WO2008087038A2 - Diagnosis and therapy of diseases relating to a disproportion of luteinizing hormone/chorionic gonadotropin receptor (lhr; lhcgr) splice variants - Google Patents

Abstract

The present invention relates to a novel exon of the LHR, namely exon 6A, as well as to mutated forms thereof. Particularly, the present invention relates to a nucleotide sequence of exon 6A, to a nucleotide sequence comprising said nucleotide sequence of exon 6A, to a nucleotide sequence comprising a nucleotide sequence of a splice variant of LHR comprising exon 6A1 to variants or fragments of said nucleotide sequences, as well as to a vector comprising said nucleotide sequences. The present invention further relates to a polypeptide comprising an amino acid sequence encoded by exon 6A or encoded by a splice variant of LHR comprising exon 6A, as well as to variants and fragments of said amino acid sequence. Furthermore, the present invention relates to a host cell genetically engineered with a nucleotide sequence of the present invention or comprising the vector of the present invention. Additionally, the present invention relates to a pharmaceutical composition comprising an inhibitor of exon 6A of LHR. Furthermore, provided herein is a method of diagnosing a disease in a patient which is characterized by an increase of the amount of LHR mRNA comprising exon 6A, to a method for treating, ameliorating or preventing such a disease, as well as to a use of the nucleotide sequence, the vector, the polypeptide or the host cell as provided or the inhibitor of exon 6A as defined in the context of the invention for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of such a disease. The present invention also refers to a method for treating, ameliorating or preventing a disease which is characterized by an increase of the amount of LHR mRNA comprising exon 6A and to a use of the compounds of the invention and an inhibitor of exon 6A for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of such a disease.

Description

Diagnosis and therapy of diseases relating to a disproportion of luteinizing hormone/chorionic gonadotropin receptor (LHR; LHCGR) splice variants

The present invention relates to a novel exon of the LHR, namely exon 6A, as well as to mutated forms thereof. Particularly, the present invention relates to a nucleotide sequence of exon 6A, to a nucleotide sequence comprising said nucleotide sequence of exon 6A, to a nucleotide sequence comprising a nucleotide sequence of a splice variant of LHR comprising exon 6A, to variants or fragments of said nucleotide sequences, as well as to a vector comprising said nucleotide sequences. The present invention further relates to a polypeptide comprising an amino acid sequence encoded by exon 6A or encoded by a splice variant of LHR comprising exon 6A, as well as to variants and fragments of said amino acid sequence. Furthermore, the present invention relates to a host cell genetically engineered with a nucleotide sequence of the present invention or comprising the vector of the present invention. Additionally, the present invention relates to a pharmaceutical composition comprising an inhibitor of exon 6A of LHR. Furthermore, provided herein is a method of diagnosing a disease in a patient which is characterized by an increase of the amount of LHR mRNA comprising exon 6A, to a method for treating, ameliorating or preventing such a disease, as well as to a use of the nucleotide sequence, the vector, the polypeptide or the host cell as provided or the inhibitor of exon 6A as defined in the context of the invention for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of such a disease. The present invention also refers to a method for treating, ameliorating or preventing a disease which is characterized by an increase of the amount of LHR mRNA comprising exon 6A and to a use of the compounds of the invention and an inhibitor of exon 6A for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of such a disease.

Luteinizing hormone (LH) and chorionic gonadotropin (CG) play an essential role in male sexual differentiation. Their action is mediated by the LH/CG receptor (LHCGR; also referred to herein as LHR), a G protein-coupled receptor expressed in Leydig and in granulosa-lutein cells. The human LHR gene (ID 3973), consists of 11 exons and 10 introns and spans 67 kb on chromosome 2 p21. Ten of the 11 exons encode the extracellular domain, while exon 11 encodes the seven transmembrane and the intracellular domain (Ascoli, Endocr Rev. 23: 141-74, 2002). Several inactivating LHR mutations were described in patients with 46,XY disorder of sex development (46,XY DSD) due to Leydig cell hypoplasia (LCH), an autosomal recessive disease characterized by a female phenotype in subjects with 46,XY karyotype (Themmen, Endocr. Rev. 21 : 551-583, 2000). However, in a substantial number of patients (50%) with the classical symptoms of LCH no mutations of the LHR are found (Zenteno, J. Clin. Endocrinol. Metab. 84: 3803-3806, 1999; Richter-Unruh, Clin. Endocrinol. (Oxf). 56:103-12, 2002). This raises the hypothesis that genomic defects in LHR regions not considered so far may cause LCH.

Thus, the technical problem underlying the present invention is the provision of reliable means and methods for the diagnostic or therapeutic intervention of diseases associated with an impaired/improper function of LHR.

The technical problem is solved by provision of the embodiments characterized in the claims.

The present invention relates to a novel exon of the LHR, denoted and herein referred to as "exon 6A", as well as to splice variants of the LHR comprising "exon 6A".

As documented herein below and in the appended examples, it was surprisingly found that an imbalance between the amount of LHR splice variants comprising exon 6A and the amount of LHR splice variants lacking exon 6A leads to an impaired/improper function of the LHR, and hence to a disease associated with an impaired/improper function of the LHR, like, e. g., LCH. Moreover, it was found out that the balance between the amount of splice variants of the LHR comprising exon 6A and the amount of splice variants of the LHR lacking exon 6A depends on the degree to which exon 6A is spliced into the mature transcript of the LHR gene. These surprising results are based on the following particular findings of the present invention:

First, it was found that the LHR gene comprises the alternative exon 6A within the sequence of intron 6 of the LHR. In the prior art, it was not known that there is a further exon of the LHR existing at all, not to mention that this exon is comprised in the sequence of intron 6 of the LHR.

Second, it was found that the proper function of the LHR depends on the degree to which the alternative exon 6A is spliced into the maturing gene product of the LHR gene.

Third, it was found that the degree of splicing-in the alternative exon 6A depends on one ore more particular mutations in the LHR gene, particularly in exon 6A.

Fourth, it was found that the usage of the alternative exon 6A is highly regulated.

These findings result from the experiments described in the appended Examples which are summarized in the following:

For the first time, it has been shown that there is a primate-specific bona fide exon, namely exon 6A, within the LHR gene, particularly within intron 6 thereof. Exon 6A displays composite characteristics of an internal or terminal exon and possesses stop codons triggering nonsense-mediated decay (NMD) of LHR mRNA. Transcripts including exon 6A are physiologically highly expressed in human testis and in granulosa cells and result in an intracellular, truncated LHR protein of 209 amino acids (SEQ ID NO: 10). Moreover, mutations of exon 6A were found in three patients with unexplained LCH. Functional studies revealed a dramatic increase in the expression of the mutated internal exon 6A transcripts, indicating aberrant NMD resulting in altered ratios of LHR transcripts preventing LHR function. In view of the findings provided herein, the genomic organization of the LHR gene is redefined and leads to a new corresponding model that enables completely new insights into a complex network of receptor regulation, particularly of LHR regulation (see, for example, Fig. 6).

In view of the above, the present invention, inter alia, provides the possibility to diagnose a disease, like, e. g., Leydig Cell Hypoplasia (LCH) or (other) disorders of sexual development (DSD), associated with an improper function of the LHR due to an increased amount of LHR splice variants comprising exon 6A. The diagnostic means and methods of this invention are particularly useful for diagnosing such diseases, the diagnosed symptoms of which lead to the assumption that a dysfunction of the LHR may occur, but for which the reason(s) of the diagnosed symptoms (for example (a) certain mutation(s) in the LHR gene) are (were) unknown (for example unexplained LCH).

The term "exon 6A" as used herein, refers to a novel exon of the LHR/LHCGR gene in addition to the already known 11 exons of this gene. This novel exon lies within that part of the LHR gene which was formally known as intron 6. Exon 6A can be spliced into the LHR mRNA resulting in LHR splice variants comprising exon 6A. Particularly provided herein are three possible splice variants of the LHR comprising exon 6A₁ namely two splice variants with an internal exon 6A and one truncated splice variant with a terminal exon 6A. The two splice variants with an internal exon 6A result from a short and a long version of exon 6A being spliced into the LHR mRNA.

Exon 6A is shown herein to specifically occur in primates, and hence, the term "exon 6A" generally refers to exon 6A of the LHR gene from all species encompassed in the meaning of the term "primates". The skilled person is aware which animal species are comprised by the meaning of this term.

Particularly provided herein is exon 6A, and correspondingly LHR splice variants comprising it, of Homo sapiens, Pan troglodytes, Macaca fascicularis, Callithrix jacchus and Lemur coronatus. Exemplarily, the human nucleotide sequence of exon 6A and the encoded amino acid sequence (SEQ ID NOs: 1 and 9 , respectively), as well as the human nucleotide sequences of the different splice variants of the LHR comprising exon 6A and the encoded amino acid sequence (SEQ ID NOs: 5 to 8 and 10, respectively) is provided herein. Further provided is the detailed structural organisation of exon 6A including possible mutations and polymorphisms (like single nucleotide polymorphisms (SNPs)), the indication of internal STOP codons and the splicing sites of exon 6A (internal and flanking splicing sites) as well as the PoIy-A signal sequence of Exon 6A. The LHR/LHCGR lacking exon 6A, and the gene encoding it, is known in the art. It was isolated from several mammals including humans (accession No: NM_000233), chimpanzees (genomic sequences available), macaques (e. g. accession No: M001114090), marmosets (e. g. accession No: U80673), lemurs and mouse (accession No: NM-013582). In humans, LHR was firstly described in 1989 by Mc Farland (Science 245:494-9).

The particular nucleotide sequence encoding the human LHR lacking exon 6A and the corresponding amino acid sequence are exemplarily given herein below (SEQ ID NOs: 11 and 20, respectively).

The term "splice variant" as used herein, on the one hand refers to the mature mRNA, i.e. the mRNA resulting from the pre-mRNA (also known and referred to herein as hnRNA) by the event known in the art as "splicing", and, on the other hand to the polypeptide/protein encoded by said mature mRNA. Additionally, this term means that at least two different mature mRNAs, and hence, at least two different encoded polypeptides/proteins can result from one pre-mRNA. Accordingly, the term "splice variant of the LHR" as used herein refers to LHR mRNA comprising or lacking the nucleotide sequence of exon 6A, or parts thereof, as well as to the polypeptide/protein encoded by said LHR mRNA. Particularly, the term "splice variant of the LHR comprising exon 6A" refers to LHR mRNA comprising the nucleotide sequence of exon 6A, or parts thereof, as well as to the polypeptide/protein encoded by said LHR mRNA.

In view of the above, the term "exon 6A" or "exon 6A of the LHR gene" as used in the context of the present invention refers to an exon of the LHR gene in addition to the known exons 1 to 11 of the LHR gene. As mentioned above, this "exon 6A" lies within the formerly known intron 6 of the LHR gene, i.e. it comprises a part of this intron 6. This part is flanked by the 5'-end and the 3'-end of intron 6, herein also referred to as 'left' and 'right' portion, respectively, of intron 6, or as "intron 6A" and "intron 6B", respectively. Particularly, the term "exon 6A" as used herein refers to exon 6A as a part of the LHR gene, and hence, to the nucleotide sequence of exon 6A (or fragments thereof) as well as to a polypeptide (or fragments thereof) encoded by said nucleotide sequence. "Nucleotide sequence of exon 6A" thereby, inter alia, refers to the DNA sequence of exon 6A (like the genomic DNA sequence of exon 6A) or the corresponding RNA sequence of exon 6A (like the mRNA sequence of exon 6A) or to any other nucleotide sequences of exon 6A, for example the other nucleotide sequences as defined herein below.

In a preferred embodiment of the present invention, the term "exon 6A" particularly refers to a mutated form of exon 6A or to a form of exon 6A carrying (a) polymorphism(s). Such particular variants of exon 6A are described herein below. A non-limiting example of such a variant is exon 6A of the human LHR gene carrying at least one of the mutations A21 C and G22C in its nucleotide sequence (SEQ ID NO:

1 ).

The detailed structural organisation of exon 6A is given in the following, exemplarily and not limiting based on the structural organisation of exon 6A of the human LHR gene (also referred to herein as human exon 6A). It is of note that the skilled person is readily in the position to transfer the structural organisation or any other herein defined characteristics of exon 6A of the human LHR to an exon 6A of any other LHR. Thereby, the skilled person is also able to find out, for example, such particular structural features that are specific for exon 6A of the human LHR (for example specific mutations or polymorphisms).

The complete nucleotide sequence of exon 6A of the human LHR is shown in SEQ ID NO: 1. The corresponding amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 9. In this context, the first nucleotide of human exon 6A is the last nucleotide of a codon which consists of the last two nucleotides from exon 6 (CT) and said first nucleotide from exon 6A (C). Accordingly, the open reading frame of exon 6A continues with full codons from position 2 of SEQ ID NO: 1 to position 94 of SEQ ID NO: 1. The nucleotide sequence of the coding region of human exon 6A is also shown in SEQ ID NO: 4, including the stop codon TAG. Human exon 6A shows a poly-A tail and a polyadenylation signal sequence comprising the nucleotide stretch AATACA, both of which can be seen in Fig. 1 B. Human exon 6A further shows two internal stop codons, namely TAG at position 92 to 94 of SEQ ID NO: 1 and TGA at position 98 to 100 of SEQ ID NO: 1. Moreover, human exon 6A shows three splicing sites, one flanking splicing site at its 5'-end and two (alternative) internal splicing sites between nucleotide position 159 and 160 of SEQ ID NO: 1 and between nucleotide position 207 and 208 of SEQ ID NO: 1. Alternative splicing at the flanking splicing site at the 5'-end and either one of the other internal splicing site leads to a long or a short variant of human exon 6A, the nucleotide sequences of which are depicted in SEQ ID NO: 2 and 3, respectively. As mentioned above, human exon 6A is flanked by the 'left' and the 'right' portion of formerly known intron 6. The nucleotide sequences of this 'left' and 'right' portion of formerly known intron 6 are depicted in SEQ ID NOs: 12 and 13, respectively.

In a first aspect, the present invention relates to the nucleotide sequence of exon 6A, or a variant or a fragment thereof. Particularly, it is envisaged that the nucleotide sequence of exon 6A, or the variant or the fragment thereof, is selected from the group consisting of the following nucleotide sequences:

(a) a nucleotide sequence as depicted in any one of SEQ ID NOs: 1 to 4;

(b) a nucleotide sequence as depicted in any one of SEQ ID NOs: 1 to 4 having at least one nucleotide exchange. Said nucleotide sequence may correspond to a nucleotide sequence that can be spliced into the LHR mRNA, particularly between exon 6 and exon 7 of the LHR mRNA, preferably under physiologic conditions;

(c) a nucleotide sequence as depicted in any one of SEQ ID NOs: 1 to 4 having at least one of the following nucleotide exchanges: A21C; G22C; T63C; A117G; and T212G;

(d) a nucleotide sequence encoding a polypeptide as depicted in SEQ ID NO: 9;

(e) a nucleotide sequence encoding a polypeptide as depicted in SEQ ID NO: 9 having at least one amino acid exchange. Said nucleotide sequence may correspond to a nucleotide sequence that can be spliced into the LHR mRNA, particularly between exon 6 and exon 7 of the LHR mRNA, preferably under physiologic conditions;

(f) a nucleotide sequence encoding a polypeptide as depicted in SEQ ID NO: 9 having at least one of the following amino acid exchanges: E7A; E7D; and M21T;

(I) a nucleotide sequence which is capable of hybridizing to the nucleotide sequence of any one of (a), (c), (d) and (T). Said nucleotide sequence may have at least one nucleotide exchange corresponding to any one of the nucleotide or amino acid exchanges as defined in (c) or (f), said nucleotide sequence may start with a nucleotide sequence capable of hybridizing to the 5^'-end and/or may end with a nucleotide sequence capable of hybridizing to the 3^'-end of the nucleotide sequence of any one of (a), (c), (d) and (f) and/or said nucleotide sequence may span a splicing site of exon 6A;

(m) a nucleotide sequence being homologous to the nucleotide sequence of any one of (a), (c), (d) and (f). Said nucleotide sequence may have at least one nucleotide exchange corresponding to any one of the nucleotide or amino acid exchanges as defined in (c) or (f), said nucleotide sequence may start with a nucleotide sequence being homologous to the 5^'-end and/or may end with a nucleotide sequence being homologous to 3^'-end of the nucleotide sequence of any one of (a), (c), (d) and (f) and/or said nucleotide sequence may span a splicing site of exon 6A;

(n) a fragment of the nucleotide sequence of any one of (a) to (m). Said fragment may have at least one nucleotide exchange corresponding to any one of the nucleotide or amino acid exchanges as defined in (c) or (f), said fragment may start with a nucleotide sequence corresponding to the 5^'-end and/or may end with a nucleotide sequence corresponding to the 3^'-end of the nucleotide sequence of any one of (a) to (m) and/or said fragment may span a splicing site of exon 6A; and

(o) the complementary strand of the nucleotide sequence of any one of (a) to (n).

The meanings of the terms "polypeptide" and "nucleotide sequence" are well known in the art, and are, if not otherwise defined herein, used accordingly in the context of the present invention. For example, "nucleotide sequence" as used herein refers to all forms of naturally occurring or recombinantly generated types of nucleic acids and/or nucleotide sequences as well as to chemically synthesized nucleic acids/nucleotide sequences. This term also encompasses nucleic acid analogs and nucleic acid derivatives such as, e. g., locked DNA, PNA, oligonucleotide tiophosphates and substituted ribo-oligonucleotides. Furthermore, the term "nucleotide sequence" also refers to any molecule that comprises nucleotides or nucleotide analogs. Preferably, the term "nucleotide sequence" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The "nucleotide sequence" in the context of the present invention may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded. "Nucleotide sequence" also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.

Furthermore, the term "nucleotide sequence" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., US 5525711 , US 4711955, US 5792608 or EP 302175 for examples of modifications). The nucleotide sequence may be single- or double-stranded, linear or circular, natural or synthetic, and, if not otherwise defined, without any size limitation. For instance, the nucleotide sequence may be genomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Colestrauss, Science, 1996, 1386-1389). Said nucleotide sequence may be in the form of a plasmid or of viral DNA or RNA. "Nucleotide sequence" may also refer to (an) oligonucleotide(s), wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included.

In the context of the present invention, a nucleotide sequence that "corresponds" or "corresponding" to another nucleotide sequence is, if not otherwise defined explicitly, a nucleotide sequence (basically) representing the same length and sequence information than the other nucleotide sequence, but, for example, using different kind of nucleotides, like, e.g., U (uridine) instead of T (thymidine). Particularly, this refers to an RNA (e.g. an mRNA or hnRNA) that "corresponds" to a DNA (e.g. gDNA or cDNA) representing the same sequence information or, vice versa, to a DNA that "corresponds" to an RNA representing the same sequence information.

The term "variant of exon 6A" or "exon 6A variant" as used herein refers to exon 6A having at least one nucleotide exchange (when "exon 6A" refers to a nucleotide sequence) or at least one amino acid exchange (when "exon 6A" refers to an amino acid sequence). Preferably, a "variant of exon 6A" of this invention has (at least one of) the same properties exon 6A has. For example, it is envisaged herein that the "variant of exon 6A" can, like exon 6A itself, also be spliced into the (maturing) mRNA of the LHR, preferably under physiological conditions.

The term "nucleotide exchange" as used herein means that one nucleotide of a nucleotide sequence is replaced by a different nucleotide. The number of nucleotides exchanged in exon 6A to form an "exon 6A variant" may be at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 150, wherein the lower values are preferred.

The term "amino acid exchange" as used herein means that one amino acid of an amino acid sequence is replaced by a different amino acid.

The number of amino acids exchanged in exon 6A to form an "exon 6A variant" may be at least 1 , at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 15, or at least 20, wherein the lower values are preferred.

In a particular aspect of this invention, the term "nucleotide exchange" or "amino acid exchange" refers to a mutation or a polymorphism, for example to a mutation or a polymorphism that also occurs in vivo. Examples of mutations of exon 6A are mutations corresponding to the mutations A21 C or G22C of the nucleotide sequences of human exon 6A as depicted in SEQ ID NO: 1 to 4 and to the resulting mutations E7A or E7D of the amino acid sequence of exon 6A as depicted in SEQ ID NO: 9. It is clear that the above mutations A21C or G22C can also be denoted with respect to the LHR nucleotide sequence comprising exon 6A. These mutations would then be denoted as mutations A557C or G558C of the nucleotide sequences coding for human LHR comprising exon 6A (for example as depicted in SEQ ID NO: 5 to 8). Examples of polymorphisms of exon 6A are polymorphisms corresponding to the polymorphisms T63C, A117G or T212G of the nucleotide sequences of human exon 6A as depicted in SEQ ID NO: 1 to 4 and to the resulting polymorphism M21T of the amino acid sequence of exon 6A as depicted in SEQ ID NO: 9. It is clear that the above polymorphisms T63C, A117G or T212G can also be denoted with respect to the LHR nucleotide sequence comprising exon 6A. These polymorphisms would then be denoted as polymorphisms T599C, A653G or T748G of the nucleotide sequences coding for human LHR comprising exon 6A (for example as depicted in SEQ ID NO: 5 to 8).

Accordingly, in one specific embodiment, the term "variant of exon 6A" refers to a nucleotide sequence of exon 6A having one or more nucleotide exchanges corresponding to the nucleotide exchanges A21C, G22C, T63C, A117G or T212G of the nucleotide sequences of human exon 6A as depicted in SEQ ID NO: 1 to 4. Moreover, the term "variant of exon 6A" refers to an amino acid sequence of exon 6A having one or more amino acid exchanges corresponding to the amino acid exchanges E7A, E7D or M21T of the amino acid sequence of human exon 6A as depicted in SEQ ID NO: 9.

In the context of the present invention, when a first nucleotide/amino acid exchange is "corresponding" to a second nucleotide/amino acid exchange, it is preferably meant that said first nucleotide/amino acid exchange is the same than the second nucleotide/amino acid exchange. However, it is clear that the nucleotide/amino acid sequences of exons 6A of different LHRs (for example LHRs from different species) or of different variants of exon 6A may, inter alia, vary particularly at those positions where the exemplified human exon 6A has (a) nucleotide/amino acid exchange(s). Accordingly, (a) nucleotide/amino acid exchange(s) of an exon 6A other than the human exon 6A or of an exon 6A variant may differ from that (those) of human exon 6A, but may still cause the same effect (for example may still lead to an increased splicing of exon 6A into the LHR mRNA). For example, the absolute nucleotide/amino acid position of the nucleotide/amino acid exchange(s) may (slightly) differ from that of human exon 6A, depending on, for example, the absolute length of exon 6A. Moreover, the specific nucleotides/amino acids to be exchanged may differ from those of human exon 6A, as long as the nucleotide/amino acid exchanges result in the same exchange on amino acid level and/or cause the same effect as the "corresponding" nucleotide/amino acid exchanges of human exon 6A. For example, a nucleotide exchange "corresponding" to any one of the nucleotide exchanges A21C, G22C, T63C, A117G or T212G and/or to any one of the amino acid exchanges E7A, E7D or M21T of human exon 6A may be any nucleotide exchange that results in any one of the amino acid exchanges E7A, E7D or M21T of exon 6A, or a variant thereof (or the same amino acid exchanges at corresponding positions in exon 6A, or a variant thereof), and/or that causes the same effect than any one of the nucleotide/amino acid exchanges A21C, G22C, T63C, A117G or T212G and E7A, E7D or M21T, respectively, of human exon 6A. For example, an amino acid exchange "corresponding" to any one of the amino acid exchanges E7A, E7D or M21T of human exon 6A may be any amino acid exchange that results in any one of the amino acids A at position 7, D at position 7 or T at position 21 of exon 6A, or a variant thereof, and/or that causes the same effect as any one of the nucleotide/amino acid exchanges A21C, G22C or T63C and E7A, E7D or M21T, respectively, of human exon 6A. For example, an amino acid exchange "corresponding" to any one of the nucleotide exchanges A21C, G22C or T63C of human exon 6A may be any amino acid exchange that result from any one of the nucleotide exchanges A21 C, G22C or T63C or from nucleotide exchanges "corresponding" thereof; and/or that causes the same effect as any one of the nucleotide/amino acid exchanges A21C, G22C or T63C and E7A, E7D or M21T, respectively, of human exon 6A.

A non-limiting example of an "effect" caused by a nucleotide/amino acid exchange in the context of the present invention is an increased splicing of exon 6A into the maturing mRNA of the LHR gene.

By his common general knowledge and the teaching provided herein, the skilled person is readily in the position to find out such nucleotide/amino acid exchanges of an exon 6A other than the human exon 6A or of exon 6A variants that "correspond" to the herein described nucleotide/amino acid exchanges of human exon 6A.

The term "variant of exon 6A" or "exon 6A variant" as used herein further refers to a nucleotide sequence being homologous to or being capable of hybridizing to a nucleotide sequence of exon 6A, for example of human exon 6A. In a first preferred embodiment, this nucleotide sequence may have at least one nucleotide exchange corresponding to any one of the nucleotide/amino acid exchanges A21C, G22C, T63C, A117G and T212G and E7A, E7D and M21T, respectively. In a second preferred embodiment, this nucleotide sequence may start with a nucleotide sequence being homologous or capable of hybridizing to the 5'-end and/or may end with a nucleotide sequence being homologous or capable of hybridizing to the 3^'-end of a nucleotide sequence of exon 6A, or a variant thereof. In a third preferred embodiment, this nucleotide sequence may span a splicing site of exon 6A, i.e. this nucleotide sequence may be homologous or capable to hybridize to such nucleotide sequence stretch of a nucleotide sequence of exon 6A that comprises at least one splicing site of exon 6A.

In the context of the present invention the term "capable of hybridizing" means that hybridization can occur between one nucleotide sequence and another (complementary) nucleotide sequence. Thereby, the term "hybridization" means hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA. In an especially preferred embodiment, the term "hybridization" means that hybridization occurs under the following conditions: Hybridization buffer: 2 x SSC; 10 x Denhardt solution (Fikoll 400 + PEG +

BSA; ratio 1 :1 :1); 0.1% SDS; 5 mM EDTA; 50 mM

Na₂HPO₄;

250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or

0.25 M of sodium phosphate buffer, pH 7.2;

1 mM EDTA

7% SDS

Hybridization temperature T = 60⁰C Washing buffer: 2 x SSC; 0.1 % SDS

Washing temperature T = 60⁰C.

Nucleotide sequences which are capable to hybridize with the nucleotide sequences disclosed in connection with the invention can for instance be isolated from genomic libraries or cDNA libraries of bacteria, fungi, plants or animals. Preferably, such polynucleotides are from animal origin, particularly preferred from an animal belonging to the taxonomic group of primates , more preferably from a human being. Alternatively, such nucleotide sequences can be prepared by genetic engineering or chemical synthesis. Such nucleotide sequences being capable of hybridizing may be identified and isolated by using the nucleotide sequences described herein or parts or reverse complements thereof, for instance by hybridization according to standard methods (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA). Nucleotide sequences comprising the same or substantially the same nucleotide sequences as indicated in SEQ ID NOs: 1 to 8, or parts/fragments thereof, can, for instance, be used as hybridization probes. The fragments used as hybridization probes can also be synthetic fragments which are prepared by usual synthesis techniques, and the sequence of which is substantially identical with that of a nucleotide sequence according to the invention.

The molecules being capable of hybridizing with the nucleotide sequences of the invention also comprise fragments, derivatives and allelic variants of the herein- described nucleotide sequences of exon 6A. In a preferred embodiment fragments are understood to mean parts of nucleotide sequences which are long enough to encode the described LHR polypeptide, preferably long enough to encode a polypeptide showing the biological activity of an LHR polypeptide as provided and described herein. In a further preferred embodiment, the term "derivative" or "variant" means that the sequences of these molecules differ from the sequences of the herein-described nucleotide sequences in one or more positions, but still show a high degree of homology to these sequences, preferably within the preferred ranges of homology mentioned below. Preferably, the degree of homology can determined by comparing the respective sequence with the nucleotide sequence of SEQ ID NOs: 1 to 8.

The meaning of the term "homologous" and "homology", respectively, particularly with respect to two nucleotide sequences or amino acid sequences/polypeptides to be compared, is also known in the art. These terms are used herein accordingly. For example, the tern "homology'V'homologous" is used herein in the context of a nucleotide sequence or amino acid sequence/polypeptide which has a homology, that is to say a sequence identity, of at least 50%, of at least 60%, preferably of at least 70%, more preferably of at least 80%, even more preferably of at least 90% and particularly preferred of at least 95%, especially preferred of at least 98% and even more preferred of at least 99% to another, preferably entire, nucleotide sequence or amino acid sequence/polypeptide. Herein, "another nucleotide sequence or amino acid sequence/polypeptide" particularly refers to a nucleotide sequence or amino acid sequence/poiypeptide of exon 6A, for example of human exon 6A. Methods for sequence comparison, particularly nucleotide or amino acid sequence comparison, and hence, determination of homology are well known in the art. For example, the degree of homology can be determined conventionally using known computer programs such as the DNASTAR program with the ClustalW analysis. This program can be obtained from DNASTAR, Inc., 1228 South Park Street, Madison, Wl 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 OAS UK (support@dnastar.com) and is accessible at the server of the EMBL outstation. When using the Clustal analysis method to determine whether a particular sequence is, for instance, 90% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

Preferably, the degree of homology of two nucleotide sequences, like two hybridizing nucleotide sequences, is calculated over their complete length, preferably of their coding sequences.

If the two nucleotide sequences to be compared by sequence comparisons differ in length, the degree of homology refers to the shorter sequence and that part of the longer sequence that matches the shorter sequence. In other words, when the sequences which are compared do not have the same length, the degree of homology preferably either refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence. In this context, the skilled person is readily in the position to determine that part of a longer sequence that "matches" the shorter sequence. Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding nucleotide sequences or polypeptides (e.g. encoded thereby). Nucleotide/amino acid sequences which are homologous to the herein- described particular nucleotide/amino acid sequences and represent derivatives/variants of these sequences are normally variations of these sequences which, preferably, represent modifications having the same biological function. They may be either naturally occurring variations, for instance sequences from other ecotypes, varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. Allelic variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques. Deviations from the above-described sequences may have been produced, e.g., by deletion, substitution, insertion and/or recombination. The polypeptides encoded by the different variants of the nucleotide sequences of the invention or the variants of the polypeptides of the present invention preferably exhibit certain characteristics they have in common. These include for instance biological activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc. The biological activity of a polypeptide of the invention, for example the capacity to bind a binding partner of the LHR, like LH, can be tested in corresponding testing assays, for example by using LH or a suitable modified form thereof. Such assays can easily be established by the skilled person based on his common general knowledge and the teaching provided herein.

The meanings of the terms "5'-end" and "3'-end" are also known in the art, and are, if not otherwise defined herein, used accordingly in the context of the present invention. In one preferred embodiment, "5'-end" particularly means a stretch of nucleotides starting with the first, i.e. the most 5'-terminal, nucleotide of a nucleotide sequence followed by a nucleotide stretch of said nucleotide sequence lying downstream (i.e. in 5' → 3' direction) in direct proximity to said first nucleotide. Correspondingly, "3'-end" particularly means a stretch of nucleotides ending with the last, i.e. the most 3'-terminal, nucleotide of a nucleotide sequence preceded by a nucleotide stretch of said nucleotide sequence lying upstream (i.e. in 3¹ → 5¹ direction) in direct proximity to said last nucleotide.

For example, particularly with respect the nucleotide sequence of exon 6A or a variant thereof, the "5'-end" or the "3'-end" or the above mentioned stretches of nucleotides being the "5'-end" or the "3'-end" in the context of the present invention may be of at least 3, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, at least 50, at least 100 or at least 150 nucleotides in length. With respect to longer nucleotide sequences, e.g., that of the LHR splicing variants as disclosed herein, the "5'-end" or the "3'-end" or those stretches of nucleotides may, for example, furthermore be of at least 200, at least 500, at least 1000 or at least 1500 nucleotides in length.

It is particularly envisaged herein, that a nucleotide sequence that "starts" or "ends" with a nucleotide sequence capable of hybridizing to the 5'-end or 3'-end, respectively, of another nucleotide sequence, indeed "starts" or "ends" with a nucleotide hybridizing to, or at least lying vis-a-vis of, the first or the last nucleotide, respectively, of this other nucleotide sequence. "Lying vis-a-vis" in this context means lying at the position complementary to said first or last nucleotide in a double stranded hybridization product formed or to be formed by two single stranded nucleotide sequences.

Moreover, it is particularly envisaged herein, that a nucleotide sequence that "starts" or "ends" with a nucleotide sequence having a certain homology to the 5'-end or 3'- end, respectively, of another nucleotide sequence, indeed "starts" or "ends" with a nucleotide being, or at least replacing, the first or the last nucleotide, respectively, of this other nucleotide sequence.

In the context of the present invention, the term to "span" or "spanning" a splicing site means, firstly, to overlap a splicing site, i.e. that the splicing site lies internally, within, e.g., a nucleotide sequence, of the present invention, and, secondly, to start or end with a splicing site, i.e. that the splicing site lies 5¹- or 3'-terminally, e.g., of a nucleotide sequence of the present invention. The term "splicing site" particularly refers to a splicing site of exon 6A, e.g. a splicing site as defined herein with respect to human exon 6A or a corresponding splicing site of another exon 6A. In analogy to the mutations of exon 6A, the skilled person is readily in the position to find out "corresponding" splicing sites, e.g. by performing sequence comparison(s).

The term "spliceable" or "can be spliced", in particular "spliceable in the LHR mRNA" or "can be spliced into the LHR mRNA", as used herein means recognizable as an exon by the splicing machinery, for example by the spliceosome, i.e. comprising at least two splicing sites recognizable by the splicing machinery, which can result in that the nucleotide sequence (e.g. hnRNA sequence) between those splicing sites is spliced into the (maturing) mRNA.

The meaning of "splicing site" is known in the art. For example, known/common splicing sites are AG as an acceptor or GT as a donor site.

The term "physiologic conditions" as used herein are such conditions usually present in a living organism, like a living cell. For example such conditions are those, present during the event of splicing in said living organism, i.e. those, under which the splicing machinery of a living cell (e.g. the (assembly of the) spliceosome) works. Such conditions are known in the art or can be determined by a person skilled in the art. Without being bound by theory, such "conditions" are those present in a typical living cell and determined by the corresponding characteristic cell parameters, like the pH, the charge, the osmolarity (e.g. the salt/ion concentration), or a hormonal stimulus etc. As a non-limiting example "physiological conditions" in the context of the present invention are characterized by a pH of about 7.4 and/or an osmolarity of about 300 mM.

The term "fragment of exon 6A" or "exon 6A fragment" as used herein refers to a partial nucleotide or amino acid sequence of exon 6A, or a variant thereof, for example of human exon 6A, or a variant thereof.

In a first preferred embodiment, this partial nucleotide or amino acid sequence may have at least one nucleotide/amino acid exchange corresponding to any one of the nucleotide/amino acid exchanges A21C, G22C, T63C, A117G and T212G and E7A, E7D and M21T, respectively. Thereby, it is clear that particularly this partial amino acid sequence cannot have an amino acid exchange corresponding to any one of the nucleotide exchanges A117G and T212G, since these nucleotide exchanges are located outside the coding sequence of exon 6A. In a second preferred embodiment, this partial nucleotide or amino acid sequence may start with the 5^'-end of exon 6A, or with a variant thereof, and/or may end with the 3 ^'-end of exon 6A, or with a variant thereof.

In a third preferred embodiment, this partial nucleotide or amino acid sequence, or a variant thereof, may span a splicing site of exon 6A or of a variant thereof. With respect to a nucleotide sequence of exon 6A, or to a variant thereof, the term "fragment" or "variant" as used herein exemplarily means a nucleotide sequence being of at least 7, at least 10, at least 15, at least 20, at least 30, at least 50, at least 100, at least 150, at least 200 or at least 250 nucleotides in length. With respect to longer nucleotide sequences, e.g. nucleotide sequences comprising exon 6A, like, e.g., the corresponding nucleotide sequences of the LHR splicing variants, the term "fragment" or "variant" is envisaged to, exemplarily, further encompass nucleotide sequences of at least 30, at least 50, at least 100, at least 150, at least 200, at least 300 or at least 500 amino acids in length.

With respect to an amino acid sequence of exon 6A, or a variant thereof, the term "fragment" or "variant" as used herein exemplary means an amino acid sequence being of at least 5, at least 7, at least 10, at least 15, at least 20 or at least 25 amino acid residues in length. With respect to longer amino acid sequences, e.g. amino acid sequences comprising those of exon 6A, or a variant thereof, like, e.g. the corresponding amino acid sequences of the LHR splicing variants, the term "fragment" or "variant" is envisaged to, exemplarily, further encompass amino acid sequences of at least 30, at least 50, at least 100, at least 150, at least 200, at least 300 or at least 500 amino acids in length.

In a preferred embodiment, a "fragment" of exon 6A, or a variant thereof, or a "variant of exon 6A/exon 6A variant" (particularly a "fragment" of exon 6A, or a variant thereof, or a "variant of exon 6A/exon 6A variant" as defined herein) is envisaged to show (an) exon 6A function(s), for example (an) exon 6A function(s) as described herein. Particularly, such function may, e.g., be the capability to be spliced into the (maturing) mRNA of the LHR, preferably under physiologic conditions, and/or the capability to impair the balance between the different splice variants of the LHR. Moreover, one particular function of the exon 6A "fragment" as provided, particularly in form of an antisense nucleotide sequence, is envisaged to be an inhibition of exon 6A, e.g. an inhibition as described and defined herein.

In a further preferred embodiment, a "fragment" or "variant" of a nucleotide sequence or amino acid sequence comprising the nucleotide and amino acid sequence, respectively, of exon 6A, is envisaged to show (an) exon 6A function(s), for example (an) exon 6A function(s) as described herein, or (a) function of a splicing variant of the LHR comprising exon 6A. Particularly, such function may, e.g., be the capability to be degenerated by NMD, the capability to impair the balance between the splice variants of the LHR and/or the capability to be translated into a truncated form of the LHR (e.g. into a soluble truncated form of the LHR).

Inter alia, the present invention particularly relates to a nucleotide sequence, for example a recombinant nucleotide sequence, consisting of the nucleotide sequence of exon 6A as provided herein, or a variant or a fragment thereof, and, optionally, (a) nucleotide sequence(s) heterologous thereto. Said nucleotide sequence of exon 6A, or said variant or said fragment thereof may have

(a) attached to its 5^'-end a nucleotide sequence of at least 1 nucleotide of the 3^'- end of the 'left' portion of formerly known intron 6 (intron 6A) flanking exon 6A in upstream position, or a variant of this nucleotide sequence; or the complementary strand of this nucleotide sequence; and/or

(b) attached to its 3^'-end a nucleotide sequence of at least 1 nucleotide of the 5^'- end of the 'right' portion of formerly known intron 6 (intron 6B) flanking exon 6A in downstream position, or a variant thereof; or the complementary strand thereof.

Thereby, it is envisaged, that the nucleotide sequence attached to the 5'-end ends with the last, i.e. the most 3'-terminal, nucleotide of the 'left' portion of formerly known intron 6 of the LHR gene preceded by a nucleotide stretch of said 'left' portion lying upstream (i.e. in 3' → 5' direction) in direct proximity to said last nucleotide. Moreover, it is envisaged that the nucleotide sequence attached to the 3'-end starts with the first, i.e. the most δ'-terminal, nucleotide of the 'right' portion of the formerly known intron 6 of the LHR gene followed by a nucleotide stretch of said 'right' portion being downstream (i.e. in 5' → 3' direction) in direct proximity to said first nucleotide. For example, the nucleotide sequence attached to the 5'-end or 3'-end of the nucleotide sequence of exon 6A may be at least the last and first, respectively, 1 , 2, 3, 5, 7, 10, 15, 20, 30, 50, 100, 150, 300, 500, 1000 or 1500 nucleotides of the 'left' and 'right', respectively, portion of intron 6 of the LHR gene, or a variant of this nucleotide sequence.

The maximum length of the nucleotide sequence attached to the 5'-end or 3'-end of a nucleotide sequence of exon 6A thereby is envisaged to be the length of the entire 'left¹ and 'right', respectively, portion of intron 6 of the LHR gene or of a variant thereof, preferably the length of the entire 'left' and 'right', respectively, portion of intron 6 of the LHR gene minus one nucleotide.

Particularly with respect to the human LHR/LHR gene, this means that the maximum length of the nucleotide sequence attached to the 5'-end or 3'-end of the nucleotide sequence of human exon 6A is envisaged to be 1700 nucleotides, preferably 1699 nucleotides, and 7400 nucleotides, preferably 7399 nucleotides, respectively.

In a second aspect, the present invention relates to a nucleotide sequence comprising the nucleotide sequence of exon 6A, or to a variant or a fragment thereof. Particularly, it is envisaged that the nucleotide sequence comprising exon 6A, or a variant or a fragment thereof, is selected from the group consisting of:

(a) an mRNA corresponding to a nucleotide sequence comprising the nucleotide sequence of exon 6A or of a variant or of a fragment thereof;

(b) a cDNA derived from the mRNA of (a);

(c) a fragment of (a) or (b), comprising a fragment corresponding to the nucleotide sequence of exon 6A, or to a variant or to a fragment thereof;

(d) a nucleotide sequence comprising the nucleotide sequence of exon 6A, or of a variant or of a fragment thereof. Said nucleotide sequence of exon 6A, or of a variant or of a fragment thereof, may have at least one nucleotide exchange corresponding to any one of the particular nucleotide exchanges as defined herein and/or said nucleotide sequence may start with the 5^'-end and/or may end with the 3 ^'-end of a nucleotide sequence of exon 6A, or of a variant thereof;

(e) a fragment of the nucleotide sequence of (d) comprising a fragment of the nucleotide sequence of exon 6A, or of a variant thereof. Said fragment of the nucleotide sequence of exon 6A, or of a variant thereof, may have at least one nucleotide exchange corresponding to any one of the particular nucleotide exchanges as defined herein and/or said fragment may start with the 5 ^'-end and/or may end with the 3 ^'-end of the nucleotide sequence of exon 6A, or of a variant thereof;

(f) a nucleotide sequence which is capable of hybridizing to the nucleotide sequence of any one of (a) to (e). Said nucleotide sequence may particularly comprise a nucleotide sequence which is capable of hybridizing to the nucleotide sequence of exon 6A, or to a variant thereof, and, optionally, a nucleotide sequence which is capable of hybridizing to the nucleotide sequence encoding the LHR lacking exon 6A, or to a variant of the LHR lacking exon 6A;

(g) a nucleotide sequence being homologous to the nucleotide sequence of any one of (a) to (e). Said nucleotide sequence may comprise a nucleotide sequence being homologous to the nucleotide sequence of exon 6A, or to a variant thereof, and, optionally, a nucleotide sequence being homologous to the nucleotide sequence encoding the LHR lacking exon 6A, or to a variant of the LHR lacking exon 6A; and

(h) the complement strand of the nucleotide sequence of any one of (a) to (g).

Preferably, the nucleotide sequence of (f) as defined above, is capable of hybridizing to a nucleotide sequence of the LHR comprising the nucleotide sequence of exon 6A, or to a variant thereof and spans a splicing site of exon 6A or of a variant thereof. This means that said nucleotide sequence of (T) preferably is capable of hybridizing to a nucleotide sequence representing the 3'-end of exon 6 (directly) followed by the 5'-end of exon 6A and/or to the 3'-end of exon 6A (directly) followed by the 5'-end of exon 7, of the nucleotide sequence of the LHR comprising the nucleotide sequence of exon 6A, or to a variant thereof. The analogous applies with respect to the homologous nucleotide sequence of (g), mutatis mutandis.

Moreover, the nucleotide sequence of (T) as defined above may particularly comprise a nucleotide sequence which is capable of hybridizing to a nucleotide sequence of exon 6A or of a variant thereof, said nucleotide sequence of exon 6A or of said variant thereof, may

(i) have at least one nucleotide exchange corresponding to any one of the particular nucleotide or amino acid exchanges as defined herein;

(ii) start with a nucleotide sequence capable of hybridizing to the 5^'-end and/or end with a nucleotide sequence capable of hybridizing to the 3 ^'-end of the nucleotide sequence of exon 6A or of a variant thereof; and/or

(iii) span a splicing site of exon 6A or of a variant thereof.

Furthermore, the nucleotide sequence of (g) as defined above may particularly comprise a nucleotide sequence being homologous to the nucleotide sequence of exon 6A or of a variant thereof, said nucleotide sequence of exon 6A or of said variant thereof, may (i) have at least one nucleotide exchange corresponding to any one of the particular nucleotide or amino acid exchanges as defined herein; (ii) start with a nucleotide sequence being homologous to the 5^'-end and/or end with a nucleotide sequence being homologous to 3^'-end of the nucleotide sequence of exon 6A or of a variant thereof; and/or (iii) span a splicing site of exon 6A or of a variant thereof.

One particular preferred embodiment of the present invention relates to a nucleotide sequence comprising the herein defined nucleotide sequence of exon 6A, or a variant or a fragment of this nucleotide sequence (for example a variant or a fragment of this nucleotide sequence as defined herein), wherein said nucleotide sequence does not comprise genomic DNA of the LHR gene or a variant or a fragment of the genomic DNA of the LHR gene. The definitions given herein with respect to the terms "variant" and "fragment" apply here mutatis mutandis. In a third aspect, the present invention relates to a nucleotide sequence comprising a nucleotide sequence of a splice variant of the LHR comprising exon 6A, or of a variant or a fragment thereof. Particularly, it is envisaged that said nucleotide sequence of a splice variant of the LHR comprising exon 6A, or of a variant or a fragment thereof is selected from the group consisting of:

(a) a nucleotide sequence of a splice variant of the LHR comprising exon 6A, for example of a splice variant of the human LHR comprising exon 6A as depicted in any one of SEQ ID NOs: 5 to 8;

(b) the nucleotide sequence of (a) having at least one nucleotide exchange corresponding to any one of the particular nucleotide exchanges as defined herein;

(c) a fragment of the nucleotide sequence of (a) or (b). Said fragment may comprise a nucleotide sequence consisting of the 3'-end of exon 6 (e.g. of human exon 6 as depicted in SEQ ID NO: 14) followed by the 5'-end of the nucleotide sequence of exon 6A and/or a nucleotide sequence consisting of the 3'-end of the nucleotide sequence of exon 6A followed by the 5'-end of exon 7 (e.g. of human exon 7 as depicted in SEQ ID NO: 15);

(d) a nucleotide sequence having the degenerated code of the nucleotide sequence of any one of (a) to (c);

(e) a nucleotide sequence which is capable of hybridizing to the complementary strand of the nucleotide sequence of any one of (a) to (d). Said nucleotide sequence may comprise a nucleotide sequence corresponding to a nucleotide sequence consisting of the 3'-end of exon 6 (e.g. human exon 6 as depicted in SEQ ID NO: 14) followed by the 5'-end of the nucleotide sequence of exon 6A and/or a nucleotide sequence consisting of the 3'-end of the nucleotide sequence of exon 6A followed by the 5'-end of exon 7 (e.g. human exon 7 as depicted in SEQ ID NO: 15);

(T) a nucleotide sequence being homologous to the nucleotide sequence of any one of (a) to (d). Said nucleotide sequence may comprise a nucleotide sequence corresponding to a nucleotide sequence consisting of the 3'-end of exon 6 (e.g. human exon 6 as depicted in SEQ ID NO: 14) followed by the 5'- end of the nucleotide sequence of exon 6A and/or a nucleotide sequence consisting of the 3'-end of the nucleotide sequence of exon 6A followed by the 5'-end of exon 7 (e.g. human exon 7 as depicted in SEQ ID NO: 15); and (g) the complementary strand of the nucleotide sequence of any one of (a) to (f).

In general, the nucleotide sequences of the present invention can be obtained, for instance, from natural sources or may be produced synthetically or by recombinant techniques, such as PCR, and include modified or derivatized nucleotide sequences as can be obtained by applying techniques described in the pertinent literature.

In one particular aspect, the present invention relates to recombinant nucleotide sequences comprising the nucleotide sequence of the invention described herein. The term "recombinant nucleotide sequence" refers to a nucleotide sequence which contains in addition to a nucleotide sequence of the invention as described above at least one further heterologous coding or non-coding nucleotide sequence. The term "heterologous" means that said nucleotide sequence originates from a different species or from the same species, however, from another location in the genome than said added nucleotide sequence or is synthetic. The term "recombinant" implies that nucleotide sequences are combined into one recombinant nucleotide sequence by the aid of human intervention. The recombinant nucleotide sequence of the invention can be used alone or as part of a vector.

For instance, the recombinant nucleotide sequence may encode the polypeptide of exon 6A, or the LHR comprising the exon 6A polypeptide, fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. The marker sequence may for example be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.) or a His tag, which provide for convenient purification of the fusion polypeptide. Another suitable marker sequence may be the HA tag which corresponds to an epitope derived from influenza hemagglutinin polypeptide (Wilson, Cell 37 (1984), 767). As a further example, the marker sequence may be glutathione-S-transferase (GST) which, apart from providing a purification tag, enhances polypeptide stability, for instance, in bacterial expression systems.

In a preferred embodiment, the recombinant nucleotide sequences of the present invention may further comprise expression control sequences operably linked to the nucleotide sequence comprised in the recombinant nucleotide sequence. More preferably, these nucleotide sequences are expression cassettes. The term "operatively linked" or "operably linked", as used throughout the present description, refers to a linkage between one or more expression control sequences and the coding region in the nucleotide sequence to be expressed in such a way that expression is achieved under conditions compatible with the expression control sequence.

Expression comprises transcription of the heterologous nucleotide sequence, preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic as well as in eukaryotic cells, preferably in animal cells, are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals and the like. Examples are given further below in connection with explanations concerning vectors. In the case of eukaryotic cells, expression control sequences may comprise poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers.

In one embodiment, the present invention further relates to a vector consisting of the nucleotide sequence of the present invention and (a) nucleotide sequence(s) heterologous thereto.

In a first aspect of this embodiment, the definition of the term "heterologous" as given above applies, mutatis mutandis.

In another aspect of this embodiment, the term "heterologous" means not corresponding to the LHR gene or being or encoding an expression product thereof. In an preferred embodiment, the "heterologous sequences" being comprised in the vector of the present invention may be expression control sequences operably linked to said nucleotide sequence of the present invention being contained in the vector provided herein. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in prokaryotic or eukaryotic cells. The expression of the nucleotide sequence of the invention in prokaryotic or eukaryotic cells, for instance in Escherichia coli, may particularly be interesting because it permits a more precise characterization of the biological activities of the encoded polypeptide. In particular, it is possible to express these polypeptides in such prokaryotic or eukaryotic cells which are free from interfering polypeptides. In addition, it is possible to insert different mutations into the nucleotide sequences by methods usual in molecular biology (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA), leading to the synthesis of polypeptides possibly having modified biological properties. In this regard it is on the one hand possible to produce deletion mutants in which the nucleotide sequences are produced by progressive deletions from the 5' or 3' end of the coding DNA sequence, and said nucleotide sequences lead to the synthesis of correspondingly shortened polypeptides.

On the other hand, the introduction of point mutations is also conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide.

For genetic engineering in prokaryotic cells, the nucleotide sequences of the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.

The term "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In a preferred embodiment, the vectors of the invention are suitable for the transformation of cells, like fungal cells, cells of microorganisms such as yeast or bacterial cells or animal cells. In a particularly preferred embodiment such vectors are suitable for use in gene therapy. In one aspect of the invention, the vector as provided is suitable for stable transformation of an organism, and hence is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, for example a promoter as defined herein, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a nucleotide sequence desired to be expressed. The DNA sequence naturally controlling the transcription of the corresponding gene, e.g. the promoter sequence of the LHR gene, can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp- lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481 ; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), IpI ₁ rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-^β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

Examples of vectors suitable to comprise the nucleotide sequences of the present invention to form the vector of the present invention are known in the art. For example, such vectors may be suitable for gene therapy, i.e. the vector of the present invention may also be a gene transfer and/or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes or nucleic acid constructs into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper. Circ. Res. 79 (1996), 911-919; Anderson. Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser. Circ. Res. 77 (1995), 1077-1086; Wang. Nature Medicine 2 (1996), 714-716; WO 94/29469: WO 97/00957; Schaper, Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389 (1997), 239-242 and references cited therein.

Nucleic acid molecules being or encoding the herein disclosed inhibitors (or enhancers/agonists) of exon 6A, said inhibitors (or enhancers/agonists) itself or the vectors as disclosed herein may therefore be particularly designed for gene therapy approaches. Said compounds may also be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, baculoviral systems or systems based on vaccinia virus or Semliki Forest Virus can be used as eukaryotic expression system for said compounds disclosed in the context of the invention.

For gene therapy, various viral vectors which can be utilized are, for example, adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can also incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the inserted polynucleotide sequence.

The present invention further relates to a polypeptide comprising an amino acid sequence encoded by the nucleotide sequence of exon 6A, or a variant or a fragment of said amino acid sequence. Particularly, it is envisaged that said amino acid sequence is selected from the group consisting of:

(a) an amino acid sequence encoded by exon 6A (e.g. an amino acid sequence as depicted in SEQ ID NO: 9);

(b) an amino acid sequence encoded by exon 6A (e.g. an amino acid sequence as depicted in SEQ ID NO: 9) having at least one amino acid exchange. Said amino acid sequence may be encoded by a nucleotide sequence that can be spliced in the LHR mRNA, preferably under physiologic conditions;

(c) an amino acid sequence encoded by exon 6A (e.g. an amino acid sequence as depicted in SEQ ID NO: 9) having at least one amino acid exchange corresponding to any one of the particular amino acid exchanges as defined herein;

(d) fragments of the amino acid sequence of any one of (a) to (c); and

(e) an amino acid sequence being homologous to the amino acid sequence of any one of (a), (c) and (d).

The definition of the terms "fragment(s)" and "homologous" as given herein above apply here, mutatis mutandis.

Additionally, the present invention relates to a polypeptide encoded by the nucleotide sequences of the present invention, particularly by nucleotide sequences of the present invention of (i) an exon 6A or a variant or a fragment thereof, or (ii) a splicing variant of the LHR comprising exon 6A, or of a variant or of a fragment thereof.

Furthermore, the present invention relates to a polypeptide encoded by the nucleotide sequence corresponding to the LHR gene comprising exon 6A or to an expression product thereof, i.e. to a polypeptide encoded by the nucleotide sequence of a splicing variant of the LHR comprising exon 6A, or by a variant or by a fragment of said nucleotide sequence. The polypeptide of the present invention may, e.g., be a naturally purified product or a product of chemical synthetic procedures or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptide of the present invention may be glycosylated or may be non-glycosylated. The polypeptide of the invention may include, but also lack, an initial methionine amino acid residue. The polypeptide according to the invention may be further modified to contain additional chemical moieties not normally part of the polypeptide. Those derivatized moieties may, e.g., improve the stability, solubility, the biological half life or absorption of the polypeptide. The moieties may also reduce or eliminate any undesirable side effects of the polypeptide and the like. An overview for these moieties can be found, e.g., in Remington's Pharmaceutical Sciences (18^th ed., Mack Publishing Co., Easton, PA (1990)). Polyethylene glycol (PEG) is an example for such a chemical moiety which has been used for the preparation of therapeutic polypeptides. The attachment of PEG to polypeptides has been shown to protect them against proteolysis (Sada et al., J. Fermentation Bioengineering 71 (1991), 137-139). Various methods are available for the attachment of certain PEG moieties to polypeptides (for review see: Abuchowski et al., in "Enzymes as Drugs"; Holcerberg and Roberts, eds. (1981), 367-383). Generally, PEG molecules are connected to the polypeptide via a reactive group found on the polypeptide. Amino groups, e.g. on lysines or the amino terminus of the polypeptide are convenient for this attachment among others.

In a further embodiment, the present invention relates to a host cell, genetically engineered with the nucleotide sequence of the present invention or comprising the vector or the polypeptide of the present invention.

Generally, the host cell of the present invention may be a prokaryotic or eukaryotic cell, comprising the nucleotide sequence, the vector and/or the polypeptide of the invention or a cell derived from such a cell and containing the nucleotide sequence, the vector and/or the polypeptide of the invention. In a preferred embodiment, the host cell comprises, for example due to genetic engineering, the nucleotide sequence or the vector of the invention in such a way that it contains the nucleotide sequences of the present invention integrated into the genome. For example, such host cell of the invention, but also the host cell of the invention in general, may be a bacterial, yeast, fungus, plant, animal or human cell.

In one particular aspect, the host cell of the present invention is capable to express or expresses the nucleotide sequence of this invention. An overview of examples of different expression systems to be used for generating the host cell of the present invention, for example the above-described particular one, is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths, (Methods in Molecular Biology 75 (1997), 427-440).

The transformation or genetically engineering of the host cell with a nucleotide sequence or the vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. Moreover, the host cell of the present invention is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.

One further embodiment of the present invention refers to an inhibitor of exon 6A (of the LHR gene) and to a pharmaceutical composition comprising an inhibitor of exon 6A (of the LHR gene). Moreover, the present invention relates to a pharmaceutical composition comprising a nucleotide sequence, a vector, a polypeptide and/or a host cell of the present invention

The term "inhibitor of exon 6A (of the LHR gene)" refers to a compound that is capable to prevent the generation of, or is capable to reduce an increased amount of, LHR splice variants (e.g. LHR mRNA) comprising exon 6A. Hence, such a compound is capable to prevent/restore an imbalance, i.e. prevent an imbalance or restore the balance, between the amount of LHR splice variants comprising exon 6A and the amount of LHR splice variants lacking exon 6A.

For example, and without being bound by theory, an "inhibitor of exon 6A" as referred to herein may be a compound (i) that prevents the splicing of exon 6A into the maturing mRNA of the LHR, (ii) that prevents the expression of an LHR gene having a mutation which results in an increased splicing of exon 6A into the LHR mRNA, (iii) that reduces LHR mRNA comprising exon 6A, particularly truncated LHR mRNA terminally carrying exon 6A, or (iv) that prevents the generation of (i.e. the translation of) or that reduces or inhibits the activity of a truncated LHR terminally carrying exon 6A (i.e. carrying the amino acid sequence encoded by exon 6A).

A particular inhibitor of exon 6A in the context of the present invention may be one selected from the group consisting of:

(a) a binding molecule specifically recognizing nucleotide sequences of exon 6A, polypeptides encoded by said nucleotide sequences of exon 6A, nucleotide sequences of LHR variants comprising exon 6A and/or polypeptides encoded by said nucleotide sequences of LHR variants, or variants or fragments of said nucleotide sequences or polypeptides (herein also referred to as "target molecules");

(b) a nucleic acid molecule which specifically introduces an insertion of a heterologous sequence or a mutation into exon 6A of the LHR gene via in vivo mutagenesis; and

(c) a nucleic acid molecule specifically reducing the expression of mRNA comprising exon 6A by cosuppression.

Generally, the binding molecule of the present invention may be selected form the group consisting of antibodies, affybodies, trinectins, anticalins, aptamers, antisense nucleotide sequences (like antisense DNAs or antisense RNAs), PNAs, and the like. Based on prior art literature, the person skilled in the art is familiar with obtaining specific binding molecules that may be useful in the context of the present invention. These molecules are directed and bind specifically to or specifically label the above- described target molecules. Non-limiting examples of suitable binding molecules may be selected from aptamers (Gold, Ann. Rev. Biochem. 64 (1995), 763-797)), aptazymes, RNAi, shRNA, RNAzymes, ribozymes (see e.g., EP-B1 0 291 533, EP- A1 0 321 201 , EP-B1 0 360 257), antisense DNA, antisense oligonucleotides, antisense RNA, siRNA, antibodies (Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988), affibodies (Hansson, lmmunotechnology 4 (1999), 237-252; Henning, Hum Gene Ther. 13 (2000), 1427- 1439), lectins, trinectins (Phylos Inc., Lexington, Massachusetts, USA; Xu, Chem. Biol. 9 (2002), 933), anticalins (EPB1 1 017 814) and the like.

The term "specifically recognizing" is meant to refer to the high affinity antibodies or other binding molecules known in the prior art typically have for the target molecule against which they were prepared.

Advantageously, the term "specifically recognizing" refers to a specificity of the binding molecule that allows a distinction between the above described target molecules, like nucleotide sequences of exon 6A, polypeptides encoded by said nucleotide sequences of exon 6A, nucleotide sequences of LHR variants comprising exon 6A and/or polypeptides encoded by said nucleotide sequences of LHR variants, and molecules not targeted by the binding molecule(s), like nucleotide sequences of LHR variants lacking exon 6A or polypeptides encoded by said nucleotide sequences of LHR variants, in the sense that the binding molecule does not show a significant cross-reactivity with the latter molecules. The person skilled in the art is able to prepare such distinctive binding molecules, for example by selecting mainly or exclusively nucleotide sequences of exon 6A or amino acid sequences encoded thereof as a target for the binding molecule to be prepared.

Binding molecules according to the invention can, inter alia, be used for detecting the presence, absence or amount of the target molecule of the invention in a sample, in particular in the framework of methods and uses described herein. The binding molecules may furthermore be used for isolating the target molecules from a biological source material.

Moreover and most preferred, the binding molecules according to the invention may be used to inhibit exon 6A, what, for example, means to reduce the amount or inhibit the activity of the target molecules as defined herein. In a preferred particular embodiment, a binding molecule in accordance with the invention comprises or is a nucleotide sequence of the present invention.

A particular binding molecule in the context of the present invention may be one selected from the group consisting of:

(a) an antisense nucleotide sequence, for example an antisense oligonucleotide, specifically binding to mRNA (or hnRNA) comprising exon 6A; like, for example, an antisense oligonucleotide suitable for "exon skipping" of exon 6A;

(b) an antibody specifically binding to mRNA comprising exon 6A or to a truncated variant of the LHR terminally comprising the polypeptide encoded by exon 6A;

(c) a siRNA specifically binding to mRNA comprising exon 6A;

(d) an aptamer specifically binding to mRNA comprising exon 6A; and

(e) a ribozyme specifically recognizing mRNA comprising exon 6A.

The most preferred inhibitor of exon 6A in the context of the present invention is an antisense oligonucleotide suitable for "exon skipping" of exon 6A.

The meaning of "exon skipping" is known in the art and this term is used correspondingly in the context of the present invention. For example, the technology of "exon shipping" is described in Gebski (2003, Human Molecular Genetics, 12, 15,

1801-1811 ).

Without being bound by theory, "exon skipping" takes advantage of (an) oligonucleotide(s) that hybhdize(s)/bind(s) to the primary transcript of a gene (also referred to herein as pre-mRNA or hnRNA) in a manner that a certain exon of a gene, e.g. exon 6A of the LHR gene, is not spliced into the resulting mRNA, for example, but without being bound by theory, because of an interference of the hybridized/bound oligonucleotide(s) with the splicosome assembly.

Based on his common general knowledge and the teaching provided herein, the skilled person is readily in the position to generate antisense oligonucleotides

"suitable" for skipping of exon 6A, i.e. antisense oligonucleotides that hybridize/bind to hnRNA transcribed from the LHR gene in a manner that exon 6A is not spliced into the resulting mRNA of the LHR. For example, but not limiting, "suitable" in this context means that the antisense oligonucleotide hybridizes/binds to such part of the hnRNA that spans a splicing site of exon 6A, preferably a splicing site at the 5'-end of exon 6A. An example of such an antisense oligonucleotide (SEQ ID NO: 16), as well as the corresponding complement strand of the LHR hnRNA, particularly that part of the LHR hnRNA corresponding to exon 6A, (SEQ ID NO: 17) is given herein. The oligonucleotide(s) as described above and provided herein may particularly be of the same length than the "fragment(s)" with respect to a nucleotide sequence as described and provided herein.

As mentioned above, one particular binding molecule in the context of the present invention is envisaged to be an antisense nucleotide sequence, i.e. a nucleotide sequence complementary to those target molecules defined herein being nucleotide sequences. Preferably, the antisense nucleotide sequence in the context of the present invention is an antisense RNA sequence. In a particular embodiment, the antisense nucleotide sequence in accordance with the invention comprises or is an antisense nucleotide sequence corresponding to a nucleotide sequence of the present invention.

As used herein, the term "antisense nucleotide sequence corresponding to a nucleotide sequence" means that the antisense nucleotide sequence provides the same sequence information than the nucleotide sequence to which it "corresponds", but that this sequence information is implied in that strand of a nucleotide sequence which is complement of the sense strand of said nucleotide sequence.

Particularly, the target molecules of the antisense nucleotide sequence may be transcripts of the LHR gene (e.g. mRNA or hnRNA) comprising exon 6A. Since these transcripts may include nucleotide sequences corresponding to (an) intron sequence(s) of the LHR gene (particularly when the transcript is an hnRNA), it is also envisaged herein that the antisense nucleotide sequence in the context of the present invention may, at least partially, be antisense with respect to (an) intron sequence(s) of the LHR gene.

The antisense nucleotide sequences as provided and described herein may have a length of at least 15, preferably of more than 50, more preferably of more than 100, even more preferably of more than 200 and most preferably of more than 500 nucleotides. However, antisense nucleotide sequences that usually are employed in the art are shorter than 5000 nucleotides or even shorter than 2500 nucleotides, and so the antisense nucleotide sequences as provided herein are intended to be.

In general, the antisense technology to reduce the amount of a desired target molecule (e.g. an mRNA) by means of an antisense effect is known in the art. (see also : Antisense oligonucleotides: from design to therapeutic application. Chan, Clin Exp Pharmacol Physiol. 2006 May-Jun;33(5-6):533-40).

For this purpose, usually a nucleotide sequence (or a part thereof) corresponding to the target nucleotide sequence is linked in antisense orientation with a promoter ensuring the transcription and possibly with a termination signal ensuring the termination of the transcription as well as the polyadenylation of the transcript.

The siRNA technology as also intended to be employed in the context of the present invention is also well known in the art. Based on his common general knowledge and the teaching provided herein, the skilled person is readily in the position to find out siRNAs suitable to bind the corresponding target molecule(s), and hence, suitable to reduce products of the LHR gene which comprise exon 6A. In a particular embodiment, an siRNA as provided and described herein comprises or is a nucleotide sequence corresponding to the nucleotide sequence of the present invention, particularly to nucleotide sequence fragments of the present invention.

As mentioned above, one particular binding molecule in the context of the present invention is an antibody specific for/specifically binding the target molecules as defined herein, particularly the mRNA comprising exon 6A, or a variant or a fragment thereof, or the truncated variant of the LHR terminally comprising the polypeptide encoded by exon 6A, or a variant or a fragment thereof.

The antibody useful in the context of the present invention can be, for example, polyclonal or monoclonal. The term "antibody" also comprises derivatives or fragments thereof which still retain the binding specificity.

The target molecules according to the invention, its fragments or other derivatives thereof, or cells expressing them, can be used as an immunogen to produce antibodies thereto. The present invention in particular also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

In the context of the present invention, the term "antibody" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules substantially retaining binding specificity. Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules, like chimeric and humanized antibodies. The term also relates to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')₂. The term "antibody" also comprises bifunctional antibodies, trifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.

Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Antibodies directed against a polypeptide according to the present invention can be obtained, e.g., by direct injection of the target molecule into an animal or by administering the target molecule to an animal, preferably a non-human animal. The antibody so obtained will then bind the target molecule itself. In this manner, even a fragment of the target molecule can be used to generate antibodies binding the whole target molecule, as long as said binding is "specific" as defined above.

Particularly preferred in the context of the present invention are monoclonal antibodies. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kόhler and Milstein Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Thus, the antibody derivatives can also be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specifically recognizing the polypeptide of the invention. Also, transgenic animals may be used to express humanized antibodies to the polypeptide of the invention. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97- 105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Accordingly, also phage antibodies can be used in the context of this invention.

In accordance with the present invention, the term "aptamer" means nucleic acid molecules that can specifically bind to target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold (1995), Ann. Rev. Biochem 64 , 763-797).

In a preferred embodiment, the antibody or aptamer as provided and described herein is envisaged to specifically bind to a nucleotide sequence or to the polypeptide of the present invention, particularly, at least partially, to such parts thereof that are of exon 6A origin.

As a non-limiting example, the antibody or aptamer as provided and described herein is envisaged to bind to one of the following epitopes of the amino acid sequence encoded by exon 6A:

(a) CDEAIKELTLKEKREN (SEQ ID NO: 18); and, preferably,

(b) EKRENMDWNDSEMKR (SEQ ID NO: 19).

It is clear that for the purpose of antibody/aptamer production, peptides can be employed that represent (at least partially) an epitope of the amino acid sequence encoded by exon 6A and, optionally, that can further be modified according to the corresponding general practice well known in the art. One example of such a modified peptide is a peptide comprising the amino acid sequence CEKRENMDWNDSEMKR (SEQ ID NO: 21). The skilled person is readily in the position deduce which modification(s) of a peptide representing an epitope of the amino acid sequence encoded by exon 6A are suitable in order to generate the antibodies/aptamers provided and described in the context of the present invention. Another particular binding molecule provided and described herein is a ribozyme, particularly such a ribozyme that specifically recognizes and cleaves the target nucleotide sequences as defined herein. The ribozyme technology is also well known in the art. Generally, ribozymes are known to be catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques it is possible to alter the specificity of ribozymes. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of two different groups of ribozymes. The first group is made up of ribozymes which belong to the group I intron ribozyme type. The second group consists of ribozymes which, as a characteristic structural feature, exhibit the so- called "hammerhead" motif. The specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule.

In order to produce nucleotide sequences encoding a ribozyme which specifically cleaves the target nucleotide sequence(s) as defined herein, for example, a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are homologous to sequences of the target nucleotide sequence(s). Sequences encoding the catalytic domain may for example be the catalytic domains of the satellite DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224) or that of the satellite DNA or the TobR virus (Steinecke, EMBO J. 11 (1992), 1525-1530; Haseloff, Nature 334 (1988), 585-591). The DNA sequences flanking the catalytic domain are preferably derived from the above-described target nucleotide sequences. The general principle of the expression of ribozymes and the method is described, for example, in EP-B1 0 321 201.

In one embodiment, the inhibitor of exon 6A is a nucleic acid molecule that leads to a reduction of the target molecules via in vivo mutagenesis. Thereby, an insertion of a heterologous sequence or a mutation into nucleotide sequences comprising exon 6A, or into the nucleotide sequence of exon 6A itself, leads to an inhibition of exon 6A, i.e. reduction of the target molecules. Generally, methods of "in vivo mutagenesis" (also known as "chimeroplasty") are known in the art. In such methods, a hybrid RNA/DNA oligonucleotide (chimeroplast) is introduced into cells (WO 95/15972; Kren, Hepatology 25 (21997), 1462-1468; Cole-Stauss, Science 273 (1996), 1386- 1389). A part of the DNA component of the RNA/DNA oligonucleotide is homologous to a nucleotide sequence occurring endogenously in the cell and encoding a corresponding protein, but displays a mutation or comprises a heterologous part which lies within the homologous region. Due to base pairing of the regions of the RNA/DNA oligonucleotide which are homologous to the endogenous sequence with these sequences, followed by homologous recombination, the mutation or the heterologous part contained in the DNA component of the oligonucleotide can be introduced into the cell genome. This leads to a reduction of the activity, i.e. expression, of the gene, into which the heterologous part or the mutation has been introduced.

In view of the above, it is clear that the nucleic acid molecule causing in vivo mutagenesis comprises a heterologous sequence or a sequence carrying a mutation flanked by parts of the nucleotide sequence of exon 6A or variants thereof.

In a further embodiment of the invention, the inhibitor of exon 6A is a nucleic acid molecule that leads to a reduction of the target molecules by a cosuppression effect. "Cosuppression effect" means that the synthesis of a nucleotide sequence, particularly of an RNA, in a living cell reduces the expression of a gene being homologous to said nucleotide sequence. The general principle of cosuppression and corresponding methods are well known to the person skilled in the art and are described, for example, in Pal-Bhadra (Cell 90, 1997), 479-490) and Birchler (Nature Genetics 21 (1999), 148-149).

In a particular embodiment, the nucleic acid molecule causing a cosuppression effect comprises a nucleotide sequence of exon 6A or of variants or of fragments thereof.

The pharmaceutical composition of the present invention may particularly be designed for gene therapy applications. The technique of gene therapy is well known and has already been described above. All what has been said there also applies in connection with the pharmaceutical composition. For example, nucleic acid molecules, vectors or nucleic acid constructs being or encoding the herein disclosed inhibitors or enhancers/agonists of exon 6A are, in the pharmaceutical composition in accordance with this invention, preferably in a form which allows their introduction, expression and/or stable integration into cells of an individual to be treated.

It is envisaged herein that the pharmaceutical composition of this invention or to be prepared in accordance with this invention, optionally may comprise a pharmaceutically acceptable carrier and/or diluent and/or that the inhibitor of exon 6A may optionally be used or administered to a patient in need in combination with a pharmaceutically acceptable carrier.

Examples of suitable pharmaceutically acceptable carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. The resulting pharmaceutical compositions can be administered to the subject at a suitable dose, i.e. a dose leading to a pharmaceutically active amount of the compound to be employed/used herein at its desired site of effect.

Administration of the pharmaceutical composition to be prepared in accordance with the present invention or of the inhibitor of exon 6A to be administered to a patient in need may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration (for example as effected by inhalation) or, preferably, by direct administration (for example injection) into the testis. As mentioned above, one particular mode to "administrate" the pharmaceutical composition of the present invention are gene therapy approaches.

The dosage regimen of the inhibitor of exon 6A to be employed/used herein or the pharmaceutical composition comprising it will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A person skilled in the art is aware of and is able to test the relevant doses, the compounds to be used in terms of the present invention are to be administered in. Since the testis is an organ located outside of the body, it, for example, represents an ideal target for direct intratesticular application of, for example, antisense oligonucleotides or other compounds comprised in the pharmaceutical compositions of this invention. By this, the LHR expression and usage of exon 6A can be directly altered. Without being bound by theory, suppression of exon 6A usage would then restore a normal functioning LHR. Otherwise, in an exemplified systemic approach, the compound could, for example, be chemically linked to LH which then specifically binds to the LHR of the Leydig or Theca cells. After incorporation of the complex it can then excert its intracellular function by inhibiting exon 6A usage. A similar approach of a hormone-linked compound has been recently shown for the related hormone FSH (Mruk, Nat Med. 2006;12(11):1323-8).

A preferred subject/patient in the context of the present invention is a mammalian subject/patient, more preferably a primate subject/patient, most preferably a human being, preferably in need of medical intervention, either in form of treatment or in from of amelioration.

Another embodiment of the present invention relates to a diagnostic composition comprising a nucleotide sequence, a vector, a polypeptide and/or a host cell of the present invention. In a further preferred embodiment, the present invention relates to a diagnostic composition comprising a binding molecule as defined herein.

In another further preferred embodiment, the present invention relates to a diagnostic composition comprising probes or, preferably, primers capable of selectively hybridizing to nucleotide stretches carrying (a) mutation(s) or polymorphism(s) of exon 6A.

The diagnostic composition of this invention is particularly useful in diagnosing a disease as defined and described herein. The present invention furthermore relates to a kit comprising the a nucleotide sequence, a vector, a polypeptide and/or a host cell of the present invention, or comprising the pharmaceutical or diagnostic composition of the present invention. Said Kit is particularly useful for the diagnostic or therapeutic applications disclosed herein.

In a further preferred aspect of the present invention, the kit or the pharmaceutical or diagnostic composition of the present invention may further comprise or be provided with (an) instruction manual(s) which guide the skilled person how to antagonize/inhibit exon 6A (or how to agonize/enhance exon 6A), i. e. how to diagnose, treat or ameliorate a disease as defined herein in accordance with the present invention. Particularly, said instruction manual(s) may comprise guidance how to use or apply the herein provided methods of diagnosing, treating or ameliorating such a disease.

To this end, the kit and/or pharmaceutical or diagnostic composition of the present invention may further comprise the substances/chemicals and/or equipment suitable for the corresponding diagnostic or therapeutic assessment/intervention which are useful for a protocol for antagonizing/inhibiting of exon 6A (or for agonizing/enhancing of exon 6A) and, optionally, a solvent, diluent and/or buffer for stabilizing and/or storing the inventions compounds.

The present invention further relates to a method of diagnosing a disease, wherein said disease is characterized by an increased amount of LHR gene products comprising exon 6A (for example LHR mRNA comprising exon 6A), compared to the amount of LHR gene products comprising exon 6A (for example LHR mRNA comprising exon 6A) in a control patient, e.g. a patient not suffering from said disease.

In one aspect, it is envisaged that said method comprises the following steps: (a) determining the amount of LHR gene products comprising exon 6A in a patient and/or in a sample derived from said patient; and (b) comparing the amount of LHR gene products comprising exon 6A with the amount of LHR gene products comprising exon 6A in a control patient and/or in a sample derived from a control patient.

In one embodiment, the above method further comprises the step of

(c) determining whether a patient exhibits a mutation or a polymorphism in exon 6A.

Alternatively, the method of diagnosing a disease, wherein said disease is characterized by an increased amount of LHR gene products comprising exon 6A (for example LHR mRNA comprising exon 6A) compared to the amount of LHR gene products comprising exon 6A (for example LHR mRNA comprising exon 6A) in a control patient, comprises the step of:

(a) determining whether a patient exhibits a mutation or a polymorphism in exon 6A.

The meaning and scope of "mutation or a polymorphism in exon 6A" is explained in great detail herein. It will be understood that "determining whether a patient exhibits a mutation or a polymorphism" includes that a sample is obtained from a patient and the mutation or the polymorphism in exon 6A is determined in said sample.

A suitable "sample" as employed in accordance with this invention includes, but is not limited to, (a) biological or medical sample(s), like, e.g. (a) sample(s) comprising cell(s) or tissue(s). For example, such (a) sample(s) may comprise(s) biological material of biopsies. The meaning of "biopsies" is known in the art. For instance, biopsies comprise cell(s) or tissue(s) taken, e. g. by the attending physician, from a patient/subject as mentioned herein. Exemplarily, but not limiting, the biological or medical sample to be analysed in context of the present invention is or is derived from blood, plasma, white blood cells, urine, semen, sputum, cerebrospinal fluid, lymph or lymphatic tissues or cells, muscle cells, heart cells, nerve cells, cells from spinal cord, brain cells, liver cells, kidney cells, cells from the intestinal tract, cells from the testis (e. g. Leydig cells and granulosa-lutein cells), cells from the urogenital tract, colon cells, skin, bone, bone marrow, placenta, amniotic fluid, hair, hair and/or follicles, stem cells (embryonal, neuronal, and/or others) or primary or immortalized cell lines (lymphocytes, macrophages, or cell lines). The biological or medical sample as defined herein may also be or be derived from biopsies, for example biopsies derived from testis tissue or from tissue of the urogenital tract.

As used herein, the term "LHR gene product" refers to any product resulting from expression of the LHR gene. Particularly, such products are the primary gene products also known as transcripts (comprising primary transcripts (pre- mRNA/hnRNA) and secondary transcripts (mRNA/mature mRNA)) as well as the secondary gene products also known as (poly-)peptides/proteins. The preferred "LHR gene product", to be employed in the context of the above described method of diagnosing is LHR mRNA (for example human LHR mRNA). However, it is generally envisaged that also other LHR gene products may be employed, for example the truncated LHR protein as provided and described herein or genomic LHR DNA.

An "LHR gene product comprising exon 6A" is, inter alia, envisaged to particularly refer to an LHR mRNA comprising a nucleotide sequence of exon 6A or to a truncated form of the LHR terminally comprising an amino acid sequence encoded by exon 6A. These and similar gene products of an LHR gene are exhaustively described herein above.

In a particularly preferred embodiment, the term "exon 6A" as used in the context of the method for diagnosing as described above refers to a certain variant of exon 6A, particularly a mutated form of exon 6A. This mutated form is preferably envisaged to carry one or more of the particular mutations as defined herein, more preferably one or more of the mutations corresponding to the mutations A21G or G22C of human exon 6A (depicted in SEQ ID NO: 1).

In a most preferred embodiment, the term "exon 6A" used with respect to a patient in which a disease is intended to be diagnosed refers to such a certain variant of exon 6A, whereas the term "exon 6A" used with respect to a control patient refers to a wild-type form of exon 6A not being such a certain variant. In the context of the present invention, the term "increased amount" of LHR gene products comprising exon 6A as used herein means a higher amount compared to the amount of a (healthy) control patient. Particularly, an "increased amount" in the context of the present invention may be an amount being at least 1.3 fold, at least 1.5 fold, at least 1.7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at last 4 fold, at least 5 fold, at least 6 or at least 10 fold higher, as compared to the amount of a control patient. Thereby, the higher values are preferred.

Based on his common general knowledge and the teaching provided herein, the skilled person is readily in the position to determine, whether the amount of LHR gene products comprising exon 6A is "increased" or not. For example, this determination can be put into practice by determining the amount of LHR gene products comprising exon 6A in a control patient or a group of control patients and, subsequently, compare the amount of LHR gene products comprising exon 6A as determined in a patient, in which a disease as defined herein is desired to be diagnosed, with the corresponding amount in the control (group of) patient(s).

The term "control patient" as used in the context of the present invention refers to a patient having a balanced relation between LHR gene products comprising exon 6A and LHR gene products lacking exon 6A, and hence, a proper functioning LHR and/or a normal endocrine profile. The skilled person is aware what a "normal endocrine profile" is. For example, an endocrine profile is "normal", when the levels of the reproductive hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), chorionic gonadotropin (CG) and/or testosterone, are within the normal range. For example, "normal range" in this context is the range of the level of a reproductive hormone that has been defined for healthy women and/or men. This range is known by the skilled person, and can, for example, be deduced by consulting clinical endocrine textbooks (like, e. g., Andrology Textbook by Nieschlag and Behre; Springer press 2.ed. 2001). Particular non limiting examples for the "normal range" of hormone levels of men is 2-10 U/l for LH, 1-7 U/l for FSH and >12 nmol/l for testosterone.

For example, an endocrine profile is not "normal", when the level of at least one of the above described hormones is above or below the above-defined ranges. A preferred "control patient" in the context of the present invention is a patient, preferably a healthy patient, that is age- and gender-matched with respect to the patient in which the herein defined disease is desired to be determined. Thereby, "age-matched" not only means the same age, for example in terms of at least % years, at least ^ΛA years, at least ³A years, at least 1 year, at least 2 years or at least 5 years, but also means being in the same stadium of development. For example, "stadium of development" in this context means stadium of an unborn baby, stadium of a newborn baby, stadium of a baby, stadium of an infant, stadium of a child, stadium of puberty/adolescence, stadium of an adult or stadium of aging, but, also, stadium of pregnancy or stadium of menopause. "Gender-matched" as used herein, means having the same sex, with respect to the phenotype and/or, preferably, with respect to the genotype.

In the context of the present invention, it is clear that the skilled person is readily in the position to find out how to determine the amount of gene products, particularly of the gene products as defined herein, and how to determine whether a patient exhibits a mutation or a polymorphism in exon 6A, by his common general knowledge and the teaching provided herein.

For example, if the gene product is a nucleic acid molecule like an mRNA, determination can be performed by taking advantage of northern blotting techniques or PCR techniques, like, for example, quantitative PCR techniques. These and other suitable methods for quantitating (specific) nucleic acid molecules like mRNA are well known in the art and are, for example, described in Sambrook and Russell (2001 , Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA). For example, if the gene product is a protein, determination can be performed by taking advantage of western blotting techniques, and the like. These and other suitable methods of quantitating (specific) proteins are also well known in the art and are, for example, also described in Sambrook and Russell (2001 , Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA). If a mutation or a polymorphism is to be detected in the context of the present invention, advantage can be taken of, for example, hybridizing techniques/PCR techniques using probes/primers representing the mutation or polymorphism to be detected or, for example, of sequencing approaches, e.g. nucleotide or protein sequencing approaches. These and other suitable methods of detecting a mutation or a polymorphism are also well known in the art.

In one particular embodiment of the method for diagnosing as described above, it is envisaged to take advantage of the binding molecule, preferably the antibody or antisense molecule as defined herein. Particularly the step of determining the amount of LHR gene products comprising exon 6A in a patient as performed in the context of the method of diagnosing of the present invention is envisaged to comprise the use of such binding molecules.

In a further embodiment, the present invention relates to a method for treating, ameliorating or preventing a disease which is diagnosed or diagnosable by the methods of diagnosing of the present invention. Said method for treating, ameliorating or preventing is envisaged to comprise the step of administering to a patient the pharmaceutical composition or the inhibitor (or enhancer) of exon 6A as provided and described herein.

Furthermore, the present invention relates to the use of the nucleotide sequences, the vector, the polypeptide, the host cell or the inhibitor of exon 6A as provided and described herein for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of a disease which is diagnosed or diagnosable by the method of diagnosing of the present invention.

In another embodiment, the present invention relates to a method for treating, ameliorating or preventing a disease which is characterized by an increased amount of LHR gene products comprising exon 6A (for example LHR mRNA comprising exon 6A). Said method for treating, ameliorating or preventing is envisaged to comprise the step of administering to a patient the pharmaceutical composition or the inhibitor of exon 6A as provided and described herein.

Additionally, the present invention relates to the use of the nucleotide sequences, the vector, the polypeptide, the host cell or the inhibitor of exon 6A as provided and described herein for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of a disease which is characterized by an increased amount of LHR gene products comprising exon 6A (for example LHR mRNA comprising exon 6A).

Generally, as referred to herein, a disease to be diagnosed or which is diagnosable by the method of diagnosing of the present invention, but also a disease to be treated, ameliorated or prevented in accordance with the present invention, may, without being bound by theory, any disease or disorder (particularly any DSD) coming along with a dysfunction of the LHR, and hence, also any disease due to a testosterone deficit. For example, such diseases may be diseases selected from the group consisting of: cryptorchidism, idiophatic hypogonadotrophic hypogonadism, late onset hypogonadism, hermaphrodism, male sterility, male impotence and prostate carcinoma. For example, such diseases are described in Ivell (Molecular Human Reproduction, 9, No. 4, 175-181 , 2003), Bhagavath (Fertil Steril. 2006, 85(3): 106-13), Sperling ("LOH (late onset hypogonadism) Oder "aging male"" in "Der Urologe", Springer Medizin Verlag 2006, online publication) and Themmen (Reproduction (2005), 30, 263-274). Also envisaged to be encompassed by the meaning of the term "disease or disorder coming along with a dysfunction of the LHR" is an improper preceding puberty.

In a particularly preferred embodiment of the present invention, the increased amount of LHR gene products comprising exon 6A, by which the disease in the context of the present invention is characterised, is caused by at least one mutation in exon 6A, particularly by at least such a mutation in exon 6A which corresponds to one of the particular nucleotide or amino acid exchanges as defined herein (for example the mutations A21C and C22C of human exon 6A (SEQ ID NO: 1)).

In the context of the present invention, it is also envisaged to diagnostically or therapeutically assess

(i) diseases associated with an impaired/improper function of the LHR and which come along with or are due to an enhanced activity of the LHR and/or an LH hypersensitivity; or (ii) diseases, the therapeutic assessment of which requires a reduction of the normal activity of the LHR and/or a reduction of the normal LH sensitivity.

"Enhanced activity of the LHR" in this context means that the activity of the LHR is higher than the normal activity of the LHR, i. e. the activity of the LHR in a (healthy) control patient. "Higher" in this context, for example, means at least 1 ,5 fold, at least 2 fold, at least 2,5 fold, at least 3 fold, at last 4 fold, at least 5 fold, at least 6 fold, at least 10 fold or at least 100 fold higher, as compared to the activity of the LHR in a control patient.

"LH hypersensitivity" in this context means that the sensitivity of the LHR is higher than the normal sensitivity of the LHR, i. e. the sensitivity of the LHR in a (healthy) control patient. "Higher" in this context, for example, means at least 1 ,5 fold, at least

2 fold, at least 2,5 fold, at least 3 fold, at last 4 fold, at least 5 fold, at least, at least 10 fold or at least 100 fold higher, as compared to the sensitivity of the LHR in a control patient.

"Reduction" of the normal activity of the LHR and/or a "Reduction" of the normal LH sensitivity in context of the above described diagnostically or therapeutically assessment, for example, means a reduction of at least 5%, at least 10%, at least

20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98% or at least

99% of the normal activity of the LHR or the normal LH sensitivity.

As mentioned above, "normal" activity of the LHR or "normal" LH sensitivity means the activity of the LHR or the LH sensitivity in a (healthy) control patient.

In a particularly preferred embodiment, the enhanced activity of the LHR and/or the LH hypersensitivity to be diagnostically or therapeutically assessed may be due to an decreased amount of LHR gene products comprising exon 6A. In this context, the term "decreased amount" of LHR gene products comprising exon 6A means a lower amount compared to the amount of a control patient. Particularly, an "decreased amount" in this context may be an amount being at least 1.3 fold, at least 1.5 fold, at least 1.7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at last 4 fold, at least 5 fold, at least 6 or at least 10 fold lower, as compared to the amount of a control patient.

Based on his common general knowledge and the teaching provided herein, the skilled person is readily in the position to determine, whether the amount of LHR gene products comprising exon 6A is decreased or not. For example, this determination can be put into practice by determining the amount of LHR gene products comprising exon 6A in a control patient or a group of control patients and, subsequently, compare the amount of LHR gene products comprising exon 6A as determined in a patient, in which a disease as defined herein is desired to be diagnosed, with the corresponding amount in the control (group of) patient(s). The definitions of the term "control patient" given herein-above with respect to the exon 6A antagonism/inhibition also apply here with respect to the exon 6A agonism/enhancement, mutatis mutandis.

In one particular aspect, the above embodiment relates to means and methods for agonizing/enhancing exon 6A, i.e. for enhancing the exon 6A function/activity.

Therefore, the present invention also relates to

(i) an enhancer/agonist of exon 6A (of the LHR gene);

(ii) a pharmaceutical composition comprising such an enhancer/agonist;

(iii) a method of diagnosing a disease in a patient, wherein said disease is characterized by an decreased amount of LHR gene products comprising exon 6A and/or an increased amount of LHR gene products lacking exon 6A;

(iv) a method for treating, ameliorating or preventing a disease which is diagnosable by the method of (iii); and

(v) a method for treating, ameliorating or preventing a disease which is characterised by an decreased amount of LHR gene products comprising exon 6A and/or an increased amount of LHR gene products lacking exon 6A.

The term "enhancer/agonist of exon 6A (of the LHR gene)" in this context refers to a compound that is capable to improve the generation of, or is capable to increase an decreased amount of, LHR splice variants (e.g. LHR mRNA) comprising exon 6A, and hence, capable to prevent/restore an imbalance between the amount of LHR splice variants comprising exon 6A and the amount of LHR splice variants lacking exon 6A.

For example, and without being bound by theory, an "enhancer/agonist of exon 6A" as referred to herein may be a compound that leads to the splicing of exon 6A into the maturing mRNA of the LHR, that leads the expression of an LHR gene having a mutation which results in an increased splicing of exon 6A into the LHR mRNA, that increases LHR mRNA comprising exon 6A, particularly truncated LHR mRNA terminally carrying exon 6A, or that leads to the generation of (i.e. the translation of) or that enhances the activity of a truncated LHR terminally carrying exon 6A (i.e. carrying the amino acid sequence encoded by exon 6A). Particular examples of an "enhancer/agonist of exon 6A" as employed herein are given below. In a preferred embodiment an "enhancer/agonist of exon 6A" as employed herein may be a nucleotide sequence, a polypeptide, a vector or a host cell of the present invention, particularly when these compounds refer to a form of exon 6A, the splicing into the LHR mRNA of which is increased.

In the context of the exon 6A agonism/enhancement as described herein, the term "increased amount" of LHR gene products lacking exon 6A as used herein means a higher amount compared to the amount of a control patient. Particularly, an "increased amount" in the context of the present invention may be an amount being at least 1.3 fold, at least 1.5 fold, at least 1.7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at last 4 fold, at least 5 fold, at least 6 or at least 10 fold higher, as compared to the amount of a control patient.

In the context of the exon 6A agonism/enhancement as described herein, the term "decreased amount" of LHR gene products comprising exon 6A as used herein means a lower amount compared to the amount of a control patient. Particularly, an "decreased amount" in the context of the present invention may be an amount being at least 1.3 fold, at least 1.5 fold, at least 1.7 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at last 4 fold, at least 5 fold, at least 6 or at least 10 fold lower, as compared to the amount of a control patient.

Without being bound by theory, the diseases to be assessed by enhancing/agonizing exon 6A are such diseases, where the balance between the amount of LHR gene products comprising exon 6A and the amount of gene products lacking exon 6A is disturbed in that the latter gene products are enhanced.

For example, such diseases may be diseases selected from the group consisting of polycystic ovary syndrome and breast cancer, and are described in Franks (Int J Androl. 2006, 29(1): 278-85) and Piersma (The Journal of Clinical Endocrinology & Metabolism, 91 (4): 1470-1476).

The definition of terms given herein-above with respect to exon 6A antagonism/inhibition also apply to the terms correspondingly used in the context of these embodiments referring to exon 6A agonism/enhancement. Without being bound by theory, the truncated form of LHR terminally comprising exon 6A may act as a soluble binding partner for the lutenizing hormone (LH), and thereby as a competitive inhibitor of the LHR, for example in the extracellular space, like, for instance, the blood serum.

Accordingly, in one specific, non-limiting aspect of the present invention, this truncated form of the LHR, or a variant or a fragment thereof (e.g. a variant or a fragment thereof still having the function/activity of said truncated form) is employed as a modulator, particularly as an antagonist/inhibitor of the LHR. This and other antagonistic/inhibitory function against the LHR can be reached by any approach agonizing/enhancing exon 6A of the LHR gene, for example an approach that leads to an increased splicing of exon 6A into the (maturing) mRNA. As a non limiting example, this splicing in can be obtained by introducing one or more nucleotide exchanges into the nucleotide sequence of wild-type exon 6A, for example nucleotide exchanges corresponding to A21C or G22C of the human exon 6A (SEQ ID NO:1). The skilled person is readily in the position to put this approach into practice and to find out further approaches how to agonize/enhance exon 6A based on his common general knowledge and the teaching provided herein.

In one particular aspect of the present invention the preferred subject/patient to be subjected to the herein provided means and methods for diagnosing, treating, ameliorating or preventing a disease as defined herein is a human being. For the herein provided means and methods for diagnosing, treating, ameliorating or preventing that take advantage of antagonize/inhibit exon 6A/exon 6A function, the preferred subject/patient is a male. For the herein provided means and methods for diagnosing, treating, ameliorating or preventing that take advantage of agonize/enhance exon 6A/exon 6A function, the preferred subject/patient is a female.

In a further preferred embodiment of the present invention, the term exon 6A as used in the context of the pharmaceutical composition, the methods or the uses of the present invention refers to an exon 6A or a variant or a fragment thereof that has a nucleotide sequence as provided and described herein above. These and other embodiments are disclosed and encompassed by the description and examples of the present invention. All of the publications, patents and patent applications referred to in the specification in order to illustrate the invention are hereby incorporated by reference in their entirety. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on the Internet, for example under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi. ch/biology/research_tools. html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.google.de. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

Furthermore, the term "and/or" when occurring herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1. Identification and characterization of exon 6A.

A. Upper part: Schematic representation of the composite internal/terminal exon 6A and its location in the LHCGR gene. The asterisk indicates the translational stop codon. 5'SS indicates the 5' splicing sites. Middle part: Structure of the LHCGR mRNA when exon 6A is acting as a terminal exon. Lower part: Structure of the LHCGR mRNA when exon6A is acting as an internal exon.

B. cDNA nucleotide sequence and putative amino acid sequence of exon 6A. The three different variants of exon 6A are indicated by the grey line (internal short and long exon 6A) and black line (terminal exon 6A). Stop codons are underlined and the poly-adenylation signal is boxed. The 3' and 5' splice sites are indicated by small arrows and the position of the identified mutations by bold arrows. Numbering of nucleotides is according to the LHR mRNA where the translational initiation codon ATG is considered is +1 (A).

Figure 2. Exon 6A is a bona fide exon.

Exon trap experiments with exon 6A. A genomic fragment of intron 6 of the human LHCGR gene including exon 6A and 150 bp upstream and downstream of it was cloned into the pSPL3 expression vector. COS7 cells were transiently transfected with this construct and RT-PCR performed. Primers for the detection of either internal exon 6A (A) or terminal exon 6A (B) are indicated by the arrows. A representative gel electrophoresis is shown below each expected PCR product. RT-PCR results of COS7 cells transfected with either the pSPL3 construct containing exon 6A (lane 1) or empty pSPL3 vector (lane 2) or untransfected COS7 cells (lane 3) are shown. RNA fidelity was confirmed by the detection of β actin mRNA. The results show that exon 6A is recognized as a bona fide exon and is spliced to gives rise to two internal and one terminal variant mRNA as predicted by the primary genomic sequence.

Figure 3. Tissue specific expression of exon 6A mRNA.

Quantification of LHCGR transcripts in different human tissues by TaqMan® RT- PCR. mRNA extracted from human testis and granulosa cells and from a commercially available adrenal gland preparation was analyzed. After normalization against 18S RNA, results were quantified taking the testis transcript without exon 6A as the reference, which was assigned an arbitrary unit (AU) value = 1. Results are shown as mean ± SEM of three independent experiments. The amplicon without exon 6A represents the transcript which results in the mature, full-length LHCGR protein. Transcripts containing the terminal exon 6A are highly expressed in all tissues analyzed and show a 6-fold higher expression compared to those without exon 6A in testis and adrenal gland. In comparison, the expression level of the internal exon 6A transcripts is much lower in every tissue.

Figure 4. Mutation and stop codons effect exon 6A mRNA expression.

Analysis of internal exon 6 A inclusion in exon trap experiments showing that removal of the two stop codons (stop) or the presence of the A557C mutation significantly increases inclusion of the internal exon 6A variants. Upper panel: The expression of the terminal and internal variants was analyzed after transfection in COS7 cells and RT-PCR of pSPL3 constructs containing the wild type (WT) exon 6A (along with 150 bp 5' and 3' of it: see Fig. 2) or exon 6A after removal of the two stop codons (stop) or the introduction of the A557C mutation. Lower panel: densitometric quantification (in arbitrary units, AU) of internal exon 6A variants RT-PCR results (both short and long variant together) of three to four independent experiments, as indicated in the columns. Results are expressed as mean ± SEM. p = 0.0294 (Kruskall-Wallis test). Results suggest that removal of the stop codons or a mutation in the SRp55 site reduces nonsense mediated decay of the internal exon 6A transcripts.

Figure 5. The A557C mutation highly increases the expression of the exon 6A short internal variant.

A 9.5 kb genomic DNA fragment encompassing exon 6 to exon 7 of the LHCGR gene from one patient carrying the A557C mutation and one control (wild type) was cloned into the pTarget expression vector and transiently transfected in COS7 cells. Expression of each variant was quantified by TaqMan® RT-PCR. Results of three independent experiments (mean ± SEM) are expressed in arbitrary units (AU) after relative quantification against the respective wild type variant which was arbitrarily given an AU value = 1.

Figure 6. Transcriptional network of the LHCGR gene.

Hypothetical model of the transcriptional network of the LHCGR gene including the novel exon 6A in physiological conditions (hollow arrows) or in case of mutation (filled arrows). The primary transcript of the LHCGR gene includes exon 6A₁ which can then be skipped in the mature mRNA giving rise to the full length LHCGR protein, or is spliced into the terminal or internal exon 6A variants. Since exon 6A contains a stop codon (asterisk), the terminal variant may be translated into a truncated LHCGR protein which remains mainly trapped within the cell and might hypothetically interact with the mature, full length protein. Conversely, the stop codon in the internal 6A variants is recognized by the nonsense mediated decay (NMD) pathway which actively eliminates these transcripts. Under physiological conditions, both the mature transcript without exon 6A and the terminal 6A variant are present in roughly equal amounts in the cells normally expressing the LHCGR (hollow arrows of equal size). In case of mutation(s) of exon 6A, the NMD surveillance mechanism is probably inadequate to eliminate the internal exon 6 variants, which accumulate in the cell and alter the relative abundance of the various transcripts, possibly resulting in an excessive increase of truncated LHCGR protein incompatible with "normal" receptor function.

Figure 7.

A. Upper part: Genomic and cDNA nucleotide sequence comparison of exon 6A from human (H. sapiens), chimpanzee (P. troglydytes), cynomolgus monkey (M. fascicularis), marmoset (C. jacchus), lemur (E. coronatus). Identical nucleotides are indicated by an asterisk. Sequences encompassing internal exon 6A are indicated by the grey line, while terminal exon 6A is indicated by a black line. Typical exon elements are given in bold italics or are boxed (polyA signal). Lower part: Comparison of the deduced amino acid sequence of exon 6A

B. Evolutionary pedigree of primate lineage evolution after Singer (J. MoI. Biol. 341 : 883-6, 2004). The approximate time of DNA insertion is indicated by the arrows. Time scale in million of years (mya)

C. Revised organization of the genomic region between exon 6 and 7 of the human, monkey and mouse LHCGR gene. The exonic size is given above and the intronic size below the genomic elements.

Figure 8.

Revised complete genomic organization of the human LHCGR gene now consisting of 12 exons. Exon sizes are given above the boxes, intron sizes below.

Figure 9. lmmunfluoresence localization of HA-tagged LHCGR proteins in transiently transfected COS7 cells. A: HA-tagged, wild type terminal exon 6A LHCGR. B: HA- tagged, A557C terminal exon 6A LHCGR. C: full-length, HA-tagged LHCGR protein. Left column: permeabilized cell, right column: non-permeabilized cells Both truncated LHCGR variants, wild type and mutated, show a predominantly intracellular localization (left and right column A and B), while the full-length receptor is localized at the cell membrane (right columc C).

Figure 10.

A. Sequencing results of the compound heterozygous patient displaying the exon 6A and exon 11 mutations.

B. TaqMan® RT-PCR quantification of internal exon 6 A inclusion in exon trap experiments using the wild type or G558C exon 6A constructs. The expression of internal variants was analyzed after transfection in COS7 cells and RT-PCR of pSPL3 constructs containing the wild type (WT) exon 6A (along with 150 bp 5' and 3' of it: see Fig. 2) or exon 6A after introduction of the G558C mutation as described in Fig. 4. Densitometric quantification (in arbitrary units, AU) of internal exon 6A variants RT-PCR results (both short and long variant together) of two independent experiments. Results are mean ± SEM.

Figure 11.

Graphic representation of the distribution of the T748G (T212G) SNP in a group of healthy fertile men and a group of patients suffering from maldescendent testes. Given is number of patients which are homozygous T or G and the number of patients being heterozygous at this position (N). Significant differences were observed in the group of patients displaying the T genotype.

The Examples illustrate the invention.

Example 1 :

Patients and Methods

Patients:

Sixteen patients with complete LH resistance (male pseudohermaphroditism) due to

Leydig cell hypoplasia type 1 gave written informed consent for the study and were investigated. All patients had karyotype 46,XY, a blind ending vagina, no breast development and testicular structures in the abdomen (Richter-Unruh et al., 2002).

All patients had been previously screened for LHCGR mutations and found to be negative. Control group:

Forty-one fertile men with normal LH and testosterone levels and normal spermatogenesis were included as a control group. The institutional review board approved the study and written informed consent was obtained from each participant.

Case reports:

A 20-year-old Turkish woman was referred to the gynaecologist because of primary amenorrhea. She was the first daughter of a sib ship of three children from consanguineous parents. She presented with a lack of breast development, normal pubic and axillary hair, blind ending vagina (4 cm), and normal labia majora and minora. No clitoris hypertrophy was seen. She was obese (122 kg, 173 cm). Testes were palpable in the inguinal regions, no mullerian derivates were found by ultrasound. Karyotyping was performed and revealed a 46,XY karyotype. Hormone analysis showed verylow serum testosterone levels (0.1 nmol/l) which could be not stimulated by hCG treatment (maximal testosterone levels: 0.1 nmol/l). Serum levels of LH and FSH were elevated (LH 15.8 IU/I, FSH 13.6 IU/I) and responsive to stimulation with GnRH (LH 69.8 IU/I, FSH 20.1 IU/I).

At the age of 26 years her sister, younger by 6 years, was referred to the Department of Endocrinology of the University of Essen also because of primary amenorrhea. She presented exactly the same phenotype as her older sister: 46, XY karyotype, blind ending vagina (6 cm), inguinal gonads, obesity (126 kg, 176 cm), low estradiol level (21 pg/ml), low testosterone levels (0.06 nmol/l, increasing to 1.9 nmol/l after 7500 IE hCG), elevated level of gonadotropins (LH 24.3 IU/I, FSH 20.1 IU/I) responsive to GnRH (LH 98.0 IU/I, FSH 29.6 IU/I). Gonadectomy was performed and histological evaluation revealed complete Sertoli-cell-only syndrome with thickened tubule walls and focally disorganized tubules. Leydig cells appeared immature and their number was not evidently changed when compared to a normal testis. In both affected sisters the previously performed genetic analysis of the LHCGR gene did not give any hint of mutations or other genetic alterations in other candidate genes.

A third patient was analyzed at the age of 21 months. She was born from unrelated parents and presented female phenotype with labial synechia. Gonads were palpable in both labia majora and a karyotype 46,XY was found. Postnatal testosterone levels were low and the girl was gonadectomised at the age of two month. Histology showed fibrotic testis tissue. A heterozygous mutation leading to a Thr461 lle substitution in exon 11 of the LHCGR gene, resulting in complete inactivation (data not shown), was found.

Cells and tissues:

Human testis tissues were obtained from patients undergoing orchidectomy because of prostate cancer, while granulosa cells were obtained from women undergoing assisted reproduction. Written consent was obtained from the patients to use their material for scientific research. In addition, human adrenal and testis RNA was purchased from commercial sources (Biocat, Heidelberg, Germany)

Testis material:

Snap frozen testes were obtained from different primate species within the frame of a project on efficacy of spermatogenesis funded by the German Research Foundation (Gromoll, Bio.l Reprod. 69: 75-80, 2003; WE1167/4-1/2; HO1391/4-1). Tissue from the cynomolgus monkey (Macaca fascicularis) was obtained from primate colonies (Gromoll, 2003, Biol Reprod 69:75-80). All experiments were conducted according to the German Law on Animal Care and Experimentation. The cynomolgus cDNA testis library which was screened for the LHCGR cDNA has been described elsewhere (Gromoll, Hum. MoI. Genet. 8:2017-24, 1999).

Genomic DNA and RNA isolation:

Genomic DNA was isolated from EDTA blood samples using the Flexigene DNA DNA isolation kit (Hilden, Germany). RNA was isolated using Ultraspec (Biotecx, Houston, TX).

Exon trap experiments:

Analysis of exon 6A was performed using the exon trapping system (Invitrogen, Karlsruhe, Germany). Plasmid constructs were generated by amplification of a DNA fragment from human genomic DNA using the specific primers Ex6a 150 fw. 5^'- CGCTCGAGCCTGCCCTCCTCGGCCTCCCAAAG-3^' (SEQ ID NO: 22) and LHCGR Ex6a 150 rev: 5^'-CGCGGATCCCTTTATAAGCAGCCGGTAGAGCTG-S^' (SEQ ID NO: 23) containing the restriction site Xho1 within the forward primer or BamH1 for the reverse primer. The fragment was amplified using the following PCR program: 94°C 50 sec, 64⁰C 50 sec, 72° 1.30 min for 35 cycles. The obtained amplicon was restricted by Xho1 and BamH1 and cloned into the pSPL3 vector. Sequence fidelity was confirmed by DNA sequencing

Transient transfection and RT-PCR:

COS-7 cells were seeded on Petri-dishes and cultured to 40-50 % confluency. 12 μg plasmid DNA/ dish was used for transfection by lipofectamine (Invitrogen, 12μL/dish). 6h later the transfection was stopped by adding DMEM- medium supplemented with 20% FCS. 48 h later the cells were lyzed using Ultraspec (Biotecx, Houston, TX) and RNA isolated according to the manufacturer's protocol. Reverse transcription was performed using the vector specific primers SA2 (δ^'-TCTGAGTCACCTGGACAACC- 3^'; SEQ ID NO: 24) exon 6A rev (δ^'-GTAACATGACAATTATACATG-S^'; SEQ ID NO:

25) or β actin primer as a control for RNA integrity. Subsequent PCR was performed with the primer combination SD6 (δ^'-ATCTCAGTGGTATTTGTGAGC-S¹; SEQ ID NO:

26) /SA2 or SD6/exon 6A rev and β actin for/rev using the following program: 94°C for 50 sec, 60⁰C for 50 sec, 72°C for 1.30 min for 35 cycles. The PCR reactions were subjected to 2% agarose gel-electrophoresis and documented using the Multi-Image Light system (Biozym, Oldendorf, Germany)

In-vitro mutagenesis:

Mutations were introduced using the Quick Change site-directed mutagenesis kit from Stratagene, Heidelberg Germany. Sequence fidelity was confirmed by DNA sequencing.

Real-time quantitative PCR for the relative gene expression of LHCGR variants: The commercially available TaqMan® assays #HS20896337_m1 (LHCGR; Applied Biosystems, Darmstadt, Germany) was used to detect the known LHCGR transcripts (without exon 6A). For the relative quantification of the LHCGR terminal exon 6A and internal exon 6A variants specifically designed assays were used. The terminal exon 6A assay is directed towards the 3' region of exon 6A, while the internal exon 6A variants are detected by an assay spanning the 5'region of exon 6A to exon 7 of the LHCGR gene. The primers and probes of the assays designed ad hoc are listed below.

LHCGR terminal exon 6A: for. primer S^'-CAGAGGACTCTCTTTTATATCACTGGATTC-a^' (SEQ ID NO: 27) rev. primer δ^'-TGGTCACAGCTTTGTAACATGACAA-S^' (SEQ ID NO: 28)

TaqMan® MGB FAM-labelled probe: δ^'-ACCAAGGATACCAATTTT-S^' (SEQ ID NO:

29)

LHCGR internal exon 6A short: for primer δ'-CTCTGAAATGAAGAGATAGATGTGAAGCA-S' (SEQ ID NO: 30) rev primer δ'-GCATGACTTTGTACTTCTTCAAATCCAT-S' (SEQ ID NO: 31) TaqMan® MGB FAM-labelled probe: δ'-TTCCATATAGTTTGCAATTTT-S' (SEQ ID NO: 32)

LHCGR internal exon 6A long: for primer 5'-CAGAGGACTCTCTTTTATATCACTGGATTC-S' (SEQ ID NO: 33) rev primer δ'-TGACTTTGTACTTCTTCAAATCCATTTCCA-S' (SEQ ID NO: 34) TaqMan® MGB Fam-labelled probe 5'-ACTGCCTTTGTATAGTACTTTTA-S' (SEQ ID NO: 35)

LHCGR exon6/7 without exon 6A: for primer 5'-ACCACCATACCAGGAAATGCTTTT-S' (SEQ ID NO: 36) rev primer δ'-AAAGATTCAGTGTCGTCCCATTGA-S' (SEQ ID NO: 37)

TaqMan® MGB Fam-labelled probe δ'-CAAGGGATGAATAATGAATCTGT-S' (SEQ

ID NO: 38)

Reverse transcription of 2 μg total RNA/tissue was performed using random hexamer primers and Superscript Il enzyme according to the manufacturer's protocol (Invitrogen, Karlsruhe, Germany).

TaqMan® PCR was performed using the following amplification profile: an initial step consisting of 50⁰C for 2 min and 95° for 10°min followed by 40 cycles at 95°C for 15 sec and 6O⁰C for 1 min. PCR efficiency was verified with different cDNA concentrations yielding linear amplification. For the detection of the LHCGR variants 200ng cDNA/reaction were used

The relative quantification of gene expression was analyzed according to Livak et al. (Livak, Methods 25: 402-408, 2001) using the 2^^CT method. Normalization of RNA content was performed using 18S TaqMan® gene expression assay (Hs # 99999901 ; Applied Biosystems). The mRNA levels of the known LHCGR (without exon 6A) in the testis tissue were used to calibrate by relative quantification the expression levels of the other transcripts.

LHCGR mini-gene construct:

The genomic region encompassing exon 6, exon 6A and exon 7 with a total length of 9534 bp was amplified from genomic DNA from one patient and one normal healthy male using the Expand Long PCR kit by Roche (Mannheim Germany). The following two primers have been used (exon 6 for 5'- GTGATAACTTACACATAACCACCATACCAGG-3' (SEQ ID NO: 39) and exon 7 rev. 5'-GTCAGTGTCGTCCCATTGAATGCATGAC-3^I (SEQ ID NO: 40)) applying the PCR conditions recommended by the manufacturer. The obtained amplicons were cloned into the pTarget expression plasmid (Promega, Germany) and the sequence fidelity confirmed by DNA sequencing.

Protein expression of the full-length LHCGR and of the LHCGR terminal exon 6A variant:

The protein expression studies of LHCGR exon 6A were performed using a human HA-tagged, full length LHCGR cDNA cloned into the pcDPS expression plasmid (kindly provided by Dr. T. Gudermann, University of Marburg, Germany). The HA tag is directly located after the LHCGR signal peptide and can be visualized by immunofluorescence using a HA specific antibody (sc 7392 , Santa Cruz, CA). The LHCGR exon 6A variant was generated by a restriction enzyme-based cloning strategy, using Hind III and Spe1 , thereby removing most of the LHCGR cDNA except the most N-terminal part, which subsequently was joined to an LHCGR exon 6A variant treated with the same restriction enzymes beforehand. Both constructs were transiently transfected into COS-7 cells and immunofluoresence was detected by using the HA antibody followed by a monoclonal lgG2a antibody (Sigma, Deisenhofen, Germany) and fluorescence was imaged by an Zeiss Axioscope microscope.

Sequence analysis:

Sequence alignments were performed using the ClustalW programme (http://www.ebi.ac.uk/Tools/). Exon splicing binding sites were identified using the exon splicing enhancer (ESE) finder software (http://rulai.cshl.edu/tools/ESE/). Other searches were performed using the toolbox of the Alternative Splicing Database project (http://www.ebi.ac.uk/asd/). The chimpanzee LHCGR sequences were extracted from www.ncbi.nlm.nih.gov/qenome database under

>ref/NW_103889_12/Ptr2a_WGA2400_1.

Example 2:

A human testis cDNA library and human granulosa cell mRNA was interrogated for LHCGR transcripts. Two novel LHCGR mRNA variants were cloned, both consisting of exon 1-6 followed either by an additional, unknown terminal sequence ending with a poly-A tail, or by part of such a sequence continuing with exons 7-11 (Fig.1A). A BLAST search yielded a perfect match to the intronic region between exon 6 and 7 of the human LHCGR gene (ID 3973; from nucleotide position 33900 to 34200). Inspection revealed the presence of a 3' splice acceptor site (AG) and a 3' poly adenylation signal (AATAAA) indicative of a terminal exon (Sheets, Nucleic Acids Res. 18: 5799-805, 1990). Moreover, two internal 5' splice sites were evident which, together with the 3' acceptor site, give rise to a 159 bp (short) or to a 207 bp (long) internal exon (Fig.1 B). Therefore, the human LHCGR gene contains a previously unrecognized, additional, putative exon within intron 6 which can be spliced into the novel LHCGR transcripts following two different routes. This new exon was designated as exon 6A.

Example 3: Exon trap experiments with the human genomic region surrounding exon 6A yielded two transcripts including either 159 bp (short ) or 207 bp (long) of the internal exon 6A, indicating specific splicing (Fig. 2A, lane 1). A different primer set revealed the expression of the terminal exon 6A transcript as well (Fig. 2B, lane 1). Thus, exon 6A is a novel bona fide composite internal/terminal exon within the LHCGR gene. The expression pattern of the known LHCGR mRNA and of the newly discovered transcripts was analyzed by realtime RT-PCR in human tissues. As shown in Fig. 3, high mRNA levels including exon 6A were detected in granulosa cells and in the testis, where the terminal variant expression is about 5 times higher than that of the known, mature mRNA without exon 6A. In addition, low levels of exon 6A transcripts were consistently detected in adrenal tissues. mRNA levels for the two internal exon 6A mRNA variants were significantly lower compared to the known LHCGR without exon 6A (Fig. 3).

Example 4:

The genomic region including exon 6A is perfectly conserved in the chimpanzee. Moreover, the corresponding genomic region in the macaque, marmoset monkey and lemur as representative species of the principal primate lineages were cloned and sequenced (Gromoll, Bio.l Reprod. 69: 75-80, 2003). Both the internal and the terminal exon 6A are conserved in these primates but are absent in rodents (Fig. 7). Therefore this novel exon is confined to primates, where it is highly conserved, indicating strong functional constraints. These data suggest that the genomic organization of the LHCGR gene should be revised to include exon 6A and that the mRNA transcript without exon 6A encoding for the known LHCGR protein represents a splicing variant of the revised LHCGR gene (Fig. 8). It is tempting to speculate that the appearance of exon 6A in the primate LHCGR gene is connected with the appearance of CG and its function in pregnancy recognition and maintenance (Maston, MoI. Biol. Evol. 19: 320-35. 2002; Singer, J. MoI. Biol. 341 : 883-6, 2004).

Example 5:

Exon 6A maintains phase 2 splicing typical of the LHCGR gene. However, it contains two stop codons (Fig 1) and encodes 30 amino acids (pi = 4.5) with no significant sequence homology to any other known protein by FASTA and BLAST search. The incorporation of either terminal or internal exon 6A into the transcripts results in a truncated LHCGR protein of 209 amino acids. The expression of such protein using a HA-tagged LHCGR-exon 6A vector in transiently transfected COS7 cells revealed a predominantly cytosolic localization (Fig. 9). The presence of stop codons qualifies the LHCGR transcript containing the internal exon 6A as a target for nonsense mediated decay (NMD), a recently described protein-mediated surveillance mechanism that selectively degrades nonsense mRNAs thereby regulating protein expression (Nakamura, MoI. Endocrinol. 18:, 1461-70, 2004). Indeed, elimination of the stop codons by in-vitro mutagenesis significantly increased the internal exon 6A inclusion rate in transiently transfected COS7 cells (Fig. 4) Similar results were obtained when transfected cells were treated with cycloheximide, a strong protein synthesis inhibitor, for 4 h (data not shown). These results indicate that exon 6A might function as novel regulator of LHCGR mRNA levels and transcript pattern. Upon inclusion of the internal exon 6A, a NMD mediated process results in degradation of the LHCGR transcript, while inclusion of terminal exon 6A may result in an intracellular, truncated LHCGR protein. Whether the truncated 209 amino acid protein occurs naturally in testis and ovary, remains presently unknown.

Example 6:

16 clinically well characterized patients with the overt phenotype of LCH and no mutations in LHCGR gene for mutations in exon 6A were screened. Apart from several SNPs present also in an appropriately sized control group (Table 1), three patients from two families with mutations of exon 6A were identified. In two sisters with LCH a homozygous A to C mutation at position 557 within exon 6A was found, leading to an amino acid change from GIu (GAG) to Ala (GCG) (Fig. 1 B). The two patients were from consanguineous parents, who carried the same heterozygous mutation. Immunofluorescence analysis of the mutated LHCGR terminal exon 6A protein variant was uninformative (Fig. 9). A heterozygous G to C transversion at position 558 of exon 6A was found in another patient with LHC from an independent family who was concomitantly carrier of a heterozygous inactivating mutation in exon 11 of the LHCGR. The two mutations were located on different alleles (compound heterozygosis) (Fig. 10). According to the exon splicing enhancer finder (ESE) software, the A557C mutation strengthens and the G558C mutation abolishes a binding site for the splicing factor SRp55 (consensus sequence TGCGTC, mutation sites underlined). The effects of the mutations on the LHCGR transcript pattern were further analyzed in vitro. Exon trap experiments revealed a 4-fold higher expression level of the A557C exon 6A (Fig. 4) and a 6-fold increase of the A558G (Fig. 10) internal variants compared to wild type. The entire LHCGR genomic region from exon 6 to exon 7 from one patient with the A557C exchange and from one control were cloned and expressed in COS7 cells and the transcript pattern quantified by realtime RT-PCR. The mutation did not induce any change in the expression level of the transcript without exon 6a (Fig. 5). However, the transcript containing the mutated, terminal exon 6A showed 2.5-fold higher levels compared to the wildtype. The long internal exon 6A variant was about 5-fold more expressed while for the short internal exon 6A variant the expression level changed dramatically, with about 3000-fold increase compared to the wildtype (Fig. 5). These data suggest that the mutation in exon 6A specifically alters the splicing pattern of the LHCGR gene with a drastic increase of transcripts not contributing to the generation of the mature, full-length receptor protein. This dramatic change probably engulfs the NMD machinery and/or alters the pattern of protein translation and transportation to the cell surface resulting in an insufficient amount of full-length, mature LHCGR protein at the cell surface.

The resulting phenotype, i.e. LCH and 46,XY DSD of exon 6A mutations is illuminating and supportive for a strong regulatory function of exon 6A. It is proposed that a complex network including the novel transcripts described herein and involving NMD regulates the LHCGR expression both at the transcriptional and translational level (Fig. 6). The spectrum of LHCGR transcripts includes forms with and without exon 6A, but only the LHCGR mRNA transcript lacking exon 6A will lead to a functional protein. The levels of such a "normal" transcript and its translation probably depend on the correct function of two distinct regulatory avenues processing the LHCGR mRNA including exon 6A. On one side the variant in which exon 6A acts as terminal exon may give rise to an intracellular protein potentially capable of hormone binding and/or of interaction with the mature LHCGR protein. On the other side the correct processing of the LHCGR transcripts without exon 6A may depend on the elimination by NMD of the variants including the internal exon 6A. This complex pattern of LHCGR transcripts form a network which, considering the strong physiological constraints of LH/CG action, must be tightly regulated. It is postulated that the strict regulation of this network is crucial for establishing the correct ratio between the transcripts which, in turn allows proper LHCGR functioning. This view is supported by the fact that the internal and terminal exon 6A transcripts are physiologically detected along with the 'normal' LHCGR mRNA in testis and granulosa cells. Should exon 6A be mutated, the NMD machinery might be unable to process the transcripts efficiently and, if a substantial part of them is translated, the functional LHCGR protein will be competing with the truncated LHCGR protein derived from terminal and internal exon 6 A variants (Lewis, PNAS 100: 189-192, 2003). Via dimerization of the full-length LHCGR with the truncated exon 6A variant, this might result in improper receptor trafficking (Park, PNAS 102:, 8793-8794, 2005).

Example 7:

Single nucleotide polymorphism in the LHCGR exon 6A

Upon screening of exon 6A in the 16 patients, three single nucleotide polymorphism (SNP) were identified. The first one represents a non-synonymous SNP at nucleotide position 599 (starting from exon 1 , rs 4637137, SNP database) of the LHCGR changing ATG (Met) into ACG (Thr). The other two SNPs are located at nt position A653G (rs 4490239, SNP database) and at nt position T748G (not found in the SNP database) within the non-coding region of exon 6A. Determination of the different SNPs in a group of patients suffering from testis maldescensus (n= 33) and in a cohort of 41 healthy males revealed significant differences concerning the distribution of the previously not described T748G SNP (see Fig. 11) in the patient group. The other SNPs T599C and A653G appear to be in linkage disequilibrium (data not shown).

Example 8:

Further studies on patients lacking "classical" genetic alterations in the LHCGR will shed new light on the pathophysiological role of exon 6A in LH/CG function and in clinical conditions of LH resistance. The present invention refers to the following nucleotide and amino acid sequences: SEQ ID No. 1 :

Nucleotide sequence of exon 6A of the human LHCGR gene (complete, but lacking poly-A tail):

CTCTGAAATGAAGAGATAGATGTGAAGCAAAAGAAGAGATCATCTCAGAGGACTCTCTTTTATATCACTGGATTC

TTGTCATGTTACAAAGCTGTGACCATGTAGCTTCATCTCAGATTAATGCAATCAAAAGAATACAGAGCAGCAACC

SEQ ID No. 2:

Nucleotide sequence of exon 6A of the human LHCGR gene (long splice variant):

CCCATGGCAAAATTGTGATGAGGCAATAAAGGAGCTCACCCTTAAAGAAAAAAGAGAAAACATGGATTGGAATGA CTCTGAAATGAAGAGATAGATGTGAAGCAAAAGAAGAGATCATCTCAGAGGACTCTCTTTTATATCACTGGATTC TAAAAATTGGTATCCTTGGTGCACTGCCTTTGTATAGTACTTTTACTTTGTGTAGAT

SEQ ID No. 3:

Nucleotide sequence of exon 6A of the human LHCGR gene (short splice variant):

CTCTGAAATGAAGAGATAGATGTGAAGCAAAAGAAGAGATCATCTCAGAGGACTCTCTTTTATATCACTGGATTC TAAAAATTG

SEQ ID No. 4:

Nucleotide sequence of exon 6A of the human LHCGR gene (coding region):

CCCATGGCAAAATTGTGATGAGGCAATAAAGGAGCTCACCCTTAAAGAAAAAAGAGAAAACATGGATTGGAATGA CTCTGAAATGAAGAGATAG

SEQ ID No. 5:

Nucleotide sequence of truncated human LHCGR ending with complete exon 6A

(exons 1 to exon 6A (complete, but lacking poly-A tail):

1 ATGAAGCAGCGGTTCTCGGCGCTGCAGCTGCTGAAGCTGCTGCTG

46 CTGCTGCAGCCGCCGCTGCCACGAGCGCTGCGCGAGGCGCTCTGC

91 CCTGAGCCCTGCAACTGCGTGCCCGACGGCGCCCTGCGCTGCCCC 136 GGCCCCACGGCCGGTCTCACTCGACTATCACTTGCCTACCTCCCT 181 GTCAAAGTGATCCCATCTCAAGCTTTCAGAGGACTTAATGAGGTC 226 ATAAAAATTGAAATCTCTCAGATTGATTCCCTGGAAAGGATAGAA 271 GCTAATGCCTTTGACAACCTCCTCAATTTGTCTGAAATACTGATC 316 CAGAACACCAAAAATCTGAGATACATTGAGCCCGGAGCATTTATA 361 AATCTTCCCCGATTAAAATACTTGAGCATCTGTAACACAGGCATC 406 AGAAAGTTTCCAGATGTTACGAAGGTCTTCTCCTCTGAATCAAAT 451 TTCATTCTGGAAATTTGTGATAACTTACACATAACCACCATACCA 496 GGAAATGCTTTTCAAGGGATGAATAATGAATCTGTAACACTCCCA 541 TGGCAAAATTGTGATGAGGCAATAAAGGAGCTCACCCTTAAAGAA 586 AAAAGAGAAAACATGGATTGGAATGACTCTGAAATGAAGAGATAG 632 atgtgaagcaaaagaagagatcatctcagaggactctcttttatat 678 cactggattctaaaaattggtatccttggtgcactgcctttgtata 724 gtacttttactttgtgtagatgtaattttacatgtataattgtcat 770 gttacaaagctgtgaccatgtagcttcatctcagattaatgcaatc 816 aaaagaatacagagcagcaacc

SEQ ID No. 6:

Nucleotide sequence of human LHCGR internally comprising the long splice variant of exon 6A (The START codon is underlined):

ACTCAGAGGCCGTCCAAGACACTGGCAAGCCGCAGAAGCCCAGTTCGCCGGCCATGAAGCAGCGGTTCTC

GGCGCTGCAGCTGCTGAAGCTGCTGCTGCTGCTGCAGCCGCCGCTGCCACGAGCGCTGCGCGAGGCGCTC

TGCCCTGAGCCCTGCAACTGCGTGCCCGACGGCGCCCTGCGCTGCCCCGGCCCCACGGCCGGTCTCACTC

GACTATCACTTGCCTACCTCCCTGTCAAAGTGATCCCATCTCAAGCTTTCAGAGGACTTAATGAGGTCAT

AAAAATTGAAATCTCTCAGATTGATTCCCTGGAAAGGATAGAAGCTAATGCCTTTGACAACCTCCTCAAT

TTGTCTGAAATACTGATCCAGAACACCAAAAATCTGAGATACATTGAGCCCGGAGCATTTATAAATCTTC

CCCGATTAAAATACTTGAGCATCTGTAACACAGGCATCAGAAAGTTTCCAGATGTTACGAAGGTCTTCTC

CTCTGAATCAAATTTCATTCTGGAAATTTGTGATAACTTACACATAACCACCATACCAGGAAATGCTTTT

CAAGGGATGAATAATGAATCTGTAACACTCCCATGGCAAAATTGTGATGAGGCAATAAAGGAGCTCACCC

TTAAAGAAAAAAGAGAAAACATGGATTGGAATGATCTGAAATGAAGAGATAGATGTGAAGCAAAAGAAGA

GATCATCTCAGAGGACTCTCTTTTATATCACTGGATTCTAAAAATTGGTATCCTTGGTGCACTGCCTTTG

TATAGTACTTTTACTTTGTGTAGATCAAACTATATGGAAATGGATTTGAAGAAGTACAAAGTCATG

CATTCAATGGGACGACACTGACTTCACTGGAGCTAAAGGAAAACGTACATCTGGAGAAGATGCACAATGG

AGCCTTCCGTGGGGCCACAGGGCCGAAAACCTTGGATATTTCTTCCACCAAATTGCAGGCCCTGCCGAGC

TATGGCCTAGAGTCCATTCAGAGGCTAATTGCCACGTCATCCTATTCTCTAAAAAAATTGCCATCAAGAG

AAACATTTGTCAATCTCCTGGAGGCCACGTTGACTTACCCCAGCCACTGCTGTGCTTTTAGAAACTTGCC

AACAAAAGAACAGAATTTTTCACATTCCATTTCTGAAAACTTTTCCAAACAATGTGAAAGCACAGTAAGG

AAAGTGAATAACAAAACACTTTATTCTTCCATGCTTGCTGAGAGTGAACTGAGTGGCTGGGACTATGAAT

ATGGTTTCTGCTTACCCAAGACACCCCGATGTGCTCCTGAACCAGATGCTTTTAATCCCTGTGAAGATAT

TATGGGCTATGACTTCCTTAGGGTCCTGATTTGGCTGATTAATATTCTAGCCATCATGGGAAACATGACT

GTTCTTTTTGTTCTCCTGACAAGTCGTTACAAACTTACAGTGCCTCGTTTTCTCATGTGCAATCTCTCCT

TTGCAGACTTTTGCATGGGGCTCTATCTGCTGCTCATAGCCTCAGTTGATTCCCAAACCAAGGGCCAGTA

CTATAACCATGCCATAGACTGGCAGACAGGGAGTGGGTGCAGCACTGCTGGCTTTTTCACTGTATTCGCA

AGTGAACTTTCTGTCTACACCCTCACCGTCATCACTCTAGAAAGATGGCACACCATCACCTATGCTATTC

ACCTGGACCAAAAGCTGCGATTAAGACATGCCATTCTGATTATGCTTGGAGGATGGCTCTTTTCTTCTCT

AATTGCTATGTTGCCCCTTGTCGGTGTCAGCAATTACATGAAGGTCAGTATTTGCTTCCCCATGGATGTG

GAAACCACTCTCTCACAAGTCTATATATTAACCATCCTGATTCTCAATGTGGTGGCCTTCTTCATAATTT

GTGCTTGCTACATTAAAATTTATTTTGCAGTTCGAAACCCAGAATTAATGGCTACCAATAAAGATACAAA

GATTGCTAAGAAAATGGCAATCCTCATCTTCACCGATTTCACCTGCATGGCACCTATCTCTTTTTTTGCC

ATCTCAGCTGCCTTCAAAGTACCTCTTATCACAGTAACCAACTCTAAAGTTTTACTGGTTCTTTTTTATC

CCATCAATTCTTGTGCCAATCCATTTCTGTATGCAATATTCACTAAGACATTCCAAAGAGATTTCTTTCT

TTTGCTGAGCAAATTTGGCTGCTGTAAACGTCGGGCTGAACTTTATAGAAGGAAAGATTTTTCAGCTTAC

ACCTCCAACTGCAAAAATGGCTTCACTGGATCAAATAAGCCTTCTCAATCCACCTTGAAGTTGTCCACAT

TGCACTGTCAAGGTACAGCTCTCCTAGACAAGACTCGCTACACAGAGTGTTAACTGTTACATCAGTAACT GCATTATTGAATTGTTCTTAAACCTGTAAAAAAAAATTACCTGTACCAGTAATTTTAACATAAAGGGTTG GATTTAGGAAATTATTTATTTTTAGGTACATTAGGCAAGAGACCTCTACCTAGTAGAAAGTGTAGTCTAT GACCACTGCCACACTAAAAACTATTTGTCATTGTTACATGGCATAAATACTGAAGTTGAGAGTGTTTAGA AATTTTTATAGAAATTTTGACACAGTAATTTTGTTTGATGAATCTTTTAAAAAACTGAGGAGGTATTTTG CATATCTTTTTTTTCATTTTCGTAATTTGTATTGCATTCTATAAAAATATTAGTTCATAACAGATCAGAA ATTTAAAATAACTGGCCTTTTTCCTCAGGTAGTTTGAAAAACACACTCTAGAGATGCACTGTCCAATCCG GTAGCCACTAGCCACATGTGGCTAAATTAAAATTAAATAAAATGAGAAATGTAGTTTCTCAGTTGCACTA GCCACGTTTCAAGTTCTCAATGGCTACGTGTGACTAGTGCTTACCATACTGGACAGCACAGACACAGAAT ATTTTCATCACCACAGAAAGTTCTATCTGTTCTATTATAGAGACTTTTATCTATGCCCTATCTGGATTCT ACTTATTTATAATTTAAGGTAAACATCTGAAAGCACATTTCAGCCTATTTGCTTAGTGAAACATTAAGCT GTAGACTGTAAACTCCTCGTGAGTAGGAACCCTGTCTCAGTGCATTTTGTTTTCCTGCTTCCTACCTCAA GATCTTGGCAATGGTACACTACAAATGTGCTGAGTTAGAATTACTCTGAAGTTATGAAACATATAATGAA AACAATTTTTTCTAGAGCTTATATTTTTATTTGAATGAAATAAAATGTTTAAATATTTAAAAATAAAAAA AAAAAAAAA

SEQ ID No. 7:

Nucleotide sequence of human LHCGR internally comprising the short splice variant of exon 6A (The START codon is underlined):

ACTCAGAGGCCGTCCAAGACACTGGCAAGCCGCAGAAGCCCAGTTCGCCGGCCATGAAGCAGCGGTTCTC

GGCGCTGCAGCTGCTGAAGCTGCTGCTGCTGCTGCAGCCGCCGCTGCCACGAGCGCTGCGCGAGGCGCTC

TGCCCTGAGCCCTGCAACTGCGTGCCCGACGGCGCCCTGCGCTGCCCCGGCCCCACGGCCGGTCTCACTC

GACTATCACTTGCCTACCTCCCTGTCAAAGTGATCCCATCTCAAGCTTTCAGAGGACTTAATGAGGTCAT

AAAAATTGAAATCTCTCAGATTGATTCCCTGGAAAGGATAGAAGCTAATGCCTTTGACAACCTCCTCAAT

TTGTCTGAAATACTGATCCAGAACACCAAAAATCTGAGATACATTGAGCCCGGAGCATTTATAAATCTTC

CCCGATTAAAATACTTGAGCATCTGTAACACAGGCATCAGAAAGTTTCCAGATGTTACGAAGGTCTTCTC

CTCTGAATCAAATTTCATTCTGGAAATTTGTGATAACTTACACATAACCACCATACCAGGAAATGCTTTT

CAAGGGATGAATAATGAATCTGTAACACTCCCATGGCAAAATTGTGATGAGGCAATAAAGGAGCTCACCC

TTAAAGAAAAAAGAGAAAACATGGATTGGAATGATCTGAAATGAAGAGATAGATGTGAAGCAAAAGAAGA

GATCATCTCAGAGGACTCTCTTTTATATCACTGGATTCTAAAAATTGCAAACTATATGGAAATGGATTTG

AAGAAGTACAAAGTCATG

CATTCAATGGGACGACACTGACTTCACTGGAGCTAAAGGAAAACGTACATCTGGAGAAGATGCACAATGG

AGCCTTCCGTGGGGCCACAGGGCCGAAAACCTTGGATATTTCTTCCACCAAATTGCAGGCCCTGCCGAGC

TATGGCCTAGAGTCCATTCAGAGGCTAATTGCCACGTCATCCTATTCTCTAAAAAAATTGCCATCAAGAG

AAACATTTGTCAATCTCCTGGAGGCCACGTTGACTTACCCCAGCCACTGCTGTGCTTTTAGAAACTTGCC

AACAAAAGAACAGAATTTTTCACATTCCATTTCTGAAAACTTTTCCAAACAATGTGAAAGCACAGTAAGG

AAAGTGAATAACAAAACACTTTATTCTTCCATGCTTGCTGAGAGTGAACTGAGTGGCTGGGACTATGAAT

ATGGTTTCTGCTTACCCAAGACACCCCGATGTGCTCCTGAACCAGATGCTTTTAATCCCTGTGAAGATAT

TATGGGCTATGACTTCCTTAGGGTCCTGATTTGGCTGATTAATATTCTAGCCATCATGGGAAACATGACT

GTTCTTTTTGTTCTCCTGACAAGTCGTTACAAACTTACAGTGCCTCGTTTTCTCATGTGCAATCTCTCCT

TTGCAGACTTTTGCATGGGGCTCTATCTGCTGCTCATAGCCTCAGTTGATTCCCAAACCAAGGGCCAGTA

CTATAACCATGCCATAGACTGGCAGACAGGGAGTGGGTGCAGCACTGCTGGCTTTTTCACTGTATTCGCA

AGTGAACTTTCTGTCTACACCCTCACCGTCATCACTCTAGAAAGATGGCACACCATCACCTATGCTATTC

ACCTGGACCAAAAGCTGCGATTAAGACATGCCATTCTGATTATGCTTGGAGGATGGCTCTTTTCTTCTCT

AATTGCTATGTTGCCCCTTGTCGGTGTCAGCAATTACATGAAGGTCAGTATTTGCTTCCCCATGGATGTG

GAAACCACTCTCTCACAAGTCTATATATTAACCATCCTGATTCTCAATGTGGTGGCCTTCTTCATAATTT

GTGCTTGCTACATTAAAATTTATTTTGCAGTTCGAAACCCAGAATTAATGGCTACCAATAAAGATACAAA

GATTGCTAAGAAAATGGCAATCCTCATCTTCACCGATTTCACCTGCATGGCACCTATCTCTTTTTTTGCC

ATCTCAGCTGCCTTCAAAGTACCTCTTATCACAGTAACCAACTCTAAAGTTTTACTGGTTCTTTTTTATC

CCATCAATTCTTGTGCCAATCCATTTCTGTATGCAATATTCACTAAGACATTCCAAAGAGATTTCTTTCT

TTTGCTGAGCAAATTTGGCTGCTGTAAACGTCGGGCTGAACTTTATAGAAGGAAAGATTTTTCAGCTTAC

ACCTCCAACTGCAAAAATGGCTTCACTGGATCAAATAAGCCTTCTCAATCCACCTTGAAGTTGTCCACAT

TGCACTGTCAAGGTACAGCTCTCCTAGACAAGACTCGCTACACAGAGTGTTAACTGTTACATCAGTAACT

GCATTATTGAATTGTTCTTAAACCTGTAAAAAAAAATTACCTGTACCAGTAATTTTAACATAAAGGGTTG

GATTTAGGAAATTATTTATTTTTAGGTACATTAGGCAAGAGACCTCTACCTAGTAGAAAGTGTAGTCTAT

GACCACTGCCACACTAAAAACTATTTGTCATTGTTACATGGCATAAATACTGAAGTTGAGAGTGTTTAGA

AATTTTTATAGAAATTTTGACACAGTAATTTTGTTTGATGAATCTTTTAAAAAACTGAGGAGGTATTTTG CATATCTTTTTTTTCATTTTCGTAATTTGTATTGCATTCTATAAAAATATTAGTTCATAACAGATCAGAA ATTTAAAATAACTGGCCTTTTTCCTCAGGTAGTTTGAAAAACACACTCTAGAGATGCACTGTCCAATCCG GTAGCCACTAGCCACATGTGGCTAAATTAAAATTAAATAAAATGAGAAATGTAGTTTCTCAGTTGCACTA GCCACGTTTCAAGTTCTCAATGGCTACGTGTGACTAGTGCTTACCATACTGGACAGCACAGACACAGAAT ATTTTCATCACCACAGAAAGTTCTATCTGTTCTATTATAGAGACTTTTATCTATGCCCTATCTGGATTCT ACTTATTTATAATTTAAGGTAAACATCTGAAAGCACATTTCAGCCTATTTGCTTAGTGAAACATTAAGCT GTAGACTGTAAACTCCTCGTGAGTAGGAACCCTGTCTCAGTGCATTTTGTTTTCCTGCTTCCTACCTCAA GATCTTGGCAATGGTACACTACAAATGTGCTGAGTTAGAATTACTCTGAAGTTATGAAACATATAATGAA AACAATTTTTTCTAGAGCTTATATTTTTATTTGAATGAAATAAAATGTTTAAATATTTAAAAATAAAAAA AAAAAAAAA

SEQ ID No.8:

Nucleotide sequence of truncated human LHCGR ending with the coding region of exon 6A (exon 1 to exon 6A (coding region)):

1 ATGAAGCAGCGGTTCTCGGCGCTGCAGCTGCTGAAGCTGCTGCTG

46 CTGCTGCAGCCGCCGCTGCCACGAGCGCTGCGCGAGGCGCTCTGC

91 CCTGAGCCCTGCAACTGCGTGCCCGACGGCGCCCTGCGCTGCCCC 136 GGCCCCACGGCCGGTCTCACTCGACTATCACTTGCCTACCTCCCT 181 GTCAAAGTGATCCCATCTCAAGCTTTCAGAGGACTTAATGAGGTC 226 ATAAAAATTGAAATCTCTCAGATTGATTCCCTGGAAAGGATAGAA 271 GCTAATGCCTTTGACAACCTCCTCAATTTGTCTGAAATACTGATC 316 CAGAACACCAAAAATCTGAGATACATTGAGCCCGGAGCATTTATA 361 AATCTTCCCCGATTAAAATACTTGAGCATCTGTAACACAGGCATC 406 AGAAAGTTTCCAGATGTTACGAAGGTCTTCTCCTCTGAATCAAAT 451 TTCATTCTGGAAATTTGTGATAACTTACACATAACCACCATACCA 496 GGAAATGCTTTTCAAGGGATGAATAATGAATCTGTAACACTCCCA 541 TGGCAAAATTGTGATGAGGCAATAAAGGAGCTCACCCTTAAAGAA 586 AAAAGAGAAAACATGGATTGGAATGACTCTGAAATGAAGAGATAG

SEQ ID No.9:

Amino acid sequence encoded by exon 6A of the human LHCGR gene:

PWQNCDEAIKELTLKEKRENMDWNDSEMKR

SEQIDNo.10:

Amino acid sequence of truncated human LHCGR ending with the amino acid sequence encoded by exon 6A (Amino acid sequence encoded by exons 1 to 6A (coding region)):

M K Q R F S A L Q L L K L L L L L Q P P L P R A L R E A L C P E P C N C V P D G A L R C P G P T A G L T R L S L A Y L P V K V I P S Q A F R G L N E V

I K I E I S Q I D S L E R I E

A N A F D N L L N L S E I L I

Q N T K N L R Y I E P G A F I

N L P R L K Y L S I C N T G I

R K F P D V T K V F S S E S N

F I L E I C D N L H I T T I P

G N A F Q G M N N E S V T L P

W Q N C D E A I K E L T L K E

K R E N M D W N D S E M K R

SEQ ID No. 11 :

Nucleotide sequence encoding human LHCGR (lacking exon 6A) (The START codon is underlined):

ACTCAGAGGCCGTCCAAGACACTGGCAAGCCGCAGAAGCCCAGTTCGCCGGCCATGAAGCAGCGGTTCTC GGCGCTGCAGCTGCTGAAGCTGCTGCTGCTGCTGCAGCCGCCGCTGCCACGAGCGCTGCGCGAGGCGCTC TGCCCTGAGCCCTGCAACTGCGTGCCCGACGGCGCCCTGCGCTGCCCCGGCCCCACGGCCGGTCTCACTC GACTATCACTTGCCTACCTCCCTGTCAAAGTGATCCCATCTCAAGCTTTCAGAGGACTTAATGAGGTCAT AAAAATTGAAATCTCTCAGATTGATTCCCTGGAAAGGATAGAAGCTAATGCCTTTGACAACCTCCTCAAT TTGTCTGAAATACTGATCCAGAACACCAAAAATCTGAGATACATTGAGCCCGGAGCATTTATAAATCTTC CCCGATTAAAATACTTGAGCATCTGTAACACAGGCATCAGAAAGTTTCCAGATGTTACGAAGGTCTTCTC CTCTGAATCAAATTTCATTCTGGAAATTTGTGATAACTTACACATAACCACCATACCAGGAAATGCTTTT CAAGGGATGAATAATGAATCTGTAACACTCAAACTATATGGAAATGGATTTGAAGAAGTACAAAGTCATG CATTCAATGGGACGACACTGACTTCACTGGAGCTAAAGGAAAACGTACATCTGGAGAAGATGCACAATGG AGCCTTCCGTGGGGCCACAGGGCCGAAAACCTTGGATATTTCTTCCACCAAATTGCAGGCCCTGCCGAGC TATGGCCTAGAGTCCATTCAGAGGCTAATTGCCACGTCATCCTATTCTCTAAAAAAATTGCCATCAAGAG AAACATTTGTCAATCTCCTGGAGGCCACGTTGACTTACCCCAGCCACTGCTGTGCTTTTAGAAACTTGCC AACAAAAGAACAGAATTTTTCACATTCCATTTCTGAAAACTTTTCCAAACAATGTGAAAGCACAGTAAGG AAAGTGAATAACAAAACACTTTATTCTTCCATGCTTGCTGAGAGTGAACTGAGTGGCTGGGACTATGAAT ATGGTTTCTGCTTACCCAAGACACCCCGATGTGCTCCTGAACCAGATGCTTTTAATCCCTGTGAAGATAT TATGGGCTATGACTTCCTTAGGGTCCTGATTTGGCTGATTAATATTCTAGCCATCATGGGAAACATGACT GTTCTTTTTGTTCTCCTGACAAGTCGTTACAAACTTACAGTGCCTCGTTTTCTCATGTGCAATCTCTCCT TTGCAGACTTTTGCATGGGGCTCTATCTGCTGCTCATAGCCTCAGTTGATTCCCAAACCAAGGGCCAGTA CTATAACCATGCCATAGACTGGCAGACAGGGAGTGGGTGCAGCACTGCTGGCTTTTTCACTGTATTCGCA AGTGAACTTTCTGTCTACACCCTCACCGTCATCACTCTAGAAAGATGGCACACCATCACCTATGCTATTC ACCTGGACCAAAAGCTGCGATTAAGACATGCCATTCTGATTATGCTTGGAGGATGGCTCTTTTCTTCTCT AATTGCTATGTTGCCCCTTGTCGGTGTCAGCAATTACATGAAGGTCAGTATTTGCTTCCCCATGGATGTG GAAACCACTCTCTCACAAGTCTATATATTAACCATCCTGATTCTCAATGTGGTGGCCTTCTTCATAATTT GTGCTTGCTACATTAAAATTTATTTTGCAGTTCGAAACCCAGAATTAATGGCTACCAATAAAGATACAAA GATTGCTAAGAAAATGGCAATCCTCATCTTCACCGATTTCACCTGCATGGCACCTATCTCTTTTTTTGCC ATCTCAGCTGCCTTCAAAGTACCTCTTATCACAGTAACCAACTCTAAAGTTTTACTGGTTCTTTTTTATC CCATCAATTCTTGTGCCAATCCATTTCTGTATGCAATATTCACTAAGACATTCCAAAGAGATTTCTTTCT TTTGCTGAGCAAATTTGGCTGCTGTAAACGTCGGGCTGAACTTTATAGAAGGAAAGATTTTTCAGCTTAC ACCTCCAACTGCAAAAATGGCTTCACTGGATCAAATAAGCCTTCTCAATCCACCTTGAAGTTGTCCACAT TGCACTGTCAAGGTACAGCTCTCCTAGACAAGACTCGCTACACAGAGTGTTAACTGTTACATCAGTAACT GCATTATTGAATTGTTCTTAAACCTGTAAAAAAAAATTACCTGTACCAGTAATTTTAACATAAAGGGTTG GATTTAGGAAATTATTTATTTTTAGGTACATTAGGCAAGAGACCTCTACCTAGTAGAAAGTGTAGTCTAT GACCACTGCCACACTAAAAACTATTTGTCATTGTTACATGGCATAAATACTGAAGTTGAGAGTGTTTAGA AATTTTTATAGAAATTTTGACACAGTAATTTTGTTTGATGAATCTTTTAAAAAACTGAGGAGGTATTTTG CATATCTTTTTTTTCATTTTCGTAATTTGTATTGCATTCTATAAAAATATTAGTTCATAACAGATCAGAA ATTTAAAATAACTGGCCTTTTTCCTCAGGTAGTTTGAAAAACACACTCTAGAGATGCACTGTCCAATCCG GTAGCCACTAGCCACATGTGGCTAAATTAAAATTAAATAAAATGAGAAATGTAGTTTCTCAGTTGCACTA GCCACGTTTCAAGTTCTCAATGGCTACGTGTGACTAGTGCTTACCATACTGGACAGCACAGACACAGAAT ATTTTCATCACCACAGAAAGTTCTATCTGTTCTATTATAGAGACTTTTATCTATGCCCTATCTGGATTCT ACTTATTTATAATTTAAGGTAAACATCTGAAAGCACATTTCAGCCTATTTGCTTAGTGAAACATTAAGCT GTAGACTGTAAACTCCTCGTGAGTAGGAACCCTGTCTCAGTGCATTTTGTTTTCCTGCTTCCTACCTCAA GATCTTGGCAATGGTACACTACAAATGTGCTGAGTTAGAATTACTCTGAAGTTATGAAACATATAATGAA AACAATTTTTTCTAGAGCTTATATTTTTATTTGAATGAAATAAAATGTTTAAATATTTAAAAATAAAAAA AAAAAAAAA

SEQ ID No. 12:

Nucleotide sequence of the ^'left^' portion of intron 6 of the human LHCGR gene

("intron 6A"; intron sequence between exon 6 and exon 6A):

TCTCATGTTTTACCACATTCCTCACTCACTGGTGACATTCTTCTTAACCTAAAATGCTAAGGGTGAAGGGAGTTG TGTAAGATCACAGCTGGGTCATATGTTCACATGACAACCAACCAACCAATTCAGTTTAACCAAACAGGCCCTGAG

CACAGTCTAGAGAGGAGTGGCTTTCCTGGCTTAAAAAGAGCATAGAAATTTCAAGATTTCAACAGAGAAGAGTTT GGAAGGATATTCTCCAAGTAGGAAAAGTAGTTGATCAATGGACCTTGCTCTGTCTCTCTCATTTTCGCCTTCCTC

ATGTGTTTTTATTTGACAGTTACTCGTCAGGTTACCAGGCTATCCCTTTCATGGTGTTTCCTAAGGCCTCACTAT TCAAACTATGAGCCATGATCTTCAGCATCTTCTGAGAGCTGGTGTATAAGTCCATTCTCACATTGCTATAAAGAA CTACCTGAGACTGGGTAATTTATAAAGAAAAGAGGTTTTAATTGGCTCACAATTCTGCAGGCTTTGCAGGAAGCA TGATTCTGGCATCTGCTCAGCTTCTGGGGAGGTCTCAGGAAACTTACAATACTGATGGAAGGTGAAGGGTGAGCC

CATGAGAACTCTATCACAAGAACAGCACTAGGGGGATGGTGCTAAACCATCCATGAGAAACCACCCCCATGATCC AGTCACCTCCCACCAGGCCCCACCTCCAACACTGGGGATTATAATTTGTAATGCAATTTTGTGGAAACACAGATC TAAACCATATCCTCTAGTTAGAAATAGCTCACCGCAGACCTACTAACTCAGAATATGCTTTTTACAAGATCCCAA GTGATTCACATGTACATTAAAGTTTGAGGAGCACTCCACTAAGATAACTCAACCCATGTTCTTCACAGGACACTT GCCAGACTTCCTTCCCTCTGGAATCCCTGCCATACATCTTCACATGGGAGCCCTCCACTGCCGTACCATTCAGAA

CACCCCCCGGCCTCATTTCTTTATACCTAAAGCCCAGTGACCCTGATTTAATTCTGCTCCTAAAATCCATATCCC TCTCACTCTTTCTGCTTTGGTTACAG

SEQ ID No. 13:

Nucleotide sequence of the ^"right^' portion of intron 6 of the human LHCGR gene

("intron 6B"; intron sequence between exon 6A and exon 7):

GGTAACTATGAGCTCACAAGACTGGGCCACCCCACGAGACTCAAAGAGTAACCAAAGATTCCTTCTCACACATCA

AGAAAAAGAGTGCATGCCAGTAGGGTGTAATTTCCCCACTCTGAAGTTAATAAAGATTGAGGCTCCGTCTCTCCC

CATTTATTTGCCTGGCACTTGTTCTCTGCTGAATGGGCCCCTCTGTAAGCAAAGGATCGTGTGGAGCAGCCTGCT

AATGAAAGCAAGCCTTCTCTTTTCATTATTAGTGGTATTTGAGATTTTGCTTTTTGTAGAACCTGTCTTGCCCTC

AGTGGGGCTAATGCTAAAACTATGCATTATGCTTTTATGTCCCTTTATAGCCACTTTGGTTGTTCATTAAATCTC CTCAAGAGGCCACCTTGAAATGCCCCTCCTTCCCAAATAAGCAGAATTTAGCTGTGCCACCTTCAAAATATTCTA GGTGATTGGTCACACCAGTCTCAAAAAAAGAGACAATCTTGATTCACATCTTAACTCCACAAAGAAATGGAAAGG TCATAGCAATTAATTTTCTAAGACCCTACAGAGAAGTGATGTCTGGAAAGGCATGATGATTCCAGGCTGCACCCA TGACAAGCCACAGTAGAGAGACTGCTGGGTCTCTCTACTGTGTTGAAATAGGGAGTCTGGGCTTCAGTCCTGGCT CTGTGTCTAACCAGCCAAGCAGCCTTAGTCAGGTCATCCCACTTTGACATAGCTCACTGGGTTCAAGCGATTCTC CTGCCTCAGCCTCCCAAGTAGCTGGGACCACAGGCGCCCACCACCATGCCCGGCTAATTTTTTGTATTTTTAATA

AAGTGCTGGGATTACAGGCGTGAGCCACCATGCCCGGCCTAGAATCCTTTATTGTTATCATTCTTTCTGATGCCT

AAAACTCAGTGCTGCTGAGAGGCAGGAGGCACAGCAATACAGGGAGAGGGATCCATTGCTATAGAGACTATGAAC

ATCTAACCACTGCCGGCACATCCACTAGAGAAGTGTCTTCATACGTAAGGAATTATTCAGCTATAAAAGGACTTC

GTCATTTGAAAAAAAAAATTGCTTACAAATATACCACCATATTTTCTAAAAGATATCTTCTAAAACGGTATGCTT TGCCTTTTAGCAATGGGTCCACTTTTTTGAGACCCTGAGTATCTGTAAACATAAATTAAGCTTTCTTCTTCAAAA TCATCCAGAAACCAGGTGATCCAAAAGGAAACTATAGAAGAATGTCTAATACTGTTAATATTAGAATCCCAGAAG

AGAACCTAGACAATGCTTTAAAGTTAGGTACAGCTATTGTAATAGAGAAGTTGGGGGAGGCGAAAAATCTTCATT

GGAAAAAAGTTATCTGAAGTGACTCTCCAGATGTTTTTTTAAGGGCATTAAAAGCATTGTGCAAGACTGACAGCA

CAAGCAGGACTGAGGCTATTCAGAAAAAAAAAAAAAAAAAAAAAAAGACATGGAAAATGTCTAAAGGCAAAAAAG TCCATGACGCTAGTACAGACCAAAGGTGTTTGAGAGCCTTCAGCACGTGAGTACATTTAGTGCAGCCCAGGCCAC

AATGCTTGTATTTCACAGAAATCCTGTCCAGAGCATGGAAAGGGAGATCGTTTACCTCTGTTCTTGTCAGAAGGC

CTTATAAATGTTTAACATATATTTACATTTTCAGTCCTTACAATAGCTTTATGAGGTTGATACTATTATCATACT

GGGATTCAAGCAATTGGGTTCAGTGCCTGCAGACTTAACCACAAGGGTACACCTGTGACACTGTGAGTCTAACAG

ACAACTGTTAGCAGCTTTGGTGAAAACAAAGCAAGGGCTATCTTTTCTTCATCTACTCCATGTGTAGTGCGATCA

CCCCATGGGAATGATGAGAACAGTATCATCCTATGACATACCCTAGGGCAAAGGTTCTCAAACTGGCATACTGAA GGCTCACTAGGAGAGCTTGTTATAAAAGTGCATATTTTTGGATACCACTATTAGTTTCTGATTTAATAGGTCTGA GTCTGCATTTTTGACAAGCAACTCCAGGTGATTCTAATTCAGTGGATGGAGGAGAAAGTTTTGACAAATACCGGG CAAGAGTCTAAGAAATCAAACTTAAGGAAGTTCAGAAAAAGAGTGTGATTCCTTTATCTAAAAGAGACTGCTATA AAGGAATTCAACCCCTAAAATGACAGGAGACTGCCAAAGACTAACATCCTAGGTGCAAAATTTTAAAAAATTCTG GTGAAATTGAGCTTCTAAGGAAACAACAAGGTTGTGTATAATTATGCAAGACAACAGGCATCAACCCTGTACCTA TGTTCTCAGATTCCACAATATAGAAGGGGAAGAAAACTACTGAGAGTGGCATCCAGTAATCCAACATGGTAGAGC TAGTATGTGAAACTGATGATTGGCTTTGCCTCCATTGCTTTATCATCATCTGACCCATGATTATATTTTATGTTT TTATTGTTTGCCTCCTCCAGAGAAACATATCCTCCTGTATCTAGAACAGTGCCTGGCACCTGGGAGTATTACAGT AAATATACATTAAATGAATGGGATGACCAAATGAGAATATGTAAGATGAATTTCTTACCATAAATCATAACCAAA CAATTTTGAAGGCTACTGACTTAGATGGAAATATAAGTTATTTGAGAAAGGCCTGTGGTGTGATGTAAAGTTAAG CTCCTTGTATCTGCCAAGACCCTAGCTTCTCTTTGCAGGGTTGCTGGAAGCTGGCCAGGGACAAGCTGTGGGGTG TAACTTGAGAAATGGTTCAGAAACATGCTTCAGATTCTGCAATATAGAAAGGGATGAAAACTATTGAAAGTGGCA TCCAGTAATCCAATATGGGAGAGCTAGTATGTGAAGCTGTAAACTCTGAAGAATCTCAGAAGAAAGCATATGAGA GCCTGGGAAACCATGGAAGCCTCCCCTTCCCGGGGAAGGGCAAAGCTGCTGCTTTACTCAGGCTATTGACTGAGC TAGGCACCAGGCATTCTGTGAGGCCCCAGGAGTTTAAGAAATGAATTAAATATTCTCCCCTGCCCTCTTTGAACT GACTCTAACGAGGAGACTTAAGAATTATTTTGTAATCTCTAGTTATATTTTCTGAATTTCAGAGCTTAAATATTA TACTTCAACATGAGTCACACCTTTATTTATATGTTGGTTTGTCTCAGCTGTGTTGTGGGTTGGTGGAAGGAGACC ACACATACATACACACAGAGTACATACATGCTGTTGATGTTACACACATACTCACACCCCACAAAGTGAAGCTCC ATGCTCATTTTGTTTAACAAAGACTAGAGAGGCCTTGCAGACAACAGCTACCTGGAGCAGGAACAAGTGAAGCAT

TAGGGTGTTGGCAGATATAAGGAGTTTAACTTTACATCACACCACAGCCCTTTCTCACGTGGGTTTTACATCATC ATTCACAAAGAATTTATACAAATATTTCCATCTAAGGCAGTAGCTTTCAAACTTGTTTGGCTATGATTTATGGTA AGAAATTCCCATTTGGTCATCCAATTTATTTAATATATATTTACTGTAATACTCCCAGGTGCCACGCACTGTTCT

TAAAGCAACGGAGGCAAAGCAAATCAGGGAAGGAGTATAGGGAAAGTAGGGGGAAACAGGAAATTGGTGTTTTAA

TGAGGATATCAGTGGAGGAAAAGTAAAAAGAATCAGCAAAATTGATGTGCTGGATGTAGCCCCAGACAAAGCATA TCCTAGTCCCATTTATGCCAGTTGTAGGCTTTAGACAAGTCACTTTCCCTCACTGAGCCTATTTCCTTCTTTGCA

TATACCATAAAAAATGGTGGGATAAAGTTTCATAAACATTAATACCCTAATCACTTGCAAAATGCTCTAATACTT

AAAGGACTGTTTACAGACATTGAAATCTTCTTGGTTGTGGAGCCAAATCTGACATCAGCAAAACCACATACTCAT

CCATATGCCCACACCGGCCTGCAAAACCAGAGGAAAGGCTTAAGTGTTAAACATCTCTGGCCTTGGATCCCAGCT ATGCCACTGTTTTCCTTTGACATTAAGCAAGCTACATAACCACACTGAGGGTTAGGCTTCTTGTCGGTATAATGG AAATGATGATGAGGATTAAATCATAAAATAAACATAGAGTGGCTGATGAAGTATCTAGCACTTAATAGACACTTA

CAGGAGGAAAAATGTGTTTCCAAACTGTTTCTTTCAGTTTCTTCCCATTCCTAAACCCTCTCCTCCCTCCTTCAG

SEQ ID No. 14:

Nucleotide sequence of exon 6 of the human LHCGR gene:

GGAAATTTGTGATAACTTACACATAACCACCATACCAGGAAATGCTTTTCAAGGGATGAATAATGAATCTGTAAC ACT

SEQ ID No. 15: Nucleotide sequence of exon 7 of the human LHCGR gene:

CAAACTATATGGAAATGGATTTGAAGAAGTACAAAGTCATGCATTCAATGGGACGACACTGACTTCACT

SEQ ID No. 16:

Antisense oligonucleotide putatively suitable for exon 6A skipping (complement strand to SEQ ID No. 17):

GATTTTTAACCATAGGAACCACGTGA

SEQ ID No. 17:

Partial nucleotide sequence of exon 6A of the human LHCGR gene:

CTAAAAATTGGTATCCTTGGTGCACT

SEQ ID No. 18:

Partial sequence of the amino acid sequence encoded by exon 6A of the human

LHCGR gene (putative epitope):

CDEAIKELTLKEKREN

SEQ ID No. 19:

Partial sequence of the amino acid sequence encoded by exon 6A of the human

LHCGR gene (putative epitope):

EKRENMDWNDSEMKR

SEQ ID No. 20:

Amino acid sequence of the human LHCGR:

MKQRFSALQLLKLLLLLQPPLPRALREALCPEPCNCVPDGALRC PGPTAGLTRLSLAYLPVKVIPSQAFRGLNEVIKIEISQIDSLERIEANAFDNLLNLSE

ILIQNTKNLRYIEPGAFINLPRLKYLSICNTGIRKFPDVTKVFSSESNFILEICDNLH ITTIPGNAFQGMNNESVTLKLYGNGFEEVQSHAFNGTTLTSLELKENVHLEKMHNGAF RGATGPKTLDISSTKLQALPSYGLESIQRLIATSSYSLKKLPSRETFVNLLEATLTYP SHCCAFRNLPTKEQNFSHSISENFSKQCESTVRKVNNKTLYSSMLAESELSGWDYEYG FCLPKTPRCAPEPDAFNPCEDIMGYDFLRVLIWLINILAIMGNMTVLFVLLTSRYKLT VPRFLMCNLSFADFCMGLYLLLIASVDSQTKGQYYNHAIDWQTGSGCSTAGFFTVFAS ELSVYTLTVITLERWHTITYAIHLDQKLRLRHAILIMLGGWLFSSLIAMLPLVGVSNY MKVSICFPMDVETTLSQVYILTILILNVVAFFIICACYIKIYFAVRNPELMATNKDTK IAKKMAILIFTDFTCMAPISFFAISAAFKVPLITVTNSKVLLVLFYPINSCANPFLYA IFTKTFQRDFFLLLSKFGCCKRRAELYRRKDFSAYTSNCKNGFTGSNKPSQSTLKLST LHCQGTALLDKTRYTEC

SEQIDNo.21: Partial sequence of the amino acid sequence encoded by exon 6A of the human LHCGR gene (putative epitope) supplemented with cysteine (C) at its N-terminus:

CEKRENMDWNDSEMKR

Claims

A nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence as depicted in any one of SEQ ID NOs: 1 to 4;

(b) a nucleotide sequence as depicted in any one of SEQ ID NOs: 1 to 4 having at least one nucleotide exchange, wherein said nucleotide sequence corresponds to a nucleotide sequence that can be spliced into the LHR mRNA;

(c) a nucleotide sequence as depicted in any one of SEQ ID NOs: 1 to 4 having at least one of the following nucleotide exchanges: A21C; G22C; T63C; A117G; and T212G;

(d) a nucleotide sequence encoding a polypeptide as depicted in SEQ ID NO: 9;

(e) a nucleotide sequence encoding a polypeptide as depicted in SEQ ID NO: 9 having at least one amino acid exchange, wherein said nucleotide sequence corresponds to a nucleotide sequence that can be spliced into the LHR mRNA;

(f) a nucleotide sequence encoding a polypeptide as depicted in SEQ ID NO: 9 having at least one of the following amino acid exchanges: E7A; E7D; and M21T;

(I) a nucleotide sequence which is capable of hybridizing to the nucleotide sequence of any one of (a), (c), (d) and (f), wherein said nucleotide sequence has at least one nucleotide exchange corresponding to any one of the nucleotide or amino acid exchanges as defined in (c) or (f) and/or wherein said nucleotide sequence starts with a nucleotide sequence capable of hybridizing to the 5 ^'-end and/or ends with a nucleotide sequence capable of hybridizing to the 3^'-end of the nucleotide sequence of any one of (a), (c), (d) and (f);

(m) a nucleotide sequence having at least 60% homology to the nucleotide sequence of any one of (a), (c), (d) and (f), wherein said nucleotide sequence has at least one nucleotide exchange corresponding to any one of the nucleotide or amino acid exchanges as defined in (c) or (f) and/or wherein said nucleotide sequence starts with a nucleotide sequence having at least 60% homology to the 5^'-end and/or ends with a nucleotide sequence having at least 60% homology to 3^'-end of the nucleotide sequence of any one of (a), (c), (d) and (f);

(n) a fragment of the nucleotide sequence of any one of (a) to (m), wherein said fragment has at least one nucleotide exchange corresponding to any one of the nucleotide or amino acid exchanges as defined in (c) or (T) and/or wherein said fragment starts with a nucleotide sequence corresponding to the 5 ^'-end and/or ends with a nucleotide sequence corresponding to the 3^'-end of the nucleotide sequence of any one of (a) to (m); and

(o) the complementary strand of the nucleotide sequence of any one of (a) to (n).

2. A nucleotide sequence consisting of the nucleotide sequence of claim 1 and, optionally, (a) nucleotide sequence(s) heterologous thereto, wherein said nucleotide sequence of claim 1 has:

(a) attached to its 5^'-end a nucleotide sequence of at least 1 nucleotide of the 3^'-end of SEQ ID NO: 12 or a variant of said nucleotide sequence; or the complementary strand thereof; and/or

(b) attached to its 3 ^'-end a nucleotide sequence of at least 1 nucleotide of the 5^'-end of SEQ ID NO: 13 or a variant of said nucleotide sequence; or the complementary strand thereof.

3. A nucleotide sequence selected from the group consisting of:

(a) an mRNA corresponding to a nucleotide sequence comprising the nucleotide sequence of any one of claim 1(a) to (f);

(b) a cDNA derived from the mRNA of (a);

(c) fragment of (a) or (b), comprising a fragment corresponding to the nucleotide sequence of any one of claims 1 (a) to (f);

(d) a nucleotide sequence comprising the nucleotide sequence of any one of claims 1(a) to (f), wherein said nucleotide sequence of any one of claims 1 (a) to (T) has at least one nucleotide exchange corresponding to any one of the nucleotide exchanges as defined in claim 1 (c) and/or wherein said nucleotide sequence starts with the 5^'-end and/or ends with the 3^'-end of the nucleotide sequence of any one of claims 1 (a) to

(f);

(e) a fragment of the nucleotide sequence of (d) comprising a fragment of the nucleotide sequence of any one of claims 1(a) to (f), wherein said fragment of the nucleotide sequence of any one of any one of claims 1 (a) to (f) has at least one nucleotide exchange corresponding to any one of the nucleotide exchanges as defined in claim 1(c) and/or wherein said fragment starts with the 5^'-end and/or ends with the 3^'-end of the nucleotide sequence of any one of claims 1(a) to (T);

(f) a nucleotide sequence which is capable of hybridizing to the nucleotide sequence of any one of (a) to (e), wherein said nucleotide sequence comprises a nucleotide sequence which is capable of hybridizing to the nucleotide sequence of any one of claim 1(a), (c), (d) and (f) and a nucleotide sequence which is capable of hybridizing to the nucleotide sequence as depicted in SEQ ID NO. 11 ;

(g) a nucleotide sequence having at least 60% homology to the nucleotide sequence of any one of (a) to (e), wherein said nucleotide sequence comprises a nucleotide sequence having at least 60% homology to the nucleotide sequence of any one of claim 1(a), (c), (d) and (f) and a nucleotide sequence having at least 60% homology to the nucleotide sequence as depicted in SEQ ID NO. 11 ; and

(h) the complementary strand of the nucleotide sequence of any one of (a) to (g).

4. A nucleotide sequence comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence as depicted in any one of SEQ ID NOs: 5 to 8;

(b) a nucleotide sequence as depicted in any one of SEQ ID NOs: 5 to 8 having at least one nucleotide exchange corresponding to any one of the nucleotide exchanges as defined in claim 1(c); (c) a fragment of the nucleotide sequence of (a) or (b), said fragment comprises a nucleotide sequence consisting of the 3'-end of SEQ ID NO: 14 followed by the 5'-end of the nucleotide sequence of claim 1 (a) and/or a nucleotide sequence consisting of the 3'-end of the nucleotide sequence of claim 1 (a) followed by the 5'-end of SEQ ID NO: 15;

(d) a nucleotide sequence having the degenerated code of the nucleotide sequence of any one of (a) to (c);

(e) a nucleotide sequence which is capable of hybridizing to the complementary strand of the nucleotide sequence of any one of (a) to (d), said nucleotide sequence comprises a nucleotide sequence corresponding to a nucleotide sequence consisting of the 3'-end of SEQ ID NO: 14 followed by the 5'-end of the nucleotide sequence of claim 1 (a) and/or a nucleotide sequence consisting of the 3'-end of the nucleotide sequence of claim 1(a) followed by the 5'-end of SEQ ID NO: 15;

(T) a nucleotide sequence having at least 60% homology to the nucleotide sequence of any one of (a) to (d), said nucleotide sequence comprises a nucleotide sequence corresponding to a nucleotide sequence consisting of the 3'-end of SEQ ID NO: 14 followed by the 5'-end of the nucleotide sequence of claim 1(a) and/or a nucleotide sequence consisting of the 3'- end of the nucleotide sequence of claim 1 (a) followed by the 5'-end of SEQ ID NO: 15; and

(g) the complementary strand of the nucleotide sequence of any one of (a) to

(f).

5. A vector consisting of the nucleotide sequence of any one of claims 1 to 4 and (a) nucleotide sequence(s) heterologous thereto.

6. A polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence as depicted in SEQ ID NO: 9;

(b) an amino acid sequence as depicted in SEQ ID NO: 9 having at least one amino acid exchange, wherein said amino acid sequence is encoded by a nucleotide sequence that can be spliced in the LHR mRNA;

(c) an amino acid sequence as depicted in SEQ ID NO: 9 having at least one amino acid exchange corresponding to any one of the nucleotide exchanges as defined in claim 1(f);

(d) fragments of the amino acid sequence of any one of (a) to (c); and

(e) an amino acid sequence having at least 60% homology to the amino acid sequence of any one of (a), (c) and (d).

7. A polypeptide encoded by the nucleotide sequence of any one of claim 4(a) to (f).

8. A host cell, genetically engineered with the nucleotide sequence of any one of claims 1 to 4 or comprising the vector of claim 5 or the polypeptide of claim 6 or 7.

9. A pharmaceutical composition comprising of the nucleotide sequence of any one of claims 1 to 4, the vector of claim 5, the polypeptide of claim 6 or 7, the host cell of claim 8 or an inhibitor of exon 6A of the LHR.

10. The pharmaceutical of claim 9, wherein said inhibitor is selected from the group consisting of:

(a) an antisense nucleotide sequence, specifically binding to mRNA or hnRNA (pre-mRNA) comprising exon 6A;

(b) an antibody specifically binding to mRNA comprising exon 6A;

(c) a siRNA specifically binding to mRNA comprising exon 6A;

(d) an aptamer specifically binding to mRNA comprising exon 6A;

(e) a nucleic acid molecule which specifically introduces an insertion of a heterologous sequence or a mutation into exon 6A of the LHR gene (via in vivo mutagenesis);

(f) a nucleic acid molecule specifically reducing the expression of mRNA comprising exon 6A by cosuppression; and

(g) a ribozyme specifically recognizing mRNA comprising exon 6A.

11. The pharmaceutical composition of claim 10, wherein said antisense nucleotide sequence comprises an antisense nucleotide sequence corresponding to the nucleotide sequence of any one of claims 1 to 4.

12. The pharmaceutical composition of claim 10, wherein said siRNA comprises a nucleotide sequence corresponding to the nucleotide sequence of any one of claims 1 to 4.

13. The pharmaceutical composition of claim 10, wherein said antibody or aptamer specifically binds to the nucleotide sequence according to any one of claims 1 to 4 or to the polypeptide of claim 6 or 7.

14. The pharmaceutical composition of claim 10, wherein said nucleic acid molecule of (e) comprises said heterologous sequence flanked by parts of the nucleotide sequence of claim 1.

15. The pharmaceutical composition of claim 10, wherein said nucleic acid molecule of (f) comprises the nucleotide sequence of any one of claims 1 to 4.

16. A method of diagnosing a disease in a patient which is characterized by an increased amount of LHR mRNA comprising exon 6A, compared to the amount of LHR mRNA comprising exon 6A in a control patient, wherein said method comprises the following steps:

(a) determining the amount of said LHR mRNA comprising exon 6A in a patient and/or in a sample derived from said patient; and

(b) comparing the amount of said LHR mRNA comprising exon 6A with the amount of LHR mRNA comprising exon 6A in a control patient and/or in a sample derived from said control patient; and/or the step of determining whether a patient exhibits a mutation or a polymorphism in exon 6A.

17. A method for treating, ameliorating or preventing a disease which is diagnosable by the method of claim 16, said method comprises administering to a patient the pharmaceutical composition of any one of claims 9 to 15.

18. Use of the nucleotide sequence of any on of claims 1 to 4, the vector of claim 5, the polypeptide of claim 6 or 7, the host cell of claim 8 or the inhibitor as defined in any one of claims 9 to 15 for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of a disease which is diagnosable by the method of claim 16.

19. A method for treating, ameliorating or preventing a disease which is characterized by an increased amount of LHR mRNA comprising exon 6A, said method comprises administering to a patient the pharmaceutical composition of any one of claims 9 to 15.

20. Use of the nucleotide sequence of any on of claims 1 to 4, the vector of claim 5, the polypeptide of claim 6 or 7, the host cell of claim 8 or the inhibitor as defined in any one of claims 9 to 15 for the preparation of a pharmaceutical composition for the treatment, amelioration or prevention of a disease which is characterized by an increased amount of LHR mRNA comprising exon 6A.

21. The method or the use of any one of claims 16 to 20, wherein said increased amount of LHR mRNA comprising exon 6A is caused by at least one mutation in exon 6A.

22. The method or the use of claim 21 , wherein said increased amount of LHR mRNA comprising exon 6A is caused by at least one mutation in exon 6A which corresponds to one of the nucleotide exchanges as defined in claim 1(c) and/or to one of the amino acid exchanges as defined in claim 1(f).

23. The method or the use of any one of claims 16 to 22, wherein said increased amount of LHR mRNA comprising exon 6A is at least 2 fold when compared to a control patient.

24. The method or the use of claim 23, wherein said increased amount of LHR mRNA comprising exon 6A is at least 4 fold when compared to a control patient.

25. The method or the use of claim 24, wherein said increased amount of LHR mRNA comprising exon 6A is at least 6 fold when compared to a control patient.

26. The pharmaceutical composition of any one of claims 9 to 15 or the method or the use of any one of claims 16 to 25, wherein said exon 6A has a nucleotide sequence as depicted in any one of claims 1(a) to 1 (f).