SUPPRESSORS OF PREMATURE TERMINATION CODONS AS THERAPEUTICS AND METHODS FOR THEIR USE

TECHNICAL FIELD

This invention relates to therapeutic compounds and compositions, and methods for their use in the treatment or amelioration of various indications, including medical conditions associated with premature termination codons (PTCs) in RNA, including various cancers. In particular the invention relates to therapies and methods of treatment that would at least partially restore translation of full-length protein products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. US Provisional 62/232,789 filed 25 September 2015.

BACKGROUND

Genomics advances will soon make it routine to identify the precise molecular lesions responsible for many of the rare genetic diseases that afflict our population.

Unfortunately, most of these diseases have no treatments, in Canada about 30% of patients die in childhood, and it is exceedingly difficult to develop disease-specific treatments because of the small number of patients for each disease and the high cost of developing new drugs. About 10% of disease-causing mutations are nonsense mutations that introduce a PTC.

The European Organization for Rare Diseases estimates that there are at least 5,000 rare genetic diseases, defined as affecting less than 1 in 2,000 people and the genes for about 4,000 have been identified (Online Mendelian Inheritance in Man database). Rare genetic diseases are believed to affect 5-6% of the population, or about 25 million people in the EU, 16 million in the USA and 1.8 million in Canada. It is estimated that 95% of rare genetic diseases have no specific treatment. Furthermore, genetic diseases that would not have the rare classification, also have nonsense mutations. It is estimated that 20.3% of the ~ 43,000 disease-associated single-base pair substitutions affecting gene coding regions that are cataloged in the Human Gene Mutation Database (HGMD 2007 - Mort M. et al. 2008) are PTCs.

For nearly all these diseases, about 10-11% of patients have nonsense point mutations. These mutations change an amino acid codon to a PTC (i.e. UAA, UAG and UGA). PTCs may result in decreased mRNA stability via nonsense-mediated mRNA decay (NMD), as well as production of some truncated non-functional protein, if any protein is produced. Compounds that allow insertion of an amino acid at a PTC, without affecting normal termination codons, can enable production of functional full-length protein. This approach, termed both nonsense mutation suppression and PTC read- through, offers the possibility of developing a single treatment for large numbers of patients across multiple diseases. In reality, a proportion of these patients would likely not benefit from such a therapy, for example those children born with irreversible neurological damage. Nevertheless, for 50% of rare genetic diseases, the onset of disease occurs in childhood and progressively worsens, and these patients are the ones who stand to benefit most from nonsense suppression therapy.

The therapeutic potential of nonsense suppression is not limited to inherited disorders. Nonsense mutations also occur in tumour suppressor genes in about 10% of cases of sporadic cancer, which affects 40% of the population and is far from rare. To illustrate, the R213X mutation in protein p53 is present in 1% of all human cancers. This corresponds to about 220,000 cases worldwide (Hoe, K.K. Verma, C.S. and Lane, D.P. 2014) that could theoretically benefit from nonsense suppression therapy. A further 70 cancer- driver tumour suppressor genes have been identified (Vogelstein B, et al. 2013). Tumour sequencing and mutation analysis is not yet routine for cancer diagnosis. However, the concept of personalized medicine has taken huge steps in the cancer field and it is anticipated that identifying nonsense mutations in cancer will become routine in the next decade.

Accordingly, the targeting of nonsense mutations could eliminate the "rare" element of rare genetic diseases in some cases where the genetic disease is caused at least in part by a nonsense mutation and nonsense suppression may also be of use in the treatment of some cancers.

Compounds that enable PTC read-through, offer the possibility of using the same treatment for large numbers of patients across multiple diseases based on the mechanism of the PTC and not the particular gene having the PTC.

High concentrations of aminoglycoside antibiotics were shown 30 years ago to induce PTC read-through in some yeast genes (Singh A et al. 1979) and in a reporter gene in mammalian cells (Burke JF and Mogg AE. 1985). The potential for using gentamicin to treat cystic fibrosis patients with a PTC in the CFTR gene was shown when gentamicin was used to induce CFTR protein expression from the endogenous gene in a patient- derived bronchial epithelial cell line (Bedwell DM et al. 1997), recovery of function in mice bearing the human CFTR G542X transgene (Du M et al. 2002) and increases in CFTR chloride conductance in patients (Clancy JP et al. 2001; and Wilschanski M et al. 2003). Similarly, paromomycin, geneticin (G418) and PTC124 (3-[5-(2-fluorophenyl)- [i,2,4]oxadiazole-3-yl]-benzoic acid) are all reported to have nonsense suppressive properties (Karijolich J, and Yu, Y-T 2014). In all cases the improvement was small and patient response was variable (Linde L et al. 2007). The lack of potency, the recognized renal and otic toxicities of high dose gentamicin and the need for intravenous or intramuscular administration likely limited its further development.

Read-through by gentamicin was demonstrated in mdx mice (Barton-Davis ER et al. 1999) with a PTC introduced into the mouse dystrophin gene to model human Duchenne Muscular Dystrophy (DMD). The first small trial in DMD patients showed no effect of gentamicin. Two others showed dystrophin expression in some patients (Malik V et al. 2010) but the level of expression was insufficient for patient improvement. Again, dose- limiting toxicities prevented further development.

Major efforts have been put into developing aminoglycoside derivatives with reduced toxicity e.g. (Shulman E et al. 2014; and Xue X et al. 2014) and discovering non- aminoglycoside RT compounds such as RTC13, RTC14, GJ71, GJ72 and PTC124 (Gatti RA. 2012; and Welch EM et al. 2007). These compounds increased protein production in several cell culture and animal disease models, but often at the limit of detection by western blotting for endogenous gene expression and with variable responses between genes, cell lines, and PTC mutations.

Furthermore, there are numerous approaches to read-through therapy. For example, read-through drugs, suppressor tRNAs, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay (Keeling, K. M. et al. 2104).

PTC124 (Translarna™) is the sole new compound to have entered clinical trials. It is orally bioavailable and has a good safety profile compared with aminoglycosides.

PTCi24's PTC RT activity has been challenged based on artifactual activity in luciferase reporter assays of the type used for its discovery and lack of demonstrable RT activity in other reporter assays (McElroy SP et al. 2013). Nevertheless, it has shown activity in higher model systems, including increased dystrophin expression and muscle function in the mdx mouse (Welch EM et al. 2007) and CFTR protein expression and improved chloride conductance in the intestine of the G542X-I1CFTR mouse (Du M et al. 2008). Recently, a phase 3 clinical trial in CFTR (Kerem E. 2014) and a phase 2b trial in DMD patients (Bushby K et al. 2014) both failed to reach statistical significance. However, retrospective analyses hinted at signs of efficacy in subgroups of authorization for DMD treatment in the European Union, conditional upon completion of a phase 3 trial (mid- 2015) and submission of additional safety and efficacy data (Ryan NJ. 2014). Overall, currently available RT compounds suffer two major limitations: they display low activity, typically inducing less than 5% of wild-type (wt) protein levels; and they show unpredictable activity in only a small subset of genetic disease systems tested.

SUMMARY

This invention is based in part on the discovery that compounds described herein suppress premature termination codons. Specifically, compounds identified herein, show the ability to read through premature stop codons.

In accordance with one embodiment, there is provided a pharmaceutical composition including 1) a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure

of Formula II:

,NH ₂ OH .OH

wherein M may be , or ^Ν ^ ; and 2) a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.

In accordance with a further embodiment, there is provided a pharmaceutical composition including 1) a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula I:

wherein R may be OH or NH ₂ ; M may be

when R is NH ₂ ; and 2) a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier.

In accordance with a further embodiment, there is provided a method of treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, the method including administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with a PTC in RNA, wherein the compound has the structure of

Formula II: wherein M may be ; to a subject in need thereof.

Alternatively, the method may have a compound of Formula I.

In accordance with a further embodiment, there is provided a method of promoting read-through of a premature termination codon (PTC) in a RNA sequence, , the method including administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with a PTC in RNA, wherein the compound has the structure of Formula II:

wherein M may be ; to a subject in need thereof. Alternatively, the compound may be of Formula I.

In accordance with a further embodiment, there is provided a method of promoting production of a functional protein in a cell, the protein encoded by a nucleotide sequence comprising a premature termination codon (PTC), the method comprising contacting the cell with an effective amount of a compound having the

structure of Formula II: II

.NH ₂ .OH ,OH

wherein M may be ^ν ^ ^Λ , *f or ^v>t ; to a subject in need thereof. Alternatively, the compound may be of Formula I.

In accordance with a further embodiment, there is provided a compound, wherein

the compound has the structure:

In accordance with a further embodiment, there is provided a pharmaceutical composition, the pharmaceutical composition comprising: a compound having the

structure ; and a steroid.

In accordance with a further embodiment, there is provided a method of treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, the method including administering a compound, or a pharmaceutically acceptable salt thereof, in an amount effective to treat or ameliorate a medical condition associated with a PTC in RNA, wherein the compound has the structure of

on with a steroid, to a subject in need thereof. In accordance with a further embodiment, there is provided a use of a compound, or a pharmaceutically acceptable salt thereof, in an amount effective for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula II:

wherein M may be Alternatively, the compound may be of Formula I.

In accordance with a further embodiment, there is provided a use of a compound in the manufacture of a medicament for treatment or amelioration of a medical condition associated with premature termination codons (PTCs) in RNA, wherein the compound has the structure of Formula II:

,ΝΗ, \ ^OH OH

and wherein M may be Y , or . Alternatively, the compound may be of Formula I.

In accordance with a further embodiment, there is provided a commercial package comprising: (a) a compound having the structure of Formula II:

wherein M may be ; and (b) instructions for treating or ameliorating a medical condition associated with premature termination codons (PTCs) in RNA. Alternatively, the compound ma be of Formula I.

The compound may be selected from one or more of the following:

The compound may be selected from one or more of the following:

The compound may be selected from one or more of the following:

The medical condition may be selected from one or more of the conditions listed in TABLE l or TABLE 2. The medical condition may be selected from TABLE l or TABLE 2. The medical condition may be selected from TABLE l. The medical condition may be selected from TABLE 2.

The medical condition may be selected from the group consisting of: central nervous system disease; peripheral nervous system disease; neurodegenerative disease; autoimmune disease; DNA repair disease; inflammatory disease; collagen disease; kidney disease; pulmonary disease; eye disease; cardiovascular disease; blood disease; metabolic disease; neuromuscular diseases; neoplastic disease; and any genetic disorder caused by nonsense mutation(s).

The medical condition may be selected from the group consisting of: ataxia- telangiectasia; muscular dystrophy; Duchenne muscular dystrophy; Dravet syndrome; myotonic dystrophy; multiple sclerosis; infantile neuronal ceroid lipofuscinosis;

Alzheimer's disease; Tay-Sachs disease; neural tissue degeneration; Parkinson's disease; chronic rheumatoid arthritis; lupus erythematosus; graft-versus-host disease; primary immunodeficiencies; severe combined immunodeficiency; DNA Ligase IV deficiency; Nijmegen breakage disorders; xeroderma pigmentosum (XP); rheumatoid arthritis; hemophilia; von Willebrand disease; thalassemia (for example; β-thalassemia); familial erythrocytosis; nephrolithiasis; osteogenesis imperfecta; cirrhosis; neurofibroma; bullous disease; lysosomal storage diseases; Hurler's disease; familial cholesterolemia; cerebellar ataxia; tuberous sclerosis; immune deficiency; cystic fibrosis; familial

hypercholesterolemia; pigmentary retinopathy; retinitis pigmentosa; amyloidosis;

atherosclerosis; giantism; dwarfism; hypothyroidism; hyperthyroidism; aging; obesity; diabetes mellitus; familial polycythemia; Niemann-Pick disease; epidermolysis bullosa; Marfan syndrome; Becker muscular dystrophy (BMD); spinal muscular atrophy; cancer; and any genetic disorder caused by nonsense mutation(s).

The medical condition may be cancer. The cancer may be of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, blood, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals. The cancer may be sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood- born tumor or multiple myeloma. The cancer may be acute lymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, or multiple myeloma.

The premature termination codon may be UGA or UAG. The premature termination codon may be UGA. The premature termination codon may be UAG. The premature termination codon may be UAA. The method may further include the administration of a steroid to the subject. The steroid may be selected from one or more of the following: Medroxyprogesterone; Betamethasone; Dexamethasone; Beclomethasone; Budesonide; Clobetasol propionate; Cortisone acetate; Flumethasone Pivalate; Fluticasone Propionate; Hydrocortisone; Methylprednisolone; Paramethasone; Prednisolone; Prednisone; Triamcinolone;

Danazol; Fludrocortisone; Mifepristone; Megestrol acetate; and Progesterone.

The compound may be OH

The compound may be

The compound may be

The compound may be

The compound may be

The compound may be The compound may be

The compound may be

The compound may be

The compound may be

The compound may be

The compound may be

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE l shows the structures of Gentamicins Ci, Cia, C2, C2a, C2b, B, Bi, A, G418,

X2, Sisomicin, Garamine and Ring C, as well as the structure of some of the steroids tested in combination with G418.

FIGURE 2 shows the induction of PTC read-through by gentamicin Bl and X2 using the 96-well plate immunofluorescence assay, wherein those not shown on the graph had no read-through activity.

FIGURE 3A shows the induction of full-length p53 by gentamicin Bl, gentamicin X2,

G418 and gentamicin measured by western analysis, where the intensity of the full-length (FL) and truncated p53 (TR) bands is shown relative to the intensity of the truncated p53 band seen in untreated cells and is displayed under the lanes.

FIGURE 3B shows the induction of PTC read-through by G418, gentamicin, gentamicin

Bi and gentamicin X2 using western analysis, wherein the amount of full- length p53 observed in FIGURE 3A was plotted versus the concentration of the different compounds on a log scale.

FIGURE 4 shows the induction of full-length P53 by gentamicin G418 in combination with a steroid (A) Dexamethasone; and (B) Betamethasone and Medroxyprogesterone Acetate (Medroxy Pro).

FIGURE 5 shows the induction of premature termination codon (PTC) readthrough by gentamicin Bl and gentamicin X2.

FIGURE 6 shows induction of PTC readthrough at TGA, TAG and TAA termination codons by gentamicin Bl.

FIGURE 7 shows induction of PTC readthrough in variety of cancer cell lines -

SW900; NCI-H1688; ESS-i; SK-MES-l; HCC1937; H1299; and HCT116.

FIGURE 8 shows induction of PTC readthrough in a mouse in vivo assay.

FIGURE 9 shows induction of PTC readthrough by Gentamicin Bl in cells derived from patients with rare genetic diseases, wherein Panels A and B show Neuronal Ceroid Lipofuscinosis; Panel C shows Duchenne Muscular Dystrophy; Panel D shows Schimke Immuno-Osseous Dysplasia; and Panel E shows Recessive Dystrophic Epidermolysis Bullosa. DETAILED DESCRIPTION

In some embodiments, the compounds described herein may be used to treat or ameliorate various indications, including medical conditions associated wdth premature termination codons (PTCs) in RNA, including various cancers. The various conditions may be found in TABLE l.

TABLE l - Medical Conditions Associated with PTC

Medical Condition Associated with PTC Gene

symbol

Pk synthase deficiency (p phenotype) A4GALT

Triple-A syndrome AAAS

Ichthyosis, harlequin ABCA12

Ichthyosiform erythroderma, congenital, nonbullous ABCA12

Fatal surfactant deficiency ABCA3

Fundus flavimaculatus, late onset ABCA4

Stargardt disease ABCA4

Intrahepatic cholestasis, familial progressive 2 ABCB11

Intrahepatic cholestasis of pregnancy ABCB4

Intrahepatic cholestasis, familial progressive ABCB4

Dubin-Johnson syndrome ABCC2

Pseudoxanthoma elasticum ABCC6

Pseudoxanthoma elasticum, autosomal recessive ABCC6

Pseudoxanthoma elasticum, autosomal dominant ABCC6

Hyperinsulinism ABCC8

Hypoglycaemia, persistent hyperinsulinaemic ABCC8

Adrenoleukodystrophy ABCDi

Sitosterolaemia ABCG5

Sitosterolaemia ABCG8

Chanarin-Dorfman syndrome ABHD5

Medium chain acyl CoA dehydrogenase deficiency ACADM

Very long chain acyl-CoA dehydrogenase deficiency ACADVL

Alpha actin 3 deficiency ACTN3

Haemorrhagic telangiectasia 2 ACVRLi

Adenosine deaminase deficiency ADA

Weill-Marchesani syndrome ADAMTS10

Thrombotic thrombocytopaenic purpura ADAMTS13

Upshaw-Schulman syndrome ADAMTS13

Geleophysic dysplasia ADAMTSL2

Ectopia lentis, isolated form ADAMTSL4

Dyschromatosis symmetrica hereditaria ADAR

Parkinson disease, association with ADHiC

Glycogen storage disease 3 AGL

Glycogen storage disease 3a AGL

Renal tubular dysgenesis AGT

Hyperoxaluria AGXT

Molar tooth sign & superior vermian dysplasia AHIi

Joubert syndrome AHIi

Pituitary adenoma AIP

Leber congenital amaurosis IV AIPLi

APECED AIRE

Adenylate kinase deficiency AKi Wilson disease ATP7B

Intrahepatic cholestasis, familial progressive ATP8B1

Intrahepatic cholestasis, benign recurrent ATP8B1

ATRX syndrome ATRX

3-methylglutaconic aciduria type l AUH

Diabetes insipidus, neurohypophyseal AVP

Diabetes insipidus, central AVP

Diabetes insipidus, nephrogenic AVPR2

Tooth agenesis and colorectal cancer AXIN2

B3GALNT1 deficiency (P2K phenotype) B3GALNT1

Cholinesterasaemia BCHE

Butyrylcholinesterase variant BCHE

Maple syrup urine disease BCKDHA

Maple syrup urine disease BCKDHB

Oculofaciocardiodental syndrome BCOR

Bestrophinopathy BESTi

Cleft lip and palate BMP4

Juvenile polyposis syndrome BMPRiA

Polyposis, juvenile intestinal BMPRlA

Pulmonary hypertension, primary BMPR2

Pulmonary arterial hypertension BMPR2

Pulmonary hypertension, primary BMPR2

Breast cancer BRCAi

Breast and/or ovarian cancer BRCAl

Breast cancer BRCA2

Breast and/or ovarian cancer BRCA2

Berardinelli-Seip lipodystrophy BSCL2

Bartter syndrome with sensorineural deafness BSND

Biotinidase deficiency BTD

Agammaglobulinaemia BTK

Premature chromatid separation syndrome BUBiB

Complement CiS deficiency CiS

Complement C3 deficiency C3

Complement C5 deficiency C5

Complement C7 deficiency C7

Complement C8 alpha-gamma deficiency C8A

Carbonic anhydrase deficiency CA2

Cone-rod synaptic disorder CABP4

Episodic ataxia 2 CACNAiA

Night blindness, congenital stationary, incomplete CACNAlF

Muscular dystrophy, limb girdle CAPN3

Ventricular tachycardia, polymorphic CASQ2

Hypercalcaemia, hypocalciuric CASR

Berardinelli-Seip lipodystrophy CAVi

Joubert syndrome CC2D2A

Joubert syndrome CC2D2A

Cerebral cavernous malformations CCM2

CD36 deficiency CD36

Hyper-IgM syndrome CD40LG

Cromer blood group CD55

Agammaglobulinaemia CD79B

Hyperparathyroidism, primary CDC73 Gastric cancer CDHi

Usher syndrome id CDH23

Hypotrichosis with juvenile macular dystrophy CDH3

Rett syndrome, atypical CDKL5

Pituitary and parathyroid tumours CDKNlB

Melanoma CDKN2A

Hypotrichosis simplex of the scalp CDSN

Bardet-Biedl syndrome CEP290

Leber congenital amaurosis CEP290

Cholesterol ester transfer protein deficiency CETP

Drusen, basal laminar CFH

Cystic fibrosis CFTR

Congenital absence of vas deferens CFTR

Elevated sweat chloride concentration CFTR

CHARGE syndrome CHD7

Choroideraemia CHM

Frontotemporal dementia CHMP2B

Fetal akinesia deformation sequence disorder CHRND

Congenital myasthenic syndrome CHRNE

Slow channel myasthenic syndrome CHRNE

Macular corneal dystrophy, type l CHST6

Immunodeficiency CIITA

Myotonia congenita CLCNi

Myotonia, Becker CLCNi

Myotonia CLCNi

Low molecular weight proteinuria CLCN5

Dent disease CLCN5

Dent (Japan) disease CLCN5

Bartter syndrome 4, digenic CLCNKA

Bartter syndrome 3 CLCNKB

Neuronal ceroid lipofuscinosis, juvenile CLN3

Neuronal ceroid lipofuscinosis, late infantile CLN5

Neuronal ceroid lipofuscinosis, late infantile CLN6

Retinitis pigmentosa CNGAi

Achromatopsia CNGB3

Congenital disorder of glycosylation Ilh COG8

Metaphyseal chondrodysplasia, Schmid COL10A1

Stickler syndrome, without eye involvement COL11A2

Epidermolysis bullosa COL17A1

Epidermolysis bullosa, junctional COL17A1

Epidermolysis bullosa, atrophic benign COL17A1

Osteogenesis imperfecta I COLlAl

Osteogenesis imperfecta COL1A1

Ehlers-Danlos syndrome VII COL1A2

Stickler syndrome COL2A1

Spondyloperipheral dysplasia COL2A1

Ehlers-Danlos syndrome TV COL3A1

Alport syndrome COL4A3

Alport syndrome COL4A5

Ullrich congenital muscular dystrophy COL6A1

Myosclerosis myopathy COL6A2

Ullrich congenital muscular dystrophy COL6A3 Epidermolysis bullosa COL7A1

Epidermolysis bullosa dystrophica COL7A1

Endplate acetylcholinesterase deficiency COLO

Aceruloplasminaemia with diabetes CP

Aceruloplasminaemia CP

Coproporphyria CPOX

Harderoporphyria CPOX

Coproporphyria CPOX

Carbamoyl phosphate synthetase I deficiency CPSi

Carnitine palmitoyltransferase l deficiency CPTiA

Leber congenital amaurosis CRBi

Mental retardation, non-syndromic, autosomal recessive CRBN

Rubinstein-Taybi syndrome CREBBP

Crisponi syndrome CRLFl

Congenital cataract CRYAA

Cataract, autosomal dominant CRYBBi

Cataract CRYGC

Cataract, pediatric CRYGD

Cataract CRYGD

Cystinosis CTNS

Pancreatitis, chronic CTRC

Papillon-Lefevre syndrome CTSC

Pycnodysostosis CTSK

3-M syndrome CUL7

Methaemoglobinaemia 2 CYB5R3

Methaemoglobinaemia CYB5R3

Chronic granulomatous disease CYBA

Chronic granulomatous disease CYBB

Trichoepithelioma, multiple familial CYLD

Adrenal hyperplasia CYP11B1

Steroid-ii beta-hydroxylase deficiency CYPiiBi

Adrenal hyperplasia CYP11B1

Steroid-ii beta-hydroxylase deficiency CYP11B1 i7-alpha-hydroxylase/i7,20-lyase deficiency CYP17A1

Glaucoma, primary congenital CYPiBi

Adrenal hyperplasia CYP21A2

Non-classic 21-hydroxylase deficiency CYP21A2

Adrenal hyperplasia CYP21A2

Pseudovitamin D-deficiency rickets CYP27B1

Null allele CYP2A13

Cytochrome P450 deficiency CYP2D6

CYP2G deficiency, association with CYP2G2P

Null allele CYP4A22

Bietti crystalline corneoretinal dystrophy CYP4V2

Spastic paraplegia CYP7B1

Maple syrup urine disease DBT

Immunodeficiency, severe combined DCLREiC

Subcortical band heterotopia DCX

Double cortex syndrome DCX

Usher syndrome 2 DFNB31

Progressive hearing loss, autosomal recessive DFNB59

Mitochondrial DNA depletion syndrome DGUOK Smith-Lemli-Opitz syndrome DHCR.7

Spondylocostal dysostosis DLL3

Muscular dystrophy, Duchenne DMD

Dystrophinopathy DMD

Muscular dystrophy, Becker DMD

Primary ciliary dyskinesia and situs inversus DNAHii

Primary ciliary dyskinesia DNAHs

Primary ciliary dyskinesia DNAIl

Primary ciliary dyskinesia DNAI2

Systemic lupus erythematosus DNASEi

Immunodeficiency, centromeric instability and facial anomalies DNMT3B syndrome

Dihydropyrimidine dehydrogenase deficiency DPYD

Receptor deficiency DRD5

Striate palmoplantar keratoderma DSGi

Cardiomyopathy, arrhythmogenic right ventricular DSG2

Dilated cardiomyopathy, woolly hair, keratoderma DSP

Dentinogenesis imperfecta Shields type II DSPP

Hypothyroidism DUOX2

Hypothyroidism, transient DUOX2

Hypothyroidism, transient DU0X2

Hypothyroidism DUOX2

Hypothyroidism DUOXA2

Smith-McCort dysplasia DYM

Dyggve-Melchior-Clausen syndrome DYM

Muscular dystrophy, limb girdle DYSF

Miyoshi myopathy DYSF

Chondrodysplasia punctata, X-linked EBP

CHILD syndrome EBP

Lipoid proteinosis ECMi

Ectodermal dysplasia EDA

Ectodermal dysplasia, hypohidrotic EDAR

Waardenburg-Hirschsprung disease EDNRB

ABCD syndrome EDNRB

Craniofrontonasal syndrome EFNBi

Erythrocytosis EGLNi

Mental retardation EHMTi

Wolcott-Rallison syndrome EIF2AK3

Leukoencephalopathy with vanishing white matter EIF2B4

Prostate cancer ELAC2

Supravalvular aortic stenosis ELN

Amelogenesis imperfecta, hypoplastic ENAM

Haemorrhagic telangiectasia 1 ENG

Idiopathic infantile arterial calcification ENPPl

Prostate cancer, increased risk, in African Americans, association with EPHB2

Erythrocytosis EPOR

Xeroderma pigmentosum (B) ERCC3

Xeroderma pigmentosum/Cockayne syndrome ERCC3

Cockayne syndrome ERCC8

SC Phocomelia ESC02

Glutaricacidaemia 2a ETFA

Electron transfer flavoprotein deficiency ETFA Multiple exostoses EXTi

Multiple exostoses EXT2

Branchio-oto-renal / branchiootic syndrome EYAi

Branchio-oto-renal syndrome EYAi

Factor XI deficiency F11

Factor XIII deficiency F13A1

Factor V deficiency F5

Factor VII deficiency F7

Haemophilia A F8

Haemophilia B F9

Tyrosinaemia 1 FAH

Amelogenesis imperfecta, hypoplastic local FAM83H

Amelogenesis imperfecta, hypocalcified FAM83H

Fanconi anaemia FANCA

Fanconi anaemia FANCC

Fanconi anaemia FANCG

Cytochrome c oxidase deficiency FASTKD2

Marfan syndrome FBNi

Ectopia lentis FBNl

Fibrillinopathy FBNi

Kindler syndrome FERMTi

Aflbrinogenaemia FGA

Dysfibrinogenaemia FGA

Hypofibrinogenaemia FGB

Aflbrinogenaemia FGB

Charcot-Marie-Tooth disease 4H FGD4

Lacrimo-auriculo-dento-digital syndrome FGFio

Kallmann syndrome FGFRi

Aflbrinogenaemia FGG

Leiomyomatosis and renal cell cancer FH

Fumarase deficiency FH

Cutaneous leiomyomatosis FH

Muscular dystrophy, Fukuyama FKTN

Pneumothorax, primary spontaneous FLCN

Birt-Hogg-Dub syndrome FLCN

Ichthyosis vulgaris FLG

Ichthyosis vulgaris flglO.2

Heterotopia, periventricular FLNA

Myopathy, myofibrillar FLNC

FMOi variant FMOi

FMO2 variant FM02

Trimethylaminuria FMO3 fmo6 variant FM06P

Axenfeld-Rieger & Peters' anomaly FOXCi

Axenfeld-Rieger anomaly FOXCi

Lymphoedema-distichiasis FOXC2

Aphakia, congenital, primary FOXE3

ACD/MPV with cardiovascular malformations FOXFi

Blepharophimosis/ptosis/epicanthus inversus syndrome F0XL2

Developmental verbal dyspraxia FOXP2

Follicle-stimulating hormone deficiency FSHB

Mental retardation FTSJi Fucosidosis FUCAl

H antigen, Bombay phenotype FUTi

H antigen, para-Bombay phenotype FUTi

Non-secretor phenotype FUT2

Fucosyltransferase deficiency FUT2

Fucosyltransferase deficiency FUT6

Friedreich ataxia FX

Exudative vitreoretinopathy FZD4

Glycogen storage disease la G6PC

Glucose-6-phosphate dehydrogenase deficiency G6PD

Glycogen storage disease 2 GAA

Krabbe disease GALC

Galactosaemia epimerase deficiency GALE

Mucopolysaccharidosis IVa GALNS

Tumoural calcinosis GALNT3

Galactosaemia GALT

Giant axonal neuropathy GAN

Hypoparathyroidism, deafness and renal dysplasia GATA3

Gaucher disease 2 GBA

Glycogen storage disease 4 GBEl

Dystonia, dopa-responsive GCHi

Diabetes, NIDDM GCK

Diabetes, MODY2 GCK

Diabetes, MODY GCK

Congenital cataract GC T2

Demyelinating peripheral neuropathy GDAPl

Charcot-Marie-Tooth disease 4A GDAPi

Charcot-Marie-Tooth disease, autosomal recessive GDAPl

Brachydactyly, type C GDF5

Laron dwarfism GHR

Growth hormone insensitivity GHR

Growth hormone deficiency GHRHR

Growth hormone deficiency, isolated GHSR

Oculodentodigital dysplasia GJAi

Charcot-Marie-Tooth disease GJBi

Deafness, autosomal recessive 1 GJB2

Deafness GJB2

Deafness, non-syndromic, autosomal dominant GJB3

Pelizaeus-Merzbacher-like disease GJC2

Glycerol kinase deficiency GK

Fabry disease GLA

Gangliosidosis GMi GLBl

Hyperglycinemia, non-ketotic GLDC

Hyperglycinaemia, non-ketotic GLDC

Hyperglycinemia, non-ketotic GLDC

Hyperglycinaemia, non-ketotic GLDC

Pallister-Hall syndrome GLI3

Greig cephalopolysyndactyly syndrome GLI3

Postaxial Polydactyly A/B GLI3

Hyperekplexia GLRAi

Gangliosidosis GM2 GM2A

Albright hereditary osteodystrophy GNAS Progressive osseous heteroplasia GNAS

Mucolipidosis II GNPTAB

Mucopolysaccharidosis Hid GNS

Bernard-Soulier syndrome GPiBA

Giant platelet disorder GPiBB

Bernard-Soulier syndrome GP9

Simpson-Golabi-Behmel syndrome GPC3

Glucosephosphate isomerase deficiency GPI

Albinism, ocular GPR143

Febrile and afebrile seizures GPR98

Hyperoxaluria II GRHPR

Frontotemporal dementia GRN

Alzheimer disease GRN

Glutathione synthetase deficiency GSS

Leber congenital amaurosis GUCY2D

Mucopolysaccharidosis VII GUSB

Hypoglycaemia, hyperinsulinaemic HADH

Thalassaemia alpha HBA2

Thalassaemia beta HBB

Microphthalmia, syndromic 7 HCCS

Tay-Sachs disease HEXA

Sandhoff disease HEXB

Haemochromatosis HFE

Haemochromatosis HFE2

Alkaptonuria HGD

Mucopolysaccharidosis IIIC HGSNAT

HLA-A null allele HLA-A

HLA-B null allele HLA-B

Holocarboxylase synthetase deficiency HLCS

Porphyria, acute intermittent HMBS

HMG-CoA lyase deficiency HMGCL

3-hydroxy-3-methylglutaric aciduria HMGCL

HMG-CoA lyase deficiency HMGCL

Diabetes, MODY3 HNFiA

Diabetes, MODY HNFiB

GCKD with early-onset diabetes HNFiB

Diabetes, MODYi HNF4A

Hand-foot-genital syndrome HOXA13

Tyrosinaemia 3 HPD

Lesch-Nyhan syndrome HPRTl

Hypoxanthine guanine phosphoribosyltransferase deficiency HPRTl

Hermansky-Pudlak syndrome HPSi

Hermansky-Pudlak syndrome HPS4

Atrichia with papular lesions HR

Congenital atrichia HR

Adrenal hyperplasia HSD3B2

Cataract, autosomal recessive hsf4b

Schwartz-Jampel syndrome type 1 HSPG2

CARASIL HTRAl

Mucopolysaccharidosis II IDS

Scheie syndrome IDUA

Hurler syndrome IDUA Reduced activity IFIHi

Growth retardation IGFiR

Acid-labile subunit deficiency IGFALS

Spinal muscular atrophy with respiratory distress 1 IGHMBP2

Spinal muscular atrophy with respiratory distress l IGHMBP2

Spinal muscular atrophy with respiratory distress l IGHMBP2

Incontinentia pigmenti IKBKG

Incontinentia pigmenti, familial IKBKG

Mental retardation, X-linked IL1RAPL1

Immunodeficiency, severe combined IL2RG

Immunodeficiency, severe combined IL7R

Leprechaunism INSR

Insulin resistance INSR

Insulin resistance A INSR

Senior-Loken syndrome 5 IQCBl

Van der Woude syndrome IRF6

Popliteal pterygium syndrome IRF6

Diabetes, type 2 ISLi

Glanzmann thrombasthenia ITGA2B

Leukocyte adhesion deficiency ITGB2

Glanzmann thrombasthenia ITGB3

Epidermolysis bullosa with pyloric atresia ITGB4

Alagille syndrome JAGi

Immunodeficiency, severe combined JAK3

Kallmann syndrome KALi

Atrial fibrillation KCNA5

Miscarriage and intrauterine foetal loss KCNH2

Long QT syndrome KCNH2

Hyperinsulinism KCNJ11

Long QT syndrome KCNQi

Cone dystrophy with supernormal rod ERG KCNV2

Mental retardation, X-linked KDM5C

Kell blood group variation KEL

K(null) phenotype KEL

Cornea plana 2 KERA

Goldberg-Shprintzen syndrome KIAA1279

Piebaldism KIT

Prostate cancer KLF6

Cerebral cavernous malformations KRITi

Dowling-Degos disease KRT5

Epidermolysis bullosa, Dowling-Meara KRT5

Epidermolysis bullosa simplex KRT5

Epidermolytic hyperkeratosis KRTio

Epidermolysis bullosa, Koebner KRT14

Naegeli syndrome KRT14

Dermatopathia pigmentosa reticularis KRT14

Hydrocephalus, X-linked LiCAM

L-2-Hydroxyglutaric aciduria L2HGDH

Muscular dystrophy, merosin deficient LAMA2

Laminin alpha 2 chain deficiency, partial LAMA2

Epidermolysis bullosa, Herlitz LAM A3

Laryngo-onycho-cutaneous syndrome LAMA3 Cardiomyopathy, dilated LAMA4

Epidermolysis bullosa, Herlitz LAMB3

Epidermolysis bullosa, junctional LAMB3

Epidermolysis bullosa, Herlitz LAMC2

Epidermolysis bullosa, junctional LAMC2

Danon disease LAMP2

Pelger-Huet anomaly LBR

Leber congenital amaurosis LCA5

Lecithi cholesterol acyltransferase deficiency LCAT

Lactase deficiency, congenital LCT

Lactate dehydrogenase deficiency LDHB

Hypercholesterolemia LDLR

Left-right axis malformation LEFTY2

Osteopoikilosis LEMD3

Leydig cell hypoplasia LHCGR

Pseudohermaphroditism LHCGR

Wolman syndrome LIPA

Factor V and factor VIII deficiency, combined LMANl

Factor V and factor VIII deficiency, combined LMANi

Muscular dystrophy, limb girdle LMNA

Muscular dystrophy, Emery-Dreifuss LMNA

Cardiomyopathy, dilated LMNA

Nail patella syndrome LMXiB

Lipoprotein lipase deficiency LPL

Hypertriglyceridaemia LPL

Lipoprotein lipase deficiency, association with LPL

Deafness, non-syndromic lrtomt2

Oligodontia LTBP3

Chediak-Higashi syndrome LYST

Hypospadias MAMLDi

Mannosidosis, alpha MAN2B1

Mannosidosis, beta, lysosomal MANBA

Obesity, autosomal dominant MC4R

3-methylcrotonyl-CoA carboxylase deficiency MCCCl

3-methylcrotonyl-CoA carboxylase deficiency MCCC2

Methylmalonic aciduria MCEE

Factor V and Factor VIII deficiency, combined MCFD2

Mucolipidosis IV MCOLNl

Rett syndrome MECP2

Myocardial infarction MEF2A

Mediterranean fever, familial MEFV

Multiple endocrine neoplasia l MENi

Spondylocostal dysostosis MESP2

Neuronal ceroid lipofuscinoses, late infantile MFSD8

Opitz G/BBB syndrome MIDI

Bardet-Biedl syndrome MKKS

Colorectal cancer, non-polyposis MLHi

Colorectal cancer, young-onset MLHi

Colorectal cancer MLHi

Gastrointestinal cancer MLHi

Lynch syndrome-associated breast cancer MLHi

Colorectal cancer, early onset MLHi Methylmalonic aciduria MMAB

Methylmalonic aciduria, cblB type MMAB

Fetomaternal alloimmunisation MME

Osteolysis, idiopathic, Saudi type MMP2

Currarino syndrome MNXi

Xanthinuria, type 2 MOCOS

Amegakaryocytic thrombocytopaenia, congenital MPL

Mercaptopyruvate sulphurtransferase deficiency, association with MPST

Mitochondrial DNA depletion syndrome, hepatocerebral MPV17

Charcot-Marie-Tooth disease lb MPZ

Charcot-Marie-Tooth disease 1 MPZ

Ataxia telangiectasia-like disease MRE11A

Mitochondrial respiratory chain disorder MRPS16

Atopy MS4A2

Colorectal cancer, non-polyposis MSH2

Colorectal cancer, non-polyposis MSH6

Prostate cancer MSRi

Witkop syndrome MSXi

Homocystinuria MTHFR

Methylenetetrahydrofolate reductase deficiency MTHFR

Homocystinuria MTHFR

Myotubular myopathy MTMl

Methionine synthase deficiency MTR

Methionine synthase reductase deficiency MTRR

Abetalipoproteinaemia MTTP

Methylmalonic aciduria MUT

Mevalonic kinase deficiency MVK

Hyperimmunoglobulin D and periodic fever syndrome MVK

Hyperimmunoglobulin D and periodic fever syndrome MVK

Cardiomyopathy, hypertrophic MYBPC3

Cardiomyopathy, hypertrophic MYBPC3

Feingold syndrome MYCN

Hearing impairment MYH14

Cardiomyopathy, hypertrophic MYH7

May-Hegglin anomaly MYH9

Deafness, non-syndromic, autosomal recessive MYO15A

Sensorineural deafness, nonsyndromic MYOiA

Microvillus inclusion disease MYO5B

Deafness, autosomal dominant 22 MY06

Deafness, autosomal recessive MY06

Usher syndrome lb MYO7A

Sanfilippo syndrome B NAGLU

Fertility defects NBN

Chronic granulomatous disease NCFi

Chronic granulomatous disease NCF2

Norrie disease NDP

Mitochondrial complex I deficiency NDUFAF2

Complex 1 deficiency NDUFS4

Nemaline myopathy NEB

Charcot-Marie-Tooth disease NEFL

Sialidosis NEUi

Sialidosis 2 NEUi Neurofibromatosis l NFi

Neurofibromatosis 2 NF2

Ectodermal dysplasia, anhidrotic with immune deficiency NFKBIA

Myoclonic epilepsy of Lafora NHLRCi

Ichthyosis, autosomal recessive NIPAL4

Cornelia de Lange syndrome NIPBL

Benign hereditary chorea NKX2-1

Hypothyroidism NKX2-1

Periodic fever syndrome NLRP12

Familial cold autoinflammatory syndrome NLRP3

Primary ciliary dyskinesia NME8

Stapes ankylosis with broad thumb and toes NOG

Niemann-Pick disease C NPCi

Niemann-Pick type C2 disease NPC2

Nephronophthisis 1 NPHPi

Nephronophthisis 3 NPHP3

Nephronophthisis 4 NPHP4

Congenital nephrotic syndrome, Finnish type NPHSi

Nephrotic syndrome NPHSi

Nephrotic syndrome, steroid resistant NPHS2

Nephrotic syndrome NPHS2

Acromesomelic dysplasia, Maroteaux type NPR2

Adrenal hypoplasia NRoBi

Enhanced S cone syndrome NR2E3

Pseudohypoaldosteronism 1 NR3C2

XY sex reversal, without adrenal failure NR5A1

Sotos syndrome NSDi

CHILD syndrome NSDHL

Pain insensitivity, congenital NTRKi

Gyrate atrophy OAT

Albinism, oculocutaneous II OCA2

Lowe oculocerebrorenal syndrome OCRL

Oral-facial-digital syndrome 1 OFDi

Optic atrophy 1 OPAi

Mental retardation syndrome, X-linked OPHNi

X-linked cone dystrophy orfi5

Atrophic macular degeneration orfl5

Retinitis pigmentosa, X-linked orfis

Osteopetrosis, autosomal recessive OSTMi

Ornithine transcarbamylase deficiency OTC

Ornithine transcarbamylase deficiency OTC

Ornithine transcarbamylase deficiency OTC

Deafness, autosomal recessive 9 OTOF

Deafness, non-syndromic OTOF

Lissencephaly, isolated PAFAHiBi

Subcortical band heterotopia PAFAH1B1

Phenylketonuria PAH

HARP syndrome PANK2

Pantothenate kinase-associated neurodegeneration PANK2

Spondyloepiphyseal dysplasia PAPSS2

Parkinsonism, juvenile, autosomal recessive PARK2

Renal hypoplasia PAX2 Waardenburg syndrome PAX3

Aniridia PAX6

Oligodontia PAX9

Hyperphenylalaninaemia PCBDi

Propionic acidaemia PCCA

Propionic acidaemia PCCB

Usher syndrome if PCDH15

Epilepsy and mental retardation limited to females PCDH19

Schizophrenia PCMi

Obesity and impaired prohormone processing PCSKl

Low LDL cholesterol PCSK9

Cerebral cavernous malformation PDCDio

Retinitis pigmentosa PDE6B

Pyruvate dehydrogenase deficiency PDHAl

Pyruvate dehydrogenase complex deficiency PDHX

Pyruvate dehydrogenase phosphatase deficiency PDPl

Prolidase deficiency PEPD

Zellweger syndrome PEXi

Peroxisome biogenesis disorder PEXi

Neonatal adrenoleukodystrophy PEXio

Zellweger syndrome H PEX13

Zellweger syndrome PEX14

Zellweger syndrome, complementation group D PEX16

Rhizomelic chondrodysplasia punctata PEX7

Glycogen storage disease 7 PFKM

Rickets, hypophosphataemic PHEX

X-linked mental retardation & cleft lip/palate PHF8

Phosphorylase kinase deficiency PHKAl

Liver glycogenosis 1 PHKA2

Liver glycogenosis PHKB

Fibrosis of extraocular muscles type 2 PHOX2A

Central hypoventilation syndrome PHOX2B

Parkinson disease, early-onset PINKi

Axenfeld-Rieger syndrome PITX2

Polycystic kidney disease 1 PKDi

Polycystic kidney disease 2 PKD2

Polycystic kidney disease PKHDi

Pyruvate kinase deficiency PKLR

Haemolytic anaemia PKLR

Pyruvate kinase deficiency PKLR

Ectodermal dysplasia/ skin fragility syndrome PKPi

Infantile neuroaxonal dystrophy 1 PLA2G6

Epidermolysis bullosa with pyloric atresia PLEC

Muscular dystrophy with epidermolysis bullosa PLEC

Epidermolysis bullosa simplex PLEC

Plasminogen deficiency PLG

Ehlers-Danlos syndrome VI PLODi

Pelizaeus-Merzbacher disease PLPi

Spastic paraplegia PLPl

Congenital disorder of glycosylation la PMM2

Turcot syndrome PMS2

PNPO deficiency PNPO Alpers syndrome POLG

Xeroderma pigmentosum, variant POLH

Xeroderma pigmentosum, variant POLH

Obesity POMC

Walker-Warburg syndrome POMTl

Focal dermal hypoplasia PORCN

Pituitary hormone deficiency POU1F1

Partial lipodystrophy PPARG

Porphyria, variegate PPOX

Neuronal ceroid lipofuscinosis, juvenile PPTi

Neuronal ceroid lipofuscinosis, infantile PPTi

Neuronal ceroid lipofuscinosis, late infantile PPTi

Haemophagocytic lymphohistiocytosis, familial PRFi

Perforin deficiency PRFl

Camptodactyly-arthropathy-coxa vara-pericarditis PRG4

Carney complex PRKARiA

Azoospermia PRM2

Protein C deficiency PROC

Hypogonadotropic hypogonadism PROKR2

Hypogonadotropic hypogonadism PROPl

Protein S deficiency PROSi

High myopia PRPH

Pattern dystrophy PRPH2

Pancreatitis, protection against PRSSi

Deierine-Sottas syndrome PRX

Charcot-Marie-Tooth disease 4 PRX

Gaucher disease, atypical PSAP

Nevoid basal cell carcinoma syndrome PTCHi

Cowden disease PTEN

Hypertension PTGIS

Osteochondrodysplasia, Blomstrand, type l PTHiR

Mitochondrial myopathy and sideroblastic anaemia PUSi

McArdle disease PYGM

Dihydropteridine reductase deficiency ODPR

Acrocephalopolysyndactyly, type II RAB23

Immunodeficiency, severe combined RAG2

Immunodeficiency, severe combined, B cell -ve RAG2

Omenn syndrome RAG2

Smith-Magenis syndrome RAIi

Anophthalmia RAX

Retinoblastoma RBl

RAPADILINO syndrome RECOL4

Spastic paraplegia 31 REEPi

Hirschsprung disease RET

MHC class II deficiency RFXANK

Retinitis pigmentosa RHO

Ribonuclease L deficiency RNASEL

Brachydactyly, type B R0R2

Robinow syndrome, autosomal recessive R0R2

Brachydactyly, type B R0R2

Retinitis pigmentosa RPl

Retinitis pigmentosa, X-linked RP2 Leber congenital amaurosis RPE65

Retinitis pigmentosa, X-linked RPGR

Diamond-Blackfan anaemia RPS24

Coffin-Lowry syndrome RPS6KA3

Mitochondrial DNA depletion syndrome RRM2B

Retinoschisis, X linked juvenile RSi

Platelet disorder, familial RU Xi

Cleidocranial dysplasia RUNX2

Townes-Brocks syndrome SALLi

Goldenhar syndrome SALLi

Townes-Brocks syndrome SALLi

Okihiro syndrome SALL4

Tumoural calcinosis, normophosphataemic SAMD9

Chylomicron retention disease SARiB

Cleft palate, osteoporosis and cognitive defects SATB2

Charcot-Marie-Tooth disease 402 SBF2

Action myoclonus-renal failure syndrome SCARB2

Myoclonic epilepsy of infancy SCNiA

Dravet syndrome or Dravet syndrome C or Dravet syndrome B SCNlA

Generalized epilepsy with febrile seizures plus SCNiA

Intractable epilepsy SCNlA

Intractable epilepsy and mental decline SCN2A

Brugada syndrome SCN5A

Cardiac conduction disease SCN5A

Channelopathy-associated insensitivity to pain SCN9A

Cardioencephalomyopathy, fatal infantile SCO2

Cytochrome c oxidase deficiency SCO2

Leigh syndrome SDHA

Phaeochromocytoma SDHB

Paraganglioma, autosomal dominant 3 SDHC

Paraganglioma SDHD

SEPN-related myopathy SEPNi

Antitrypsin alpha 1 deficiency SERPINAi

Venous thromboembolic disease SERPINAio

Thyroxine-binding globulin deficiency SERPINA7

Antithrombin deficiency SERPINCi

Deep vein thrombosis SERPINCi

Angioneurotic oedema SERPINGl

Surfactant protein B deficiency SFTPB

Muscular dystrophy, limb girdle SGCD

Myoclonus dystonia SGCE

Muscular dystrophy, limb girdle SGCG

Sanfilippo syndrome A SGSH

Lymphoproliferative syndrome, X-linked SH2D1A

Holoprosencephaly SHH

Leri- Weill dyschondrosteosis SHOX

JK-null variant SLC14A1

Cataract, juvenile with microcornea and renal glucosuria SLC16A12

Monocarboxylate transporter 8 deficiency SLC16A2

Salla disease SLC17A5

Sialic acid storage disease, infantile SLC17A5

Megaloblastic anaemia, thiamine responsive SLC19A2 Organic cation transporter deficiency SLC22A4

Intrahepatic cholestasis, neonatal SLC25A13

HHH syndrome SLC25A15

Diarrhoea, congenital chloride SLC26A3

Glucose transporter l deficiency syndrome SLC2A1

Fanconi-Bickel syndrome SLC2A2

Hereditary hypophosphataemic rickets with hypercalciuria SLC34A3

Acrodermatitis enteropathica SLC39A4

Cystinuria SLC3A1

Spherocytosis SLC4A1

Corneal endothelial dystrophy 2 SLC4A11

Proximal renal tubular acidosis SLC4A4

Glucose / galactose malabsorption SLC5A1

Renal glucosuria SLC5A2

Iodide transport defect SLC5A5

Hyperekplexia SLC6A ^< 5

Creatine deficiency SLC6A8

Lysinuric protein intolerance SLC7A7

Cystinuria, type I/III SLC7A9

Mai de Meleda SLURPl

Juvenile polyposis syndrome SMAD4

Pulmonary arterial hypertension SMAD9

Schimke immuno-osseous dysplasia SMARCALi

Schimke immuno-osseous dysplasia SMARCALi

Spinal muscular atrophy SMNi

Niemann-Pick disease SMPDi

Amyotrophic lateral sclerosis SODi

Sclerosteosis SOST

PCWH SOXio

Shah-Waardenburg syndrome and neuropathy SOXio

Hypotrichosis-Lymphoedema-Telangiectasia SOX18

Anophthalmia, hearing loss and brain abnormalities SOX2

Anophthalmia-oesophageal-genital syndrome SOX2

Campomelic dysplasia SOX9

Spastic paraplegia SPAST

Spastic paraplegia, autosomal dominant SPAST

Retiniitis pigmentosa, juvenile SPATA7

Leber congenital amaurosis IV SPATA7

Spastic paraplegia, autosomal recessive SPG11

Spastic paraplegia with thin corpus callosum SPG11

Netherton syndrome SPINK5

Neurofibromatosis l-like syndrome SPREDi

Legius syndrome SPREDi

Cafe-au-lait macules SPREDi

Pyropoikilocytosis SPTAi

Spherocytosis SPTB

Steroid-5 alpha-reductase deficiency SRD5A2

XY sex reversal SRY

Gonadal dysgenesis SRY

Amish infantile epilepsy syndrome ST3GAL5

Congenital lipoid adrenal hyperplasia STAR

Growth hormone insensitivity STAT5B Gonadotrophin-independent precocious puberty STK11

Peutz-Jeghers syndrome STK11

Microphthalmia STRA6

Haemophagocytic lymphohistiocytosis, familial STX11

Glutaric aciduria 3 SUGCT

Sulphite oxidase deficiency SUOX

Leigh syndrome SURFi

Epilepsy SYNi

Schizophrenia syngric

Corneal dystrophy, gelatinous drop-like TACSTD2

Tyrosinaemia 2 TAT

Barth syndrome TAZ

Cardiomyopathy, X-linked infantile TAZ

Amyotrophic lateral sclerosis TBKl

ACTH deficiency, isolated TBX19

Congenital heart disease TBX20

Cleft palate and ankyloglossia TBX22

Ulnar-mammary syndrome TBX3

Holt-Oram syndrome TBX5

Osteopetrosis, autosomal recessive TCIRGl

Transcobalamin II deficiency TCN2

Treacher-Collins syndrome TCOFi

Haemochromatosis TFR2

Goitre with hypothyroidism TG

Goitre, simple TG

Holoprosencephaly TGIFi

Ichthyosis, congenital, autosomal recessive TGMi

Ichthyosis, lamellar TGMi

Dystonia THAPi

Thyroid hormone resistance THRB

Epidermodysplasia verruciformis TMC6

Epidermodysplasia verruciformis TMC8

Enteropeptidase deficiency TMPRSS15

Microcytic anaemia & iron deficiency TMPRSS6

Microcytic anaemia & iron deficiency TMPRSS6

Li-Fraumeni syndrome TP53

Multiple cancers TP53

Osteosarcoma TP53

Adrenocortical carcinoma TP53

Split-hand/ split-foot malformation TP63

Nemaline myopathy TPM3

Goitrous hypothyroidism TPO

Neuronal ceroid lipofuscinosis, late infantile TPPi

Spondyloepiphyseal dysplasia tarda TRAPPC2

Deafness, non-syndromic TRIOBP

Hypomagnesaemia with secondary hypocalcaemia TRPM6

Tricho-rhino-phalangeal syndrome I TRPSi

Tuberous sclerosis TSCi

Tuberous sclerosis TSC2

Hypothyroidism TSHB

Hyperthyroidism TSHR

Tibial muscular dystrophy TTN Cardiomyopathy, dilated ttntvn2b

Saethre-Chotzen syndrome TWISTi

Baller-Gerold syndrome TWISTi

Albinism, oculocutaneous l TYR

Albinism, oculocutaneous lA TYR

Albinism, oculocutaneous 3 TYRPi

Hypotrichosis, Marie Unna type U2hr

Angelman syndrome UBE3A

Crigler-Najjar syndrome 1 UGT1A1

Crigler-Naiiar syndrome 2 UGTlAi

Haemophagocytic lymphohistiocytosis, familial UNC13D

Mental retardation UPF3B

Porphyria, hepatoerythropoietic UROD

Porphyria, cutanea tarda UROD

Porphyria, erythropoietic UROS

Usher syndrome lc USHiC

Usher syndrome lg USHlG

Usher syndrome 2a USH2A

Retinitis pigmentosa, recessive, no hearing loss USH2A

Usher syndrome 2 USH2A

Rickets, vitamin D resistant VDR

Von Hippel-Lindau syndrome VHL

Cerebellar hypoplasia and quadrupedal locomotion VLDLR

Dysequilibrium syndrome VLDLR

Chorea-acanthocytosis VPS13A

Cohen syndrome VPS13B

Von Willebrand disease 3 VWF

Von Willebrand disease VWF

Von Willebrand disease 2n VWF

Wiskott-Aldrich syndrome WAS

Wolfram syndrome WFSl

Neuropathy, hereditary sensory, type II wnkitv3

Odonto-onycho-dermal dysplasia WNTloA

Tetra-amelia WNT3

Werner syndrome WRN

Wilms tumour WTi

Renal dysfunction & renal blastema WTi

Xanthinuria, type 1 XDH

Xeroderma pigmentosum (A) XPA

Xeroderma pigmentosum (C) XPC

Posterior polymorphous corneal dystrophy ZEBi

Mowat- Wilson syndrome ZEB2

Cardiac malformation ZIC3

Situs abnormality ZIC3

Mental retardation, X-linked ZNF674

TABLE 2 - Short List of Medical Conditions Associated with PTC

Medical Condition Associated with PTC Gene symbol

Muscular Dystrophy (Duchenne or Becker) DMD

Chronic granulomatous disease CYBB or NCFi or NCF2

Late infantile neuronal ceroid lipofuscinosis TPPi Neuronal ceroid lipofuscinosis (juvenile, infantile or late PPTi

infantile)

Neuronal ceroid lipofuscinosis (juvenile or late or late CLN3, CLN5, CLN6, or infantile) MFSD8

Frontotemporal dementia GRN or CHMP2B

Epidermolysis bullosa (dystrophic/dystrophica, COL7A1 or COL17A1

junctional, atrophic benign)

Rett syndrome MECP2

Congenital disorder of deglycosylation (Ilh or la) COG8 or PMM2 or NGLYi

Cystic fibrosis CFTPv

Schimke immuno-osseous dysplasia SMARCALi

Adenomatous polyposis coli APC

Li-Fraumeni syndrome TP53

Sporadic cancer various tumour suppressor genes including TP53

The codon changes resulting in all of the above medical conditions are well known in the art and new codon changes that result in PTC are still being discovered.

Nevertheless, there is an expectation that the compounds described herein will have some degree of readthrough activity for all such PTCs.

Compounds as described herein may be in the free form or in the form of a salt thereof. In some embodiment, compounds as described herein may be in the form of a pharmaceutically acceptable salt, which are known in the art (Berge S. M. et al., J. Pharm.

Sci. (1977) 66(i):i-i9). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound

(salts which retain the biological effectiveness and/or properties of the parent compound and which are not biologically and/or otherwise undesirable). Compounds as described herein having one or more functional groups capable of forming a salt may be, for example, formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt with, for example, a pharmaceutically acceptable organic or inorganic acid.

Pharmaceutically acceptable salts may be derived from, for example, and without limitation, acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, diethylacetic acid, digluconic acid, dodecylsulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemisulfonic acid, heptanoic acid, hexanoic acid, hydrochloric acid, hydrobromic acid, hydriodic acid, 2- hydroxyethanesulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-napthalenesulfonic acid,

naphthalenedisulphonic acid, p-toluenesulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphoric acid, picric acid, pimelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acidic functional groups may be capable of forming

pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example, and without limitation, inorganic bases based on alkaline metals or alkaline earth metals or organic bases such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion-exchange resins.

Pharmaceutically acceptable salts may be derived from, for example, and without limitation, a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine,

isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, glucamine, methylglucamine, theobromine, purines, piperazine, piperidine, procaine, N- ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, Ν,Ν-dimethylaniline, N-methylpiperidine, morpholine, N- methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N,N- dibenzylphenethylamine, l-ephenamine, Ν,Ν'-dibenzylethylenediamine or polyamine resins. In some embodiments, compounds as described herein may contain both acidic and basic groups and may be in the form of inner salts or zwitterions, for example, and without limitation, betaines. Salts as described herein may be prepared by conventional processes known to a person skilled in the art, for example, and without limitation, by reacting the free form with an organic acid or inorganic acid or base, or by anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that preparation of salts may occur in situ during isolation and purification of the compounds or preparation of salts may occur by separately reacting an isolated and purified compound. In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, polymorphs, isomeric forms) as described herein may be in the solvent addition form, for example, solvates. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent in physical association the compound or salt thereof. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.

In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, isomeric forms) as described herein may include crystalline and amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms, or a combination thereof. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound.

Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and/or solubility. Those skilled in the art will appreciate that various factors including recrystallization solvent, rate of crystallization and storage temperature may cause a single crystal form to dominate.

A PTC read-through compound may provide a therapeutic benefit if the compound permits read-through of a PTC in a protein coding sequence to produce the full length protein. Wherein the full length protein may have sequence variations and may not be the same as the native protein. Generally, the full length protein produced by the read- through is functional and can stand in for the wild-type protein. In some cases, as little as 5% of the normal total amount of the full length protein, wherein the total amount of protein, is what a subject not having the medical condition associated with the PTC would normally produce. However, depending on the medical condition associated with the PTC, as little as 1% of the normal total amount of the full length protein may be sufficient to have a therapeutic benefit. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 1% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 2% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read- through compound provides read-through of a PTC in a protein coding sequence to produce at least about 3% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 4% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 5% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read- through compound provides read-through of a PTC in a protein coding sequence to produce at least about 6% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 7% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 8% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read- through compound provides read-through of a PTC in a protein coding sequence to produce at least about 9% of the normal total amount of the full length protein a therapeutic benefit may be achieved. Provided that the PTC read-through compound provides read-through of a PTC in a protein coding sequence to produce at least about 10% of the normal total amount of the full length protein a therapeutic benefit may be achieved.

Alternatively, a PTC read-through compound may provide a therapeutic benefit if the compound permits sufficient read-through of a PTC in a protein coding sequence to provide some therapeutic benefit to the subject or achieve some therapeutic result. The therapeutic benefit may be determined functionally by measuring some therapeutic result. A therapeutic result may result from a therapeutically effective amount or a prophylactically effective amount of the compound, and may include, for example, reduced tumor size, increased life span, a delay of symptom onset or disease onset, increase metabolic efficiency or increased life expectancy. A therapeutically effective amount of a compound or a prophylactically effective amount of a compound may vary according to the disease state, age, sex, other health factors unrelated to or related to the disease and weight of the subject, and the ability of the compound to elicit a desired response in the subject.

Furthermore, the read-through efficiently may be greater at TGA than TAG, and in some circumstances there may be no read-through at TAA. Accordingly, treatments may be tailored to particular stop codons.

In some embodiments, compounds and all different forms thereof (e.g. free forms, salts, solvates, polymorphs, protonated forms) as described herein include isomers such as geometrical isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof, and are not limited by the description of the formula illustrated for the sake of convenience.

For example,

In some embodiments, compounds may include analogs, isomers, stereoisomers, or related derivatives. In some embodiments the compounds may be used in conjunction with another compound to form a pharmaceutical composition.

In some embodiments, pharmaceutical compositions as described herein may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more carriers, excipients or diluents acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers, excipients or diluents (used interchangeably herein) are those known in the art for use in such modes of administration.

Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of skill in the art are described in Remington: the Science & Prac ce of Pharmacy by Alfonso Gennaro, 20 ^th ed., Lippencott Williams & Wilkins, (2000).

Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

Compounds or pharmaceutical compositions as described herein or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, etc. Also, implants may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.

An "effective amount" of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced tumor size, increased life span or increased life expectancy. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (for example, smaller tumors, increased life span, increased life expectancy or prevention of the progression of the medical condition associated with premature termination codons). Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

In some embodiments, compounds and all different forms thereof as described herein may be used, for example, and without limitation, in combination with other treatment methods for at least one indication selected from the group set out in TABLE 1 or TABLE 2.

In general, compounds as described herein should be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the compositions. Some compounds as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Toxicity may be evaluated by examining a particular compound's or composition's specificity across cell lines or in an animal model.

Compounds as described herein may be administered to a subject. As used herein, a "subject" may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk of having a medical condition associated with premature termination codons (PTCs).

As used herein, a "medical condition associated with premature termination codons" may be defined as any medical condition caused in whole or in part by a nonsense codon, which may result in decreased mRNA stability as well as protein truncation resulting in a non-functional protein, which in turn may directly or indirectly result in the medical condition. For example, the medical condition associated with premature termination codons may be selected from TABLE l or TABLE 2.

There are about 5000 or so such genetic diseases which may be grouped into broad categories, as follows: an autoimmune disease; a blood disease; a collagen disease; diabetes; a neurodegenerative disease; a cardiovascular disease; a pulmonary disease; or an inflammatory disease; a neoplastic disease or central nervous system disease. One third of the cases of genetic inherited diseases involve a premature termination codon (PTC) (Frischmeyer PA and Dietz HC 1999). In most cases, the primary mechanism whereby a nonsense mutation has an effect is through the degradation of that mRNA by a surveillance mechanism called nonsense-mediated mRNA decay (NMD) (see: Chang YF et al. 2007; Isken 0 and Maquat LE 2007; Rebbapragada I and Lykke-Andersen J 2009; Rehwinkel J et al. 2006; and Muhlemann O et al. 2008).

Diagnostic methods for various medical conditions associated with premature termination codons are known in the art. Depending on the condition genetic diagnostics may be readily available or may be determined with directed sequencing. For example, the medical condition may be selected from the group consisting of central nervous system diseases, ataxia-telangiectasia, muscular dystrophy, Duchenne muscular dystrophy, Dravet syndrome, myotonic dystrophy, multiple sclerosis, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neural tissue degeneration, Parkinson's disease, autoimmune diseases, chronic rheumatoid arthritis, lupus erythematosus, graft-versus-host disease, primary immunodeficiencies, severe combined immunodeficiency, DNA Ligase Γν deficiency, DNA repair disorders, Nijmegen breakage disorders, xeroderma pigmentosum (XP), inflammatory diseases, rheumatoid arthritis, blood diseases, hemophilia, von Willebrand disease, thalassemia (for example, β- thalassemia), familial erythrocytosis, nephrolithiasis, collagen diseases, osteogenesis imperfecta, cirrhosis, neurofibroma, bullous disease, lysosomal storage disease, Hurler's disease, familial cholesterolemia, cerebellar ataxia, tuberous sclerosis, immune deficiency, kidney disease, lung disease, cystic fibrosis, familial hypercholesterolemia, pigmentary retinopathy, retinitis pigmentosa, amyloidosis, atherosclerosis, giantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, diabetes mellitus, familial

polycythemia, Niemann-Pick disease, epidermolysis bullosa, Marfan syndrome, neuromuscular diseases, Becker muscular dystrophy (BMD), spinal muscular atrophy, cancer, and any genetic disorder caused by nonsense mutation(s). Furthermore, where the medical condition associated with a premature termination codon is a cancer, the cancer may be selected from one or more of cancer is of the head and neck, eye, skin, mouth, throat, esophagus, chest, bone, blood, lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, brain, intestine, heart or adrenals. Alternatively, the cancer may be selected from sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, a blood- born tumor or multiple myeloma. Tests for determining whether a PTC is involved in the condition are known to those of ordinary skill in the art.

Compounds tested and to be tested are set out below in TABLES A and B respectively.

The Gentamicin complex or Gentamicin C complex as used herein includes gentamicin Cl, gentamicin Cia, and gentamicin C2 (~8o% of complex) and are reported to have the most significant antibacterial activity. The remaining ~20% of the complex is made up of Gentamicins A, B, X, et al. The exact compositions may vary between different production runs and based on the producer.

Various alternative embodiments and examples of the invention are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

MATERIALS AND METHODS

P53 PTC Read-through in a Human Cell Line.

Compounds were tested for PTC read-through in human cells, wherein mammary carcinoma HDQ-Pi cells homozygous for TGA (R213X) in exon 6 of the TP53 gene (Wang et al., 2000) were selected on the basis of convincing evidence of read-through by the aminoglycoside G418 (Floquet, C. et al. 2011). Western analysis using a quantitative automated capillary electrophoresis system showed that HDQ-Pi cells express very low levels of truncated P53 and no full-length P53 and that 50 μΜ G418 induces the formation of full-length p53 while also increasing truncated p53 levels as reported (Floquet, C. et al. 2011). Nuclear localization sequences and a tetramerization domain located in the P53 C- terminus contribute to retaining p53 in the nucleus (Shaulsky, G et al. 1990; Liang, S.H and Clark, M.F. 2001) and p53 truncated at R213 lacks these sequences. To enable analysis of P53 R213X read-through at high throughput an automated 96-well fluorescence microscopy assay was established to detect and quantitate nuclear Ρ53 signal. G418 induced a concentration-dependent increase in nuclear p53 consistent with read-through induction. During 72 h exposure, 50 uM G418 induced nuclear 53 expression in 9% of cells while 250 uM G418 induced nuclear 53 expression in nearly all cells.

Automated p53 Immunofluorescence 96-well Plate Assay

HDQ-Pi cells cultured in DMEM containing 10% FBS and lx Gibco™ antibioticantimicotic were seeded at 4000 per well of PerkinElmer View™ 96-well plates. The next day, the medium was replaced with fresh culture medium containing the compounds to be tested. After 72 h, the culture medium was removed by aspiration, the cells were fixed with 3% paraformaldehyde, 0.3% Triton X-ioo and 1.5 g/ml Hoechst 33323 in phosphate-buffered saline pH 7.2 (PBS) for 20 min at room temp. The cells were rinsed once with PBS and incubated for 2 h at room temp with a blocking solution of 3% BSA in PBS. The blocking solution was removed by aspiration and cells were incubated with o.l μg/ml DO-i p53 mouse monoclonal P53 antibody (Santa Cruz™) in blocking solution for 90 min at room temp. The wells were washed once with PBS for 5 min and the cells were incubated with Alexa 488-conjugated goat anti-mouse antibody (Invitrogen Life Technologies A11029™) in blocking buffer for 90 min at room temp. The wells were washed once with PBS for 5 min, 75 μΐ PBS was added, the plates were covered with a black adherent membrane and stored at 4°C overnight. Nuclear P53 immunofluorescence intensity was measured using a Cellomics ArrayScan VTI™ automated fluorescence imager.

Briefly, images were acquired with a 20X objective in the Hoechst™ and GFP (XF53) channels. Images of 15 fields were acquired for each well, corresponding to -2000 cells.

The Compartment Analysis bioapplication was used to identify the nuclei and define their border. The nuclear Alexa 488™ fluorescence intensity was then measured and expressed as average nuclear fluorescence intensity or % positive nuclei, using as a threshold the fluorescence intensity of nuclei from untreated cells (50-75, depending on experiment). Automated Electrophoresis Western Analysis Assay

HDQ-Pi cells were seeded at 100,000 cells per well of TC-treated 6-well plates.

The next day, the medium was replaced with fresh medium containing compounds to be tested and were incubated for 48 to 96 h. The medium was removed by aspiration, cell monolayers were rinsed with 1 ml ice-cold PBS. Cells were lysed in 80 μΐ lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton Xioo™, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate supplemented with fresh 1 mM Na3V04, l mM dithiothreitol and lx complete protease inhibitor cocktail (Roche Molecular Biochemicals™)). Lysates were pre-cleared by centrifugation at 18,000 g for 15 min at 4°C. Supernatants were collected, protein was quantitated using the Bradford assay and lysates were adjusted to l mg/ml protein. Capillary electrophoresis and western analysis conditions were carried out with manufacturer's reagents according to the user manual (ProteinSimple WES™). Briefly, 5.6 μΐ of cell lysate was mixed with 1.4 μΐ fluorescent master mix and heated at 95°C for 5min. The samples, blocking reagent, wash buffer, DO-l p53 antibody (0.5 μg/ml) and vinculin antibody (1:2000, R&D clone 728526), secondary antibody and chemiluminescent substrate were dispensed into the microplate provided by the manufacturer. The electrophoretic separation and immunodetection was performed automatically using default settings. The data was analyzed with inbuilt Compass™ software (Proteinsimple™). The truncated and full- length p53 peak intensities were normalized to that of the vinculin peak, used as a loading control. Results are shown as pseudo blots and as electropherograms.

In some instances, a traditional western blotting procedure was used (e.g. FIGURE 4), using the same antibodies.

Compounds Tested

Gentamicin, gentamicin A, B, Bi, Ci, Cia, C2, C2a, C2b, X2, sisomicin, as well as gentamicin fragments garamine and ring C (see FIGURE 1) were obtained from MicroCombiChem. G418 was from Sigma™. Betamethasone, dexamethasone and medroxyprogesterone acetate were from Sigma™. Gentamicin Bi was purchased from MicroCombiChem™ (catalogue # MCC3436). Gentamicin X2 was from TokuoE (catalogue # G036). G418 from Life Technologies (catalogue # 11811-023). Gentamicin from Sigma (catalogue # G1264).

Immunofluorescence P53 Testing

Methods for FIGURE 5: Panels A to D: Human HDQ-Pi breast carcinoma cells with a homozygous R213X nonsense mutation in the TP53 gene were exposed to three different batches of pharmaceutical gentamicin sulfate or to major and minor gentamicin components purified from pharmaceutical gentamicin, for 72 h. The cells were then fixed, DNA was stained with Hoechst™ 33323 and nuclear p53 was detected by immunofluorescence labeling using Santa Cruz DO-i p53 antibody. The p53-positive nuclei were determined using a Cellomics™ VTI 96-well imager as described in Baradaran-Heravi et al. (2016). The percent p53-positive nuclei is a measure of the extent of PTC readthrough. Panels E and F: HDQ-Pi cells were exposed to the gentamicin batches, gentamicin Bi or gentamicin X2 for 72 h and subjected to Ρ53 Western analysis using Santa Cruz™ DO-i Ρ53 antibody as described in Baradaran-Heravi et al. (2016) to measure formation of truncated P53 and full-length Ρ53, where full-length p53 is the PTC readthrough product. The y axis in Panel F shows the full-length P53 signal intensity, expressed as chemiluminescence units.

Premature Stop Codon Testing with Genatamicin Bi

Methods for FIGURE 6: NCI-H1299 human non-small cell lung carcinoma cells were transiently transfected with p53 expression constructs bearing a TGA, TAG or TAA nonsense mutation at amino acid position 213. Cells exposed to transfection reagent only (mock) or transiently transfected with a WT p53 expression were included as controls. The cells were exposed to the indicated concentrations of gentamicin Bi or USP gentamicin sulfate (Sigma™) for 48 h and the formation of truncated p53 and full-length P53 (readthrough product) was determined as described in Baradaran-Heravi et al. (2016). The amounts of full-length P53 and truncated p53 are expressed relative to the amount of full-length (for WT) or truncated P53 in untreated cells.

Premature Stop Codon Testing with Genatamicin Bi

Methods for FIGURE 7: Different human cancer cell lines with homozygous TP53 nonsense mutations (i.e. SW900; NCI-H1688; ESS-i; SK-MES-i; HCC1937; H1299; and HCT116) were exposed to the indicated concentrations of gentamicin Bi or G418 for 3 days, 6 days or 13 days, as indicated and the formation of truncated Ρ53 (lower arrowhead) and full length 53 (upper arrowhead, readthrough product) was determined as described in Baradaran-Heravi et al. (2016). The nonsense mutations are indicated under the cell line names. Vinculin, which migrates around 116 kDa, was used as a protein loading control.

Induction of PTC Readthrough in vivo

Methods for FIGURE 8: Two million NCI-H1299 human non-small cell lung carcinoma cells stably expressing a TP53 expression construct bearing the R213X (TGA) nonsense mutation were implanted subcutaneously on the lower back of immunocompromised NRG (NOD-RagJ""" IL2rg ^nuU ) mice. Panel A: When the tumour xenografts reached a size of approximately 0.2-0.5 cubic centimeters, the mice were injected intraperitoneally with saline, gentamicin Bi or USP gentamicin sulfate at the indicated doses for 5 consecutive days. 72 hours after the last injection, the mice were sacrificed and the amounts of truncated P53 (TR-P53) and full-length P53 (FL-P53) were determined by western analysis as described in Baradaran-Heravi et al. (2016). Panel B: When the tumour xenografts reached a size of approximately 0.2-0.5 cubic centimeters, the mice were injected intraperitoneally once with saline, gentamicin Bi or USP gentamicin sulfate. 48 hours after the last injection, the mice were sacrificed and the amounts of truncated p53 and full-length P53 were determined by western analysis. The amounts of full-length P53 relative to saline-treated mice are indicated under each lane. Vinculin was used as a protein loading control.

Induction of PTC Readthrough by Gentamicin Bi in Cells Derived from Patients with Rare Genetic Diseases.

Methods for FIGURE 9: Panels A and B: GM16485 primary fibroblasts derived from a Neuronal Ceroid Lipofuscinosis patient with compound heterozygous nonsense mutations in the TPPi (tripeptidylpeptidase I) gene (R127X/R208X) were exposed to 25 g/ml gentamicin Bi or 100 g/ml gentamicin for up to 10 days. Cell lysates were prepared and TPPi enzyme activity was determined as in Lojewski et al. (2014) with modifications: Lysates were diluted 1:5 in 50 mM sodium acetate pH 4.0 and pre- incubated at 37°C for l h. After pre-incubation, 20 μg of total protein from GM16485 lysates or 5 μg of total protein from lysates of fibroblasts from unaffected individuals (WT) was incubated in 150 μΐ of 50 mM sodium acetate pH 4.0 containing a final concentration of 62.5 μΜ Ala-Ala-Phe-7-amido-4-methylcoumarin for 2 h at 37°C.

Fluorescence was measured using a TECAN Infinite M200™ spectrophotometer with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Assays were carried out under conditions where product formation was linear with respect to protein concentration and time. TPPi activity was expressed relative to the average activity of untreated primary fibroblasts from two unaffected individuals (WT) (Panel A). For panel B, the same cell extracts were analysed for formation of TPPi by automated capillary electrophoresis western analysis using the Abeam™ ab54685 a-TPPi antibody as in Baradaran-Heravi et al (2016). Extracts from WT fibroblasts were also analysed, using 20% of the amount of protein used for GM16485.

Panel C: HSKooi myoblasts derived from a Duchenne Muscular Dystrophy patient with nonsense mutation {DMD: E2035X) were differentiated into myotubes and exposed to the indicated concentrations of gentamicin Bi or gentamicin for 3 days and dystrophin expression level was determined by automated capillary electrophoresis western analysis using Abeam™ abi52 _/ 7 a-dystrophin antibody. Extracts from WT myotubes were also analyzed, using 5% of the amount of protein used for DMD cells. Beta-actin was used as a loading control.

Panel D: SD123 fibroblasts from a patient with Schimke Immuno-Osseous Dysplasia, with a homozygous SMARCALi nonsense mutation (R17X) were exposed to the indicated concentrations of gentamicin Bl or gentamicin for 6 days and SMARCALi levels were determined by western blotting using an anti SMARCALi antibody provided by Dr. Cornelius Boerkoel (University of British Columbia). Extracts from WT fibroblasts were also analyzed, using 10% of the amount of protein used for SIOD cells. Beta-actin was used as a loading control.

Panel E: EB14 keratinocytes from a patient with Recessive Dystrophic Epidermolysis Bullosa, with a homozygous Q251X nonsense mutation on the COL7A1 gene were incubated with the indicated concentrations of gentamicin Bl or gentamicin for 72 h and cellular collagen 7 was measured by western blotting using EMD Millipore 234192 collagen 7 antibody. Extracts from WT keratinocytes were also analyzed, using 10% of the amount of protein used for EB14 cells.

Proposed Synthesis of Compounds

Synthesis of the gentamicin analogues (i.e. see Table B) is proposed via a- glycosylation of the pseudo-disaccharide comprising garosamine linked to deoxystreptamine, either chemically or enzymatically. This would require access to the selectively protected disaccharide in which the alcohol to be glycosylated is free while the other alcohols and amines are protected. One route to this disaccharide would involve first protection of all amines with a suitable blocking group known to one skilled in the art (Cbz, Boc etc), then protection of the syn-diol within the streptamine moiety using Ley's reagent. Subsequent protection of the remaining alcohols and selective removal of the Ley protecting group would leave the pseudo-disaccharide with two free alcohols. Glycosylation of this under conditions for generating 1,2-syn linked product (a -gluco in this case) would likely generate a mixture of the two glycosides from which the one of interest could be separated and protecting groups removed.

Alternatively the direct enzymatic glycosylation of the pseudo-disaccharide comprising garosamine linked to deoxystreptamine may be carried out using a variety of a-glycoside phosphorylases, a-glucosidases (run in trans-glycosylation mode) or available a-glucosyl transferases may prove successful. Large libraries of such enzymes are being assembled making such "screening approaches" feasible. If successful this synthesis may provide a remarkably simple and scalable synthetic route. EXAMPLES

EXAMPLE l: 53 Read-through Assay

Gentamicin, gentamicin A, B, Bi, Ci, Cia, C2, C2a, C2b, X2, sisomicin, as well as gentamicin fragments garamine and ring C (see FIGURE l) were tested for PTC read- through using the 96-well plate assay. For comparison, G418, a related aminoglycoside that is known to be potent inducer of PTC read-through was used as a positive control. G418 is not an approved drug.

As shown in FIGURE 2 Gentamicin did not induce PTC read-through at the concentrations tested, which did not exceed 200 uM. However, it is active at 3 mM as shown in FIGURE 3A. Gentamicin A, B, Ci, Cia, C2, C2a, C2b, sisomicin, garamine and ring C showed no activity whatsoever (data not shown). G418 showed activity in the 25- 200 μΜ concentration range. Gentamicin X2 showed activity, but it was less potent than G418. Gentamicin Bi showed strong activity, slightly more potent than G418. Therefore, the PTC read-through activity of gentamicin drug is due mostly to the presence of the minor components Bi and X2. Similarly, FIGURE 3B shows the induction of PTC read- through by G418, gentamicin, gentamicin Bi and gentamicin X2 using western analysis, wherein the amount of full-length Ρ53 observed in FIGURE 3A was plotted versus the concentration of the different compounds on a log scale.

The 96-well plate assay results were confirmed using western analysis as shown in FIGURE 3A, wherein HDQ-Pi cells contain very small amounts of p53 protein truncated at R213, and no full-length P53. Induction of PTC read-through causes the appearance of full length p53. Western analysis was performed using an automated quantitative capillary electrophoresis western system. The results confirm the 96-well plate assays and show that gentamicin Bi induces the appearance of full length p53 and that is more potent than G418 or X2. The activity of gentamicin at 3 mM is shown for comparison.

This result is important for medical applications of PTC read-through.

Gentamicin is known to be nephrotoxic and ototoxic. (Kohlhepp S. J. et al. 1984) have examined the nephrotoxicity of the major gentamicins C, Cia and C2 and found that nephrotoxicity was caused mainly by C2. Although it is not yet know to what extent gentamicin Bi might be nephrotoxic or ototoxic, it is anticipated that treatment of patients with gentamicin Bi would induce PTC read-through at lower doses than treatment with gentamicin, which typically contains only 0.5-3% Bi (MicroCombiChem™, personal communication). Treatment with gentamicin Bi instead of gentamicin should achieve both higher PTC read-through and lower toxicity via omission of toxic gentamicin

C2. Monitoring of gentamicin plasma concentrations is recommended to avoid toxicity. A cursory search indicates that plasma levels of gentamicin are typically between l and 12 g/ml (2-24 μΜ) and that concentrations above about 10 uM should be avoided during long-term treatment. The concentrations of gentamicin Bi showing read-through (3 μΜ and higher) are within this range.

EXAMPLE 2: 53 Read-through Assay with Steroids

As shown in FIGURE 4, G418 showed much improved PTC read-through at a concentration of 25 μΜ in combination with Dexamethasone (5 μΜ), Betamethazone (5 μΜ) or Medroxyprogesterone acetate (Medroxy ρπ))(5μΜ), whereas Dexamethasone, Betamethazone alone and Medroxy pro alone showed no read-through activity.

EXAMPLE 3 : p53 Read-through Assay with Steroids

The results presented in FIGURE 5 show that two gentamicin batches display low PTC readthrough activity at 1 mg/ml while a third batch was inactive (see FIGURES 5A, B, E and F - batch 2). The results also show that gentamicin Bi and gentamicin X2 display potent PTC readthrough activity (see FIGURES 5C-F).

EXAMPLE 4: Read-through Assay Comparing Stop Codons

The results in FIGURE 6 show that gentamicin Bi at 50 μg/ml and 100 μg/ml are induce PTC readthrough at all three premature termination codons (i.e. TGA, TAG and TAA). However, there appears to be a slight decrease in readthrough with the TAA stop codon.

EXAMPLE 5: Read-through Assays Comparing Cell Types

FIGURE 7 shows that gentamicin Bi can induce PTC readthrough in a variety of cancer cell lines having nonsense mutations at different positions in the TP53 gene (i.e. SW900; NCI-H1688; ESS-i; SK-MES-i; HCC1937; H1299; and HCT116). Gentamicin Bi consistently showed readthrough of the stop codons in various cancer cell lines.

EXAMPLE 6: In Vivo Read-through Assays

As shown in FIGURE 8 gentamicin Bi can induce premature termination codon readthrough in a tumour xenograft in vivo. Gentamicin Bi showed readthrough as low as 50 mg/kg (see FIGURE 8A), at 200 mg/kg and at 40omg/kg (see FIGURE 8B), whereas no readthrough was detected for gentamicin. No toxicity was observed for Bi but 400 mg/kg gentamicin induced acute toxicity and the mice had to be sacrificed shortly after administration, as denoted by the asterisks.

EXAMPLE 7: Induction of PTC Readthrough by Gentamicin Bi in Cells Derived from Patients with Rare Genetic Diseases FIGURE 9A and B show GM16485 primary fibroblasts derived from a Neuronal Ceroid Lipofuscinosis patient with heterozygous nonsense mutations in the TPPi

(tripeptidylpeptidase I) gene (R127X/R208X) where the fibroblasts were exposed to 25 μg/ml gentamicin Bi or 100 μg/ml gentamicin for up to 10 days and before the fluorescence of cell extracts were measured for TPPi activity was expressed relative to the average activity of untreated primary fibroblasts from two unaffected individuals (WT) (A). FIGURE 9B, shows the same cell extracts analysed for formation of TPPi by automated capillary electrophoresis western analysis. FIGURE 9C shows HSK001 myoblasts derived from a Duchenne Muscular Dystrophy patient with nonsense mutation (DMD: E2035X) were differentiated into myotubes and exposed to the indicated concentrations of gentamicin Bi or gentamicin for 3 days and subsequent dystrophin expression levels were determined by automated capillary electrophoresis western analysis as compared to WT myotubes and loading control. FIGURE 9D shows SD123 fibroblasts from a patient with Schimke Immuno-Osseous Dysplasia, with a homozygous SMARCALi nonsense mutation (R17X) exposed to the indicated concentrations of gentamicin Bi or gentamicin for 6 days before the SMARCALi levels were determined by western blotting as compared to WT fibroblasts and loading control. FIGURE 9E shows EB14 keratinocytes from a patient with Recessive Dystrophic Epidermolysis Bullosa, with a homozygous Q251X nonsense mutation on the COL7A1 gene incubated with the indicated concentrations of gentamicin Bl or gentamicin for 72 h prior to cellular collagen 7 measurement by western blotting as compared to WT keratinocytes. In all of the tested genetic diseases gentamicin Bi induced readthrough.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. References

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