Abstract:
Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) is essential for opening the CFTR chloride channel. A short segment containing many negatively charged amino acids (817-838, NEG2) within the regulatory (R) domain of CFTR is a critical regulator of the chloride channel activity. An isolated NEG2 polypeptide may be expressed as a separate sequence that stimulates CFTR channel openings at lower concentrations, but that inhibits CFTR channel openings at higher concentrations. Residues in the NEG2 sequence were substituted to produce a polypeptide that exerts only an activating effect on CFTR. One such polypeptide is the Q4N2NEG2 polypeptide. Exogenous Q4N2NEG2 exerts stimulatory effects on both wild-type and mutant G551D CFTR function, without exhibiting inhibitory activity at any concentration.

Description:
[0001]    This application is a non-provisional of and claims priority to U.S. Provisional Application Serial No. 60/323,724, filed Sep. 21, 2001, the disclosure of which is expressly incorporated herein. 
     
    
       [0002] This invention was made with government support under RO1 HL/DK 49003, P30 DK27651 and RO1 DK51770 awarded by the National Institute of Health. The government has certain rights in the invention 
     
    
     
       TECHNICAL FIELD OF THE INVENTION  
         [0003]    This invention is related to the field of cystic fibrosis. More particularly, it is related to the area of therapeutic treatments and drug discovery for treating cystic fibrosis.  
         BACKGROUND OF THE INVENTION  
         [0004]    Defects in CFTR, a chloride channel located in the apical membrane of epithelial cells, are associated with the common genetic disease, cystic fibrosis (Quinton, 1986, Welsh and Smith, 1993, Zielenski and Tsui, 1995). CFTR is a 1480 amino acid protein that is a member of the ATP binding cassette (ABC) transporter family (Riordan et al., 1989, Higgins, 1992). Each half of CFTR contains a transmembrane domain and a nucleotide binding fold (NBF), and the two halves are connected by a regulatory, or R domain. The R domain is unique to CFTR and contains several consensus PKA phosphorylation sites (Cheng et al., 1991, Picciotto et al., 1992). Opening of the CFTR channel is controlled by PKA phosphorylation of serine residues in the R domain (Tabcharani et al., 1991, Bear et al., 1992) and ATP binding and hydrolysis at the NBFs (Anderson et al., 1991, Gunderson and Kopito, 1995). Phosphorylation adds negative charges to the R domain, and introduces global conformational changes reflected by the reduction in the α-helical content of the R domain protein (Dulhanty and Riordan, 1994). Thus, electrostatic and/or allosteric changes mediated by phosphorylation are likely to be responsible for interactions between the R domain and other CFTR domains that regulate channel function (Rich et al., 1993, Gadsby and Naim, 1994).  
           [0005]    Rich et al., 1991 showed that deletion of amino acids 708-835 from the R domain (ΔR-CFTR), which removes most of the PKA consensus sites, renders the CFTR channel PKA independent, but the open probability of ΔR-CFTR is one-third that of the wild type channel and does not increase upon PKA phosphorylation (Ma et al., 1997, Winter and Welsh, 1997). Thus, it is possible that deletion of the R domain removes both inhibitory and stimulatory effects conferred by the R domain on CFTR chloride channel function. This conclusion is supported by studies that show that addition of exogenous unphosphorylated R domain protein (amino acids 588-858) to wt-CFTR blocks the chloride channel (Ma et al., 1996), suggesting that the unphosphorylated R domain is inhibitory. Conversely, exogenous phosphorylated R domain protein (amino acids 588-855 or 645-834) stimulated the ΔR-CFTR channel, suggesting that the phosphorylated R domain is stimulatory (Ma et al., 1997, Winter and Welsh, 1997). Therefore, it appears that the manifest activity (stimulatory or inhibitory) depends on the phosphorylation state of the R domain.  
           [0006]    About 25% of the known 700 mutations in CFTR produce a mutant CFTR protein which is properly transported to the apical membrane of epithelial cells but have only low level, residual channel activity. There is a need in the art for agents which can boost the level of channel activity in those mutants having low level activity.  
         SUMMARY OF THE INVENTION  
         [0007]    These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment of the invention an isolated polypeptide is provided. The polypeptide comprises an amino acid sequence of SEQ ID NO: 6 wherein the polypeptide retains a net negative charge of 1-8. More preferably the variant of said CFTR protein has the sequence of SEQ ID NO: 1.  
           [0008]    In another embodiment of the invention a method is provided for activating a CFTR protein. An effective amount of the polypeptide is administered to a cell comprising a CFTR protein that forms a cAMP regulated chloride channel. The polypeptide comprises the sequence of SEQ ID NO: 6. The CFTR protein is consequently activated. More preferably, the polypeptide has the sequence of SEQ ID NO: 1.  
           [0009]    According to another embodiment of the invention a method is provided for activating a CFTR protein. An effective amount of a polypeptide is contacted with a CFTR protein in a lipid bilayer wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 6. The CFTR protein is thereby activated. More preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.  
           [0010]    In another embodiment of the invention a method is provided for synthesizing a CFTR-related polypeptide. Units of one or more amino acid residues are linked to form a polypeptide comprising the amino acid sequence of SEQ ID NO: 6. More preferably, the polypeptide has the sequence of SEQ ID NO: 1.  
           [0011]    In another embodiment of the invention an isolated polypeptide is provided. The polypeptide comprises the amino acid sequence of SEQ ID NO: 2.  
           [0012]    In yet another embodiment of the invention a nucleic acid molecule is provided. The nucleic acid comprises a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 2.  
           [0013]    In another embodiment of the invention a method of activating a CFTR protein is provided. A nucleic acid comprising a sequence encoding a polypeptide according to SEQ ID NO: 2 is administered to a cell comprising the CFTR protein, whereby the polypeptide is expressed and the CFTR protein is activated.  
           [0014]    These and other embodiments of the invention, which will be apparent to those of skill in the art, provide the art with reagents and tools for enhancing function of cAMP regulated chloride channels that are defective in cystic fibrosis patients. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0015]    [0015]FIG. 1A and 1B and  1 C: Demonstration of increase in open probability of CFTR channel with addition of the Q4 N2 NEG2 peptide.  
         [0016]    (FIG. 1A) Single channel trace of the CFTR channel before addition of peptide.  
         [0017]    (FIG. 1B) Single channel trace after addition of Q4 N2 NEG2 peptide (4 μM).  
         [0018]    (FIG. 1C) Summary of five separate experiments. Addition of Q4N2 NEG2 peptide increases the Po by about two-fold. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0019]    It is a discovery of the present inventors that the channel inhibitory properties of the R domain of CFTR protein can be separated from the channel activating properties. Thus activating polypeptides can be used to treat CFTR defective cells, without concern for inhibition at certain concentrations. Activating polypeptides may also be used to enhance the activity of normal CFTR, including that delivered by gene transfer.  
         [0020]    A polypeptide for use in treating CFTR-defective cells contains a 22 amino acid sequence, GLXISXXINXXXLKXXFFXXXX, as shown in SEQ ID NO: 6. The amino terminal residue is acetylated and the carboxy terminal residue is amidated. The residue X, at positions 3, 6, 7, 10, and 11 is either glutamic acid or glutamine; at position 12 is aspartic acid or asparagine; at position 15 is glutamic acid or glutamine; at position 16 is cysteine or serine; at positions 19 or 20 is aspartic acid or asparagine; at position 21 is methionine or norleucine; at position 22 is either glutamic acid or glutamine. The amino acid residue at position 16 is more preferably serine. The amino residue at position 21 is more preferable norleucine. The polypeptide of SEQ ID NO: 6 has a net negative charge. The net negative charge is preferably within the ranges of 1-8, 2-8, 3-8, 4-8, 5-8, 6-8, or 7-8.  
         [0021]    The polypeptide more preferably has the sequence of SEQ ID NO: 1, GLEISEQINQQNLKQSFFNDLE, wherein L at position 21 is norleucine. The amino terminal residue of the polypeptide is preferably acetylated and the carboxy terminal residue is preferably amidated.  
         [0022]    The polypeptide may also be present in a composition with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those in the art. Pharmaceutically acceptable carriers include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. The composition can also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Buffering agents include Hanks&#39; solution, Ringer&#39;s solution, or physiologically buffered saline.  
         [0023]    It may be desirable that the polypeptide be fused to another polypeptide to provide additional functional properties. For example, fusion to another protein such as keyhole limpet hemocyanin can be used to increase immunogenicity. Another desirable fusion partner is a membrane-penetrating peptide. Such peptides include VP-22 (SEQ ID NO: 3), as well as the peptides shown in SEQ ID NO: 4 and SEQ ID NO: 5. Such peptides can be used to facilitate the uptake of the polypeptide by target cells. The polypeptides of the invention may also be fused to proteins that cause specific targeting to lung epithelial cells. For instance, the peptide THALWHT directs DNA to human airway epithelial cells. Single chain antibody variable domains may be used to do the same.  
         [0024]    A CFTR protein can be activated by the polypeptide. The CFTR protein can be in a cell, preferably in the cell membrane and the CFTR protein forms a cAMP-regulated chloride channel. An effective amount of a polypeptide that comprises the sequence of SEQ ID NO: 6 can be administered to the cell, and administration of the polypeptide activates the CFTR protein. The polypeptide administered more preferably comprises the sequence of SEQ ID NO: 1.  
         [0025]    The cells may be any cells that contain or express a CFTR protein. The cells may naturally express the CFTR protein, such as lung epithelial cells, or the cells may express the CFTR protein after transient or stable transformation. The cells may be primary cells isolated from individuals that express a wild-type CFTR protein, or may be primary cells isolated from individuals that express a mutant CFTR protein. The cells may also be of a stable cell line. The cells may also exist in the body.  
         [0026]    The CFTR protein is a wild type or a mutant CFTR protein. The mutant CFTR protein is a CFTR protein that is expressed by the cells and that is transported to the cell surface. The mutant CFTR protein also forms a cAMP-regulated chloride channel. The mutant CFTR protein may contain alterations that are known and characterized, or may contain alterations that have not yet been discovered. A mutant CFTR protein that fails to undergo full activation is a CFTR protein that does not conduct ions to the same degree as wild-type CFTR. The mutant CFTR protein may not conduct ions at all. The mutant protein may also conduct ions to a similar extent as wild type CFTR but be present in the membrane in substantially lower amounts than is true for normal individuals.  
         [0027]    Activated is defined as any increase in conductance by the CFTR protein. An increase in conductance may result when the opening of the CFTR channel occurs with greater frequency than previously observed. An increase in CFTR conductance may result when the duration of opening is increased each time the CFTR channel opens. An increase in conductance may also result due to greater ability to conduct ions each time the CFTR protein channel is open. The increase in open probability of the CFTR protein is preferably at least 25%, at least 50%, at least 75%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, or at least 300%.  
         [0028]    An effective amount is any amount of polypeptide that is sufficient to activate the CFTR protein, as activate is defined above. Preferably, the polypeptide is administered to achieve a concentration of 0.5 to 14 μM. More preferably, the polypeptide is administered to achieve a concentration of 4-6 μM.  
         [0029]    The polypeptide may be administered by any means acceptable in the art. For instance, the polypeptide may be administered in vitro, or to cells in culture, by addition to the medium. The polypeptide may be administered in vivo, to a patient, by any route including intravenous, intrathecal, oral, intranasal, transdermal, subcutaneous, intraperitoneal, parenteral, topical, sublingual, or rectal. Most preferably, the polypeptide is administered to a patient in an aerosol.  
         [0030]    The aerosolized polypeptide can be co-administered with an expression vector that encodes wild type CFTR protein. An expression vector may be linear DNA that encodes wild type CFTR protein, or a plasmid or human artificial chromosome that expresses wild type CFTR protein. The vector may be administered as naked DNA or may be administered complexed to lipid molecules such as with liposomes, short polypeptides such as the THALWHT polypeptide, or polycations such as polylysine, with or without stabilizing agents and/or receptor ligands. The DNA may also be administered in a viral vector. Viral vectors are known in the art. Several nonlimiting examples include retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus. The gene encoding the wild type CFTR protein may additionally comprise a promoter sequence to drive expression of the CFTR gene. Any promoter known in the art may be used. Promoters include strong promoters such as the promoters of cytomegalovirus, SV40, or Rous sarcoma virus. The promoter may also be a tissue specific promoter. Preferably the tissue specific promoter is a lung specific promoter. Lung specific promoters include the promoters of surfactant protein A, keratin 18, Du Clara cell secretory protein, and the promoter of CFTR.  
         [0031]    A CFTR protein can also be activated by applying an effective amount of a polypeptide to a CFTR protein in a lipid bilayer. The polypeptide comprises the amino acid sequence of SEQ ID NO: 6. The polypeptide more preferably comprises the amino acid sequence of SEQ ID NO: 1. Activating a CFTR protein in a lipid bilayer is useful to the art for screening agents for the treatment of cystic fibrosis.  
         [0032]    A CFTR protein in a lipid bilayer may be a CFTR protein that is expressed in cells in culture. The cells may express the CFTR protein without manipulation, or may be stably or transiently transfected to express the CFTR protein. The lipid bilayer may also be such artificial preparations as, without limitation, a microsome preparation, a lipid-bilayer vesicle preparation, or liposomes. The polypeptide may be applied to the protein by its addition to cell culture media, or solution in which the lipid bilayers are maintained. A change in conductance may be measured by any means known in the art, such as patch clamping.  
         [0033]    A CFTR activating polypeptide can be synthesized by sequentially linking units of one or more amino acid residues to form a polypeptide comprising the amino acid sequence of SEQ ID NO: 6. Preferably the polypeptide has the amino acid sequence of SEQ ID NO: 1. Synthesis of the CFTR polypeptide can be performed using solid-phase synthesis, liquid-phase synthesis, semisynthesis, or enzymatic synthesis techniques. Preferably the polypeptides are synthesized by solid-phase synthesis. More preferably the peptides are synthesized by F-moc synthesis.  
         [0034]    The polypeptide of the invention may alternatively comprise the sequence of SEQ ID NO: 2, GLEISEQINQQNLKQSFFNDME. The polypeptide of SEQ ID NO: 2 is not modified. It is similar to the sequence of SEQ ID NO: 1, but for a methionine at position 21, rather than a norleucine. Like SEQ ID NO: 1 and SEQ ID NO: 6, it may be fused to a membrane penetrating polypeptide.  
         [0035]    Nucleic acid molecules comprise a nucleotide sequence that encodes the polynucleotide sequence of SEQ ID NO: 2. One of skill in the art will recognize that many sequences will encode the polypeptide, as more than one codon can specify a given amino acid. The nucleic acid may further comprise regulatory sequences that enhance the expression of the polypeptide. Promoters may be strong constitutive promoters, as discussed above, or may be tissue-specific promoters. Preferably the tissue-specific promoter is a lung-specific promoter. The nucleic acid molecules may further comprise a vector. The vector can be any suitable vector for the delivery of the polynucleotide sequence into the lungs of a patient, resulting in expression of the polypeptide in the lungs of the patient.  
         [0036]    A CFTR protein can be activated by expression of a polynucleotide. A nucleic acid comprising a sequence encoding a polypeptide according to SEQ ID NO: 2 is administered to a cell comprising the CFTR protein. The polypeptide is expressed and the CFTR protein is thereby activated. The polynucleotide may be administered by any acceptable means in the art. Preferably the polynucleotide is administered as an aerosol.  
         [0037]    The administration of the polypeptides of the present invention are most useful in treatment of a class of mutations that encode CFTR proteins that are properly delivered to the plasma membrane but that are residually or minimally active. Minimally or residually active CFTR proteins have the ability to mediate or modulate channel conductance. However, channel conductance is insufficient to sustain the healthy, not cystic fibrotic phenotype. Residually or minimally active includes proteins for which the activity of the CFTR can be recorded but may be at a level that is barely detectable. This invention will also be useful for CFTR mutants that are, to a large extent, misprocessed and thus reach the plasma membrane in much lower quantities than normally processed CFTR, and for CFTR mutants that are, to a large extent, improperly spliced, but retain production of some properly spliced CFTR. Known mutants of CFTR are listed in Table 1. In addition to its utility in the activation of mutant forms of CFTR, this invention will be a useful adjunct to gene therapy for cystic fibrosis. By enhancing the per-CFTR molecule chloride transport activity, this peptide will increase the chloride transport activity obtained at any level of expression of CFTR, thereby increasing its effective efficacy.  
                               TABLE 1                       Name   Nucleotide_change   Exon   Consequence   Reference                   −816C − &gt;T   C to T at −816   5′   promoter mutation?   Bienvenu et al. (NL#60)               flanking       −741T − &gt;G   T to G at −741   5′   promoter mutation?   Bienvenu et al. (NL#59)               flanking       −471delAGG   deletion of AGG from −471   5′   promoter mutation?   Grade et al. 1994               flanking       −363C/T   C to T at −363   5′   promoter mutation   Zielenski et al. 1999*               flanking       −102T − &gt;A   T to A at −102   5′   regulatory mutation?   Claustres et al. (NL#69)               flanking       −94G − &gt;T   G to T at −94   5′   promoter mutation?   Claustres et al. (NL#70)               flanking       −33G − &gt;A   G to A at −33   5′   promoter mutation?   Claustres et al (NL#67)               flanking       132C − &gt;G   C to G at 132   1   altered translation   Claustres et al (NL#67)                   initiation?       P5L   C to T at 146   1   Pro to Leu at codon 5   Chillón et al. (NL#59)       S10R   C to A at 160   1   Ser to Arg at codon 10   Hughes et al. (NL#65)       S13F   C to T at 170   1   Ser to Phe at 13   Cao et al. (NL#69)       185 + 1G − &gt;T   G to T at 185 + 1   intron 1   mRNA splicing defect   Férec 1998*       185 + 4A − &gt;T   A to T at 185 + 4   intron 1   mRNA splicing defect?   Culard et al. 1994                   (CBAVD)       186 − 13C − &gt;G   C to G at 186 − 13   intron 1   mRNA splicing defect?   Férec et al. (NL#50)       W19C   G to T at 189   2   Trp to Cys at 19   Macek et al. (NL#62)       G27E   G to A at 212   2   Gly to Glu at 27   Bienvenu et al. 1994a       R31C   C to T at 223   2   Arg to Cys at 31   Costes et al. (NL#56)       R31L   G to T at 224   2   Arg to Leu at 31   Zielenski et al. 1995       232del18   Deletion of 18 bp from 232   2   Deletion of 6 aa from   Faucz et al. (NL#69)                   Leu34 to Gln39       S42F   C to T at 257   2   Ser to Phe at 42   Férec et al. 1995       D44G   A to G at 263   2   Asp to Gly at 44   Fanen et al. 1992       A46D   C to A at 269   2   Ala to Asp at 46   Andoniadi et al. (NL#64)       279A/G   A to G at 279   2   No change (Leu at 49)   Bienvenu et al. (NL#69)       I50T   T to C at 280   2   Ile to Thr at codon 50   Casals et al. (NL#65)       S50P   T to C at 280   2   Ser to Pro at 50   Casals et al. (NL#65)       S50Y   C to A at 281   2   Ser to Tyr at 50   Zielenski et al. (NL#63)                   (CBAVD)       296 + 3insT   insertion of T after 296 + 3   intron 2   mRNA splicing defect?   Casals et al. 1998*       296 + 1G − &gt;T   G to T at 296 + 1   intron 2   missense; mRNA   Walker et al. 2000*                   splicing defect?       296 + 1G − &gt;C   G to C at 296 + 1   intron 2   mRNA splicing defect   Tzetis et al. (NL#64)       296 + 2T − &gt;C   T to C at 296 + 2   intron 2   mRNA splicing defect   Férec et al. (NL#63)       296 + 9A − &gt;T   A to T at 296 + 9   intron 2   mRNA splicing defect?   Zielenski et al. (NL#68)       296 + 12T − &gt;C   T to C at 296 + 12   intron 2   mRNA splicing defect?   Cuppens et al. (NL#53)       297 − 28insA   insertion of A after 297 − 28   intron 2   mRNA splicing defect?   Scheffer &amp; Dijkstra                       (NL#60)       297 − 3C − &gt;A   C to A at 297 − 3   intron 2   mRNA splicing defect?   Zielenski et al. (NL#70)       297 − 3C − &gt;T   C to T at 297 − 3   intron 2   mRNA splicing defect?   Bienvenu et al. (NL#55)       297 − 2A − &gt;G   A to G at 297 − 2   intron 2   mRNA splicing defect   Schwarz et al (NL#67)       297 − 10T − &gt;G   C to G at 297 − 10   intron 2   splice mutation?   Zielenski et al. 1999*       297 − 12insA   insertion of A at 297 − 12   intron 3   splice mutation?   Girodon et al. 1999*       E56K   G to A at 298   3   Glu to Lys at 56   Dörk et al. (NL#69)       W57G   T to G at 301   3   Trp to Gly at 57   Ferrari et al. (NL#47)       W57R   T to C at 301   3   Trp to Arg at 57   Malone et al. (NL#69)       D58N   G to A at 304   3   Asp to Asn at 58   Dörk et al. (NL#69)       D58G   A to G at 305   3   Asp to Gly at 58   Claustres et al. 2000*       E60K   G to A at 310   3   Glu to Lys at 60   Claustres et al. 2000*       E60L   G to A at 310   3   Glu to Leu at 60   Casals et al. 2000*       N66S   A to G at 328   3   Asn to Ser at 66   Cashman et al. (NL#55)       P67L   C to T at 332   3   Pro to Leu at 67   Hamosh et al. (NL#54)       K68E   A to G at 334   3   Lys to Glu at 68   Kilinc et al. (NL#70)       K68N   A to T at 336   3   Lys to Asn at 68   Dörk &amp; Tümmler                       (NL#48)       A72T   G to A at 346   3   Ala to Thr at 72   Pacheco et al. 1999*       A72D   C to A at 347   3   Ala to Asp at 72   Le Gall et al. (NL#68)       R74W   C to T at 352   3   Arg to Trp at 74   Claustres et al. 1993       R74Q   G to A at 353   3   Arg to Gln at 74   Malone et al. 2000*       R75L   G to T at 356   3   Arg to Leu at 75   Costes et al. (NL#55)       W79R   T to C at 367   3   Trp to Arg at 79   Macek et al. (NL#56)       G85E   G to A at 386   3   Gly to Glu at 85   Zielenski et al. 1991b       G85V   G to T at 386   3   Gly to Val at 85   Casals et al. (NL#67)       F87L   T to C at 391   3   Phe to Leu at 87   Bienvenu et al. 1994c       L88S   T to C at 395   3   Leu to Ser at 88   Malone et al. (NL#51)       Y89C   A to G at 398   3   Tyr to Cys at 89   Seia et al. 1999*       L90S   T to C at 401   3   Leu to Ser at 90   Férec 1998*       G91R   G to A at 403   3   Gly to Arg at 91   Guillermit et al. 1993       405 + 1G − &gt;A   G to A at 405 + 1   intron 3   mRNA splicing defect   Dörk et al. 1993e       405 + 3A − &gt;C   A to C at 405 + 3   intron 3   mRNA splicing defect?   Hamosh et al. (NL#54)       405 + 4A − &gt;G   A to G at 405 + 4   intron 3   mRNA splicing defect?   Ghanem et al. 1994       406 − 10C − &gt;G   C to G at 406 − 10   intron 3   mRNA splicing defect?   Greil et al. (NL#55)       406 − 6T − &gt;C   T to C at 406 − 6   intron 3   mRNA splicing defect?   Claustres et al. 1993       406 − 3T − &gt;C   T to C at 406 − 3   intron 3   mRNA splicing defect?   Kilinc et al. (NL#70)       406 − 2A − &gt;G   A to G at 406 − 2   intron 3   mRNA splicing defect   Dörk et al. (NL#69)       406 − 2A − &gt;C   A to C at 406 − 2   intron 3   mRNA splicing defect   Costes et al. (NL#60)       406 − 1G − &gt;C   G to C at 406 − 1   intron 3   mRNA splicing defect   Bonizzato et al. 1992       406 − 1G − &gt;A   G to A at 406 − 1   intron 3   mRNA splicing defect   Wang et al. 1998*       406 − 1G − &gt;T   G to T at 406 − 1   intron 3   mRNA splicing defect   Bienvenu et al. (NL#55)       E92K   G to A at 406   4   Glu to Lys at 92   Nunes et al. 1993       A96E   C to A at 419   4   Ala to Glu at 96   Férec 1998*       Q98R   A to G at 425   4   Gln to Arg at 98   Romey et al. 1995       P99L   C to T at 428   4   Pro to Leu at 99   Schwartz &amp;                       Holmberg (NL#50)       I105N   T to A at 446   4   Ile to Asn at 105   Claustres et al. 2000*       S108F   C to T at 455   4   Ser to Phe at 108   Seydewitz et al. 1995       Y109N   T to A at 457   4   Tyr to Asn at 109   Schaedel et al. 1998*       Y109C   A to G at 458   4   Tyr to Cys at 109   Schaedel et al. 1994       D110H   G to C at 460   4   Asp to His at 110   Dean et al. 1990       D110Y   G to T at 460   4   Asp to Tyr at 110   Casals et al. 2000*       D110E   C to A at 462   4   Asp to Glu at 110   Seia et al. 1999*       P111A   C to G at 463   4   Pro to Ala at 111   Férec et al. (NL#69)       P111L   C to T at 464   4   Pro to Leu at 111   Claustres et al. (NL#62)       delta E115   3 bp deletion of 475-477   4   deletion of Glu at 115   Chillón et al. 1995                       (NL#61)       E116Q   G to C at 478   4   Glu to Gln at 116   Walker et al. 2000*       E116K   G to A at 478   4   Glu to Lys at 116   Costes et al. (NL#60)       R117C   C to T at 481   4   Arg to Cys at 117   Dörk et al. 1994b       R117H   G to A at 482   4   Arg to His at 117   Dean et al. 1990       R117P   G to C at 482   4   Arg to Pro at 117   Feldmann et a. (NL#64)       R117L   G to T at 482   4   Arg to Leu at 117   Férec et al. 1995       A120T   G to A at 490   4   Ala to Thr at 120   Chillón et al. 1994       I125T   T to C at 506   4   Ile to Thr at 125   Mittre (NL#70)       G126D   G to A at 509   4   Gly to Asp at 126   Wagner et al 1994       L137R   T to G at 542   4   Leu to Arg at 137   Chevalier-Porst &amp; Bozon                       (NL#70)       L137H   T to A at 542   4   Leu to His at 137   Wallace (NL#69)       L138ins   insertion of CTA, TAC or ACT   4   insertion of leucine at   Dörk et al. (NL#69)           at nucleotide 544, 545 or 546       138       H139R   A to G at 548   4   His to Arg at 139   Férec et al. 1995       P140S   C to T at 550   4   Pro to Ser at 140   Férec et al. (NL#61)       P140L   C to T at 551   4   Pro to Leu at 140   Tzetis et al. (NL#70)       A141D   C to A at 554   4   Ala to Asp at 141   Gouya et al. (NL#65)       H146R   A to G at 569   4   His to Arg at 146   Bienvenu et al. (NL#68)                   (CBAVD)       I148T   T to C at 575   4   Ile to Thr at 148   Bozon et al. 1994       I148N   T to A at 575   4   Ile to Asn at 148   Casals et al. (NL#69)       G149R   G to A at 577   4   Gly to Arg at 149   Mercier et al. 1995       M152V   A to G at 586   4   Met to Val at 152   Edkins &amp; Creegan                   (mutation?)   (NL#54)       M152R   T to G at 587   4   Met to Arg at 152   Yoshimura 1998*       591del18   deletion of 18 bp from 591   4   deletion of 6 a.a. from   Varon &amp; Reis (NL#64)       A155P   G to C at 595   4   Ala to Pro at 155   Zielenski et al. (NL#70)       S158R   A to C at 604   4   Ser to Arg at 158   Girodon et al. 1999*       Y161N   T to A at 613   4   Tyr to Asn at 161   Claustres et al. 2000*       Y161D   T to G at 613   4   Tyr to Asp at 161   Zielenski et al. 1999*       Y161S   A to C at 614 (together with   4   Tyr to Ser at 161   Andrew et al. 1999*           612T/A)       K162E   A to G at 616   4   Lys to Glu at 162   Tzetis et al. (NL#70)       621G − &gt;A   G to A at 621   4   mRNA splicing defect   Mackova et al. (NL#64)       621 + 1G − &gt;T   G to T at 621 + 1   intron 4   mRNA splicing defect   Zielenski et al. 1991b       621 + 2T − &gt;C   T to C at 621 + 2   intron 4   mRNA splicing defect   Schwarz et al. (NL#66)       621 + 2T − &gt;G   T to G at 621 + 2   intron 4   mRNA splicing defect   Claustres et al. 1993       621 + 3A − &gt;G   A to G at 621 + 3   intron 4   mRNA splicing defect   Tzetis et al. (NL#70)       622 − 2A − &gt;C   A to C at 622 − 2   intron 4   mRNA splicing defect   Cuppens et al. 1993       622 − 1G − &gt;A   G to A at 622 − 1   intron 4   mRNA splicing defect   Zielenski et al. (NL#66)       L165S   T to C at 626   5   Leu to Ser at 165   Férec et al. (NL#51)       K166Q   A to G at 628   5   Lys to Gln at 166   Macek et al.                       (NL#62; #66)       R170C   C to T at 640   5   Arg to Cys at 170   Férec et al. (NL#62)       R170G   C to G at 640   5   Arg to Gly at 170   Claustres et al. (NL#49)       R170H   G to A at 641   5   Arg to His at 170   Brownsell et al. 2001*       I175V   A to G at 655   5   Ile to Val at 175   Romey et al. 1994a       I177T   T to C at 662   5   Ile to Thr at 177   Bienvenu et al. (NL#68)       G178R   G to A at 664   5   Gly to Arg at 178   Zielenski et al. 1991b       Q179K   C to A at 667   5   Gln to Lys at 179   Zhang &amp; Wong 2000*       N186K   C to A at 690   5   Asn to Lys at 186   Claustres &amp; Carles                       (NL#70)       N187K   C to A at 693   5   Asn to Lys at 187   Arduino et al. 1998*       D192N   G to A at 706   5   Asp to Asn at 192   Costes et al. (NL#62)       delta D192   deletion of TGA or GAT from   5   deletion of Asp at 192   Feldmann et al. (NL#66)           706 or 707       D192G   A to G at 707   5   Asp to Gly at 192   Audrézet et al. 1994       E193K   G to A at 709   5   Glu to Lys at 193   Ferrari et al. (NL#62); et                       al. Mercier et al. 1995       711 + 1G − &gt;T   G to T at 711 + 1   intron 5   mRNA splicing defect   Zielenski et al. 1991b       711 + 3A − &gt;C   A to C at 711 + 3   intron 5   mRNA splicing defect   Macek MJr et al.                       (NL#61)       711 + 3A − &gt;G   A to G at 711 + 3   intron 5   mRNA splicing defect   Petreska et al. 1994       711 + 3A − &gt;T   A to T at 711 + 3   intron 5   mRNA splicing defect?   Casasl et al. (NL#67)       711 + 5G − &gt;A   G to A at 711 + 5   intron 5   mRNA splicing defect   Bisceglia et al. 1994       711 + 34A − &gt;G   A to G at 711 + 34   intron 5   mRNA splicing defect?   Tzetis et al. (NL#68)       712 − 1G − &gt;T   G to T at 712 − 1   intron 5   mRNA splicing defect   Chillón et al. (NL#59)       G194V   G to T at 713   6a   Gly to Val at 194   Férec 1998*       A198P   G to C at 724   6a   Ala to Pro at 198   Walker et al. 1999*       H199Y   C to T at 727   6a   His to Tyr at 199   Dörk &amp; Tümmler                       (NL#45)       H199Q   T to G at 729   6a   His to Gln at 199   Dean et al. (NL#28)       V201M   G to A at 733   6a   Val to Met al 201   Férec 1998*       P205S   C to T at 745   6a   Pro to Ser at 205   Chillón et al. 1993b       L206W   T to G at 749   6a   Leu to Trp at 206   Claustres et al. 1993       L206F   G to T at 750   6a   Leu to Phe at 206   Férec et al. (NL#69)       A209S   G to T at 757   6a   Ala to Ser at 209   Férec 1998*       E217G   A to G at 782   6a   Glu to Gly at 217   Zielenski et al. (NL#70)       Q220R   A to G at 791   6a   Gln to Arg at 220   Férec 1998*       C225R   T to C at 805   6a   Cys to Arg at 225   Fanen et al. 1992       L227R   T to G at 812   6a   Leu to Arg at 227   Ghanem et al. (NL#59)       V232D   T to A at 827   6a   Val to Asp at 232   Costes et al. (NL#60)                   (CBAVD)       Q237E   C to G at 841   6a   Gln to Glu at 237   Costes et al. (NL#62)       G239R   G to A at 847   6a   Gly to Arg at 239   Zielenski et al. (NL#60)       G241R   G to A at 852   6a   Gly to Arg at 241   Férec et al. (NL#69)       M243L   A to C at 859   6a   Met to Leu at 243 (ATG   Yoshimura 1999*                   to CTG)       M244K   T to A at 863   6a   Met to Lys at 244   Claustres et al. (NL#64)       R248T   G to C at 875   6a   Arg to Thr at 248   Scheffer et al. (NL#70)                   (CBAVD)       875 + 1G − &gt;C   G to C at 875 + 1   intron   mRNA splicing defect   Zielenski et al. (NL#58)               6a       875 + 1G − &gt;A   G to A at 875 + 1   intron   mRNA splicing defect   Duarte et al. (NL#63)               6a       876 − 14del12   deletion of 12 bp from 876 − 14   intron   mRNA splicing defect?   Audrézet et al. 1993a               6a       876 − 10del8   deletion of 8 bp from 876 − 10   intron   mRNA splicing defect?   Costes et al. (NL#46, 47)               6a       876 − 3C − &gt;T   C to T at 876 − 3   intron   splicing mutation?   Chevalier-Porst &amp; Bozon               6a       1999*       R258G   G to A at 904   6b   Arg to Gly at 258   Mercier et al. 1995       V920L   G to T at 289   15   Val to Leu at 920   Girodon et al. 1999*       M265R   T to G at 926   6b   Met to Arg at 265   Schwarz et al. (NL#65)       E278del   deletion of AAG from 965   6b   deletion of Glu at 278   Casals et al. (NL#70)       N287Y   A to T at 991   6b   Asn to Tyr at 287   Shrimpton &amp; Borowitz                       (NL#69)       994del9   deletion of TTAAGACAG   6b   mRNA splicing defect   Zielenski et al. (NL#70)           from 994       1002 − 3T − &gt;G   T to G at 1002 − 3   intron   mRNA splicing defect   Mackova et al. (NL#64)               6b       E292K   G to A at 1006   7   Glu to Lys at 292   Bienvenu et al. (NL#68)       R297W   C to T at 1021   7   Arg to Trp at 297   Dörk et al. (NL#69)       R297Q   G to A at 1022   7   Arg to Gln at 297   Graham et al. 1991       A299T   G to A at 1027   7   Ala to Thr at 299   Férec 1999*       Y301C   A to G at 1034   7   Tyr to Cys at 301   Constantinou-Deltas                       (NL#58)       S307N   G to A at 1052   7   Ser to Asn at 307   Onay &amp; Kirdar (NL#70)       A309D   C to A at 1058   7   Ala to Asp at 309   Ferrari et al. (NL#64)       A309G   C to G at 1058   7   Ala to Gly at 309   Bienvenu et al. (NL#68)       delta F311   deletion of 3 bp between 1059   7   deletion of Phe310, 311   Meitinger et al. 1993           and 1069       or 312       F311L   C to G at 1065   7   Phe to Leu at 311   Férec et al. 1992       G314R   G to C at 1072   7   Gly to Arg at 314   Nasr et al. (NL#56)       G314V   G to T at 1073   7   Gly to Val at 324   Chevalier-Porst &amp; Bozon                       (NL#70)       G314E   G to A at 1073   7   Gly to Glu at 314   Golla et al. 1994       F316L   T to G at 1077   7   Phe to Leu at 316   Férec 2000*       V317A   T to C at 1082   7   Val to Ala at 317   Férec et al. (NL#55)       L320V   T to G at 1090   7   Leu to Val at 320 CAVD   Bienvenu et al (NL#67)       L320F   A to T at 1092   7   Leu to Phe at 320   Macek et al. (NL#64)       V322A   T to C at 1097   7   Val to Ala at 322   Férec et al. (NL#63)                   (mutation?)       L327R   T to G at 1112   7   Leu to Arg at 327   Ravnik-Glavac et al.                       (NL#53)       R334W   C to T at 1132   7   Arg to Trp at 334   Estivill et al. 1991       R334L   G to T at 1133   7   Arg to Leu at 334   Dörk et al. (NL#69)       R334Q   G to A at 1133   7   Arg to Gln at 334   Férec et al. (NL#65)       I336K   T to A at 1139   7   Ile to Lys at 336   Cuppens et al. 1993       T338I   C to T at 1145   7   Thr to Ile at 338   Saba et al. 1993       E474K   G to A at 1152   10   Glu to Lys at 474   Girodon et al. 1999*       L346P   T to C at 1169   7   Leu to Pro at 346   Constantinou (NL#58)       R347C   C to T at 1171   7   Arg to Cys at 347   Férec et al. (NL#56)       R347H   G to A at 1172   7   Arg to His at 347   Cremonesi et al., 1992       R347P   G to C at 1172   7   Arg to Pro at 347   Dean et al. (NL#6)       R347L   G to T at 1172   7   Arg to Leu at 347   Audrézet et al. 1993a       M348K   T to A at 1175   7   Met to Lys at 348   Audrézet et al. 1993b       A349V   C to T at 1178   7   Ala to Val at 349   Audrézet et al. 1993a       R352W   C to T at 1186   7   Arg to Trp at 352   Byrne et al. (NL#69)       R352Q   G to A at 1187   7   Arg to Gln at 352   Cremonesi et al. 1992       Q353H   A to C at 1191   7   Gln to His at 353   Férec et al. (NL#65)       Q359K/T360K   C to A at 1207 and C to A at   7   Glu to Lys at 359 and Thr   Shoshani et al. 1992           1211       to Lys at 360       Q359R   A to G at 1208   7   Gln to Arg at 359   Férec 1999*       W361R(T − &gt;C)   T to C at 1213   7   Trp to Arg at 361   Bienvenu et al. (NL#56)       W361R(T − &gt;A)   T to A at 1213   7   Trp to Arg at 361   Telleria &amp; Alonso 1998*       S364P   T to C at 1222   7   Ser to Pro at 364   Hamosh et al. (NL#54)       L365P   T to C at 1226   7   Leu to Pro at 365   Casals et al. 2000*       1243ins6   insertion of ACAAAA after   7   insertion of Asp and Lys   Shackleton et al (NL#67)           1243       after Lys370       1248 + 1G − &gt;A   G to A at 1248 + 1   intron 7   mRNA splicing defect   Schwarz et al. (NL#58)       1249 − 29delAT   deletion of AT from 1249 − 29   intron 7   mRNA splicing defect?   Zielenski et al. (NL#69)       1249 − 27delTA   deletion of TA at 1249 − 27   intron 7   mRNA splicing defect?   Egan et al. (NL#70)       1249 − 5A − &gt;G   A to G at 1249   intron 7   mRNA splicing defect?   Bienvenu et al. (NL#62)       L375F   A to C at 1257   8   Leu to Phe at 375   Jézéquel (NL#65)                   (CUAVD)       E379X   G to T at 1267   8   Glu to Stp at 379   Glaeser &amp; Mehnert                       2000*       L383S   T to C at 1280   8   Leu to Ser at 383   Casals et al. (NL#69)       T360R   C to G at ?   7   Thr to Arg at 360   Férec 1998*       V392A   T to C at 1307   8   Val to Ala at 392 CAVD   Bienvenu et al (NL#67,                       NL#68)       V392G   T to G at 1307   8   Val to Gly at 392   Zielenski et al. Larder et                       al. (NL#70)       M394R   T to G at 1313   8   Met to Arg at 394   Férec 1998*       A399V   C to T at 1328   8   Ala to Val at 399   Yoshimura &amp; Azuma                       2000*       E403D   G to C at 1341   8   Glu to Asp at 403   Férec 1999*       1341G − &gt;A   G to A at 1341   8   ?   Telleria &amp; Alonso 1998*       1341G − &gt;A   G to A at 1341   8       Telleria 1999*       1341 + 1G − &gt;A   G to A at 1341 + 1   intron 8   mRNA splicing defect   Dörk et al. (NL#69)       1341 + 18A − &gt;C   A to C at 1341 + 18   intron 8   mRNA splicing defect?   Claustres et al. (NL#60)       1342 − 11TTT − &gt;G   TTT to G at 1342 − 11   intron 8   mRNA splicing defect?   Dörk &amp; Tümmler                       (NL#59)       1342 − 2A − &gt;C   A to C at 1342 − 2   intron 8   mRNA splicing defect   Dörk et al. 1993b       1342 − 1G − &gt;C   G to C at 1342 − 1   intron 8   mRNA splicing defect   Cutting &amp; Curristin (NL                       #30)       E407V   A to T at 1352   9   Glu to Val at 407   Zielenski et al. 1999*       N418S   A to G at 1385   9   Asn to Ser at 418   Sava et al. (NL#64)       G424S   G to A at 1402   9   Gly to Ser at 424   Bienvenu et al. 2000*       D443Y   G to T at 1459   9   Asp to Tyr at 443   Bienvenu et al. (NL#63)       I444S   T to G at 1463   9   Ile to Ser at 444   Zielenski et al. 1999*       Q452P   A to C at 1487   9   Gln to Pro at 452   Claustres et al. (NL#70)       delta L453   deletion of 3 bp between 1488   9   deletion of Leu at 452 or   Dörk et al (NL#67)           and 1494       454       A455E   C to A at 1496   9   Ala to Glu at 455   Kerem et al. 1990       V456F   G to T at 1498   9   Val to Phe at 456   Dörk et al. 1994a       G458V   G to T at 1505   9   Gly to Val at 458   Cuppens et al. 1990       1524 + 6insC   insertion of C after 1524 + 6,   intron 9   mRNA splicing defect?   Bienvenu et al. (NL#61)           with G to A at 1524 + 12       1525 − 1G − &gt;A   G to A at 1525 − 1   intron 9   mRNA splicing defect   Dörk et al. 1993a       S466L   C to T at 1529   10   Ser to Leu at 466   Costes et al. (NL#66)                   (CBAVD)       G480S   G to A at 1570   10   Gly to Ser at 480   Kawasoe et al. 2001*       G480C   G to T at 1570   10   Gly to Cys at 480   Smit et al 1991       G480D   G to A at 1570   10   Gly to Asp at 480   Hawworth et al. (NL#66)       H484Y   C to T at 1582   10   His to Tyr at 484   Casals et al. (NL#69)                   (CBAVD?)       H484R   A to G at 1583   10   His to Arg at 484   Férec 1998*       S485C   A to T at 1585   10   Ser to Cys at 485   Andrew et al. 1999*       C491R   T to C at 1603   10   Cys to Arg at 491   Chevalier-Porst &amp; Bozon                       (NL#70)       S492F   C to T at 1607   10   Ser to Phe at 492   Férec et al. 1992       Q493R   A to G at 1610   10   Gln to Arg at 493   Savov et al. 1994a       P499A   C to G at 1627   10   Pro to Ala at 499   Arduino et al. (NL#68)                   (CBAVD)       T501A   A to G at 1633   10   Thr to Ala at 501   Claustres et al. 1999*       I502T   T to C at 1637   10   Ile to Thr at 502   Chevalier-Porst &amp; Bozon                       (NL#70)       E504Q   G to C at 1642   10   Glu to Gln at 504   Baranov (NL#34, #35)       I506L   A to C at 1648   10   Ile to Leu at 506   Zielenski et al. (NL#70)       delta 1507   deletion of 3 bp between 1648   10   deletion of Ile506 or   Kerem et al. 1990;           and 1653       Ile507   Schwarz et al. 1991       I506S   T to G at 1649   10   Ile to Ser at 506   Deufel et al. 1994       I506T   T to C at 1649   10   Ile to Thr at 506   Desgeorges et al. 1995       delta F508   deletion of 3 bp between 1652   10   deletion of Phe at 508   Rommens et al., Riordan           and 1655           et al., Kerem et al. 1989       F508S   T to C at 1655   10   Phe to Ser at 508   Férec 1998*       D513G   A to G at 1670   10   Asp to Gly at 513   Bienvenu et al. (NL#70)                   (CBAVD)       Y517C   A to G at 1682   10   Tyr to Cys at 517   Arduino et al. (NL#70)       V520F   G to T at 1690   10   Val to Phe at 520   Jones et al. 1992       V520I   G to A at 1690   10   Val to Ile at 520   Malone et al. (NL#60)       1706del16   16 bp deletion from 1706   10   deletion of spice site       1706del17   deletion of 17 bp ftom 1706   10   deletion of splice site   Leoni et al. 1993       E527Q   G to C at 1711   10   Glu to Gln at 527   Byrne et al. (NL#70)       E527G   A to G at 1712   10   Glu to Gly at 527   Benetazzo et al. (NL#70)       1716 − 1G − &gt;A   G to A at 1716 − 1   intron   mRNA splicing defect   Jordanova et al. (NL#69)               10       E528D   G to T at 1716   10   Glu to Asp at 528 (splice   Girodon et al. 1999*                   mutation?)       1716 + 2T − &gt;C   T to C at 1716 + 2   intron   mRNA splicing defect   Claustres et al. (NL#68)               10       1717 − 8G − &gt;A   G to A at 1717 − 8   intron   mRNA splicing defect?   Savov et al. 1994a               10       1717 − 3T − &gt;G   T to G at 1717 − 3   intron   mRNA splicing defect?   Férec et al. (NL#68)               10       1717 − 2A − &gt;G   A to G at 1717 − 2   intron   mRNA splicing defect   Hawworth et al (NL#67)               10       1717 − 1G − &gt;A   G to A at 1717 − 1   intron   mRNA splicing defect   Kerem et al. 1990               10       1717 − 9T − &gt;A   T to A at 1717 − 9   intron   mRNA splicing   Vouk &amp; Komel 1999*               10   mutation?       D529H   G to C at 1717   11   Asp to His at 529   Férec 1998*       A534E   C to A at 1733   11   Ala to Glu at 534   Audrézet et al. 1993a       I539T   T to C at 1748   11   Ile to Thr at 539   Chomel &amp; Kitzis                       (NL#66)       G544S   G to A at 1762   11   Gly to Ser at 544   Férec et al. (NL#61)       G544V   G to T at 1763   11   Gly to Val at 544   Claustres et al. (NL#69)                   (CBAVD)       S549R(A − &gt;C)   A to C at 1777   11   Ser to Arg at 549   Sangiuolo et al. 1990       S549N   G to A at 1778   11   Ser to Asn at 549   Cutting et al. 1990a       S549I   G to T at 1778   11   Ser to Ile at 549   Kerem et al. 1990       S549R(T − &gt;G)   T to G at 1779   11   Ser to Arg at 549   Kerem et al. 1990       G550R   G to A at 1780   11   Gly to Arg at 550   Férec et al. (NL#66)       G551S   G to A at 1783   11   Gly to Ser at 551   Strong et al. 1991       G551D   G to A at 1784   11   Gly to Asp at 551   Cutting et al. 1990a       Q552K   C to A at 1786   11   Gln to Lys   Faucz et al. (NL#69)       R553G   C to G at 1789   11   Arg to Gly at 553   Férec et al. (NL#59)       R553Q   G to A at 1790   11   Arg to Gln at 553   Dörk et al. 1991b                   (associated with delta                   F508;       R555G   A to G at 1795   11   Arg to Gly at 555   Zielenski et al 1999*       I556V   A to G at 1798   11   Ile to Val at 556   Ghanem et al. (NL#50)                   (mutation?)       L558S   T to C at 1805   11   Leu to Ser at 558   Maggio et al. (NL#31)       A559T   G to A at 1807   11   Ala to Thr at 559   Cutting et al. 1990a       A559E   C to A at 1808   11   Ala to Glu at 559   Girodon et al. 1999*       R560K   G to A at 1811   11   Arg to Lys at 560   Férec et al. 1992       R560T   G to C at 1811   11   Arg to Thr at 560;   Kerem et al. 1990                   mRNA splicing defect?       1811 + 1G − &gt;C   G to C at 1811 + 1   intron   mRNA splicing defect   Petreska et al. (NL#50)               11       1811 + 1.6kbA − &gt;G   A to G at 1811 + 1.2 kb   intron   creation of splice donor   Chillón et al. 1995               11   site       1811 + 18G − &gt;A   G to A at 1811 + 18   intron   mRNA splicing defect?   Teng et al. (NL#65)               11       1812 − 1G − &gt;A   G to A at 1812 − 1   intron   mRNA splicing defect   Chillón et al. 1994               11       R560S   A to C at 1812   12   Arg to Ser at 560   Costes et al. (NL#54)       A561E   C to A at 1814   12   Ala to Glu at 561   Duarte et al. (NL#55)       V562L   G to C at 1816   12   Val to Leu at 562   Hughes et al. (NL#65)       V562I   G to A at 1816   12   Val to Ile at 562   Feldmann et al (NL#67)       Y563D   T to G at 1819   12   Tyr to Asp at 563   Hamosh et al. (NL#54)       Y563N   T to A at 1819   12   Tyr to Asn at 563   Kerem et al. (NL#13)       Y563C   A to G at 1821   12   Tyr to Cys at 563   Delhaize C (NL#67)       L568F   G to T at 1836   12   Leu to Phe at 568   Dörk et al. (NL#69)                   (CBAVD?)       Y569D   T to G at 1837   12   Tyr to Asp at 569   Malone et al. (NL#65)       Y569H   T to C at 1837   12   Tyr to His at 569   Costes et al. (NL#52)       Y569C   A to G at 1838   12   Tyr to Cys at 569   Plaseska et al. (NL#45)       LS71S   T to C at 1844   12   Leu to Ser at 571   Savov et al. (NL#60)       D572N   G to A at 1846   12   Asp to Asn at 572   Férec et al. (NL#59)       P574H   C to A at 1853   12   Pro to His at 574   Kerem et al. 1990       G576A   G to C at 1859   12   Gly to Ala at 576   Sarginson et al. (NL#69)                   (CAVD)       Y577F   A to T at 1862   12   Tyr to Phe at 577   Dörk et al (NL#67)       D579Y   G to T at 1867   12   Asp to Tyr at 579   Harris et al. (NL#63)       D579G   A to G at 1868   12   Asp to Gly at 579   Ferrari et al. (NL#53)       D579A   A to C at 1868   12   Asp to Ala at 579   Pacheco et al. (NL#70)       T582I   C to T at 1877   12   Thr to Ile at 582   Claustres et al (NL#67)       T582R   C to G at 1877   12   Thr to Arg at 582   Casals et al. (NL#55)       S589N   G to A at 1898   12   Ser to Asn at 589   Scheffer et al. (NL#68)                   (mRNA splicing defect?)       S589I   G to T at 1898   12   Ser to Ile at 589   Schwarz et al. 1999*                   (splicing?)       1898 + 1G − &gt;T   G to T at 1898 + 1   intron   mRNA splicing defect   Morris (NL#62)               12       1898 + 1G − &gt;C   G to C at 1898 + 1   intron   mRNA splicing defect   Cuppens et al. 1993               12       1898 + 1G − &gt;A   G to A at 1898 + 1   intron   mRNA splicing defect   Strong et al. 1992               12       1898 + 3A − &gt;C   A to C at 1898 + 3   intron   mRNA splicing defect?   Mercier et al. 1995               12       1898 + 3A − &gt;G   A to G at 1898 + 3   intron   mRNA splicing defect?   Ferrari et al. (NL#35)               12       1898 + 5G − &gt;T   G to T at 1898 + 5   intron   mRNA splicing defect   Zielenski et al. 1995               12       1898 + 5G − &gt;A   G to A at 1898 + 5   intron   mRNA splicing defect   Férec et al. (NL#69)               12       1898 + 73T − &gt;G   T to G at 1898 + 73   intron   mRNA splicing defect?   Smit et al. (NL#37)               12       R600G   A to G at 1930   13   Arg to Gly at 600   Bienvenu et al. (NL#69)       I601F   A to T at 1933   13   Ile to Phe at 601   Schwarz et al. (NL#68)       V603F   G to T at 1939   13   Val to Phe at 603   Zielenski et al. (NL#70)       T604I   C to T at 1943   13   Thr to Ile at 604   Girodon et al. 1999*       1949del84   deletion of 84 bp from 1949   13   deletion of 28 a.a.   Granell et al. 1992                   (Met607 to Gln634)       H609R   A to G at 1958   13   His to Arg at 609   Bienvenu et al. (NL#69)       L610S   T to C at 1961   13   Leu to Ser at 610   Férec et al. (NL#52)       A613T   G to A at 1969   13   Ala to Thr at 613   Liechti-Gallati (NL#68)       D614Y   G to T at 1972   13   Asp to Tyr 614   Girodon et al. 1999*       D614G   A to G at 1973   13   Asp to Gly at 614   Audrézet et al. 1993b       I618T   T to C at 1985   13   Ile to Thr at 618   Macek et al. (NL#62)       L619S   T to C at 1988   13   Leu to Ser at 619   Dörk et al. 1991       H620P   A to C at 1991   13   His to Pro at 620   Haworth et al. (NL#66)       H620Q   T to G at 1992   13   His to Gln at 620   Dörk and Sturhmann                       (NL#68)       G622D   G to A at 1997   13   Gly to Asp at 622   Zielenski et al. (NL#68)                   (oligospermia)       G628R(G − &gt;A)   G to A at 2014   13   Gly to Arg at 628   Fanen et al. 1992       G628R(G − &gt;C)   G to C at 2014   13   Gly to Arg at 628   Cuppens et al. 1993       L633P   T to C at 2030   13   Leu to Pro at 633   Haworth et al. (NL#62)       L636P   T to C at 2039   13   Leu to Pro at 636   Bombieri et al. (NL#70)       D648V   A to T at 2075   13   Asp to Val at 648   Férec et al. (NL#44)       D651N   G to A at 2083   13   Asp to Asn at 651   Bombieri et al.(NL#70)       T665S   A to T at 2125   13   Thr to Ser at 665   Férec et al. (NL#63)       E672del   deletion of 3 bp between 2145-   13   deletion of Glu at 672   Claustres et al. (NL#69)           2148       K683R   A to G at 2180   13   Lys to Arg at 683   Chevalier-Porst &amp; Bozon                       2000*       F693L(CTT)   T to C at 2209   13   Phe to Leu at 693   Audrézet et al. 1993b       F693L(TTG)   T to G at 2211   13   Phe to Leu at 693   Meyer et al. 2001*       K698R   A to G 2225   13   Lys to Arg at 698   Férec et al. (NL#69)       E725K   G to A at 2305   13   Glu to Lys at 725   Tzetis et al. (NL#70)       P750L   C to T at 2381   13   Pro to Leu at 750   Chevalier-Porst &amp; Bozon                       2000*       V754M   G to A at 2392   13   Val to Met al 754   Wallace (NL#69)       T760M   C to T at 2411   13   Thr to Met al 760   Zielenski et al. 1999*       R766M   G to T at 2429   13   Arg to Met al 766   Glavac et al. (NL#66)       N782K   C to A at 2478   13   Asn to Lys at 782   Girodon et al. 1999*       R792G   C to G at 2506   13   Arg to Gly at 792   Glavac et al. (NL#66)       A800G   C to G at 2531   13   Ala to Gly at 800   Mercier et al. 1995       E822K   G to A at 2596   13   Glu to Lys at 822   Mercier et al. 1993a       E826K   G to A at 2608   13   Glu to Lys at 826   Bombieri et al (NL#67)       2622 + 1G − &gt;T   G to T at 2622 + 1   intron   splice mutation   Girodon et al. 1999*               13       2622 + 1G − &gt;A   G to A at 2622 + 1   intron   mRNA splicing defect   Audrézet et al. 1993a               13       2622 + 2del6   deletion of TAGGTA from   intron   mRNA splicing defect   Zielenski et al. (NL#70)           2622 + 2   13       D836Y   G to T at 2638   14a   Asp to Tyr at 836   Ghanem &amp; Goossens                       (NL#47)       R851L   G to T at 2684   14a   Arg to Leu at 851   Casals et al. (NL#68)       C866Y   G to A at 2729   14a   Cys to Tyr at 866   Audrézet et al. (NL#41)       L867X   T to A at 2732   14a   Leu to Stop at 867   Haworth et al. (NL#69)       2751G − &gt;A   G to A at 2751   14a   mRNA splicing defect?   Wagner et al. (NL#65)       2751 + 2T − &gt;A   T to A at 2751 + 2   intron   mRNA splicing defect   Antoniadi et al. (NL#68)               14a       2751 + 3A − &gt;G   A to G at 2751 + 3   intron   mRNA splicing defect?   Casals et al. (NL#65)               14a   (CBAVD)       2752 − 26A − &gt;G   A to G at 2752 − 26   intron   mRNA splicing defect?   Tzetis et al. (NL#66)               14a       2752 − 1G − &gt;T   G to T at 2752 − 1   intron   mRNA splicing defect   Férec et al. (NL#65)               14a       2752 − 1G − &gt;C   G to C at 2752 − 1   intron   splice mutation   Dubourg &amp; Blayau               14a       1999*       T908N   C to A at 2788   14b   Thr to Asn at 908   Férec et al. (NL#69)       2789 + 2insA   insertion of A after 2789 + 2   intron   mRNA splicing defect?   Dubourg et al. (NL#70)               14b   (CAVD)       2789 + 3delG   deletion of G at 2789 + 3   intron   mRNA splicing defect   Macek et al. (NL#63)               14b       2789 + 5G − &gt;A   G to A at 2789 + 5   intron   mRNA splicing defect   Highsmith et al. 1990               14b       2790 − 2A − &gt;G   A to G at 2790 − 2   intron   mRNA splicing defect   Marigo et al. (NL#61)               14b       2790 − 1G − &gt;C   G to C at 2790 − 1   intron   mRNA splicing defect   Schwartz et al. (NL#54)               14b       2790 − 1G − &gt;T   C to T at 2790 − 1   intron   mRNA splicing defect   Bienvenu et al. (NL#63)               14b       Q890R   A to G at 2801   15   Gln to Arg at 890   Casals et al. 1998*       D891G   A to G at 2804   15   Asp to Gly at 891   Kilinc et al. (NL#70)       S895T   G to T at 2816   15   Ser to Thr at 895   Férec 1999*       T896I   C to T at 2819   15   Thr to Ile at 896   Lázaro et al. 2000*       N900T   G to A at 2831   15   Asn to Thr at 900   Férec 1999*       2851A/G   A or G at 2851   15   Ile or Val at 907   Claustres et al. 2000*       S912L   C to T at 2867   15   Ser to Leu at 912   Ghanem et al. 1994       Y913C   A to G at 2870   15   Tyr to Cys at 913   Vidaud et al. 1990       Y917D   T to G at 2881   15   Tyr to Asp at 917   Schwarz et al. (NL#69)       Y917C   A to G at 2882   15   Tyr to Cys at 917   Edkins &amp; Creegan                       (NL#60)       I918M   T to G at 2886   15   Ile to Met al 918   Girodon et al. 1999*       Y919C   A to G at 2888   15   Tyr to Cys at 919   Savov et al. 1994a       V920M   G to A at 2890   15   Val to Met al 920   Bienvenu et al. (NL#63)       D924N   G to A at 2902   15   Asp to Asn at 924   Girodon et al. 1999*       L927P   T to G at 2912   15   Leu to Pro at 927   Hermans et al. 1994       F932S   T to C at 2927   15   Phe to Ser at 932   Férec 1999*       R933S   A to T at 2931   15   Arg to Ser at 933   Dörk et al. (NL#69)                   (CBAVD)       V938G   T to G at 2945   15   Val to Gly at 938   Dörk et al. (NL#69)                   (CAVD)       H939D   C to G at 2947   15   His to Asp at 939   Férec et al. (NL#54)       H939R   A to G at 2948   15   His to Arg at 939   Férec et al. (NL#69)       S945L   C to T at 2966   15   Ser to Leu at 945   Claustres et al. 1993       K946X   A to T at 2968   15   Lys to Stop at 946   Haworth et al. (NL#69)       H949Y   C to T at 2977   15   His to Tyr at 949   Ghanem et al. 1994       H949R   A to G at 2978   15   His to Arg at 949   Férec et al. (NL#65)       M952T   T to C at 2987   15   Met to Thr at 952   Zielenski et al. 1999*       M952I   G to C at 2988   15   Met to Ile at 952   Girodon et al (NL#67)                   CBAVD mutation?       M961I   G to T at 3015   15   Met to Ile at 961   Malone et al. 2000*       L967S   T to C at 3032   15   Leu to Ser at 967   Zielenski et al. (NL#70)                   (oligospermia?)       G970R   G to C at 3040   15   Gly to Arg at 970   Cuppens et al. 1993       3040 + 2T − &gt;C   T to C at 3040 + 2   intron   mRNA splicing defect   Poncin (NL#69)               15       3041 − 1G − &gt;A   G to A at 3041 − 1   intron   mRNA splicing defect   Malone et al (NL#67)               15       G970D   G to A at 3041   16   Gly to Asp at 970   Vassilakis et al. (NL#69)       L973F   TC to AT at 3048 and 3049   16   Leu to Phe at 973   Dörk and Sturhmann                   CBAVD)   (NL#68)       L973P   T to C at 3050   16   Leu to Pro at 973   Férec 1998*       S977P   T to C at 3061   16   Ser to Pro at 977   Dörk et al. (NL#51)       S977F   C to T at 3062   16   Ser to Phe at 977   Férec et al. (NL#69)       D979V   A to T at 3068   16   Asp to Val at 979   Feldmann et al. (NL#68)       D979A   A to C at 3068   16   Asp to Ala at 979   Dörk and Sturhmann                   (CBAVD?)   (NL#68)       I980K   T to A at 3071   16   Ile to Lys at 980   Bienvenu et al. (NL#62)       D985H   G to C at 3085   16   Asp to His at 985   Claustres &amp; Guittard                       (NL#70)       D985Y   G to T at 3085   16   Asp to Tyr at 985   Bienvenu et al. (NL#63)       I991V   A to G at 3103   16   Ile to Val at 991   Bombieri et al. 2000*       D993Y   G to T at 3109   16   Asp to Tyr at 993   Claustres et al (NL#67)       F994C   T to G at 3113   16   Phe to Cys at 994   Claustres et al. (NL#70)       3120G − &gt;A   G to A at 3120   16   mRNA splicing defect   Zielenski et al. 1994       3120 + 1G − &gt;A   G to A at 3120 + 1   intron   mRNA splicing defect   Macek et al. (1997)               16       3121 − 2A − &gt;T   A to T at 3121 − 2   intron   mRNA splicing defect   Férec et al. 1995               16       3121 − 2A − &gt;G   A to G at 3121 − 2   intron   mRNA splicing defect   Macek et al. (NL#60)               16       3121 − 1G − &gt;A   G to A at 3121 − 1   intron   mRNA splicing defect   Feldmann et al (NL#67)               16       L997F   G to C at 3123   17a   Leu to Phe at 997   Kabra et al. (NL#69)       3131del15   deletion of 15 bp from 3130,   17a   deletion of Val at 1001 to   Wallace &amp; Tassabehji           3131, or 3132       Ile at 1005   (NL#61)       I1005R   T to G at 3146   17a   Ile to Arg at 1005   Dörk et al. 1994b       A1006E   C to A at 3149   17a   Ala to Glu at 1006   Férec et al. 1995       V1008D   T to A at 3155   17a   Val to Asp at 1008   Casals et al. (NL#70)       A1009T   G to A at 3157   17a   Ala to Thr at 1009   Bombieri et al. 2000*       P1013L   C to T at 3169   17a   Pro to Leu at 1013   Onay et al. (NL#69)       Y1014C   A to G at 3173   17a   Tyr to Cys at 1014   Bozon (NL#70)       P1021S   C to T at 3193   17a   Pro to Ser at 1021   Casals et al. (NL#69)                   (CBAVD)       3195del6   deletion of AGTGAT from   17a   deletion of Val1022 and   Claustres et al. 1994           3195 to 3200       Ile1023       3196del54   deletion of 54 bp from 3196   17a   deletion of 18 aa from   Desgeorges et al.                   codon 1022   (NL#65)       3199del6   deletion of ATAGTG from   17a   deletion of Ile at 1023   Bozon (NL#70)           3199       and Val at 1024       I1027T   T to C at 3212   17a   Ile to Thr at 1027   Andrew et al. 2001*       M1028R   T to G at 3215   17a   Met to Arg at 1028   Lázaro et al. 2000*       M1028I   G to T at 3216   17a   Met to Ile at 1028   Onay et al (NL#69)       Y1032C   A to G at 3227   17a   Tyr to Cys at 1032   Dörk et al. (NL#69)                   (CBAVD)       I1366T   T to C at 4229   22   Iso to Thr at 1366   Férec 1999*       3271delGG   deletion of GG at 3271   17a   framshift for exon 17b,   Wang 1998*                   loss of splice site       3271 + 1G − &gt;A   G to A at 3271 + 1   intron   mRNA splicing defect   Mercier et al. 1994               17a       3271 + 1delGG   deletion of GG at 3271 + 1   intron   mRNA splicing defect   Wang et al. 1998*               17b       3272 − 26A − &gt;G   A to G at 3272 − 26   intron   mRNA splicing defect?   Fanen et al. 1992               17a       3272 − 9A − &gt;T   A to T at 3272 − 9   intron   mRNA splicing defect?   Chomel et al (NL#67)               17a       3272 − 4A − &gt;G   A to G at 3272 − 4   intron   mRNA splicing defect?   Kanvakis (NL#63)               17a       3272 − 1G − &gt;A   G to A at 3272 − 1   intron   mRNA splicing defect   Mercier et al. 1993b               17a       G1047D   G to A at 3272   17b   Gly to Asp at 1047 and   Teng et al. (NL#68)                   mRNA splicing defect?                   (CBAVD?)       F1052V   T to G at 3286   17b   Phe to Val at 1052   Mercier et al. 1993b       T1053I   C to T at 3290   17b   missense mutation   Bienvenu et al. 1998*       T1053I   C to T at 3290   17b   Thr to Ile at 1053   Bienvenu et al. (1998)                   (CBAVD?)       H1054D   C to G at 3292   17b   His to Asp at 1054   Férec et al. 1993       T1057A   A to G at 3301   17b   Thr to Ala at 1057   Ghanem et al. (NL#68)       K1060T   A to C at 3311   17b   Lys to Thr at 1060   Casals et al. 1995                       (NL#61)       G1061R   G to C at 3313   17b   Gly to Arg at 1061   Mercier et al. 1993b       L1065F   C to T at 3325   17b   Leu to Phe at 1065   Tzetis et al. (NL#70)       L1065R   T to G at 3326   17b   Leu to Arg at 1065   Casals et al (NL#67)       L1065P   T to C at 3326   17b   Leu to Pro at 1065   Ghanem et al. 1994       R1066S   C to A at 3328   17b   Arg to Ser at 1066   Férec et al. (NL#65)       R1066C   C to T at 3328   17b   Arg to Cys at 1066   Fanen et al. 1992       R1066H   G to A at 3329   17b   Arg to His at 1066   Férec et al. 1992       R1066L   G to T at 3329   17b   Arg to Leu at 1066   Mercier et al. 1993b       A1067T   G to A at 3331   17b   Ala to Thr at 1067   Férec et al. 1992       A1067D   C to A at 3332   17b   Ala to Asp at 1067   Girodon et al. 1999*       G1069R   G to A at 3337   17b   Gly to Arg at 1069   Savov et al. 1994a       R1070W   C to T at 3340   17b   Arg to Trp at 1070   Macek et al. (NL#58)       R1070Q   G to A at 3341   17b   Arg to Gln at 1070   Mercier et al. 1993b       R1070P   33341 G to C   17b   Arg to Pro at 1070   Shrimpton &amp; Borowitz       Q1071P   A to C at 3344   17b   Gln to Pro at 1071   Ghanem et al. 1994       Q1071H   G to T at 3345   17b   Glu to His at 1071   Clasutres et al. 2000*       P1072L   C to T at 3347   17b   Pro to Leu at 1072   Bombieri et al. (NL#70)       F1074L   T to A at 3354   17b   Phe to Leu at 1074   Casals et al. (NL#65)       L1077P   T to C at 3362   17b   Leu to Pro at 1077   Bozon et al. 1994       H1085R   A to G at 3386   17b   His to Arg at 1085   Mercier et al. 1993b       T1086I   C to T at 3389   17b   Thr to Ile at 1086   Bienvenu et al (NL#67)       N1088D   A to G at 3394   17b   Asn to Asp at 1088   Zielenski et al. (NL#70)       Y1082H   T to C at 3406   17b   Tyr to His at 1082   Egan et al. (NL#69)       L1093P   T to C at 3410   17b   Leu to Pro at 1093   Wine et al. (NL#69)       L1096R   T to G at 3419   17b   Leu to Arg at 1096   Claustres &amp; Guittard                       1998*       W1098R   T to C at 3424   17b   Trp to Arg at 1098   Zielenski et al. 1995       Q1100P   A to C at 3431   17b   Gln to Pro at 1100   Nunes et al. (NL#55)       M1101R   T to G at 3434   17b   Met to Arg at 1101   Mercier et al. 1993b       M1101K   T to A at 3434   17b   Met to Lys at 1101   Zielenski et al. 1993       S1118F   C to T at 3485   17b   Ser to Phe at 1118   Férec 1998*       S1118C   C to G at 2485   17b   Ser to Cys at 1118   Zielenski et al. 1999*       G1123R   G to C at 3499   17b   Gly to Arg at 1123   Wallace &amp; Tassabehji                   mRNA splicing defect?   (NL#60)       3499 + 2T − &gt;C   T to C at 3499 + 2   intron   mRNA splicing defect   Creegan &amp; Edkins               17b       (NL#64)       3499 + 3A − &gt;G   A to G at 3499 + 3   intron   mRNA splicing defect?   Haworth et al. (NL#68)               17b       3499 + 6A − &gt;G   A to G at 3499 + 6   intron   mRNA splicing defect?   Férec et al. (NL#65)               17b       3500 − 2A − &gt;G   A to G at 3500 − 2   intron   mRNA splicing defect   Vidaud et al. (NL#70)               17b       E1123del   Deletion of AAG at 3504-   18   deletion of Glu at 1123   Ellis (NL#70)           3506       G1127E   G to A at 3512   18   Gly to Glu at 1127   Bienvenu et al. (NL#63)       3523A − &gt;G   A to G at 3523   18   Ile to Val at 1131   Giorgi et al. 1999*       A1136T   G to A at 3538   18   Ala to Thr at 1136   Férec 2000*       M1137V   A to G at 3541   18   Met to Val at 1137   Zielenski et al. (NL#59)       M1137R   T to G at 3542   18   Met to Arg at 1137   Duarte et al. (NL#65)       I1139V   A to G at 3547   18   Ile to Val at 1139   Teng et al. 1994       delta M1140   deletion of 3 bp between 3550   18   deletion of Met al 1140   Férec et al. (NL#64)           and 3553       M1140K   T to A at 3551   18   Met to Lys at 1140   Férec 1998*       T1142I   C to T at 3557   18   Thr to Ile at 421   Lázaro et al. 2000*       V1147I   G to A at 3571   18   Val to Ile at 1147   Kilinc et al. (NL#70)       N1148K   C to A at 3576   18   Asn to Lys at 1148   Casals et al. 2000*       D1152H   G to C at 3586   18   Asp to His at 1152   Highsmith et al. (NL#49)       V1153E   T to A at 3590   18   Val to Glu at 1153   Dörk et al. (NL#68)                   (CBAVD)       D1154G   A to G at 3593   18   Asp to Gly at 1154   Costes et al. (NL#64)                   (CBAVD)       3600G − &gt;A   G to A at 3600   18   mRNA splicing defect   Zielenski et al. 1994       3600 + 2insT   insertion of T after 3600 + 2   intron   mRNA splicing defect?   Zielenski et al. (NL#70)               18       3600 + 5G − &gt;A   G to A at 3600 + 5   intron   mRNA splicing defect?   Bienvenu et al. (NL#66)               18       3601 − 20T − &gt;C   T to C at 3601 − 20   intron   mRNA splicing mutant?   Kabra et al. (NL#69)               18       3601 − 17T − &gt;C   T to C at 3601 − 17   intron   mRNA splicing defect?   Audrézet et al. 1993a               18       3601 − 2A − &gt;G   A to G at 3601 − 2   intron   mRNA splicing defect   Dörk et al. 1993a               18       S1159P   T to C at 3607   19   Ser to Pro at 115p   Macek et al. (NL#55)       S1159F   C to T at 3608   19   Ser to Phe at 1159   Férec 1999*       D1168G   A to G at 3635   19   Asp to Gly at 1168   Macek et al. (NL#58)       K1177R   A to G at 3662   19   Lys to Arg at 1177   Baralle et al. (NL#61)       3696G/A   G to A at 3696   18   No change to Ser at 1188   Malone et al. 1999*       V1190P   T to A at 3701   19   Val to Pro at 1190   Glavac et al. (NL#64)       3750delAG   deletion of AG from 3750   19   frameshift   Mercier et al. 1993a       3755delG   deletion of G between 3751 and   19   frameshift   Claustres et al. (NL#70)           3755       M1210I   G to A at 3762   19   Met to Ile at 1210   Nukiwa &amp; Seyama                       (NL#55)       V1212I   G to A at 3766   19   Val to Ile at 1212   Macek et al. (NL#55)       L1227S   T to C at 3812   19   Leu to Ser at 1227   Dubourg &amp; David                       (NL#70)       E1228G   A to G at 3815   19   Glu to Gly at 1228   Kilinc et al. 2000*       I1230T   T to C at 3821   19   Ile to Thr at 1230   Claustres &amp; Maugard                       (NL#69)       I1234V   A to G at 3832   19   Ile to Val at 1234   Claustres et al. 1992b       S1235R   T to G at 3837   19   Ser to Arg at 1235   Cuppens et al. 1993       G1237S   G to A at 3841   19   Gly to Ser at 1237   Casals et al. 2000*       Q1238R   A to G at 3845   19   Gln to Arg at 1238   Férec C et al. (NL#58)       3849G − &gt;A   G to A at 3849   19   mRNA splicing defect?   Cutting et al. 1992       3849 + 1G − &gt;A   G to A at 3849 + 1   intron   mRNA splicing defect   Greil et al. 1993               19       3849 + 4A − &gt;G   A to G at 3849 + 4   intron   mRNA splicing defect?   Ronchetto et al. 1992               19       3849 + 10kbC − &gt;T   C to T in a 6.2 kb EcoRI   intron   creation of splice   Highsmith et al. 1994           fragment 10 kb from 19   19   acceptor site       3849 + 5G − &gt;A   G to A at 3849 + 5   intron   mRNA splicing defect?   Kilinc et al. (NL#70)               19       3850 − 3T − &gt;G   T to G at 3850 − 3   intron   mRNA splicing defect   Dörk et al. 1993a               19       3850 − 1G − &gt;A   G to A at 3850 − 1   intron   mRNA splicing defect   Audrézet et al. 1993a               19       V1240G   T to G at 3851   20   Val to Gly at 1240   Zielenski et al. 1999*       G1244V   G to T at 3863   20   Gly to Val at 1244   Savov et al. 1994b       G1244E   G to A at 3863   20   Gly to Glu at 1244   Devoto et al. 1991       T1246I   C to T at 3869   20   Thr to Ile at 1246   Férec et al. (NL#64)                   (mutation?)       G1247R   G to A at 3871   20   Gly to Arg at 1247   Casals et al. (NL#69)       G1249R   G to A at 3877   20   Gly to Arg at 1249   Dijkstra et al. 1994       G1249E   G to A at 3878   20   Gly to Glu at 1249   Greil et al. 1994       S1251N   G to A 3884   20   Ser to Asn at 1251   Kälin et al. 1992a;                       Mercier et al. 1993a       T1252P   A to C at 3886   20   Thr to Pro at 1252   Wallace (NL#69)       S1255P   T to C at 3895   20   Ser to Pro at 1255   Lissens et al. 1992       S1255L   C to T at 3896   20   Ser to Leu at 1255   Bienvenu et al. (NL#69)       F1257L   T to G at 3903   20   Phe to Leu at 1257   Férec 1998*       delta L1260   deletion of ACT from either   20   deletion of Leu at 1260 or   Hermans et al. 1994           3909 or 3912       1261       3922del10 − &gt;C   deletion of 10 bp from 3922   20   deletion of Glu1264 to   Schwarz et al.(NL#69)           and replacement with 3921       Glu1266       I1269N   T to A at 3938   20   Ile to Asn at 1269   McDowell et al. (NL#66)       D1270N   G to A at 3940   20   Asp to Asn at 1270   Dean et al. 1991       W1282G   T to G at 3976   20   Trp to Gly at 1282   Faucz et al. (NL#69)       W1282R   T to C at 3976   20   Trp to Arg at 1282   Ivaschenko et al. 1993       W1282C   G to T at 3978   20   Trp to Cys at 1282   Férec et al. (NL#69)       R1283M   G to T at 3980   20   Arg to Met al 1283   Cheadle et al. 1992       R1283K   G to A at 3980   20   Arg to Lys at 1283   Chevalier &amp; Bozon                       (NL#54)       F1286S   T to C at 3989   20   Phe to Ser at 1286   Dorval et al. 1993       Q1291R   A to G at 4004   20   Gln to Arg at 1291   Dörk et al. 1994b       Q1291H   G to C at 4005   20   Gln to His at 1291;   Jones et al. 1992                   mRNA splicing defect (?)       4005 + 1G − &gt;A   G to A at 4005 + 1   intron   mRNA splicing defect   Férec et al. 1992               20       4005 + 2T − &gt;C   T to C at 4005 + 2   intron   mRNA splicing defect   Boman (NL#69)               20       4006 − 61del14   deletion of 14 bp from 4006 − 61   intron   mRNA splicing defect?   Friedman et al. (NL#59)           to 4006 − 47   20       4006 − 19del3   deletion of 3 bp from 4006 − 19   intron   mRNA splicing defect?   Naseem et al. (NL#36)               20       4006 − 14C − &gt;G   C to G at 4006 − 14   intron   mRNA splicing defect?   Poncin (NL#69)               20       4006 − 8T − &gt;A   T to A at 4006 − 8   intron   mRNA splicing defect?   Chevalier-Porst &amp; Bozon               20       (NL#70)       4006 − 4A − &gt;G   A to G at 4006 − 4   intron   mRNA splicing defect?   Chomel et al. (NL#68)       V1293I   G to A at 4009   21   Val to Ile at 1293   Férec et al. (NL#69)       T1299I   C to T at 4028   21   Thr to Ile at 1299   Liechti-Gallati (NL#68)       F1300L   T to C at 4030   21   Phe to Leu at 1300   Poncin (NL#69)       N1303H   A to C at 4039   21   Asn to His at 1303   Claustres et al. 1992b       N1303I   A to T at 4040   21   Asn to Ile at 1303   Lissens et al. (NL#66);                       Férec et al. (NL#66)       N1303K   C to G at 4041   21   Asn to Lys at 1303   Osborne et al. 1991       D1305E   T to A at 4047   21   Asp to Glu at 1305   Claustres et al. (NL#69)       Q1313K   C to A at 4069   21   Gln to Lys at 1313   Malone et al. (NL#68)       V1318A   T to C at 4085   21   Val to Ala at 1318   Férec 1998*       E1321Q   G to C at 4093   21   Glu to Gln at 1321   Férec et al. (NL#64)       4096 − 28G − &gt;A   G to A at 4096 − 28   intron   mRNA splicing defect?   Claustres et al. (NL#68)               21       4096 − 3C − &gt;G   C to G at 4096 − 3   intron   mRNA splicing defect?   Claustres et al. (NL#69)               21       L1335P   T to C at 4136   22   Leu to Pro at 1335   Zielenski et al. (NL#70)       F1337V   T to G at 4138   22   Phe to Val at 1337   Scheffer et al. (NL#70)                   (CBAVD)       L1339F   C to T at 4147   22   Leu to Phe at 1339   Girodon et al. 1999*       G1349S   G to A at 4177   22   Gly to Ser at 1349   Yoshimura 1999*       G1349D   G to A at 4178   22   Gly to Asp at 1349   Beaudet et al. 1991       K1351E   A to G at 4183   22   Lys to Glu at 1351   Dörk et al. (NL#69)                   (CBAVD)       Q1352H*   G to C at 4188   22   Gln to His at 1352   Nukiwa &amp; Seyama                       (NL#55)       R1358S   A to T at 2406   22   Arg to Ser at 1358   Férec 1999*       A1364V   C to T at 4223   22   Ala to Val at 1364   Claustres et al (NL#67)                   CBAVD       D1377H   G to C at 4261   22   Asp to His at 1377   Costes et al. (NL#56)       L1388Q   T to A at 4295   23   Leu to Gln at 1388   Dörk et al. (NL#68)                   (CBAVD)       V1397E   T to A at 4322   23   Val to Glu at 1397   Petreska et al. 1994       E1409V   A to T at 4358   23   Glu to Val at 1409   Claustres et al. (NL#55)       Q1412X   C to T at 4366   23   Gln to Stop at 1412   Wallace &amp; Tassabehji                       (NL#60)       4374 + 10T − &gt;C   T to C at 4374 + 10   intron   splicing?   Férec 1998*               23       4374 + 1G − &gt;A   G to A at 4374 + 1   intron   mRNA splicing defect   Fanen et al. 1992               23       4374 + 1G − &gt;T   G to T at 4374 + 1   intron   mRNA splicing defect   Dörk et al.(NL#38)               23       4375 − 1G − &gt;C   G to C at 4375 − 1   intron   splicing mutation   Chevalier-Porst &amp; Bozon               23       1999*       R1422W   C to T at 4396   24   Arg to Trp at 1422   Claustres et al. (NL#70)       S1426P   T to C at 4408   24   Ser to Pro at 1426   Férec 1999*       D1445N   G to A at 4465   24   Asp to Asn at 1445   Antoniadi et al. (NL#69)       R1453W   C to T at 4489   24   Arg to Trp at 1453   Yoshimura 1999*       CFTRdele14a   deletion of &gt;= 1.2 kb including   14a   aberrant mRNA splicing   Egan et al. (NL#68)           exon 14a       CFTRdele19   deletion of 5.3 kb, removing   19   ?   Girodon et al. 1999           exon 19       2104insA + 2109 −   insertion of A at 2104, deletion   13   ?   Girodon et al. 1999*       2118del10   of 10 bp at 2109       CF25kbdel   Complex   intron 3   ?   Shackleton et al. (NL#           deletion/rearrangement           70)                  
 
         [0038]    The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.  
       EXAMPLES  
       [0039]    Development of a Polypeptide that Exerts Only an Activating Effect on CFTR  
         [0040]    The activating peptide of Q4N2NEG2 was created by substituting glutamine residues for glutamic acid residues at four sites and asparagines for aspartic acid residues at two sites of the authentic NEG2 peptide sequence GLEISFEINEEDLKECFFDDME (SEQ ID NO: 7). In addition, a serine residue was substituted for cysteine, to prevent peptide dimerization, and norleucine was substituted for methionine, to prevent oxidation. These changes create a peptide with reduced chemical reactivity and high predicted helical structure, confirmed by circular dichroism, as well as reduced net negative charge (from −9 to −3). Attempts to eliminate negative charge completely resulted in an insoluble peptide. When this peptide was added to the cis (intracellular) side of CFTR channels captured in the planar lipid bilayer, at concentration ranging 0.5 to 14 μM, marked dose-related stimulation of channel activity was observed. At concentrations of 4-6 μM Po of CFTR doubles. No inhibitory activity was seen in any experiment at any concentration of peptide.  
         [0041]    Q4N2NEG2 Polypeptide Stimulates Wild-type CFTR Protein.  
         [0042]    To test whether the Q4N2NEG2 polypeptide is responsible for increasing the open probability of the CFTR channel, synthetic Q4N2NEG2, a 22 amino acid peptide, was added to the cis-intracellular side of single CFTR channels captured in the planar lipid bilayer (FIG. 1). The diary plot of open probability as a function of time shows the activity of a single wt-CFTR channel during the course of the experiment (FIG. 1A). During stimulation, the open probability doubles and more transitions are observed between the open and closed states (FIG. 1B). The open probability observed in 5 experiments at 4 μM concentration Q4N2NEG2 is shown to be increased by about two-fold in the graph (FIG. 1C).  
         [0043]    Q4N2NEG2 Polypeptide Stimulates Mutant G551D CFTR Protein.  
         [0044]    The Q4 N2 NEG2 peptide sequence has been tested on one mutant form of CFTR, G551D, which reaches the plasma membrane. In the planar lipid bilayer, Q4N2NEG2 increased the open probability of G551 by about threefold. Thus, this peptide is useful to stimulate channel activity in mutant forms of CFTR that reach the plasma membrane.  
         [0045]    The NEG2 Polypeptide can be Rendered Inhibitory to CFTR  
         [0046]    The NEG2 sequence can also be rendered inhibitory, with no stimulatory activity, by scrambling the sequence such that the resulting peptide is predicted to not have helical tendencies, as confirmed by circular dichroism measurements, but retains the full net negative charge of −9. This peptide, called scrambled NEG2, inhibits channel activity by about 90% at 6 μM concentration, with no stimulation observed at any concentration. In addition, insertion of a proline residue into the middle of the NEG2 sequence also results in a peptide which inhibits channel activity by about 60%, but does not stimulate. Proline residues are known to disrupt helical structures.  
         [0047]    Methods Used In Examples  
         [0048]    Subcloning of CFTR Gene  
         [0049]    The wt CFTR cDNA was subcloned into an Epstein-Barr virus-based episomal eukaryotic expression vector, pCEP4 (Invitrogen, San Diego, Calif.), between the Nhe1 and Xho1 restriction sites.  
         [0050]    Expression of CFTR in HEK 293 Cells  
         [0051]    A human embryonic kidney cell line (293-EBNA HEK; Invitrogen) was used for transfection and expression of the CFTR proteins (Ma et al., 1997, Ma et al., 1996, Xie et al., 1995). The HEK-293 cell line contains a pCMV-EBNA vector, which constitutively expresses the Epstein-Barr virus nuclear antigen-1 (EBNA-1) gene product and increases the transfection efficiency of Epstein-Barr virus-based vectors. The cells were maintained in Dulbecco&#39;s Modified Eagle Medium with 10% FBS and 1% L-glutamine. Geneticin (G418, 250 (g/ml) was added to the cell culture medium to maintain selection of the cells containing the pCMV-EBNA vector. Lipofectamine reagent (Life Technologies, Inc) in Optimem media (serum-free) was used to transfect the HEK-293 cells with pCEP4(wt). After 5 hours, serum was added to the media (10% final serum concentration). Twenty-four hours after transfection, the transfection media was replaced with fresh media. The cells were harvested two days after transfection and microsomal membrane vesicles were prepared for single channel measurements in the lipid bilayer reconstitution system.  
         [0052]    Vesicle Preparation from Transfected HEK 293 Cells  
         [0053]    HEK-293 cells transfected with pCEP4(CFTR) were harvested and homogenized using a combination of hypotonic lysis and Dounce homogenization in the presence of protease inhibitors (Ma et al., 1997, Ma et al., 1996, Xie et al., 1995). Microsomes were collected by centrifugation of postnuclear supernatant (4500×g, 15 min) at 100,000×g for 20 min and resuspended in a buffer containing 250 mM sucrose, 10 mM HEPES, pH 7.2. The membrane vesicles were stored at −75° C. until use.  
         [0054]    Reconstitution of CFTR Channels in Lipid Bilayer Membranes  
         [0055]    Lipid bilayer membranes were formed across an aperture of ˜200 (m diameter with a mixture of phosphatidylethanolamine:phosphatidylserine:cholesterol in a ratio of 5:5:1. The lipids were dissolved in decane at a concentration of 33 mg/ml. The recording solutions contained: cis (intracellular), 200 mM CsCl, 1 mM MgCl 2 , 2 mM ATP, and 10 mM HEPES-Tris (pH 7.4); trans (extracellular), 50 mM CsCl, 10 mM HEPES-Tris (pH 7.4). Vesicles (1-4 (1) containing wild-type CFTR were added to the cis solution. The PKA catalytic subunit was present at a concentration of 50 units/ml in the cis solution unless noted otherwise. Single channel currents were recorded with an Axopatch 200A patch clamp unit (Axon Instruments). The currents were sampled at 1-2.5 ms/point. Single channel data analyses were performed with pClamp and TIPS softwares.  
       REFERENCES  
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         [0059]    Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J., and Smith, A. E. (1991). Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66, 1027-1036.  
         [0060]    Cotten, J. F. and Welsh, M. J. (1997). Covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 272, 25617-25622.  
         [0061]    Dulhanty, A. M. and Riordan, J. R. (1994). Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. Biochemistry 22, 4072-4079.  
         [0062]    Gadsby, D. C. and Nairn, A. C. (1994). Regulation of CFTR channel gating. Trends Biochem. Sci. 19, 513-518.  
         [0063]    Geourjon, C. and Deleage, G. (1995). SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. CABIOS 11, 681-684.  
         [0064]    Gunderson, K. L. and Kopito, R. R. (1995). Conformational states of CFTR associated with channel gating: the role of ATP binding and hydrolysis. Cell 82, 231-239.  
         [0065]    Higgens, C. F. (1992). ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8, 67-113.  
         [0066]    Ma, J. and Davis, P. B. (1998). What we know and what we do not know about cystic fibrosis transmembrane conductance regulator. Clinics in Chest Medicine 19, 459-471.  
         [0067]    Ma, J., Tasch, J. E., Tao, T., Zhao, J., Xie, J., Drumm, M. L., and Davis, P. B. (1996). Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein. J. Biol. Chem. 271, 7351-7356.  
         [0068]    Ma, J., Zhao, J., Drumm, M. L., Xie, J., and Davis, P. B. (1997). Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel. J. Biol. Chem. 272, 28133-28141.  
         [0069]    Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992). Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267, 12742-12752.  
         [0070]    Quinton, P. M. (1986). Missing Cl −  conductance in cystic fibrosis. Am. J. Physiol. 251, C649-C652.  
         [0071]    Rich, D. P., Berger, H. A., Cheng, S. H., Travis, S. M., Saxena, M., Smith, A. E., and Welsh, M. J. (1993). Regulation of the cystic fibrosis transmembrane conductance regulator Cl −  channel by negative charge in the R domain. J. Biol. Chem. 268, 20259-20267.  
         [0072]    Rich, D. P., Gregory, R. J., Anderson, M. P., Manavalan, P., Smith, A. E., and Welsh, M. J. (1991). Effect of deleting the R domain on CFTR-generated chloride channels. Science 253, 205-207.  
         [0073]    Riordan, J., Rommens, J., Kerem, B.-S., Noa, A., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M., Iannuzzi, M., Collins, F., and Tsui, L.-C. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073.  
         [0074]    Rost, B. and Sander, C. (1993). Prediction of protein structure at better than 70% accuracy. J. Mol. Biol. 232, 584-599.  
         [0075]    Rost, B. and Sander, C. (1994). Combining evolutionary information and neural networks to predict protein secondary structure. Proteins 19, 55-72.  
         [0076]    Tabcharani, J. A., Chang, X.-B., Riordan, J. R. and Hanrahan, J. W. (1991). Phosphorylation-regulated C− channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352, 628-631.  
         [0077]    Tao, T., Xie, J., Drumm, M. L., Zhao, J., Davis, P. B., and Ma, J. (1996). Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel. Biophys. J. 70, 743-753.  
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         [0079]    Welsh, M. J. and Smith, A. E. (1993). Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73, 1251-1254.  
         [0080]    Winter, M. C. and Welsh, M. J. (1997). Stimulation of CFTR activity by its phosphorylated R domain. Nature 389, 294-296.  
         [0081]    Xie, J., Drumm, M. L., Ma, J., and Davis, P. B. (1995). Intracellular loop between transmembrane segments IV and V of cystic fibrosis transmembrane conductance regulator is involved in regulation of chloride channel conductance state. J. Biol. Chem. 270, 28084-28091.  
         [0082]    Zielenski, J. and Tsui, L. C. (1995). Cystic fibrosis: genotypic and phenotypic variations. Annu. Rev. Genetics 29, 777-807.  
     
       
       
         1 
         
           
             7  
           
           
             1  
             22  
             PRT  
             homo sapiens  
             
               MOD_RES  
               (1)..(1)  
               ACETYLATION  
             
           
            1 

Gly Leu Glu Ile Ser Glu Gln Ile Asn Gln Gln Asn Leu Lys Gln Ser 
1               5                   10                  15 

Phe Phe Asn Asp Leu Glu 
            20 

 
           
             2  
             22  
             PRT  
             homo sapiens  
           
            2 

Gly Leu Glu Ile Ser Glu Gln Ile Asn Gln Gln Asn Leu Lys Gln Ser 
1               5                   10                  15 

Phe Phe Asn Asp Met Glu 
            20 

 
           
             3  
             301  
             PRT  
             homo sapiens  
           
            3 

Met Thr Ser Arg Arg Ser Val Lys Ser Gly Pro Arg Glu Val Pro Arg 
1               5                   10                  15 

Asp Glu Tyr Glu Asp Leu Tyr Tyr Thr Pro Ser Ser Gly Met Ala Ser 
            20                  25                  30 

Pro Asp Ser Pro Pro Asp Thr Ser Arg Arg Gly Ala Leu Gln Thr Arg 
        35                  40                  45 

Ser Arg Gln Arg Gly Glu Val Arg Phe Val Gln Tyr Asp Glu Ser Asp 
    50                  55                  60 

Tyr Ala Leu Tyr Gly Gly Ser Ser Ser Glu Asp Asp Glu His Pro Glu 
65                  70                  75                  80 

Val Pro Arg Thr Arg Arg Pro Val Ser Gly Ala Val Leu Ser Gly Pro 
                85                  90                  95 

Gly Pro Ala Arg Ala Pro Pro Pro Pro Ala Gly Ser Gly Gly Ala Gly 
            100                 105                 110 

Arg Thr Pro Thr Thr Ala Pro Arg Ala Pro Arg Thr Gln Arg Val Ala 
        115                 120                 125 

Thr Lys Ala Pro Ala Ala Pro Ala Ala Glu Thr Thr Arg Gly Arg Lys 
    130                 135                 140 

Ser Ala Gln Pro Glu Ser Ala Ala Leu Pro Asp Ala Pro Ala Ser Thr 
145                 150                 155                 160 

Ala Pro Thr Arg Ser Lys Thr Pro Ala Gln Gly Leu Ala Arg Lys Leu 
                165                 170                 175 

His Phe Ser Thr Ala Pro Pro Asn Pro Asp Ala Pro Trp Thr Pro Arg 
            180                 185                 190 

Val Ala Gly Phe Asn Lys Arg Val Phe Cys Ala Ala Val Gly Arg Leu 
        195                 200                 205 

Ala Ala Met His Ala Arg Met Ala Ala Val Gln Leu Trp Asp Met Ser 
    210                 215                 220 

Arg Pro Arg Thr Asp Glu Asp Leu Asn Glu Leu Leu Gly Ile Thr Thr 
225                 230                 235                 240 

Ile Arg Val Thr Val Cys Glu Gly Lys Asn Leu Leu Gln Arg Ala Asn 
                245                 250                 255 

Glu Leu Val Asn Pro Asp Val Val Gln Asp Val Asp Ala Ala Thr Ala 
            260                 265                 270 

Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr Glu Arg Pro Arg Ala 
        275                 280                 285 

Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro Val Glu 
    290                 295                 300 

 
           
             4  
             27  
             PRT  
             homo sapiens  
           
            4 

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 
1               5                   10                  15 

Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 
            20                  25 

 
           
             5  
             16  
             PRT  
             homo sapiens  
           
            5 

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 
1               5                   10                  15 

 
           
             6  
             22  
             PRT  
             homo sapiens  
             
               CDS  
               (1)..(11)  
               Xaa=Glu or Gln  
             
           
            6 

Gly Leu Xaa Ile Ser Xaa Xaa Ile Asn Xaa Xaa Xaa Leu Lys Xaa Xaa 
1               5                   10                  15 

Phe Phe Xaa Xaa Xaa Xaa 
            20 

 
           
             7  
             22  
             PRT  
             homo sapiens  
           
            7 

Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys 
1               5                   10                  15 

Phe Phe Asp Asp Met Glu 
            20