used: 
     Mutated allele primer: 5′ TTC TAA CCC CCA CCC CCA CTG CCG 3′ 
     Wild-type allele primer: 5′ TTC TAA CCC CCA CCC CCA CTG CCA 3′ 
     Common reverse primer: 5° CAA AAG CAG AGA AGA ACA GGC AGG 3′ 
     PCR Product=176 base pairs 
     The common primer is used in combination with either one of the other primers, the 3′ end of which is the specific nucleotide that is different in the mutated allele. The GALT gene DNA from an individual with only the wild-type form of the gene will only produce the appropriately sized product with the wild-type primer and none with the mutated allele primer. A heterozygous individual will produce the appropriately sized product with both primers, and a homozygous individual will not produce any product with the wild-type primer but will yield the appropriately sized product with the mutated allele primer. 
     C. Hybridization Techniques 
     When two DNA strands associate through base-pairing, it is referred to as hybridization. Hybridization is strongest when the two strands are exactly complementary. The stability of a hybrid is also a function of temperature, the ionic concentration of the medium, the length of the two strands, and the nucleotide composition, i.e. % A−T or % G−C. Under identical conditions, two strands that are not exactly complementary, differing by even one nucleotide, will be less stable and will disassociate at a temperature at which exactly complementary hybrids remain paired. 
     This property of mismatched hybrids provides a method to detect mutations in the GALT gene. The GALT sequence in the DNA of the subject, e.g. a newborn or a patient needing a diagnosis, is amplified, using techniques known in the art, such as polymerase chain reaction or cDNA synthesis, to produce copies of the gene which are then immobilized on a support, such as a nylon membrane (see Sambrook, et al.). The DNA on the support is incubated with a solution containing a labeled oligonucleotide from Example 1 at a temperature that promotes formation of hybrids. The support is then washed at a temperature at which only exact hybrids would be stable. The presence of the label is then measured; if the subject&#39;s DNA had the mutation corresponding to the oligonucleotide used in the assay, the label would be detected on the support. 
     Many subjects&#39; DNA can be tested in a microtiter plate format. Additionally, a pool of probes, containing all or a subset of the mutations listed in Table 1A or 1B, can be used simultaneously to make an initial qualitative determination of whether the subject&#39;s DNA contains any mutations in the GALT gene. Parallel hybridizations using oligonucleotides for the normal sequence corresponding to the mutated sequence can be performed to determine zygosity. It is also possible to put a unique and distinguishable label on each probe, so that one could determine the presence of different mutations in a single hybridization mixture. 
     Example 2 
     GALT Mismatched Hybrids Assay 
     To use multiple probes in a single hybridization format, an assay was developed by determining an optimal length for the probes so that the same temperature changes would result in the melting of the mismatched hybrids. Oligonucleotides between 9 and 25 nucleotides could satisfy this requirement, and those of 21 nucleotides, with the mutation at position 11, (see table 3) were found to be especially well-suited to a single temperature for disassociation of the hybrid. In addition, the use of tetramethyl ammonium chloride (TMAC) to the hybridization buffer allowed the hybridization temperature to be standardized at 55 C for all probes. 
     Hybridization Buffers and Techniques: 
     Amplified DNA in 5 μl aliquots are spotted on a prewetted positively charged nylon membrane, denatured (using 0.5M NaOH, 2.0M NaCl, 25 mM EDTA) and UV-crosslinked. The membranes are prehybridized at 55 ° C. for 30 minutes in a hybridization buffer (3.0M TMAC, 0.6% SDS, 1.0 mM EDTA, 10 mM Na 3 PO 4 , pH 6.8, 5×Denhardt&#39;s solution, 40 μg/ml yeast RNA) followed by overnight hybridization in the same buffer and the same temperature containing 1-2 × 10   6  cpm/ml  32 P-end labeled oligonucleotides. Unlabeled oligonucleotides corresponding to the normal sequence are used at 10× concentration of the labeled mutant sequence in the hybridization. Similarly, in detecting the normal alleles, unlabeled oligonucleotides corresponding to the mutant sequence are used at 10× concentration of the normal sequence in the hybridization. Filters are then rinsed one time for 20 minutes at room temperature with wash buffer (3.0M TMAC, 0.6% SDS, 1.0 mM Na 3 PO 4 , pH 6.8), then washed again in wash buffer at 55 ° C. for 30 minutes. The membranes are sealed in plastic and set for autoradiography overnight. 
     
       
         
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Mutation 
                 Sequence 
                 Sequence ID NO: 
               
               
                   
               
             
             
               
                 Y209C 
                 CAGCAGGCCTGTAAGAGTCAG 
                 6 
               
               
                 S135L 
                 CACCCCTGGTTGGATGTAACG 
                 1 
               
               
                 Q188R 
                 CCCCACTGCCGGGTAAGGGTG 
                 2 
               
               
                 L195P 
                 AGCAGTTTCCCGCCAGATATT 
                 3 
               
               
                 K285N 
                 TCTTGACCAATTATGACAACC 
                 4 
               
               
                 N314D 
                 GGCCAACTGGGACCATTGGCA 
                 5 
               
               
                   
               
             
          
         
       
     
     Example 3 
     Screening of a Galactosemia-Positive Patient for Mutation Type(s) 
     It is sometimes desirable to determine the exact nature of the mutation or mutations that are contributing to a patient&#39;s galactosemia. Such a determination will become critical as gene therapy techniques are implemented to correct these deficiencies. One example for analyzing a single patient&#39;s GALT gene for the presence of any or all of the known mutations in the GALT gene is provided herein. Synthetic oligonucleotides carrying each of the mutations to be screened are immobilized individually onto a support, such as a membrane, for example, as “dots” on a nitrocellulose membrane. A sample of the patient&#39;s labeled, amplified GALT gene (see Example 1 above for methods of amplification) is hybridized to all the dots on the membrane under conditions that promote only the hybridization of exactly matched sequences. The membrane is washed to remove mismatched and nonspecific hybridizing material, and the membrane is exposed to the appropriate detection system for the label, e.g. film, in the case of a radioactive label, to reveal which synthetic oligonucleotides are hybridizing to the patients&#39; DNA. 
     Alternatively, detection of a GALT gene mutation can be accomplished in solution, without the need for an immobilization support. An example of a solution-based analysis is the detection of a GALT gene mutation or a wild-type GALT gene by determining the melting temperature of the hybrid of the amplified DNA and the specific oligonucleotide (Ririe, K M, Rasmussen et al. 1997 Product differentiation by analysis of DNA melting curves during the polymerase chain reaction,  Anal Biochem  245(2): 154-60; Wittwer, C T, M G Herrmann et al. 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification,  Biotechniques  22(1): 130-1, 134-8). The melting temperature (Tm) of a hybrid is determined empirically by monitoring the absorbance of the solution as the temperature of the solution is raised incrementally. The Tm is typically defined as the temperature at which half of a single hybrid population is melted. When several different hybrids are present in the solution, there will be several different Tm values, representing the melting of each hybrid. The particular Tm value is determined by the G+C content of the hybrids, their length, and the ionic concentration of the solution. Mismatched hybrids will denature at a lower temperature than exactly matched hybrids. One or more of the probes can be hybridized to the amplified DNA and the melting curves of hybrids can be determined against small increments in temperature. Discrimination between multiple mutations can be achieved by using oligonucleotides of differing lengths or by attaching different detectable moieties, such as fluorescent, chemiluminescent, bioluminescent, or radioactive molecules, biotin, or antibody ligands, to the oligonucleotides. 
     A specific application of this approach, known as fluorescence energy transfer, can be performed using a Lightcycler®, a device manufactured by Roche Diagnostics Ltd., a division of Roche Molecular Biochemicals, Mannheim, Germany (see Wittwer, C T, K M Ririe et al. 1997 The LightCycler®: a microvolume multisample fluorimeter with rapid temperature control.  Biotechniques  22(1): 176-81). For example, to detect mutation Q 188R, the following primers are synthesized: 
     Amplification Forward Primer: 5′ CTT TTG GCT TAA CAG AGC TCC G 3′ 
     Amplification Reverse Primer: 5′ TTC CCA TGT CCA CAG TGC TGG 3′ 
     Anchor probe: 5′ GTC CCC GAG GTC ACC CAA AGA ACC G 3′ 
     Detection wild-type probe: 5′ GGT GAC GGt CCA TTC CCA CA 3′ 
     Detection mutated allele probe: 5′ GGT GAC GGt CCA TTC CCA CA 3′ 
     (The lower case letter represents the nucleotide that is different between the mutated and wild-type alleles). The anchor probe forms one half of the detection probe set and carries a fluorescence quencher. The other half of the probe set has the allele-specific sequence. When the allele specific probe and the anchor probe are hybridized the fluorescence is quenched. Then, during melting at the Tm for the allele specific probe the hybrid melts and fluorescence is observed. In this example, the Tm for the hybrid formed with the wild-type probe is 56 C, while the Tm of the hybrid formed with the mutated allele probe is 65C. 
     Example 4 
     ELISA-like Detection of Hybridization 
     The hybridization of the DNA oligonucleotide probe to the amplified GALT DNA sequences can also be detected using an enzyme-linked immunodetection assay, which can be generally called an ELISA. The amplified DNA is labeled during the amplification reaction with digoxigenin, then hybridized with biotin-labeled probes. Under the hybridization conditions described above, a hybrid of the biotin-labeled probe and the digoxigenin-labeled DNA forms. These hybrids are introduced into avidin-coated microtiter plates, and the biotin-labeled probe is adsorbed to the plate. Digoxigenin-labeled DNA that is hybridized to the bound biotinylated probe is detected using an enzyme-coupled anti-digoxigenin antibodies. Detection signals other than an enzyme can be used to detect the anti-digoxigenin antibodies, as will be recognized by those skilled in the art. Examples of other detection signals include bioluminescent proteins, chemiluminescent or fluorescent labels, or radioactive labels. 
     Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention. The texts of the references herein cited are hereby incorporated in their entirety by reference. 
     
       
         
           
             15 
           
           
             1 
             21 
             DNA 
             Homo sapiens 
           
            1
cacccctggt tggatgtaac g                                               21 
           
             2 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            2
ccccactgcc gggtaagggt g                                               21 
           
             3 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            3
agcagtttcc cgccagatat t                                               21 
           
             4 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            4
tcttgaccaa ttatgacaac c                                               21 
           
             5 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            5
ggccaactgg gaccattggc a                                               21 
           
             6 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            6
cagcaggcct gtaagagtca g                                               21 
           
             7 
             4286 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            7
gaattccgga tcaaatgaat gattgcagca agcaagtcct gtaggcatcc tggagcccaa     60
ggattctgca gtaggcagct ttcacagagg ttcttccagt gtagtggctc tagctctggg    120
tgaagtagga tcatcaatgt cggcccccag ggttcacagc tgttctgagc cccgccccct    180
ggtggcagcc gacgggagtc agtcagtcac gtgctggcgg ctggccaatc atcgggggcg    240
gcgcggggag gggtggtgtg gacggagaaa gtgaaaggtg aggcacggcc ctgcagattt    300
tccagcggat cccccggtgg cctcatgtcg cgcagtggaa ccgatcctca gcaacgccag    360
caggcgtcag aggcggacgc cgcagcagca accttccggg caaacggtaa ctgcaccgcg    420
gcagggactc gctggggcgc ggagccgagc cctccccttc cttaggaagc tttcgtcccc    480
ctccgaaggt tggaacgctc atcccgagcc agaccgacaa ggcgtacagt ctgcaggcct    540
ctacgagcag caggccaatt ggcgctggga aagtccaatc ctgggcctct agctcctgag    600
cgggacaggg ccgagagggc gctcccgagc ttgggcctgc tggtgggtga gacccaggag    660
agagggagct agagggggga gctctgagga ctgatcttga ctgtctgccc ccagaccatc    720
agcatatccg ctacaacccg ctgcaggatg agtgggtgct ggtgtcagct caccgcatga    780
agcggccctg gcagggtcaa gtggagcccc agcttctgaa gacagtgccc cgccatgacc    840
ctctcaaccc tctgtgtcct ggggccatcc gagccaacgg agaggtaagc ctgtagagcc    900
ctgcatctgc aggctgggcc acggggagta gttccctctt agaactgtcc tccacccaca    960
ggatagtgaa cctccttctg ggtcatatcc caccaagctt tttggtcccc tagggtgggc   1020
cttccctact cccttgtagc ctgtccagtc tttgaagccc accaggtaac tggtggtatg   1080
gggcagtgag tgcttctagc ctatccttgt cggtaggtga atccccagta cgatagcacc   1140
ttcctgtttg acaacgactt cccagctctg cagcctgatg cccccagtcc aggtaacctg   1200
gctccaactg ctgctgggga ggagggtggc tagacctctt gagggacttc tgctgcagag   1260
atgctgagtg atactccttt acctcaggac ccagtgatca tccccttttc caagcaaagt   1320
ctgctcgagg agtctggtaa ctatggattt cccctcttac aactttcaaa ccagagttgg   1380
agactcagca ttggggttcg ccctgcccgt agcacagcca agccctacct ctcggttatc   1440
ttttctcccg tcaccaccca gtaaggtcat gtgcttccac ccctggtcgg atgtaacgct   1500
gccactcatg tcggtccctg agatccgggc tgttgttgat gcatgggcct cagtcacaga   1560
ggagctgggt gcccagtacc cttgggtgca ggtttgtgag gtcgcccctt cccctggatg   1620
ggcagggagg gggtgatgaa gctttggttc tggggagtaa catttctgtt tccacagggt   1680
gtggtcagga gggagttgac ttggtgtctt ttggctaaca gagctccgta tccctatctg   1740
atagatcttt gaaaacaaag gtgccatgat gggctgttct aacccccacc cccactgcca   1800
ggtaagggtg tcaggggctc cagtgggttt cttggctgag tctgagccag cactgtggac   1860
atgggaacag gattaatgga tgggacagag gaaatatgcc aatgatgtgg aggcttggag   1920
gtaaaggacc tgcctgttct tctctgcttt tgccccttga caggtatggg ccagcagttt   1980
cctgccagat attgcccagc gtgaggagcg atctcagcag gcctataaga gtcagcatgg   2040
agagcccctg ctaatggagt acagccgcca ggagctactc aggaaggtgg gagagagcca   2100
agccctgtgt ccccaaggag tccctaactt tcttatccca tgagagaggt gtgtaaagga   2160
gaaagctaga ggtgaactag tagagagaga cttgctagga ggccttagca ataatccagt   2220
aatctaaagg aaagatgatg gtgacttaga ctcgggtggt tagtggtaga ggtggtgaga   2280
agacatcaga tcctgggcac attcttttct tctgcttccc ttgcctattt gctgaccaca   2340
ctccggctcc tatgtcacct tgatgacttc ctatccattc tgtcttccta ggaacgtctg   2400
gtcctaacca gtgagcactg gttagtactg gtccccttct gggcaacatg gccctaccag   2460
acactgctgc tgccccgtcg gcatgtgcgg cggctacctg agctgacccc tgctgagcgt   2520
gatggtcagt ctcccaagta ggatcctggg gctaggcact ggatggaggt tgctcccagt   2580
agggtcagca tctggacccc aggctgagag tcaggctctg attccagatc tagcctccat   2640
catgaagaag ctcttgacca agtatgacaa cctctttgag acgtcctttc cctactccat   2700
gggctggcat ggtgaggctt ttcaagtacc tatatttagc cccaacacca tttctgggct   2760
cctgggctca gcctagtgaa ctgcaacctc aaaggagcaa gccttgaaac agttgctggg   2820
ggaagtggcc agagtagaga tgctgggact gagggtggag cagcaaactt ggtgaaacta   2880
catctccaat gtgctttcta atctcctgcc agctcttctc aagcagggga tcctgggaga   2940
tgtagttttc agatacctgg ttgggtttgg gagtaggtgc taacctggat aactgtaaaa   3000
gggctctctc tccccactgt ctctcttctt tctgtcaggg gctcccacag gatcagaggc   3060
tggggccaac tggaaccatt ggcagctgca cgctcattac taccctccgc tcctgcgctc   3120
tgccactgtc cggaaattca tggttggcta cgaaatgctt gctcaggctc agagggacct   3180
cacccctgag caggtcagga ctcagaacag tctggcgtct ccagactctc acatgcagta   3240
tgtgcaggca cctgatactt ctgttgccct tgtgctccag tcattgcaca aggcagaaac   3300
agctctggca ggaagggact gccaaagtta ggagccctag ggcctggaag gagagtatgg   3360
tcctcagatc ccccttctct cctgcttcct ccagggaacc caacagtcat gaccctgata   3420
gtttcccata acaacctggg cattccttgg gactcaggag ctgctaaact ctttcatccc   3480
ctggtggctt cagcagtcct tatcaccagc ctcacaatcc cacaggccca cccccagtgg   3540
gcctgtggca ttcatatttc atattcatat ttcaaaccac aatatccagc aaaatgtctc   3600
ctgagcaccc agaactccat accatcggcc gggtgtggtg gctcatgcct taatcccagc   3660
actttgggag gtcaagatgg gaggattgct tgagcccaga agttcgagac tagcctggga   3720
aacataggaa gccctcgtct ctacaaaaaa aatttaaaaa gttagccagg tatggtggca   3780
tatacgatgc tttgtggtcc cagatacttg ggaggctgag ataggatcac ttgggcccag   3840
gagtttgagg ctgcagtgag ccatcatcat ggcatcattg cattccagcc tgggcaacag   3900
agcaagacct cgtctcaaaa aaaaaaaaaa aaatgaagtc catgccacca ttcttggcag   3960
cccagccctt atcctcctta attgctccct gtcccttttc caggctgcag agagactaag   4020
ggcacttcct gaggttcatt accacctggg gcagaaggac agggagacag caaccatcgc   4080
ctgaccacgc cgaccacagg gccttgaatc cttttttgtt ttcaacagtc ttgctgaatt   4140
aagcagaaag ggccttgaat cctggcctgg aatttgggca gatatagcat taataaaact   4200
gtgcatctca aacttttatc acatactcta atatcagagg agtgtgaacc ttcagagatc   4260
tagggttaaa agctaaaggc atagct                                        4286 
           
             8 
             1617 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            8
taaggtcatg tgcttccacc cctggtcgga tgtaacgctg ccactcatgt cggtccctga     60
gatccgggct gttgttgatg catgggcctc agtcacagag gagctgggtg cccagtaccc    120
ttgggtgcag gtttgtgagg tcgccccttc ccctggatgg gcagggaggg ggtgatgaag    180
ctttggttct ggggagtaac atttctgttt ccacagggtg tggtcaggag ggagttgact    240
tggtgtcttt tggctaacag agctccgtat ccctatctga tagatctttg aaaacaaagg    300
tgccatgatg ggctgttcta acccccaccc ccactgccag gtaagggtgt caggggctcc    360
agtgggtttc ttggctgagt ctgagccagc actgtggaca tgggaacagg attaatggat    420
gggacagagg aaatatgcca atgatgtgga ggcttggagg taaaggacct gcctgttctt    480
ctctgctttt gccccttgac aggtatgggc cagcagtttc ctgccagata ttgcccagcg    540
tgaggagcga tctcagcagg cctataagag tcagcatgga gagcccctgc taatggagta    600
cagccgccag gagctactca ggaaggtggg agagagccaa gccctgtgtc cccaaggagt    660
ccctaacttt cttatcccat gagagaggtg tgtaaaggag aaagctagag gtgaactagt    720
agagagagac ttgctaggag gccttagcaa taatccagta atctaaagga aagatgatgg    780
tgacttagac tcgggtggtt agtggtagag gtggtgagaa gacatcagat cctgggcaca    840
ttcttttctt ctgcttccct tgcctatttg ctgaccacac tccggctcct atgtcacctt    900
gatgacttcc tatccattct gtcttcctag gaacgtctgg tcctaaccag tgagcactgg    960
ttagtactgg tccccttctg ggcaacatgg ccctaccaga cactgctgct gccccgtcgg   1020
catgtgcggc ggctacctga gctgacccct gctgagcgtg atggtcagtc tcccaagtag   1080
gatcctgggg ctaggcactg gatggaggtt gctcccagta gggtcagcat ctggacccca   1140
ggctgagagt caggctctga ttccagatct agcctccatc atgaagaagc tcttgaccaa   1200
gtatgacaac ctctttgaga cgtcctttcc ctactccatg ggctggcatg gtgaggcttt   1260
tcaagtacct atatttagcc ccaacaccat ttctgggctc ctgggctcag cctagtgaac   1320
tgcaacctca aaggagcaag ccttgaaaca gttgctgggg gaagtggcca gagtagagat   1380
gctgggactg agggtggagc agcaaacttg gtgaaactac atctccaatg tgctttctaa   1440
tctcctgcca gctcttctca agcaggggat cctgggagat gtagttttca gatacctggt   1500
tgggtttggg agtaggtgct aacctggata actgtaaaag ggctctctct ccccactgtc   1560
tctcttcttt ctgtcagggg ctcccacagg atcagaggct ggggccaact ggaacca      1617 
           
             9 
             20 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            9
tgtagtggct ctagctctgg                                                 20 
           
             10 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            10
tatcagatag ggatacggag c                                               21 
           
             11 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            11
gtcaggaggg agttgacttg g                                               21 
           
             12 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            12
gcccttttac agttatccag g                                               21 
           
             13 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            13
tcaagcaggg gatcctggga g                                               21 
           
             14 
             21 
             DNA 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            14
atctctgaag gttcacactc c                                               21 
           
             15 
             379 
             PRT 
             Homo sapiens 
             
               Description of Artificial Sequence/Note = 
             
           
            15
Met Ser Arg Ser Gly Thr Asp Pro Gln Gln Arg Gln Gln Ala Ser Glu
 1               5                  10                  15
Ala Asp Ala Ala Ala Ala Thr Phe Arg Ala Asn Asp His Gln His Ile
            20                  25                  30
Arg Tyr Asn Pro Leu Gln Asp Glu Trp Val Leu Val Ser Ala His Arg
        35                  40                  45
Met Lys Arg Pro Trp Gln Gly Gln Val Glu Pro Gln Leu Leu Lys Thr
    50                  55                  60
Val Pro Arg His Asp Pro Leu Asn Pro Leu Cys Pro Gly Ala Ile Arg
65                  70                  75                  80
Ala Asn Gly Glu Val Asn Pro Gln Tyr Asp Ser Thr Phe Leu Phe Asp
                85                  90                  95
Asn Asp Phe Pro Ala Leu Gln Pro Asp Ala Pro Ser Pro Gly Pro Ser
            100                 105                 110
Asp His Pro Leu Phe Gln Ala Lys Ser Ala Arg Gly Val Cys Lys Val
        115                 120                 125
Met Cys Phe His Pro Trp Ser Asp Val Thr Leu Pro Leu Met Ser Val
    130                 135                 140
Pro Glu Ile Arg Ala Val Val Asp Ala Trp Ala Ser Val Thr Glu Glu
145                 150                 155                 160
Leu Gly Ala Gln Tyr Pro Trp Val Gln Ile Phe Glu Asn Lys Gly Ala
                165                 170                 175
Met Met Gly Cys Ser Asn Pro His Pro His Cys Gln Val Trp Ala Ser
            180                 185                 190
Ser Phe Leu Pro Asp Ile Ala Gln Arg Glu Glu Arg Ser Gln Gln Ala
        195                 200                 205
Tyr Lys Ser Gln His Gly Glu Pro Leu Leu Met Glu Tyr Ser Arg Gln
    210                 215                 220
Glu Leu Leu Arg Lys Glu Arg Leu Val Leu Thr Ser Glu His Trp Leu
225                 230                 235                 240
Val Leu Val Pro Phe Trp Ala Thr Trp Pro Tyr Gln Thr Leu Leu Leu
                245                 250                 255
Pro Arg Arg His Val Arg Arg Leu Pro Glu Leu Thr Pro Ala Glu Arg
            260                 265                 270
Asp Asp Leu Ala Ser Ile Met Lys Lys Leu Leu Thr Lys Tyr Asp Asn
        275                 280                 285
Leu Phe Glu Thr Ser Phe Pro Tyr Ser Met Gly Trp His Gly Ala Pro
    290                 295                 300
Thr Gly Ser Glu Ala Gly Ala Asn Trp Asn His Trp Gln Leu His Ala
305                 310                 315                 320
His Tyr Tyr Pro Pro Leu Leu Arg Ser Ala Thr Val Arg Lys Phe Met
                325                 330                 335
Val Gly Tyr Glu Met Leu Ala Gln Ala Gln Arg Asp Leu Thr Pro Glu
            340                 345                 350
Gln Ala Ala Glu Arg Leu Arg Ala Leu Pro Glu Val His Tyr His Leu
        355                 360                 365
Gly Gln Lys Asp Arg Glu Thr Ala Thr Ile Ala
    370                 375