WO2019229113A1 - Detection of tubb1 mutations for diagnosing thyroid dysgenesis - Google Patents

Abstract

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. The inventors identified three novel TUBB1 gene mutations that co-segregated with TD in three affected families. TUBB1 (Tubulin, Beta 1 Class VI) encodes a member of the β-tubulin protein family. In mice, Tubb1 knock-out in vivo disrupted microtubule integrity by preventing β1-tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused hyperaggregation of human platelets. The data highlight unexpected roles for β1-tubulin in thyroid development and function and in platelet physiology. Accordingly, the present invention relates to a method of diagnosing thyroid dysgenesis in a subject, comprising detecting a mutation in the TUBB1 gene in a sample obtained from said subject, wherein detecting the presence of a mutation in the TUBB1 gene is considered to be indicative of thyroid dysgenesis.

Description

DETECTION OF TUBB1 MUTATIONS FOR DIAGNOSING THYROID

DYSGENESIS

FIELD OF THE INVENTION:

The present invention relates to a method for diagnosing or predicting thyroid dysgenesis by detecting a mutation in Tubulin, Beta 1 Class VI ( TUBB1 ).

BACKGROUND OF THE INVENTION:

Thyroid dysgenesis (TD) is a feature in 65% of patients with congenital hypothyroidism (CH), which is the most common neonatal endocrine disorder (1/3,500 neonates)^1,2. TD includes a vast spectrum of developmental thyroid anomalies encompassing athyreosis, thyroid ectopia, hypoplasia of an orthotopic gland, and hemiagenesis^1,3. During embryogenesis, the midline thyroid anlage appears on embryonic day E.8.5 in mice and at 3 gestational weeks (GW) in humans. The midline anlage and ultimobranchial bodies migrate and fuse in the definitive pretracheal position on El 3.5 in mice and at 7 GW in humans^4,5. The cells differentiate into thyrocytes organized in follicles or C-cells ⁶. Abnormalities at any step of thyroid development may result in TD with variable degrees of hypothyroidism or rarely normal thyroid function⁷. Studies of sporadic and familial TD covering a wide clinical spectrum identified mutations in eight genes: PAX8, NKX2-1, FOXE1, NKX2-5, TSHR, GLIS3, NTN1, and BOREALIN ^8-14. However, mutations in these genes are found in only 5% of all patients with TD and identification of causative mutations remains a challenging task.

TUBB1 (Tubulin, Beta 1 Class VI) encodes a member of the b-tubulin protein family. b-tubulins are one of two core protein families that heterodimerise a/b-tubulin dimers, which assemble into microtubules, one of the major cytoskeletal structures. Microtubules are involved in crucial processes such as cell division, the growth polarity, and migration; intracellular trafficking; and cell communication^17,18. The bΐ isotype of tubulin (TUBB1) has been described as specifically expressed in platelets and megakaryocytes and involved in proplatelet formation and platelet release¹⁹. Three mutations in the intermediate and the C-terminal domain of TUBB1 have been identified in patients with a rare autosomal dominant disease congenital macrothrombocytopenia, in which impaired microtubule assembly results in low platelet counts and macroplatelets^20-22. Patients with these TUBB1 mutations had mild bleeding, which could be due to thrombocytopenia. Features reported to date in Tubbl -knockout mice include thrombocytopenia and spherical platelets²³, but not thyroid abnormalities. SUMMARY OF THE INVENTION:

The present invention relates to a method for diagnosing or predicting thyroid dysgenesis by detecting a mutation in Tubulin, Beta 1 Class VI ( TUBB1 ). In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. The inventors identified three novel TUBB1 gene mutations that co- segregated with TD in three affected families. TUBB1 (Tubulin, Beta 1 Class VI) encodes a member of the b-tubulin protein family. In mice, Tubbl knock-out in vivo disrupted microtubule integrity by preventing bΐ -tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused hyperaggregation of human platelets. The data highlight unexpected roles for bΐ -tubulin in thyroid development and function and in platelet physiology.

Accordingly, the first object of the present invention relates to a method of diagnosing thyroid dysgenesis in a subject, comprising detecting a mutation in the TUBB1 gene in a sample obtained from said subject, wherein detecting the presence of a mutation in the TUBB1 gene is considered to be indicative of thyroid dysgenesis.

As used herein, the term“thyroid dysgenesis” has its general meaning in the art and refers to a permanent thyroid hormone deficiency present from birth and resulting from an abnormality in the development of thyroid. Thyroid dysgenesis comprises e.g. thyroid ectopy, athyreosis and thyroid hypoplasia.

As used herein, the term“subject” is preferably a human. More particularly, the subject is a new-bom or a foetus. As used herein, a“new-born” is a child who is less than 2 weeks old, particularly less than 1 week old, more particularly less than 3 days old. Thus, when the method is method is performed“just after birth”, it means that the method is performed within 2 weeks, particularly 1 week, more particularly 3 days after birth.

As disclosed above, thyroid dysgenesis results from an abnormality of the development of thyroid during embryo development. The present invention is thus particularly interesting for detecting thyroid dysgenesis just after birth, i.e. wherein the subject is a new-bom. In some embodiments, the method according to the present invention is performed just after birth and after having suspected thyroid dysgenesis during embryo development, e.g. by observing an abnormality in the development of the embryo’s thyroid during an ultrasonography, particularly obstetric ultrasonography. In some embodiments, the method according to the present invention is performed in a child, preferably a new-born, whose parents (1 or both of them) present thyroid dysgenesis. Particularly, the method according to the present invention is performed in a child, preferably a new-born, whose parents (1 or both of them) are identified has displaying a mutation in the TUBB1 gene.

The method according to the present invention is interesting for detecting an abnormality in the thyroid development at the embryo stage. In some embodiments, the present invention relates to a prenatal method for diagnosing or predicting thyroid dysgenesis in a foetus by detecting a mutation in the TUBB1 gene in a sample obtained from said foetus or from said foetus’s mother.

In some embodiments, TD is suspected by routine screening which shows thyroid stimulating hormone (TSH) elevation and low T4 level. Other tests, such as thyroid radionuclide uptake and scan, thyroid sonography, or serum thyroglobulin determination may be carried out before performing the method of the present invention.

As used herein, the term“TUBB1” has its general mean in the art and refers to gene that encodes a member of the beta tubulin protein family: tubulin beta 1 class VI. Beta tubulins are one of two core protein families (alpha and beta tubulins) that heterodimerize and assemble to form microtubules tubulin beta 1 class VI is encoded by the TUBB1 gene (available under the reference ENSG00000101162 (gene) in the Ensembl Gene Database) or referenced as GENE ID : 81027 in the NCBI database. An exemplary nucleic acid sequence is represented by SEQ ID NO: l and an exemplary amino acid sequence is represented by SEQ ID NO:2.

SEQ ID NO: 1

1 ggggcagtat tctgtgttga gggaggaaaa acactccctt ccaaaagcat gacaggcaga

61 aagcagagaa gggccaggac tggctgaggg cggggagctg ggcctctggg gtggacacac

121 ccttggtcac attgtgaggg tagcttggtt ggccagtccc accactgcag tgaccacagt

181 tgtgttgggc tcacaccagt gaaccgaagc tctggattct gagagtctga ggattccgtg

241 aagatctcag acttgggctc agagcaagga tgcgtgaaat tgtccatatt cagattggcc

301 agtgtggcaa ccagatcgga gccaagttct gggagatgat tggtgaggaa cacgggatcg

361 acttggctgg gagcgaccgc ggggcctcgg ccttgcagct ggagagaatc agcgtgtact

421 acaacgaagc ctacggtagg aaatatgtgc cccgagcagt cttggtggac ctagaacctg

481 ggacgatgga cagcattcga tctagcaaat taggagctct ctttcaaccc gacagttttg

541 tccatggtaa ctctggggct ggcaacaact gggccaaagg ccactacacg gagggagccg

601 agctgatcga gaatgtccta gaggtggtga ggcacgagag tgagagctgt gactgcctgc

661 agggcttcca gatcgtccac tccctgggcg ggggcacagg ctccgggatg ggcactctgc

721 tcatgaacaa gattagagag gagtacccgg accggatcat gaattccttc agcgtcatgc

781 cttctcccaa ggtgtcggac actgtggtgg agccctacaa cgcggttctg tctatccacc

841 agctgattga gaatgcagat gcctgtttct gcattgacaa tgaggccctc tatgacatct

901 gcttccgtac cctgaagctg acgacaccca cctatgggga tctcaaccac ctagtgtcct

961 tgaccatgag cggcataacc acctccctcc ggttcccggg tcagctcaac gcagacctgc

1021 gcaagctggc ggtgaacatg gtccccttcc cccgcctgca cttctttatg cccggctttg

1081 ccccactcac ggcccagggc agccagcagt accgagccct ctccgtggcc gagctcaccc

1141 agcagatgtt cgatgcccgc aataccatgg ctgcctgtga cctccgccgt ggccgctacc

1201 tcacagtggc ctgcattttc cggggcaaga tgtccaccaa ggaagtggac cagcaactgc

1261 tctccgtgca gaccaggaac agcagctgct ttgtggagtg gattcccaac aacgtcaagg

1321 tggctgtctg cgacatcccg ccccgggggc tgagcatggc cgccaccttc attggcaaca 1381 acacggccat ccaagagatc tttaataggg tctctgagca tttctcagcc atgttcaaaa 1441 ggaaagcttt tgtgcactgg tacaccagcg aagggatgga cataaacgaa tttggggaag 1501 ctgaaaataa catccatgat ttggtatccg agtaccaaca atttcaagat gccaaagcag 1561 ttctagagga agatgaagag gtcacggagg aggcagaaat ggagccagaa gataagggac 1621 attaactgtg agagaagctg tgccgcggag tcgcttacag aacagtttct cattagatga 1681 gtgtttctcc tgcagcactc caaaacccac tctgcactgc agcacagtga atgatatgca 1741 ctcaccatta gcttcgacac agggactgag ggagacaggt ggggagcagc tgacaggcat 1801 tagggtcttt gctgacatct actaaccttg aagagtttga tgttcagtgc atacttatta 1861 acttaaaaaa atagcaaatt tattgtaaag tgctcccttt gtttcaaagt gtttgccagg 1921 catccagact acacgtgtgg atttgcaggg agccactgga gttggtgtta catttttata 1981 ctttagcagc actgataggc accctggaat cctcacttgg tatccgaggg ctactaagac 2041 tctttcctta ggttctttcc tctgagcaaa cactgactgg catcctgctt tccagtgcct 2101 gccagcctcc agaagagcca ggtgcctgac tagtacatgg ggagctacag agccaaggtc 2161 aatgtgagtc aacatccact agaaatatcc atgttgtgta gacctgtgca tacaacatgc 2221 taactggaaa agaggaaaaa agaaaagcca cagtcctctc cacaaaaata cctggtccaa 2281 acaagaaaaa caaaaagaca agcaaaacta aagaactgca gtcttctgat ctttatttct 2341 gaagagctag cctttaacat atatgtttat atagtttaaa tttcttacta ctgttagatc 2401 ccaggaattc attaataatc atccttggct ttccttttaa aggctatttt gaaatggtct 2461 tttcactttc attcagtcat caccccccaa aatgctctgc agcctctctg ctctttgaga 2521 aagggcacac catgcgctcg gcaaccattc aaatgcagga attaagcagc aatggctgca 2581 gtgtccttct cagttatgga ggacatcgtc tcattaggga acttttacag ttcaaattaa 2641 tttgcagaag ttgccataaa tgtttgcata atgacatagc tttaagcact acatgatttt 2701 aatctgctca cattataaca ggaccaaata cacaagagcg taatcaaatc atctgtaact 2761 tcttaattac agtttaccta tttctgacat gcagcactgc catctcttcc agcaccatca 2821 gggttttaat ggccctctag aattaccact gagatacact atttgatcca tggataaccg 2881 gtaatgggaa aatgctccga ccctcaatgc agtaaatatt tacttgcagg caactgggtt 2941 ctcatctctt gatttgcttt tgtaatcagc aataataaaa tagcaggtag atggatgaca 3001 gttgctcatt ctgagaaact tcactctttt cacttatgca tcacgaggaa ataactaaaa 3061 tacataccaa gagaaaaata ccttgccatc ggatcatcaa caagtcttct atttacaaac 3121 ttcaaaaaac acaaaacaac attcatgttt taaatgcttt ctacttgtgg ttcaagaagc 3181 actagattta gtaagaaact ctacctatat acttagtttg aagttagtaa cttcctgaga 3241 tgctaaagac ttacagcctg cgattataca aggatttaca catgcttcct ctggtgcttt 3301 acttcccaaa cctaaaaaag caatgaaata gatgtaagga aggagggatt taaacctttt 3361 aaaaaacttt tgctgactta tattactgta aagatttgtt tgctcaatag taatcattaa 3421 actacaaagt aattcaattt taaatggcaa aattgcttta tttcagacta aataaattcc 3481 ttttcttgaa gcctaa

SEQ ID NO:2:

1 mreivhiqig qcgnqigakf wemigeehgi dlagsdrgas alqlerisvy yneaygrkyv

61 pravlvdlep gtmdsirssk lgalfqpdsf vhgnsgagnn wakghytega elienvlevv 121 rhesescdcl qgfqivhslg ggtgsgmgtl lmnkireeyp drimnsfsvm pspkvsdtvv 181 epynavlsih qlienadacf cidnealydi cfrtlklttp tygdlnhlvs ltmsgittsl 241 rfpgqlnadl rklavnmvpf prlhffmpgf apltaqgsqq yralsvaelt qqmfdarntm 301 aacdlrrgry ltvacifrgk mstkevdqql lsvqtrnssc fvewipnnvk vavcdipprg 361 lsmaatfign ntaiqeifnr vsehfsamfk rkafvhwyts egmdinefge aennihdlvs 421 eyqqfqdaka vleedeevte eaemepedkg h

As used herein, the term "mutation" has its general meaning in the art and refers to any detectable change in genetic material, e.g. DNA, RNA, cDNA, or in an amino acid sequence encoded by such a genetic material. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered any gene as well as protein mutations, in which the amino- acid structure of the protein is altered. Generally a mutation is identified in a subject by comparing the sequence of a nucleic acid or of a polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population. Typically, mutations are accessible in the Single Nucleotide Polymorphism Database (dbSNP), which is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI).

The present inventors have identified different mutations of the TUBB1 gene involved in thyroid dysgenesis. One of these mutations is a missense homozygous TUBB1 mutation (c.479C>T, p.Pl60L, rs759l 17911) which is a substitution of a cytosine residue into a thymine residue at position 479 of the TUBB1 gene. This mutation encodes for the substitution of the proline residue at position 160 by a leucine residue in the mature protein. Another mutation identified by the present inventors is a heterozygous TUBB1 mutation (c.3 l8C>G, p.Yl06X) which is the substitution of a cytosine residue into a guanine residue at position 318 of the TUBB1 gene. This mutation leads to the creation a premature stop codon at amino acid 106. Another mutation identified by the inventors is a heterozygous frameshift TUBB1 mutation (c.35delG, p.Cysl 2Lcufs* 12, rs77324804) that created a premature stop codon at amino acid 23. All three mutations are located in the first part of TUBB1, i.e., in the N-terminal domain needed for guanosine triphosphate (GTP) activity, i.e. the domain ranging from the amino acid residue at position 1 to the amino acid residue at position 206 in the protein. All three TUBB 1 mutations identified lead to nonfunctional a/b -tubulin dimers that cannot be incorporated into microtubules. The mutations lead to an early abnormal proliferation of progenitors, delayed thyroid migration, defective thyroid tissue differentiation, and impaired thyroid hormone release. More interestingly, the mutations also affect bΐ -tubulin expression in platelets and result in abnormally large platelet size, probably as a consequence of proplatelet abnormal formation. Moreover, the p.Pl60L and r.U106C mutations induce significant basal platelet activation and hyperaggregation in response to agonists, whereas the c.35delG mutation does not seem to affect platelet function.

As used herein, the term“sample” refers to sample liable to contain nucleic acid molecules including mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and/or hybridization with a probe, for instance. The nucleic acid sample may be obtained from any cell source or tissue biopsy. Non-limiting examples of cell sources available include without limitation blood cells, buccal cells, epithelial cells, fibroblasts, or any cells present in a tissue obtained by biopsy. Cells may also be obtained from body fluids, such as blood, plasma, serum, lymph, etc. In some embodiments, the sample is an amniotic fluid sample. The sample may further be any biological sample wherein foetal DNA may be detected. For instance, in this case, the biological sample may be a sample obtained from the mother but wherein foetal DNA can be found. As disclosed e.g. in Hixson et al (J Lab Autom; 20(5):562-73, 2015) or in the reference EP patent N° 0994963, foetal DNA is detectable in maternal serum or plasma samples. Abnormalities in the foetus genetic material can thus be detected by directly analysing the foetal DNA present in the mother’s blood. Thus, in some embodiments, the sample according to the present invention is a maternal serum or plasma sample.

DNA may be extracted using any methods known in the art, such as described in Sambrook et ah, 1989. RNA may also be isolated, for instance from tissue biopsy, using standard methods well known to the one skilled in the art such as guanidium thiocyanate- phenol-chloroform extraction.

TUBB1 mutations may be detected in a RNA or DNA sample, preferably after amplification. For instance, the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a mutated site or that enable amplification of a region containing the mutated site. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a particular TUBB1 mutation. Otherwise, RNA may be reverse- transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to identify a mutation in TUBB1 sequence. Actually numerous strategies for genotype analysis are available (Antonarakis et ah, 1989; Cooper et ah, 1991; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR test for the mutation. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFFP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase- PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et ah, 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing; sequencing using a chip-based technology; and real- time quantitative PCR. Preferably, DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base substitution mutations. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized. Therefore, useful nucleic acid molecules, in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the regions where the mutations are located. Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides. In some embodiments, the TUBB1 mutations of the present invention may be detected by Next Generation Sequencing or NGS. As used herein, the term "Next Generation Sequencing" or“NGS” refers to a relatively new sequencing technique as compared to the traditional Sanger sequencing technique. For review, see Shendure et al, Nature Biotech., 26(10): 1135-45 (2008), which is hereby incorporated by reference into this disclosure. For purpose of this disclosure, NGS may include cyclic array sequencing, micro electrophoretic sequencing, sequencing by hybridization, among others. By way of example, in a typical NGS using cyclic-array methods, genomic DNA or cDNA library is first prepared, and common adaptors may then be ligated to the fragmented genomic DNA or cDNA. Different protocols may be used to generate jumping libraries of mate-paired tags with controllable distance distribution. An array of millions of spatially immobilized PCR colonies or "polonies" is generated with each polonies consisting of many copies of a single shotgun library fragment. Because the polonies are tethered to a planar array, a single micro liter- scale reagent volume can be applied to manipulate the array features in parallel, for example, for primer hybridization or for enzymatic extension reactions. Imaging-based detection of fluorescent labels incorporated with each extension may be used to acquire sequencing data on all features in parallel. Successive iterations of enzymatic interrogation and imaging may also be used to build up a contiguous sequencing read for each array feature.

The mutation may be also detected at a protein level (e.g. for loss of function mutation) according to any appropriate method known in the art. In particular a biological sample, such as a tissue biopsy, obtained from a subject may be contacted with antibodies specific of a mutated form of TUBB1 protein, i.e. antibodies that are capable of distinguishing between a mutated form of TUBB1 and the wild-type protein, to determine the presence or absence of a TUBB1 specified by the antibody. The antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies. Whereas polyclonal antibodies may be used, monoclonal antibodies are preferred for they are more reproducible in the long run. Procedures for raising“polyclonal antibodies” are also well known. Alternatively, binding agents other than antibodies may be used for the purpose of the invention. These may be for instance aptamers, which are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library.

In some embodiments, the method according to the present invention is performed after having suspected thyroid dysgenesis during embryo development and treated the foetus thereby identified as presenting an abnormality in the thyroid development. An example of treatment which can be administered to a foetus identified as presenting an abnormality in the development of thyroid is intra-amniotic injection of Levothyroxine. Such treatment is e.g. disclosed in Leger et al (ESPE-PES-SLEP-JSPE-APEG-APPES-ISPAE; Congenital Hypothyroidism Consensus Conference Group. European Society for Paediatric Endocrinology consensus guidelines on screening, diagnosis, and management of congenital hypothyroidism. J Clin Endocrinol Metab. 2014 Feb;99(2):363-84) and in Ribault et al (French Fetal Goiter Study Group. Experience with intraamniotic thyroxine treatment in nonimmune fetal goitrous hypothyroidism in 12 cases. J Clin Endocrinol Metab. 2009 Oct;94(lO):373 l-9).

In some embodiments, when it is concluded that the subject suffers from TD, a therapy is administered. Levothyroxine (l-thyroxine) is the therapy of choice. Treatment is typically initiated in any infant with a positive screening result. The dose and timing of thyroid hormone replacement are important in achieving optimal neurocognitive outcome. Thus, the goal of treatment should be to restore the serum T4 to > 129 mmol/L (> 10 pg/dl) as rapidly as possible. The recommended initial l-thyroxine dose set forth by the American Academy of Pediatrics (AAP) and the European Society for Paediatric Endocrinology (ESPE) is 10-15 mcg/kg per day. In term infants this amounts to an average of 37.5 to 50 meg per day.

Since the subjects harbouring said mutations are also at risk of having thrombosis, in particular subject harbouring the p.Pl60L or r.UIObC mutations, a prophylactic treatment mays also be prescribed. Typically said treatment may consist in antagonists of platelet activation and particularly of ADP receptors such as thienopyridines (eg, clopidogrel, prasugrel) or non-thienopyridine ADP inhibitors (eg, ticagrelor), or any newer anti-platelet agent (eg, inhibitors of the thrombin receptor).

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Molecular genetics

a) Pedigrees of three families with TUBB1 mutations. Family Fl has three affected individuals with homozygous mutations, family F2 has two affected individuals with heterozygous mutations, and family F3 has two affected individuals with heterozygous mutations. Thus, all seven patients (Pl to P7) carry at least one mutated allele and have thyroid dysgenesis (TD) and macroplatelets. The patients are represented with filled symbols and unaffected family members with open symbols. N, not mutated, m, mutated

b) Location of TUBB1 mutations in the cDNA and of the corresponding changes in the protein. Exons are represented by boxes numbered from 1 to 4. The black box represents the protein domain responsible for encoding guanosine triphosphate (GTP) and the box points represents the domain for microtubule-associated protein (MAP) binding. The arrows show the consequences of the three TUBB1 mutations in our patients, all of which are in the GTP domain.

EXAMPLE:

Methods:

Subjects

A consanguineous family in which two children had TD and CH and another had TD with normal thyroid function was studied. Subsequently, genetic testing was performed in 270 patients (184 girls and 86 boys) with CH and TD (ectopic thyroid gland, n=l67; athyreosis, n=77; hemi-agenesis, n=20; and thyroid hypoplasia, n=6). This study was approved by our institutional review board and informed consent were collected.

Detection of mutations in humans

Genomic DNA was isolated from whole blood. Exome capture and sequencing were performed at the genomics platform of the Imagine Institute. WES libraries were prepared from 3 Lig genomic DNA per individual, which was sheared by ultrasonication (Covaris S220 Ultrasonicator, Wobum, MA, USA). Exome capture was performed using the SureSelect Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA). The resulting libraries were sequenced on a HiSeq 2,500 HT device (Illumina, San Diego, CA, USA) according to the manufacturer’s recommendations. Paired-end (2x130) 76-bp reads were generated and mapped on the human reference genome. More than 97% of the exome was covered at least 30 times. Raw data were analysed as described⁵⁴, using an in-house software system (Polyquery). The variant prioritisation strategy was as follows: (i) selection of functional (protein-altering) variants (removal of intergenic and 375’ UTR variants, non-splice related intronic variants, and synonymous variants); (ii) variants with a frequency below 1% in public databases (dbSNP, 1,000 Genomes, EVS, ExAC; release date, January 2018), and (iii) variants previously identified in fewer than five individuals contributing 11,811 in-house exomes (data not shown).

The HypothySeq NGS panel included 78 genes known to be associated with CH (TD; dyshormonogenesis; defects in thyroid hormone (TH) transport proteins; and inborn errors in TH membrane transport, metabolism, or action) and candidate genes validated in animal models (mouse and zebrafish knock-out models) or by microarray assays but not yet validated in humans. This panel was previously validated using controls including samples from positive controls with known thyroid disease-causing mutations, to assess sensitivity (false-negative rate); and from healthy individuals screened by WES for another research study, to test specificity (false-positive rate). Genomic DNA libraries were created using SureSelectXT Target Enrichment Reagent Kit (Agilent Technologies) and subjected to custom targeted DNA panel enrichment. In the 78 genes associated with CH, 1006 regions of interest were captured by the corresponding l20-bp cRNA baits, using SureDesign software (Agilent Technologies) (H. sapiens, hgl9, GRCh37, February 2,009). The 233,l03-bp targeted DNA regions (protein coding exons of the main iso form and supplementary coding exons of each gene, including 25- bp flanking intronic sequences) were sequenced on Illumina HiSeq 2,500 (Illumina). This step generated 2x130 paired-end reads. Bioinformatic analyses included alignment against the reference genome, variant calling and annotation, and copy number variation (CNV) detection. All data were integrated in the dedicated interface Polydiag developed by the bioinformatics platform at the IMAGINE Institute to check coverage of the targeted regions, to sort and filter the called variants by impact and frequency, and to identify relevant candidate mutations and/or CNVs for molecular diagnosis.

Sanger sequencing was performed to validate and segregate the identified TUBB1 (NM_030773) variants (3,500xL Genetic Analyzer, Thermo Fisher Scientific, Waltham, MA, USA). Primer sequences and PCR conditions are available on request.

Burden test Rare variant burden testing was performed for the TUBB1 gene using the CAST collapsing method⁵⁵ in 270 patients with TD, including 193 with ectopia, hemi-agenesis or hypoplasia and 77 with athyreosis. Logistic regression was performed to compare the prevalence of deleterious variants in the patients with TD and in 406 Caucasian controls from the 1,000 Genomes project phase 3⁵⁶. Disruptive in- frame, frame-shift, missense, splice- acceptor, splice-donor, start-lost, stop-gained, or stop-lost variants were considered deleterious. All deleterious variants with a minor allele frequency <1% in the ExAC database were included in the analysis. We first studied all 270 TD patients then only the 193 TD patients without athyreosis.

Human thyroid tissue samples

After approval by our institutional review board of the experimental design and protocols, embryonic thyroid tissue was obtained from products of elective termination of pregnancy and adult thyroid tissues from patients undergoing thyroid surgery.

Animals

Tubbl ^/ mice were previously generated by replacing exons 3 and 4, encoding amino acids 56 to 451, with a neomycin-resistance gene cassette as previously described²³. Tubbl^+/ mice on a mixed l29/Sv-BALB/c background were interbred with C57BL/6J mice over ten generations to generate homozygous null mutants (Tubbl· ) with the C57BL/6J background (Bb.CG-b 1 tubulin™). All experiments were conducted in accordance with French regulations and were approved by the Strasbourg regional ethics committee for animal experimentation (C.R.E.M.E.A.S., CEEA 35). Thyroids at different embryonic stages from El3.5 to E17.5 and adult thyroids at 3 months of age were obtained from wild-type and Tubbl mice and microdissected as described previously³⁴.

Assays on mouse serum samples

Aortic blood samples were collected from 3-month-old wild-type and Tubbl mice. Radioimmunoassays were used to measure serum TSH and serum total T4 after iodothyronine extraction (Dr. S. Refetoff, Chicago, IL, USA) as previously described⁵⁷.

Flow cytometry of mouse thyroid cells

Mouse thyroid tissue from El 7.5 embryos (15 pooled thyroids per sample) and adults (4 thyroids per sample) were microdissected, cleansed of fat and connective tissue, and placed in ice-cold phosphate-buffer saline (PBS) containing 2% foetal calf serum (FCS). Cells were prepared and sorted by flow cytometry as previously described²⁸. Briefly, single-cell suspensions were obtained by enzymatic digestion with 1 mg/mL collagenase/dispase and 2 Lig/mL DNase I (Roche Diagnostics, Basel, Switzerland) at 37°C for 20 min. The cells were then centrifuged with PBS containing 2% FCS and stained with cell surface markers for 20 min. Finally, the cells were acquired on a BD FACSAria II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The following monoclonal antibodies were used: EpCam/CD326 (G8.8), PDGFRa/CDl40a (APA5), CD45 (30-F11), and Pecam/CD3 l (390) from BioLegend (San Diego, CA, USA); and CD41 from Abeam (Cambridge, UK). The secondary antibody was goat anti-rabbit A647 from Life Technologies (Carlsbad, CA, USA). Each pool of sorted cells was collected in RLT buffer from the Qiagen RNeasy MicroKit (Qiagen, Valencia, CA, USA) for RNA extraction experiments.

RNA extraction and quantitative RT-PCR

The thyroids were microdissected and immediately snap-frozen and stored at -80°C. Total RNA of sorted cells or thyroid tissue was isolated using the Qiagen RNeasy Microkit or Minikit (Qiagen). The Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) was used for reverse transcription of 250 ng of each RNA sample. The synthesised cDNA was diluted to 1/20, and 5 mE was used for each PCR reaction. Each reaction consisted of TaqMan Universal PCR Master Mix or SybrGreen PCR Master Mix (Thermo Fisher Scientific) and primers. Peptidylpropyl isomerase A served as an endogenous control. Real-time PCR was performed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The data were analysed using the comparative cycle threshold method and reported as the fold change in gene expression, normalised for a calibrator of value 1.

Immunohistochemistry and quantification

Human or mouse tissues were fixed by immersion in 3.7% buffered formalin then embedded in paraffin. Subsequently, 4 pm-thick sections were mounted on StarFrost adhesive slides (Knittel Glaser, Braunschweig, Germany) and processed for immunohistochemistry, as previously described³⁴. The primary antibodies were used at the following dilutions: rabbit antibody to human or mouse bΐ -tubulin, 1 : 1 000 (donated by Frangois Fanza), rabbit anti- Ecadherin, 1 : 100 (Becton Dickinson), mouse anti-TG, 1 : 100 (DakoCytomation, Glostrup, Denmark), rabbit anti-Nkx2-l, 1 :2500 (Biopat, Italy), mouse anti-T4, 1 : 10 000 (AbD Serotec, Raleigh, NC, USA), rabbit anti-calcitonin, 1 :400 (DakoCytomation), mouse anti-Ki67, 1 :20 (Becton Dickinson), and rabbit anti-KDEF, 1 : 1500 (Thermo Fisher Scientific). The fluorescent secondary antibodies were Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 488 goat anti mouse antibodies (1/400, Thermo Fisher Scientific). The nuclei were stained using the Hoechst 33 342 fluorescent stain (0.3 mg/mL; Thermo Fisher Scientific). Photographs were taken using a fluorescence microscope (Leitz DMRB; Leica, Wetzlar, Germany) and digitised using a chilled 3CCD camera (C5810; Hamamatsu Photonics, Hamamatsu City, Japan).

The sections were then analysed using Image J l.32s (freeware, www.rsbweb.nih.gov/ij) as previously described^34,58. The Nkx2-l -positive surface areas per section allowed us to draw the total thyroid surface area in pm². The surface areas positive for calcitonin and T4, two markers of late thyroid differentiation, were normalised for total thyroid surface area. For stained surface quantification, we used one of every two sections at E9.5 and El 1.5, one of every five sections at E13.5, and five sections per adult thyroid (3 months of age). We determined the surface area to obtain an estimate of the total stained surface for each thyroid and each marker. Proliferation of Nkx2-l -positive cells at E9.5 was estimated by counting Ki67-positive nuclei among Nkx2-l -positive cells on every other section throughout the entire tissue sample at E9.5. At least three thyroids were analysed per genotype. The results are reported as mean±SEM.

For Nkx2-l staining of adult mouse thyroid glands, the first immunohistochemistry steps were as described above. After application of the primary antibody, the sections were incubated with biotinylated secondary antibody for 1 hour. Immunostaining was then performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. The sections were then incubated in 3,39- diaminobenzidine tetrahydrochloride and counterstained with hemalum-eosin.

Western Blot studies of mouse thyroid tissue

Proteins prepared from mouse thyroid tissue collected in RIPA buffer and sonicated were quantified using the BCA protein assay (Thermo Fisher Scientific). Then, 20 pg of total protein was separated on BisTris polyacrylamide gel with a 4%-l2% gradient (Thermo Fisher Scientific) and transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with the primary antibodies mouse anti-Chop (1 :1,000, Cell Signalling Technology, Danvers, MA, USA) or rabbit anti-actin (Sigma- Aldrich) antibodies, followed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies. Binding of secondary antibodies was revealed using the Amersham ECL Prime Detection Reagent Kit (GE Healthcare, Chicago, IL, USA). The protein bands on the membranes were scanned with the ImageQuant LAS 4,000 Station (GE Healthcare) then analysed using ImageJ 1 32s to determine the protein levels, with actin protein serving as an internal control.

Molecular modelling of the P160L mutated protein

The wild-type human TUBB1 sequence (accession number: Q9H4B7) was downloaded from the UniProt database, and the P160L mutation introduced into it. Both the wild-type and mutant TUBB1 sequences were modelled using Modeller 9.18 software⁵⁹ with PDB 4I4T chain B as the template⁶⁰. The models were analysed using PyMOL visualisation software⁶¹.

Electron microscopy

Samples were fixed for 1 hour in 3% glutaraldehyde in PBS buffer, washed, and embedded in Epon. 90 nm sections were collected on nickel grids and contrasted with uranyl acetate and lead citrate. Acquisitions were performed with a Gatan Orius 1000 CCD camera (Gatan, Pleasanton, CA, United States) on a JEOL 1011 transmission electron microscope (JEOL, Tokyo, Japan).

Plasmids, cell cultures, transfection, and immunofluorescence

We used the phumanTUBBl-tagged-Myc vector described by Kunishima et al²⁰. Mutant P 160 L- 77/55/ was generated using a PCR-based site-directed mutagenesis method as described previously, using the Stratagene QuikchangeVR kit (Agilent Technologies)⁶². Nthy (Nthy-ori 3.1) immortalised human thyroid cell lines were cultured as previously described⁶³. The Nthy cells were plated at 0.4· l0⁵/well on poly-L-lysine-coated slides in l2-well plates 24 h before transfection then transfected with 500 ng of vectors containing wild-type or P160L mutant TUBB1 using XtremeGENE-HP-DNA, as recommended by the manufacturer (Roche Applied Science, Penzberg, Germany). After 24 h, cells were used for immunofluorescence as already described⁶⁴. The cells were washed with pre-warmed PHEM buffer, fixed with 4% PFA, 0.2% glutaraldehyde, and 0.5% Triton, and permeabilised with PBS-Triton 0.1%. Immunostaining was performed with rabbit anti-Myc antibody (1 :500, Cell Signaling Technology) and mouse anti-a-tubulin (DM1 A, 1 : 1,000, Sigma- Aldrich, Saint-Louis, MI, USA) then with Alexa Fluor 647 goat anti-rabbit and Alexa Fluor 555 goat anti-mouse antibodies (1 :400, Thermo Fisher Scientific).

Human megakaryocytes and proplatelet formation

CD34⁺ cells were isolated from peripheral blood using an immunomagnetic technique (Miltenyi Biotec, Bergisch Gladbach, Germany). The remaining population was cultured at 37°C in 5% C0₂ in Iscove Modified Dulbecco’s Medium (IMDM; Thermo Fisher Scientific) supplemented with 15% BIT 9500 serum substitute (Stemcell Technologies, Vancouver, Canada), a-monothioglycerol (Sigma-Aldrich), and liposomes (phosphatidylcholine, cholesterol, and oleic acid; all from Sigma-Aldrich), in the presence of human recombinant stem cell factor (SCF, 20 ng/mL, Miltenyi Biotec) and human thrombopoeitin (50 nM, Miltenyi Biotec) added once on day 0 to the culture medium, followed by 20 nM thrombopoeitin alone on day 6 with no further SCF addition. For proplatelet formation assays, megakaryocytes were plated on a BSA-coated surface (chamber slide, Ibidi, Martinsried, Germany) on day 10. On day 13 or 14, the megakaryocytes were fixed using 4% paraformaldehyde and stained for b- tubulin.

Preparation of washed platelets

To obtain human platelets, venous blood from healthy donors or patients was collected in 10% ACD/A buffer (75 mM sodium citrate, 44 mM citric acid, 136 mM dextrose, pH 4.5). Platelets were washed as previously described⁶⁵ in the presence of apyrase (100 mU/mL) and prostaglandin El (1 mM) to minimise platelet activation. Platelet counts in patients and controls were adjusted to similar levels (3 Ί0⁸ platelets/mL) in Tyrode's buffer (137 mM NaCl, 2 mM KC1, 0.3 mM NaH₂P0₄, 1 mM MgCh, 5.5 mM glucose, 5 mM N-2-hydroxyethylpiperazine- N’-2-ethanesulfonic acid, 12 mM NaHCCE, and 2 mM CaCl₂, pH 7.3).

Platelet aggregation

Platelet aggregation was monitored by measuring light transmission through a stirred suspension of washed platelets (3 · l0⁸/mL) at 37°C using a Chrono-Log aggregometer (Chrono- Log Corporation, Havertown, PA, USA), as previously described⁶⁶. Platelet aggregation was triggered by ADP and type I collagen.

Flow cytometry of human platelets

Whole blood from healthy donors or patients was diluted in PBS to obtain a platelet concentration of 2.5 · l0⁷/mL. Diluted whole blood was then incubated with phycocrythrin (PE)- anti-human CD62P (clone AK-4; eBioscience, Thermo Fisher Scientific) or fluorescein isothiocyanate (FITC) anti-human activated api,b^ integrin (PAC1; Becton Dickinson) for 20 minutes at room temperature. The samples were then analysed directly with an Accuri C6 flow cytometer (Becton Dickinson).

Western blotting study of human platelets

Washed platelets (300 uL; 3 - l0⁸/mL) were lysed in Laemmli sample buffer (10 mM HEPES, 2% SDS, 10% glycerol, 5 mM EDTA). The proteins were separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, which were incubated with the primary antibodies rabbit anti-a tubulin (Clone EP1332Y; Merck Millipore, Billerica, MA, USA) or rabbit anti-b! -tubulin antibody (donated by Frangois Lanza). Immunoreactive bands were visualised with enhanced chemiluminescence detection reagents (Perbio Science, Thermo Fisher Scientific) using a G:BOX Chemi XT 16 Image System then quantified using Gene Tools version 4.03.05.0 (Syngene, Cambridge, UK).

Statistics Results are reported as mean±SEM for the number of experiments indicated in the figure legends. Continuous data were compared using the two-tailed Student’s /-test or, for multiple comparisons, one-way A OVA followed by Dunnett’s test, as indicated in the figure legends. Statistical analyses were performed using GraphPad Prism4 (GraphPad, La Jolla, CA, USA).

Results:

Identification of TUBB1 mutations in a family (FI) with thyroid dysgenesis (TD)

Family Fl is a consanguineous family of Algerian descent. The parents are first cousins (1.1 , 1.2) with five children including two females (II.1 [patient Pl] and II.2 [patient P2]) with CH. Both patients were bom at full term and diagnosed with CH by routine neonatal screening (Fig. la), which showed thyroid stimulating hormone (TSH) elevation (164 and 177 LiIU/mL in Pl and P2, respectively). On days 13 and 11, TSH was 67 and 202 uIU/mL in Pl and P2, respectively (normal: 0.3-7 uIU/mL), and free thyroxine (T4) was 14 and 13.3 pmol in Pl and P2, respectively (normal: 9.5-25 pmol) (Data not shown). L-thyroxine therapy was initiated. I¹²³ scintigraphy showed thyroid ectopia in both siblings. Another sibling (II.5, P3), aged 12 years, had thyroid hypoplasia (thyroid volume, 3.1 mL; normal: 7±3 mL) with a pyramidal lobe and normal thyroid function tests. The parents had normal thyroid function and two other siblings (II.3 and II.4) had normal thyroid function but were not able to undergo thyroid ultrasonography.

To look for genetic causes of CH in Pl and P2, we performed whole exome sequencing (WES) using the variant filtering and prioritisation strategy. Using the recessive transmission model, WES identified a novel missense homozygous TUBB1 mutation (c.479C>T, p.Pl60L, rs759117911) in both siblings with CH (Pl and P2) and in the sibling with thyroid hypoplasia (P3) (Data not shown). Both parents and sibling II.3 were carriers. The remaining sibling (II.4) did not carry the mutation. WES identified no variants in genes known to be associated with TD or thyroid dyshormonogenesis.

Search for TUBB1 mutations in a cohort with thyroid dysgenesis (TD) and congenital hypothyroidism (CH)

After identification of the above-described novel TUBB1 mutation, we used targeted next generation sequencing (NGS) to assess TUBB1 in a cohort of 270 patients with CH and TD. In a second family (F2) with a father (1.2) of Moroccan and a mother (1.1) of French descent, a female with CH and thyroid gland ectopia (P4, II.1) had a heterozygous TUBB1 mutation (c.3 l8C>G, r.U106C) (Fig. la). CH was diagnosed upon routine neonatal screening (TSH, 250 mΐu/mL) and confirmed on day 15 (TSH, 1,100 qfU/mL; free T4, 3.5 pmol/L; and free T3, 2.45 pmol/L). Thyroid scintigraphy showed an ectopic thyroid. The father carried the same heterozygous mutation but was not able to undergo thyroid ultrasonography. In a paternal aunt (1.3, P5), an evaluation at 26 years of age for obesity and depression showed mild hypothyroidism (TSH, 6.6 uIU/mL; normal, 0.1-5.5 mΐu/mL; free T4, 8.7 pmol/L; normal, 9.8- 23.1 pmol/L). Thyroid ultrasonography and scintigraphy showed thyroid hemi-agenesis with absence of the left lobe.

In a third family (F3), a patient (II.1, P6) with CH and an ectopic thyroid was shown by targeted NGS to have a heterozygous frameshift TUBB1 mutation (c.35delG, p.Cys 12Lcufs* 12, rs77324804) that created a premature stop codon at amino acid 23 (Fig. la). CH was diagnosed neonatally based on TSH elevation (476 mΐu/mL) and low free T4 and T3 levels (8 pmol/L and 5.6 pmol/L, respectively). L-thyroxine therapy was started at 11 days of age. Both parents were of French descent. The father (1.1, P7) carried the same heterozygous mutation and had normal thyroid function with mild thyroid lobe asymmetry by ultrasonography (right lobe, 6.9 mL; left lobe, 5 mL). The other siblings and mother had normal thyroid function and morphology and did not carry the mutation.

By targeted NGS, neither P4 nor P6 had any variants in genes known to cause CH (with TD or dyshormonogenesis).

In the Exome Aggregation Consortium (ExAC) database, estimated allele frequencies are 0.000008 for the c.479C>T mutation (rs759l 17911, 20:57598961 C/T) and 0.000025 for the c35delG (rs773248042, 20:57594611 TG/T) mutation. Neither mutation has been reported in homozygous form. The c.3 l8C>G variation has not been reported in public databases. The in silico prediction tools, Polyphen-2, SIFT, predict that c.479C>T is probably damaging and deleterious, respectively, with a CADD score of 32 (damaging: >l5)²⁴. The other two mutations create a premature stop codon and have CADD score of 35.

A Burden test was applied to determine whether TUBB1 was significantly enriched in rare variants in the 270 patients with CH and TD versus 406 Caucasian controls from the 1,000 Genomes project. The patients had athyreosis, ectopia, hemi-agenesis, or hypoplasia. Compared to the control group, the TD group had a significantly higher proportion of individuals exhibiting at least one rare functional variant (p= 0.0227). None of the patients in our cohort with TUBB1 mutations had athyreosis. Performing the Burden test after excluding the 77 patients with athyreosis increased the significance of the difference of TD group (n=l93) versus controls 6/^=0.0095 ). The three amino acids affected by the TUBB1 mutations are strictly conserved across species, from humans to zebrafish, and across all b-tubulins (data not shown). All three mutations were located in the first part of TUBB1, i.e., in the N-terminal domain needed for guanosine triphosphate (GTP) activity (Fig. lb). The c.3 l8C>G and c.35delG mutations created a premature stop codon, thereby removing the intermediate and C-terminal domains required for microtubule-associated protein (MAP) binding.²⁵

In sum, we identified three TUBB1 mutations in three independent families of patients with CH and TD chiefly consisting in thyroid gland ectopia.

bΐ-tubulin is expressed in the developing thyroid in humans and mice

b 1 -tubulin expression has so far been reported only in megakaryocytes and platelets^26,27. Our finding of TUBB1 mutations in patients with TD prompted us to look for bΐ -tubulin expression in thyroid tissue. In human thyroid tissue, TUBB1 mRNA was expressed at 8, 10, and 12 GW and in adulthood (data not shown). In mouse thyroid tissue, Tubbl was expressed at E13.5 and strongly at E15.5, E17.5, and adulthood (data not shown). To refine our study of Tubbl expression, we used cells sorted from mice thyroid tissue based on well-accepted markers²⁸. As expected, expression was strongest in platelets sorted using the specific megakaryocyte lineage marker CD41 (data not shown). However, Tubbl was also expressed in EpCam-positive epithelial-cell populations containing thyrocytes, at El 7.5 and adulthood (data not shown).

Similarly, in human thyroid tissue, immunohistochemistry showed bΐ -tubulin expression in the cytoplasm of thyroglobulin (TG)-producing thyrocytes at 12 GW (data not shown). Comparable findings were obtained with mouse thyroid tissue (data not shown). These data established that bΐ -tubulin is expressed in thyrocytes.

Functional in vitro analysis of disease-causing mutations

To further investigate the implication of TUBB1 gene mutations in thyroid disease, we transfected the mutations into the Nthy cell line. Only the c479C>T (p.Pl60L) mutation could be studied in this model. The c3 l8C>G (p.Yl06X) and c.35delG (p.Cys l 2Lcufs* 12) mutations created premature stop codons that yielded truncated proteins that cannot form functional a/b- tubulin dimers, and thus cannot get incorporated into microtubules^25,29. The pathogenic role of these mutations is thus loss-of-fimction.

Structural modelling of p.P160L mutant bΐ-tubulin

To assess the consequences of the P160L mutation on bΐ -tubulin function, the sole missense mutation that should give rise to a full-length protein, we first compared the configurations of the mutated and wild-type proteins. The P160L mutation is located at the end of helix H4 (data not shown). In wild-type bΐ -tubulin, the proline residue stabilises loop H4- S5 and places R156 in a position that promotes a salt bridge interaction with D197 in the b- sheet S6. Furthermore, R162 in loop H4-S5 and N195 in the b-sheet S6 establish hydrogen bonds with R156. Mutation of pro line 160 to leucine might affect the loop conformation of the H4-S5 loop and thus disrupting the interaction network mediated by R156. Hence, the P160L mutation is most likely affecting the conformation of the B -tubulin, which could lead to dysfunctions of the a/b-tubulin dimer.

In vitro consequences of the p.P160L bΐ -tubulin mutation on microtubule incorporation

Following the predictions from the modelling, we examined the ability of bΐ -tubulin P160L protein to incorporate within the microtubule network in the Nthy cell line. We transfected Nthy cells with the wild-type and P160L mutant then compared the distribution and location of the protein by double-label immunofluorescence (data not shown). While the overexpressed wild-type bΐ -tubulin coassembled incorporates into microtubules, not incorporation was seen for bΐ -tubulin P160L. Thus, the P160L mutation clearly affects the capacity of bΐ -tubulin to incorporate into microtubules, which is most likely related to defects on the structural level, thus forming a dysfunctional a/B-tubulin dimer.

In summary, all three TUBB1 mutations identified here lead to nonfunctional a/B-tubulin dimers that cannot be incorporated into microtubules.

Tubbl knock-out in mice affects thyroid development and function

The phenotype of patients bearing TUBB1 mutations suggests that bΐ -tubulin may contribute to thyroid development and function. We assessed this hypothesis in Tubbl knock out mice ( TubbT ).

As expected, we found significant increases in expression levels of the other b-tubulin iso forms (Tubb2a, Tubb5, Tubb2b, and Tubb3) in E17.5 thyroids of TubbT^/ mice compared to wild-type mice (data not shown). Tubb2b and Tubb3 expression levels were also increased in adult TubbT mice. In addition, Tuba3 and Tuba4 expression in TubbT mouse thyroids were diminished at El 7.5 and in adults. Interestingly, these compensatory changes in expression levels seen in the thyroid gland mirror those described in platelets of TubbT mice (data not shown)²³.

Thyroid gland development and differentiation

Thyroid gland morphology in mice To study thyroid gland development from the early stages of budding and migration of the median anlage (MA) and ultimobranchial bodies (UB) to the late stages of differentiation, we used immunohistochemistry, surface quantification, and quantitative PCR (data not shown). For most of the experiments, we used Nkx2-l as a marker of progenitor and differentiated thyroid cells. At E9.5, thyroid anlage surface area and proliferation ratio were significantly greater in TubbT¹ mice than in wild-type mice (data not shown. At El 1.5, thyroid migration was slightly delayed in 3 of 6 TubbT¹ embryos, a few Nkx2-l -positive cells were visible along the migration tract of the mutants, and thyroid surface area was not different between mutant and wild-type embryos (data not shown). During late thyroid development, at E13.5, fusion of the median anlage and ultimobranchial bodies was slightly delayed in 3 of 4 TubbT ^/_ embryos (data not shown). After E13.5, Tubbl ¹ thyroids were significantly hypoplastic (data not shown). Moreover, at El 7.5, a supplementary pyramidal lobe was visible near the normal lobe in 2 of 6 embryos (data not shown). In keeping with this finding, patient P3 in family 1 described above, who was homozygous for the p.Pl60L mutation, had a hypoplastic thyroid with a pyramidal lobe. In sum, the thyroid phenotype of TubbT¹ mice indicates that bΐ -tubulin is required for normal thyroid migration and morphology.

Thyroid gene ontogeny in mice

We used quantitative PCR to assess the ontogeny of genes involved in thyroid function and development (data not shown). Expression levels of mRNAs for Tg, thyroid peroxidase (Tpo), TSH receptor (Tshr), and Calca), late differentiation markers, were significantly decreased at E15.5, as were those of Tg, Tpo, and Calca at E17.5, in TubbT^/ compared to wild- type embryos. Significant decreases were also observed at E17.5 in the TubbT^/ embryos in the early thyroid development markers Nkx2-l, Pax8, and Foxel . These results indicate impaired thyroid gland development and differentiation in Tubb ^/_ mice.

Endocrine signature at completion of thyroid gland development

T4-positive thyroid surface area relative to total thyroid surface area was shown by immunohistochemistry to be significantly increased at E17.5 in TubbT ^/_ versus wild-type embryos (data not shown). Calcitonin-positive thyroid surface area relative to total thyroid surface area was also significantly greater in the mutants. Thus, final thyroid differentiation was abnormal in TubbT ^/_ embryos, with increases in T4 and calcitonin that probably reflected impaired hormone secretion.

These results establish a central role for bΐ -tubulin in thyroid development and differentiation.

Thyroid gland function and structure in adult mice We compared thyroid hormone status in adult Tubbl ' and wild-type mice. At 3 months of age, serum TSH levels were higher and T4 levels lower in Tubbl ¹ than in wild-type mice, suggesting hypothyroidism in the mutants data not shown). These results are consistent with the finding of CH in our patients carrying TUBB1 mutations.

In sum, the increase in T4-positive surface area combined with low serum T4 suggest trapping of T4 hormone within the thyrocyte cytoplasm.

Finally, we examined thyroid structure in adult mice (3 months of age). Surface area was not different between the Tubbl ¹ and wild-type thyroids (data not shown). However, the thyroid tissue was disorganised in the mutants with large regions without organized follicles (data not shown). When we used electron microscopy to examine thyrocyte ultrastructure (data not shown), we found larger numbers of dense and rod-shaped vesicles in Tubbl ¹ versus wild-type thyrocytes. These data suggest that the T4 retention in El 7.5 thyroids of Tubbl ¹ mice was related to impaired T4 release responsible for hypothyroidism. Marked endoplasmic reticulum (ER) dilation was seen in Tubbl ¹ compared to wild-type thyrocytes (data not shown), indicating ER stress, which was confirmed by the findings of increased Chop and XBPs expression by quantitative PCR and of increased Chop protein levels by western blotting in Tubbl ¹ thyroids (data not shown). By immunohistochemistry, the ER marker KDEL co- localized with TG in Tubbl ¹ and wild-type mice but was especially abundant in disorganized areas of adult Tubbl ¹ thyroids (data not shown).

These data indicated thyroid tissue disorganization, vesicle accumulation, and ER stress in TubbT^/ mice.

In sum, our findings in Tubbl-/- mice demonstrate a complex mechanism of hypothyroidism involving early abnormal proliferation of progenitors, delayed thyroid migration, defective thyroid tissue differentiation, and impaired thyroid hormone release. The abnormal thyroid migration may explain the thyroid phenotype found in patients carrying TUBB1 mutations, especially those with thyroid gland ectopia. Additional contributors to hypothyroidism are structural disorganization of the thyroid tissue and retention of thyroid hormone.

Analysis of human platelets of patients bearing TUBB1 mutations

Until now, bΐ -tubulin expression had been reported only in the megakaryocyte lineage¹⁹. TUBB1 mutations have been previously reported to cause macrothrombocytopenia^{20- 22}. We therefore studied the platelets of the above-described patients with TUBB1 mutations and thyroid gland abnormalities.

Haematological parameters Thrombocytopenia was a feature in patients with TUBB1 mutations studied by other groups^20-22. However, when we used an automated haematology analyser to measure the platelet count and mean platelet volume (MPV) in our seven patients, we consistently found normal platelet counts. In contrast, MPV was above the normal range in Pl and near the upper limit of normal in P4 and P7. A blood smear analysis (data not shown) demonstrated variations in platelet size, with the presence of macroplatelets. Moreover, parents 1.1 and 1.2 of the family Fl had MPV values at the top of the normal range (10.2 fL and 1 1.3 fL, respectively; normal, 7.5-11.2 fL) with many macroplatelets. Electron microscopy confirmed the large platelet size (data not shown).

The increased platelet size could be related to defects in their biogenesis. To study this we cultured megakaryocytes from patients Pl and P4 from peripheral blood progeniors and analysed proplatelet formation (data not shown). Interestingly, shaft thickness and coiled element diameter of the future platelets were significantly increased compared to controls, indicating that the TUBB1 mutations affected proplatelet formation and platelet size.

Functional analysis of human platelets

To investigate whether the TUBB1 mutations affected platelet function, we first quantified bΐ -tubulin expression in the platelets of our patients and found significant decreases ( O.OOl), of45.0±3.7% and 36.5±3.4% in Pl and P3 (Fl), respectively; 45.0±3.8% in P4 (F2); and 29. l±3.3% and 30.6±6.2% in P6 and P7 (F3), respectively. Expression of a-tubulin was normal (data not shown). The bΐ -tubulin antibody used in our study detects only the C- terminal part of the protein. Consequently, in the patients of families F2 and F3, whose mutations created premature stop codons, only the wild-type protein was detected. In the Fl patients, who were homozygous for the mutation, the results suggested either diminished protein expression or protein instability.

A discoid shape is a normal feature of circulating or non-activated platelets. By electron microscopy, discoid platelets were less numerous in Pl and P3 (Fl) (data not shown), consistent with either abnormal platelet sphericity or platelet activation. We therefore assessed basal platelet activation, using flow cytometry to investigate the platelet activation markers P- selectin exposure and integrin api,b ^ activation in whole blood. Both markers were significantly increased in Pl and P3, indicating abnormal platelet activation. In P6 and P7 (family 3), in contrast, the marker levels were comparable to those in controls (data not shown). We were unable to evaluate P4 (F2). Finally, we investigated platelet aggregation upon activation by ADP or collagen, two key platelet agonists (data not shown). Aggregation in response to ADP was normal in P6 and P7 (F3) but increased in Pl and P3 (Fl) and in P4 (F2) compared to controls (data not shown). Similarly, with collagen, platelet aggregation was normal in P6 and P7 but was increased in Pl and P4, even with low doses. These abnormal platelet aggregations were not related to hypothyroidism or patient’s treatment by L-Thyroxine, since they were not found in three patients without TUBB1 mutations but under the same treatment (data not shown). Moreover, it should be noted that these results were confirmed by at least three independent investigations for Pl and P3 and two for P6. The other patients were studied once. None of the 9 controls exhibited similar hyperaggregation profil,

In sum, these results indicate that p.Pl60L (Fl), r.UIObC (F2), and c.35delG (F3) TUBB1 mutations affect bΐ -tubulin expression in platelets and result in abnormally large platelet size, probably as a consequence of proplatelet abnormal formation. Moreover, the p.Pl60L mutation induces significant basal platelet activation and hyper aggregation in response to agonists, whereas the c.35delG mutation does not seem to affect platelet function.

Discussion:

We identified three TUBB1 mutations in patients with TD and macroplatelets. In a consanguineous family (Fl), two females with CH and thyroid gland ectopia had the same homozygous TUBB1 mutation, and their brother had thyroid gland hypoplasia with normal function. Of 270 patients with CH and TD, two probands from unrelated families had heterozygous TUBB1 mutations. All patients with heterozygous or homozygous TUBB1 mutations had TD and macroplatelets. Thyroid gland ectopia was the most common form of TD, but thyroid asymmetry, thyroid hypoplasia, and thyroid hemi-agenesis were seen also. All three TUBB1 mutations impaired bΐ -tubulin protein function.

This is the first time that CH with TD was associated with macroplatelets and we demonstrate in our study that TUBB1 mutations are the common cause bΐ -tubulin expression has heretofore been described as confined to megakaryocytes and platelets. Our findings demonstrate that bΐ -tubulin is also expressed in developing and adult thyroid tissue. Furthermore, CH and TD were found in humans with TUBB1 mutations and in Tubbl mice. These data indicate that normal thyroid development and function require bΐ -tubulin incorporation into the microtubule network. Furthermore, our patients with TUBB1 mutations had normal platelet counts contrasting with abnormal platelet morphology, and two of the three mutations (p.Pl60L and r.U106C) were associated with abnormal platelet function. In contrast, in previous studies, humans with TUBB1 mutations and Tubbl mice had macrothrombocytopenia but no reported thyroid disorders^20-22.

The previously reported TUBB1 mutations affect the intermediate or C-terminal domain of bΐ -tubulin, whereas the three novel mutations described here modify the N-terminal domain. Strikingly, all three mutations are loss-of- function mutations: two of the mutations lead to truncated bΐ -tubulin, which cannot form functional a/B-tubulin dimers and thus cannot be incorporated into microtubules. The third is a missense mutation (P160L), which, despite expressing full-length B-tubulin, still does not allow the formation of functional a/B-tubulin dimers to be integrated into the microtubules network. Hence, the three TUBB1 mutations were deleterious for the bΐ -tubulin function, which is confirmed on the functional level as their phenotypes are similar to those of the Tubbl-/- mouse. Consequently, Tubbl ² mice emerged as a useful tool for studying the impact of impaired bΐ -tubulin function on the thyroid gland.

Our experiments in Tubbl ² mice provided the first evidence of a role for bΐ -tubulin in TD and hypothyroidism. The thyroid glands of Tubbl ² mice exhibited deficient proliferation at E9.5, abnormal migration at El 1.5 and E13.5, and hormone secretion failure at E17.5 and adulthood. All these mechanisms require normal microtubule organisation and function. The idea that microtubule functions are fine-tuned, and thus adapted to specific cellular role by the expression of specific tubulin isotypes is an emerging concept known as the‘tubulin code’¹⁷. Tubulin mutations have been more and more linked to different human disorders, in particular to neurological disorders³⁰. Microtubules are essential for mitosis to unfold normally³¹. Here we show that invalidation of B-tubulin isotype Tubbl perturbs mitosis of progenitors at E9.5, thus inducing hyperproliferation. Mitotic phenotypes have already described during neuronal development in mice with loss of another B-tubulin isotype, TiibbS'². Moreover, normal thyroid development requires that progenitor proliferation occur during a specific time window³³. Early proliferation at E9.5, as described by our group in HesT¹ mice³⁴, results in abnormal thyroid development, probably via impairment of the pool of progenitors dedicated to thyroid development. Furthermore, microtubules are required for cell orientation during migration³⁵. Our data in the Tubbl-/- mice show a delay of thyroid progenitors cells migration during thyroid development, which again is mirrored by Tubb5-/- that show impaired neuronal migration³². In our patients with TUBB1 mutations, thyroid gland ectopia was the most common form of TD (4/7 patients), further supporting a role for bΐ -tubulin in thyroid gland migration. Finally, endosome/lysosome trafficking is taking place on microtubules^36-38. Endosome-to-lysosome transport of thyroid hormones via a vesicular transport system has been described in the thyroid gland^39,40. That thyroid microtubule integrity is required for thyroid hormone secretion was established by studies done in the l970s and l980s^41,42. While so far nothing is known on the precise role of tubulin isotypes and microtubules organization in trafficking of thyroid hormone vesicles, there is strong evidence that transport along microtubules is affected by the tubulin code⁴³. Our data are the first to demonstrate that a specific tubulin isotype, b 1 -tubulin, is required for proper vesicle trafficking and thyroid hormone release into the bloodstream, and they extend our knowledge about the association between microtubules and secretion vesicles in the thyroid gland. We found that thyroid hormone retention suggested in ER stress in the thyroid gland. Previous studies demonstrated ER stress with activation of the unfolded protein response in association with reduced TG synthesis and TG accumulation within the ER^44,45. bΐ -tubulin is the most divergent isotype of tubulin, and its incorporation into microtubule is expected to change the properties of these filaments. Losing bΐ -tubulin thus certainly alters microtubule properties, and our data provide strong evidence that these particular properties are essential for adequate thyroid development and function.

Our patients with TUBB1 mutations had macroplatelets but no thrombocytopenia, in contrast to earlier reports^20-22. The abnormal platelet size is further evidence that TUBB1 mutations adversely affect platelet morphology probably as a consequence of proplatelets coiled element diameter increase, as already suggested. In these previous described mutations, only the p.R3 l8W mutation has been investigated in platelet aggregations and it did not induce defect of platelet function²⁰. For the other mutations (p.F260S and p.Q423X)^21,22, no bleeding tendency have been reported. An unexpected finding in our patients with p.Pl60L and p.Yl06X mutations was basal activation and exaggerated aggregation of platelets, which had not been described previously in patients with TUBB1 mutations or in Tubbl mice. Whether these platelet function abnormalities result in clinical symptoms such as thrombosis requires evaluation, particularly in older patients who may be at increased risk. Platelets play an important pathophysiological role in thrombosis and inflammation in atherosclerosis. Large platelets have greater haemostatic and enzymatic potency and contain larger amounts of prothrombotic molecules chemokines and growth factors affecting both pathophysiological processes⁴⁶ and increased MPV was previously reported to be associated to cardiovascular disease⁴⁷. Thyroid dysfunction can affect the haemostatic balance^48,49, with clinical effects that vary across thyroid disorders⁵⁰. Moreover, MPV correlated positively with the TSH level⁵¹. A TUBB1 -mutation screening study in patients with hypothyroidism and altered MPV and/or a history of thrombotic disease would be of interest. The clinical symptoms to accompany platelet hyperaggregability include unexplained arterial thrombosis, complications during pregnancy, and less frequently, venous thromboembolism. Patients bearing a TUBB1 mutation with cardiovascular risk factors could be monitored and the question arises whether antiplatelet drugs might be effective as treatment for and/or prophylaxis⁵².

The c.35delG mutation in P6 and P7 did not cause hyperaggregation. The patients with the c.35delG mutation had the same thyroid phenotype as other families with other mutations. At least one patient per family had thyroid gland ectopia with CH. Neither thyroid nor platelet phenotype severity correlated with the type of mutation. Further investigations are needed to elucidate the difference between the effects of c.35delG and pl60L/pYl06X on platelet function.

TUBB1 mutations constitute a model of dominant inheritance of CH with TD. Most known mutations responsible for TD and previously described TUBB1 mutations causing macrothrombocytopenia are also dominant^{10,13,20 22,53}. Our data indicate high penetrance for platelet alterations and variable expressivity for TD, ranging from TD without CH to TD with CH and leading to variable types of TD (ectopia, hypoplasia, hemi-agenesis, or asymmetric thyroid gland). No patient had athyreosis. TUBB1 mutations were found in only 1.1% of our cohort with CH and TD patients. The Burden test showed enrichment in rare TUBB1 variants carriers in the cohort versus controls. Our data increment the number of causative genes for thyroid dysgenesis but with a novel phenotype associating platelets disorder.

Taken together, our data indicate heretofore unsuspected roles for a specific isotype of b-tubulin, Tubbl, in thyroid development and function, while also confirming its importance for microtubule integrity and platelet function. Loss-of-fimction of TUBB1 mutations impair bΐ -tubulin incorporation into microtubules. Our results confirm that normal thyroid-cell proliferation and thyroid migration are essential to thyroid gland development. Thyroid hormone secretion requires bΐ -tubulin incorporation into the microtubules, suggesting a specific function of this tubulin isotype in intracellular transport of vesicles. Thus, our work provides novel insights into the role of the Tubbl isotype in thyroid physiopathology and in platelet function and therefore expands the spectrum of the rare pediatric diseases related to tubulin mutations and microtubule malfunction.

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Claims

CLAIMS:

1. A method of diagnosing thyroid dysgenesis in a subject, comprising detecting a mutation in the TUBB1 gene in a sample obtained from said subject, wherein detecting the presence of a mutation in the TUBB 1 gene is considered to be indicative of thyroid dysgenesis.

2. The method of claim 1 wherein the subject is a new-born or a foetus.

3. The method of claim 1 wherein the sample is a blood sample.

4. The method of claim 1 wherein the mutation is located in the N-terminal domain needed for guanosine triphosphate (GTP) activity, i.e. the domain ranging from the amino acid residue at position 1 to the amino acid residue at position 206 in the protein (SEQ ID NO:2).

5. The method of claim 1 wherein the mutation leads to nonfunctional a/B-tubulin dimers that cannot be incorporated into microtubules.

6. The method of claim 1 wherein the mutation leads to an early abnormal proliferation of progenitors, delayed thyroid migration, defective thyroid tissue differentiation, and impaired thyroid hormone release.

7. The method of claim 1 wherein the mutation affects bΐ 1 -tubulin expression in platelets and result in abnormally large platelet size, probably as a consequence of proplatelet abnormal formation.

8. The method of claim 1 wherein the mutation is a missense homozygous TUBB1 mutation (c.479C>T, p.Pl60L, rs759l 17911) which is a substitution of a cytosine residue into a thymine residue at position 479 of the TUBB1 gene.

9. The method of claim 1 wherein the mutation is a heterozygous TUBB1 mutation (c.3 l8C>G, p.Yl06X) which is the substitution of a cytosine residue into a guanine residue at position 318 of the TUBB1 gene

10. The method of claim 1 wherein the mutation is a heterozygous frameshift TUBB1 mutation (c.35delG, p.Cysl 2Lcufs* 12, rs77324804) that created a premature stop codon at amino acid 23.