WO2014095922A1

WO2014095922A1 - Method of diagnosing or prognosing, or treating friedreich ataxia

Info

Publication number: WO2014095922A1
Application number: PCT/EP2013/076976
Authority: WO
Inventors: Alexandra HENRION-CAUDE; François CARTAULT
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Centre Hospitalier Felix Guyon (Saint-Denis)
Priority date: 2012-12-17
Filing date: 2013-12-17
Publication date: 2014-06-26

Abstract

The invention concerns an in vitro method of diagnosis or prognosis of Friedreich's ataxia (FRDA) in an individual, by determining whether the individual harbours at least one variation in the 3' untranslated region (UTR) of the frataxin (FXN) gene. The invention further provides an isolated polynucleotide comprising at least one variation in the 3'UTR of the FXN gene and a polynucleotide of interest which is not frataxin (FXN) gene, the vector and a host cell comprising such isolated polynucleotide. Also provided is an inhibitor of a microRNA (miRNA) which specifically interacts with a variant 3'UTR of the FXN gene for use as a medicament, in particular for the treatment of FRDA.

Description

Method of diagnosing or prognosing, or treating Friedreich Ataxia

The invention concerns an in vitro method of diagnosis or prognosis of Friedreich's ataxia (FRDA) in an individual, by determining whether the individual harbours at least one variation in the 3' untranslated region (UTR) of the frataxin (FXN) gene. The invention further provides an isolated polynucleotide comprising at least one variation in the 3'UTR of the FXN gene and a polynucleotide of interest which is not frataxin (FXN) gene, the vector and a host cell comprising such isolated polynucleotide. Also provided is an inhibitor of a microRNA (miRNA), which specifically interacts with a variant 3'UTR of the FXN gene for use as a medicament, in particular for the treatment of FRDA.

BACKGROUND TO THE INVENTION

Friedreich's ataxia (FRDA) is the most frequent hereditary ataxia in Western European descent. This progressive autosomal recessive disorder is primarily characterized by neurodegeneration and cardiomyopathy. The onset of symptoms typically occurs around puberty, but earlier and later onsets also exist. FRDA is caused by a deficit of frataxin, a mitochondrial protein involved in iron metabolism and sensitivity to oxidative stress. The most common disease mutation is an expansion of trinucleotide GAA repeats within the first intron of the frataxin (FXN) gene (Campuzano V, et al. (1996) Science 271 : 1423-1427). The vast majority of patients are homozygous for the GAA expansion harbouring 70 to 1700 repeats, while unaffected individuals usually present 6 to 36 repeats. Expanded alleles were reported to derive from a single founder chromosome. Therefore, a major risk haplotype at the FXN, constituted by several markers, was defined as strongly associated both to expansion and "premutation" intermediates.

In vitro and in vivo data suggest that the expansion mutation results in partial transcriptional inhibition of the FXN gene in FRDA, leading to decreased frataxin. In patients homozygous for the expansion mutation, residual levels of frataxin protein were inversely correlated with the size of the GAA repeat on the smaller allele. However, some of those patients presented higher residual frataxin level than expected from their expansion size, suggesting that beside transcriptional regulation multiple mechanisms may regulate the expression of frataxin. So far, very little is known about the regulation of the stability of frataxin transcript and protein. Beyond the proposed post-translational regulation of frataxin by recombinant human erythropoietin and by the ubiquitin- proteasome system, the post-transcriptional mechanisms controlling frataxin expression are poorly studied. BRIEF SUMMARY OF THE INVENTION

The inventors provide the first systematic analysis of the FXN 3'-UTR in patients with Friedreich ataxia and its regulation on frataxin levels. The inventors have identified seven single nucleotide polymorphisms (SNP) which define several haplotypes, one of them reaching about 89% homozygosity in a cohort of patients versus 24% for the controls. This haplotype (called "FRDA-3'-UTR") completes the founder haplotype for the GAA repeats in Friedreich ataxia, and may thus represents a new molecular marker for FRDA.

Moroever, the inventors further established that the FRDA-3'-UTR haplotype was associated with specific post-transcriptional regulation that likely influences frataxin level. Indeed, the inventors provide the evidence of a miRNA-based regulation of FRDA-causal gene, frataxin, but also unravel the specificity of regulation through the 3'-UTR that is achieved by the FRDA-3'-UTR haplotype, highly associated to FRDA.

The invention concerns an in vitro method of diagnosis or prognosis of Friedreich's ataxia (FRDA) in an individual, which comprises:

a) determining whether the individual harbours at least one variation in the 3' untranslated region (UTR) of the frataxin (FXN) gene; and

b) deducing that the individual has developed or is at risk of developing FRDA if said at least one variation is present, or that the individual is not at risk of developing FRDA if said at least one variation is absent.

The invention further provides an isolated polynucleotide comprising the FRDA- 3'UTR or a fragment thereof and a polynucleotide of interest wherein said polynucleotide does not comprise frataxin (FXN) gene. Also provided are a vector comprising said isolated polynucleotide and a host cell comprising the vector or the isolated polynucleotide of the invention.

Also provided is a method for screening for a compound suitable for the treatment and/or prevention of Friedreich's ataxia (FRDA) comprising the steps of:

a) contacting a candidate compound with a polynucleotide comprising the 3' untranslated region (UTR) of the frataxin (FXN) gene harbouring at least one variation or a fragment thereof, and a microRNA (miRNA) targeting said 3' UTR of the FXN gene harbouring at least one variation, or said fragment thereof;

b) identifying as a compound suitable for the treatment and/or prevention of a frataxin inactivation-related disease the candidate compound which inhibits the interaction of said miRNA with the 3' UTR of the FXN gene harbouring at least one variation, or said fragment thereof.

The invention further provides an inhibitor of a microRNA (miRNA) which specifically interacts with a 3'UTR of the FXN gene or a fragment thereof including at least one variation selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043, for use as a medicament.

Also provided is an inhibitor of a microRNA (miRNA) which specifically interacts with a 3'UTR of the FXN gene or a fragment thereof including at least one variation selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043, for use for treating Friedreich's ataxia (FRDA).

The invention also concerns a pair of primers suitable for amplifying the FRDA 3' untranslated region (UTR) of the frataxin (FXN) gene, or a fragment thereof including at least one variation selected from the group consisting of rs10890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, or their complementary sequences, for use for diagnosing or prognosing Friedreich's ataxia (FRDA).

The invention further provides a probe specifically hybridizing to a region of the FRDA 3' untranslated region (UTR) of the frataxin (FXN) gene or a fragment thereof including at least one variation selected from the group consisting of rs10890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, or the sequence complementary to said region, for use for diagnosing or prognosing Friedreich's ataxia (FRDA).

The invention further provides a method for preventing and/or treating Friedreich's ataxia (FRDA) in an individual, the method comprising:

- performing the method of diagnosis or prognosis of Friedreich's ataxia (FRDA) as defined above, and

- when deducing said individual has developed or is at risk of developing Friedreich's ataxia (FRDA), administering a suitable treatment to said individual.

DETAILED DESCRIPTION OF THE INVENTION Method of diagnosis or prognosis of Friedreich's ataxia (FRDA)

The inventors have developed an in vitro method of diagnosis or prognosis of Friedreich's ataxia (FRDA) in an individual, which comprises: a) determining whether the individual harbours at least one variation in the 3' untranslated region (UTR) of the frataxin (FXN) gene; and

b) deducing that the individual has developed or is at risk of developing FRDA if said at least one variation is present or that the individual is not at risk of developing FRDA if said at least one variation is absent.

"Frataxin protein" refers to a nuclear-encoded mitochondrial protein coded in humans by the FXN gene localized on chromosome 9 (gi|224589821 :71650479- 71715094 Homo sapiens chromosome 9, GRCh37.p10 Primary). An exemplary sequence of FXN gene is shown in SEQ ID NO: 1 . The function of this enzyme (EC 1 .1 6.3.1 ) is not entirely clear, but it seems to be involved in promotion of the biosynthesis of heme and assembly and repair of iron-sulfur clusters by delivering Fe(2+) to proteins involved in these pathways. This protein is found in cells throughout the body, with the highest levels in the heart, spinal cord, liver, pancreas, and muscles used for voluntary movement (skeletal muscles). Within cells, frataxin is found in mitochondria. Defects in FXN are the cause of FRDA. In the normal version of the Frataxin (FXN) gene, a sequence of DNA (labeled "GAA") motif within intron 1 is repeated between 6 and 36 times this expansion is named "GAA repeats". In the defective FXN gene, the GAA repeats occur 70 to 1700 times. FRDA is well known to one of skill in the art and is notably an autosomal recessive, progressive degenerative disease characterized by neurodegeneration and cardiomyopathy; it is the most common inherited ataxia. Acute FRDA according to the invention refers to a FRDA with severe symptoms.

Alternative splicing of FXN gene results in multiple transcript variants:

- a first transcript "variant 1 " (name FXN-001 , NM_000144.4 NCBI database, SEQ ID NO: 23) coding for frataxin Isoform 1 (Q16595-1 , NP_000135.2 Gl:31077081 NCBI database, SEQ ID NO: 2) of 210 amino acids,

- a transcript "variant 2" (name FXN-004, NM_181425.2; SEQ ID NO: 24) coding for the frataxin Isoform 2 (NP_852090.1 Gl:31742516) of 196 amino acids,

- a third transcript "variant 3" (name FXN-003, ENST00000498653) coding for the isoform 3 of frataxin of 135 amino acids.

The untranslated region (or UTR) refers to either of the two regions immediately adjacent to the coding sequence on a strand of mature mRNA. When it is found on the 5' side, it is called the 5' UTR (or leader sequence), or if it is found on the 3' side, it is called the 3' UTR (or trailer sequence). The reference sequence of the 3'UTR of the FXN gene, as used herein, is shown in SEQ ID NO: 35, preferably from position 1 to position 1451 of SEQ ID NO: 35. As compared with the transcript variant sequences and the gene sequence described herein, the 3'UTR is the 3' extremity of sequence SEQ ID NO: 23 starting at position 854 (positions 854-7168 of SEQ ID NO: 23, preferably positions 854- 2304 of SEQ ID NO: 23), or the 3' extremity of SEQ ID NO: 24 starting at position 862 (positions 862-7176 of sequence SEQ ID NO: 24, preferably positions 862-2312 of SEQ ID NO: 24). The 3'UTR of FXN is also located at positions 37201 to 43515 of SEQ ID NO: 1 , preferably at positions 37201 to 38651 of SEQ ID NO: 1 . The reference sequence SEQ ID NO: 35 contains a G at position 798, whereas SEQ ID NO:1 , SEQ ID NO:23 and SEQ ID NO:24 contain a T at the corresponding position (position 37998 of SEQ ID NO :1 , position 1651 of SEQ ID NO:23 or position 1659 of SEQ ID NO:24).

All nucleotide positions variation in the present specification are given in reference to the reference sequence SEQ ID NO: 35 or transcript variant 1 (SEQ ID NO: 23) or variant 2 (SEQ ID NO: 24), as specified.

According to the invention "frataxin protein" means isoform 1 , 2 or 3 of frataxin protein.

As will be clear to one of skill in the art, the expression "determining whether the individual harbours at least one variation of the 3' untranslated region (UTR) of the frataxin (FXN) gene" refers to determining the presence or absence of at least one variation, such as a substitution (in particular a single nucleotide variation (SNV) also called single nucleotide polymorphism (SNP, for example T>C), a deletion (del; for example GTT>-) or an insertion (ins, for example ->GTT), at a site of polymorphism, i.e. a site of the genome which is not identical in all individuals, by comparison with the 3'UTR sequence as shown in SEQ ID NO: 35. It is then deduced that the individual has developed or is at risk of developing FRDA if said at least one variation is present. Reference "C>T" hereunder such as for example in "C>T at position 246 of SEQ ID NO: 35" means that at position 246 of SEQ ID NO: 35, a C is replaced by a T.

Preferably, in the method of the invention, it is determined whether the individual harbours at least one variation in a fragment of the 3'UTR of the FXN gene corresponding to nucleotides at positions 854 to 7146 of SEQ ID NO: 23 (or 862 to 7154 of sequence SEQ ID NO: 24); or at positions 854 to 2326 of SEQ ID NO: 23 (or at positions 862 to 2334 of sequence SEQ ID NO: 24), i.e. a fragment of the 3'UTR consisting of nucleotides at positions 1 to 1473 of SEQ ID NO: 35; or at positions 854 to 2304 of SEQ ID NO: 23 (or at positions 862 to 2312 of sequence SEQ ID NO: 24), i.e. a fragment of the 3'UTR consisting of nucleotides at positions 1 to 1451 of SEQ ID NO: 35; or corresponding to nucleotides at positions 900 to 1700 of SEQ ID NO:23 (or 908 to 1708 of sequence SEQ ID NO: 24) or 1098 to 1652 of SEQ ID NO: 23 (or 1 106 to 1660 of SEQ ID NO: 24). In the transcript variant 3 (FXN-003), the 3'UTR ends at position 343 of SEQ ID NO:35, and in the transcript variant 1 (FXN-001 ), the 3'UTR ends at position 1451 of SEQ ID NO:35. Accordingly, in an embodiment, it is determined whether the individual harbours at least one variation in a fragment of the 3'UTR consisting of nucleotides at positions 1 to 343 of SEQ ID NO:35 or at positions 1 to 1451 of SEQ ID NO:35.

In one embodiment, a fragment of the 3'UTR of the FX N gene may be of 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, 120, 200, 250, 300, 350, 500, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6300 contiguous nucleotides of a sequence identical (or complementary) to sequence SEQ ID NO: 35. Preferably, said fragment comprises position 633 of SEQ ID NO:35 (or position 1486 of SEQ ID NO: 23 or position 1494 of SEQ ID NO: 24) and/or bases 78-80 of SEQ ID NO:35 (or bases 931 -933 of SEQ ID NO: 23 or 939-941 of SEQ ID NO: 24) and/or position 246 of SEQ ID NO:35 (or position 1099 of SEQ ID NO: 23 or position 1 107 of SEQ ID NO: 24) and/or position 423 of SEQ ID NO:35 (or position 1276 of SEQ ID NO: 23 or position 1284 of SEQ ID NO: 24) and/or position 540 of SEQ ID NO:35 (or position 1393 of SEQ ID NO: 23 or position 1401 of SEQ ID NO: 24) and/or position 622 of SEQ ID NO:35 (or position 1475 of SEQ ID NO: 23 or position 1483 of SEQ ID NO: 24) and/or position 798 of SEQ ID NO:35 (position 1651 of SEQ ID NO: 23 or position 1659 of SEQ ID NO: 24).

In one embodiment, said method of diagnosis or prognosis of Friedreich's ataxia (FRDA) comprises the step of determining whether the individual harbours at least one variation in the 3'UTR of FXN gene selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043 (NCBI dbSNP database) and deducing that the individual has developed or is at risk of developing FRDA if said at least one variation is present.

As intended herein, the expression "the individual is at risk of developing FRDA" notably means that the individual is predisposed to develop the FRDA or that the individual presents an increased risk of developing FRDA with respect to a general population of individuals or to a population of individuals which do not harbour the above- defined at least one variation of the 3'UTR of FXN gene.

Said variations are located in the 3'UTR of FXN gene and can be positioned according to the sequence of FXN gene SEQ ID NO: 1 or the transcript variant 1 (of sequence SEQ ID NO: 23) or the transcript variant 2 (of sequence SEQ ID NO: 24) of FXN gene or the reference sequence SEQ ID NO:35. The following polymorphisms have notably been identified in the 3'UTR of FXN gene:

- C/T at position 1486 of SEQ ID NO: 23 or 1494 of SEQ ID NO: 24 or 633 of SEQ ID NO:35;

- TTG/- (deletion of TTG) at positions 931 -933 of SEQ ID NO: 23 or 939-941 of

SEQ ID NO: 24 or 78-80 of SEQ ID NO:35;

- C/T at position 1099 of SEQ ID NO: 23 or 1 107 of SEQ ID NO: 24 or 246 of SEQ ID NO:35;

- A/G at position 1276 of SEQ ID NO: 23 or 1284 of SEQ ID NO: 24 or 423 of SEQ ID NO:35;

- T/C at position 1393 of SEQ ID NO: 23 or 1401 of SEQ ID NO: 24 or 540 of SEQ ID NO:35;

- A/G at position 1475 of SEQ ID NO: 23 or 1483 of SEQ ID NO: 24 or 622 of SEQ ID NO:35; and

- T/G at position 1651 of SEQ ID NO: 23 or 1659 of SEQ ID NO: 24 or 798 of SEQ

ID NO:35.

According to the invention, the "at least one variation in the 3' untranslated region (UTR) " denotes at least one nucleotide variation compared to SEQ ID NO:35, SEQ ID NO: 23 or SEQ ID NO: 24, said at least one variation is selected from the group consisting of:

- C>T at position 633 of SEQ ID NO:35, or at position 1486 of SEQ ID NO: 23 or at position 1494 of SEQ ID NO: 24 (rs4744808);

- a deletion of bases TTG at positions 78-80 of SEQ ID NO:35, or at position 931 -933 of SEQ ID NO: 23 or 939-941 of SEQ ID NO: 24 (rs60033969);

- C>T at position 246 of SEQ ID NO:35, or at position 1099 of SEQ ID NO: 23 or at position 1 107 of SEQ ID NO: 24 (rs10890);

- A>G at position 423 of SEQ ID NO:35, or at position 1276 of SEQ ID NO: 23 or at position 1284 of SEQ ID NO: 24 (rs4745577);

- T>C at position 540 of SEQ ID NO:35; or at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 (rs4744806);

- A>G at position 622 of SEQ ID NO:35, or at position 1475 of SEQ ID NO: 23 or at position 1483 of SEQ ID NO: 24 (rs4744807); and

- G>T at position 798 of SEQ ID NO:35 (T is present at position 1651 of SEQ ID NO: 23 and at position 1659 of SEQ ID NO: 24) (rs1 1 145043). Preferably, at least 2, 3, 4 or 5 of the above variations are determined. For example, 2, 3, 4, 5, 6 or 7 of the above variations are determined.

In an embodiment it is determined if the individual harbours the following variations in the 3' untranslated region (UTR) of the FAN gene:

i) C>T at position 246 of SEQ ID NO:35, or at position at position 1099 of

SEQ ID NO: 23 or at position 1 107 of SEQ ID NO: 24; or a deletion of bases TTG at positions 78-80 of SEQ ID NO:35, or at position 931 -933 of SEQ ID NO: 23 or 939-941 of SEQ ID NO: 24, and ii) A>G at position 423 of SEQ ID NO:35, or at position 1276 of

SEQ ID NO: 23 or at position 1284 of SEQ ID NO: 24, and iii) T>C at position 540 of SEQ ID NO:35; or at position 1393 of

SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24, and iv) C>T at position 633 of SEQ ID NO:35, or at position 1486 of

SEQ ID NO: 23 or at position 1494 of SEQ ID NO: 24; or

A>G at position 622 of SEQ ID NO:35, or at position 1475 of SEQ ID NO: 23 or at position 1483 of SEQ ID NO: 24, and v) G>T at position 798 of SEQ ID NO:35.

Detection of polymorphisms according to the invention may be done by techniques well known to one of skill in the art and notably by Allele Specific Hybridization, Primer Extension, Allele Specific Oligonucleotide Ligation or sequencing (Kwok PY, Chen X. Curr Issues Mol Biol. 2003 Apr;5(2):43-60; Review), or by RT-PCR of transcripts.

In one embodiment, the method of the invention further comprises determining if the individual is homozygote for said at least one variation and, if homozygote, deducing that the individual has developing or is at risk of developing an acute FRDA.

In one embodiment, said method further comprises determining if the individual is homozygote for at least rs4744806 and rs1 1 145043 variations or preferably at least for i) rs10890 or rs60033969, and ii) rs4745577, iii) rs4744806, iv) rs4744808 or rs4744807, and v) rs1 1 145043 variations. Variations rs10890 and rs60033969 being associated, determination of homozygosity can be made indifferently for rs10890 and rs60033969. Variations rs4744808 and rs4744807 are also associated, and can be determined indifferently.

In one embodiment, said method further comprises determining in a fragment of the 3'UTR of FXN comprising nucleotides 1 to 1473 of SEQ ID NO: 35 (or 854 to 2326 of sequence SEQ ID NO: 23 or 862 to 2334 of sequence SEQ ID NO: 24), preferably nucleotides 1 to 1451 of SEQ ID NO: 35 (or 854 to 2304 of sequence SEQ ID NO: 23 or 862 to 2312 of sequence SEQ ID NO: 24) , if the individual is homozygote for at least a C at position 540 of SEQ ID NO:35; or at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24, and a T at position 798 of SEQ ID NO:35 or at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24, or in particular at least for:

i) a T at position 246 of SEQ ID NO:35, or at position 1099 of

SEQ ID NO: 23 or at position 1 107 of SEQ ID NO: 24 or a deletion of bases TTG at positions 78-80 of SEQ ID NO:35, or at position 931 -933 of SEQ ID NO: 23 or 939-941 of SEQ ID NO: 24,

ii) a G at position 423 of SEQ ID NO:35, or at position 1276 of

SEQ ID NO: 23 or at position 1284 of SEQ ID NO: 24,

iii) a C at position 540 of SEQ ID NO:35; or at position 1393 of

SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24,

iv) a T at position 633 of SEQ ID NO:35, or at position 1486 of

SEQ ID NO: 23 or at position 1494 of SEQ ID NO: 24 or a G at position 622 of SEQ ID NO:35, or at position 1475 of SEQ ID NO: 23 or at position 1483 of SEQ ID NO: 24, and

v) a T at position 798 of SEQ ID NO:35, or at position 1651 of

SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24.

In another embodiment, said method further comprises determining if the individual is homozygote for the T-G-C-T-T haplotype.

According to the invention, a sequence which comprises the "T-G-C-T-T haplotype" is a sequence that differs from positions 854 to 2326, preferably from positions 854 to 2304, or more preferably from positions 1098 to 1652 of sequence SEQ ID NO: 23 only by the presence of:

ii a T at position 1099 of SEQ ID NO: 23;

ii) a G at position 1276 of SEQ ID NO: 23;

iii) a C at position 1393 of SEQ ID NO: 23; and

iyi a T at position 1486 of SEQ ID NO: 23.

As compared with SEQ ID NO:35, the "T-G-C-T-T haplotype" differs from positions 1 to 1473 of SEQ ID NO: 35, preferably from position 1 to 1451 of SEQ ID NO: 35, only by the presence of:

ii a T at position 246 of SEQ ID NO:35;

ii) a G at position 423 of SEQ ID NO:35;

iiil a C at position 540 of SEQ ID NO:35; i i a T at position 633 of SEQ ID NO:35; and

Yl a T at position 798 of SEQ ID NO:35.

Such sequence may be a sequence comprising or consisting of SEQ ID NO: 34 or SEQ ID NO: 36, preferably a sequence comprising or consisting of SEQ ID NO: 34 or of positions 1 to 1451 of SEQ ID NO: 36.

In the examples described hereafter, the few patients that did not harbour the T-G- C-T-T haplotype displayed less severe forms of FRDA. Accordingly, an individual found to be harbouring the T-G-C-T-T haplotype is at risk of developing a severe form of FRDA.

According to another embodiment, said method further comprises measuring the level of expression of hsa-miR-1 24-3p (miRbase) (SEQ ID NO: 13) and/or of frataxin protein (SEQ ID NO: 2) and/or determining the presence or absence of a GAA repeats of preferably more than 70 GAA repeats, within the FXN gene (SEQ ID NO: 1 ).

Methods and techniques for the identification and sizing of the GAA repeats are well known to one of skill in the art, such identification and sizing may be performed by PCR and separation on agarose gel using primers flanking the GAA repeat for example, by using the following primers GAA-104F: 5'-GGCTTAAACTTCCCACACGTGTT-3'; (SEQ ID NO: 32) and GAA-629R: 5'-AGGACCATCATGGCCACACTT-3' (SEQ ID NO: 33) (Filla A. et al Am J Hum Genet. 1996 September; 59(3): 554-560).

In one embodiment, the level of expression of said gene(s) is determined by detecting transcription product(s) and/or translation product(s) of said gene(s). Level of expression of gene can be performed by methods which are well known to the person skilled in the art, including in particular quantitative methods involving reverse transcriptase PCR (RT-PCR), such as real-time quantitative RT-PCR (qRT-PCR), and methods involving the use of DNA arrays (macroarrays or microarrays) and In Situ hybridizations.

Level of expression of gene(s) may further be assessed by using immunologic methods such as detection using polyclonal or monoclonal antibodies. Suitable immunologic methods include enzyme linked immunoassays (ELISA), sandwich, direct, indirect, or competitive ELISA assays, enzyme linked immunospotassays (ELIspot), radio immunoassays (RIA), flow-cytometry assays (FACS), immunohistochemistry, Western Blot, fluorescence resonance energy transfer (FRET) assays, protein chip assays using for example antibodies, antibody fragments, receptor ligands or other agents binding the proteins coded by said genes. The level of frataxin protein (SEQ ID NO: 2) can be detected using a capture- sandwich immunoassay which can include using an anti-frataxin polypeptide antibody. An anti-frataxin polypeptide antibody can be labeled for detection, for example, with a radioactive molecule, a fluorescent molecule, or a bioluminescent molecule. Frataxin polypeptides can also be detected indirectly using a labeled antibody that binds to an anti- frataxin polypeptide antibody that binds to a frataxin polypeptide. An anti-frataxin antibody can be a polyclonal or monoclonal antibody. For example, monoclonal anti-human frataxin polypeptide antibodies are commercially available, e.g., from MitoSciences Inc., clone #17A1 1 AC7.

Isolated polynucleotide, Vector and host cells

The invention further provides an isolated polynucleotide comprising a sequence of 3'UTR of FXN gene having at least one variation, preferably selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043 and a polynucleotide of interest, wherein said polynucleotide does not comprise frataxin (FXN) gene, preferably the frataxin (FXN) gene of sequence SEQ ID NO: 1 .

The term "nucleic acid" will generally refer to at least one molecule or strand of DNA, RNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A," guanine "G," thymine "T," and cytosine "C") or RNA (e.g. A, G, uracil "U," and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide." The term "oligonucleotide" refers to at least one molecule of between about 3 and about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of greater than about 100 nucleobases in length. These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially or fully complementary to the at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double- stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a strand of the molecule.

A nucleic acid may be made by any technique known to one of ordinary skill in the art. Non-limiting examples of synthetic nucleic acid, particularly a synthetic oligonucleotide, include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such described by Froehler et at., 1986 via deoxynucleoside H-phosphonate intermediates. A non-limiting example of enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCR™ or the synthesis of oligonucleotides. A non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells (see for example, Sambrook et al. 2000). A nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al. 2000). The nucleic acid molecule is preferably isolated, which means that it is essentially free of other nucleic acids. Essentially free from other nucleic acids means that the nucleic acid molecule is at least about 90%, preferably at least about 95% and, more preferably at least about 98% free of other nucleic acids. Preferably, the molecule is essentially pure, which means that the molecule is free not only of other nucleic acids, but also of other materials used in the synthesis and isolation of the molecule. Materials used in synthesis include, for example, enzymes. Materials used in isolation include, for example, gels, such as SDS-PAGE. The molecule is at least about 90% free, preferably at least about 95% free and, more preferably at least about 98% free of other nucleic acids and such other materials.

The polynucleotide of interest may be a gene of interest such as a gene coding for a protein of interest or a reporter gene. The term "reporter" refers to a gene product that can be easily measured when it is fused to transcriptional control elements, such as those in promoters, and that "reports" the effect of a signalling cascade or experimental conditions on gene expression in living cells. The reporter gene may be for example, green fluorescent or luminescent reporter proteins such as green fluorescent protein (GFP) or luciferase, enzymatic reporters such as alkaline phosphatase, or colorimetric reporters such as lacZ, antibiotics gene (ampicillin, kanamycin, or chloramphenicol resistance). The reporter gene may further be a gene of beta-galactosidase (beta-gal), alkaline phosphatase, horseradish peroxidase (HRP) or growth hormone (GH).

In certain embodiments, a "gene" refers to a nucleic acid that is transcribed. In certain aspects, the gene includes regulatory sequences involved in transcription, or message production or composition. In particular embodiments, the gene comprises transcribed sequences that encode for a protein, polypeptide or peptide. The gene further comprises transcribed sequences which have a regulatory function such as UTR sequences. In other particular aspects, the gene comprises a nucleic acid, and/or encodes a polypeptide or peptide-coding sequences of a gene that is defective or mutated in a neurodegenerative disorder. In keeping with the terminology described herein, an "isolated gene" may comprise transcribed nucleic acid(s), regulatory sequences, coding sequences, or the like, isolated substantially away from other such sequences, such as other naturally occurring genes, regulatory sequences, polypeptide or peptide encoding sequences, etc. In this respect, the term "gene" is used for simplicity to refer to a nucleic acid comprising a nucleotide sequence that is transcribed, and the complement thereof. In particular aspects, the transcribed nucleotide sequence comprises at least one functional protein, polypeptide and/or peptide encoding unit. As will be understood by those in the art, this functional term "gene" includes both genomic sequences, RNA or cDNA sequences, or smaller engineered nucleic acid segments, including nucleic acid segments of a non-transcribed part of a gene, including but not limited to the non-transcribed promoter or enhancer regions of a gene. Smaller engineered gene nucleic acid segments may express, or may be adapted to express using nucleic acid manipulation technology, proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or such like.

The isolated polynucleotide comprising the 3'UTR sequence of the invention or a fragment thereof and the polynucleotide of interest which is not frataxin (FXN) gene may be gene expression cassettes, gene expression constructs and gene expression systems.

The 3'UTR sequence of the invention may be located upstream of a terminator sequence present in the gene expression cassette, gene expression construct or gene expression system. Where the polynucleotide of interest is a gene encoding a protein of interest, the 3'UTR sequence of the invention may be located downstream of said gene. Thus, the 3'UTR sequence of the invention may be located between a gene encoding a protein of interest and a terminator sequence.

Most preferably the 3'UTR sequence of the invention is immediately downstream of the ORF of the gene (after the stop codon) and upstream of the terminator sequence.

In one embodiment, said isolated polynucleotide comprises a sequence of the 3'UTR of the FXN gene which comprises at least a C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 or at position 540 of SEQ ID NO:35, and a T at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 or at position 798 of SEQ ID NO:35.

In another embodiment, the invention provides an isolated polynucleotide comprising the FRDA-3' untranslated region (UTR) or a fragment thereof and a polynucleotide of interest, wherein said isolated polynucleotide does not comprise frataxin {FXN) gene (SEQ ID NO: 1 ). According to the invention, the "FRDA-3'UTR" comprises or consists of sequence SEQ ID NO: 34 or SEQ ID NO: 36, preferably of SEQ ID NO: 34 or positions 1 to 1451 of SEQ ID NO: 36.

A fragment of FRDA-3'UTR may be of 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, 120, 200, 250, 300, 350, 500, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6300 b of contiguous nucleotides of a sequence identical (or complementary) to sequence SEQ ID NO: 34. A fragment of FRDA-3'UTR may be of 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, 120, 200, 250, 300, 350, 500, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6300 b of contiguous nucleotides of a sequence identical (or complementary) to sequence SEQ ID NO: 23 or SEQ ID NO: 24 which comprises at least one variation selected from the group consisting of T at position 1486 of SEQ ID NO: 23 or at position 1494 of SEQ ID NO: 24 (rs4744808); a deletion of bases 931 -933 of SEQ ID NO: 23 or 939-941 of SEQ ID NO: 24 (rs60033969); T at position 1099 of SEQ ID NO: 23 or at position 1 107 of SEQ ID NO: 24 (rs10890); G at position 1276 of SEQ ID NO: 23 or at position 1284 of SEQ ID NO: 24 (rs4745577); C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 (rs4744806); G at position 1475 of SEQ ID NO: 23 or at position 1483 of SEQ ID NO: 24 (rs4744807); G at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 (rs1 1 145043). Preferably, said fragment comprises a sequence identical (or complementary) to sequence SEQ ID NO: 23 or SEQ ID NO: 24 which comprises C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 (rs4744806) and G at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 (rs1 1 145043).

A fragment of FRDA-3'UTR may be a sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 34, a fragment of SEQ ID NO: 36 (corresponding to variation rs10890, rs4745577, rs4744806, rs4744807 and rs1 1 145043), SEQ ID NO: 26 (corresponding to variation rs60033969), SEQ ID NO: 27 (corresponding to variation rs10890), SEQ ID NO: 28 (corresponding to variation rs4745577), SEQ ID NO: 30 (corresponding to variation rs4744806), SEQ ID NO: 29 (corresponding to variation rs4744807), SEQ ID NO: 25 (corresponding to variation rs4744808) and SEQ ID NO: 31 (corresponding to variation rs1 1 145043).

A fragment of FRDA-3'UTR may be at least 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, 120, 200, 250, 300, 350, 500, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500 or 6000 nucleotides in length. A fragment of FRDA-3'UTR may be at most 20, 30, 40, 50, 60, 70, 80, 90, 1 00, 1 15, 120, 200, 250, 300, 350, 500, 800, 900, 1000, 1 500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000 or 6300 nucleotides in length.

For example, a fragment of FRDA-3'UTR is at least 1 5, 20, 30, 40, 50, 60, 70, 80, 90, 1 00, 1 1 5, 1 20, 200, 250, 300, 350, 500, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500 or 6000 nucleotides in length and at most 20, 30, 40, 50, 60, 70, 80, 90, 1 00, 1 1 5, 120, 200, 250, 300, 350, 500, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000 or 6300 nucleotides in length.

A preferred isolated polynucleotide according to the invention comprises:

- the FRDA-3' untranslated region (UTR) or a fragment of at least 500 nucleotides thereof, and

- a polynucleotide of interest,

wherein said isolated polynucleotide does not comprise frataxin (FXN) gene (SEQ ID NO: 1 ).

The gene expression systems of the invention may thus be used to express a protein of interest in a host organism or host cell. In this case, the protein of interest may also be heterologous to the host organism or host cell in question i.e. introduced into the cells in question using genetic engineering, i.e. by human intervention. A heterologous gene in an organism may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence

Gene expression systems will typically be operably linked to promoter and terminator sequences. In embodiments of the invention, the promoter may be an inducible promoter.

Thus, gene expression systems may further comprise a termination sequence and the gene encoding a protein of interest may be located between the enhancer sequence and the termination sequence, i.e. downstream (3') of the enhancer sequence and upstream (5') of the termination sequence.

The gene expression system may be in the form of an expression construct or expression cassette.

Thus the invention further provides an expression cassette comprising:

(i) a promoter, operably linked to, (ii) an enhancer sequence as described above,

(iii) a gene of interest it is desired to express and,

(iv) a terminator sequence.

According to the invention, the terminator sequence comprises the 3'UTR sequence of the invention or a fragment thereof including at least one variation selected from the group consisting of rs10890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, preferably the FRDA-3'UTR or a fragment thereof. More preferably, the terminator sequence comprises the sequence SEQ ID NO: 36 or a fragment thereof.

Also is provided a vector comprising said isolated polynucleotide of the invention or an expression cassette which comprises the 3'UTR of the invention or a fragment thereof.

In its broadest sense, a vector is any vehicle capable of facilitating the transfer of a nucleic acid to a host cells so that the protein coded by said nucleic acid can be expressed by the host cell. The vector generally transports a nucleic acid to host cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. The vector optionally includes the above-described expression cassette sequence to regulate the expression of the gene of interest in host cells. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the 3'UTR sequences of the invention or a fragment thereof.

Also is provided a host cell comprising the vector or the isolated polynucleotide of the invention or at least one copy of the FXN gene wherein the 3' untranslated region (UTR) is the FRDA-3'UTR or a 3'UTR comprising at least one variation, preferably selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043 or a fragment thereof.

In another embodiment, the host cell comprises at least one copy of the FXN gene wherein the 3'UTR comprises at least a C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 and a T at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 or preferably at least a T-G-C-T-T haplotype.

Preferably, the host cell comprises at least one copy of the FXN gene wherein the 3'UTR has the sequence SEQ ID NO: 36, preferably wherein the 3'UTR has a sequence consisting of positions 1 to 1451 of SEQ ID NO: 36. Said human FXN gene may further comprises in the intron 1 , 60 to 1700 GAA repeats.

A "host cell" is a cell that has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule such as the isolated polynucleotide of the invention. Host cells containing the transformed polynucleotide are referred to as "transgenic" host cells. Said host cells may be used to obtain organisms which are not human or may be obtained from said organisms. The host cell may be a prokaryotic cell (bacteria or cyanobacteria) or an eukaryotic cell (e.g. fungi, algae, yeast, plant, mammalian or insect cells).

Method for screening for compounds suitable for the treatment and/or prevention of Friedreich's ataxia (FRDA)

b) identifying as a compound suitable for the treatment and/or prevention of FRDA the candidate compound which inhibits the interaction of said miRNA with the 3' UTR of the FXN gene harbouring at least one variation, or said fragment thereof.

The fragment used in step a) also preferably comprises said at least one variation.

According to one embodiment, said method further comprises the step of administering said candidate compound to a cellular model or to a non-human animal model of a frataxin inactivation-related disease such as FRDA or to the host cell of the invention.

A cellular model or a non-human animal model of a frataxin inactivation-related disease such as FRDA may be a host cell of a non-human animal model tansfected with the isolated polynucleotide or the vector of the invention.

A cellular model or a non-human animal model may comprise at least one copy of the entire human FXN gene with 60 to 1000 GAA repeats. In one embodiment, the cellular model or non-human animal model comprises at least one copy of the FXN gene wherein the 3' untranslated region (UTR) is the FRDA- 3'UTR or a 3'UTR of the FXN gene which comprises at least one variation, preferably selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043 or a fragment thereof.

In another embodiment, the cellular model or non-human animal model comprises at least one copy of the FXN gene wherein the 3'UTR comprises at least a C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 or at position 540 of SEQ ID NO:35, and a T at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 or at position 540 of SEQ ID NO:35, or preferably at least a T-G-C-T-T haplotype. Preferably, the cellular model or non-human animal model comprises at least one copy of the FXN gene wherein the 3'UTR has the sequence SEQ ID NO: 36, preferably wherein the 3'UTR has a sequence consisting of positions 1 to 1451 of SEQ ID NO: 36, or wherein the fragment of such 3'UTR comprises the sequence SEQ ID NO: 34.

Said human FXN gene may further comprises in the intron 1 , 60 to 1700 GAA repeats.

Inhibitor of microRNAs for use for preventing and/or treating Friedreich's ataxia (FRDA)

The invention further provides an inhibitor of a microRNA (miRNA), which miRNA specifically interacts with a 3' untranslated region (UTR) of the frataxin (FXN) gene including at least one variation selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043, for use as a medicament.

Also provided is an inhibitor of a microRNA (miRNA), which miRNA specifically interacts with a 3' untranslated region (UTR) of the frataxin (FXN) gene including at least one variation selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043, for use for preventing and/or treating Friedreich's ataxia (FRDA).

By "treatment of FRDA" or "treating FRDA", it is meant herein a therapeutic use, i.e. on an individual having FRDA.

By "prevention of FRDA" or "preventing FRDA", it is meant herein a prophylactic use, i.e. on an individual at risk of developing FRDA. The term "treatment" includes a treatment leading to complete cure of FRDA, a treatment to cure or relieve the symptoms of the disease, such as heart condition and diabetes, a treatment that slows down the progression of FRDA and/or a treatment prolonging the survival of the individual having FRDA.

The term "prevention" includes a treatment for delaying the onset of FRDA and/or of the symptoms of FRDA, such as heart condition and diabetes.

"MicroRNAs" or "miRNAs" are endogenously encoded RNAs that are about 22- nucleotide-long, that post-transcriptionally regulate target genes and are generally expressed in a highly tissue-specific or developmental-stage-specific fashion. One can design and express artificial miRNAs based on the features of existing miRNA genes. The miRNA may be hsa-miR-559 (MIMAT0003223 SEQ ID NO: 14), hsa-miR-589 (Acc N ° MIMAT0004799 SEQ ID NO: 15), hsa-miR-1270-1 (Acc N ° MIMAT0005924 SEQ ID NO: 16 ), hsa-miR-620 (Acc N ° MIMAT0003289 SEQ ID NO: 17), hsa-miR-522 (Acc N ° MIMAT0002868 SEQ ID NO: 18), hsa-miR-299-3p (Acc N ° MIMAT0000687 SEQ ID NO: 19), hsa-miR-506 (Acc N ° MIMAT0002878 SEQ ID NO: 20), hsa-miR-124-3p (Acc N ° MIMAT0000422 SEQ ID NO: 13, SEQ ID NO:21 ) aid hsa-miR-624 (Acc N ° MIMAT0004807SEQ ID NO: 22).

Inhibitor of a microRNAs refers to nucleic acid-based molecules that suppress miRNA function. Synthetic miRNA inhibitor may be designed by incorporating the reverse complement of the mature miRNA (the target site) and may be chemically modified to prevent any cleavage induced by the RNA-induced silencing complex (RISC), to enhance binding affinity and provide resistance to nucleolytic degradation. The miRNA inhibitor may be in particular a locked nucleic acid (LNA) that hybridise to complementary nucleic acids (Petersen M. and Wengel J. (2003). Trends Biotechnol. 21 , 74 - 81 ). An inhibitor of a miRNA may be chemically modified and/or single stranded nucleic acids designed to specifically bind to and inhibit a specific miRNA molecule. These inhibitors can be introduced into cells using transfection or electroporation. An inhibitor of the miRNA hsa- miR-124-3p of sequence SEQ ID NO: 13 (mature form) may be the complementary sequence of its mature form.

Examples of miRNA inhibitor may be a fragment of the 3'UTR of the FXN gene comprising at least one variation selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043, preferably selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043. In one embodiment, the miRNA inhibitor is selected from the group consisting of sequences comprising or consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31 .

Primers and probes for use for diagnosing or prognosing Friedreich's ataxia (FRDA) The invention also concerns a pair of primers suitable for amplifying the FRDA 3' untranslated region (UTR) of the frataxin (FXN) gene, or a fragment thereof including at least one variation selected from the group consisting of rs10890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, or their complementary sequences, for use for diagnosing or prognosing Friedreich's ataxia (FRDA).

The invention also relates to the use of a pair of primers suitable for amplifying the

FRDA 3' untranslated region (UTR) of the frataxin (FXN) gene or a fragment thereof, said FRDA 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of rs10890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, or their complementary sequences, for diagnosing or prognosing Friedreich's ataxia (FRDA).

Primers may comprise, consist or consist essentially of a nucleotide sequence of 17 to about 200 consecutive nucleotides located upstream and downstream of a position selected from the group consisting of 1099, 931 -933, 1276, 1393, 1486, 1475, 1651 of SEQ ID NO: 23 or the complement thereof.

Primers may comprise, consist or consist essentially of a nucleotide sequence of

17 to about 200 consecutive nucleotides selected from the nucleotide sequence at position 854 to 7168 of SEQ ID NO: 23, preferably at positions 854 to 2304 of SEQ ID NO: 23, or the complement thereof, preferably from sequence located between the positions 854 to 2000 or 900 to 1700 of SEQ ID NO: 23 or the complement thereof.

In one embodiment said primers are selected from the group consisting of

SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and the complement thereof,

Primers may also comprise these nucleotide sequences located at their extreme 3' end, and further comprise unrelated sequences or sequences derived from the mentioned nucleotide sequences, but comprising mismatches.

The invention further provides a probe specifically hybridizing to a region of the FRDA 3' untranslated region (UTR) of the frataxin (FXN) gene including at least one variation selected from the group consisting of rs10890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, or the sequence complementary to said region, for use for diagnosing or prognosing Friedreich's ataxia (FRDA).

The invention also relates to the use of a probe specifically hybridizing to a region of the FRDA 3' untranslated region (UTR) of the frataxin (FXN) gene including at least one variation selected from the group consisting of rs1 0890, rs60033969, rs4745577, rs4744806, rs4744808, rs4744807 and rs1 1 145043, or the sequence complementary to said region, for diagnosing or prognosing Friedreich's ataxia (FRDA).

In one embodiment, the probe according to the invention comprises or consists (or consists essentially) of a sequence which corresponds (or is complementary to) a sequence having between 80% and 1 00% sequence identity with sequence SEQ ID NO: 34 or SEQ ID NO: 36 or positions 1 to 1451 of SEQ ID NO: 36. Preferably, the probe comprises or consists (or consists essentially) of a sequence which corresponds (or is complementary to) a sequence having between at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleic acid sequence identity with sequence SEQ ID NO: 34 or SEQ ID NO: 36 or positions 1 to 1451 of SEQ ID NO: 36 as disclosed herein. Nucleic acid sequence identity can be calculated by methods well-known to one of skill in the art. The percentage of identity may be calculated by performing a pairwise global alignment based on the Needleman-Wunsch alignment algorithm to find the optimum alignment (including gaps) of two sequences along their entire length, for instance using Needle, and using the BLOSUM62 matrix with a gap opening penalty of 10 and a gap extension penalty of 0.5.

Probes can be used for diagnosing or prognosing Friedreich's ataxia (FRDA) by identifying at least one variation in biological samples. Contacting nucleic acid of a biological sample, with the probe, under conditions which allow hybridization of the probe with its corresponding fragment in the nucleic acid, results in the formation of a nucleic acid/probe hybrid. The formation of this hybrid can be detected (e.g., via labeling of the nucleic acid or probe), whereby the formation of this hybrid indicates the presence of variation. Such identification methods based on hybridization with a specific probe (either on a solid phase carrier or in solution) have been described in the art. The specific probe is preferably a sequence which, under optimized conditions, hybridizes specifically to a region within the 5' or 3' flanking region of at least one variation and preferably also comprising part of the foreign DNA contiguous therewith (hereinafter referred to as "specific region"). Preferably, the specific probe comprises a sequence of between 50 and 800 bp, preferably of 60 and 500 bp or 100 to 350 bp which is at least 80%, preferably between 80 and 85%, more preferably between 85 and 90%, especially preferably between 90 and 95%, most preferably between 95% and 100% identical (or complementary) to the nucleotide sequence of a specific region. Preferably, the specific probe will comprise a sequence of about 15 to about 100, or 25 to about 80 contiguous nucleotides identical (or complementary) to SEQ ID NO: 34, or SEQ ID NO: 36, or positions 1 to 1451 of SEQ ID NO: 36.

In one embodiment, said probes have about 15 to about 800 contiguous nucleotides identical (or complementary) to sequence SEQ ID NO: 34 or SEQ ID NO: 36 or positions 1 to 1451 of SEQ ID NO: 36.

In another embodiment, said probes have about 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, 120, 200, 250, 300, 350, 800 contiguous nucleotides identical (or complementary) to SEQ ID NO: 23 or SEQ ID NO: 24 which comprises a T at position 1486 of SEQ ID NO: 23 or at position 1494 of SEQ ID NO: 24 (rs4744808); a deletion of bases 931 -933 of SEQ ID NO: 23 or 939-941 of SEQ ID NO: 24 (rs60033969); a T at position 1099 of SEQ ID NO: 23 or at position 1 107 of SEQ ID NO: 24 (rs10890); a G at position 1276 of SEQ ID NO: 23 or at position 1284 of SEQ ID NO: 24 (rs4745577); a C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 (rs4744806); a G at position 1475 of SEQ ID NO: 23 or at position 1483 of SEQ ID NO: 24 (rs4744807); a G at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 (rs1 1 145043).

In one embodiment said probe comprises a fragment of 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 15, 120, 200, 250, 300, 350 bp of contiguous nucleotides of a sequence identical (or complementary) to sequence SEQ ID NO: 34, SEQ ID NO: 23 or SEQ ID NO: 24 which comprises a C at position 1393 of SEQ ID NO: 23 or at position 1401 of SEQ ID NO: 24 (rs4744806) and a G at position 1651 of SEQ ID NO: 23 or at position 1659 of SEQ ID NO: 24 (rs1 1 145043).

In one embodiment said probe comprises a fragment of a sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 34 or SEQ ID NO: 36 (corresponding to variation rs10890, rs4745577, rs4744806, rs4744807 and rs1 1 145043), SEQ ID NO: 26 (corresponding to variation rs60033969), SEQ ID NO: 27 (corresponding to variation rs10890), SEQ ID NO: 28 (corresponding to variation rs4745577), SEQ ID NO: 30 (corresponding to variation rs4744806), SEQ ID NO: 29 (corresponding to variation rs4744807), SEQ ID NO: 25 (corresponding to variation rs4744808) and SEQ ID NO: 31 (corresponding to variation rs1 1 145043). In a preferred embodiment, said probe is labelled, thus allowing its detection.

The probe may be labelled by a marker according to any method well-known in the art. For example, the probe may be labelled with a marker selected in the group consisting of a fluorescent molecule, a luminescent molecule, a radioactive molecule and/or an enzyme.

Kit of diagnosis or prognosis of Friedreich's ataxia (FRDA)

The present invention also relates to a kit comprising:

- a pair of primers suitable for amplifying the FRDA 3' untranslated region (UTR) (SEQ ID NO: 36, preferably positions 1 to 1451 of SEQ ID NO: 36) of the frataxin (FXN) gene or a fragment thereof, said 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID

NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), or their complementary sequences, and

- optionally, a packaging material.

The present invention also relates to the use of a pair of primers suitable for amplifying the FRDA 3' untranslated region (UTR) (SEQ ID NO: 36, preferably positions 1 to 1451 of SEQ ID NO: 36) of the frataxin (FXN) gene or a fragment thereof, said 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), or their complementary sequence, for the manufacture of a kit intended for the diagnosis or prognosis of Friedreich's ataxia (FRDA).

The pair of primers that may be used in the kit are preferably as defined above.

The present invention also relates to a kit comprising:

- a probe specifically hybridizing to a region of the FRDA 3' untranslated region (UTR) (SEQ ID NO: 36, preferably positions 1 to 1451 of

SEQ ID NO: 36) of the frataxin (FXN) gene including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), or the sequence complementary to said region, and

- optionally, a packaging material.

The present invention also relates to the use of a probe specifically hybridizing to a region of the FRDA 3' untranslated region (UTR) (SEQ ID NO: 36, preferably positions 1 to 1451 of SEQ ID NO: 36) of the frataxin (FXN) gene or a fragment thereof, said 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), or the sequence complementary to said region, for the manufacture of a kit intended for the diagnosis or prognosis of Friedreich's ataxia (FRDA).

The probe that may be used in the kit is preferably as defined above.

The probe is preferably a labelled probe.

Method for preventing and/or treating Friedreich's ataxia (FRDA)

The present invention also relates to a method for preventing and/or treating Friedreich's ataxia (FRDA) in a patient in need thereof, the method comprising a step of administering a suitable treatment to said patient.

In a preferred embodiment, the present invention relates to a method for preventing and/or treating Friedreich's ataxia (FRDA) in an individual in need thereof, the method comprising:

- performing the method of diagnosis or prognosis of Friedreich's ataxia (FRDA), as defined above, and

- when deducing said individual has developed or is at risk of developing Friedreich's ataxia (FRDA), administering a suitable treatment to said individual. A suitable treatment may comprise a treatment increasing the level of frataxin, an antioxidant treatment, an iron chelator treatment, an anti-diabetes treatment and/or a heart condition treatment.

A treatment increasing the level of frataxin may be human erythropoietin and/or an inhibitor of a microRNA (miRNA) as defined above, which miRNA specifically interacts with a 3' untranslated region (UTR) of the frataxin (FXN) gene including at least one variation selected from the group consisting of rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043.

Non-limiting examples of antioxidant treatment are glutathione, vitamin C, vitamin E, beta-carotene, coenzyme Q10, Idebenone, mitoquinone and their combinations.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Schematized representation of the genomic structure of the FXN gene.

The pathogenic expansion of the GAA repeat within intron 1 is indicated by a triangle, exons by grey boxes, respectively dark when translated and light when untranslated. Short genetic variations are indicated as black bars. The FXN 3'-UTR region, which was sequenced is highlighted by a square bracket.

Figure 2: Distribution of the most common haplotypes. Genotype frequencies of haplotypes in cases, controls and the replication study cases (RS-cases) are plotted as pie charts for the most common haplotypes of the FXN 3'-UTR (TGCTT, CATCG, CATCT). Haplotype TATTT was uniquely found among RS-cases.

Figure 3: Functional assessment of FRDA-3'-UTR versus WT-3'-UTR. U20S (black bars) and HEK-293 (grey bars) cells were transfected with lucif erase reporter gene system, respectively 150 ng of empty plasmid or plasmid WT-3'-UTR or plasmid FRDA-3'- UTR. Histograms show the Renilla luciferase activity (normalized to firefly luciferase and to the mock transfected cells) following transfection of each plasmid into both cell lines. All results represent mean ± SEM of three independent experiments, each in triplicate. ^*P < 0.05, Student-t test.

Figure 4: miRNAs targeting of FRDA-3'-UTR: Schematic representation of the nine miRNAs predicted to target FXN 3'-UTR dependengly on the haplotype: WT-3'-UTR (upper panel) or FRDA-3'-UTR (lower panel). The SNPs genotyped in the frataxin 3'-UTR are indicated in bold letters. Double arrows indicate whether the SNP is targeted by the same miRNA. Crossed double arrows indicates that the SNP is targeted by different miRNAs dependingly on the allele.

Figure 5: miRNAs targeting of FRDA-3'-UTR: Histograms illustrate the Renilla luciferase activity (normalized to firefly luciferase) following co-transfection of 10nM miR-124-3p mimic with 150ng of the indicated reporters into HEK 293 cells (n=3). All results represent mean ± SEM of three independent experiments, each in triplicate. ^*P < 0.05, Student-t test. EXAMPLES

EXAMPLE 1: Material and methods

Ethics Statement

For each patient written informed consent was obtained according to the French Ethics Committee, and all procedures were approved by the Necker Hospital reviewing board.

Subjects

Patients of either sex diagnosed with FRDA were determined at the molecular level as being homozygous for GAA expansion. Number of repeats ranged from 330 to 1500. One cohort was pediatric (n=57) with patients followed by the Necker Children's Hospital. The other cohort was adult (n=47) with patients followed by the CHR Felix Guyon, Saint-Denis, La Reunion, France. Control subjects of either sex were patients genetically tested at the Necker Children's Hospital for diseases non-related to FRDA (n=58).

PCR sequencing and markers

The sequence of human F /V 3'-UTR (bases 854 to 7168 of SEQ ID NO: 23, bases 862 to 7168 of SEQ ID NO: 24) was retrieved from NCBI (NM_000144 of SEQ ID NO: 23 and NM_181425 of SEQ ID NO: 24) and Ensembl genome browser (ENST00000377270, ENST00000498653, ENST00000396366 and ENST00000484259). Variations of the FXN locus were retrieved from the dbSNP database, build 129 (http://www.ncbi.nlm.nih.gov/SNP). In addition to short genetic variations of the 3'-UTR, rs3829062, also referred to as ITR3 due to location in intron 3 of the FXN gene, was also genotyped. This SNP has been previously shown to be associated with FRDA (Cossee M, et al. (1997) Evolution of the Friedreich's ataxia trinucleotide repeat expansion: founder effect and premutations. Proc Natl Acad Sci USA 94: 7452-7457). Genomic DNA was kindly provided by Pr Jean-Paul Bonnefont, Hopital Necker, Paris, and the Centre de Ressources Biologiques, CHR Felix Guyon, Saint-Denis, La Reunion. For genotyping, the sequences of primers used are as follows:

- FXN F, 5'-CCGCAGAGCTCACTAAAGC-3' (SEQ ID NO: 3);

- FXN R, 5'-ATTCATTTTCCCTCCTGGAA-3' (SEQ ID NO: 4);

- FXN_2F, 5'-TGTCGAAAGCAACTCACACG-3' (SEQ ID NO: 5);

- FXN_2R, 5'-GAACTATGTCTAGGACCAGG-3' (SEQ ID NO: 6);

- FXN_3F, 5'-TGTCCAGGGAGACCTAGTGC-3' (SEQ ID NO: 7);

- FXN_3R, 5'-AGGTTGCTTGACAGGACCAC-3' (SEQ ID NO: 8);

- FXN_4F, 5'-ATGGTTGATTCCCAGCATTC-3'(SEQ ID NO: 9); - FXN_4R, 5'-CAACCTCCACCTCTGGGTTC-3'(SEQ ID NO: 10);

- ITR3_F, 5' - A AA ATG G AAG C ATTTG GT AATC A-3 ' ( S E Q ID NO: 1 1 );

- ITR3_R, 5'-AGTGAACTAAAATTCTTAGAGGG-3'(SEQ ID NO: 12).

PCR amplification was performed using reagents from Roche Diagnostics with an annealing temperature range of 55-62 °C. PCR product were sequenced using Big Dye Terminator v3.1 (Life Technologies) according to manufacturer's instructions.

Cell culture, transfection and luciferase reporter assay

HEK-293 and U20S cell lines were obtained from ATCC (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 °C at an atmosphere of 5% C0₂. Dual luciferase reporters were generated by inserting the FA7V 3'-UTR carrying either the FRDA haplotype (FRDA-3'-UTR) or the alternative haplotype configuration (WT-3'- UTR) between the Not\ and Xho\ sites of the psiCHECK2 plasmid (Promega). To assess the functionality of F /V 3'-UTR, 150 ng of the so-obtained constructs, i.e. plasmid FRDA- 3'-UTR and plasmid WT-3'-UTR, respectively, were transfected in either cell lines using Fugene HD (Roche Diagnostics) according to manufacturer's instructions. Cells transfected with empty psiCHECK2 vector were used as a control. To assess the regulation of FXN by hsa-miR-124, HEK-293 cells were transfected with a Fugene HD (Roche Diagnostics)-complexed mixture of 10nM miRIDIAN mimic hsa-miR-124-3p (Acc N ° MI0000443 SEQ ID NO: 13) (Dharmacon) and 150ng d plasmid DNA (either plasmid FRDA-3'-UTR or plasmid WT-3'-UTR). The miRIDIAN mimic negative control 1 was used as a negative control. In all transfection experiments, cells were lysed with Passive Lysis Buffer (Promega) thirty-six hours post-transfection and luciferase levels were analyzed using the Dual Luciferase reporter assay (Promega) on a Centro LB960 Microplate Luminometer (Berthold).

Computational prediction of miRNA targets and miRNA expression.

miRNA sequences were retrieved from miRBase registry, release 13. miRNA targeting analysis on the FXN 3'-UTR region from both FRDA patient and control were performed using miRDB (http://mirdb.org/miRDB/) and our in-house developed tool MiRiFix (http://mirifix.com). MiRiFix integrates predictions from Diana microT 3.0 (Maragkakis M et al. (2009) Nucleic Acids Res 37: W273-276. doi:10.1093/nar/gkp292), Target Scan 5.1 (Friedman RC et al (2009) Genome Res 19: 92-105. doi:10.1 101/gr.082701 .108), microRNA.org (2008 release; (John B et al. (2004) PLoS Biol 2: e363. doi:10.1371/journal.pbio.0020363)) and PicTar (Lall S et al. (2006) Curr Biol 16: 460-471 . doi:10.1016 j.cub.2006.01 .050), as well as RegRNA (Huang H-Y et al (2006) Nucleic Acids Res 34: W429-434. doi:10.1093/nar/gkl333), Rna22 (Miranda KC et al. (2006) Cell 126: 1203-1217. doi:10.1016/j.cell.2006.07.031 ), FindTar3 (http://bio.sz.tsinghua.edu.cn/) and MiRTif, which is a support vector machine-based miRNA-target filtering-system to distinguish true predicted target sites from false ones (Yang Y et al (2008) BMC Bioinformatics 9 Suppl 12: S4. doi:10.1 186/1471 -2105-9-S12-S4). miRNA expression profiling data were accessed through MirZ (Hausser J et al. (2009) Nucleic Acids Res 37: W266-272. doi:10.1093/nar/gkp412) and mimiRNA (Ritchie Wet al (2010) Bioinformatics 26: 223-227. doi:10.1093/bioinformatics/btp649). Statistical analysis

Chi-square tests were performed to compare the allelic distribution of SNPs between FRDA patients and controls. Student-t test was used to validate the statistical significance in luciferase reporter assays. EXAMPLE 2: RESUL TS

Association between SNPs in the 3'-UTR of frataxin and FRDA

To assess the existence of variants in the 3'-UTR of the FXN gene, the inventors sequenced 1 .5 kb of the gene 3' terminus, which encompassed the 1451 bp of its 3'-UTR region, in a cohort of 57 FRDA patients and 58 controls (Figure 1 ). No mutations were found. Nineteen short genetic variations, which were already annotated in the dbSNP database were explored in our cohort in addition to rs3829062 (ITR3), a marker from the FRDA risk haplotype, already found to be associated to FRDA. Of those 19, 12 variations were monomorph whilst seven variations, namely rs60033969, rs10890, rs4745577, rs4744806, rs4744807, rs4744808 and rs1 1 145043 were biallelic markers as in the HapMap CEU population panel (Figure 1 ).

The inventors calculated allelic frequencies of those polymorphic 3'-UTR variations in our cohorts of FRDA patients and controls (Table 1 ).

Complete association was found between rs60033969 and rs10890, and between rs4744807 and rs4744808, respectively. Thus, results for rs60033969 and rs4744807 were not presented. All typed SNPs were significantly associated to FRDA with rs4744806 being even more strongly associated than ITR3 (Table 1 ).

Patients were mostly homozygous for all SNPs with frequencies ranging from 93 to 98.2% among the different SNPs (Table 2). Conversely, homozygosity was under-represented among controls, ranging from 15.5 to 46.5% (Table 2). These SNPs from the 3'-UTR (rs10890, rs4745577, rs4744806, rs4744808 and rs1 1 145043) defined several haplotypes (Table 3), which were differently distributed between cases and controls (Figure 2).

Frequency i² P-value

Cases, Controls,

n = 1 14 (%) n = 1 16 (%)

rs3829062

(ITR3)

C 1 13 (99.1 ) 71 (61 .2) 51 .66 6.58 x 10^"13

T 1 (0.9) 45 (38.8)

rs10890

C 4 (3.5) 53 (45.7) 54.878 1 .28 x 10^"13

T 1 10 (96.5) 63 (54.3)

rs4745577

G 109 (95.6) 63 (54.3) 52.013 5.51 x 10^"13

A 5 (4.4) 53 (45.7)

rs4744806

C 1 1 1 (97.4) 63 (54.3) 57.871 2.80 x 10^"14

T 3 (2.6) 53 (45.7)

rs4744808

T 108 (94.7) 60 (51 .7) 54.024 1 .98 x 10^"13

c 6 (5.3) 56 (48.3)

rs1 1 145043

G 2 (1 .8) 40 (34.5) 41 .259 1 .33 x 10^"10

T 1 12 (98.2) 76 (65.5) Table 1 . Genetic association of ITR3 and SNPs of the FXN 3'-UTR with FRDA haplotype in cases versus controls

The inventors found that the vast majority of patients, i.e. 89%, were homozygous for the T-G-C-T-T haplotype versus 24% of control subjects (Figure 2). Of note, genotype frequencies of the haplotypes in our control cohort comply with Hardy-Weinberg equilibrium proportions, and were closest to the ones of Western European descent population available from 1000 Genomes project.

Forty-seven adult patients from Reunionese island, referred to as the replication study cases (RS-cases), were similarly genotyped (Table 2). 94% of this population was found to be homozygous for the T-G-C-T-T haplotype (Figure 2). This result further emphasizes our finding that the 3'-UTR haplotype, which the inventors termed FRDA-3'-UTR for simplicity, expands the FRDA risk haplotype to the 3'-UTR. Frequency

Cases, RS-cases, Controls, n = 57 (%) n = 47 (%) n = 58 (%) rs10890

CC 1 (1.8%) 1 (2.1%) 11 (19%)

TT 54 (94.7%) 45 (95.8%) 16 (27.6%)

CT 2 (3.5%) 1 (2.1%) 31 (53.4%) rs4745577

GG 53 (93%) 44 (93.6%) 16 (27.6%)

AA 1 (1.8%) 1 (2.1%) 11 (19%)

GA 3 (5.2%) 2 (4.3%) 31 (53.4%) rs4744806

CC 55 (96.4%) 44 (93.6%) 16 (27.6%)

TT 1 (1.8%) 1 (2.1%) 11 (19%)

CT 1 (1.8%) 2 (4.3%) 31 (53.4%) rs4744808

TT 53 (93%) 45 (95.8%) 15 (25.9%)

CC 2 (3.5%) 1 (2.1%) 13 (22.4%)

TC 2 (3.5%) 1 (2.1%) 30 (51.7%) rs11145043

GG 1 (1.8%) 1 (2.1%) 9 (15.5%)

TT 56 (98.2%) 46 (97.9%) 27 (46.5%)

GT 0 (0%) 0 (0%) 22 (38%)

Table 2. Genotype frequencies of the five SNPs for the F /V3'-UTR in cases, cases from the replication study (RS-cases), and controls.

Haplotype Frequency

rs 10890- rs4745577-rs4744806- Cases, Controls

rs4744808-rs11145043 n =114, (%) n =116, (%)

T-G-C-T-T 106 (93) 59 (50.9)

C-A-T-C-G 2(1.8) 37 (31.9)

C-A-T-C-T 1 (0.9) 15(12.9)

T-G-C-C-T 2(1.8) 1 (0.9)

C-G-C-C-T 1 (0.9) 0(0)

T-A-C-T-T 2(1.8) 0(0)

T-G-C-C-G 0(0) 3 (2.6)

C-A-T-T-T 0(0) 1 (0.9)

Table 3. Frequencies of the haplotypes comprising the five SNPs of the F /V3'-UTR Functionality assessment of the FRDA haplotype and miRNA targeting predictions

To investigate what type of regulation confers the FRDA-3'-UTR haplotype to the frataxin protein level, the inventors transfected the FRDA-3'-UTR as compared to the alternative 3'-UTR-haplotype configuration, i.e. C-A-T-C-G, referred to as the WT-3'-UTR. The inventors repeatedly found in U20S as in HEK293 cells that the FRDA-3'-UTR affected the levels of frataxin through a significant decrease of 1 .2-fold (Figure 3, for U20S P-value = 0.001 ; for HEK293 P-value = 0.027; n=3). Thus, the FXN 3'-UTR harboured at the homozygous state by the vast majority of patients may contribute to lower levels of frataxin in addition to the effect of the expanded mutation. Subsequently, the inventors reasoned that the difference observed in the regulatory potency of FRDA haplotype versus control haplotype might at least partially be due to a distinct miRNA targeting. In order to test the hypothetical involvement of miRNAs in the regulation of frataxin, the inventors first screened miRNA target sites of the FXN 3'-UTR. To this purpose, the inventors used our in-house developed bioinformatic tool MiRiFix, and found prediction for 19 miRNAs at 15 target sites. Five of those 15 target sites overlapped one of the genotyped variations (Table 4). Based on our predictions, nine miRNAs differentially targeted the FXN gene, dependingly on the allele: hsa-miR-559 (MIMAT0003223 SEQ ID NO: 14), hsa-miR-589 (SEQ ID NO: 15), hsa-miR-1270 (SEQ ID NO: 16), hsa- miR-620 (SEQ ID NO: 17), hsa-miR-522 (SEQ ID NO: 18), hsa-miR-299-3p (SEQ ID NO: 19), hsa-miR-506 (SEQ ID NO: 20), hsa-miR-124-3p (SEQ ID NO: 13) and hsa-miR-624 (SEQ ID NO: 22) (Table 4, Figure 4A). In particular, the inventors found that variations rs4744806 and rs1 1 145043 created novel target sites for hsa-miR-522, hsa- miR-506, hsa-miR-624 and hsa-miR-124 in the FRDA-3'-UTR, which were not predicted with WT-3'-UTR (Figure 4A). Finally, using training sets of experimentally validated predictions, each of those miRNA:FX7V duplexes was confirmed but hsa-miR-1270 (Table 4). Our results suggest that miRNA targeting of FX7V 3'-UTR is differentially affected by the different haplotypes. MirTif

SNP Targeted Predicted miRNA

allele Result Score SVM

rs60033969 deletion hsa-miR-559 True 1 .6667198

GTT hsa-miR-559 True 1 .6667198

rs4745577 G hsa-miR-589 True 0.056741344

A

hsa-miR-1270 False

0.025866646

A hsa-miR-620 True 1 .4955749

rs4744806 C hsa-miR-522 True 0.086234009

T

rs4744807 G - - -

A hsa-miR-299-3p True 0.1 1721095

rs1 1 145043 T hsa-miR-506 True 1 .4271538

T hsa-miR-124-3p True 1 .2504418

T hsa-miR-624 True 3.1861664

G - - -

Table 4. Computational analysis of miRNA targeting on FA7V 3'-UTR

3' -UTR of frataxin as a target of miRNA regulation

The inventors searched for the miRNA(s) whose pattern of expression showed more consistency with the phenotype. To this end, the inventors searched for the miRNAs that were endogenous to either cell lines used for functional assessment of the 3'-UTR. Intersecting both patterns from public databases revealed miR-124 as the only common miRNA. The inventors thus chose to assess the regulation by hsa-miR-124. Interestingly, miR-124 is also known as the most abundant miRNA in the nervous system. Overexpression of hsa-miR-124 in HEK-293 cells led to a reproducible and significant 32%-decrease of luciferase activity in cells transfected with plasmid FRDA-3'-UTR compared to cells transfected with plasmid WT-3'-UTR (Figure 4B, P-value = 0.001 14; n=3). These data are consistent with the computational findings of a specific targeting of miR-124 at the level of FRDA-3'-UTR, suggesting a post-transcriptional regulation of frataxin mediated by differential miRNA targeting.

Claims

1. An in vitro method of diagnosis or prognosis of Friedreich's ataxia (FRDA) in an individual, which comprises:

a) determining whether the individual harbours at least one variation in the 3' untranslated region (UTR) (SEQ ID NO:35) of the frataxin (FXN) gene; and b) deducing that the individual has developed or is at risk of developing FRDA if said at least one variation is present, or that the individual is not at risk of developing FRDA if said at least one variation is absent.

2. The method according to claim 1 , comprising:

- determining the presence or absence of at least one variation selected from the group consisting of:

- a deletion of bases TTG at positions 78-80 of SEQ ID NO:35 (rs60033969),

- a T at position 246 of SEQ ID NO:35 (rs10890),

- a G at position 423 of SEQ ID NO:35 (rs4745577),

- a C at position 540 of SEQ ID NO:35 (rs4744806),

- a G at position 622 of SEQ ID NO:35 (rs4744807),

- a T at position 633 of SEQ ID NO:35 (rs4744808) and

- a T at position 798 of SEQ ID NO:35 (rs1 1 145043),

in the individual;

- deducing that the individual has developed or is at risk of developing FRDA if said at least one variation is present or that the individual is not at risk of developing FRDA if said at least one variation is absent.

3. The method according to claim 1 or 2, wherein said method further comprises determining if the individual is homozygote for said at least one variation and, if homozygote, deducing that the individual has developed or is at risk of developing an acute FRDA.

4. The method according to any one of claims 1 to 3, wherein said method further comprises determining in a fragment of the 3'UTR comprising nucleotides 1 to 1451 of SEQ ID NO: 35, if the individual is homozygote for at least a C at position 540 of SEQ ID NO:35 and a T at position 798 of SEQ ID NO:35.

5. The method according to any one of claims 1 to 4, wherein said method further comprises determining in a fragment of the 3'UTR comprising nucleotides 1 to 1451 of SEQ ID NO: 35, if the individual is homozygote at least for:

il a T at position 246 of SEQ ID NO:35;

HI a G at position 423 of SEQ ID NO:35;

Nil a C at position 540 of SEQ ID NO:35;

i i a T at position 633 of SEQ ID NO:35; and

Yl a T at position 798 of SEQ ID NO:35.

6. The method according to claims 1 to 5, wherein said method further comprises measuring the level of expression of hsa-miR-124 (SEQ ID NO: 13) and/or of frataxin protein (SEQ ID NO: 2) and/or determining the presence or absence of a GAA repeats, preferably of more than 70 GAA repeat, within FXN gene (SEQ ID NO: 1 ).

7. An isolated polynucleotide comprising:

- a polynucleotide of interest,

8. A vector comprising the isolated polynucleotide of claim 7.

9. A host cell comprising the vector of claim 8 or the isolated polynucleotide of claim 7 or at least one copy of the FXN gene wherein the 3' untranslated region (UTR) is the FRDA- 3'UTR comprising or consisting of positions 1 to 1451 of SEQ ID NO: 36.

10. A method for screening for a compound suitable for the treatment and/or prevention of

Friedreich's ataxia (FRDA) comprising the steps of:

b) identifying as a compound suitable for the treatment and/or prevention

FRDA the candidate compound which inhibits the interaction of said miRNA with the 3' UTR of the FXN gene harbouring at least one variation, or said fragment thereof.

11. An inhibitor of a microRNA (miRNA) which specifically interacts with a 3' untranslated region (UTR) (SEQ ID NO:35) of the frataxin {FXN) gene or a fragment thereof, said 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), for use as a medicament.

12. An inhibitor of a microRNA (miRNA) which specifically interacts with a 3' untranslated region (UTR) (SEQ ID NO:35) of the frataxin {FXN) gene or a fragment thereof, said 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), for use for treating Friedreich's ataxia (FRDA).

13. The inhibitor of a miRNA for the use according to claim 1 1 or 12, wherein said miRNA is selected from the group consisting of hsa-miR-559, hsa-miR-589, hsa-miR1270, hsa- miR620, hsa -miR-522, hsa-miR299-3p, hsa-miR-506, hsa-miR-124 and hsa-miR-624.

14. A pair of primers suitable for amplifying the FRDA 3' untranslated region (UTR) of the frataxin {FXN) gene comprising or consisting of positions 1 to 1451 of SEQ ID NO: 36 or a fragment thereof, said 3' untranslated region (UTR) or fragment thereof including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), or their complementary sequences, for use for diagnosing or prognosing Friedreich's ataxia (FRDA).

15. A probe specifically hybridizing to a region of the FRDA 3' untranslated region (UTR) of the frataxin {FXN) gene comprising or consisting of positions 1 to 1451 of SEQ ID NO: 36 including at least one variation selected from the group consisting of a deletion of bases TTG at positions 78-80 of SEQ ID NO: 35 (rs60033969), a T at position 246 of SEQ ID NO: 35 (rs10890), a G at position 423 of SEQ ID NO: 35 (rs4745577); a C at position 540 of SEQ ID NO: 35 (rs4744806), a G at position 622 of SEQ ID NO: 35 (rs4744807), a T at position 633 of SEQ ID NO: 35 (rs4744808) and a T at position 798 of SEQ ID NO: 35 (rs1 1 145043), or the sequence complementary to said region, for use for diagnosing or prognosing Friedreich's ataxia (FRDA).

16. A method for preventing and/or treating Friedreich's ataxia (FRDA) in an individual, the method comprising:

- performing the method of diagnosis or prognosis of Friedreich's ataxia (FRDA), according to any one of claims 1 to 6, and